Induction of Apoptosis and Antiangiogenesis Effects of Pinostrobin from Kaempferia pandurata

Roxb against Induction of Fibrosarcoma Mice Results Benzopiren

Adi Parwata1, Sukardiman2, Mulja Hadi Santosa3, Alit Widhiartini4

1) Nature Materials Study Group, Laboratory of Organic Chemistry, Department of Chemistry,

Faculty Mathematics and Natural Sciences, Udayana University, Denpasar, Bali. 2,3) Laboratory of Phytochemistry and Pharmacognosy Faculty Pharmacy Airlangga University,

Surabaya 4) Section of Pharmacy, Faculty of Medicine, University of Udayana, Jalan PB Sudirman,

Denpasar

ABSTRACS

Induction of apoptosis and antiangiogenesis effects of Pinostrobin from Kaempferia pandurata

Roxb against Fibrosarkoma mice results benzopiren induction has been done. Examination or

surgery begins taking tissue fibrosarcoma in mice infected and weigh fibrosarcoma obtained .

Fibrosarcoma tissues were then stored in containers that have contained 10 % formalin .

Weighing results showed that the concentration of pinostrobin oral 80 mg / kg can inhibit the

growth of fibrosarcoma with a gram weight of 68.62 % and a cancer drug ( control + ) there is

resistance 95.95 % compared to the negative control which is only given orally CMC – Na.

Furthermore done shooting patohistologi tissue fibrosarcoma with HE staining with a light microscope

with 400x magnification . The results obtained showed many chromatin (polikromatin) which prove the

damage caused by having fibrosarcoma cells.Immunohistochemical assay showed oral pinostrobin

concentration 80 mg / kg body weight can increase the expression of p53 to apoptosis induction could

take place and the decreased expression of VEGF angiogenesis which proves the existence of

barriers .

Conclusion : Oral pinostrobin concentration 80 mg / kg body weight can reduce 68.62 % by weight

of fibrosarcoma , increase the expression of p53 and decreased the expression of VEGF. This means

pinostrobin potentially be developed as a cancer chemotherapeutic agent .

(Key Word : Pinostrobin, fibrosarcoma, apoptosis, antiangiogenesis, p53 dan VEGF)

*Induction of Apoptosis and

Antiangiogenesis Effects of

Pinostrobin from Kaempferia pandurata Roxb against

Induction of Fibrosarcoma Mice

Results Benzopiren *

I Made Oka Adi Parwata

Ida Ayu Alit Widhiartini

Prof. Dr. Drs. Sukardiman, Apt. MSi

Icowobas 2015

Surabaya, 2015

Apoptosis and Angiogenesis?

• Apoptosis is programmed cell death by the organsof the body that are involved in this event as thecytoplasm, cytochrome, apoptosis and macrophagebody while angiogenesis is multiplication of newblood vessels.

Background

Apoptosis• Apoptotic function is to selectively eliminate

unwanted cells

• If the DNA repair mechanisms can not overcomethe damage caused by radiation or cytotoxic drugs,kill itself through apoptosis

• Morphological of apoptosis include cell shrinkage,chromatin condensation and fragmentation,formation of blisters on the cell apoptosis andpragmentasi into objects and objects phagocytosisby healthy cells macrofag, as shown by thefollowing picture

Apoptosis and angiogenesis actually

occurs normally in the human body such as the

development of babies become children,

children become adults, and so on but if

apoptosis and angiogenesis occurs without

control or uncontrollable there will be mutations

that cause malignant cancer. When cancer

develops, the expression of p53 is going down

while the expression of VEGF, COX-2 and MMP-

9 will go up

This event can be prevented with drugs

from nature, one of them with pinostrobin from

rhizomes Kaempferia pandurata Roxb

Angiogenesis

• Angiogenesis is the formation of new blood from existing blood vessels

• In the healthy human body, angiogenesis very strictly controlled by molecules endogenous angiogenic and angiostatik

• Angiogenesis also gives access to the tumor cells to spread to other body tissues / metastasis, as shown by the following picture

Inducer apoptosis and antiangiogenesis

from natural

• There are so many compounds of natural

ingredients that have been proven

experimentally in vitro anticancer such as

curcumin (from Curcuma), Omega-3 (salmon

oil, Isoflavones (soybeans/ Glycine (L). Merr

atau Glycine max.

• The experimental results indicate that these

compounds can be increased of p53 and

decreased of VEGF , COX-2 and MMPs, so

that it can be regarded as a compound that

can inducer apoptosis and antiangiogenesis

Pinostrobin

• How does the pinostrobin?

• whether these compounds increase / induceapoptosis and inhibit angiogenesis so that itcan be said as an inducer of apoptosis andantiangiogenesis.

• Do pinostrobin can be increase p53

expression and decreased the expression of

VEGF, COX-2 and MMPs-9

• “this is the topic of this research”

• as shown in the following research scheme :

APOPTOSIS ANGIOGENESIS

ANTIANGIOGENESIS

cancer

(Fibrosarcoma)

uncontrollable

PINOSTROBIN?

C

COX-2 VEGF MMPs-9

COX-2 VEGF MMPs-9

- curcumin- PGV-1- Omega -3- Isoflavon

INHIBITOR

Research purposes

Determining the induction or induced ofapoptosis through increased p53 expressionand angiogenesis barriers pinostrobincompounds from rhizomes Kaempferiapandurata Roxb to VEGF (VascularEphidermal Growth Factor) expression, incancer cells Fibrosarkoma Mice ResultsInduction Benzopirena

This study aimed is determine effect of compound

pinostrobin to apoptosis and angiogenesis, whether

pinostrobin can stimulate or induce apoptosis and inhibit

angiogenesis so pinostrobin later can be regarded as a basic

ingredient of drugs to stimulate or induce apoptosis and

inhibit angiogenesis or antiangiogenesis. Pinostrobin further

can be developed as an anticancer drug

Pinostrobin compound can be said to stimulate or induce

apoptosis when it may increase the expression of p53 and

can be regarded as angiogensis inhibitor or

antiangiogenesis when it compound can decrease the

expression of VEGF and MMPs and reduce the activity of the

enzyme COX-2

Procedure or steps of research

20-40 mice

Mice with fibrosarcoma (V=100 mm3)

Fibrosarcoma (Weighed)

Expression of p53, COX-2 and VEGF

G III (S=13,33 gr)G II (p=80 mg)G I (CMC-Na)

Induced with benzopiren (0,3 % w/v in oleum

olivarum/0,2 ml for mice, two days as much as 5-8 times

treatment

IHC test for p53,COX-2 , VEGF and MMPs-9

After treatment about 2 month

Induced Apoptosis and antiangiogenesis

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Manufacture of fibrosarcoma in mice12

After 1 month will be formed fibrosarcoma, and after

approximately 2 months fibrosarcoma will have a volume

of approximately 100 mm3, as shown by the following

picture

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After fibrosarcoma volume reached 100 mm3 mice,

divided into 3 groups :

1. group I (negative control) that without treatment,

2. group II pinostrobin given 80 mg / kg and

3. the third group was given the anticancer drug

(cyclophosphamide).

This treatment is carried out for approximately 45-60

days.

Furthermore, mice were sacrificed / dissected to be

taken fibrosarcoma and to weighed, and then preserved

in Formaldehyde solution.

The final step, fibrosarcoma treatment results were then

analyzed by immunohistochemical methods to see the

expression of p53, VEGF, MMPs and COX-2 21

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The results of surgery fibrosarcoma 12

the results surgery of fibrosarcoma in all the mice who have received treatment

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treatment

weighing of fibrosarcoma (gram )

I II III IV rata-rata

CMC-Na [ Control (-)] 4,2765 4,1665 4,1095 4,1383 4,1727

Pinostrobin 80 mm/kg BW 1,0665 1,0605 1,1198 1,1027 1,0874

Siklofosfamid [Control (+)] 0,1709 0,1021 0,2553 0,1021 0,1576

Table of weighing of fibrosarcoma cells after treatment

The results show that Pinostrobin 80 mg / kg BW can be inhibit

the growth of fibrosarcoma

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Phatohistology of fibrosarcoma

• Before analyzed by Imunohistochemistry, checked of fibrosarcoma, whether it really is cell fibrosarcoma, it can be seen patohistologi fibrosarcoma with HE staining system, as shown by the following picture

Phatohistology of fibrosarcoma with HE staining

theorytical

the results

of analysis

The process of making preparat for IHC 12

Analysis p53 with IHC

• preparations are already colored analyzed by IHC. p53 expression was analyzed by light mikrokop with magnification of 400 times. the image will be visible holes or wells with a certain color which in this analysis brown color. Count and total the number of holes / wells are visible, as shown by the following picture

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Sel tanpa apoptosis Sel dengan apoptosis

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Analysis VEGF with IHC

• Preparations are already colored andincubated will show the number of bloodvessels are formed with a light microscopewith a magnification of 400 times

• Results of analysis of p53 and VEGF expressioncan be seen in the following picture and table

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Analysis of antiangiogenesis

a. Angiogenesis b. Antiangiogenesis

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No Jenis Sampel

Amount of holes

(p53)

Amount of

blood vessels

(VEGF)

(+) (-) (+) (-)

1. CMC-Na (C-) 287 122 342 161

2.Pinostrobin 80

mg/KgBB 299 107 299 107

3. Obat Kanker (C+) 265 250 270 237

Results of Analysis p53 and VEGF expression by IHC

The analysis showed that Pinostrobin induced

Apoptosis through the increase in the expression of

p53 and to inhibit of angiogenesis through reduced

expression of VEGF

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Conclusion• Histopathological analysis results fibrosarcoma cells by HE

staining in getting that there are a lot of chromatin(polikromatin) on fibrosarcoma as evidence of damage tonormal cells.

• Pinostrobin oral concentration of 80 mg / kg body weight can reduce 68.62% fibrosarcoma

• Pinostrobin oral concentration of 80 mg / kg body weight can increase the expression of p53 so that apoptosis can take place and decreased the expression of VEGF signaling can be inhibited angiogenesis.

• The analysis showed that pinostrobin can be developed into a natural cancer drug

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Solution of benzopiren• Benzopiren concentrations were injected 0.3% w / v in oleum olivarum

Once the injection volume was 0.2 mL / mice so that the levels once injection was 0.2 mL x 0,003 mg = 0.0006 mg or 0.0006 mg/20 gr BW

or 30 mg / kg BWThe injection volume for the mice weighed more than 20 Induction done as much as 5x 2 days (10 days)The calculation is as follows benzopiren concentration60/50 x 165 mg = 198 mg benzopiren0.2 mL x 5 (doses) x 60 = 60 ml oleum olivarum

*Thank you for your attention!*

Dr. Drs. I Made Adi Oka Parwata, M.Si.,

Nature Product Study Group, Department of Chemistry

Udayana of University