阿霉素诱导乳腺癌细胞P-gp高表达的代谢性分子机理研究

Acquired resistance to chemotherapy drugs greatly challenged the treatment of cancer in clinical. Studies already demonstrated that profound metabolic reprogramming occurred in cancer cells, while our preliminary study suggested that adriamycin-resistant breast cancer cells, characterized by high expression of P-gp, have significantly altered pharmacokinetic behavior, which greatly contributed to the occurrence of drug resistance. Moreover, its own metabolic patterns were also distinctly different from the sensitive phenotype cells, especially retarded turnover of amino acids and nucleic acids, aberrant glutathione cycle and accelerated metabolism of glycerol. However, the relationship between the metabolic rearrangement and drug resistance remains unclear. Hence, on the basis of prvious studies in vitro, this proposal intends to further performe research on breast cancer patients sensitive or resistant breast cancer to adriamycin chemotherapy through metabolomics approach so as to clarify metabolic characteristics and metabolic response to adriamycin under the resistant phenotype. Biomarkers and key pathways closely associated with resistance will be screened and identified by metabolic pathways/network analysis, and further validated with molecular biology methods. We will also assess alterations in the efficacy of adriamycin after interference of metabolic pathways to seek potential targets for reversal of chemoresistance. In summary, combining metabolomics-informed targeted gene/protein investigation and pharmacokinetic-assisted study of adriamycin-resistant bio-system allowed for more comprehensive interrogation of mechanisms involved in the acquired resistance, which will be a solid support for the research of chemo-resistance and provide a new approach for the development of sensitizing agents.

化疗药引起肿瘤耐药给临床肿瘤治疗带来了严峻挑战。研究证实肿瘤细胞常出现与众不同的代谢重排，前期研究显示: 以P-gp高表达为最显著特征的阿霉素耐药型乳腺癌细胞中阿霉素的细胞药代行为发生显著变化而极大贡献于耐药的发生；且其自身代谢模式也显著区别于敏感细胞，突出表现在耐药细胞中氨基酸和核酸的利用率减慢、谷胱甘肽循环异常而甘油代谢加快，但代谢重排与耐药之间的关系尚不清楚。项目计划在体外前期研究基础上，以代谢组学为基础在乳腺癌病人中初步探索阿霉素耐药表型的代谢特征和药物应答规律。借助于代谢通路、网络分析和药代-代谢组关联性分析等筛选出耐药相关的关键通路及标志物，并整合分子生物学和药代动力学研究手段予以证实。在此基础上考察调控代谢通路尤其是胱氨酸循环对阿霉素药效的影响。总之，本项目以代谢组学为导向，揭示与阿霉素耐药相关的代谢性分子机制，为解决化疗耐药难题和发现潜在增敏靶点提供依据。

化疗药物引起肿瘤耐药给临床肿瘤治疗带来了严峻挑战，研究证实肿瘤细胞常出现与众不同的代谢重排，但敏感细胞和耐药细胞的代谢规律和特征的研究报道鲜见。本项目以代谢组学为导向，探索阿霉素耐药表型的代谢特征和药物应答规律，揭示与阿霉素耐药相关的代谢性分子机制。研究发现耐药细胞自身代谢模式显著区别于敏感细胞，突出表现在氧化还原系统的重构，包括胱氨酸循环和谷胱甘肽合成、甘油代谢以及磷酸戊糖通路，但代谢重排与耐药间的关系尚不清楚。因此项目拟借助于代谢通路、网络分析和药代-代谢组关联性分析等筛选出耐药潜在的代谢性标记物，并寻求潜在的增敏靶点。我们的研究表明阿霉素能时间和剂量依赖性地下调胱氨酸转运体SLC7A11的表达和活性，使GSH合成减少而ROS增多从而诱导P-gp的高表达，而ROS对于P-gp的诱导作用可以被ROS清除剂NAC所拮抗。基于乳腺癌细胞模型，我们首次发现SLC7A11转运体调控、胱氨酸补充或剥夺明显影响乳腺癌细胞P-gp的表达与功能，在培养基中充分补充胱氨酸可以明显降低耐药型乳腺癌细胞MCF-7R中P-gp的表达与功能，提示胱氨酸是潜在的耐药标记物而SLC7A11可作为潜在的增敏靶点，为增敏基于P-gp表达与功能的多药耐药提供新的方法和策略。在MCF-7荷瘤鼠模型中，采用补充胱氨酸的手段，一方面可显著增强阿霉素的抗肿瘤药效，另一方面可有效降低阿霉素所致的肝脏毒性。此外，外源性胱氨酸补充能够剂量依赖的调控乳腺癌细胞谷胱甘肽合成关键限速酶GCLC和GSS的转录及翻译水平，进而影响细胞对药物治疗的敏感性。同时我们初步探索GCLC和GSS的上游调控信号通路Keap1-Nrf2与药物敏感之间的关系。在MCF-7A耐药细胞模型中，沉默Keap1基因表达能够显著增加肿瘤细胞对药物治疗的敏感性，而在MCF-7S敏感细胞模型中，沉默Nrf2基因则能够显著的降低肿瘤细胞对药物治疗的敏感性。可见，Keap1-Nrf2信号通路介导的代谢重排与耐药之间密切相关，从而为寻求新的增敏靶点提供线索，为胱氨酸的临床合用提供数据支持。