Jin J, Huang M, Wei HL, Liu GT. Mechanism of 5-fluorouracil required resistance in human hepatocellular carcinoma cell line Bel
7402.
World J Gastroenterol 2002;
8:1029-34. [PMID:
12439919 PMCID:
PMC4656374 DOI:
10.3748/wjg.v8.i6.1029]
[Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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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