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

ESTABLISHMENT A SUBCUTANEOUS TUMOR MODEL BY HepG2 CELL LINE IN MICE TO STUDY ANTI-TUMOR EFFECT OF LIPOSOME PACLITAXEL Dr.. Hepatocellular carcinoma HCC: one of the leading causes of canc

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ESTABLISHMENT A SUBCUTANEOUS TUMOR MODEL BY

HepG2 CELL LINE IN MICE TO STUDY ANTI-TUMOR EFFECT

OF LIPOSOME PACLITAXEL

Dr Trương Công Trị MSc Trần Thị Như Nguyện Pharm Trần Thị Phương Uyên

MSc Nguyễn Bá Thọ

UNIVERSITY OF MEDICINE AND PHARMACY AT HO CHI MINH CITY

Faculty of Pharmacy

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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

INTRODUCTION

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

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In vivo model of HCC (xenograft model)

METHODS - RESULTS

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Zhang et al

(2007)

Male BALB/c nude mice, s.c

injection of 0,1 ml (107 cells/ml)

After 10 days, tumors with diameter from 3 - 5 mm

Hagiwara et al

(2007)

Male BALB/c nude mice, s.c

injection of 106 HepG2 cells/mouse

After 7 days, 100% mice had tumors with diameter from 5-10 mm

Chen et al

(2011)

Kunming mice, s.c injection of 106 HepG2 cells/mouse

16th day, 100% mice developed tumors

Culturing HepG2

cell line

s.c injection

on nude/SCID mice

Treatment

Immunodeficiency induced by CYP

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Studying immunodeficiency-induced model on Swiss albino

Monitor mortality rate and ability to maintain immunodeficiency

male

Swiss albino

8-10 week

32 ± 2 g

n = 6

day 3 day 3

day 3 day 1

i.p saline

i.p cyclophosphamid 100mg/kg; 0,1ml/10g i.p doxorubicine 10mg/kg

day 1

CYP

DOX

• Collect blood from tail vein every 1 – 2 days

• Count leucocytes (Neubauer chamber)

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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

Studying immunodeficiency-induced model on Swiss albino

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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

Studying immunodeficiency induced model on Swiss albino

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 Results (Mean ± SEM) analyzed by student’t test/Mann-Whitney

 p < 0,05

Establish a model of HCC in SCID mice

Control

n= 6 (PBS, sc)

HCC-induced

n= 30 (HepG2, sc)

Treatment

(5-FU, iv 20mg/kg)

Pathology control

(iv, NaCl 0.9%)

CYP

n = 36

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Day 12, visible tumors on 17/30 mice (56.7%)

Tumor size (mm 3 ) (Mean ± SEM) Pathology

control (n = 7)

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 ##

Pathology control 5-FU treatment

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Day 12 Day 14 Day 16 Day 18

Subcutaneous tumors of pathology control group

Pathology control Treatment

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Pathology control 5 – FU treatment

• Cloudy white tumor between skin and muscle tissue with formation of new blood vessels

 Local inflammation in the surrounding skin

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Pathology control 5 – FU treatment

• 6/6 overproduction of fibroblasts,

abnormal cells undergoing mitosis,

big nucleus

• 4/6 central necrosis, infiltration of

inflammatory cells

• 6/6 overproduction of fibroblasts,

dysplastic cells (fibroblast)

• Peripheral necrosis, infiltration of inflammatory cells

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Mice with sc tumors

Pathology

Size mesuring

Positive control

NaCl 0,9%

0.1 ml/10 g PXT-Monta 1.5 10 mg/kg 20 mg/kg5-FU • 5-FU 20 mg/kg

• PXT-Monta 10 mg/kg

iv once a day, for 5 continuous days

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Ip CYP

Sc 10 6 HepG2 cells/mouse

Day 19: Tumors collecting, sample preparing, histology analyzing

Study the anti-tumor effect of liposome paclitaxel formulation

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Mean percentage of size difference compare to day 12 ± SEM (%)

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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5-FU + PTX-Monta 5-FU

PTX-Monta Pathology control Study the anti-tumor effect of liposome paclitaxel formulation

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 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

 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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