蛋白磷酸酶2A在NO供体诱导肝癌细胞凋亡中的调节作用

Nitric oxide (NO) is a signaling molecule with a broad spectrum of actions in carcinogenesis, cell cycle regulation, and apoptosis processes. Protein phosphatase 2A (PP2A) is widely described as a tumor suppressor and takes part in the majority of the cellular pathways to induce apoptosis. The MAPK, PI3K-Akt and Wnt/β-catenin signaling pathway is a meeting point between the NO donor and PP2A.Our previous study indicated that NO released from NO donor could induce human hematoma HepG2 cells apoptosis through a MAPK-mediated mitochondrial pathway. Treatment with okadaic acid (an inhibitor of PP2A) prior to NO donor was found to partly reverse NO donor-induced apoptosis. To investigate the molecular mechanisms of NO donor-induced apoptosis of hepatoma cells from PP2A aspects, this study will establish the human hematoma cells and hepatocellular carcinoma in rats combined with RNAi technology and specific inhibitor/agonists that can regulate the expression of PP2A. Diazeniumdiolate -based derivatives JS-K as NO donor will be used to detect the changes of PP2A in the regulation of apoptosis which is closely related MAPK, PI3K-Akt and Wnt/β-catenin signaling pathway, etc, which will provide a new entry point for the anti-tumor effect of NO donor.

一氧化氮（Nitric Oxide, NO）在肿瘤的发生、发展中具有重要的调节作用；而蛋白磷酸酶2A（protein phosphatase 2A, PP2A）作为肿瘤抑制因子，通过调控多种信号通路可引起细胞凋亡。而MAPK、PI3K-Akt和Wnt/β-catenin等信号通路是两者的交汇点。前期研究发现，NO供体能提高HepG2肝癌细胞内NO水平，激活MAPK等信号通路而诱导细胞凋亡；PP2A抑制剂冈田酸可减弱NO供体对肝癌细胞的凋亡诱导作用。本项目旨从PP2A角度探讨NO供体诱导肝癌细胞凋亡的分子机制，采用偶氮鎓二醇盐衍生物JS-K作为NO供体，结合RNAi技术和特异性抑制剂/激动剂，建立可调控PP2A表达的肝癌细胞系和动物模型，围绕PP2A检测与其在凋亡调控中密切相关的MAPK、PI3K-Akt和Wnt/β-catenin等信号通路的变化，为研究NO供体的抗肿瘤作用提供新的切入点。

蛋白磷酸酶2A（PP2A）作为肿瘤抑制因子，参与细胞周期和凋亡等过程。NO供体在细胞内释放大量NO而诱导细胞凋亡。MAPK、PI3K-AKT和Wnt/β-catenin等信号通路是两者的交汇点。本项目采用偶氮鎓二醇盐衍生物JS-K作为NO供体，建立可调控PP2A表达的肝癌细胞系，围绕PP2A检测与其在凋亡调控中密切相关的信号通路的变化。结果显示：（1）JS-K对SMMC-7721和HepG2细胞可显著抑制其增殖、诱导细胞凋亡、增加PP2A表达。（2）JS-K可释放大量NO，引起染色质凝聚、核碎裂、线粒体膜电位下降，下调Bcl-2而上调Bax，活化cleaved-caspase-9/3和cleaved-PARP诱导细胞凋亡；增加PP2A-C的表达，使β-catenin、c-Myc和p-Bcl-2（Ser70）的表达减少。（3）JS-K处理后引起PP2A活性增强，可下调抗凋亡蛋白如p-Bcl-2（Ser70），Bcl-2，Bcl-xL和Mcl-1表达，而上调Bim，Bad，Bax，Bak等促凋亡蛋白表达，从而激活线粒体途径诱导细胞凋亡。（4）JS-K处理后引起PP2A活性增强，对MEK-ERK信号轴呈现抑制作用，使p-MEK1/2和p-ERK1/2表达减少，p90RSK，Elk-1，c-Myc和c-Fos磷酸化水平减少。（5）JS-K处理后引起PP2A的活性增强，减少PI3K和p-AKT蛋白表达而上调p53蛋白表达；显著下调Cyclin B1蛋白；增加p-Cdk1水平；使p-Chk2水平升高，p-Cdc25C水平增加，p21和p27表达增加，最终使细胞阻滞于G2/M期。（6）JS-K处理后引起PP2A活性增强，使p-GSK-3β的水平减少，明显下调胞浆中β-catenin蛋白水平；使降解复合物中的Axin1表达增加，引起β-catenin入核减少；核内TCF、LEF1的蛋白水平下调；下游靶蛋白c-Myc、Cyclin D1、Survivin水平下调。（7）JS-K可明显抑制原发性肝癌大鼠肿瘤的生长，抑制胶原的生成，诱导PP2A-C活化后，使β-catenin，c-Myc和p-Bcl-2（Ser70）蛋白表达下降；抑制MEK，ERK和p90RSK的磷酸化；下调p-AKT和Cyclin B1蛋白表达，上调p53蛋白表达。结果阐释了PP2A在NO供体诱导肝癌细胞凋亡中的作用。