Effects of 5-fluorouracil on biological characteristics and drug resistance mechanisms of liver cancer cell line PLC/RAF/5
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摘要:
目的研究化疗药物5-氟尿嘧啶(5-Fu)反复作用肝癌细胞株PLC/RAF/5后生物学特性的变化,探讨耐药肝癌细胞与肝癌干细胞之间的相关性。方法用低浓度的5-Fu(1μg/ml)反复多次作用于人肝癌细胞株PLC/RAF/5后建立PLC/RAF/5/5-Fu细胞株,观察两种细胞的形态学差异。用CCK-8法检测不同浓度的5-Fu(0、0.25、0.5、1、1.5、2μg/ml)对两种细胞增殖的抑制作用;在不同浓度的5-Fu(0.5、1、2μg/ml)作用48 h后,流式细胞仪分析不同浓度下两种细胞的凋亡情况;检测两种细胞中侧群细胞的比例;平板克隆集落实验比较两种细胞的克隆形成能力;Western Blot检测两种细胞中Bax、Bcl-2、ABCG2和Fox M1蛋白的表达情况。计量资料组间比较采用t检验。结果用化疗药物5-Fu成功建立PLC/RAF/5/5-Fu细胞株。PLC/RAF/5/5-Fu细胞比PLC/RAF/5细胞体积大,突起减少,呈长梭形改变,折光性增强。与亲本细胞相比,PLC/RAF/5/5-Fu细胞对5-Fu增殖抑制作用的敏感性降低,细胞周期G0/G1期比例降低,而S期和G2...
Abstract:Objective To study the changes in biological characteristics of a liver cancer cell line PLC / RAF /5 after repeated exposure to a chemotherapy drug,5-fluorouraci( 5-FU),and to investigate the relationship between drug-resistant liver cancer cells and liver cancer stem cells. Methods A low concentration of 5-FU( 1 μg / ml) was used to treat the human liver cancer cell line PLC / RAF /5 repeatedly to establish the PLC / RAF /5 /5-FU cell line. Morphological differences between the two types of cells were observed. The inhibitory effects of different concentrations of 5-FU( 0,0. 25,0. 5,1,1. 5,and 2 μg / ml) on the proliferation of the two types of cells were determined using the CCK-8 assay. Apoptosis of the two types of cells after exposure to different concentrations of 5-FU( 0. 5,1,and 2 μg / ml) for 48 h was analyzed using flow cytometry. The proportions of side population cells in both types of cells were measured using flow cytometry. The colony-forming ability was compared between the two types of cells by the plate colony-forming assay. The expression of Bax,Bcl-2,ABCG2,and Fox M1 proteins in both types of cells was examined by Western blot. Between-group comparison was performed by t test. Results The PLC / RAF /5 /5-FU cell line was successfully established using the chemotherapy drug 5-FU. Compared with the PLC / RAF /5cells,the PLC / RAF /5 /5-FU cells had a larger volume,fewer protrusions,a changed shape of a long shuttle,and enhanced refractivity.Moreover,compared with the parent cells,the PLC / RAF /5 /5-FU cells had a significantly lower sensitivity to the inhibitory effect of 5-FU on proliferation,a significantly lower proportion of cells at the G0 / G1 phase of the cell cycle,significantly higher proportions of cells at the S and G2 / M phases,significantly higher resistance to apoptosis,a significantly higher proportion of side population cells,and significantly enhanced proliferation( P < 0. 05). According to the results of Western blot assay,the expression of Bcl-2,Fox M1,and ABCG2 proteins in the PLC / RAF /5 /5-FU cells was significantly increased( P < 0. 01),while the expression of Bax protein in the PLC / RAF /5 cells was significantly increased( P < 0. 01). Conclusion Liver cancer stem cells are probably responsible for drug resistance of the PLC / RAF /5-FU cell line. Exposure to a low concentration of 5-FU( 1 μg / ml) can enrich liver cancer stem cell-like cells.
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Key words:
- fluorouracil /
- liver neoplasms /
- cell line,tumor /
- neoplastic stem cells /
- drug resistance,neoplasm
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