抑制泛素-蛋白酶体-survivin-自噬环路增效有丝分裂灾难克服肿瘤细胞多药耐药的机制研究

Multidrug resistance (MDR) of tumor cells, resulting from apoptosis-resistance, remains a major obstacle to cancer treatment. The evasion of apoptosis-resistance pathway through non-apoptotic death pathway could overcome MDR in tumors. Thus, mitotic catastrophe (MC) acts as a novel cell death mode against MDR. In previous study, we found that BZML was a novel colchicine binding site inhibitor with potent anti-cancer activity against MDR in non-small-cell lung cancer through MC. However, autophagy could block the occurrence and development of MC. Meanwhile, the expression of survivin protein was significantly increased during MC treatment. Therefore, we proposed that, in BZML-treated A549/Taxol cells, the accumulation of ubiquitinated survivin protein could be enhanced by the inhibition of proteasome during MC. Subsequently, it could increase Beclin-1 protein expression to induce autophagy, which further suppresses proteasome in return and hinders cell death during MC. In the present study, based on the BZML-induced MC process, the molecular mechanisms underlying survivin-induced autophagy hindering cell death under MC was elucidated by using small molecule inhibitors, siRNA interference technology and protein–protein interaction analysis. Xenograft NSCLC models induced by A549/Taxol cells were established to confirm that the inhibition of autophagy and/or survivin accumulation in tumor cells could enhance the efficiency of MC in vivo. This project is expected to provide a theoretical basis for clinical applying of MC and a new strategy to solve the problem of MDR in cancer treatment.

凋亡耐受介导的多药耐药（MDR）是肿瘤治疗的主要障碍。规避凋亡耐受途径，诱导非凋亡死亡方式能克服MDR，故有丝分裂灾难（MC）成为克服肿瘤细胞MDR的新策略。申请人前期研究发现，秋水仙碱位点抑制剂BZML通过诱导MC克服非小细胞肺癌的MDR，但自噬阻碍了MC的发生和发展。同时，survivin蛋白在MC过程中增加。故提出假设：在BZML诱导A549/Taxol细胞MC过程中，蛋白酶体的抑制导致泛素化survivin蛋白积累，而后通过Beclin-1诱导自噬，又进一步抑制蛋白酶体，还阻碍发生MC的细胞死亡。因此，本项目基于BZML诱导的MC过程，采用小分子抑制剂、siRNA干扰技术及蛋白互作分析等，阐明由survivin蛋白增加诱导的自噬，阻碍发生了MC的细胞死亡的分子机制。建立裸鼠移植瘤模型，确证抑制自噬和survivin能增强MC的体内作用，为实现MC的临床应用提供理论基础和治疗新策略。

凋亡耐受介导的多药耐药（MDR）是肿瘤治疗的主要障碍。规避凋亡耐受途径，诱导非凋亡死亡方式能够克服MDR，故诱导有丝分裂灾难（MC）成为克服肿瘤细胞MDR的新策略。本项目以新型秋水仙碱位点抑制剂BZML诱导MC克服A549/Taxol细胞MDR过程为研究对象，以期深入研究MC发生、发展的分子机制，阐明自噬是如何延缓/阻碍发生了MC的细胞死亡，为增强MC克服肿瘤细胞MDR提供新策略。研究发现，BZML不仅能够靶向抑制微管蛋白聚合，还通过直接或间接诱导DNA损伤发挥抗肿瘤作用。通过评价BZML在多种肿瘤细胞株中的作用，明确了药物诱导MC与P-gp的表达无关。重要的是，在BZML诱导MC克服A549/Taxol细胞MDR过程中，自噬的诱导导致UPS被抑制，泛素化survivin蛋白的表达显著增加，且主要分布于细胞核中。同时，过表达survivin蛋白不能介导A549细胞对BZML产生凋亡耐受，也不能促使BZML诱导A549细胞发生MC，故survivin不是介导A549/Taxol细胞凋亡耐受的主要原因。但是，异常增加的survivin蛋白通过与Beclin-1蛋白相互作用，诱导细胞自噬，自噬又进一步抑制UPS，形成UPS-survivin-自噬环路，最终延缓甚至阻碍了发生MC的细胞死亡。此外，UPS的抑制导致p53蛋白增加，促进发生MC细胞的衰老。同时，survivin蛋白的核积累还能够维持BZML诱导的A549/Taxol细胞衰老，促进发生MC的肿瘤细胞存活。进一步地，靶向抑制survivin蛋白/自噬能够加速发生MC的肿瘤细胞死亡，增效MC克服肿瘤细胞MDR。综上所述，抑制survivin蛋白/自噬能够增效MC克服肿瘤细胞MDR。