一种耐5-氟尿嘧啶的肝癌细胞系的特性及耐药机制

Characterization and resistance mechanisms of a 5-fluorouracil- resistant hepatocellular carcinoma cell line.

作者信息

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

机构信息

Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China.

出版信息

Asian Pac J Cancer Prev. 2012;13(9):4807-14. doi: 10.7314/apjcp.2012.13.9.4807.

DOI:10.7314/apjcp.2012.13.9.4807

PMID:23167424

Abstract

PURPOSE

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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.

摘要

目的

人类肝细胞癌（HCC）对细胞毒性药物的化疗耐药性，尤其是内在性或获得性多药耐药（MDR），仍然是HCC治疗中的一项重大挑战。在本研究中，使用一种对5-氟尿嘧啶（5-FU）耐药的人类HCC细胞系来确定HCC多药耐药中涉及的可能机制。

方法

通过连续培养亲本BEL-7402细胞建立BEL-7402/5-FU细胞，模拟临床上的化疗模式。通过MTT法测定生长曲线和对细胞毒性药物的化学敏感性。细胞计数后计算倍增时间、集落形成率和贴壁率。在光学和电子显微镜下评估形态学改变、核型形态和超微结构。通过流式细胞术测量细胞周期分布和药物外排泵活性。此外，通过免疫细胞化学检测BEL-7402/5-FU细胞中与多药耐药相关的潜在基因的表达。

结果

与亲本细胞相比，BEL-7402/5-FU细胞的倍增时间延长，有丝分裂指数、集落形成效率和黏附能力降低，药物外排泵活性下降。耐药细胞倾向于成簇生长，并且发生了明显的超微结构变化。BEL-7402/5-FU细胞在S期和G2/M期的比例增加，同时G0/G1期减少。BEL-7402/5-FU的多药耐药表型可能部分归因于通过多药耐药蛋白1（MRP1）增加药物外排泵活性、胸苷酸合成酶（TS）的过表达、通过增加Bcl-xl/Bax比值对凋亡的抵抗以及由E-钙黏蛋白（E-cad）介导的细胞内黏附。P-糖蛋白（P-gp）在BEL-7402/5-FU的多药耐药中可能起有限作用。