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 .
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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
Apoptosis and Angiogenesis?
•
Apoptosis
is programmed cell death by the organs
of the body that are involved in this event as the
cytoplasm, cytochrome, apoptosis and macrophage
body while
angiogenesis
is multiplication of new
blood vessels.
Apoptosis
•
Apoptotic function is to selectively eliminate
unwanted cells
•
If the DNA repair mechanisms can not overcome
the damage caused by radiation or cytotoxic drugs,
kill itself through apoptosis
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
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 /
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
Pinostrobin
•
How does the pinostrobin?
•
whether these compounds increase / induce
apoptosis and inhibit angiogenesis so that it
can be said as an inducer of apoptosis and
antiangiogenesis.
•
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
APOPTOSIS
ANGIOGENESIS
ANTIANGIOGENESIS
cancer
(
Fibrosarcoma
)
uncontrollable
PINOSTROBIN?
C
COX-2
VEGF
MMPs-9
COX-2
VEGF
MMPs-9
-curcumin
- PGV-1
- Omega -3
- Isoflavon
Research purposes
Determining the induction or induced of
apoptosis through increased p53 expression
and
angiogenesis
barriers
pinostrobin
compounds
from
rhizomes
Kaempferia
pandurata
Roxb
to
VEGF
(Vascular
Ephidermal Growth Factor) expression, in
cancer
cells
Fibrosarkoma
Mice
Results
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
Procedure or steps of research
20-40 mice
Mice with fibrosarcoma (V=100 mm
3)
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
After 1 month will be formed fibrosarcoma, and after
approximately 2 months fibrosarcoma will have a volume
of approximately 100 mm
3, as shown by the following
picture
After fibrosarcoma volume reached 100 mm
3
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
the results surgery of fibrosarcoma in all the mice who have received treatment
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
[image:23.720.26.693.104.326.2]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
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
Phatohistology of fibrosarcoma with HE staining
theorytical
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
Sel tanpa apoptosis
Sel dengan apoptosis
Analysis VEGF with IHC
•
Preparations
are
already
colored
and
incubated will show the number of blood
vessels are formed with a light microscope
with a magnification of 400 times
•
Results of analysis of p53 and VEGF expression
can be seen in the following picture and table
Analysis of antiangiogenesis
a. Angiogenesis
b. Antiangiogenesis
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
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 to
normal 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
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 BW
The 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 concentration
60/50 x 165 mg = 198 mg benzopiren
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Thank you for your attention
!*
Dr. Drs. I Made Adi Oka Parwata, M.Si.
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