Characterization and Resistance Mechanisms of A 5-fluorouracil-resistant Hepatocellular Carcinoma Cell Line

Wei Gu,Chang-Quan Ling,Bin-Bin Cheng,Fan-Fu Fang,Bai Li

doi:10.7314/apjcp.2012.13.9.4807

Abstract

The chemoresistance of human hepatocellular carcinoma (HCC) to cytotoxic drugs, especially intrinsic or acquired multidrug resistance (MDR), still remains a major challenge in the management of HCC. In the present study, possible mechanisms involved in MDR of HCC were identified using a 5-fluorouracil (5-FU) -resistant human HCC cell line. BEL-7402/5-FU cells were established through continuous culturing parental BEL-7402 cells, imitating the pattern of chemotherapy clinically. Growth curves and chemosensitivity to cytotoxic drugs were determined by MTT assay. Doubling times, colony formation and adherence rates were calculated after cell counting. Morphological alteration, karyotype morphology, and untrastructure were assessed under optical and electron microscopes. The distribution in the cell cycle and drug efflux pump activity were measured by flow cytometry. Furthermore, expression of potential genes involved in MDR of BEL-7402/5-FU cells were detected by immunocytochemistry. Compared to its parental cells, BEL-7402/5-FU cells had a prolonged doubling time, a lower mitotic index, colony efficiency and adhesive ability, and a decreased drug efflux pump activity. The resistant cells tended to grow in clusters and apparent changes of ultrastructures occurred. BEL-7402/5-FU cells presented with an increased proportion in S and G2/M phases with a concomitant decrease in G0/G1 phase. The MDR phenotype of BEL-7402/5-FU might be partly attributed to increased drug efflux pump activity via multidrug resistance protein 1 (MRP1), overexpression of thymidylate synthase (TS), resistance to apoptosis by augmentation of the Bcl-xl/Bax ratio, and intracellular adhesion medicated by E-cadherin (E-cad). P-glycoprotein (P-gp) might play a limited role in the MDR of BEL-7402/5-FU. Increased activity or expression of MRP1, Bcl-xl, TS, and E-cad appear to be involved in the MDR mechanism of BEL-7402/5-FU.

Highlights

Hepatocellular carcinoma (HCC) is currently the sixth most common type of cancer and the third leading cause of cancer-related death in the world
The MDR phenotype of BEL-7402/5-FU might be partly attributed to increased drug efflux pump activity via multidrug resistance protein 1 (MRP1), overexpression of thymidylate synthase (TS), resistance to apoptosis by augmentation of the B-cell lymphoma-extra large (Bcl-xl)/Bax ratio, and intracellular adhesion medicated by E-cadherin (E-cad)
Cells were harvested with trypsin after twice washes with cold phosphate buffered saline (PBS) and resuspended rapidly with 400 μl PBS, and intracellular fluorescence of FLU was immediately measured with flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 520 nm

Summary

Introduction

Hepatocellular carcinoma (HCC) is currently the sixth most common type of cancer and the third leading cause of cancer-related death in the world. More than 747000 new cases of HCC and 695000 deaths were estimated to have occurred in 2008. In China, more than 401000 new patients (53.7% of the world) are diagnosed with HCC and more than 371000 patients (53.4% of the world) are killed by the terrible disease annually (Ferlay et al, 2010). Even worse are approximately 80% of patients diagnosed initially with an advanced stage where the lesions were more often unresectable. Nearly half of patients who underwent hepatectomy would have a recurrent or metastatic disease within two years. Effective therapeutic approaches are urgently desired for patients with HCC (El-Serag, 2011; Livraghi et al, 2011)

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Results

Conclusion