ESTABLISHMENT A SUBCUTANEOUS TUMOR MODEL BY HepG2 CELL LINE IN MICE TO STUDY ANTI-TUMOR EFFECT OF LIPOSOME PACLITAXEL

UNIVERSITY OF MEDICINE AND PHARMACY AT HO CHI MINH CITY
Faculty of Pharmacy

ESTABLISHMENT A SUBCUTANEOUS TUMOR MODEL BY
HepG2 CELL LINE IN MICE TO STUDY ANTI-TUMOR EFFECT
OF LIPOSOME PACLITAXEL

Research Team:

Asso.Prof. Đỗ Thị Hồng Tươi
Dr. Trương Công Trị
MSc. Trần Thị Như Nguyện
Pharm. Trần Thị Phương Uyên
MSc. Nguyễn Bá Thọ
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INTRODUCTION
Hepatocellular carcinoma (HCC): one of the leading causes of cancer deaths (ranked
3rd for men, 6th for women) (Ferlay, Bray, 2010)
- 2007, over 700.000 cases in the world
- 2008, over 725.000 (Southeast Asia: 75.000 new cases)
- Vietnam: high percentage of HCC patients, high cost of medications (imported)
Paclitaxel:
- Antiproliferation & death induction against human HCC (in vitro);
- Combination with DOX reduced HCC tumor size (in vivo)
- Poor solubility and permeability  poor bioavailability
- Liposomal paclitaxel: enhance solubility, permeability and targeting specificity

Establish a model of HCC using HepG2 cell line in mice
Study the anti-tumor effect of liposome paclitaxel formulation

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METHODS - RESULTS
In vivo model of HCC (xenograft model)
Culturing HepG2
cell line

s.c. injection
on nude/SCID mice

Treatment

Immunodeficiency
induced by CYP

Zhang et al
(2007)

Male BALB/c nude mice, s.c. injection After 10 days, tumors with diameter
of 0,1 ml (107 cells/ml)
from 3 - 5 mm

Hagiwara et al
(2007)

Male BALB/c nude mice, s.c. injection After 7 days, 100% mice had tumors
of 106 HepG2 cells/mouse
with diameter from 5-10 mm

Chen et al
(2011)

Kunming mice, s.c. injection of 10 6
HepG2 cells/mouse

16th day, 100% mice developed
tumors.
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METHODS - RESULTS
Studying immunodeficiency-induced model on Swiss albino
CYP i.p. cyclophosphamid 100mg/kg; 0,1ml/10g
DOX i.p. doxorubicine 10mg/kg

i.p. saline

male
Swiss albino
8-10 week
32 ± 2 g
n=6

CYP
day 1

CYP
day 3

CYP
day 1

CYP
day 3

DOX
day 1

CYP
day 3

• Collect blood
from tail vein
every 1 – 2
days
CYP
day 5

• Count
leucocytes
(Neubauer
chamber)

Monitor mortality rate and ability to maintain immunodeficiency
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METHODS - RESULTS
Studying immunodeficiency-induced model on Swiss albino

I.p. CYP day 1, 3, 5: weak, ruffled fur, slow movement, 2/6 blind. 1 dead on day 7.
Sharp decrease in leucocytes, lowest on day 6, followed by a slight recovery of
23% on day 21.
I.p. DOX & CYP: weak, ruffled fur, slow movement. 1 dead on day 5 and another
on day 6. Lowest number of leucocytes is recorded on day 5, followed by a rapid
recovery of 87% on day 21.
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METHODS - RESULTS
Studying immunodeficiency induced model on Swiss albino

Ip CYP day 1, 3: weak, ruffled fur, normal movement, 0 dead. Total leucocytes
were lowest on day 5, then recovered by 33% on day 21.

Choose this protocol for the study of HCC induction
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METHODS - RESULTS
Establish a model of HCC in SCID mice

CYP
i.p. 100mg/kg

Control
n= 6
(PBS, sc)

Pathology control
(iv, NaCl 0.9%)

n = 36

HCC-induced
n= 30
(HepG2, sc)

Day

1&3

5

Collect
tumors

Treatment
(5-FU, iv 20mg/kg)

12 =>16

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 Results (Mean ± SEM) analyzed by student’t test/Mann-Whitney
 p < 0,05
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METHODS - RESULTS
Establish a model of HCC in SCID mice
Day 12, visible tumors on 17/30 mice (56.7%)
 
 

Tumor size (mm3)
(Mean ± SEM)
Pathology
control (n = 7)

Pathology control

Treatment
(n = 6)

D12

9.67 ± 1.40

9.81 ± 1.60

D14

12.17 ± 2.52*

9.68 ± 1.63*

D16

15.84 ± 4.04**

7.15 ± 1.40**#

D18

17.83 ± 4.41**

4.08 ± 1.17**##

D19

17.46 ± 4.47

3.80 ± 1,10##

5-FU treatment
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METHODS - RESULTS
Establish a model of HCC in SCID mice

d12

d14

d16

d18

Pathology control
Treatment

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METHODS - RESULTS
Establish a model of HCC in SCID mice

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METHODS - RESULTS
Establish a model of HCC in SCID mice

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METHODS - RESULTS
Study the anti-tumor effect of liposome paclitaxel formulation
Ip CYP
Sc 106 HepG2 cells/mouse

Mice with sc tumors
Size mesuring

Pathology

Treatment

Positive control

NaCl 0,9%
0.1 ml/10 g

PXT-Monta 1.5
10 mg/kg

5-FU
20 mg/kg

Combination
•
•

5-FU 20 mg/kg
PXT-Monta 10 mg/kg

iv once a day, for 5 continuous days

Day 19: Tumors collecting, sample preparing, histology analyzing

Percentage of difference (%) = (Vafter – Vbefore)/Vbefore x 100
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METHODS - RESULTS
Study the anti-tumor effect of liposome paclitaxel formulation

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METHODS - RESULTS
Study the anti-tumor effect of liposome paclitaxel formulation
Mean percentage of size difference compare to day 12 ± SEM (%)
Groups

Day 14

Day 16

Day 18

Pathology control (n = 4)

5.9 ± 2.9

23.5 ± 6.8

44.6 ± 10.8

PTX-Monta (n = 3)

-0.8 ± 2.1

-8.7 ± 3.6

-17.8 ± 2.4

5-FU (n = 4)

-9.3 ± 2.2

-26.4 ± 4.4

-43.5 ± 1.5

5-FU + PTX (n = 3)

-14.4 ± 4.5

-33.4 ± 1.8

-53.1 ± 4.1

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METHODS - RESULTS
Study the anti-tumor effect of liposome paclitaxel formulation

5-FU + PTX-Monta

PTX-Monta

5-FU

Pathology control
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METHODS - RESULTS
Study the anti-tumor effect of liposome paclitaxel formulation

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METHODS - RESULTS
Study the anti-tumor effect of liposome paclitaxel formulation

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METHODS - RESULTS
Study the anti-tumor effect of liposome paclitaxel formulation

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CONCLUSIONS
 Evaluated and chose the suitable immunodeficiency-induced model of CYP 100
mg/kg, i.p. on day 1, 3.

 Successfully

establish

a

model

of

HCC

in

male

immunodeficient

Swiss albino by s.c. injection of 106 human HepG2 cells per mouse.

 In vivo antitumor effect
+ Liposomal PTX decreased tumor size less than 5-FU did.
+ Combination of liposomal PTX and 5-FU are more efficient in decreasing
tumor size than single therapies.
+ Histology: tumors in combination group are the most likely to have necrosis
and less likely to have cell abnormalities than 2 single therapy groups.
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THANK YOU FOR YOUR ATTENTION

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