自噬在紫杉醇抑制乳腺癌中的作用及其机制研究

Paclitaxel(PTX) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors, including breast cancer. Many studies have focused on the apoptosis of tumor cells induced by PTX and further inhibition of tumor progression. However, the clinical use of PTX still has many problems, including drug resistance. These problems can not be explained clearly by the current basic research. Our findings showed that promotion of apoptotic cell death by PTX was accompanied with induction of autophagy in PTX. PTX could lead to the formation of acidic vesicular organelles (AVOs), the up-regulation of Ratio of microtubule-associated protein 1 light chain 3 (LC3B) type II and type I as well as down-regulation of P62 protein expression. Meanwhile, PTX increased the rate of apoptosis cells, Bax, Caspase-9 as well as PARP-1 protein, and reduced the Bcl-2 protein. These results suggested paclitaxel promotes apoptosis and induces autophagy. Further results have shown that autophagy can repair tumor cell damage through mitochondrial autophagy and weaken the effect of its induction of apoptosis. To further elucidate the mechanism of PTX induced autophagy and to provide a practical target for the increase of the effect of PTX. In this study, we investigate the molecular mechanism of PTX induced autophagy using breast cancer cells. Furthermore, we conform whether autophagy induced by paclitaxel can reduce the effect of autophagy during the treatment of tumor using human breast cancer xenografts in nude mice. Moreover, we study whether blocking autophagy induced by paclitaxel can increase the therapeutic effect of breast cancer using transgenic mice with primary breast cancer. Our findings can elucidate the molecular basis of autophagy induced by paclitaxel during the treatment of tumor and autophagy inhibition contribute to the increased effect of paclitaxel in the treatment of tumors. The study provides a theoretical basis for further drug development and clinical application.

紫杉醇（PTX）是临床治疗乳腺癌的一线化疗药物。有关其诱导细胞凋亡进而抑制肿瘤进展的研究已很多，但是其临床使用仍然存在包括耐药性在内的诸多问题，目前的基础研究也还无法解释清楚。我们的前期研究结果表明：PTX处理形成了酸性自噬囊泡、增加微管结合蛋白LC3B-II /LC3B-I以及减少P62蛋白；同时，增加了凋亡率、Bax、Caspase-9和PARP-1蛋白、降低Bcl-2蛋白。提示PTX促进细胞凋亡同时诱导自噬。进一步的结果显示其引发的自噬通过线粒体自噬等功能修复了肿瘤细胞损伤，减弱其诱导细胞凋亡的效果。为进一步阐明PTX诱导自噬的机制，为其增效提供理论上可行的靶点。本研究拟采用细胞、裸鼠移植瘤和转基因原发乳腺癌小鼠三种模型，明确PTX诱导自噬的分子机制，找到关键节点。并尝试通过基因敲除小鼠和药物处理两种方法阻断自噬，进而评估对紫杉醇疗效的影响，为进一步的药物研发和临床应用提供理论基础。

三阴性乳腺癌症（TNBC）是乳腺癌中最具侵袭性的亚型，它常常对紫杉醇（PTX）治疗产生耐药性。自噬在PTX诱导的肿瘤细胞死亡中起着重要的细胞保护作用，近年来靶向自噬被认为是提高肿瘤化疗疗效的有效途径。但是，紫衫醇在肿瘤治疗过程中诱发的自噬是否会降低疗效及其机制尚需阐明。本研究用三阴性乳腺癌细胞、基因修饰成纤维细胞及裸鼠移植乳腺癌模型，研究PTX诱导自噬的分子机制，找到关键靶点，并评估自噬抑制剂对PTX的增效作用。得出的实验结果如下：.1. PTX可同时诱导MDA-MB-231和HCC-1937细胞的自噬和凋亡。其中，HCC-1937细胞对紫杉醇更为敏感，但是P62蛋白表达没有变化，所以我们选取MDA-MB-231细胞进行PTX诱导自噬机制的研究。PTX可增加FOXO1的表达，进而通过转录激活诱导MDA-MB-231细胞自噬，其分子机制可能与下游靶基因ATG5、PIK3C3、Beclin 1等的激活相关。用自噬抑制剂3-MA抑制MDA-MB-231细胞的自噬，可促进PTX诱导的细胞凋亡，表明了自噬抑制剂对PTX诱导三阴性乳腺癌细胞凋亡的增效作用。.2. 在三阴性乳腺癌细胞荷瘤小鼠模型的构建中，MDA-MB-231细胞移植后成功获得荷瘤小鼠模型，而HCC-1937细胞不易成瘤。在自噬抑制剂对紫杉醇是否有增效作用的体内研究中，发现PTX同样可激活肿瘤组织自噬，PTX与3-MA联用可抑制自噬，增强PTX对三阴性乳腺癌肿瘤生长的抑制作用。.3. 用基因修饰成纤维细胞模型，明确了P53、Leptin和PIK3C3基因在自噬中的作用：P53抑制自噬，Leptin和PIK3C3过表达促进自噬，其分子机制可能与自噬调控基因的上调有关。.综上所述，我们的研究结果提示，PTX在体外可诱导三阴性乳腺癌细胞发生自噬，其机制可能与FOXO1、ATG5以及Beclin1的激活有关，3-MA的联用可抑制自噬，并增强PTX诱导三阴性乳腺癌细胞凋亡的作用。PTX在三阴性乳腺癌荷瘤鼠体内，同样可诱导肿瘤组织自噬，从而导致PTX对肿瘤生长的抑制作用变差，3-MA的联用可增强PTX对肿瘤生长的抑制作用，其机制可能与3-MA抑制自噬相关基因和信号通路基因的表达有关。这些结果可为治疗乳腺癌的药物研发和临床应用提供理论基础。