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 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 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 - mm Hagiwara et al (2007) Male BALB/c nude mice, s.c injection After 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 HepG2 cells/mouse 16th day, 100% mice developed tumors 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 ± g n=6 CYP day CYP day CYP day CYP day DOX day CYP day • Collect blood from tail vein every – days CYP day • Count leucocytes (Neubauer chamber) Monitor mortality rate and ability to maintain immunodeficiency METHODS - RESULTS Studying immunodeficiency-induced model on Swiss albino I.p CYP day 1, 3, 5: weak, ruffled fur, slow movement, 2/6 blind dead on day 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 dead on day and another on day Lowest number of leucocytes is recorded on day 5, followed by a rapid recovery of 87% on day 21 METHODS - RESULTS Studying immunodeficiency induced model on Swiss albino Ip CYP day 1, 3: weak, ruffled fur, normal movement, dead Total leucocytes were lowest on day 5, then recovered by 33% on day 21 Choose this protocol for the study of HCC induction METHODS - RESULTS Establish a model of HCC in SCID mice CYP i.p 100mg/kg Control n= (PBS, sc) Pathology control (iv, NaCl 0.9%) n = 36 HCC-induced n= 30 (HepG2, sc) Day 1&3 Collect tumors Treatment (5-FU, iv 20mg/kg) 12 =>16 19  Results (Mean ± SEM) analyzed by student’t test/Mann-Whitney  p < 0,05 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 METHODS - RESULTS Establish a model of HCC in SCID mice d12 d14 d16 d18 Pathology control Treatment METHODS - RESULTS Establish a model of HCC in SCID mice 10 METHODS - RESULTS Establish a model of HCC in SCID mice 11 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 continuous days Day 19: Tumors collecting, sample preparing, histology analyzing Percentage of difference (%) = (Vafter – Vbefore)/Vbefore x 100 12 METHODS - RESULTS Study the anti-tumor effect of liposome paclitaxel formulation 13 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 14 METHODS - RESULTS Study the anti-tumor effect of liposome paclitaxel formulation 5-FU + PTX-Monta PTX-Monta 5-FU Pathology control 15 METHODS - RESULTS Study the anti-tumor effect of liposome paclitaxel formulation 16 METHODS - RESULTS Study the anti-tumor effect of liposome paclitaxel formulation 17 METHODS - RESULTS Study the anti-tumor effect of liposome paclitaxel formulation 18 CONCLUSIONS  Evaluated and chose the suitable immunodeficiency-induced model of CYP 100 mg/kg, i.p on day 1,  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 single therapy groups 19 THANK YOU FOR YOUR ATTENTION 20