, 2010, 1, 1-47 · 2 Synthesis, Characterization and Anti-Angiogenic Effects of Novel 5-Amino Pyrazole Derivatives on Ehrlich Ascites Tumor [EAT] Cells in-Vivo teins play a crucial

Journal of Cancer Therapy, 2010, 1, 1-47 Published Online March 2010 in SciRes (http://www.SciRP.org/journal/jct/).

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TABLE OF CONTENTS

Volume 1 Number 1 March 2010 Synthesis, Characterization and Anti-Angiogenic Effects of Novel 5-Amino Pyrazole Derivatives on Ehrlich Ascites Tumor [EAT] Cells in-Vivo

H. Raju, S. Chandrappa, M. K. Ramakrishna, T. S. Nagamani, H. Ananda, S. M. Byregowda, K. S. Rangappa……………………1

Adjuvant Treatment for High-Risk Operable Prostate Cancer B. Paule, N. Brion…………………………………………………………………………………………………………………10

Novel Ethyl 2-(1-aminocyclobutyl)-5-(benzoyloxy)-6-hydroxy-pyrimidine-4-carboxylate Derivatives: Synthesis and Anticancer Activities

D. Asha, C. V. Kavitha, S. Chandrappa, D. S. Prasanna, K. Vinaya, S. C. Raghavan, K. S. Rangappa……………………………21

Transfusion of Ipscs-Derived Leukocytes to Kill Cancer J. Li, Y. Cui, G. D. Gao, T. F. Yuan………………………………………………………………………………………………29

Ovarian Sex Cord-Stromal Tumors in Postmenopausal Women and Total Laparoscopical Management A. Tinelli, M. Pellegrino, V. E. Chiuri, A. Malvasi………………………………………………………………………………31

Sum-Based Meta-Analytical Enrichment of Gene Expression Data to Identify Pathway Signatures of Cancers

K. Wagholikar, P. Venkatraman, S. Vijayraghavan, C. Kumar-Sinha……………………………………………………………36

Role of Estradiol, Progestins, Insulines and Adipocytokines in Breast Cancer Promotion in Post-Menopausal Women

C. Jamin…………………………………………………………………………………………………………………………43

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Journal of Cancer Therapy, 2010, 1: 1-9 doi:10.4236/jct.2010.11001 Published Online March 2010 (http://www.scirp.org/journal/jct)

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1

Synthesis, Characterization and Anti-Angiogenic Effects of Novel 5-Amino Pyrazole Derivatives on Ehrlich Ascites Tumor [EAT] Cells in-Vivo

H. Raju1, S. Chandrappa1, M. K. Ramakrishna1, T. S. Nagamani1, H. Ananda2, S. M. Byregowda2, K. S. Rangappa1*

1Department of Studies in Chemistry, University of Mysore, Manasagangotri, India; 2Institute of Animal Health and Veterinary Biologicals, Hebbal, India. Email: [email protected], [email protected] Received November 11th, 2009; revised December 14th, 2009; accepted December 25th, 2009.

ABSTRACT

In search of synthetic chemotherapeutic substances capable of inhibiting, retarding, or reversing the process of multi-stage carcinogenesis, we synthesized a series of novel 5-amino pyrazole derivatives 11(a-h) by a nucleophilic substitution reaction and characterized by 1H nuclear magnetic resonance (NMR), liquid chromatography mass spec-trometry (LC/MS), Fourier-transform infrared (FTIR), and elemental analysis. These novel compounds were evaluated for their efficacy in inhibiting Ehrlich ascites tumor [EAT] cells in-vivo. In the present study we designed, synthesized, characterized and investigate the anti-angiogenic effects of these compounds, on Ehrlich ascites tumor [EAT] cells in-vivo. The compounds were subsequently tested for their ability to inhibit neovascularisation in chorio allantoin membrane (CAM) model. From the Structure Activity Relationship (SAR) studies, it reveals that, the substitution at N-terminal in pyrazole ring plays key role in the antitumor and anti-angiogenic effects. Keywords: 1H-Pyrazol, Aryl Isothiocyanates, Ehrlich Ascites Tumor [EAT] Cells, Anti-Angiogenesis

1. Introduction

Angiogenesis, a physiological process involving the growth of new blood vessels from pre-existing vessels, contributes to the development and progression of vari-ous pathological conditions including tumor growth and metastasis, cardiovascular diseases, inflammatory disease and psoriasis. Down-regulation of angiogenesis has been considered to be advantageous for prevention of neoplas-tic growth and inflammation. Currently, anti-angiogenic strategies are based on inhibition of endothelial cell pro-liferation, interference with endothelial cell adhesion and migration, and interference with metalloproteinases [1].

Many researchers have been trying to screen novel anti-angiogenic principles from various natural products. Tumors can make and release many chemicals that can start angiogenesis. Using a drug that targets only one of these chemicals may not have a large effect on the cancer, but combining drugs that attack different targets may prove to be more useful. Synthetic compounds are used to control the advanced stages of malignancies but most of the compounds exhibit normal tissue toxicity with

undesirable side effects and development of drug resis-tance is the major clinical problem.

Indian System of Medicine has many herbal prepara-tions with versatile medicinal properties. In common with many other low-molecular weight phenolic com-pounds, vanillin displays antimicrobial and antioxidant properties and hence has the potential for use as a food preservative [2]. The chemo preventive effects of vanillin have been attributed to various biological properties in-cluding neutralization of carcinogenic free radicals [3] and anti-angiogenesis action [4,5]. There is some evi-dence for anti-mutagenic effects of vanillin, for example in suppressing chromosomal damage induced by meth-otrexate in the Chinese hamster V79 cell line [6]. Most studies have addressed the prognostic significance of VEGF (Vascular endothelial growth factor) expression [7]. Pyrazoles and several N-substituted pyrazoles are known to possess numerous chemical, biological, medici-nal, and agricultural applications because of their versatile biological activities like antimicrobial activity [8], antitu-mor and antileukemia activity [9], antidepressant and an-ticonvulsant [10]. Amides are ubiquitous in life, as pro-

Synthesis, Characterization and Anti-Angiogenic Effects of Novel 5-Amino Pyrazole Derivatives 2 on Ehrlich Ascites Tumor [EAT] Cells in-Vivo

teins play a crucial role in virtually all biological processes such as enzymatic catalysis, transport or storage, immune protection, and mechanical support. An in-depth analysis of the comprehensive medicinal chemistry database re-vealed that the carboxamide group appears in more than 25% of known drugs. Recently we have reported the syn-thesis and anti-angiogenisis studies of bioactive heterocy-cles [11].

In the future, such drugs may blur the line between anti-angiogenesis drugs and other forms of cancer treat-ment. Researchers are now looking at many different aspects of anti-angiogenesis drugs. Better understanding of these drugs will probably make them a bigger part of cancer treatment in the future. Hence there is a need to discover novel compounds that selectively kill cancer cells. Recently, attention has been toward the drug de-rived from plant sources which are non-toxic and acces-sible to common man. Hence attempts are made to syn-thesize compounds of natural origin.

2. Materials and Methods

2.1 Chemicals and Reagents

Unless otherwise mentioned all the chemicals used in the present study were from Sigma–Aldrich, USA. Melting points were determined using SELCO-650 hot stage melt-ing apparatus and were uncorrected. Infrared (IR) spectra were recorded using a Jasco FTIR-4100 series. Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were recorded on Shimadzu AMX 400-Bruker, 400 MHz spec-trometer using CDCl3 as solvent and TMS as internal standard (chemical shift in ppm). Spin multiples are given as br s (broad singlet), d (doublet), t (triplet) and m (multiplet). Mass and purity were recorded on a LC/ MSD-Trap-XCT. Elemental (CHN) analyses were ob-tained on Vario EL III Elementar. Silica gel column chro-matography was performed using Merck 7734 silica gel (60–120 mesh) and Merck made TLC plates. Substituted (E)-1H-pyrazol-5-amine 11(a-h) derivatives were synthe-sized by the method summarized in Figure 1.

2.1.1 Synthesis of 3-Methoxybenzaldehyde (3) A solution of 4-hydroxy-3-methoxybenzaldehyde (1) (2.0 g, 6.12 mmol) in N, N-dimethyl formamide (20 mL) was taken, anhydrous potassium carbonate (4.2 g, 30.6 mmol) was added and stirred for 20-30 min and then 5-(bromomethyl)-1, 2, 3-trimethoxybenzene (2) (2.69 g, 13.4 mmol) was added. The reaction mixture was stirred for 6 h at room temperature, and monitored by TLC. Upon completion, the solvent was removed under re-duced pressure; residue was taken in water and extracted with ethyl acetate. Finally water wash was given to the organic layer and dried with anhydrous sodium sulphate. The solvent was evaporated to get crude product which

C

OH

H O

O

O+

Br

O

10(a-h)

+

CH2CNO(v)

O

O

O

O

C

O

H N

2

HN Boc

(iii)(ii)O

O

O

O

O

NH

HN Boc

O

O

O

O

O

NH

HN NH2Cl

(iv)

O

O

O

O

O

NN

H2N

1 3 4

6

7

5

8 9

(i)

(vi)

O

O

O

O

C

O

H O

H2NHN Boc+

O

O

O

O

O

NN

NHN

HR

S

11 (a-h)

NO2

Cl

Cl

F

F

(10a)=

(10b)=

(10c)=

(10d)=

(10e)=

(10f)=

(10g)=

(10h)=

R=

O

NO2

F

Cl

F

Cl

Figure 1. Reaction and reagent condition (a) K2CO3, DMF/ r.t, 20–30 min (b) EtOH /r.t, 2–3 h (iii) 10% Pd/c / H2

EtOAc, r.t, 3h (c) dichloromethane, ether in HCl, 3 h (d) EtOH/EtCOONa, reflux 80oC, 2–3 h (e) triethylamine, di-chloromethane, aryl isothiocyanates 10(a-h), r.t, 6–7 h was purified by column chromatography over silica gel (60–120 mesh) using chloroform: methanol (9:1) as an eluent.

2.1.2 Synthesis of 1H-Pyrazol-5-Amine (9) Initially mono boc protected 3-methoxy-benzylidene hydrazine (5) was synthesised by the condensation reaction of 3-methoxy-benzaldehyde (3) (1.0 g, 9.84 mmol) with mono boc protected hydrazine (4) (1.0 g, 15.26 mmol). The subsequent double bond reduction was done by using 10% Pd/c in ethanol yielded mono boc protected 3-methoxybenzyl hydrazine (6). The deprotec-tion of amine group was carried out by using HCl in ether gave free amine compound (7). Finally the key in-termediate 5-amino pyrazole (9) by the cyclisation of 3-methoxybenzyl hydrazine salt (7) (1.0 g, 5.36 mmol) and 3-cyclopropyl-3-oxopro-panenitrile (8) (0.85 g, 5.36 mmol) were taken in ethanol, and then sodium ethoxide (1.09 g, 16.0 mmol) was added. The reaction mixture was refluxed for 2–3 h. The progress of the reaction was monitored by TLC.

2.1.3 General Procedure for the Synthesis and Characterization of 5-Amino Pyrazole Derivatives 11(a-h)

To the solution of intermediate compound 9 (1 eq) in dichloromethane, triethylamine (3 eq) was added and




3

cooled to 0–5 °C in an ice bath. Respective aryl isothio- cyanate 10(a-h) (1 eq) were added at cold condition and stirred at room temperature for 6–7 h. The progress of the reaction was monitored by TLC. Upon completion of reaction water was added to reaction mixture and ex-tracted with ethyl acetate. The organic layer was washed

with 10% ammonium chloride solution followed by wa-ter wash and dried with anhydrous sodium sulphate. The solvent was evaporated and the crude product obtained was purified by column chromatography over silica gel (60–120 mesh) using hexane: ethyl acetate (8:2) as an eluent. Characterization of novel 5-amino pyrazole

Table 1. Elemental analysis, mp/(oC), physical state and yield of compounds 3, 9, 11(a–h)

Elemental analysis, Anal. Found (calcd)/%

Compound Appearance m.p/(oC) Formula yield/% C H N

3 white solid 221-226 C18H20O6 95 65.10(65.03) 6.04(6.02) - -

9 white solid 234-233 C24H29N3O5 93 65.59(65.48) 6.64(6.62) 9.56(9.51)

11a white solid 254-256 C31H32N6O9S 89 56.02(56.01) 4.85(4.82) 9.64(9.61)

11b white solid 245-246 C31H34N4O5S 91 64.79(62.76) 5.96(5.93) 9.75(9.71)

11c white solid 263-265 C31H33ClN4O5S 88 61.10(61.08) 5.46(5.43) 9.18(9.11)

11d white solid 254-255 C31H33ClN4O5S 85 61.13(61.11) 5.48(5.45) 9.17(9.14)

11e white solid 248-249 C31H32F2N4O5S 81 60.97(60.08) 5.28(5.22) 9.17(9.11)

11f white solid 255-258 C31H33FN4O5S 84 62.82(62.80) 5.61(5.61) 9.45(9.41)

11g white solid 273-275 C31H32Cl2N4O5S 79 57.85(57.83) 5.01(5.0) 8.71(8.11)

11h white solid 267-268 C31H33FN4O5S 80 62.88(62.86) 5.67(5.63) 9.42(9.41)

Table 2. 1H NMR and IR data of compounds 3, 9, 11(a–h)

Compound 1H NMR (CDCl3, 400 MHz) δ and IR (KBr, cm-1) data

3 8.95 (s, 1H, -OH), 7.92 (dd, 1H, J = 2, 8.5 Hz, Ar-H), 7.18 (s, 1H, Ar-H), 6.82 (d, 1H, J = 8.5 Hz, Ar-H), 6.35 (br s, 1H, Ar-H), 6.15(br s, 1H, Ar-H), 5.71 (m, 2H, -CH2) 3.83 (s, 3H, -OCH3), 3.70 (s, 3H, -OCH3), 3.68 (s, 6H, -OCH3). 3328, 2912, 1665, 1583, 1502, 1443, 1266, 1124, 1017.

9 6.54 (s, 1H, Ar-H), 6.51 (dd, 1H, J = 2, 8.5 Hz, Ar-H), 6.46 (s, 1H, Ar-H), 6.15 (d, 2H, J = 6.5 Hz, Ar-H), 4.99 (m, 2H, -CH2), 3.95 (br s, 2H, -NH2), 1.85 (m, 1H, -CH), 0.92 (m, 2H, -CH2), 0.71 (m, 2H, -CH2). 3448, 2998, 2942, 2626, 1667, 1616, 1516, 1506, 1257, 1129, 1024, 1003, 924, 818, 772.

11a 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH), 7.65 (s, 1H, Ar-H), 7.11 (s, 1H, Ar-H), 7.52 (s, 1H, Ar-H), 6.1 (br s, 1H, Ar-H), 5.21-5.18 (m, 2H, -CH2), 3.83 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.68 (s, 6H, -OCH3). 3448, 3318, 2998, 2902, 1665, 1584, 1506, 1455, 1414, 1276, 1123, 1027, 925, 851, 722.

11b 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH), 6.78 (s, 1H, Ar-H), 6.87 (s, 2H, Ar-H), 6.62 (s, 1H, Ar-H), 6.60 (s, 1H, Ar-H), 6.1 (br s, 1H, Ar-H), 5.21-5.18 (m, 2H, -CH2), 3.83 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.70 (s, 3H, -OCH3), 3.68 (s, 6H, -OCH3). 3001, 2929, 1665, 1598, 1583, 1506, 1272, 1127, 1028, 992, 862, 836, 778.

11c 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH), 7.52 (s, 1H, Ar-H), 6.89 (s, 1H, Ar-H), 6.56 (s, 1H, Ar-H), 6.40 (s, 1H, Ar-H), 6.1 (br s, 1H, Ar-H), 5.21-5.18 (m, 2H, -CH2), 3.83 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.68 (s, 6H, -OCH3). 3068, 2942, 1676, 1584, 1510, 1411, 1257, 1222, 1126, 1035, 913, 837, 714.

11d 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH), 6.95 (s, 1H, Ar-H), 6.63 (s, 1H, Ar-H), 6.47 (s, 1H, Ar-H), 6.34 (s, 1H, Ar-H), 6.1 (br s, 1H, Ar-H), 5.21-5.18 (m, 2H, -CH2), 3.83 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.68 (s, 6H, -OCH3). 3448, 2941, 1666, 1583, 1536, 1413, 1266, 1128, 1088, 1015, 996, 830, 730.

11e 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH), 6.72 (s, 1H, Ar-H), 6.21 (s, 1H, Ar-H), 6.18 (s, 1H, Ar-H), 6.1 (br s, 1H, Ar-H), 5.21-5.18 (m, 2H, -CH2), 3.83 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.68 (s, 6H, -OCH3). 3448, 3318, 2998, 2935, 1769, 1675, 1667, 1588, 1462, 1411, 1278, 1249, 1159, 1124, 1023, 994.

11f 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH), 6.72 (s, 2H, Ar-H), 6.44 (s, 2H, Ar-H), 6.13 (s, 1H, Ar-H), 6.1 (br s, 1H, Ar-H), 5.21-5.18 (m, 2H, -CH2), 3.83 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.70 (s, 3H, -OCH3), 3.68 (s, 6H, -OCH3). 3316, 2937, 1767, 1672, 1588, 1503, 1460, 1413, 1245, 1169, 1126, 1025, 991.

11g 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH), 6.96 (s, 1H, Ar-H), 6.41 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 6.1 (br s, 1H, Ar-H), 5.21-5.18 (m, 2H, -CH2), 3.83 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.68 (s, 6H, -OCH3). 3448, 3207, 2998, 2942, 1667, 1616, 1516, 1506, 1278, 1257, 1129, 1024, 1003, 924, 818, 772.

11h 8.20–8.16 (dd, 1H, -NH), 8.06 (s, 1H, -NH), 6.78 (s, 1H, Ar-H), 6.72 (s, 1H, Ar-H), 6.60 (s, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 6.1 (br s, 1H, Ar-H), 5.21-5.18 (m, 2H, -CH2), 3.83 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.70 (s, 3H, -OCH3), 3.68 (s, 6H, -OCH3). 3318, 2935, 1769, 1675, 1588, 1506, 1462, 1411, 1249, 1159, 1124, 1023, 994.


derivatives are tabulated in Tables 1 and 2.

2.2 Biology: in-Vivo Anti-Cancer and Angio-Inhibitory Effects of Synthetic Novel 5-Amino Pyrazole Derivatives 11(a-h)

Animals and tumor model: Inbred Swiss albino mice, 6–8 weeks old, weighing 25 ± 5 g of either sex, were used for the experiments. They were bred and maintained in the animal house, Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore, India. Ehrlich ascites tu-mor was grown in adult Swiss albino mice intraperitoneally (ip). Cell viability was tested by trypan blue exclusion as-say. Experimental animals were prepared by injecting 5 × 106 viable tumor cells into intraperitoneal cavity of Swiss mice. Tumor growth was followed by recording the animal weights. EAT cells begin their exponential growth phase from the 7th day after tumor cell injection and the animal succumbs to the ascites tumor burden on day 16–20 after injection.

Compounds: Synthetic 5-amino pyrazole derivatives 11(a-h) were used as compounds for the experiments. The compounds were weighed and dissolved in 0.1% DMSO to get required concentrations. The compound treatments were initiated on the day 7 of tumor transplantation on the advanced stage of tumor when the cells enter into exponen-tial growth period.

Animal survival: After 7 days of tumor cell injection, the animals were divided into groups of 10 each and were treated as follows: control: 0.2 ml of 0.1% DMSO was given on day 7, 9, and 11 of tumor transplantation. Compound treated groups- the compounds 11(a-h) were given to different groups of tumor bearing mice. The compound 100 mg/kg body wt was injected intraperito-neally (ip) into the mice using 26 gauge needle on day 7, 9, and 11 of tumor transplantation. All the mice were weighed on the day of tumor inoculation and at weekly intervals. Animal survival was recorded up to 40 days. The tumor response was assessed on the basis of MST and increase in life span (% ILS). Median survival time (MST) and % ILS were calculated from the mortality data within the observation period. Increase in life span was calculated by the formula. % ILS = MST of treated group - MST of control group×100

MST of control group Enhancements of life span by 25% are more over that of the control was considered as effective antitumor response [12].

Tumor growth inhibition and anti-angiogenesis: After 7 days of tumor cell injection, the animals were divided into eight groups of 10 each and the control group received 0.2 ml of 0.1% DMSO on day 7, 9, and 11 of tumor transplantation. The compounds 11(a-h) were given to eight different groups of tumor bearing mice.

The tumor inhibitory effects of the compounds on EAT cell growth were assessed by measuring cell number and ascites volume. On day 12 injected 0.5ml saline into abdomen. The control and compounds treated tumor bearing mice were sacrificed, an incision was made in the abdominal region and EAT cells along with the ascites fluid were harvested into a beaker containing 2 ml saline and centrifuged at 3000 rpm for 10 min at 4 0C. Subtracting the volume of saline added previously from the volume of the supernatant gave the volume of ascites fluid. After harvesting the EAT cells, the cells were resuspended in 0.9% saline and counted using a haemocytometer.

Changes in the morphology of EAT cells with Giemsa staining: EAT cells from the control and treated groups 11(a–h) were smeared on clean glass slides, air-dried, and fixed in a solution of methanol/acetic acid (3:1). The slides were hydrated with PBS, then stained with 0.1% Giemsa solution, and observed under compound micro-scope.

Acridine orange/ethidium bromide staining: Nuclear staining was performed according to the method of Srinivas [13]. The EAT cells collected from both control and compounds 11(a–h) treated groups were smeared on clean glass slides, air-dried, and fixed in a solution of methanol/acetic acid (3:1). The slides were hydrated with PBS, and then stained with mixture of acridine or-ange/ethidium bromide (1:1). The cells were immediately washed with PBS and viewed under fluorescent micro-scope and photographed.

Anti-angiogenic effects of the compounds in peritoneal angiogenesis: The peritoneum of the mice was cut open and the inner lining of the peritoneal cavity were exam-ined for angiogenesis in both control and compounds 11(a–h) treated tumor bearing mice and photographed.

Histopathology of mice peritoneum tissue: Peritoneal tissues from tumor bearing control mice and mice treated with compounds 11(a–h) were fixed in 10% formalin, embedded in paraffin, and 5-μm sections were routinely stained with haematoxylin and eosin. The sections were observed under low power (10X) of light microscope to identify the highly vascularized areas. The micro vessel density (MVD) was counted in 10 fields of these vascu-larized areas under high power (40X) and the average MVD/HPF was noted.

Angioinhibitory effects of the compounds on in vivo chorio allantoin membrane assay: CAM was performed according to the method of Chandru and Sharada [14]. The fertilized eggs were divided into different treatment groups. Control, the saline treated group, and compound treated groups with minimum of six eggs in each group were maintained separately and observed. The fertilized eggs were incubated for 5 days at 37 0C in a humidified and sterile atmosphere. A window was made under aseptic conditions on the egg shell to check for the proper de-


Synthesis, Characterization and Anti-Angiogenic Effects of Novel 5-Amino Pyrazole Derivatives 5on Ehrlich Ascites Tumor [EAT] Cells in-Vivo

velopment of the embryo. The windows were resealed and incubation was continued. On day 11 the windows were opened and the compounds 11(a–h) (0.1 mM) or vehicle was loaded on the cover slips separately, air-dried, and inverted over the CAM and the windows were closed. The windows were resealed and the embryo was allowed to develop further. The windows were opened and observed on day 13 and inspected for changes of MVD in the area under cover slip and exam-ined under a microscope for avascular zone and photo-graphed.

Statistical analysis: All data were analyzed by one-way ANOVA. Values of p < 0.05 were considered significant.

3. Results

3.1 Tumor Growth Inhibition of Ehrlich Ascites Tumor in-Vivo

Animal survival (Table 3): The vehicle 0.1% DMSO did not have any effect on the tumor growth. All the animals in vehicle treated controls developed tumor and died within 16–20 days. The median survival time (MST) was 18 days. All the treatments produced significant increase in MST and % ILS compared to control (p < 0.01). Three doses of 11(a-h) treatments on 7, 9, and 11 days after tumor transplantation showed effective antitumor re-sponse (>25% ILS) and resulted in 48%, 34%, and 28% ILS, respectively. However the compound 11a exhibited higher tumor inhibitory effect and showed 32 days of MST with 77% ILS. The weight changes were signifi-

Table 3. Effect of synthetic novel 5-amino pyrazole deriva-tives 11(a-h) on survival of mice bearing Ehrlich ascites tumor

Treatment groups MST (days)a ILS (%) Av. wtb changes

1. Control (0.1% DMSO)

18 -------- + 11.32

2. 11a-100 32.8** 77.7 - 4.4**

3. 11b-100 28.7** 68.2 - 3.2**

4. 11c-100 27.5** 70.6 - 2.8**

5. 11d-100 28.2** 71.9 - 2.9**

6. 11e-100 31.2** 77.2 - 4.2 **

7. 11f-100 29.6** 73.2 - 3.1**

8. 11g-100 30.3** 76.8 - 3.6 **

9. 11h-100 23.9** 63.8 - 2.3**

11(a–h)-100 indicates dose in mg/kg body wt. Vehicle or compounds were administered on day 7, 9, and 11 after tumor cell inoculation to different treatment groups of 10 animals each. aMedian survival time (MST) and ILS % was calculated from the mortality data within the observation period. bDetermined on 12th day of treatment.** Significant from control (p < 0.01).

cantly higher in compound treated groups compared to control (Table 3) indicating the effect of the compounds in preventing the tumor growth. The treated groups showed reduction in body weight due to decrease in tu-mor burden however no side effects were observed.

3.2 Ascites Volume and Cell Number (Figure 2)

The inhibitory effect of 11(a-h) on EAT cells in vivo was further examined in terms of total volume of ascites and number of cells in mice treated with vehicle or compounds. The mean value of cell number and ascites volume in con-trol animals was found to be 1820.30 ± 0.65×106 cells/ mouse (Figure 2(a)) and 9.24 ± 0.30 ml, respectively (Figure 2(b)). All the compound treatments showed sig-nificant decrease in ascites volume and cell number com-pared to control (p<0.01). The compound 11a treated group which demonstrated highest % ILS (Table 3) showed maximum reduction in cell number and ascites volume compared to other compounds treated groups.

3.3 Changes in the Morphology of EAT Cells (Figure 3)

(a)

(b)

Figure 2. Effects of synthetic compounds 11(a-h) on ascites volume and cell number of EAT bearing mice. The bar graph represents the effect of the compounds on ascites volume (a) and cell number (b). All the treatments showed a significant difference in ascites volume and cell number from the control and DMSO (p < 0.01). The error bars rep-resent standard deviation of the mean



(a)

(b)

Figure 3. Changes in the morphology of EAT cells after treatment with synthetic 5-amino pyrazole derivatives. EAT cells with or without compounds 11(a-h) treatment in-vivo were washed with PBS, fixed, and stained with Giemsa stain (a) or with mixture of acridine orange: ethidium bromide (b). The cells were viewed under compound or fluorescent mi-croscope and photographed. The apoptotic bodies and con-densed nuclei are evident in the compound treated groups The inhibitory effect of synthetic 11(a-h) on EAT cell growth may be due to induction of apoptosis. The EAT cells were stained with Geimsa or with nuclear stain (ac-ridine orange: ethidium bromide) and the slides were observed under microscope and photographed. The apoptotic bodies and nuclear condensation are evident in compound treated groups.

3.4 Inhibition of Tumor Induced Neovascularisation (Figures 4(a) and 5)

Significant inhibitions of blood vessel formation were ob-served in the peritoneal wall of compound treated mice compared to control (Figure 4(a)). The MVD (Figure 5) studies using haematoxylin and eosin stained peritoneal wall sections demonstrated significant decrease in MVD count in all the compounds 11(a–h) treated groups com-pared to control (p < 0.01). The strong angioinhibitory effect of the compounds was evident in this study.

3.5 Angioinhibitory Effects on CAM Assay (Figure 4(b))

The anti-angiogenic activity was evaluated by observing the formation of avascular zone under the cover slip (Figure 4(b)), control CAM treated with 0.1% DMSO showed no changes in vasculature. The MVD decreased in the entire compounds 11 (a-h) treated CAM model.

4. Discussion

The basic principle of cancer chemotherapy is to use drugs that have targets and preferably non overlapping toxicity. Thus a logical chemotherapeutic strategy is the

(a)

(b)

Figure 4. Suppression of angiogenesis in-vivo by compounds 11(a-h). Peritoneal lining of tumor bearing mice treated with vehicle (0.1% DMSO) and were inspected for anti-angiogenesis effects. (a) Inhibitions of angiogenesis were prominent in compound treated mice compared to control. (b) CAM assay model-compounds 11(a-h) or the vehicle was applied on the CAM of 11 days old chick em-bryo. Decreased vasculature was observed in treated groups compared to control. Dotted circles indicate the area cov-ered by the cover slip

(a)

(b)

Figure 5. Histopathology and MVD count of the peritoneal wall sections of EAT bearing mice with or without in-vivo treatment of compounds 11(a-h). The arrow indicates the blood vessel present in section (a). Haematoxylin and eosin staining of peritoneal wall sections were observed to study MVD. The bar graph shows the effect of compounds on MVD count (b) of the peritoneal section of tumor bearing mice. All the compound treatments resulted in a significant decrease in MVD count compared to control (p < 0.01). The error bars represent standard deviation of the mean


Synthesis, Characterization and Anti-Angiogenic Effects of Novel 5-Amino Pyrazole Derivatives 7on Ehrlich Ascites Tumor [EAT] Cells in-Vivo

combined use of apoptosis inducing compounds and cy-totoxic agents [15–17]. Examination of 11(a-h) aromatic regions might be critical for potential ligand-receptor bind-ing. A reasonable approach following standard medicinal chemistry design concepts was to explore compounds with systematic differences in the carbon chain connect-ing to the aromatic regions and substitution of cyclopro-pyl ring and the nitro, chloro, fluoro phenyl groups in pyrazole ring led to the synthesis of eight novel com-pounds. The results on Ehrlich ascites tumor [EAT] showed that the administration of 11(a-h) on day 7, 9, and 11 after tumor cell inoculation produced effective antitumor response (>25% ILS). The compounds also exhibited corresponding reduction in mean ascites volume and cell number. Our findings demonstrated the potent anti-angiogenic activity of the 5-amino pyrazole derivatives against EAT in-vivo. The compounds effectively reduced the ascites tumor burden and produced no side effects. Although the antitumor property of pyrazole and its synthetic derivatives on in-vivo and in-vitro studies has been reported earlier [18]. Morphology of EAT cells after compound treatments exhibited apoptotic bodies, nuclear condensation, and intra nucleosomal fragmentation. These results indicate that the compounds 11(a–h) induced inhibition of EAT cell growth was due to the induction of apoptosis in EAT cells. As the compounds are injected (ip) directly into the peritoneal cavity where the tumor is growing, the effect would be immediate and direct on the tumor cells. In the control EAT bearing mice; extensive peritoneal angiogenesis was observed which may be due to the secretion of the angiogenesis inducing factors in the ascites fluid. Involvement of VEGF in the formation of malignant ascites has been well documented [19]. Treatment of synthetic 11(a–h) to EAT bearing mice significantly decreased peritoneal angio-genesis suggesting the inhibition of the secretion of such factors and thereby preventing the formation of new blood vessels. Micro vessel density (MVD) counts have become the morphological gold standard to assess the neovasculature in tumors. MVD counts are reflective of the angioarchitectural properties of the tumor in that they are a representative of the average intercapillary distance. This is in fact an important parameter as it is the goal of an anti-angiogenic tumor therapy to reduce the intercapillary distance to a degree that it becomes rate-limiting for the growth of the tumor [20]. Haematoxylin and eosin staining of peritoneal wall tissue sections of EAT bearing mice treated or untreated with the compounds 11(a–h) were examined for the MVD count. Angiogenesis is clearly evident in the inner peritoneal lining of EAT bearing mice and it is a reliable model for in-vivo angiogenesis. Hence the peritoneal linings of treated mice verified for its effect on microvasculature when compared to untreated EAT bearing mice (Figure 4(a)). In the CAM

assay model compound 11a, 11e and 11g induced vasculature zone formation in the developing embryos. Notably, a newly formed microvessel was regressed around the compounds implanted disc (Figure 4(b)). Quantification of VEGF (Figure 6) shows that compounds 11(a-h) has dose dependent effect on secretion of VEGF under in-vivo conditions compared to the untreated EAT bearing mice. The amount of VEGF increased in untreated EAT cells over the growth period, whereas the amount of VEGF in ascites of compounds 11(a-h) treated EAT cells did not show any significant increase in the same growth period, suggesting a dose dependent inhibition of VEGF secretion upon compounds treatment in EAT cells. Fur-ther, VEGF is considered to be one of the major stimula-tors of tumor angiogenesis [21].

The compounds 11(a–h) with minor structural differ-ences has exhibited varying degree of tumor growth in-hibition and anti-angiogenic activities against EAT in-vivo. In the present findings, the compound 11a with the bioactive nitro, cyclopropoxy and methoxy groups at ortho, para and meta-positions in rings A and B showed potent in-vivo antitumor and anti-angiogenic activities against mouse tumor, Electron withdrawing groups such as dinitro, difluoro and dichloro at para and meta position in 11a, 11e and 11g showed relatively significant activity, whereas 11(c, d, f, h) groups having fewer electrons withdrawing groups of chloro (ortho), (meta) and fluoro (para), (ortho) showed moderate activity. On the other hand, as the electron withdrawing efficiency increases, the activity also increases. Compound 11b without sub-stituent on the phenyl ring of aryl isothiocyanates showed poor activity. We have briefly investigated the different structure-activity relationships (SAR) of the aryl isothio cyanates 11(a-h) functionalized derivatives with different groups added on the phenyl ring. These modifications change the potency of anti-angiogenic ac-tivity profile of the synthesised compounds. Thus the structural modifications have profound influence on an-titumor and angioinhibitory activities of compounds. From our findings we can conclude that 11a > 11e > 11g

Figure 6. Effect of compounds on in-vivo production of VEGF. The bar graph shows the amount of VEGF in ascites of compounds 11(a-h) treated EAT cells



novel 11(a-h) can be considered as promising antitumor and anti-angiogenic compounds. Thus our results show that compounds having electron withdrawing groups have more potent activity. Hence, we compare the results within same electron withdrawing nitro, fluoro and chloro group present at different position. Currently, a large variety of chemotherapeutic drugs are being used to treat cancer. Unfortunately, many compounds hold lim-ited efficacy, due to problems of delivery and penetration, and a moderate degree of selectivity for the tumor cells, thereby causing severe damage to healthy tissues. From our studies, it is clear that compound 11a, 11e and 11g has antiangiogenic effect as shown by peritoneal angio-genesis assay, chorioallantoic membrane (CAM) assay, and also from the reduction in the EAT cell number, as-cites volume, and body weight of the animals in-vivo. The above study shed light toward the identification of new anti-angiogenic molecules to the cancer therapy. Further research to know the mechanism of inhibition and the modifications of the compounds 11a, 11e and 11g to improve the potency is currently under progress in our laboratory.

5. Acknowledgement

One of the authors H.Raju is grateful to UGC Research Fellowships in Sciences for Meritorious Students Scheme (RFSMS), New Delhi for financial support under RFSMS-JRF order No. DV5/373[13]-/RFSMS/ 2008–09. The 1H and 13C, CHN, IR and other data obtained from the instruments purchased under DST-FIST and UGC-SAP (phase II) Programmes are greatly acknowl-edged.

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Adjuvant Treatment for High-Risk Operable Prostate Cancer Bernard Paule1, Nathalie Brion2,3

1AP-HP, Groupe Henri Mondor-Albert Chenevier, Service Oncologie, Créteil, France; 2Centre Hospitalier de Versailles, Hôpital André Mignot, unité Thérapeutique, Le Chesnay, France; 3UFR de Médecine Paris Ile-de-France Ouest, Université Versailles Saint Quentin en Yvelines, Guyancourt, France. Email: [email protected] Received December 14th, 2009; revised December 25th, 2009; accepted December 28th, 2009.

ABSTRACT Patients who have undergone a radical prostatectomy may have to face high risks of recurrence. The risk of recurrence is elevated due to probable occult metastatic disease at the time of diagnosis. A rationale for using multimodal ap-proach in order to minimize the chance of disease recurrence and to improve the survival of high risk patients is emerging from preclinical and clinical studies. New molecular and genetics assays, may help to select patients most likely to benefit from these approaches. In this review, we will especially discuss the potential benefits of adjuvant therapy after radical prostatectomy. This paper presents the identification of these high-risk patients; the explanation of an adjuvant treatment of residual disease after a radical prostatectomy; the clinical studies with adjuvant androgen deprivation, radiotherapy and/or chemotherapy and the microarrays analysis. This review highlights the importance of these new adjuvant treatments that aims at targeting the factor which triggers metastatic disease following a radical prostatectomy. Keywords: Adjuvant Treatment, Radical Prostatectomy, High-Risk Patients

1. Introduction According to the pre-operative d’Amico criteria, patients with localized prostate cancer (Pca) (PSA>20 ng/mL, Gleason 8-10, T2c to T4 disease) are considered to be at high risk, with recurrence rates ranged from 50 to 100 percent after a local therapy alone, especially if they are young, healthy and with a long life expectancy. For these patients, prostate cancer specific survival is significantly compromised [1] and surgery alone won’t be able to con-trol the disease. Instead, these patients can show signs of residual disease at the primary site with likely persistent androgen-dependent and independent subpopulation of malignant cells. They also have high risk to develop as-ymptomatic or symptomatic metastases. In this case, ad-juvant approach may be especially important. It is well known that, in breast or colon cancers, the use of adju-vant treatment after surgery has shown a beneficial im-provement in survival [2–6]. In Pca, randomized studies are needed to evaluate the potential effect of adjuvant therapy in these high-risk patients. The optimum adjuvant management for high-risk patients after radical prostate ctomy (RP) may consist in androgen deprivation therapy

(ADT), chemotherapy, prostate bed radiotherapy (RT) or some combination of these modalities.

2. Identifying High-Risk Patients According to CaPSURE study and using D’Amico’s cri-teria, around 20 to 30 percent of localized prostate can-cers would be at high-risk of progression [1,7] and, as well, about 30% to 35% of non metastatic prostate can-cers will eventually relapse with distant disease [8]. High-risk Pca has higher biochemical relapse or disease recurrence rate after RP. Prior surgery, the identification of such aggressive cancers can be based on, at least, three well-defined predictors of the disease extent and outcome after treatment: patients with clinical stage T3 or T4 dis-ease, a serum PSA level of above 20 ng/ml, and those with Gleason scores of 8-10 plus some 4+3 Gleason score but with negative bone scan and negative computed tomography (CT) scan of abdomen and pelvis. In addi-tion, a number of additional clinical parameters could potentially be used to identify patients with high risk of recurrence. Those includes PSA velocity of >2.0ng/mL/ year, at least 50% positive biopsies cores or either tissue cores invaded by tumor above 20% [9,10].

Adjuvant Treatment for High-Risk Operable Prostate Cancer


11

D’Amico reported in 2004 a study including 1.095 pa-tients who underwent RP and who did not receive adju-vant therapy [11]. At a median follow-up of 5.1 years, 27 of 84 deaths were attributable to prostate cancer. On mul-tivariable analysis, preoperative PSA velocity>2ng/ ml/year was associated with an increased risk of cancer specific mortality (RR: 0.8, p<0.01). This was also an increased risk of overall mortality (RR: 0.9, p=0.01). Pretreatment Gleason score 8 to 10 correlated with an increase in cancer-specific mortality (RR: 32, p=0.02). Patients with higher clinical stage were at greater risk for death from Pca (RR: 7.4, p=<0.01) and 2.0 (p=0.004) for death from any cause.

Today, clinicians may be able to better characterize high risk patients and predict the probability of Pca re-currence for each patient through the use of several re-cently developed statistical models called nomograms [12, 13]. The presence of micrometastases remains a major issue since it is likely that many high-risk Pca have mi-crometastasized at the time of diagnosis [14–16]. Though the literature in this regard is poorly documented in pros-tate cancer, upcoming methods to detect those types of microscopic diseases would help to decide appropriate therapeutic strategies [17,18]. Finally, gene expression profiling of prostate carcinoma could be an alternative means to distinguish aggressive tumor. Biology and inte-gration of gene expression signature together with clini-cal variables may improve the outcome prediction for patients treated with RP [19].

3. Adjuvant Treatment

Adjuvant treatment is defined as an additional therapy given in association with primary management. RP alone cannot be considered as an efficient curative treatment for locally advanced Pca, due to the high risk of regional or distant metastases and local failure [20,21]. In these conditions adjuvant treatment may be important so as to control the local and/or distant disease. Importantly sys-temic adjuvant therapy will not compensate for insuffi-cient local therapy.

3.1 Rationale for Adjuvant Treatment Clinical data and preclinical models provide a rationale for adjuvant therapy and notably for the concomitant ad-ministration of hormonal treatment and chemotherapy in prostate cancer.

1) In human prostate cancer xenografts, Craft et al. [22] have shown that prostate cancers contain heterogeneous mixture of cells that vary in their dependence on andro-gens for growth and survival, and that treatment with anti-androgen therapy provides a selective pressure. The latter stage of androgen independence could result from the clonal expansion of androgen-independent cells that are present at a frequency of about 1 per 105-106 andro-

gen-dependent cells. 2) Among patients treated by RP with occult distant

diseases including metastases and micrometastases, an early adjuvant hormone therapy may destroy the andro-gen-dependent residual tumour cells. By contrast, if the number of residual tumour cells is too important, the presence of many androgen-independent clones could make the hormone therapy ineffective and chemotherapy necessary. Pound et al. [8] observed that patients relaps-ing less than two years after RP had particular clinical and pathological characteristics: preoperative PSA>10 ng/ml, Gleason>7 or pT3. Survival without progression was decreased and could justify an adjuvant treatment.

3) Using Dunning R3327 rat prostatic adenocarcinoma model that creates lung metastasis on untreated recipient hosts, studies demonstrated that there was a direct rela-tionship between primary tumor size at the time of surgi-cal removal and the number of lung metastases [23] This concept is in favor of early treatment after local therapy such as RP. Theoretically, when the tumor burden of an-drogen independent cells is low, chemotherapy could be more effective. In other words, if treatment is delayed, the ability of adjuvant chemotherapy to cure the disease may be lost. These results emphasize the critical re-quirement of combining surgery and adjuvant chemo-therapy as early as possible in the treatment of occult metastases, in order to minimize the total metastatic tu-mor burden and maximize the possibility of cure. In hu-man, in recent decades, several cytotoxic agents have been tested as monotherapy in metastatic hormone re-fractory Pca with a certain success, at least in terms of PSA response and quality of life [24–33]. Even if these drugs are still deficient as to cure hormone refractory disease, the observed effects strongly support their sig-nificant activity on distant disease.

4) A study using the serially transplantable Dunning R-3327H rat prostatic adenocarcinoma has shown how changing the timing of androgen ablation alone and of hormone-chemotherapy can affect the tumor growth rate and host survival [34]. This study demonstrated three basic points: a) when either androgen ablation or cytoxan chemotherapy were given as a single agent treatment, they were both more effective when given as early as possible; b) when androgen ablation was combined with cytoxan chemotherapy, it was more effective when both therapies were begun simultaneously and as early as pos-sible; and c) when androgen ablation and cytoxan treat-ments were initiated simultaneously and early, it was possible to increase survival as compared with the groups who were given one of the two therapies alone (i.e., such simultaneous early treatment enhanced the individual therapeutic effectiveness of both treatments).

5) Preclinical data evaluating the optimal timing and combination of androgen deprivating therapy (ADT) in LNCaP and Shionogi prostate cancer xenografts reported



12

that the mice that received simultaneous hormone-chemotherapy had a significant improvement in time to treat-ment failure compared to sequential therapy. A marked lack of response to castration was observed after initial paclitaxel therapy. Moreover, transcriptional profiling identified, after paclitaxel exposure, an increased expres-sion of several survival genes known to play a role in androgen independence [35]. These findings supported simultaneous chemohormonal therapy-adjuvant trials.

3.2 Adjuvant Post RP Treatment 3.2.1 Adjuvant Hormone Therapy (HT) Messing et al. have [36] demonstrated that adjuvant HT significantly improves survival in patients with positive lymph nodes. Data were updated [37] regarding the use of immediate versus deferred ADT in patients found to have node-positive disease at the time of PR. At a median follow-up of 11.9 years, the survival results remain un-changed. The median survival for the immediate and dif-fered ADT arms, was 13.9 years (2.1-14.5) and 11.3 years (1.3-14.2) respectively. The median disease specific survival has not been reached in the immediate arm yet (2.1-14.5 years) and in the differed arm, it was 12.3 years (1.3-14.2; p: 0.0004). The data continue to support the use of ADT in node-positive disease but it is unknown whether ADT improves overall survival in high-risk pa-tients with negative lymph node. Mc Leod et al. [38] have recently published the preliminary results of a large trial evaluating the efficacy and the tolerability of bicatu-lamide (150 mg daily) as adjuvant therapy after PR or RT in patients with locally advanced prostate cancer. A total of 8.133 patients were recruited for this placebo-controlled double-blinded randomized study. With a median fol-low-up of 7.4 years, bicalutamide significantly reduced the risk of objective progression compared to placebo (HR: 0.75; IC 95%: 0.61-0.91; p: 0.004). There was no statisti-cally significant difference between the two groups in terms of overall survival after RP (HR: 1.09; IC 95%: 0.85-1.39; p: 0.51). Again, in men with locally advanced prostate cancer, by 5 years follow-up, the Study by the Scandinavian Prostate Group suggests benefits in terms of progression free survival (PFS) of adding bicalutamide to RP, RT or watchfull waiting [39]. In contrast, bicalutamide provides no benefit in patients with localized prostate can-cer but rather may decrease PFS.

In addition, the optimal duration of the hormonal ther-apy remains to be established. Adjuvant therapy has been used for duration of 2 years or 3 years in phase III trials in men with high-risk disease and the question of adverse effects should be considered in this setting.

3.2.2 Adjuvant Radiotherapy (RT) Two large randomized trials, European Organization for research and Treatment of cancer (EORTC) trial 22911 and Southwest Oncology Group (SWOG) trial 8794,

have reported on the beneficial outcome of adjuvant RT in patients with pathological risk factors after RP. The EORTC 22911 trial showed that adjuvant RT (60 Gy) was associated with improvement in biochemical pro-gression free survival (74% versus 52.6% ; p<0.0001) [40] but the impact on overall survival awaits maturation on the data. In the South Western Oncology Group (SWOG) trial 8794 [41] which randomized 425 high-risk patients to adjuvant RT after RP versus RP alone, no benefit in terms of overall survival was observed in patients as-signed to the adjuvant group. It was shown that adjuvant radiation reduced the risk of biochemical treatment fail-ure by 50% over RP alone. High-risk was defined as ex-tracapsular tumor extension, positive surgical margins, or seminal vesicle involvement. To be eligible, patients had to have histologically negative lymph nodes and a nega-tive bone scan. Adjuvant radiation to the prostate bed (60 to 64 Gy) also seemed to reduce the risk of metastatic disease and biochemical failure at all postsurgical PSA levels [42]. Of note, in this study, the pattern of treatment failure in high-risk patients was predominantly local with a surprisingly low incidence of metastatic failure. SWOG 8794 and EORTC 22911 convincingly showed that the primary risk of treatment failure was local, suggesting that the adjuvant studies treating these patients solely with systemic therapy might have limited benefits. Addi-tionally, offering adjuvant irradiation to all patients with pT3 disease could result in overtreatment for a number of patients, as it was exemplified by the fact that in the ob-servation arm of the EORTC and SWOG studies, respec-tively 52.6% and 38% of patients did not show any bio-chemical relapse. Therefore a better definition of high- risk groups is necessary to reduce the overtreatment rate of RT, side effects and care costs.

Wiegel et al. [43], in their preliminary evaluation of a Phase III study comparing RP followed by RT (60 Gy) with RP alone in patients with pT3 disease, have reported a significant improvement of relapse-free survival among patients receiving adjuvant therapy compared to the con-trol arm; particularly in patients with a preoperative PSA> 10 ng/ml, pT3b and Gleason 8 as well as positive margins.

3.2.3 Adjuvant RT and Andogen Deprivation Therapy after RP

RADICALS is a large international Phase III randomized controlled trial addressing the RT to the tumoral bed (66 Gy) and ADT after RP [44]. The first randomization, performed within the 3 months after RP (the RT timing), consists in randomizing patients to immediate RT versus salvage RT. The second randomization is performed be-fore giving RT (RT duration hormonal therapy) between no HT, short-term HT (6 months duration) and long-term HT (24 months). The primary end point will be the can-cer-specific survival (CCS), the secondary end point will be overall survival. Especially, The RADICALS trial is



13

designed to identify treatment options that could achieve an absolute increase in 10-years CCS of =>5%. 2600 patients in the RT timing randomization could detect an increase in 10-years CCS from 70 to 75% with 80% power, or from 80 to 85% with 90% power and 5% sig-nificant level. 3500 patients would be required for the HT duration randomization. The patients who have a PSA level after RP<0.2 ng/ml with some risk factors for dis-ease recurrence that is pT3, positive margins, Gleason score>6, pre-operative PSA level of >10 ng/ml or a com-bination of these criteria will be included.

3.2.4 Adjuvant Chemotherapy: In men with metastatic hormone-refractory Pca, two Phase III trials have demonstrated that chemotherapy based on taxotere is superior to that based on mitoxantrone plus prednisone [45,46]. Patients receiving docetaxel-based chemotherapy had a longer progression-free survival and a better quality of life. They improved overall survival (2 months benefit) as compared with mitoxantrone groups. Overall, the use of Docetaxel is now a standard for men with hormone refractory disease. Conversely, adjuvant chemotherapy is not standard for high risk patients after RP. Should adjuvant chemotherapy be administered? Is chemotherapy the next step? [47] These questions need to be addressed in specific trials.

Schmidt et al. [48,49] from the National Prostate Can-cer Group randomly assigned 184 patients with localized advanced prostate cancer to one of the three following arms: 2 years of oral cyclophosphamide, estramustine- phosphate for 2 years versus observation. After 10 years of follow-up, the estramustine-phosphate group had an improvement in relapse-free survival but there was no difference in overall survival.

In patients at high-risk for occult distant disease fol-lowing RP, a phase II trial of adjuvant docetaxel was performed [50]. Treatment consisted in 6 cycles of 35 mg/m2 docetaxel weekly given from 4 to 12 weeks fol-lowing RP. At a median follow-up of 29.2 months (range 1.6 to 39.2), 26 of 46 evaluable patients (60.5%) relapsed. The observed median PFS was 15.7 months (95% CI: 12.8-25.1). This PFS is longer than the normal predicted 10 months for these patients, and adjuvant docetaxel had significant but acceptable toxicity in high-risk patients. Grade III toxicity occurred in 20 patients (26%) including dyspnea in 4, fatigue and cardiac arrhythmia in 3 and diarrhea, dizziness, nausea, acute vascular leak syndrome and hyperglycemia in 2. The incidence of Grade IV tox-icity was relatively low and appeared in 3 patients. Seven patients died including 4 of prostate cancer, 1 with in-tra-abdominal bleeding during treatment and 2 of pneu-monia and sudden cardiac deaths following treatment.

The Veterans Affairs Cooperative Study 553 [51] has been designed to prospectively evaluate the efficacy of early adjuvant chemotherapy, using docetaxel and pred-

nisone added to the standard of care (i.e., surveillance with the addition of androgen deprivation at the time of biochemical relapse) for patients, who are at high risk for relapse after RP. Patients “veterans”, are stratified for PSA, Gleason score, tumor stage, and the presence of positive margins. A planned 636 patients will be accrued and randomized to one of two treatment arms: docetaxel plus prednisone administred every 3 weeks for 18 weeks or surveillance alone. Patients will then be followed for a minimum of 1 yr and a maximum of 5 yr. The study is designed with 90% power to detect a reduction in the 5-year progression rate from 60% to 45% (15% absolute difference, 25% relative difference). The estimated study Completion Date is June 2011.

3.2.5 Ajuvant Chemohormonal Therapy: Recent neoadjuvant studies have indicated that combin-ing hormonal and chemotherapy is feasible and safe (for review see ref [52]). Although the impact of chemother-apy on survival need to be proved in randomized trials, it is interesting to note that neoadjuvant studies, wherein hormonal therapy was not included, consistently showed declines in preoperative PSA level, ranging from 20% to 60% after chemotherapy. This indicated the likelihood of an antitumoral effect of these drugs in high risk patients irrespective of hormonal treatment [53–56].

In their study, Pummer et al. [57] evaluated whether patients with previously untreated advanced Pca benefit from combining total ADT with weekly epirubicin che-motherapy Patients with either metastatic (n=117) or lo-cally advanced (n=28) were randomly allocated to treat-ment with ADT by bilateral orchiectomy and flutamide 250 mg or ADT plus weekly epirubicin 25 mg/m2 i.v. for 18 weeks. At a median follow-up of 81 months, progres-sion-free survival and overall survival in the ADT and E-ADT groups were 12 and 18 months (p<0.02) and 22 and 30 months respectively (p=0.12). Subjective quality of life assessment showed no impairment of quality of life by epirubicin treatment. Objective toxicities were generally mild with either treatment. The authors con-cluded that the combination of ADT and epirubicin was well tolerated by patients with advanced Pca and resulted in a significant extension of progression-free survival.

Wang et al. [58] have randomly assigned 96 patients with clinical T3 or T4 disease or metastatic disease to mitoxantrone plus combined anfrogen bockade versus combined androgen blockade alone. In the 38 patients without metastatic disease, a higher initial objective re-sponse (95% versus 53%; p=0.0008) and median survival (80 versus 36 months; p=0.04) were observed in patients healed with mitoxantrone plus combined androgen abla-tion.

In a prospective randomized Phase II study (trial PR005) [59] of adjuvant paclitaxel and ADT versus ADT alone, 47 patients with high-risk Pca were randomized



14

after PR between paclitaxel 100 mg/m2 once a week for 8 weeks and ADT for 3 years versus ADT for 3 years. The mean age was 58 year-old [51–67], the mean PSA con-centration and Gleason score were respectively 18ng/ ml and 7.4 [7–9]. Toxicity, quality of life and functional results were compared between the two arms. With a mean follow up of 36 months, 23 patients receiving pa-clitaxel and ADT were evaluated. Toxicity and side effets were assessed using the National Cancer Institute’s Common Toxicity Criteria (version.2). Alopecia was observed in 100% of the cases. No hematologic toxicity was noted. 4 patients had neurological disorders in fin-gers (86% of grade I), 2 patients had nausea and/or vom-iting disorders (grade I and II), 2 had asthenia (grade I) and one patient developed cardiac insufficiency not due to chemotherapy. One grade III febrile neutropenia was reported. These preliminary results indicated that adju-vant paclitaxel-based chemotherapy associated with ADT was a safe and well tolerated approach.

4. Local Control and Systemic Therapy The effect of RT on survival should be considered in the context of systemic therapy, which is thought to be effec-tive against distant disease. In breast cancer, adjuvant systemic therapy reduces the likelihood of both local and distant recurrence. A subgroup analysis in the EBCTCG metanalysis of local therapy showed that the use of RT after mastectomy in node-positive patients improved 15-year survival only in patients who also received adju-vant systemic therapy and not in patients who were treated with mastectomy alone [3]. In high-risk patients for distant metastases, such as women with positive lymph nodes, RT in the absence of systemic therapy can improve survival only in the rare patients with residual local disease who have no distant dissemination. In con-trast, in node-positive patients treated with mastectomy and adjuvant systemic therapy, RT will potentially con-tribute to survival in patients in whom systemic therapy eradicates microscopic metastases but not residual local disease.

What is also pertinent for this review is recent evi-dence that, in men experiencing an increasing PSA after their primary local treatments (RP or RT), docetaxel -based systemic therapy administred every three weeks was able to reduce PSA level [60]. Authors reported a decreased > or =50% in 17 of 35 patients (48.5%) and > or =75% in seven of 35 patients (20%) with docetaxel. Again, this demonstrated the activity of chemotherapy against prostate cancer cells. In this study, chemotherapy (for up to 6 cycles) was followed by hormone therapy. In five of 33 men, the PSA remains at 0.1 ng/mL at a me-dian of 18.9 months. Herein, it is further interesting to note that three of these five men had soft tissue metasta-sis at entry but remain in complete remission.

The influence of RT on local control emphazise the

need in high-risk patients with Pca: 1) To select patients with high-risk of residual disease

after PR (pathological margin). In stage I or II breast cancer treated with breast-con-

serving surgery and RT, the patients with close margins and those with negative margins both have a rate of local recurrence (LR) of 7%. It is interesting to note that women with extensively positive margins have an LR of 27%, whereas patients with focally positive margins had an intermediate rate of LR of 14%. The use of systemic therapy adds significant effect on the risk-ratio of LR [61]. In Pca, data from the Mayo clinic indicate that 76% of patients with no positive surgical margin and 65% of patients with a single-positive margin after RP remain biochemically and clinically free from disease by 5 years; 62% with two or more positive margins had no evidence of disease by 5 years [62]. SWOG 8794 and EORTC 22911 showed evidence that the primary risk of treatment failure was local. In recent reports of EORTC 22911 us-ing the grading, staging and surgical margins status de-termined by a central pathology review, it was shown that the margin status was a stronger predictor for the magni-tude of the treatment benefit [63,64]. Thus, while the 5-year biochemical PFS rates were 67.4% (95% CI: 56.1% to 76.3%) and 76.2% (95% CI: 66.1% to 83.6%) for patients with negative margins in the control versus irradiation arm, they were 77.6% (95% CI: 68.8% to 84.2%) and 48.5% (95% CI: 39.6% to 58.9 %) for the patients with positive surgical margin in the control ver-sus irradiation arm [61]. Treatment failure is now docu-mented to be primarily in the area of the prostate fossa, and adjuvant radiation reduces both biochemical and clinical local recurrence (22% versus 8%). In the obser-vation arm of the EORTC study, the rate of clinical local treatment failure was four times the rate of systemic fail-ure. In view of these data, patients with high-risk prostate cancer after PR should be given adjuvant RT as standard treatment [42] however immediate postoperative RT might not be recommended for Pca patients with negative surgical margins.

2) To identify the patients in whom the influence of local RT on mortality will be reduced and possibly elimi-nated with systemic therapy.

In breast cancer, Marks et al. [65] suggest that benefits of local therapy on survival has an inverted-U-shaped or parabolic relationship with increasing effective systemic therapy, so that the survival benefit derived from better local therapy increases with increasing effective therapy but only to a certain threshold of effectiveness and then declines. In the SWOG study, in the observation group, the rate of clinical local failure was 24% versus 16% dis-tant metastases. By improving the local control, adjuvant RT was associated with a reduction in the proportion of patients with metastases (16 to 7%). Based on the above mentioned observations, these data also suggest that of-



15

fering these patients systemic therapy could lead to lim-ited improvement in survival [42]. The fact remains that reduction in local recurrence with systemic therapy with and without RT must be reported in further studies. The new EORTC trial 22043-30041 aims at recruiting 600 patients with a PSA level of =<0.2 ng/ml after RP and testing the value of adding 6 months of ADT to RT. The RTOG 0534 trial in patients with PSA level of 0.2-2 ng/ml after RP will compare treatment with adjuvant RT alone or with additional 5 months of ADT or similar ADT with pelvic RT.

5. Clinical Studies with Adjuvant ADT and/or Chemotherapy

The role of adjuvant therapy in the high-risk population following RP needs systematic study. From experience in colon and breast cancer, active agents in the metastatic setting seem to be more beneficial when used in patients with earlier stage disease. Prospects for long-term sur-vival following surgical treatment of localized breast cancer have significantly improved with the widespread use of adjuvant systemic chemotherapy. According to the uptaded 2005, Early Breast cancer Trialist collaborative meta-analysis, combination chemotherapy was associated with an approximate 23% reduction in the risk of breast cancer recurrence. The greatest absolute benefit was no-ticed in younger women with lymph node–positive dis-ease. In absolute terms, a 5-year course’ of a selective oestrogen receptor modulator tamoxifen for patients with oestrogen receptor-positive tumors reduces the annual breast cancer death rate by 31% largely irrespective of chemotherapy [3].

As with the use of adjuvant treatment in breast cancer, three randomized studies were planned to investigate chemohormonal adjuvant setting in patients considered to be at high-risk Pca after RP.

SWOG 9921 was intiated but it closed prematurely in January 2007 (Table 1). The primary endpoint of this trial was initially overall survival. All patients should have received 2 years of combined ADT using goserelin and bicalutamide, and half should have been treated with 6 cycles of 12 mg/m2 mitoxantrone plus 5 mg prednisone twice daily. RT to the prostate was allowed. Study ac-crual was held when three cases of leukemia (AML) were reported in the mitoxantrone-containing arm. Therefore, all patients in the chemotherapy-containing arm stopped taking mitoxantrone. Although this phase III has stopped, 2 years of ADT will be continued in active patients in both arms and it may be possible to obtain precious in-formation given the high number of patients included.

A second Phase III TAX 3501 aimed at using do-cetaxel as an adjuvant treatment for high-risk disease with an accrual goal of 2172 patients (Table 2). The pri-mary endpoint was PFS. Unfortunately, this study failed to meet its accrual target and was closed very recently.

The design was a 4 arms randomization to immediate versus delayed therapy with ADT plus or without che-motherapy. This trial was designed to provide valuable information on both hormonal therapy and chemotherapy in the adjuvant setting. In particular, it would have helped to define the optimal timing in adjuvant therapy among immediate postoperative period and delayed when in-creasing PSA was detected [66]. Of interest is that an important number of men were accrued prior to closure, and useful data on their outcomes might be possible.

A Phase II randomized trial PR005 aims at using pa-clitaxel as an adjuvant for high-risk disease (Table 3) [59]. All patients will receive 4 years of ADT using LH-RH agonist for three years and bicalutamide 50 mg for 1 month, and half will be treated with 8 weekly cycles of 100 mg/m2 paclitaxel. An approach to maximizing tumor-cell death with adjuvant chemotherapy is to use optimal doses of active chemotherapy drugs administered sequentially with a shortened scheduling interval. This approach, called “dose-dense,” increases dose in- tensity (drug delivery over time) by reducing the inter- treatment interval for chemotherapy delivery. A number of preclinical studies suggest that continuous dosing of chemotherapy with a very short interdose interval, so

Table 1. SWOG 9921 adjuvant trial in patients with Pca at high risk after RP

High-risk criteria Randomized adjuvant treatment

-Arm A: ADT 24 months

T3a,+margin and Gleason 7

T3 on biopsy N0M0

T3 b-T4 or pN1 -Arm B: ADT 24months

Or Gleason=>8 and mitoxantrone

PSA>20 ng/ml 12 mg: m2 d1+

prednisone 5mg BID

N: 1380 (to detect D1-D21 Q 3 weeks x6

A 30% survival difference)

Table 2. TAX3501 adjuvant trial in patients with Pca at high risk after RP


-Arm A: observation

Kattan nomogram

stratification progression

0-20% -ADT

20-40 % -or ADT+docetaxel

40-60%

-Arm B

N: 1696 -ADT

-ADT+docetaxel



16

Table 3. PROO5 adjuvant trial in patients with Pca at high risk after RP


Age =<67 years -Arm A: ADT 36 months

cT2-3a N0M0

pT3 b-pT4 and/or pN1 -Arm B: ADT 36 months weekly

Gleason=>7 paclitaxel 100 mg/m2

PSA>10 ng/ml

N: 178 (to detect a

20% PFS difference)

called “metronomic” scheduling, may enhance the anti- angiogenic effects of chemotherapy in breast cancer [66–71]. This current study tests a sequential dose-dense of weekly administration of paclitaxel (cumulative dose: 800 mg/m2) for which the feasibility and efficacy has been assessed previously [26,27]. In this study, frozen prostate tissue will be obtained from men undergoing RP who are enrolled in either the treatment or the control arms of the trial. These samples will be analyzed for their mRNA level expression patterns in an attempt to draw up outcome prediction models. Likewise, from these ar-ray-based methods of expression analysis, it would be ideal if we could predict the sensitivity to chemothera-peutic agents and the response to chemotherapy.

6. Microarrays Analysis Microarrays analysis has been used to characterize the molecular profiles of breast cancer [72–74]. Important advances are being made in the use of genetic analysis to determine the risk of recurrence and to predict a tumor’s responsiveness to adjuvant chemotherapy or tamoxifen in breast cancer [75–79]. These approaches may be infor-mative to determine high-risk Pca for local recurrence or distant recurrence. Given that initiation and progression of Pca involve multiple changes in gene expression, cDNA microarray technology has been recently used to identify disease-related gene expression patterns in pros-tate samples [80–82] This approach has successfully de-tected alterations in several candidate genes associated with Pca progression [83,84]. However, there is not any definitive molecular classification that can consistently and reliably predict the clinical behaviour of Pca yet. Nevertheless, gene expression profiling offers an alterna-tive means to distinguish aggressive tumor biology and may improve the accuracy of outcome prediction for pa-tients with Pca treated by RP [14,85,86]. Interestingly, Lapointe et al. [87], so as to further characterize the clinical relevance of tumor subtypes identified from a gene expression profiling, used immunohistochemistry on tissue microarrays in an independent set of 225 pros-tate tumors in order to assess two genes as surrogate

markers, namely MUC1 and AZGP1, differentially ex-pressed among indentified tumor subgroups. MUC1 and AZGP1were found to be strong predictor of tumor recur-rence. In Kaplan-Meier survival analysis, positive MUC1 staining was associated with significantly shorter time to recurrence, while strong immunostaining of AZGP1 was associated with significantly prolonged time to recur-rence. Importantly, these genes were found, in multivari-ate analysis, as additional prognostic information over and above the known risk factors of tumor grade, stage, and preoperative PSA. These genes also provided inde-pendent prognostic value, suggesting that using two genes improves the accuracy of tumor subtyping and prognostication.

Glinsky et al. [88] reported a Pca recurrence predictor algorithm that appeared suitable for stratification of pa-tients at the time of diagnosis into poor-and good-prog-nosis subsets, this, with a statistically significant differ-ence in the disease-free survival after RP. It could pro-vide additional predictive value over conventional prog-nostic factors such as PSA level and Gleason sum. Tom-kins et al. [89] reported that a majority of Pca exhibit fusions between the control region of an androgen regu-lated gene TMPRSS2 and the coding region of the ETS family of transcription factors, most frequently ERG and much less frequently ETV1 and ETV4. The fusions are associated with an increased risk of cancer progression in patients treated surgically [90,91]. Recently, a four- variable model predictive of cancer-specific outcome incorporate gene expression of topoisomerase-2a, cad-herine-10, the fusion status based ERG, ETV1 and ETVA expression and the aneuploïd status in men with high- grade Pca treated with RP was established [92]. The trial POO5 has used gene expression profiling to define sub-groups of high-risk Pca associated with good or poor outcome. The refined gene-expression signature associ-ated with metastases contained three upregulated and thirty down regulated gene (Personal communication).

Collectively, these data illustrate the potential helpful-ness of expression profiling in the identification of high-risk patients as well as in the development of new biological markers and prognostic markers.

7. Conclusions Patients with high-risk Pca after RP should be offered adjuvant RT as standard treatment. However, a policy of adjuvant RT would result in significant overtreatment: in the observation arm of the EORTC and SWOG studies, 52.6% and 38% of patients did not show any biochemical relapse. Thus, a better definition of high-risk groups is warranted to reduce the overtreatment rate of RT and to reduce the side effects and cost of this adjuvant treatment. An alternative approach to reduce the number of treated patients might be to identify subsets of patients who may



17

significantly benefit from immediate post operative RT. Further improved in local control with adjuvant systemic treatment would also enable better results. Accumulating clinical and preclinical data suggests that the use of early HT will improve the outcome in patients with high-risk localized Pca. Yet, two randomized Phase III trials evaluating the effect of adjuvant hormonal therapy with or without chemotherapy in high-risk patients after RP have been prematurely closed. Another problem is to better distinguish diseases that will be cured with local treatement only, from those that will require an adjuvant approach. Gene expression profiling of Pca offers an al-ternative means to distinguish aggressive tumor biology and may improve the accuracy of outcome prediction for patients with Pca treated by RP.

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21

Novel Ethyl 2-(1-aminocyclobutyl)-5-(benzoyloxy)- 6-hydroxy-pyrimidine-4-carboxylate Derivatives: Synthesis and Anticancer Activities

D. Asha1, C. V. Kavitha2, S. Chandrappa1, D. S. Prasanna1, K. Vinaya1, Sathees C. Raghavan2, K. S. Rangappa1

1Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, India; 2Department of Biochemistry, Indian Institute of Science, Bangalore, India. Email: [email protected]; [email protected] Received December 26th, 2009; revised February 2nd, 2010; accepted February 12th, 2010.

ABSTRACT

To explore the anticancer activity of 2, 4, 5, 6-substituted pyrimidines, several ethyl 2-(1-aminocyclobutyl)-5-(benzoyloxy)-6-hydroxy-pyrimidine-4-carboxylate derivatives associated with the different substituted aromatic/aliphatic carboxamides and sulfonamides were synthesized. Different groups and position on phenyl ring attached to the carboxamide and sulfonamide of the pyrimidine led to two set of compounds. Their chemical structures were confirmed by IR, 1H NMR and LC/MS analysis. Cytotoxicity of all the synthesized compounds were examined on human leukemia cell lines (K562 and CEM). The preliminary results showed most of the derivatives exhibited good antitumor activity. Com- pound with para chloro substitution among carboxamides and compound with meta dichloro substitution among sul-phonamides exhibited significant antitumor activity with IC50 value of 14.0 µM and 15.0 µM respectively against K562 cell line. For comparison among electron donating groups between carboxamides and sulfonamides, compounds with para tert-butyl substitution were chosen for further studies. Cell cycle analysis suggests that both tert-butyl substituted compounds are able to induce apoptosis. Keywords: Pyrimidine Derivatives, Cytotoxicity, Apoptosis, Leukemia, Cell Cycle Analysis

1. Introduction

Cancer is a terrible disease which is the leading death of the human population in some areas of the world. It is the second leading cause of death, behind cardio-vascular disease, in the United States [1]. Cancer may affect people at all ages, even fetuses, but the risk for most varieties increases with age. Leukemia is a cancer of the blood-forming cells. Leukemia is the most common childhood cancer, affecting more than 3,500 children in the United States every year. At present, there are three main methods of cancer treatment: surgery, radiation therapy, and chemotherapy. With the devel-opment of molecular biology, chemotherapy is becom-ing a more important therapeutic method. Therefore, designing new anticancer drugs with high-efficiency and broad-spectrum activity is a significant study area today.

Heterocycles are ubiquitous to among pharmacy- eutical compounds [2]. Pyrimidine moiety is an impor-tant class of nitrogen containing heterocycles widely

used as key building blocks for pharmaceutical agents. Pyrimidines have a long and distinguished history ex-tending from the days of their discovery as important constituents of nucleic acids to their current use in the chemotherapy of cancer and AIDS. The most important pyrimidine derivatives are those upon which biological organisms depend. Pyrimidine derivatives possessing anticancer activity have been reported in the literature [3–9]. Pyrimidine derivatives comprise a diverse and interesting group of drugs [2]. The subject has been discussed recently [10]. Earlier, a comprehensive re-view concerning pyrimidines has been published by Brown [11]. Pyrimidines in general are extremely im-portant for their biological activities. In addition to above-mentioned activities, pyrimidine derivatives possessing analgesic [12], antileishmanial [13], antim-icrobial [14], antifungal [15], and anti-infective have also been reported in the literature. Many other exam-ples of pyrimidine-based derivatives have been inves-tigated as potential antitumor agents, including 2-phenylamino derivatives [16–19], 4-phenylamino deriva-

Novel Ethyl 2-(1-aminocyclobutyl)-5-(benzoyloxy)-6-hydroxy-pyrimidine-4-carboxylate Derivatives: Synthesis and Anticancer Activities 22

tives [20,21], 2.4-bis(phenylamino) derivatives [22], 4.6-bis(phenylamino) derivatives [23,24], and 4-aryl- substituted derivatives [25]. In addition, the pyrimidine pharmacophore is found in several fused-ring examples of ATP-competitive protein kinase inhibitors, such as purines, quinazolines and pyrido-, pyrimido-, pyrazolo-, and pyrrolo-pyrimidines [26–28]. In continuation of our efforts to get novel and potent heterocyclic antileukemic agents [29–32], a number of novel pyrimidine derivatives were synthesized and evaluated for their anticancer activity which we wish to report in this pa-per.

2. Methods

2.1 Chemistry

Infrared (IR) spectra were recorded using a Jasco FTIR-4100 spectrometer in the wave number range of 4000-400 cm-1. Nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AM 400 MHz spec-trometer using DMSO-d6 as solvent and tetramethylsi-lane as an internal standard. The chemical shifts are ex-pressed in and the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet) and m (multi-plet). Mass and purity were recorded on a LC-MSD- Trap-XCT. The purity of the compounds was checked by thin layer chromatography (TLC). Silica gel column chromatography was performed using Merck 7734 silica gel (60-120 mesh) and Merck made TLC plates. All the reagents and chemicals were from Sigma Aldrich Chemi- cals Pvt Ltd.

2.2.1 Synthesis of Ethyl 2-(1-aminocyclobutyl)-5- (benzoyloxy)-6-hydroxypyrimidine- 4-carboxylate (1)

For the synthesis of the target key intermediate 1 the re-action sequences as reported in our previous article were followed [33]. The synthesis of compounds 3(a-i) is also reported earlier [33]. Treating cyclobutanone with am-monium chloride and sodium cyanide in methanol gave 1-isocyanocyclobutanamine. Amine group of 1-isocyanocyclobutanamine was protected by using benzyl chlo- roformate in presence of mild base sodium carbonate followed by the oxime formation by using hydroxyla- mine hydrochloride in presence of base potassium hydro- xide and methanol as a solvent to get benzyl 1-amidino- cyclobutylcarbamate. This compound on cyclisation with diethyl acetylene dicarboxylate in chloroform using triethylamine as a base for 5 hr yielded benzyl 1-(4-(ethoxycarbonyl)-5, 6-dihy-droxypyrimidin-2-yl) cyclobutylcar-bamate. Treating the above compound with benzoic an-hydride in pyridine gave benzyl 1-(4-(ethoxycarbonyl)- 5-(benzoyloxy)-6-hydroxylpyrimidin-2-yl)cyclobutyl car- bamate. Final key intermediate 1 was obtained by depro-tection of amine group of previous compound by using

Pd/C in ethyl acetate.

2.1.2 General Procedure for the Synthesis of Ethyl 2(1-aminocyclobutyl)-5-(benzoyloxy)-6-hydroxy-pyrimidine-4-carboxylate Derivatives 2(a-i)

A solution of ethyl 2-(1-aminocyclobutyl)-5-(benzoy-loxy)-6-hydroxypyrimidine-4-carboxylate (1) (1.0 eq) in dichloromethane was taken and cooled to 0-5 oC in ice bath. Triethyl amine (3.0 eq) was added to the cold mix-ture and stirred for 10 min and respective aryl carbonyl chlorides (1.0 eq) were added, the mixture was stirred and allowed at room temperature for 5 hr. The progress of the reaction was monitored by TLC. Upon completion, water was added to reaction mixture and extracted with ethyl acetate. The organic layer was washed with 10% ammonium chloride solution followed by water wash and dried with anhydrous sodium sulphate. The solvent was evaporated and the crude product obtained was purified by column chromatography over silica gel (60-120 mesh) using dichloromethane: methanol (9:1) as eluent. The IR, 1H NMR and mass spectroscopic data are given in Table 1.

2.1.3 Synthesis of Ethyl 2-(1-(3,5-dinitrobenzamido) Cyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate (2a)

The product obtained was white solid from ethyl 2-(1- aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine- 4-carboxylate 1 (0.1 g, 0.28 mmol), 3, 5-dinitrobenzoyl chloride (0.064 g, 0.28 mmol) and triethylamine (0.085 g, 0.84 mmol).

2.1.4 Synthesis of Ethyl 2-(1-(3-methoxybenzamido) Cyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate (2b)

The product obtained was white solid from ethyl 2-(1- aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine- 4-carboxylate 1 (0.1 g, 0.28 mmol), 3-methoxy-benzoyl chloride (0.048 g, 0.28 mmol) and triethylamine (0.085 g, 0.84 mmol).

2.1.5 Synthesis of Ethyl 2-(1-(2-fluorobenzamido) Cyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate (2c)

The product obtained was white solid from ethyl 2-(1- aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine- 4-carboxylate 1 (0.1 g, 0.28 mmol), 2-fluorobenzoyl chloride (0.044 g, 0.28 mmol) and triethylamine (0.085 g, 0.84 mmol).

2.1.6 Synthesis of Ethyl 2-(1-(4-tert-butylbenzamido) Cyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate (2d)

The product obtained was white solid from ethyl 2-(1- aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine- 4-carboxylate 1 (0.1 g, 0.28 mmol), 4-tert-butyl-benzoyl chloride (0.055 g, 0.28 mmol) and triethylamine (0.085 g, 0.84 mmol).



H2N N

N

OH

O

O

S

N

N

OH

O

OR

O

O

1

COOC2H5

COOC2H5

NH

C

N

N

OH

O

OR

O

COOC2H5

NH

2(a-i)

3(a-i)

(i)

(ii)

Scheme 1. Reagents and conditions: (i) Alkyl/Aryl carbonyl chloride, triethylamine, dichloromethane, 0 oC-rt, 6-8 hr; (ii) Alkyl/Aryl sulphonyl chloride, triethylamine, dichloro-methane, 0 oC-rt, 6-8 hr

2.1.7 Synthesis of Ethyl 2-(1-(2,6-difluorobenzamido)

Cyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate (2e)

The product obtained was white solid from ethyl 2-(1- aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate 1 (0.1 g, 0.28 mmol), 2, 6-difluoro- benzoyl chloride (0.049 g, 0.28 mmol) and triethylamine (0.085 g, 0.84 mmol). 2.1.8 Synthesis of Ethyl 2-(1-(3-bromobenzamido)

Cyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate (2f)

The product obtained was white solid from ethyl 2-(1- aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate 1 (0.1 g, 0.28 mmol), 3-bromobenzoyl chloride (0.061 g, 0.28 mmol) and triethylamine (0.085 g, 0.84 mmol). 2.1.9 Synthesis of Ethyl 2-(1-(4-chlorobenzamido)

Cyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate (2g)

The product obtained was white solid from ethyl 2-(1- aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate 1 (0.1 g, 0.28 mmol), 4-chlorobenzoyl chloride (0.049 g, 0.28 mmol) and triethylamine (0.085 g, 0.84 mmol). 2.1.10 Synthesis of Ethyl 2-(1-(benzamido)

Cyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate (2h)

The product obtained was white solid from ethyl 2-(1- aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate 1 (0.1 g, 0.28 mmol), benzoyl chloride (0.039 g, 0.28 mmol) and triethylamine (0.085 g, 0.84 mmol). 2.1.11 Synthesis of Ethyl 2-(1-(3-nitrobenzamido)

Cyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate (2i)

The product obtained was white solid from ethyl 2-(1- aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimi-dine -4-carboxylate 1 (0.1 g, 0.28 mmol), 3-nitrobenzoyl chloride (0.052 g, 0.28 mmol) and triethylamine (0.085 g, 0.84 mmol).

Table 1. IR, 1H NMR and mass spectroscopic data of com-pounds 2(a-i)

Com- pound

No

IR (KBr, cm-1)

1H NMR (DMSO-d6, 400 MHz) MS

(M+1) m/z

2a

3460, 2930, 2859, 2371, 1681, 1059

12.85 (s, 1H, -OH), 9.85 (s, 1H, -NH), 9.65 (s, 1H, Ar-H), 9.15 (m, 2H, Ar-H), 7.22 (m, 5H, Ar-H), 4.24 (q, 2H, -CH2), 2.82 (t, 2H, -CH2), 2.45 (t, 2H, -CH2), 1.97 (m, 2H -CH2), 1.10 (t, 3H, -CH3)

552.41

2b

3459, 2941, 2848, 2365, 1679, 1065

13.08 (s, 1H, -OH), 9.01 (s, 1H, -NH), 8.08 (t, 2H, Ar-H), 7.78 (d, 1H, Ar-H), 7.63 (m, 1H, Ar-H), 7.12 (m, 5H, Ar-H), 4.24 (q, 2H, -CH2), 3.84 (s, 3H, -OCH3), 2.82 (t, 2H, -CH2), 2.45 (t, 2H, -CH2), 1.97 (m, 2H -CH2), 1.10 (t, 3H, -CH3)

492.17

2c

3450, 2959, 2855, 2360, 1665, 1079, 790

12.89 (s, 1H, -OH), 9.80 (s, 1H, -NH), 7.78 (t, 1H, Ar-H), 7.38 (t, 1H, Ar-H), 7.85 (d, 1H, Ar-H), 7.58 (d, 1H, Ar-H), 7.17 (m, 5H, Ar-H), 4.22 (q, 2H, -CH2), 2.75 (t, 2H, -CH2), 2.42 (t, 2H -CH2), 1.91 (m, 2H, -CH2), 1.12 (t, 3H, -CH3)

480.46

2d

3462, 2955, 2848, 2358, 1669, 1045

12.56 (s, 1H, -OH), 10.25 (s, 1H, -NH), 8.88 (d, 2H, Ar-H), 7.87 (d, 2H, Ar-H), 7.44-7.50 (m, 5H, Ar-H), 4.22 (q, 2H, -CH2), 2.79 (t, 2H, -CH2), 2.50 (t, 2H, -CH2), 1.96 (m, 2H, -CH2), 1.29 (s, 9H, -(CH3)3), 1.07 (t, 3H, -CH3)

518.22

2e

3434, 2927, 2360, 1666, 1105, 794

12.52 (s, 1H, -OH), 9.85 (s, 1H, -NH), 7.91 (dd, 2H, Ar-H), 7.75 (m, 1H, Ar-H), 7.23 (m, 5H, Ar-H), 4.25 (q, 2H, -CH2), 2.76 (t, 2H, -CH2), 2.53 (t, 2H, -CH2), 1.94 (m, 2H, -CH2), 1.1 (t, 3H -CH3)

498.14

2f

3451, 2956, 2854, 2362, 1672, 1046, 763

12.95 (s, 1H, -OH), 9.20 (s, 1H, -NH), 8.24 (s, 1H, Ar-H), 8.06 (d, 1H, Ar-H), 7.88 (t, 1H, Ar-H), 7.61 (m, 5H, Ar-H), 7.48 (d, 1H, Ar-H), 4.21 (q, 2H, -CH2), 2.78 (t, 2H, -CH2), 2.48 (t, 2H -CH2), 1.95 (m, 2H, -CH2), 1.01 (t, 3H, -CH3)

541.07

2g

3458, 2951, 2836, 2355, 1668, 723

12.43 (s, 1H, -OH), 10.21 (s, 1H, -NH), 8.91 (d, 2H, Ar-H), 7.93 (d, 2H, Ar-H), 7.44-7.56 (m, 5H, Ar-H), 4.29 (q, 2H, -CH2), 2.73 (t, 2H, -CH2), 2.48 (t, 2H, -CH2), 1.94 (m, 2H, -CH2), 1.05 (t, 3H, -CH3)

496.89

2h

3450, 2960, 2849, 2356, 1679

12.79 (s, 1H, -OH), 9.31 (s, 1H, -NH), 7.52-7.85 (m, 10H, Ar-H), 4.25 (q, 2H, -CH2), 2.80 (t, 2H, -CH2), 2.49 (t, 2H, -CH2), 2.02 (m, 2H, -CH2), 1.06 (t, 3H, -CH3)

462.26

2i

3462, 2941, 2863, 2358, 1685, 1061

12.70 (s, 1H, -OH), 9.25 (s, 1H, -NH), 8.22 (s, 1H, Ar-H), 8.12 (d, 1H, Ar-H), 7.91 (t, 1H, Ar-H), 7.63 (m, 5H, Ar-H), 7.43 (d, 1H, Ar-H), 4.24 (q, 2H, -CH2), 2.81 (t, 2H, -CH2), 2.50 (t, 2H -CH2), 1.98 (m, 2H, -CH2), 1.12 (m, 3H, -CH3)

507.34


Novel Ethyl 2-(1-aminocyclobutyl)-5-(benzoyloxy)-6-hydroxy-pyrimidine-4-carboxylate Derivatives: Synthesis and Anticancer Activities


24

2.2 Biology

Human cell lines, K562 and CEM were purchased from National Center for Cell Science, Pune, India. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL of Penicillin, and 100 μg of streptomycin/ml and incubated at 37 ºC in a humidified atmosphere containing 5% CO2. Leukemia (K562 and CEM) cells growing in log phase were treated with 10, 50 and 100 μM concentrations of 2, 4, 5, 6-substituted pyrimidine derivatives 2(a-i) and 3(a-i). Since the compounds were dissolved in DMSO, it was used as vehicle control. We employed trypan blue dye exclusion and MTT assay to assess the cytotoxicity. In addition, we have performed cell cycle analysis and DNA fragmentation assay to determine the apoptosis. Each experiment was repeated a minimum of two times.

2.2.1 Cytotoxicity Assay MTT [3-(4, 5 dimethylthiazol-2-yl-2,5-tetrazolium bro-mide)] assay was used for the measurement of the cyto-toxicity of synthesized compounds 2(a-i) and 3(a-i) as described previously [19]. In brief, Exponentially grow-ing K562 or CEM cells (1×104 cells /well) were plated in duplicates and incubated with 10, 50 and 100 μM of 2(a-i) and 3(a-i). Cells were harvested after 48 and 72 h of treatment and incubated with MTT (0.5 mg/ml) at 37°C. The water soluble tetrazolium salt, [3-(4, 5-dimethylthiazol-2-yl-2,5-tetrazolium bromide)] is metabolized to the water insoluble formazan by intact mitochondrial dehy-drogenases. The formazan is then solubilized by adding detergent. The viability of the cells was estimated on the basis of formazan formed, which was detected spectro-photometrically by optical density at 570 nm. The mean absorbance of culture medium was used as the blank and was subtracted. IC50 values (concentration of compound

causing 50% inhibition of cell growth) were estimated after 72 h of compound treatment. The absorbance of vehicle cells was taken as 100% viability and the values of treated cells were calculated as a percentage of control and presented as histograms (Figure 1 and Figure 2). The antiproliferative effects of these compounds were also analyzed by trypan blue exclusion assay against both K562 and CEM cells (data not shown).

2.2.2 Cell Cycle Analysis Cellular DNA content was measured by flow cytometry. Approximately 7.5×104 cells/ml were cultured and treated with 10, 50 and 100 μM concentrations of 2e or 3f (Figure 3). Cells were harvested after 48 h of treat-ment, washed, fixed in 70% ethanol and incubated with RNase A (Sigma-Aldrich, USA). Propidium iodide (PI, 50 μg/ml, Sigma-Aldrich, USA) was added half an hour before acquiring the flow cytometric reading (FACScan, BD Biosciences, USA). A minimum of 10,000 cells were acquired per sample and histograms were analyzed by using WinMDI 2.8 software.

3. Results and Discussion

Pyrimidine ring was derivatized by substituting electron withdrawing and electron releasing groups along with cyclobutyl carboxamide 2(a-i) and sulfonamide 3(a-i) moiety at position 2 of the pyrimidine ring. In addition, we also introduced hydroxyl group at position 6, ethyl carboxylate at position 4 and phenyl carboxylate at posi-tion 5 of the pyrimidine. To investigate the cytotoxic effects of 2,4,5,6-tetrasubstituted pyrimidines on the growth of leukemia cells, a dose response study was conducted using trypan blue dye exclusion (data not shown) and MTT assay (Figure 1 and Figure 2). Results showed that, the cytotoxicity induced by the derivatives

(a) (b)

Figure 1. Cytotoxicity as measured by MTT assay. K562 cells treated with 10, 50 and 100 µM of compounds 2(a-i) (a) and 3(a-i) (b) for 48 and 72 h were harvested and used for the assay. DMSO treated cells were used as vehicle control. Data are representative of the mean of two separate experiments done in duplicate


(a) (b)

Figure 2. Cytotoxicity as measured by MTT assay. CEM cells treated with 10, 50 and 100 µM of compounds 2(a-i) (a) and 3(a-i) (b) for 48 and 72 h were harvested and used for the assay. DMSO treated cells were used as vehicle control. Data are representative of the mean of two separate experiments done in duplicate

(a) (b)

(c) (d)

Figure 3. Cell cycle analysis of K562 cells treated with 2e or 3f. K562 cells (0.75×105 cells/ml) were incubated at 37 °C with 2e or 3f (10, 50 and 100 μM). Following 48 h of incubation, cells were fixed and stained with propidium iodide and subjected to FACS analysis. Panel (a) and (b) show histograms comparing the effect of 2e and 3f at specific cell cycle stages. In both the Panels (a) and (b), the first histogram represents DMSO treated cells. Panel (c) and (d) show the quantification of cells in different stages of cell cycle followed by treatment with 2e and 3f respectively 2(a-i) and 3(a-i) was in a concentration and time depend-ent manner. Interestingly, the DMSO control corre-sponding to the highest concentrations of compounds tested did not show any significant toxic effect and it was

taken as 100%. The relative percentage was calculated for the treated compounds (Figure 1 and Figure 2). These results were further confirmed by trypan blue as-say (data not shown). Based on these results IC50 values



were calculated for 72 h and tabulated in Table 2. Com-pounds 2c, 2d, 2e, 2f, 2g and 2i in carboxamide series and compounds 3b, 3c, 3d, 3f and 3i in sulphonamide series showed good cytotoxicity. As can be seen from Table 2, the electronic property and position of the group on the phenyl ring attached to the carboxamide/sulfonamide moiety determines the activity of these compounds.

Among carboxamide derivatives 2(a-i), we found that compounds with halogen, nitro and tert-butyl as sub-stituents on phenyl ring are more cytotoxic compared to unsubstituted (2h) and methoxy substituted (2b) deriva-tives. Compound 2g with chloro at para position on the phenyl ring of carboxamide showed good activity with an IC50 of 14.0 µM against K562 cells. Among halogen sub-stitution containing sulfonamide derivatives, compound 3c with chloro groups at two meta positions showed good activity (IC50: 15.0 µM) compared to a chloro at para position (3d, IC50: 28 µM) for K562 cells. Similarly, in nitro group containing sulfonamide derivatives activity varies with the position. Compound 3b with nitro group at meta position exhibited more activity (IC50: 26.2 µM) relative to compound 3e with an ortho nitro group (IC50: 36.5 µM) and compound 3a with a para nitro (IC50: 43.1 µM) against K562 cells. Besides electron withdrawing, electron releasing groups also played a role in enhancing the activity.

Compound 3f with tert-butyl group at para position exhibited more activity than 3i containing methyl group at the same position. This could be due to the presence of three electron releasing groups (methyl groups) in 3f and only one electron releasing group (methyl group) in 3i. More importantly, alkyl group (methyl) directly attached to the sulfur atom of the sulfonamide also showed modest activity. Interestingly, compound 3g containing only phenyl ring lost the activity. This suggest that, electron releasing or electron withdrawing group on the phenyl ring, or the groups directly attached to the sulfonamide moiety contributed to the enhancement of cytotoxicity of these compounds.

Our previous study on the cytotoxic effect of thia-zolidinone derivatives suggest that, the electron donating groups on the C-terminal of the phenyl group at 4th posi-tion resulted in increasing the activity [32]. Inspired by these results, in this series, we have chosen two mole-cules 2e and 3f containing tert-butyl group at 4th position from carboxamide and sulfonamide derivatives respec-tively for further studies. Firstly, to evaluate the effect of 2e and 3f on cell cycle progression we have carried out flow cytometry analysis. Results showed that both 2e and 3f did not induce any apoptosis up to 50 µM. At 100 µM we have seen a remarkable accumulation of subG1 cells followed by the decline of G1, S and G2/M phase cells (Figure 3). Therefore, our studies further confirm that growth inhibition could be due to apoptosis.

Table 2. Structure and IC50 values of the synthesised com-pounds 2(a-i) and 3(a-i)

IC50 (μM) Compound R

K562 CEM

2a

NO2

O2N

45.2± 6.8 54.5± 7.2

2b OMe

>100 >100

2c F

27.6± 2.6 32.2± 3.8

2d

F

F

36.1± 3.4 24.3± 2.3

2e H3C

CH3H3C

40.0± 5.2 34.7± 3.0

2f

Br

50.2± 7.1 22.1± 2.1

2g Cl

14.0± 2.1 23.0± 2.4

2h

>100 >100

2i

NO2

35.5± 3.6 48.6± 7.2

3a O2N

43.1± 6.5 52.3± 7.8

3b O2N

26.2± 3.2 45.1± 6.8

3c

Cl

Cl

15.0± 1.6 25.3± 3.8

3d Cl

28.4± 4.1 30.3± 4.3

3e NO2

36.5± 4.9 42.1± 6.5

3f H3C

CH3H3C

20.0± 2.3 24.0± 3.0

3g

>100 >100

3h H3C 30.2± 4.3 32.3± 4.4

3i H3C

24.1± 3.1 20.0± 3.4



4. Conclusions

In summary, a series of 2, 4, 5, 6-substituted pyrimidine derivatives were synthesized and evaluated for antiprolif-erative activity against human leukemia cells. From the current investigation, structure–activity relationships of those compounds suggest both electron donating and elec-tron withdrawing groups on the phenyl ring attached to the sulfonamide group will determine the anticancer activity. Compounds with halogen substitution at different positions on the phenyl ring of the aryl carboxamide and sulphona-mide showed good cytotoxicity. From cell cycle analysis, it is confirmed that tert-butyl group containing derivatives able to could induce apoptosis. Further detailed investiga-tion on the structure- activity relationship should consider the substitution pattern on phenyl ring as a means to lead the discovery for more potent cytotoxic compounds. Stud-ies on the mechanism of action these compounds and modification is under progress.

5. Acknowledgements

The authors are grateful to UGC, Govt. of India for finan-cial support to KSR under the UGC-SAP (DRS-II) vide No. F. 540/10/2004-05 Programme. Asha D is grateful to SC/ST Cell, University of Mysore for JRF and UGC, New Delhi for fellowship under RFSMS order No. DV5/373[3]/RFSMS/2008-09. Sathees C Raghavan ac-knowledges the support from Lady Tata Memorial Trust international award for leukemia research (London). Prasanna DS is grateful to Council of Scientific and In-dustrial Research, New Delhi for financial support under CSIR-SRF order No. 09/119(0173)2K8 EMR-I.

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29

Transfusion of Ipscs-Derived Leukocytes to Kill Cancer

Jiang Li1*, Ying Cui2, Guodong Gao1*, Ti-Fei Yuan

1Department of Neurosurgery, Tang Du Hospital, The Fourth Military University, Xi’an, China; 2Department of Clinical Laboratory and Research center of Tang Du Hospital, The Forth Military Medical University; Xi’an, China; *Corresponding author Email: lijiang19771103@gmail, [email protected], [email protected] Received October 28th, 2009; revised December 23rd, 2009; accepted January 23rd, 2010.

ABSTRACT

Here we propose that the rejuvenation of leukocytes with iPSC technology in vitro and transfusion of cancer cell- resistant white blood cells back to human body provide a prospective therapy for cancer patients. Keywords: Ipscs, Leukocytes, Cancer

1. Introduction

60 years ago, Chester Southam injected healthy people and cancer patients with Hela cells, and found that healthy human beings are resistant to cancer cells, the ability of which was lost in cancer patients [1]. The im-portant factors that lead to the loss of innate immunity during cancer progression include the failure of leuko-cytes (white blood cells) in killing cancer cells and the fusion of cancer cell and leukocyte, which results in the metastasis of cancer cells [2]. In recent years, a sponta-neous cancer-resistant mice line was found, whose resis-tance is mediated by rapid infiltration of leukocytes and some other cells with innate immunity [3]. In these mice, the killing of injected cancer cells was completed by the migration of leukocytes to the cancer cells-injected site, the identification/recognition of specific antigens on the surface of cancer cells, as well as the release of cancer cell-killing substances, the first two steps of which do not exist in normal mice lines [3]. Additionally, aging leads to the reduction of this resistance ability, while repeated challenge with cancer cell injection can enhance the re-sistance during ageing periods. It is thus conceivable that the failed or weakened resistance of innate immunity cells, especially leukocytes, contribute to the cancer pro-gress in patients.

To restore the innate immunity, efforts must be made to re-activate the activity of white blood cells in pa-tients with cancer, or to replace those “sick” leukocytes with healthy, young leukocytes engineered in vitro, the process of which can now be completed with the re-cently emerged iPSC technology.

2. IPSC Technology

The regenerative medicine or rejuvenation therapy re-quires a large number of health cells to be placed in damaged parts of the body. However only limited sources of adult stem cells are available, and the self-repair abil-ity in mammals including human is very restricted. The use of embryonic stem cells suffers from ethical difficul-ties, and the transplantation of heterogeneous tissues of-ten meets immunological challenges. In the last two years, the successes of inducible pluripotent stem cell (iPSC) technology on human cells [4,5], including patients- sourced cells [6], provide new hopes in generating im-mune-free stem cell lines in treating human patients.

iPSC can be acquired by transfection of somatic cell with pluripotent factors-coding viral vectors, or the direct delivery of DNA plasmids into cytoplasm to promote a pluripotent fate [4,5]. Recent studies found that the tran-sient delivery of these proteins without genome integra-tion can also bring pluripotency [7,8]. Additionally, nu-merous small molecules have been identified to promote the efficiency in generating iPSCs [9,10], including natu-ral compounds such as Vitamin C [11]. Methods with further improved safety and efficiency are expected in coming years.

The iPSCs, once stabilized, can be expanded greatly with many kinds of trophic factors and cytokines. More-over, additional genetic engineering could be performed to repair potential genetic defects of the patients, or to promote the differentiation of iPSC into a defined fate. It will be therefore possible to acquire large number of cells, such as leukocytes, for repeated clinical use.

Transfusion of Ipscs-Derived Leukocytes to Kill Cancer 30

3. The Hypothesis

We hypothesize that the transfusion of healthy, cancer cell-resistant leukocytes (in future, may include other innate immunity associated cells) generated with iPSC technology can be used to treat cancer patients. More importantly, genetic modified leukocytes (such as the knockout of specific receptors [12]) could be resistant to cancer cell fusion, therefore stopping the progression of cancer cells in vivo.

However it should be also noted that white blood cells family contains several types of cells such as neutrophils, eosinophils and basophils; these cells are of diverse func-tion within the tumor microenvironment. For example, a recent study identified different subpopulations of neu-trophils (N1-N2) that may inhibit or promote the tumor growth [13]. Therefore it will be useful to genetically engineer only a subpopulation of one kind of leukocytes in reaching effective tumor control.

4. Testing the Hypothesis and Clinical Significance

There’re few technical difficulties to generate iPSCs from cancer patients and promote these cells into a leukocyte cell fate. The fibroblasts from patients’ skin tissues could be most easily harvested and induced into iPSCs with well-built protocols, while theoretically all types of so-matic cells from biopsies can be used. Some left ques-tions are the efficiency in expanding the precursor cells and directed differentiation from iPSCs into leukocytes. These procedures could benefit from protocols inducing ESCs into leukocytes [14].

It will be useful to start with the mice model, to com-pare the efficiency of healthy leukocyte transfusion with other therapies in stopping cancer progression and me-tastasis. One question to be determined is to what extent the defected resistance of white blood cells contributes to the cancer cell progress in human patients. Should one kind of cells/subpopulation of cells or cellular signaling pathways be determined to drive tumor progression in humans, surely the new therapy with genetically engi-neered iPSC-derived white blood cell transfusion would follow.

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transplantation of human cell lines,” Science, Vol. 125, No. 3239, pp. 158–160, 1957.

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[3] A. M. Hicks, G. Riedlinger, M. C. Willingham, M. A.

Alexander-Miller, C. Von Kap-Herr, M. J. Pettenati, A. M. Sanders, H. M. Weir, W. Du, J. Kim, et al., “Transferable anticancer innate immunity in spontaneous regres-sion/complete resistance mice,” Proceedings of the Na-tional Academy of Science, USA, Vol. 103, No. 20, pp. 7753–7758, 2006.

[4] J. Yu, M. A. Vodyanik, K. Smuga-Otto, J. Antosiewicz- Bourget, J. L. Frane, S. Tian, J. Nie, G. A. Jonsdottir, V. Ruotti, R. Stewart, et al., “Induced pluripotent stem cell lines derived from human somatic cells,” Science, Vol. 318, No. 5858, pp. 1917–1920, 2007.

[5] K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichi-saka, K. Tomoda, and S. Yamanaka, “Induction of pluri-potent stem cells from adult human fibroblasts by defined factors,” Cell, Vol. 131, No. 5, pp. 861–872, 2007.

[6] G. Amabile and A. Meissner, “Induced pluripotent stem cells: Current progress and potential for regenerative medicine,” Trends in Molecular Medicine, Vol. 15, No. 2, pp. 59–68, 2009.

[7] D. Kim, C. H. Kim, J. I. Moon, Y. G. Chung, M. Y. Chang, B. S. Han, S. Ko, E. Yang, K. Y. Cha, R. Lanza, et al., “Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins,” Cell Stem Cell, Vol. 4, No. 6, pp. 472–476, 2009.

[8] H. Zhou, S. Wu, J. Y. Joo, S. Zhu, D. W. Han, T. Lin, S. Trauger, G. Bien, S. Yao, Y. Zhu, et al., “Generation of induced pluripotent stem cells using recombinant pro-teins,” Cell Stem Cell, Vol. 4, No. 5, pp. 381–384, 2009.

[9] T. Lin, R. Ambasudhan, X. Yuan, W. Li, S. Hilcove, R. Abujarour, X. Lin, H. S. Hahm, E. Hao, A. Hayek, et al., “A chemical platform for improved induction of human iPSCs,” Nature Methods, Vol. 6, No. 11, pp. 805–808, 2009.

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[11] M. A. Esteban, T. Wang, B. Qin, J. Yang, D. Qin, J. Cai, W. Li, Z. Weng, J. Chen, S. Ni, et al., “Vitamin C En-hances the Generation of Mouse and Human Induced Pluripotent Stem Cells,” Cell Stem Cell, 2009.

[12] J. M. Pawelek and A. K. Chakraborty, “Fusion of tumour cells with bone marrow-derived cells: A unifying expla-nation for metastasis,” Nature Reviews Cancer, Vol. 8, No. 5, pp. 377–386, 2008.

[13] Z. G. Fridlender, J. Sun, S. Kim, V. Kapoor, G. Cheng, L. Ling, G. S. Worthen, and S. M. Albelda, “Polarization of tumor-associated neutrophil phenotype by TGF-beta: “N1” versus "N2" TAN,” Cancer Cell, Vol. 16, No. 3, pp. 183–194, 2009.

[14] M. Hannig, H. R. Figulla, H. Sauer, and M. Wartenberg, “Control of leukocyte differentiation from embryonic stem cells upon vasculogenesis and confrontation with tumour tissue,” Journal of Cellular and Molecular Medi-cine, 2008.




31

Ovarian Sex Cord-Stromal Tumors in Postmenopausal Women and Total Laparoscopical Management

Andrea Tinelli1, Marcello Pellegrino2, Vincenzo Emanuele Chiuri3, Antonio Malvasi4

1Department of Gynecology and Obstetric, Vito Fazzi Hospital, Lecce, Italy; 2Department of Pathology, Vito Fazzi Hospital, Lecce, Italy; 3Department of Oncology, Vito Fazzi Hospital, Lecce, Italy; 4Department of Gynecology and Obstetric, Santa Maria Hospital, Bari, Italy. Email: [email protected] Received October 26th, 2009; revised December 19th, 2009; accepted January 28th, 2010.

ABSTRACT

BACKGROUND: Ovarian sex-cord stromal tumors (SCST) take up 5% of the ovarian neoplasm and may develop into an ovarian mass or a haemoperitoneum. The surgical management of SCST in early-stage adult patients is not well defined. CASE REPORT: A 69 year-old postmenopausal woman was admitted for metrorrhagia, a right ovary mass and increasing pelvic pain. Preoperative clinical and instrumental examination suspected an ovarian tumor, and the laparoscopic right ophorectomy and the frozen section suggested an ovarian SCST. To fast restore and preserve woman integrity, total laparoscopic hysterectomy (TLH) plus left salpingo-ophorectomy (SO) were performed, without compli-cations in the short and long term follow-up. CONCLUSION: In the authors’ opinion, the minimally invasive manage-ment of SCST by TLH plus bilateral SO followed by a prolonged surveillance and without intensive surgical staging, could be an appropriate clinical and surgical choice in elder patient at early stage, since these tumors are slow at growth, recurring locally and only a long time after initial treatment. We suggest, after a minimally invasive treatment, a possible “wait and see” option, as in our case report. Keywords: Menopause, Laparoscopy, Ovarian Cancer, Sex Cord-Stromal Ovarian Tumors, Granulosa Cell Tumors,

Minimally Invasive Treatment.

1. Introduction

Ovarian sex-cord stromal tumors represent 5% of ovarian neoplasm cases, it can occur at any age (mean age early fifties) and generally, they present as an adnexal mass, or a haemoperitoneum, with an estrogenic pattern linked to metrorrhagia, endometrial hyperplasia or adenocarci-noma.

These tumors show a differentiation in sex cordons or in specialized stroma: the latter includes female cellular type (granulosa or theca cell), male cellular type (Sertoli or Leydig cells) and undifferentiated elements.

Cellular variety of sex-cord stromal tumor shows various degrees of differentiation, reproducing a prolifer-ating pattern present in embryogenesis [1].

2. Pathological Findings of Ovarian Sex-Cord Stromal Tumors (SCST)

Granulosa cell tumors contain two histotypes: the young type and the adult type; the latter arises frequently after

menopause, but it can also occur in puberty, associated with hyperestrogenism in 75% of total cases, leading to precocious puberty in children and metrorrhagia in adults.

Some of these cases are inactive in hormonal pattern and some are caused by androgen producers [2].

Macroscopically, sex-cord stromal ovarian tumors are capsulated, vary in size and may be solid or partially cys-tic; the cut surface may be grey-white or yellow, de-pending on lipid contents; moreover, necrosis and haem-orrhage are often present inside the capsule, with cystic compartments filled with fluid or clotted blood.

The microscopic features of sex-cord stromal ovarian tumors may be extremely changeable, even in the same tumor, with a wide variety of patterns and characteristic Call-Exner bodies may be present; they can have in some cases a microfollicular or macrofollicular growth, and in other cases trabecular or insular.

In microscopic diagnosis, cellular elements are often in the shape of coffee beans and, at an immunohysto-

Ovarian Sex Cord-Stromal Tumors in Postmenopausal Women and Total Laparoscopical Management 32

chemical level sex-cord stromal ovarian tumors express: estradiol or vimentin, inhibin, CD 99 and cytocheratin, with a reactivity to S100 marker in 50% of cases [3,4].

Currently, the gold standard treatment for SCST is by surgery, even if its surgical management for early-stage adult patients is not well defined.

3. Short Report of an Early SCST in Post-Menopausal Women Managed by Laparoscopy

A 69 year-old woman, para 1 (spontaneous delivery) with an uneventful old gynaecological history and meno-pause at 51 year’s age, was admitted to hospital for geni-tal bleeding and increasing pelvic pain. Clinical exami-nation vaginam exam revealed a palpable right adnexal mass. Transvaginal ultrasound examination revealed so-nomorphological uterine fibromatosis with increased endometrial thickness, and a right ovary mass of 8 cm in diameter, on average, with anechogenic and hypovascu-larized structure, adherent to uterine posterior wall. The ultrasound exam was confirmed by the Computer Tomo-graphy of pelvis and abdomen, performed in contrast, which reported no other diseases or anomalies. Bio-chemical markers for epithelial or germ cell ovarian tu-mors did not reveal any anomalies and patients wanted a minimally invasive treatment by laparoscopy. Thus, ba-sed on the patient’s clinical symptoms and the instru-mental examinations, diagnostic laparoscopy was per-formed, following signed detailed informed consent, based on presurgical findings to rule out malignancy. Laparoscopic access was performed using Direct Visual Access method, at the level of umbilicus, with a 10 mm diameter optical trocar (Endopath Xcel Bladeless, Ethi-con Endo-Surgery, Johnson & Johnson Company, USA) inserted through an intra umbilical vertical incision. Fol-lowing the application of carbon dioxide pneumoperito-neum, with intra-abdominal pressure maintained at 15 mmHg, the abdominal cavity was inspected through a zero-degree laparoscope (Karl Storz, Tuttlingen, Ger-many), connected to a video monitor; three supra-pubic ancillary trocars were placed as follows: one 10 mm of diameter trocar inserted in the midline, 3 cm under the umbilicus, and one in each iliac fossa (5 mm of diameter on the left side and 5 mm of diameter on the right size), laterally to inferior epigastric vessels. Before both opera-tive procedures, all pelvic structures were inspected and the abdomen was explored through the laparoscope clockwise. The pelvic-abdominal inspection showed a right ovarian cyst, with a regular surface (Figure 1), some filmy adhesions in the pouch of Douglas and be-tween the posterior uterine wall and right ovary and tube.

The uterus had multiple fibroids and the left ovary was normal; following the patient’s decision to receive mini-

Figture 1. Pelvic-abdominal laparoscopical inspection shows a large right ovarian cyst, with regular white surface, hold-ing Douglas pouch, occluding visualization of the left ovary and uterus

mally invasive treatment (previously signed on informed consent), right ovariectomy was performed by surgeons using a spill-proof endoscopic device (Endobag, Ethicon Endo-Surgery, Johnson & Johnson Company, USA). In detail, lysis of right ovarian cysts adhesions was per-formed by aqua dissection with an irrigating probe and, after this, adnexectomy was carried out with a bipolar forceps that coagulated the meso-ovarian vessels, the uterus-ovarian and infundibolopelvic ligaments, while the ovary was sustained by two Manhes forceps. The coagulated ligaments were cut with a monopolar en-doscissor and lastly, the tubal wound was irrigated and the haemostasis was re-obtained with fine bipolar scis-sors. The excised cyst, emptied in Endobag through a laparoscopic sucking-up pin, was removed via one of the trocar through a spill-proof endoscopic device, to avoid accidental leakage of the ovarian cyst content into the peritoneal cavity, which can worsen the prognosis if the cyst turns out to be malignant or can lead to peritonitis if the cyst is a dermoid or mucinous cyst. Following laparo-scopy, Dilatation & Courretage was performed, due to her history of bleeding. The surgical procedure lasted 20 minutes and intra-laparoscopic blood loss was less than 80 cc.

The frozen section histology suggested an ovarian SCST, possibly a granulosa cell tumor, while macro-scopic examinations showed a smooth right ovarian cor-tical surface. Following pathologists’ advice, in agree-ment with the patient’s preliminary decision and based on her advanced postmenopausal status and slow tumor growth, the surgeons performed a total laparoscopic hys-terectomy (TLH) with left salpingo-ophorectomy (SO), which avoided radical treatment by surgical staging, lym-phadenectomy, appendectomy and omentectomy.

Preliminarily, the authors inserted in uterus a trans-vaginal uterine manipulator (Endopath, Ethicon Endo-surgery, Inc., Cincinnati, USA) to quickly mobilize the



uterus during the procedure and activated PK System LYONS Dissecting Forceps and PK System LP Scissors (Gyrus Medical, Inc. Maple Grove, MN, USA). The left broad ligament anterior leaf was opened while the nurse pushed the uterus into retro-version; the left broad liga-ment posterior leaf was opened to allow the utero-ovar-ian ligament and tubes to be defined. This step was per-formed with PK System LYONS dissecting coagulating forceps and PK System LP monopolar scissors, followed by dissection of the same broad ligament. The uterus was pushed by the nurse towards the opposite side, to the right, using uterine manipulator traction; the left uterine vessels were isolated, the left utero-vesical pouch perito-neum was opened by dissecting and grasping coagulating forceps and the bladder was delicately pushed down-wards. The surgeons completed the uterine devasculari-zation procedure, after dissecting the left broad ligament: they closed the uterine pedicle with a 0 Vicryl, using a knot pusher after inserting a needle round the uterine vessels. The left uterine vessels were ligated in two places and incised with scissors between the knots, with final haemostasis of the uterine vessels through bipolar forceps, after checking first the position of the ureters. The same actions were performed on the right side of uterus, where the ophorectomy had been previously done. Intra-fascial hysterectomy was continued using the same dissecting coagulating forceps and monopolar scissors, coagulating the cervicovaginal vessels and opening the vagina; anterior and posterior vaginal walls were incised with monopolar scissors and haemostasis was completed through coagulating forceps. After uterine extraction, surgeons performed laparoscopic suturing of the vaginal cuff. The hysterectomy took 65 minutes and the intra-laparoscopic blood loss was 200 cc. The final examina-tion highlighted: a granulosa cell tumor of right ovary (Figure 2); the uterus showed fibroids and a cystic glan-dular hyperplasia of endometrium, the left ovary was atrophic.

The FIGO Staging of SCST was IA. The histochemi-cal analysis showed positive reaction to estrogen recep-tors, with a proliferating index (KI-67-clone MIB1) of 8% in neoplastic cells. Postoperative recovery was nor-mal, the patient was regularly discharged 3 days after laparoscopy, with no additional therapy at remission and has been followed up for the past three years with in-strumental and biochemical monitoring.

4. Discussion

In this patient, the surgeons performed a minimally invasive treatment of SCST, avoiding radical treatment by surgical staging with pelvic and para-aortic lymphadenectomy, appendectomy and omentectomy, for the following reasons:

The woman had agreed on minimally invasive treat-ment, since presurgical assessment did not show ad-

Figture 2. Histology shows adult-type sex cord-stromal tu-mors of right ovary; the inset shows tumor cells with mi-crofollicular and coffee bean features (H&E, ×20, original magnification) vanced malignancy suspect of ovarian cancer; after the frozen section results, surgeons decided the total laparo-scopical non-radical unstaged approach either for the patient’s advanced postmenopausal age, or for the biol-ogy of granulosa ovarian mass-a tumor at slow growing.

The morbidity and morbility of a radical laparoscopic approach, with pelvic and para-aortic lymphadenectomy, appendectomy and omentectomy, collide with the patient wish and with the stage and the biology of the tumor, since more than 95% of SCST are in first stage, with a mitotic index less than 3 mitosis for 10 HPF in 75% of cases [5].

A recent study by Brown et al. affirm that lymph node metastasis in ovarian SCST are rare [6].

The stage of disease is the most important prognostic factor associated with the risk of relapse: the 5-year sur-vival rate of stage I ovarian SCST is 86-96% and all other stages are between 26-46% [7].

There have been only a few case reports on distant metastases from these tumors [8,9].

Nevertheless, adult types have a better prognosis than young types, and trabecular and follicular histological pattern has a better prognosis than sarcomatoid pattern. Generally, bilateral involvement of the ovaries in SCST is observed in only 2% of cases, the opposite ovary can be preserved in younger women, while, if the uterus needs to be preserved, endometrium biopsy should be performed because the synchronous occurrence of en-dometrial adenocarcinoma is associated with estrogen secretion [10].

The differential histological diagnosis of ovarian SCST is with: undifferentiated carcinoma, small cell car-cinoma, endometrial stroma sarcoma, cellular fibroid, endometrioid carcinoma with a sex cord-like growing pattern, gonadoblastoma, melanoma or malignant mam-



malian lobular tumor metastasis [2,3]. The SCST prognosis depends on clinical stage, dimen-

sion, cellular atipia presence and tumor rupture [11]. As for recurrence, ovarian SCST can recur long after

initial treatment and locally, with an average interval of 5-10 years, with rare metastasis at distance; the longest reported interval is 37 years, therefore lifelong follow-up care is needed [12].

Factors reported to be associated with outcome include: presentation stage, age older than 40, tumor size, tumor rupture, histologic pattern, high mitotic count, and nu-clear atypicality.

The SCST recurrences are peritoneal and retro-peri-toneal masses, histologically well expressed [13].

Patients with recurrent disease or residual disease after surgery should be treated with a combination of bleomy-cin, etoposide, and cisplatin (BEP), while no evidence shows that treatment with progesterone is beneficial and the role of the radiation therapy, especially for palliation in recurrent disease in the pelvis, has yet to be proven [10,12].

A recent article examined the clinical efficacy of bevacizumab with or without concurrent chemotherapy and evaluated the angiogenic characteristics of these tu-mors; authors concluded that anti-VEGF therapy is highly effective in patients with granulosa cell tumors [13].

For the prolonged surveillance, the tumor marker fol-low-up of ovarian SCST is through inhibin, a glycopro-tein produced by granulosa cells, which may be increased in postmenopausal women with mucinous carcinomas [14].

Concludingly, this short report differs from traditional treatments since the authors propose, for the first time, a minimally invasive treatment in a SCST, for the above reasons.

Primarily, the presurgical assessment did not show advanced malignancy suspect of ovarian cancer and the frozen section results confirmed it. Then, the biology of granulosa ovarian mass, a tumor at slow growing, con-firms that lymph node metastasis of SCSTs are rare, so as the early stage of this tumor is the most important prog-nostic factor associated with the relapse risk, with a sur-vival rate of 86–96% in stage I(5-year). Nevertheless, adult types have a better prognosis than young types. Finally, the mentioned patient was an elder postmeno-pausal women of 69 years, so the morbidity and morbil-ity of a more radical laparoscopic approach collided with the patient wish and with the stage and the biology of the tumor.

Given that the literature is lack of laparoscopical treatments of SCST in adult age patients, based on our report, a prospective multicentre trial could be proposed using non-radical laparoscopy in postmenopausal pa-tients with ovarian SCST.

5. Conclusions

Generally, SCST has been treated radically in young women [15]; since there are no clear conclusions regard-ing the role of postoperative chemotherapy or radiother-apy in stage I patients and in those with complete re-sected tumor [16], TLH plus BSO not followed by lym-phadenectomy, has also proven, in our report, to be a safe and effective alternative in postmenopausal patients, with fast recovery and minimal morbidity and morbility. In view of all these findings and due to the technical ad-vances in endoscopic surgery, laparoscopic non-radical treatment of SCST could be considered a safer treatment in selected and consenting adult age patients, which agrees with a minimally invasive surgery and with a suc-cessive prolonged surveillance, also defined as “wait and see” approach, since this tumor grows slowly.

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Kavishwar Wagholikar1, Prasanna Venkatraman2, Sundararajan Vijayraghavan3§, Chandan Kumar-Sinha2*§

1Interdisciplinary School of Scientific Computing, University of Pune, Pune, India; 2Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India; 3Centre for Advanced Computing, University of Pune campus, Pune, India; *Current Ad-dress: Michigan Center for Translational Pathology, Department of Pathology, University of Michigan Ann Arbor, MI, USA; §Corresponding authors Email: [email protected], [email protected], [email protected], [email protected] Received November 13th, 2009; revised January 26th, 2010; accepted January 31st, 2010.

ABSTRACT

A new method for analysis of microarray gene expression experiments referred to as Sum-based Meta-analytical En-richment (SME) is proposed in this manuscript. SME is a combined enrichment and meta-analytical approach to infer on the association of gene sets with particular phenotypes. SME allows enrichment to be performed across datasets, which to our knowledge was not earlier possible. As a proof of concept study, this technique is applied to datasets from Oncomine, a publicly available cancer microarray database. The genes that are significantly up-/down-regulated (p-value ≤ 10-4) in various cancer types in Oncomine were listed. These genes were assigned to biological processes using GO annotations. The SME algorithm was applied to identify a list of GO processes most deregulated in 4 major cancer types. For validation we examined whether the processes predicted by SME were already documented in litera-ture. SME method identified several known processes for the 4 cancer types and identified several novel processes which are biologically plausible. Nearly all the pathways identified by SME as common to the 4 cancers were found to contribute to processes which are widely regarded as cancer hallmarks. SME provides an intuitive yet objective ‘proc-ess-centric’ interpretation of the ‘gene-centric’ output of individual microarray comparison studies. The methods de-scribed here should be applicable in the next-generation sequencing based gene expression analysis as well. Keywords: Gene Expression, Microarray, Enrichment, Meta-Analysis

1. Introduction

Microarray based gene expression profiling studies have led to the identification of hundreds of genes differen-tially expressed across diverse sample sets which has resulted in the characterization of several biomarkers and genes of interest. But the bigger promise of providing an insight into underlying biological phenomena continues to be challenging.

The problem considered here is that of whole tran-scriptome gene expression analysis by microarrays, where genes corresponding to virtually the whole ge-nome (25–30,000 genes) are simultaneously interrogated for their relative expression in a disease type (or subtype) and compared against their normal counterpart. For ex-ample, breast cancer transcriptome compared against normal breast transcriptome. This analysis provides a list

of genes measured as either over-expressed or under- expressed in the test samples relative to normal. A vari-ety of approaches have been developed in the last decade to identify gene expression features specific to different cancer types. Rhodes et al. [1] provide a categorization of these methods.

Statistics based differential analysis lists individual genes which are differentially expressed in a dataset of microarray experiments. Clustering [2,3] was the earliest attempt to further analyze this list by grouping genes on the basis of similarities in their differential expression. Both these methods are regarded as standard. In con-trast, integrated analysis methods examine data in terms of cancer signatures from other data or other types of genomic data. These include methods for meta-analysis and functional enrichment.

Sum-Based Meta-Analytical Enrichment of Gene Expression Data to Identify Pathway Signatures of Cancers 37

Meta-analysis has been defined as analysis of multiple datasets. For instance, comparative meta-profiling [4] can be used to identify signatures of genes commonly activated across datasets.

Functional enrichment attempts to interpret the list of differential expressed genes derived from one dataset using gene annotations like biological process, molecular function and cellular localizations. These methods give a formal framework for biological interpretation which was subjective in the clustering methods. These include over-representation approach (ORA) [5], functional class scoring (FCS) [6,7] and gene set enrichment analysis [8,9].

Some of the other reported methods include those based on theory of partially ordered sets [10], random forests (LeFe) [11], non-parametric pathway based re-gression models, [12] and more recently, impact analysis based on systems biology approach [13].

In this paper a new method is proposed, which in-volves a combined enrichment and meta-analytical com-parison, to infer on the association of gene sets (for ex-ample those constituting Gene Ontology, GO) with the specific phenotypes investigated in a set of microarray experiments. We refer to the method as Sum-based Meta-analytical Enrichment (SME). SME allows en-richment to be performed across datasets, which to our knowledge was not possible earlier. Our premise is that in order to implicate a ‘process’ (eg. GO Processes) in the mechanism of a pathology (eg. cancer), the various independent ‘events’ (expression changes in individual genes) constituting and/or contributing to the process serve as surrogates to studying the process itself. Greater the evidence of association of such constituent events with a pathology, greater is the confidence in the 'proc-ess-association’ hypothesis. SME is a heuristic extension of this notion. In order to calculate the significance of a gene-set phenotype association, it considers both – 1) the number of genes in the set which are differentially ex-pressed in a microarray study, and 2) the number of studies which report each of the genes as differentially expressed. Permutation testing has been used to rule-out chance associations.

As a proof of concept, this technique was applied to a publicly available cancer-microarray database (On-comine) to identify Gene Ontology processes dys- regulated in four different cancer types and an assess-ment was carried to examine whether the identified asso-ciations were already reported in literature.

2. Methods

Given a collection of microarray gene-expression datasets for a phenotype, the significance of association of a query gene-set with the phenotype, is evaluated with fol-lowing steps:

1) Determination of number of studies reporting differential expression of a gene

On each dataset in the collection, studies are per-formed for differential expression across logical group-ings of samples [14] using Student’s t-test. Study specific gene p-values (or false discovery rates) [15,16] are con-sidered to determine the number of studies which infer differential expression of a gene at a particular cut-off. The results for this step are readily available in On-comine.

2) Calculation of Sum-based Meta-analytical En-richment Score (S) for query gene-set

Genes common to the query gene-set and dataset col-lection are identified. For each of the genes in this com-mon set, we consider the number of studies in which the gene is differentially expressed. The sum of the number of such studies for all genes in the common set gives the SME score (S) of the query gene-set.

3) Calculation of significance of the SME score To evaluate the significance of the score, SME score

for a million random gene sets, each having the same number of genes as the common set (derived in previous step) is computed. The fraction of the number of random gene-sets for which the SME score is greater than or equal to the statistic for the gene-set in question, gives the s-value for the association. The term s-value is used to differentiate it from the analogous p-value, since the distribution computed in step 2 is a surrogate for the null distribution. The null-distribution cannot be accurately determined due to dearth of information about proc-ess-phenotype associations.

2.1 Mathematical Description of SME

A) Input for the method: Gi : set of genes in the collection of microarray data-

sets. Gj : the query gene set B) Steps: 1) Determination of number of studies reporting

differential expression of a gene Let Y(g) give the total number of studies across logical groupings in individual datasets which infer that a gene g is differentially ex-pressed on the basis of a p-value/q-value cut-off.

2) Calculation of SME score (S) for query gene-set

( )

( ) ( )j i

jg G G

S G Y g

3) Determination of significance of the score With the above formula, S (Gr) is calculated for very

large number of random gene sets (Gr) from Gi, such that

|Gr| = |Gj ∩ Gi |

The s-value is given by,

(1.. )

( ) ( ) /j rr n

P G X G n

where for large number n of Gr ,



1

0

r j

r

r j

when S G S G

X G

when S G S G

2.2 Case Study

Oncomine is a publicly available cancer microarray da-tabase [17]. The SME method was applied to datasets in Oncomine (Version 2.0) to identify Gene Ontology processes associated with and common to four major cancer types — breast cancer, prostate cancer, leukemia and lymphoma. Oncomine was queried for all the human genes (listed at NCBI) to obtain the number of studies reporting a gene as differentially expressed for a p-value cut-off of 10-4. All of the Gene Ontology biological process terms were listed from the GO database (http://www.geneontology.org) [18]. In GO database, processes are segregated as biological processes, cellular components and/or molecular functions and genes con-tributing to each process are annotated. For each of the GO biological processes, the contributing human genes were obtained by querying with the GO association “contributes to”. Of the 9561 GO biological processes, those which had insufficient information in oncomine were excluded. This was done by excluding processes which had less than 10 of their contributing genes listed in oncomine. Further, processes which have more than 50 genes represented in oncomine were excluded to re-move highly generic processes. 323 processes remained, and the association of each process with 4 cancer types was tested. SME was applied to obtain the s-value of association of the processes with each of the 4 cancer types and the GO processes with s-value ≤ 0.01 (em-pirically approximated) were listed.

For example, to test the association of the GO process “insulin receptor signaling pathway” (GO: 0008286) with breast cancer, a query was performed on GO data-base to get all the genes annotated to this processes with the relation “contributes to”. There are 21 such genes from which information is available for 12 genes, in On-comine. Oncomine lists the number of breast cancer studies reporting any of these genes as differentially ex-pressed (with default p-value cut-off of 10-4), in various class comparisons. These are summed (score=12) and this score is used to calculate the s-value of association between “insulin receptor signaling pathway” and “breast cancer” by forming one million random sets of 12 genes and calculating the SME score for the random sets; the number of random gene sets with an SME score equal to or more than 12 is divided by the total number of random gene sets (one million) to get the s-value of 0.000534 (Figure 1).

For validation, an assessment was made whether the processes identified for the four major cancer types have already been reported to be associated with the particular cancer types. Further, the set of processes common to the four cancer types were analyzed.

2.3 Implementation

The algorithm was implemented on a Linux (Fedora core 6) system using Perl and MySQL database, with the permutation testing module implemented in C++. A web-based front-end for calculating significance of asso-ciation of a gene-set with a particular cancer type was based on the discussed algorithm, using apache server and Support-Vector-Graphics. The tool was tested for its precision of s-value determination for a set of GO proc-esses. As seen in Figure 2, overall the standard deviation of the s-value was less than 0.0008 and decreased to-wards the extremes, which ensures the accuracy of results.

3. Results and Discussion

To facilitate the assessment similar GO processes were grouped by clustering on basis of the overlap of con-stituent gene sets.

In case of leukemia, platelet activation, hemoglobin biosynthesis and thymic T cell activation could be read-ily associated with the physiology and metabolism of blood cells and lymphocytes. A leukemia-associated CD9 glycoprotein antigen is known to have groups of N-acetyl glucosamine residues, which may explain the identification of acetyl glucosamine metabolism [19].

Involvement of sphingolipid metabolism is known in hematological malignancies and sphingolipids have been investigated as mediators of apoptosis triggered in re-sponse to anti-leukemic agents [20].

As seen for leukemia, several pathway changes listed for lymhpoma are also physiologically relevant. Some of these are associated with chemokine (interleukine) and chemokine receptor genes. These chemokines and their receptors are involved in the development and differen-tiation of immune cells. They have also been found to be

Figure 1. Significance testing




39

Figure 2. Plot of mean vs. standard deviation was obtained by multiple runs of the tool for a subset of GO processes from the study

process which is regarded as an hallmark of cancer [31]. present in cells from other carcinomas like those of colon. The receptors are GTP binding and specific association of these receptors with lymphoma, a result emerging from our study is noteworthy [21–24]. Matrix metallo-proteinases (MMPs) have been implicated in lymphomas [25] and MMPs are known for collagenolysis [26], which is identified by SME.

Regulation (dysregulation) of mitosis which grants limitless replicative potential to cancers has been aptly identified in our analysis as well. Cell cycle check point genes maintain sensitivity to growth signals in normal cells. JAK-STAT cascade is downstream of many cyto-kine and growth hormone receptors. The activation of transcription factors of the STAT (signal transducer and activator of transcription) protein family by JAK (Janus activated kinase) is reported to be constitutively activated in a many types of cancers [32]. Intracellular receptor mediated signaling pathways are known to impart car-cinogenic attribute of self sufficiency of growth signals. Regulation of the actin cytoskeleton is critically involved in endothelial cell migration required for angiogenesis [33], which is an important hallmark of cancer. Cellular protein catabolism represents the cachexia which is seen in endstage of cancers and mediators of protein catabo-lism have been targeted as cure for cancer cachexia [34]. Identification of steroid hormone receptor signaling pathway conforms to the knowledge that steroid group, among all hormones is known to act as growth factor promoting an array of different cancers [35]. In summary nearly all the identified common pathways, were found to contribute to processes which are widely regarded as cancer hallmarks.

In contrast to both the liquid tumors, the two major solid tumor types — breast and prostate showed diverse pathway associations. In the case of breast cancer, small GTPase Rho signaling pathways are known to regulate breast cancer cells [27]. The human epidermal growth factor receptor (HER-2) oncogene is known to encode a transmembrane tyrosine kinase receptor which increases invasiveness of breast cancer [28]. HER-2 has been tar-geted for therapy.

In the case of prostate cancer, androgen receptor is known to interact with an array of growth factor signal transduction events including epidermal growth factor and vascular endothelial growth factor [29]. Pathways that involve smooth muscle contraction and sugar trans-port have been shortlisted. These processes are vital to the normal functions associated with the prostate gland. The dominant association of matrix adhesion pathways may be due to their role in metastasis [30]. Bone and lymph node metastasis are common in prostate cancer.

The pathways common to the four cancers (see Figure 3) included NF-kappaB (nuclear factor-kappa-B) -mediated survival pathway. This is widely regarded as the mechanism by which cancer cells evade apoptosis, a

Apart from sharing some of the outcomes projected by other studies which have attempted to identify pathway changes, our analysis has several unique advantages due


Figure 3. Pathways common to breast cancer, prostate cancer, leukemia and lymphoma to the analytical method employed and the use of ex-perimental sets — a) A feature of the method is the large scale combined analysis of data which intrinsically pro-vides noise-reduction and highlights the consistent fea-tures associated with the phenotype. b) Most importantly all the data used for analysis are from human samples.

This obviates the analytical problems that are often asso-ciated with animal models.

3.1 Limitations

One limitation of SME is that extrapolation of the s-value of the intersection (of genes annotated to a GO process



and present in the microarray experiments) to a GO process becomes less reliable when the gap between the intersection and the annotation widens, such as for proc-esses higher in the GO hierarchy. This problem could be circumvented by excluding the processes which have a gap greater than an optimal cut-off. Also, the analysis could be made more stringent with a correction for mul-tiple hypothesis testing. In the present analysis, we have avoided both the cut-off for intersection gaps and the multiple hypothesis correction; because our analysis is exploratory in nature and processes higher in the GO hierarchy at times help to establish a context for the de-lineated children.

4. Conclusions

SME method identified several known processes for se-lecting cancer types. Moreover novel processes were delineated which are biologically plausible and have po-tential utility. Nearly all the pathways identified by SME as common to different cancers were found to contribute to processes which are widely regarded as cancer hall-marks. With the accrual of micro-array results in reposi-tories, expansion of GO database and further optimiza-tions, the method can be expected to lead to increasingly accurate output. SME makes it possible to draw infer-ences based on a large scale combined analysis of mi-croarray data by reducing noise and has an advantage in its intuitive yet objective approach.

5. Authors’ Contributions

KBW and CKS developed the algorithm and conceived the design of the case study.

KBW and VS formulated the algorithm and imple-mented the case study. They also conceived and con-ducted the automated evaluation.

PV and CKS assessed the utility of the method to pro-vide biological insight into the case study.

All authors wrote the manuscript and approved it.

6. Acknowledgements

We are thankful for the developers of Gene Ontology, Oncomine, GoPubMed and NCBI for making their data/tools available for free public access. The technical assistance given by Chitra Alavani (ISSC, University of Pune) is duly acknowledged. We thank Dr. Gadre (Di-rector, ISSC) for making computational resources avail-able for this study.

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Role of Estradiol, Progestins, Insulines and Adipocytokines in Breast Cancer Promotion in Post-Menopausal Women

Christian Jamin

AFACS 169 Bd Haussmann, Paris, France. Email: [email protected] Received December 28th, 2009; revised January 22nd, 2010; accepted February 3rd, 2010.

ABSTRACT

Estrogens and artificial progestins used in hormone replacement therapy increase breast cancer risk. This seems to be due to a promoting and not initiating effect. A synergic effect of estradiol and hyperinsulinism has been shown. Insulin plays a role in the increase of breast cancer risk when associated with android obesity, sedentariness, type II diabetes, and high glycemic index food, alcohol and trans fatty acids intake. Natural menopause induces insulin resistance and does not induce a risk decrease. The role of insulin gives a new outlook on the influence of HRT in breast cancer pro-motion: estradiol alone, which improves insulin-sensitivity, does not increase breast cancer risk. Artificial progestins associated with estrogens increase the risk, whereas estrogens associated with progesterone do not. This could be due to the fact that artificial progestins increase insulin resistance, whereas natural progesterone does not. Adipose tissue, which is an endocrine gland, is insulin dependant. Breast cancer and its seriousness are correlated to adipocytokin circulating levels such as resistin, leptin, interleukin 1, adipocyte fatty acid-binding protein, and are inversely corre-lated to the level of adiponectin. Insulin could play a synergic role with sexual steroids by a direct effect and by in-creasing adipose tissue secretions. Keywords: Breast Cancer, Estrogens, Progestins, Insulin, Hormone Replacement Therapy, Adipocytokin

1. Introduction

The fact that breast cancer mainly affects women after puberty and regresses after an ovariectomy can logically lead to the conclusion that sexual steroids play a pre-dominant part in the genesis and the promotion of breast cancer. For numerous researchers, this predominant part has become a unique part, and consequently they have come to make estrogens a scapegoat in all epidemiologi-cal and clinical observations. A few disturbing elements like the fact that breast cancer incidence still rises after menopause, whereas it decreases sharply after hormone replacement therapy (HRT) has been stopped, raise some questions about the validity of such an approach [1,2]. A new actor, insulin, might play a predominant part in the future.

2. Role of Sexual Steroids in the Initiation and/or Promotion of Post Menopausal Breast Cancer: State of the Art

A Lancet meta-analysis compiled studies on the relation-ship between HRT and breast cancer risk and found a

1,26 RR [3]. The Women Health Initiative (WHI) study confirms this increase after 5 years of treatment [4], but only among women taking HRT before the study. It is thus impossible to evaluate the necessary HRT duration before a significant risk increase appears. The Million Women Study (MWS) [5] is in accordance with the Lan-cet meta-analysis and WHI. Only women with a BMI higher than 25 kg/m² have a risk increase [4].

Globally RR is low, but since spontaneous risk in-creases with age, this low RR could induce a high attrib-utable risk over long treatment periods (10 or 15 years) [6]. When one considers the rapidity at which excess risk appears, plus the fact that this risk increase disappears rapidly when the treatment is stopped, plus the absence of in situ cancers increase, it is likely that there is a promo-tion phenomenon and not of initiation [1,2]. The arm estrogens only of WHI, as well as numerous other studies of the cohort, do not show any increase with estrogens only (RR=0,77 (0,57–1,06) [7,8]. One study with a very long follow-up is the only one to show a very late risk increase (more than 15 years) with estrogens only [9].

Role of Estradiol, Progestins, Insulines and Adipocytokines in Breast Cancer Promotion in Post-menopausal Women 44

The French E3N cohort study does not show any risk increase with the association estrogens-natural proges-terone or retroprogesterone and confirms the risk increase when estrogens and synthesis progestins are associated, whatever their type [10]. All these results are concordant with the measure of apoptosis proliferation which shows a maximum promotion with the association estrogens- MPA or NETA, an intermediate promotion with estro-gens only and a minimum promotion with the association estrogens-progesterone and retroprogesterone or tibolone [11]. A randomized study versus placebo shows a sig-nificant breast cancer risk decrease after 3 years of ti-bolone [12]. Cancers discovered during HRT have a bet-ter prognosis, which could be due to a better differenti-ated histologic form. They are almost exclusively lobular or lobulo ductal cancers, with more hormone-sensitive forms (E3N) [13–15]. Their metastatic risk evaluated over 20 years is weaker, whatever the site [16]. These favourable prognosis characteristics are observed only among women treated with estrogens associated with progestins and not among women treated with estrogens only or with the association estrogens/progesterone.

3. The Role of Hyperinsulinism in Breast Cancer

In the observational WHI study, each woman entering the study had an exhaustive hormonal biological testing. The correlations between the hormone levels of women when they entered the study and their subsequent breast cancer risk have shown that there is not one but two actors very predictive of this risk: blood levels of estradiol and insu-lin [17]. It has to be pointed out that in this study neither IGF1 nor IGFBP3 are independent risk factors. The insu-lin effect is not affected by the adjustment of estradiol level, and similarly the adjustment of insulin level does not affect the effect of estradiol. Therefore these two fac-tors are independent. As of today, three others studies have evaluated in a prospective way the insulin level as a predictive breast cancer risk factor. Two of them do not find any correlation, but they included women under HRT. The last one, which did not include women under HRT, finds the same result as observational WHI. An-other study showed that when a post-menopausal woman has breast cancer, her insulin level at the time of diagno-sis is strongly predictive of her mortality risk after 10 years [18–21].

In another study, after stratification of insulinemia in quartiles, it has been found that the insulinemia superior quartile versus the inferior quartile gives a mortality RR at 8 and of recurrence at 4 [22]. In the MA 14 study, in-sulin resistance is associated with survival reduction without recurrence [23]. Finally a preliminary work shows that when insulin receptors are measured in tumours, tumours with the most receptors have a worse

prognosis. Insulin stimulates normal and cancer cell proliferation

and has a promoting effect on breast tumours in the ani-mal. The insulin receptor is over expressed in breast can-cer, and in woman hyperinsulinism is associated with an increase in estrogen ovarian production, a decrease of estradiol carrier protein (SHBG), and therefore an in-crease of free estradiol [15,24–29]. The fixation of insulin to its receptor on breast cancer cell increases phosphati-dylinositolkynase and kinase MAP activity. Furthermore, insulin activates estrogens receptor alpha-mediated tran-scription in breast cancer cell lines, even in the absence of estradiol. In addition estradiol activates the MAPK pathway of insulin. Finally, in breast cancer cell culture medium, estradiol has a proliferative effect only in the presence of growth factors such as insulin.

This throws a different light over numerous situations that could not be explained until now. With menopause comes insulin resistance: this could explain the fact that breast cancers do not diminish at menopause, but do di-minish rapidly when HRT is stopped [1,3,30]. Over-weight is a recognised breast cancer risk factor after menopause, whereas it is rather protective before meno-pause. In this regard we know that pre-menopausal gy-noïd obesity does not alter insulin-sensitivity, whereas post-menopausal android obesity is associated with insu-lin-resistance [31].

Metabolic syndrome and its clinical marker, waist- to-hip ratio, multiplies by 2 breast cancer risk, and this independently from testosterone level. Furthermore this ratio is predictive of breast cancer mortality at the time cancer is discovered [32–35].

The level of HDL cholesterol, a marker of insulin sen-sitivity, is inversely correlated to insulinemia and breast cancer risk [35,36].

Nowadays physical activity is admitted to be a protec-tive factor from breast cancer, as well as a reduction fac-tor of breast cancer mortality [18,37,38]. This protection also exists among women with a BRCA1/2 mutation [39]. In increasing muscular mass, physical activity improves insulin-sensitivity and thus reduces circulating insulin level, including after breast cancer [40].

High trans fatty intakes are associated with a high breast cancer risk RR=1,75 (1,08–2,83) in E3N , and with a higher insulin resistance risk and diabetes [41,42]. Al-cohol, which is a recognized breast cancer risk factor after menopause, reduces insulin-sensitivity and increases insulin receptors in tumours [43]. Food glycemic index is a marker of insulinic response to their ingestion. The more food with high glycemic index a woman eats, the higher her breast cancer risk [44,45].

Type II diabetics, non insulin-dependent, have a hy-perinsulinism and a 1,2 breast cancer relative risk. Stud-ies have shown that when these type II diabetes are treated with metformin, there is a reduction of breast



cancer mortality, whereas a treatment by sulfonyl ureas or by insulin increases this mortality [46–48].

4. Interactions between Insulin Sensitivity and HRT

This estradiol/insulin synergy enlightens the relationship between HRT and breast cancer risk. We know that es-trogens given at mean doses by oral or transcutaneous routes improve insulin-sensitivity and thus diminish insu-linemia, whereas oral estrogens given at high doses and/or associated with artificial progestin, in particular MPA, increase insulin-resistance [30]. Furthermore the PEPI study has shown that contrary to MPA, natural progesterone does not alter insulin-sensitivity, which is improved by equine conjugated estrogens [49]. Thus it could be because of insulin that HRT containing artificial progestins increase breast cancer risk, that estrogens only do not increase or maybe even reduce this risk, and that, contrary to progestins, progesterone associated with es-trogens have no deleterious effect [7,10].

Moreover, deleterious effects of HRT containing arti-ficial progestin are maximum in immediate post- menopause when insulin resistance has not set in. At dis-tance from menopause, the same treatments have a weaker effect because insulin resistance linked to meno-pause has already had its own effect [50].

5. Indirect Effects of Insulin on Breast Cancer Risk: Role of Adipose Tissue and Adipocytokines

Adipose tissue secretes hormones called adipocytokines. The volume of adipose tissue is increased by insulin. Thus insulin could play a direct part on cancerous cells, but could also have an indirect effect in modifying adi-pocytokines secretions. Among these adipocytokines, resistin is higher in women with breast cancer, is associ-ated with cancer seriousness and is partly responsible for insulin-resistance. Leptin, another adipocytokine, is also high among women with breast cancer, metastatic in par-ticular, and increases aromatase activity in intra- mammary production of estrogens. Interleukin 1, also secreted by adipose tissue, has a level correlated to breast cancer risk and progression. Adipocyte fatty acid binding protein is predominantly expressed in the cytosol of ma-ture adipocytes and has been described as associated with obesity markers and obesity-related diseases. Adipocyte fatty acid binding protein is recognized to affect insulin sensitivity, lipid metabolism, and an inflammatory re-sponse associated with atherosclerosis. Adipocyte fatty acid binding protein has been recently shown to be sig-nificantly associated with breast cancer risk. Finally adi-ponectin is lowered in case of metabolic syndrome, type II diabetes and android obesity. It is low in women with breast cancer and its level is inversely associated with

node invasion. It increases mammary cancer cell apop-tosis and diminishes neo-vascularization [51–54].

6. Conclusions

Thus the insulin/estrogens couple has a synergic and in-dependent effect on breast cancer risk. Estradiol does not promote these cancers in the absence of hyperinsulinism. Insulin-dependent adipose tissue could also be one of the actors of this synergy, through adipocytokines.

This approach gives new elements for a better under-standing of the relationship between hormones and breast cancer, and might in the future originates in new thera-peutic and/or preventive strategies in breast cancer.

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Role of Estradiol, Progestins, Insulines and Adipocytokines in Breast Cancer Promotion in Post-menopausal Women


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