基于磷酸酶PP2A的斑蝥抗肝癌物质基础及分子机制的研究

As one of important traditional Chinese medicine, cantharis has been widely used in anti-tumor therapy, and series of traditional Chinese medicines had been developed from it, which exhibit superior anti-cancer activity, especially for primary hepatic carcinoma curing. Our previous studies have already verified the existence of series of cantharidate derivates. Comparing to cantharidin, magnesium cantharidate has exhibited better antitumor activity, although its anticancer molecular mechanisms is still unclear. It was reported that protein phosphatase 2A (PP2A) inhibitor, such as okadaic acid and microcystin can induce the formation of hepatic carcinoma, while cantharidate derivates, the same as a PP2A inhibitor, have shown significant antitumor activity. Further experiments showed that cantharidin and its derivates as well as okadaic acid act on the same MAPK family signal molecules (a PP2A downstream molecule), but cause the obvious different biological behavior in tumor cells. Based on the above difference,this project aims to extensively investigate the systematic change of the downstream MAPK family signal molecules and the stress effect on hepatoma cell when PP2A is inhibited by cantharidin and its derivates and by setting okadaic acid as a control, and furthermore to build an intracellular response pathway when hepatoma cells were exposed to cantharidin and its derivates, then preliminarily elucidate the anti-hepatoma mechanisms based on PP2A molecule targets and provide experimental basis for anti-hepatoma scientific connotation of cantharis.

斑蝥是一类重要的抗肿瘤虫类中药，已利用其开发了多种中药产品，抗肿瘤效果好，特别是对原发性肝癌。我们的前期研究证实抗癌中药斑蝥体内存在大量的斑蝥素盐类衍生物，其中斑蝥素酸镁比斑蝥素有更强的抗肿瘤活性，但分子机制不明。文献报道蛋白磷酸酶2A(PP2A)抑制剂冈田酸、微囊藻毒素等可诱发肝癌，而同为PP2A抑制剂的斑蝥素盐类衍生物却具有显著的抗肝癌作用？进一步的实验表明斑蝥素类物质与冈田酸都作用于PP2A的下游MAPK家族信号分子，但引起的效果不同。本项目拟在这一差异现象的基础上，以PP2A抑制剂冈田酸为对照，探讨斑蝥素类物质抑制PP2A活性后引起的下游MAPK家族信号分子的变化及在肝癌细胞内的应激效应，并比较与冈田酸的异同，进一步构建以PP2A为中心的肝癌细胞暴露于斑蝥素类物质后的细胞内应答通路，基于磷酸酶PP2A分子靶点阐明斑蝥素类物质的抗肝癌机制，为揭示中药斑蝥的抗肝癌科学内涵提供实验依据。

斑蝥是一类重要的抗肿瘤虫类中药，已利用其开发了多种中药产品，抗肿瘤效果好，特别是针对原发性肝癌。我们的前期研究已证实抗癌中药斑蝥体内存在大量的斑蝥素盐类衍生物，其中斑蝥素酸镁（Magnesium Cantharidate，MC）比斑蝥素具有更强的抗肿瘤活性，但分子机制不明。文献报道蛋白磷酸酶2A(PP2A)抑制剂冈田酸（Okadaic acid，OA）可诱发肝癌，而同为PP2A抑制剂的斑蝥素盐类衍生物却具有显著的抗肝癌作用？进一步的实验表明斑蝥素类物质与冈田酸都作用于PP2A的下游MAPK家族信号分子，但引起的肿瘤细胞的生物学行为迥然不同。本项目在这一差异现象的基础上，以经典PP2A抑制剂OA为对照，观察MC对SMMC-7721、HepG2肝癌细胞丝裂原激活蛋白激酶(MAPK)信号通路的影响；构建PP2A过表达慢病毒，观察MC在肝癌细胞SMMC-7721中抑制PP2A活性后引起的下游细胞外信号调节激酶1/2（ERK1/2）信号通路的变化及在细胞内的应激效应；构建人肝癌细胞SMMC-7721裸鼠皮下移植瘤模型，观察MC对人肝癌细胞SMMC-7721体内抑制作用，同时在体外水平研究MC对MEK/ERK和PI3K/Akt/mTOR信号通路的影响。结果表明：1. MC虽然能抑制PP2A的活性，但同时也能抑制ERK1/2磷酸化水平，且体内实验MC+PP2A-OE组瘤重与MC组无明显差异，提示PP2A不是MC的主要作用靶点，其抑制肝癌细胞增殖与PP2A下游激酶ERK通路失活密切相关。2. MC可能通过抑制MEK/ERK通路，使肝癌细胞发生G2/M期阻滞，最终诱导细胞凋亡。与此同时，MC还可能抑制PI3K/Akt/mTOR信号转导通路，下调GLUT1和LDHA蛋白表达，减少葡萄糖的摄取和糖酵解代谢产物乳酸生成量，从而使Warburg效应减弱，最终抑制肝癌细胞增殖。提示MC的抗肝癌作用与多靶效应有关。实验为进一步阐明斑蝥素酸镁的抗肝癌机制打下基础，同时也为揭示中药斑蝥的抗肝癌科学内涵提供了实验依据。