Mechanism of 5-fluorouracil required resistance in human hepatocellular carcinoma cell line Bel7402

doi:10.3748/wjg.v8.i6.1029

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Dec 15, 2002 (publication date) through Apr 29, 2024

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World Journal of Gastroenterology

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1007-9327

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

Liver Cancer

World J Gastroenterol. Dec 15, 2002; 8(6): 1029-1034
Published online Dec 15, 2002. doi: 10.3748/wjg.v8.i6.1029

Mechanism of 5-fluorouracil required resistance in human hepatocellular carcinoma cell line Bel₇₄₀₂

Jing Jin, Min Huang, Huai-Ling Wei, Geng-Tao Liu

Jing Jin, Huai-Ling Wei, Geng-Tao Liu, Department of Pharmacology, Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100050, People’s Republic of China

Min Huang, Department of Pharmacology, University of North Carolina at Chapel Hill, 7365, North Carolina, USA

Author contributions: All authors contributed equally to the work.

Correspondence to: Prof. Geng-Tao Liu, Institute of Materia Medica, 1 Xian Nong Tan Street,Beijing 100050,China. liugt@imm.ac.cn

Telephone: +86-10-63165178 Fax: +86-10-63017757

Received: March 11, 2002
Revised: June 2, 2002
Accepted: June 15, 2002
Published online: December 15, 2002

Abstract

AIM: To investigate the resistance mechanism of 5-fluorouracil (5-FU) in Bel₇₄₀₂/5-FU cells which was established in our lab by in vitro continuous stepwise exposure of human hepatocellular carcinoma (HCC) cell line Bel₇₄₀₂ to 5-FU.

METHODS: The expression of multidrug resistance-associated protein (MRP) and thymidylate synthase (TS) in Bel₇₄₀₂ cells was detected by immonocytochemistry. The fluorescein (FLU) accumulation, an index of MRP functional activity, was determined by flow cytometry. The distribution of FLU was observed by confocal laser scanning microscope. The spectrofluorometry was used to show the intracelluar content of glutathione (GSH). Cell growth inhibition was determined by MTT assay. The activity of glutathione S-transferases (GSTs) was determined by spectrophotometry.

RESULTS: A higher expression of MRP in the Bel₇₄₀₂/5-FU cells was observed by using monoclonal mouse anti-MRP antibody, MRPr-1, in comparison with Bel₇₄₀₂ cells. Bel₇₄₀₂/5-FU cells also showed a significant decrease of FLU accumulation. FLU mainly accumulated in the nucleus with a high nuclear/cytoplasmic ratio in Bel₇₄₀₂ cells, whereas there was no difference of FLU accumulation between the nucleus and cytoplasm in Bel₇₄₀₂/5-FU cells. The intracellular GSH content in Bel₇₄₀₂/5-FU cells was almost 3 folds higher than that in Bel₇₄₀₂ cells. Addition of D, L-buthione-S, R-sulfoximine (BSO) dose-dependently reduced the GSH content in Bel₇₄₀₂/ 5-FU cells, however, only a weak enhancement on the cytotoxicity of 5-FU and doxorubicin (Dox) to Bel₇₄₀₂/5-FU cells was observed. Bel₇₄₀₂/5-FU cells also exhibited 29.1% higher total GSTs activity than Bel₇₄₀₂ cells. Immunocytochemical staining by using anti-TS monoclonal antibody TS 106 showed that the level of TS in Bel₇₄₀₂/5-FU cells elevated markedly as compared with Bel₇₄₀₂ cells.

CONCLUSION: The continuous exposure of Bel₇₄₀₂ cells to 5-FU led to overexpression of TS and MRP, as well as increased intracellular GSH content and total GST activity.

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