紫草素联合shBAG3杀伤人脑胶质瘤干细胞的协同效应及机制研究

Our previous study demonstrated that there is a BAG3-dependent protective mechanism involved in the antitumor process of shikonin toward GSCs. The results showed that not only the onset time of cytotoxicity by shikonin toward GSCs is earlier, but also the antitumor effect is enhanced by combination of shBAG3 than either shikonin or shBAG3 treatment alone. Based on a series of previous work, we propose that the mechanism of the synergistic effect is attributed to the inhibition of BAG3-dependent noncanonical autophagy by combination with shBAG3, and thus, through the following pathways: ①the enhanced protein toxicity in GSCs; ②enhanced mitochondrial apoptosis due to reduced injured mitochondria clearance; ③enhanced endoplasmic reticulum stress mediated apoptosis due to reduced PolyQ clearance. This project, for the first time, not only illustrates the enhanced antitumor effect and molecular mechanism of shikonin toward GSCs through inhibiting the BAG3 relevant pro-survival mechanism by combination of shBAG3, but also illustrates that the combination therapy by which inhibits the pro-survival mechanism involved in the antitumor process of shikonin toward GSCs may provide a novel approach to enhance the antitumor effect of shikonin toward glioma.

前期研究发现紫草素在对人脑胶质瘤干细胞（GSCs）产生肿瘤杀伤作用的同时也诱导GSCs产生了BAG3相关的促存活机制；紫草素联合shBAG3不仅可使紫草素杀伤GSCs的起效时间提前，而且与单独应用紫草素或者shBAG3相比较，紫草素联合shBAG3在杀伤GSCs效果上存在协同效应。推测联合shBAG3产生协同作用的机制可能与抑制紫草素杀伤GSCs过程中诱导BAG3产生的非经典自噬有关,从而导致：①GSCs中蛋白毒性增加；②受损线粒体清除减少增强了线粒体凋亡途径；③错叠蛋白PolyQ降解减少进而增加了内质网源性凋亡途径。本研究不仅首次阐明了紫草素联合shBAG3通过靶向抑制紫草素杀伤GSCs过程中所产生的BAG3相关的促存活机制可协同增强紫草素对GSCs的肿瘤杀伤作用及分子机制，而且阐明了应用联合治疗靶向抑制紫草素杀伤GSCs过程中产生的促存活机制能够为提高紫草素抗胶质瘤的疗效提供新的途径。

脑胶质瘤是恶性度很高的颅内原发性肿瘤，约占中枢神经系统肿瘤的60%。即使联合手术、放疗及化疗等多种治疗手段，2年生存率也仅为20%，如何能够提高胶质瘤患者的疗效及生存率已经成为亟待解决的一大难题。越来越多的报道指出肿瘤内存在一小部分具有自我更新能力、多向分化潜能以及更强体内成瘤能力的细胞，称为“肿瘤干细胞（Cancer Stem Cells, CSCs）”。胶质瘤中肿瘤干细胞的存在也被证实是导致其对化疗不敏感，对放疗抵抗及胶质瘤复发的根源. 因此,靶向胶质瘤干细胞（Glioma Stem Cells, GSCs）的杀伤性治疗已经逐渐成为抗脑胶质瘤治疗的核心。.紫草素是从中药紫草中提取的萘醌类化和物，临床上具有抗炎、抗氧化、免疫调节等多种药理活性。越来越多的报道指出紫草素在多种肿瘤细胞中具有肿瘤杀伤作用。报道证明紫草素可延长晚期肺癌患者的生存期。以上的研究结果提示紫草素在用于临床抗肿瘤治疗方面具有良好的应用前景，因此深入研究紫草素的抗肿瘤作用和机制具有重要意义。.本项目明确了紫草素联合shBAG3在杀伤GSCs作用上存在协同效应。首先研究紫草素联合shBAG3对紫草素诱导GSCs产生非经典自噬的影响；同时进行体外培养与裸鼠体内种植GSCs,观察非经典自噬在紫草素杀伤GSCs过程中所产生的生物学效应，进而明确了紫草素联合shBAG3产生杀伤GSCs协同效应的机制与抑制BAG3依赖的非经典自噬有关；最后研究了紫草素联合shBAG3对紫草素作用下GSCs中泛素化蛋白、PolyQ蛋白蓄积的影响，线粒体膜电位的改变以及细胞色素c、活化caspase-9和caspase-12等蛋白的表达变化，以明确紫草素联合shBAG3增强紫草素对GSCs肿瘤杀伤作用的分子机制可能与抑制非经典自噬所产生的促存活分子机制有关。本研究不仅首次阐明了紫草素联合shBAG3通过靶向抑制紫草素杀伤GSCs过程中所产生的BAG3相关的促存活机制可协同增强紫草素对GSCs的肿瘤杀伤作用及分子机制，而且阐明了应用联合治疗靶向抑制紫草素杀伤GSCs过程中产生的促存活机制能够为提高紫草素抗胶质瘤的疗效提供新的途径。