Research Article Iminoflavones Combat 1,2 …downloads.hindawi.com/journals/bmri/2014/569130.pdfconducted as per the OECD test guideline (acute oral toxicity, xed dose procedure) [

Research ArticleIminoflavones Combat 12-Dimethyl Hydrazine-InducedAberrant Crypt Foci Development in Colon Cancer

V Ganga Prasad1 Shishir Kawade2 B S Jayashree2

Neetinkumar D Reddy1 Albi Francis1 Pawan G Nayak1 Anoop Kishore1

K Nandakumar1 C Mallikarjuna Rao1 and Rekha R Shenoy1

1 Department of Pharmacology Manipal College of Pharmaceutical Sciences Manipal UniversityManipal Karnataka 576104 India

2Department of Pharmaceutical Chemistry Manipal College of Pharmaceutical SciencesManipal University Manipal Karnataka 576104 India

Correspondence should be addressed to Rekha R Shenoy rekhashenoymanipaledu

Received 21 February 2014 Revised 13 May 2014 Accepted 19 May 2014 Published 5 June 2014

Academic Editor Gagan Deep

Copyright copy 2014 V Ganga Prasad et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of the present study was to evaluate the antitumor potential of iminoflavones in in vitro and in vivo anticancer modelsPreliminary screening in various cancer cell lines revealed four potential iminoflavones out of which IMF-8 was taken based on itsactivity against colon cancer cells This was further confirmed by observing the nuclear changes in the cells by AOEB and Hoechst33342 staining studies In vivo activity was assessed by dimethyl hydrazine-(DMH-) induced colon cancer model in rats Animalswere administered DMH (20mgkg bw for 10 weeks and 30mgkg bw ip for 10 weeks) and were supplemented with (IMF-8)iminoflavone-8 (200mgkg po for 14 days) Results showed that DMH induced 100 aberrant crypt foci (ACF) and polyps whichwere significantly reduced in the IMF-8 treated group IMF-8 significantly increased the catalase and GSH levels whereas it reducedthe TNF-120572 and IL-6 levels markedly which suggests the antioxidative and anti-inflammatory actions of flavonoids present in IMF-8The histopathological images of the IMF-8 treated colon showed no signs of mucosal crypt abscess These findings suggest that thesemi-synthetic iminoflavones IMF-8 effectively inhibit DMH-induced ACFs and colonic crypts by alleviating the oxidative stressand suppressing the inflammation

1 Introduction

Dietary flavonoids are commonly found in various itemsof plant origin In vitro flavonoids are proven to exhibitantiproliferative action for a range of cancer cell lines [1]Contemporary research has reported potent anticarcinogenicactions of flavonoids in treatment of colon cancer [2] Todaycolon cancer is the most commonly diagnosed cancer andthe second leading cause of death in the USA [3] Theglobal prevalence of colon cancer points to effective treatmentstrategies for the prevention and treatment of colon cancersApproximately 60of the drugs that are being used currentlyare natural products [4]

Huge emphasis is being given to natural products fortheir anticancer properties owing to their health benefits

and few side effects and drawbacks of chemotherapeuticagents Phenolic compounds have been shown to possessantioxidant activity [5] based on their hydroxyl groupMoreover phenolic compounds also possess a widespectrum of biological activities such as antimutagenic[6] anticarcinogenic [7] anti-inflammatory [8] andantiallergic activities [9] as well as the ability to modifygene expression [10] Importantly a flavonoid derivativeflavopiridol was widely used in traditional medicine andwas a novel semisynthetic flavone from Indian tree Wesynthesized a series of novel substituted iminoflavones(IMF) and reported its cytotoxic activity [11] Out ofthe 12 synthesized derivatives IMF-2 [4-4-[24 Dinitro-phenyl-hydrazine]-4H-chromen-2yl-benzonitrile] IMF-4

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 569130 7 pageshttpdxdoiorg1011552014569130

2 BioMed Research International

[N-[2-(4-Bromo-phenyl)-chromen-4ylidene]-N1015840-(24 dini-tro-phenyl)-hydrazine] IMF-5 [N-(24 Dinitro-phenyl)-N1015840-(2-pyridine-4yl-chromen-4ylidine)-hydrazine] and IMF-8[(4-4-[24 Dinitro-phenyl-hydrazono]-4H-chromen-2yl-phenyl)-dimethyl-amine] were found to possess a low IC

50

against Hela and HepG2 cell lines However iminoflavoneshad not been tested experimentally for their influenceon colon cancer IMF-2 -4 -5 and -8 were screened againsthuman colon cancer cell line (HCT-116) Hence we selectedan iminoflavone IMF-8 from our pilot studies to assess itspotential on dimethyl hydrazine-induced colon cancer inalbino rats of Wistar strain

2 Materials and Methods

21 Chemicals Dimethyl hydrazine MTT and SRB wereprocured from Sigma-Aldrich Co LLC St Louis MO USA5-Fluorouracil was purchased from Biochem PharmaceuticalIndustries Ltd Mumbai Maharashtra India MCF-7 MDA-MB-231 EAC and HCT-116 cell lines were procured fromNational Centre forCell Science (NCCS) PuneMaharashtraIndia Media serum and cell culture products were procuredfrom Invitrogen Bio Services India Pvt Ltd BangaloreKarnataka India

22 Synthesis of Iminoflavones A series of novel substi-tuted iminoflavones were assessed which were designed andsynthesized previously in our laboratory [11] In brief thegeneral scheme of synthesis for iminoflavones involves thefollowing steps Synthesis of chalcones involves Claisen-Schmidt condensation synthesis of flavones involves cycli-sation of chalcones and synthesis of iminoflavones involvescondensation of 24-DNP with flavone moiety

23 Preliminary In Vitro Cytotoxicity Studies Sulforhoda-mine-B and Trypan blue exclusion assays [12 13] wereperformed using HCT-116 MCF-7 MDA-MB-231 and EACcell lines Similarly AOEB and Hoechst 33342 staining [1415] were done to examine the nuclear morphological changeswithin the cells SRB assay which measures total proteincontent of the cells is directly proportional to viable cellcount Trypan blue dye exclusion assay is a well-accepteddirect method for the measurement of cell death Stainingthe cells with fluorescent dyes including acridine orangeand ethidium bromide is used in evaluating the nuclearmorphology of dead cells To confirm whether apoptosis ornecrosis has been induced by selected iminoflavones coloncancerous cells (HCT-116) were analyzed in the presence ofacridine orange and ethidium bromide staining (AOEB)staining As a control untreated cells were cultured incomplete media and stained with AOEB

The apoptotic index (AI) was calculated to confirm thatiminoflavones caused cell death via apoptosis AI is describedas the percentage of apoptotic cells and apoptotic bodieswithin the overall population of cells An apoptotic indexwas determined as the percentage of apoptotic cells atleast 200 counted cells under observation using an invertedmicroscope

Table 1 Preliminary cytotoxicity assessment in various cell lines

Treatment IC50 in 120583M (mean plusmn SEM)

HCT-116 MCF-7 MDA-MB-231 EAC

Doxorubicin 288 plusmn 002 443 plusmn 034 177 plusmn 01 2505 plusmn 09IMF-2 1052 plusmn 09 ND ND NDIMF-4 1321 plusmn 12 ND ND NDIMF-5 4179 plusmn 067 4253 plusmn 078 5913 plusmn 076 1102 plusmn 021IMF-8 278 plusmn 010 1351 plusmn 013 638 plusmn 024 366 plusmn 32Values are reported as mean plusmn SEM of three readings in triplicate ND notdeterminedHCT-116 MCF-7 and MDA-MB-231 by SRB assay (exposure 48 h)EAC by Trypan blue exclusion assay (exposure 4 h)

Table 2 Effect on the apoptotic indices by AOEB and Hoechst33342 staining

Apoptotic indices (mean plusmn SEM)Groups AOEB staining Hoechst 33342 stainingControl 37 plusmn 12 67 plusmn 088Doxorubicin 46 plusmn 173a 387 plusmn 240a

IMF-8 333 plusmn 376a 347 plusmn 296a

Values are reported as mean plusmn SEM of three readings in triplicatea119875 lt 001 when compared to the control group

24 Acute Toxicity Studies The acute oral toxicity study wasconducted as per the OECD test guideline 420 (acute oraltoxicity fixed dose procedure) [16 17] Twelve Swiss albinomice weighing 22ndash25G (11-12 weeks old) were used for thetoxicity studies Test compounds were orally administeredwith a single dose of 300 1000 and 2000mgkg bw Ani-mals were observed for possible behavioural changes suchas tremors convulsions sleep altered feeding salivationaltered somatomotor activities and diarrhoea These obser-vations were continued for a period of 14 days followingwhich animals were sacrificed to examine gross changes tothe vital organs

25 Animals and Treatments MaleWistar rats inbred at Cen-tral Animal Research Facility Manipal University ManipalIndia were used in the study Animals were acclimatizedto the experimental room having temperature 23 plusmn 3∘Ccontrolled humidity conditions 75 and 12 12 hours lightand dark cycle Rats were housed in sterile polypropylenecages containing sterile paddy husk as bedding material Theanimals were fed on autoclaved rat feed and water Eight toten weeks old male rats weighing 120 plusmn 10 g were used forthe study The animal care and handling was carried out inaccordance with guidelines issued by Institutional AnimalEthics Committee Manipal University Manipal KarnatakaIndia (number IAECKMC892012) Animals were dividedinto four groups (119899 = 6) namely normal control DMH con-trol 5-fluorouracil and IMF-8 The normal control received025 CMC po Animals were administered DMH weeklyonce at a dose of 20mgkg body weight (ip) for 10 weeksand 30mgkg bw ip for 10 weeks 5-FU was given at a dose

BioMed Research International 3

Table 3 Effect on ACF incidence number of ACFs and size of polyps (adenomas) in DMH-induced colon cancer in rats

Groups ACF incidence ()Polyps (adenomas)

Number of ACFscm2

(mean plusmn SEM)Small

(01ndash1mm)Medium(2ndash4mm)

Large(5ndash8mm)

Totalcount

Control 0 0 0 0 0 0DMH 1000 1685 plusmn 15a 4 5 4 135-FU 1000 116 plusmn 10a 3 3 1 7IMF-8 1000 125 plusmn 16a 4 5 0 8Values are reported as mean plusmn SEM of three readings in triplicatea119875 lt 001 when compared to the control group

of 10mgkg ip and IMF-8 at 200mgkg po One animalfrom each group was sacrificed and examined for aberrantcrypt foci (ACFs) polyps adenoma and adenocarcinomaafter 20 weeks After confirming the induction of colonicACF (methylene blue staining) animals were randomizedaccording to body weight and treatments started

26 Parameters Monitored in DMH Model At Day 15 theanimals were sacrificed and colons and livers were dissectedout and perfused with an ice cold saline transcardially Thetissues were blot-dried and weighed and 10 of homogenatewas prepared with ice cold (150mM) potassium chloridesolution using a homogenizer (RQT-124AD REMI groupMumbai Maharashtra India) The homogenate was used forthe following estimations namely GSH [18] catalase [19]nitrite TNF-120572 and IL-6 [20] In the DMH model ACFincidence polyps and ACF count colon lengthweight ratioand histopathological examinations were performed

27 Statistical Analysis Data were analyzed by one-wayanalysis of variance (ANOVA) and significant differencesamongst treatment groupswere evaluated by post hoc Tukeyrsquostest The results were considered significant at 119875 lt 005 Allstatistical analyses were done using Prism 503 DemoVersion(GraphPad Software Inc La Jolla CA USA)

3 Results

31 Preliminary In Vitro Cytotoxicity Studies Based on pre-liminary screening by SRB assay in HCT-116 MCF-7 MDA-MB-231 and EAC cell lines IMF-8 [dimethylamino substi-tuted iminoflavone] was selected for further studies (Table 1)In HCT-116 cells IMF-8 showed an IC

50of 278 plusmn 010 120583M in

the SRB assay Similarly IMF-8 was treated with EAC cellsfor 3 h to evaluate number of dead cells IMF-8 was found tohave an IC

50of 366 plusmn 32 120583M in the Trypan blue exclusion

assay Doxorubicin and IMF-8 showed apoptotic indices of46 plusmn 173 and 333 plusmn 376 respectively IMF-8 has comparableactivity with standard drug doxorubicin when assessed bythe AOEB staining Likewise by the Hoechst 33342 stainingdoxorubicin and IMF-8 showed apoptotic indices of 387 plusmn240 and 347 plusmn 296 respectively (Table 2)

32 Acute Toxicity Studies All the mice were alive andhealthy for 14 days IMF-8 was found to be safe up to

2000mgkg Hence the dose selected was one tenth of thesafe dose that is 200mgkg

33 Parameters Monitored in DMH Model 100 ACF inci-dence and polyps were seen in all the treatment groups exceptin normal controlTheywere found to be reduced in 5-FUandIMF-8 in comparisonwithDMHcontrol InDMHcontrol 5-FU and IMF-8 ACF count was found to be 1685 plusmn 15 116 plusmn10 and 125 plusmn 16 respectively (Table 3) Treatments with 5-FU and IMF-8 showed (119875 lt 0001) significant reductionin ACF formation as compared to DMH control The colonlengthweight ratio was found to be significantly reducedin DMH control as compared to normal control In all thetreated groups colon lengthweight ratio was increased butnot significant with DMH control except 5-FU treatment(data not shown) In all treatment groups there were nosignificant differences in liver and spleen index as comparedto normal and DMH control

34 Biochemical Investigations DMH control showed a sig-nificant decrease in endogenous antioxidants namely cata-lase GSH and a rise in nitrite content as compared to normalcontrol 5-FU and IMF-8 showed a significant increase in thecatalase activity as compared to DMH control Only IMF-8treatment was able to maintain high GSH level as comparedto DMH control IMF-8 was able to decrease nitrite contentbut not significant with DMH control whereas 5-FU showeda significant decrease in nitrite content (Figure 1)

A significant rise in TNF-120572 and IL-6 levels was noted inDMH control compared to normal control The 5-FU andIMF-8 treatments were able to decrease TNF-120572 level Therewas no significant decrease in IL-6 levels in all the treatmentgroups except 5-FU standard (Figure 2)

35 Histopathological Results In normal control colonshowed no signs of crypt abscess and dysplasia In DMH con-trol group colonic mucosa showed crypt abscess aberrantcrypt foci and nuclear enlargement with adenocarcinomaIntense methylene blue stained ACF is shown by arrowTreatment with 5-FU and IMF-8 showed no signs of mucosalcrypt abscess and exhibited a few ACFs (Figure 3)


Catalase

0

5

10

Control DMH 5-FU IMF-8

Cata

lase

(120583M

mg

of p

rote

in)

a

a b

a b

0

5

10


GSH

aa a b

GSH

(120583M

mg

of p

rote

in)

0

5

10


Nitrite content

b

a

Nitr

ite (120583

Mm

g of

pro

tein

)

Figure 1 Effects of IMF-8 on the catalase glutathione (GSH) and nitrite levels in normal control 5-FU and DMH control in albino ratsa119875 lt 005 when compared with control b119875 lt 005 when compared with DMH Data is expressed as Mean plusmn SEM where 119899 = 6

ControlDMH

5-FUIMF-8

ControlDMH

5-FUIMF-8

ab bTN

F-120572

(pg

mg

of p

rote

in)

TNF-120572 content

0

100

200

300

400

Treatments

a

IL-6

(pg

mg

of p

rote

in)

IL-6 content

0

100

200

300

400

500

b

Treatments

Figure 2 Effects of IMF-8 on the TNF-120572 and IL-6 levels in normal control 5-FU and DMH control in albino rats a119875 lt 005 when comparedwith control b119875 lt 005 when compared with DMH Data is expressed as Mean plusmn SEM where 119899 = 6

4 Discussion

Flavonoids are proven to be of potential antiproliferativeanti-inflammatory apoptotic and anticancer activity withdiverse cell systems [21] Alvocidib (Flavopiridol) belongsto a family of flavonoids and is a cyclin-dependent kinaseinhibitor under clinical development for the treatment ofchronic lymphocytic leukemia and gastric cancer [22]

Fifteen of the 55 flavone derivatives were significantlyactive against at least one of these cell cultures [23] Thefirst step in the development of a new drug as a potentialanticancer agent is the evaluation of its cytotoxic potentialin cancer cell lines In our study we evaluated a detailedanticancer potential of iminoflavones in HCT cancer cellline in vitro and in a suitable animal model in vivo Inthis direction four iminoflavones were screened for their


Normalcontrol

DMHcontrol

5-FU

IMF-8

(a) (b) (c)

Figure 3 Histopathological images of the rat colons in normal control DMH control 5-FU and IMF-8 treated groups (a) Methylene blue(10x) (b)methylene blue (40x) and (c) haematoxylin-eosin stain (40x) Arrows indicate aberrant crypt foci Normal control and 5-FU groupsshowed no signs of crypt dysplasia

cytotoxic potential against HCT cancer cell line using twodifferent assays

The specific way in which a cell reacts to its environmentvaries It varies according to the set of receptor proteinsthe cell possesses which determines the particular subsetof signals it can respond to and it varies according tothe intracellular machinery by which cell integrates andinterprets the signals it receives Hence different type ofcells may respond differently to the same compound Resultsobtained from these two basic assays demonstrated thatIMF-8 was highly active against HCT-116 Next we raisedthe question whether IMF-8 mediated cytotoxicity in thecancer cells is owing to necrosis andor apoptosis whichare key cell death processes As a control untreated cells

were cultured in complete media and stained with AOEBThe data showed that IMF-8 induced apoptosis after 24 hincubation The percentage of apoptotic cells after treatmentwas increased significantly compared to control in HCT-116 cells AI was also significantly (119875 lt 001) increasedin treatment groups when compared to untreated groupThis is suggestive of the iminoflavonersquos capability of inducingapoptosis especially those cells which are in the final stagesof apoptosis significantly Since with Hoechst stain therewas a significant rise in apoptosis with IMF-8 it shows thatthe flavone could possibly bind to DNA and would haveinterfered with DNA replication during cell division

Colon carcinogenesis is a multistage process whichinvolves initiation promotion and progression phasesThus


the multistage sequence of events has many phases forprevention and intervention Chronic inflammation is a well-recognized risk factor for development of human cancer Inthe present study DMH control animals showed initiation ofACF promotion into adenoma and the significant increasein inflammatory mediator (TNF-120572 and IL-6) levels

DMH is a chemical carcinogen known to cause coloncancer with a reproducible experimental in vivo system forstudying ldquosporadicrdquo (nonfamilial) forms of colon carcinoma[24] Metabolism of DMH leads to the formation of AOMMAM and methyl diazonium ions which involves alkylationof colonic mucosal DNA The primary metabolite of DMHthat is AOM which is responsible for methylation at the O-6position of guanine occurswithin 6 to 12 h ofDMH injection

ACFs are considered to be an earliest hallmark of coloncarcinogenesis They are the focal lesions of colonic mucosaconsisting of several enlargedaberrantelliptical shape cryptswhich can be differentiated from normal crypts [25] Consid-ering this fact we confirmed the induction of colon cancerand grouped the animals by evaluating the colonic ACFs withmethylene blue staining

The mutations in k-ras hypo-Met-DNA in ACFs leadto the development of small and large adenoma which willfurther develop into adenocarcinoma by the mismatch repairand p53mutation [26]We observed the initial lesions of ACFand development of small adenoma in DMH control groupwhereas onlyACFs andpolyps but not adenomaswere notedin the rest of the groups Also some studies showed that DMHadministration had increased levels of TNF-120572 and IL-6 anddecreased endogenous antioxidant enzyme levels [27]

The increases in ACF count and number of polypswere used to evaluate the extent of damage to the coloncaused by DMH DMH treatment showed increase in ACFcount with adenoma The standard drug 5FU and IMF-8showed better anticancer efficacy in terms of maintaininglower number of ACFs polyps lower levels of TNF-120572 IL-6significantly higher levels of catalase enzymes and to someextent higher levels of GSH enzyme as well IMF-8 showedcomparable efficacy with respect to the standard 5-FU inthis model with an additional anti-inflammatory activityand by enhancing endogenous antioxidant enzyme levelsHistopathological studies showed mucosal crypt abscesshuge number of aberrant crypt foci and nuclear enlargementwith adenocarcinoma in only DMH treated group whereasthere was less number of aberrant crypt foci with no signs ofmucosal crypt abscess in 5-FU and IMF-8 treated groups

In view of the chemical stability of compound IMF-8 a ldquodimethylaminordquo substituted iminoflavone has com-paratively better electron donating property (a lone pair ofelectrons) which confers the structure higher stability Basedon chemical stability in vitro anticancer activity reducedACF count improved endogenous antioxidant enzymesdecreased inflammatory mediator levels and histopatholog-ical findings we conclude that IMF-8 could be a promisingflavonoid for combating against colon carcinogenesis

The exact mechanism of IMF-8 was difficult to establishsince it had multifaceted actions However it is known that amajority of naturally occurring flavones show tumor growthinhibition in cancer cells through tyrosine kinase receptor

inhibition [28] But we speculate that the investigationaliminoflavone IMF-8 mediated cytotoxicity in cancer cellscould be due to apoptosis

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgment

The authors are thankful to Manipal University for providingthe necessary facilities and infrastructure to perform thisproject

References

[1] M H Castillo E Perkins J H Campbell et al ldquoThe effects ofthe bioflavonoid quercetin on squamous cell carcinoma of headand neck originrdquoThe American Journal of Surgery vol 158 no4 pp 351ndash355 1989

[2] E P Lansky and R A Newman ldquoPunica granatum (pome-granate) and its potential for prevention and treatment ofinflammation and cancerrdquo Journal of Ethnopharmacology vol109 no 2 pp 177ndash206 2007

[3] R T Greenlee M B Hill-Harmon T Murray and M ThunldquoCancer statistics 2001rdquoCa ACancer Journal for Clinicians vol51 no 1 pp 15ndash36 2001

[4] M Gordaliza ldquoNatural products as leads to anticancer drugsrdquoClinical and Translational Oncology vol 9 no 12 pp 767ndash7762007

[5] S A B E van Acker D-J van den Berg M N J L Tromp etal ldquoStructural aspects of antioxidant activity of flavonoidsrdquo FreeRadical Biology and Medicine vol 20 no 3 pp 331ndash342 1996

[6] J S Choi K Y Park S H Moon S H Rhee and H S YoungldquoAntimutagenic effect of plant flavonoids in the Salmonellaassay systemrdquo Archives of pharmacal research vol 17 no 2 pp71ndash75 1994

[7] X Zi D K Feyes and R Agarwal ldquoAnticarcinogenic effectof a flavonoid antioxidant silymarin in human breast cancercells MDA-MB 468 induction of G1 arrest through an increasein Ciplp21 concomitant with a decrease in kinase activityof cyclin- dependent kinases and associated cyclinsrdquo ClinicalCancer Research vol 4 no 4 pp 1055ndash1064 1998

[8] A Garcıa-Lafuente E Guillamon A Villares M A Rostagnoand J A Martınez ldquoFlavonoids as anti-inflammatory agentsimplications in cancer and cardiovascular diseaserdquo Inflamma-tion Research vol 58 no 9 pp 537ndash552 2009

[9] H Cheong S-Y Ryu M-H Oak S-H Cheon G-S Yooand K-M Kim ldquoStudies of structure activity relationship offlavonoids for the anti-allergic actionsrdquo Archives of PharmacalResearch vol 21 no 4 pp 478ndash480 1998

[10] E R Gilbert and D Liu ldquoFlavonoids influence epigenetic-modifying enzyme activity structure-function relationshipsand the therapeutic potential for cancerrdquo Current MedicinalChemistry vol 17 no 17 pp 1756ndash1768 2010

[11] B S Jayashree S Kawade and H Venkatachalam ldquoAnticancerevaluation of some of the novel iminoflavonesrdquoResearch Journalof Chemistry and Environment vol 18 no 1 pp 32ndash40 2014

[12] P Skehan R Storeng D Scudiero et al ldquoNew colorimetriccytotoxicity assay for anticancer-drug screeningrdquo Journal of theNational Cancer Institute vol 82 no 13 pp 1107ndash1112 1990


[13] W Strober ldquoAPPENDIX 3B trypan blue exclusion test of cellviabilityrdquo Current Protocols in Immunology 2001

[14] S Kasibhatla G P Amarante-Mendes D Finucane T BrunnerE Bossy-Wetzel and D R Green ldquoAcridine orangeethidiumbromide (AOEB) staining to detect apoptosisrdquo Cold SpringHarbor Protocols vol 3 2006

[15] W Sawicki and S Moskalewski ldquoHoechst 33342 staining cou-pledwith conventional histological techniquerdquo StainTechnologyJournal vol 64 no 4 pp 191ndash196 1989

[16] OECD ldquoTest no 425 acute oral toxicity up-and-down proce-durerdquo in OECD Guidelines for the Testing of Chemicals section4 OECD Publishing 2008

[17] S Talwar H V Jagani P G Nayak et al ldquoToxicological eval-uation of Terminalia paniculata bark extract and its protectiveeffect against CCl

4-induced liver injury in rodentsrdquo BMC

Complementary and Alternative Medicine vol 13 article 1272013

[18] M S Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[19] H Aebi ldquoCatalaserdquo in Methods of Enzymatic Analysis H UBergmeyer Ed vol 2 pp 673ndash684 2nd edition 1974

[20] W H Habig and W B Jakoby ldquoAssays for differentiation ofglutathione S-transferasesrdquoMethods in Enzymology vol 77 pp398ndash405 1981

[21] W Ren Z Qiao H Wang L Zhu and L Zhang ldquoFlavonoidspromising anticancer agentsrdquo Medicinal Research Reviews vol23 no 4 pp 519ndash534 2003

[22] T S Lin B Fischer K A Blum et al ldquoFlavopiridol (alvocidib)in chronic lymphocytic leukemiardquo Haematologica MeetingReports vol 2 no 5 pp 112ndash119 2008

[23] M Cushman and D Nagarathnam ldquoCytotoxicities of someflavonoid analoguesrdquo Journal of Natural Products vol 54 no6 pp 1656ndash1660 1991

[24] X D Jia and C Han ldquoChemoprevention of tea on colorectalcancer induced by dimethylhydrazine in Wistar ratsrdquo WorldJournal of Gastroenterology vol 6 no 5 pp 699ndash703 2000

[25] Y N Wong W C Chang M Clapper and P F EngstromldquoChemoprevention of colorectal cancerrdquo in Colorectal CancerEvidence-BasedChemotherapy StrategiesMMarkman andL BSaltz Eds pp 33ndash49 Humana Press Inc New York NY USA2007

[26] T Tanaka ldquoDevelopment of an inflammation-associated col-orectal cancer model and its application for research oncarcinogenesis and chemopreventionrdquo International Journal ofInflammation vol 2012 Article ID 658786 16 pages 2012

[27] S Umesalma and G Sudhandiran ldquoChemomodulation of theantioxidative enzymes and peroxidative damage in the colon of12-dimethyl hydrazine-induced rats by ellagicacidrdquo Phytother-apy Research vol 24 no 1 pp S114ndashS119 2010

[28] C Kanadaswami L-T Lee P-P H Lee et al ldquoThe antitumoractivities of flavonoidsrdquo In Vivo vol 19 no 5 pp 895ndash910 2005

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Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


[N-[2-(4-Bromo-phenyl)-chromen-4ylidene]-N1015840-(24 dini-tro-phenyl)-hydrazine] IMF-5 [N-(24 Dinitro-phenyl)-N1015840-(2-pyridine-4yl-chromen-4ylidine)-hydrazine] and IMF-8[(4-4-[24 Dinitro-phenyl-hydrazono]-4H-chromen-2yl-phenyl)-dimethyl-amine] were found to possess a low IC

50

against Hela and HepG2 cell lines However iminoflavoneshad not been tested experimentally for their influenceon colon cancer IMF-2 -4 -5 and -8 were screened againsthuman colon cancer cell line (HCT-116) Hence we selectedan iminoflavone IMF-8 from our pilot studies to assess itspotential on dimethyl hydrazine-induced colon cancer inalbino rats of Wistar strain

2 Materials and Methods

21 Chemicals Dimethyl hydrazine MTT and SRB wereprocured from Sigma-Aldrich Co LLC St Louis MO USA5-Fluorouracil was purchased from Biochem PharmaceuticalIndustries Ltd Mumbai Maharashtra India MCF-7 MDA-MB-231 EAC and HCT-116 cell lines were procured fromNational Centre forCell Science (NCCS) PuneMaharashtraIndia Media serum and cell culture products were procuredfrom Invitrogen Bio Services India Pvt Ltd BangaloreKarnataka India

22 Synthesis of Iminoflavones A series of novel substi-tuted iminoflavones were assessed which were designed andsynthesized previously in our laboratory [11] In brief thegeneral scheme of synthesis for iminoflavones involves thefollowing steps Synthesis of chalcones involves Claisen-Schmidt condensation synthesis of flavones involves cycli-sation of chalcones and synthesis of iminoflavones involvescondensation of 24-DNP with flavone moiety

23 Preliminary In Vitro Cytotoxicity Studies Sulforhoda-mine-B and Trypan blue exclusion assays [12 13] wereperformed using HCT-116 MCF-7 MDA-MB-231 and EACcell lines Similarly AOEB and Hoechst 33342 staining [1415] were done to examine the nuclear morphological changeswithin the cells SRB assay which measures total proteincontent of the cells is directly proportional to viable cellcount Trypan blue dye exclusion assay is a well-accepteddirect method for the measurement of cell death Stainingthe cells with fluorescent dyes including acridine orangeand ethidium bromide is used in evaluating the nuclearmorphology of dead cells To confirm whether apoptosis ornecrosis has been induced by selected iminoflavones coloncancerous cells (HCT-116) were analyzed in the presence ofacridine orange and ethidium bromide staining (AOEB)staining As a control untreated cells were cultured incomplete media and stained with AOEB

The apoptotic index (AI) was calculated to confirm thatiminoflavones caused cell death via apoptosis AI is describedas the percentage of apoptotic cells and apoptotic bodieswithin the overall population of cells An apoptotic indexwas determined as the percentage of apoptotic cells atleast 200 counted cells under observation using an invertedmicroscope

Table 1 Preliminary cytotoxicity assessment in various cell lines

Treatment IC50 in 120583M (mean plusmn SEM)

HCT-116 MCF-7 MDA-MB-231 EAC

Doxorubicin 288 plusmn 002 443 plusmn 034 177 plusmn 01 2505 plusmn 09IMF-2 1052 plusmn 09 ND ND NDIMF-4 1321 plusmn 12 ND ND NDIMF-5 4179 plusmn 067 4253 plusmn 078 5913 plusmn 076 1102 plusmn 021IMF-8 278 plusmn 010 1351 plusmn 013 638 plusmn 024 366 plusmn 32Values are reported as mean plusmn SEM of three readings in triplicate ND notdeterminedHCT-116 MCF-7 and MDA-MB-231 by SRB assay (exposure 48 h)EAC by Trypan blue exclusion assay (exposure 4 h)

Table 2 Effect on the apoptotic indices by AOEB and Hoechst33342 staining

Apoptotic indices (mean plusmn SEM)Groups AOEB staining Hoechst 33342 stainingControl 37 plusmn 12 67 plusmn 088Doxorubicin 46 plusmn 173a 387 plusmn 240a

IMF-8 333 plusmn 376a 347 plusmn 296a

Values are reported as mean plusmn SEM of three readings in triplicatea119875 lt 001 when compared to the control group

24 Acute Toxicity Studies The acute oral toxicity study wasconducted as per the OECD test guideline 420 (acute oraltoxicity fixed dose procedure) [16 17] Twelve Swiss albinomice weighing 22ndash25G (11-12 weeks old) were used for thetoxicity studies Test compounds were orally administeredwith a single dose of 300 1000 and 2000mgkg bw Ani-mals were observed for possible behavioural changes suchas tremors convulsions sleep altered feeding salivationaltered somatomotor activities and diarrhoea These obser-vations were continued for a period of 14 days followingwhich animals were sacrificed to examine gross changes tothe vital organs

25 Animals and Treatments MaleWistar rats inbred at Cen-tral Animal Research Facility Manipal University ManipalIndia were used in the study Animals were acclimatizedto the experimental room having temperature 23 plusmn 3∘Ccontrolled humidity conditions 75 and 12 12 hours lightand dark cycle Rats were housed in sterile polypropylenecages containing sterile paddy husk as bedding material Theanimals were fed on autoclaved rat feed and water Eight toten weeks old male rats weighing 120 plusmn 10 g were used forthe study The animal care and handling was carried out inaccordance with guidelines issued by Institutional AnimalEthics Committee Manipal University Manipal KarnatakaIndia (number IAECKMC892012) Animals were dividedinto four groups (119899 = 6) namely normal control DMH con-trol 5-fluorouracil and IMF-8 The normal control received025 CMC po Animals were administered DMH weeklyonce at a dose of 20mgkg body weight (ip) for 10 weeksand 30mgkg bw ip for 10 weeks 5-FU was given at a dose




Number of ACFscm2



Large(5ndash8mm)

Totalcount





3 Results


50of 278 plusmn 010 120583M in











Catalase

0

5

10


Cata

lase

(120583M

mg

of p

rote

in)

a

a b

a b

0

5

10


GSH

aa a b

GSH

(120583M

mg

of p

rote

in)

0

5

10


Nitrite content

b

a

Nitr

ite (120583

Mm

g of

pro

tein

)


ControlDMH

5-FUIMF-8

ControlDMH

5-FUIMF-8

ab bTN

F-120572

(pg

mg

of p

rote

in)

TNF-120572 content

0

100

200

300

400

Treatments

a

IL-6

(pg

mg

of p

rote

in)

IL-6 content

0

100

200

300

400

500

b

Treatments


4 Discussion




Normalcontrol

DMHcontrol

5-FU

IMF-8

(a) (b) (c)

















Acknowledgment


References




































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Number of ACFscm2



Large(5ndash8mm)

Totalcount





3 Results


50of 278 plusmn 010 120583M in











Catalase

0

5

10


Cata

lase

(120583M

mg

of p

rote

in)

a

a b

a b

0

5

10


GSH

aa a b

GSH

(120583M

mg

of p

rote

in)

0

5

10


Nitrite content

b

a

Nitr

ite (120583

Mm

g of

pro

tein

)


ControlDMH

5-FUIMF-8

ControlDMH

5-FUIMF-8

ab bTN

F-120572

(pg

mg

of p

rote

in)

TNF-120572 content

0

100

200

300

400

Treatments

a

IL-6

(pg

mg

of p

rote

in)

IL-6 content

0

100

200

300

400

500

b

Treatments


4 Discussion




Normalcontrol

DMHcontrol

5-FU

IMF-8

(a) (b) (c)

















Acknowledgment


References




































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Catalase

0

5

10


Cata

lase

(120583M

mg

of p

rote

in)

a

a b

a b

0

5

10


GSH

aa a b

GSH

(120583M

mg

of p

rote

in)

0

5

10


Nitrite content

b

a

Nitr

ite (120583

Mm

g of

pro

tein

)


ControlDMH

5-FUIMF-8

ControlDMH

5-FUIMF-8

ab bTN

F-120572

(pg

mg

of p

rote

in)

TNF-120572 content

0

100

200

300

400

Treatments

a

IL-6

(pg

mg

of p

rote

in)

IL-6 content

0

100

200

300

400

500

b

Treatments


4 Discussion




Normalcontrol

DMHcontrol

5-FU

IMF-8

(a) (b) (c)

















Acknowledgment


References




































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Normalcontrol

DMHcontrol

5-FU

IMF-8

(a) (b) (c)

















Acknowledgment


References




































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












Acknowledgment


References




































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Research Article Iminoflavones Combat 1,2 …downloads.hindawi.com/journals/bmri/2014/569130.pdfconducted as per the OECD test guideline (acute oral toxicity, xed dose procedure) [