Induction of Apoptosis and Antiangiogenesis Effects of Pinostrobin from Kaempferia pandurata Roxb against Induction of Fibrosarcoma Mice Results Benzopiren.
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
Background
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
• Morphological of apoptosis include cell shrinkage,
chromatin condensation and fragmentation,
formation of blisters on the cell apoptosis and
pragmentasi into objects and objects phagocytosis
by healthy cells macrofag, as shown by the
following 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 MMP9 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 / 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
• as shown in the following research scheme :
APOPTOSIS
COX-2
VEGF
C
MMPs-9
ANGIOGENESIS
uncontrollable
cancer
(Fibrosarcoma)
COX-2
VEGF
MMPs-9
- curcumin
- PGV-1
- Omega -3
- Isoflavon
INHIBITOR
PINOSTROBIN?
ANTIANGIOGENESIS
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
Induction 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
13
Induced with benzopiren (0,3 % w/v in oleum
olivarum/0,2 ml for mice, two days as much as 5-8 times
Mice with fibrosarcoma (V=100 mm3)
treatment
15
G I (CMC-Na)
14
G III (S=13,33 gr)
G II (p=80 mg)
After treatment about 2 month
Fibrosarcoma (Weighed)
16
22
21
24
IHC test for p53,COX-2 , VEGF and MMPs-9
23
25
Expression of p53, COX-2 and VEGF
26
Induced Apoptosis and antiangiogenesis
27
Manufacture of fibrosarcoma in mice
12
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. 12
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 of surgery fibrosarcoma
12
the results surgery of fibrosarcoma in all the mice who have received treatment
12
Table of weighing of fibrosarcoma cells after treatment
weighing of fibrosarcoma (gram )
treatment
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
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
12
12
Sel tanpa apoptosis
Sel dengan apoptosis
Analysis VEGF with IHC
12
• 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
12
12
Results of Analysis p53 and VEGF expression by IHC
No
1.
2.
3.
Jenis Sampel
CMC-Na (C-)
Pinostrobin 80
mg/KgBB
Obat Kanker (C+)
Amount of holes
(p53)
Amount of
blood vessels
(VEGF)
(+)
(-)
(+)
(-)
287
122
342
161
299
107
299
107
265
250
270
237
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
12
• 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
0.2 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
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
Background
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
• Morphological of apoptosis include cell shrinkage,
chromatin condensation and fragmentation,
formation of blisters on the cell apoptosis and
pragmentasi into objects and objects phagocytosis
by healthy cells macrofag, as shown by the
following 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 MMP9 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 / 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
• as shown in the following research scheme :
APOPTOSIS
COX-2
VEGF
C
MMPs-9
ANGIOGENESIS
uncontrollable
cancer
(Fibrosarcoma)
COX-2
VEGF
MMPs-9
- curcumin
- PGV-1
- Omega -3
- Isoflavon
INHIBITOR
PINOSTROBIN?
ANTIANGIOGENESIS
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
Induction 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
13
Induced with benzopiren (0,3 % w/v in oleum
olivarum/0,2 ml for mice, two days as much as 5-8 times
Mice with fibrosarcoma (V=100 mm3)
treatment
15
G I (CMC-Na)
14
G III (S=13,33 gr)
G II (p=80 mg)
After treatment about 2 month
Fibrosarcoma (Weighed)
16
22
21
24
IHC test for p53,COX-2 , VEGF and MMPs-9
23
25
Expression of p53, COX-2 and VEGF
26
Induced Apoptosis and antiangiogenesis
27
Manufacture of fibrosarcoma in mice
12
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. 12
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 of surgery fibrosarcoma
12
the results surgery of fibrosarcoma in all the mice who have received treatment
12
Table of weighing of fibrosarcoma cells after treatment
weighing of fibrosarcoma (gram )
treatment
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
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
12
12
Sel tanpa apoptosis
Sel dengan apoptosis
Analysis VEGF with IHC
12
• 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
12
12
Results of Analysis p53 and VEGF expression by IHC
No
1.
2.
3.
Jenis Sampel
CMC-Na (C-)
Pinostrobin 80
mg/KgBB
Obat Kanker (C+)
Amount of holes
(p53)
Amount of
blood vessels
(VEGF)
(+)
(-)
(+)
(-)
287
122
342
161
299
107
299
107
265
250
270
237
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
12
• 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
0.2 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