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Banronginsu oriental medicine Clinic, Nonhyun-dong,
Kangnam-gu, Seoul 111-24, Korea. [email protected]
2010.09.11.
TAE YOUNG HAN. OMD
Anti-tumor activity of B0052 and a novel Korean medicine,
in murine melanoma models and relations of composition
changes of primo system.
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Contents
I. Introduction
II. Preliminary study
III. Materials and methods
IV.Results and Discussion
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I.Introduction : Malignant Melanoma
Rapid growing tumor without effective therapies.
Early detection ,surgical removal is the only choice for long
term survival
Inhibition and control of metastasis is important issue.
Angiogenesis, inflammatory reaction,immune response are important
factor
Primo-vascular system may acts as an additioinal route
for cancer migration
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Clinical experience • Recurred over and over, had resection more than 8times.
• Treated with MSB0052 3 month.
• Had partial response .
2009.7 MR 2009.6.9 2009.8.18
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II.Preliminary study
• Duration;(2010.4.15 - 2010.5.6) for 20days
• Cell line : B16F10 mouse melanoma cell
• Female nude mice: (BALB-C-nu/nu) 6weeks
• cultivated in DMEM
• Injected into the skin on the back
• Grouping
A - control group B - drug treated group
• n = 3 n =3
• melanoma cell injection melanoma cell injection
• treated with water treated with MSB0052
from the same day
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Treatment start: 2010.04.15
Melanoma cells were then resuspended in 1mL of DMEM and injected of nude
mouse mice for the development of skin cancer.
Melanoma cancer cell line and in vivo treatment
6
Inocculation
Into the skin
On the back
Divide into 2
group
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2010.04.26 10days after
Animals (n = 3 animals per
group) were treated with inhalation
of MSB0052 or vehicle once a day
for 14 days, or were part of the
control group that was not subjected
to any treatment.
The treatment was started 10 days after tumor cell inoculation.
Control group MSB0052 group
7
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2010.05.06 after 20days
Control group MSB0052 group
8
Necrosis
Melanization
is apparent
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Design
• Based on the result shown
on preliminary study
• We designed 2nd study
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III. Materials and Methods
Cell culture and reagents
• B16F10 mouse melanoma cell lines were purchased
Cells were cultured in DMEM medium
Animal model • female nude mice (BALB-C-nu/nu)
aged 6 weeks old, 17-20g, n=34
Cell count (2.5×106 cells per animals)
Cell injection • Anaesthetized by inhalation of 2% isoflurance in 100% oxygen at a
flow rate of 2L min-1
under the skin into nude mice on the back (腎兪穴)
Study design •Divided into three group
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Evaluation of tarket and Histological
analysis
• Weight
• Tumor size
• Survival
• Behavior & activity
• Microscopic pathology
– Stained with heamtoxylin and eosin(H&E)
– Organs- axillary,lumbar, inguinal lymph nodes, lung
– Collect 5days(n=1) 10days (n=1), 15 days(n=3)/each group
– made into 5 μm sections
• primo-vascular study
– difference of composition between 2 group
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Divide 3 group
(a) native group
•Non injected mice •treated with water (1ml/20g) •Every day three times • n= 4 / for weighting
(b) control group
• Melanoma cell injected mice
• treated with water (1ml/20g)
• Every day three times
• n = 15
• - 5 survivol
• - 5 histologic purpose
• - 5 primo-vascular
(c) drug Tx group
• Melanoma cell injected mice
• treated with MSB0052
(30mgdrug/1mL/20g)
• every day three times
• n = 15
• -5 survival
• -5 hisologic purpose
• -5 primo-vascular
mice were randomly divided into 3 groups
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Animal model and study design
Two sets of experimental models were used:
Cell injection &
drug treatment
08.05
start
0Day 5Days 10Days 15Days
Tissue sampling Tissue sampling Tissue sampling
n=1
(each group)
n=1
(each group)
n=3
(each group)
08.10 08.15 08.20
Cell injection & drug treatment
2010.08.05 start
0Day 40 Days
2010.09.14
mice treated with water and B0052 every day three times each group
First, Histological analysis
Second, Survival analysis
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IV. Results
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Comparison of the tumor volume after 15day.
Gross findings of melanoma masses after 15 days
Control group
With water larger
& darker
Necrosis
was apparent
compare with
drug treated
group
Drug tx group
MSB0052
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Anti-tumor effect of MSB0052 in vivo..
(a) Tumor size growth curve. Tumor sizes were measured every 5 days.
There was a difference between MSB0052 group and control groups
(b) Body weight curve. There was a difference in body weight between MSB0052 and control groups
MSB0052 difference body weight and tumor growth after 20 days.
MSB0052 inhibited tumor growth in vivo
(b)
Days after B16F10 cell challenged
(a)
0
5
10
15
20
25
30
35
40
d0 d5 d10 d15 d20 d25 d30 d35
Bo
dy w
eig
ht
(g)
Native
Control
MSB0052
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
d5 d10 d15 d20 d25 d30 d35
Tu
mo
r siz
e (
mm
)
Control
MSB0052
Days after B16F10 cell challenged
Tumor size Body weight
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Tumor volume growth. There was a difference in tumor volume between MSB0052 and control groups .
Control group
With water n =1 Drug tx group
With MSB0052 n = 1 After
10 days Insize no differ-
Pathological comparison after 10 days
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Micro in Control and MSB0052-10 D: Peritumoral reaction I
Fibroblastic
proliferation &
Angiogenesis is
apparent
Control group
With water
Drug tx group
MSB0052
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Micro in Control and MSB0052-10 D: Peritumoral reaction I
Control group
With water
Drug tx group
MSB0052
Macrophages Fibroblasts, angiogenesis,
neutrophil is prominent
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Pathological comparison after 15 days
Tumor volume growth. There was a difference in tumor volume between MSB0052 and control groups .
n1
n2
n3
n1
n2
n3
15 days
Tumor size
larger ,
Penetrated
the skin
>
Control group
With water n =1 Drug tx group
With MSB0052 n = 1
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Micro in Control and MSB0052-15 D: Peritumoral reaction I
Contro group shows Increased fibroblastic proliferation and angiogenesis, necrotic change in the
tumor
Control group MSB0052 group Angiogenesis Necrotic
changes
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Control group MSB0052 group
Micro in C-15 D: Peritumoral reaction II
•Fibroblastic proliferation
•angiogenesis
•inflammatory reaction
of neutrophil is prominent
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Micro in C-15 D: Peritumoral reaction II
Increased fibroblastic proliferation
and angiogenesis, neutrophil
Control group With water
Drug tx group With MSB0052
neutrophil macrophage
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Micro in C-15 D: Peritumoral reaction II
Control group MSB0052 group
fibroblastic proliferation macrophage
and angiogenesis
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Micro in C-15 D: Peritumoral reaction III
Neutrophils Macrophages
Control group MSB0052 group
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Experiment Design
B16F10 cell 2.5x106/ml injection volume 1ml
After sampling 17 days
Animal: Nude Mouse
oral injection (every day)
A-Group B-Group C-Group D-Group
Native
water o.inj
Control
water o.inj Test
B0052 (50mg) o.inj
After tumor devolp
B0052 (50mg) o.inj
Start time
2006.28
Comparison of composition- primo vessels
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Tissue sampling 2010.07.14
A.
B.
C.
D.
Comparison of composition- primo vessels
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UPLC-Q-TOF mass analysis_ Total chromatogram at positive mode
Primo vessel
Lymph vessel
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UPLC-Q-TOF mass analysis_ Total chromatogram at negative mode
m/z 459.2009
Primo vessel
Lymph vessel
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Survival curve for tumor-bearing mice. MSB0052-treated mice was also found when
compared with the control groups.
Days after B16F10 cell challenged
Survival curve of tumor-bearing mice
0
20
40
60
80
100
120
5d 10d 15d 20d 25d 30d 35d
Su
rviv
al (%
)
Native
Control
MSB0052
17d
28d
34d
23d
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Video file
Activity between two groups
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Conclusion
MSB0052 has Anti-tumoral effect on murine melanoma
Strenghten the immune reaction like macrophage
inhibitory effect on angiogenesis
inhibitory effect on inflammatory reaction like neutrophil
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• Baatartsogt .O, ( Hankyong National University ).
• Jung Sun Yoo*, ( Seoul National University ).
• Hee Jae Ju. ( Ajou University).
• Kwang Sup Soh*, ( Seoul National University ).
• Mi Son Chun, ( Ajou University).
• Kang Duk Choi, ( Hankyong National University ).
• Choong Hwan Lee, ( Konkuk University ).
• Ji Young Kim, ( konkuk university ).
• Namhyun Jung (Korea university)
• Il Young Han, ( sonyun biophics ).
Acknowledgment:
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