多药耐药基因ABCB1和ABCG2在下咽癌FaDu细胞多药耐药中的研究

Role of ABCB1 and ABCG2 in the multidrug resistance of hypopharyngeal carcinoma FaDu cell line

目的研究多药耐药基因ABCB1和ABCG2在下咽癌FaDu细胞株及其耐紫杉醇细胞株FaDu／T中的多药耐药特征及其机制，为进一步研究下咽癌细胞的多药耐药性及逆转提供理论支持。方法以人下咽癌细胞株FaDu为亲本细胞，采用浓度梯度递增法成功建立下咽癌细胞株FaDu的耐紫杉醇细胞株FaDu／T。四甲基偶氮唑蓝法分别检测FaDu和FaDu／T对顺铂、氟尿嘧啶、多柔比星和长春新碱的多药耐药性；多药耐药基因及蛋白表达变化情况分别通过RT—PCR，Western blot和激光共聚焦检测；c—Jun氨基末端激酶（c—Jun N—terminal kinase，JNK）信号转导通路相关蛋白的表达变化通过Western blot检测。结果耐药细胞株FaDu／T比FaDu细胞有更强的多药耐药性。FaDu细胞相比，FaDu／T细胞株中多药耐药蛋白ABCB1的表达增加（t=22．42，P〈0．05），但ABCG2表达下降（t=10．06，P〈0．05）。紫杉醇初始作用于FaDu细胞后JNK信号转导通路被激活，但是在FaDu／T细胞中JNK信号转导通路呈失活状态，丝裂原活化蛋白激酶信号转导通路的激活剂茴香霉素（Anisomycin）可以使FaDu／T细胞的JNK信号通路重新激活。耐药细胞株FaDu／T中加入茴香霉素时ABCB1表达下调（F=33．72，P〈0．05），ABCG2的表达升高（F=220．16，P〈0．05），但是在预先加入JNK特异性抑制剂SP600125的FaDu／T细胞株中加入茴香霉素时，ABCB1和ABCG2的表达无明显变化（P〉0．05），提示JNK信号通路在下咽癌的多药耐药过程中具有重要的调控作用。结论下咽癌FaDu细胞的多药耐药性以多药耐药蛋白ABCB1的高表达和ABCG2的低表达为主要特征，且两种耐药蛋白的表达变化与JNK信号转导通路密切相关。

Objective To investigate the expression of multidrug resistance gene ABCB1 and ABCG2 in FaDu cells （human hypopharyngeal carcinoma cell line ） and the multidrug resistance （MDR） cell lines FaDu/T transformed from FaDu cells by taxol and underlying mechanisms of MDR. Methods The multidrug resistance sensitivities of FaDu and FaDu/T to cisplatin （DDP）, 5-fluorouracil （5-FU）, doxorubicin （Dox）, and vincristine （VCR） were examined by methyl-thiazolyl-tetrazolium （MTF） assay. The mRNA and protein expressions of multidrug resistance genes ABCB1 and ABCG2 were analysed with RT-PCR, Western blot and laser confocal microscopy. JNK signal proteins were detected through Western blot. Results The multidrug resistance of FaDu/T cells to Taxol, DDP, 5-FU, ADM and VCR was more than that of FaDu cells. The expression of ABCB1 in FaDu/T cells was significantly higher than that in FaDu cells （ t = 22.42 ,P 〈 0. 05 ）, but the expression of ABCG2 in FaDu/T cells was significantly lower than that in FaDu cells （t = 10. 06,P 〈0. 05）. JNK signal was inhibited in FaDu or FaDu/T cells and the inhibited JNK was reactivated by taxol or anisomycin （an activator for MAPK signal transduction pathways ）. Anisomycin down-regulated the expression of ABCB1 （F = 33.72, P 〈 0. 05 ） and up-regulated the expression of ABCG2 （F =220. 16,P 〈 0. 05 ） in FaDu/T cells, but not in FaDu/T ceils pretreated by JNK inhibitor SP600125 （P 〉 0. 05）. Conclusion The overexpression of ABCB1 and the down-regulation of ABCG2 in FaDu/T cells were the main features of MDR in hypopharyngeal carcinomas, in which JNK signal transduction pathways could play an important role.