胱氨酸及其SLC7A11转运体调控乳腺癌细胞P-糖蛋白高表达耐药机理研究

Continual use of antitumor drugs (such as adriamycin) potentially induces over-expression of p-gp and multiple drug resistance, which usually lead to the failure of cancer chemotherapy. However, the biochemical process and underlying mechanisms remain unclear. Our previous study reveals that adriamycin distinctly induces metabolic reprogramming, an imbalance of oxidation-anti-oxidation and significant elevation of ROS. While scavenge of ROS markedly decreases the expression and function of P-gp, indicating that the imbalance of redox state and the increased ROS play a key role in promoting P-gp expression and the occurrence of drug resistance. Further study suggests adriamycin primarily inhibits SLC7A11 (in charge of cystine uptake), reduces intracellular cystine(source material of GSH) and biosynthesis of glutathione (GSH); and consequently the cells show a rather low capacity of anti-oxidation and hence increased level of ROS. Thanks for our recent advance in the successful transfection of stable over-expression of SLC7A11 in sensitive MCF-7S and resistant MCF-7R cell lines, and the tumor-bearing mice with MCF-7S and MCF-7R cells, in this project, we are devoted in evaluating the regulatory effect of cystine and its transporter SLC7A11 on P-gp expression and function, and further exploring the key signal pathways/moledules involved in ROS induced P-gp over-expression. Hopefully, this study will clarify the crucial factors and underlying mechanism contributing to the induction of over-expressed P-gp and chemo-resistance. We are looking forward to approaching a potential strategy to reverse P-gp over-expression and the relevant chemo-resistance.

抗肿瘤药物(如阿霉素)反复使用可诱导P-gp高表达和多药耐药，导致肿瘤化疗失败，其引起P-gp高表达机理不明。前期代谢特征研究发现阿霉素诱导MCF-7乳腺癌细胞P-gp高表达同时，出现代谢重排、氧化-抗氧化失衡和ROS明显升高；而清除ROS可显著降低P-gp表达和功能，提示肿瘤细胞氧化-抗氧化失衡和ROS增加是造成P-gp高表达和耐药的关键原因。深入研究发现阿霉素通过抑制胱氨酸摄取转运体SLC7A11，导致胞内胱氨酸减少和谷胱甘肽合成下降，是导致细胞抗氧化能力下降和ROS上升的关键原因。在已成功建立稳定高表达SLC7A11乳腺癌细胞株及MCF-7接种荷瘤鼠基础上，计划在细胞和整体动物水平重点研究胱氨酸及其转运体SLC7A11调控对P-gp表达与功能的影响，进一步探索ROS诱导P-gp高表达中的关键信号通路/分子，为阐明抗肿瘤药物诱导P-gp高表达和多药耐药机理，发现逆转耐药方法提供依据。

前期研究揭示阿霉素刺激ROS生成增加和清除减少，影响细胞氧化还原平衡，导致Pgp高表达及产生耐药。本研究证实：胱氨酸剥夺，或其转运体SLC7A11(xCT)下调明显提高细胞内ROS水平、促进MCF-7乳腺癌细胞 P-gp 表达与功能上调，诱导细胞耐药产生。相反，xCT过表达或补充胱氨酸产生反向调控作用，并显著增强阿霉素抗肿瘤药效，在整体动物荷瘤鼠上得到验证。提示xCT/胱氨酸是调控肿瘤细胞耐药的潜在靶点，细胞ROS清除能力下降是造成Pgp高表达和细胞耐药的关键因素。进一步考察不同细胞毒药物对肿瘤细胞P-gp表达的影响，发现3种细胞毒物质虽增加胞内ROS生成，但对xCT没有明显影响，其刺激P-gp上调效应明显弱于阿霉素，提示肿瘤细胞耐药同时依赖于ROS生成及其清除能力。考虑到除胱氨酸外，蛋氨酸及其转硫途径也对一些人体细胞合成GSH、维持细胞氧化还原平衡发挥重要作用，进一步研究蛋氨酸及其转硫途径对乳腺癌细胞耐药的重要性。采用代谢组学进行研究，结果显示4种常见乳腺癌细胞与2种正常乳腺细胞存在显著的代谢差异，突出表现在半胱氨酸和蛋氨酸代谢通路，转硫途径关键酶-胱硫醚γ裂解酶（CTH）上调，提示乳腺癌获得了独特的利用蛋氨酸转硫途径合成GSH、清除ROS能力。代谢流实验表明，MCF-7R耐药细胞也出现蛋氨酸代谢重排: 蛋氨酸利用减少、CTH活性与转硫途径的活性降低。蛋氨酸剥夺或CTH沉默后，细胞ROS升高、P-gp表达与功能显著上调，MCF-7S敏感型逐步转变为MCF-7R耐药表型。相反，当补充蛋氨酸或CTH过表达时，MCF-7R细胞ROS下降，P-gp下调，耐药表型转变为敏感表型，上述调控作用在荷瘤鼠上也得到证实。提示胱氨酸及其转运体xCT、蛋氨酸及其CTH调控肿瘤细胞的P-gp表达/功能，对逆转/诱导肿瘤细胞耐药发挥重要作用，调控肿瘤细胞GSH合成对逆转耐药具有潜在的临床转化价值。