repositori.unud.ac.id fileapoptosis and angiogenesis actually occurs normally in the human body such...
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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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22 24
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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
12
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
12
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
12
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
12
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
12
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