三氧化二砷诱导雄激素非依赖前列腺癌凋亡MAPK通路及VK3协同应用的研究

Although arsenic trioxide (As2O3) is a toxic chemical, it also is a broad-spectrum antineoplastic drug. Until now there are not effective drugs to treat androgen independent prostate cancer (hormone-refractory prostate cancer, HRPCa), there is an urgent need to identify effective agents against this disease .In 2004, we firstly reported As2O3 can treat HRPCa through cell aptosis in China. The mechanism remains unclear yet, and even side effects of the higher doses As2O3 restrict its clinical application moreover. Therefore, we have been devoted to exploring the mechanism of As2O3 in treating HRPCa and trying to find an assistant agent to decrease its doses and adverse side effects while increasing the efficacy of treatment in recent years. We recently confirmed that As2O3 treated HRPCa through JNK (c-Jun N-terminal kinase) protein phosphorylation,which is a kind of protein involved in the MAPK（mitogen-activated protein kinase ）signal transduction pathway .We also observed the effects of combination As2O3 with a novel assist agent (vitamin K3),when administered simultaneously, the antineoplastic activity on HRPCa(PC-3 cell) could be enhanced obviously ,showed a synergistic action in inhibiting growth of HRPCa. This project is plan to detect the main proteins involved in MAPK signal transduction pathway,including ERK(extracellular signal-regulated kinase),P38(P38 mitogen-activated protein kinase) and JNK protein ,downstream protein (such as Bcl-2,Survivin,Livin,Bax,etc. )and oxidative stress changes under the action of As2O3 alone and combination with VK3 by cell culture, flow cytometry,molecular biology and used LC proteomics technique. So that we can discover the mechanism of As2O3 alone and VK3 combination with As2O3 in treating HRPCa.We also hope the application of the synergistic actions of combination with VK3 maybe break the higher doses bottleneck of As2O3 alone, so that this old small molecule chemical can be wide used to treat HRPCa and other solid malignant tumors in the future.

As2O3虽是一种有毒物质，但其用作抗癌药物的前景极其广阔。申请人于2004年在国内率先报导其可以治疗至今仍无有效药物可以控制的雄激素非依赖性前列腺癌（HRPCa），但是机制尚不清楚，而且高剂量As2O3的毒副作用更是限制其临床应用的瓶颈，申请人近年来一直致力于探索As2O3治疗HRPCa的机制，并尝试寻找一种助剂与As2O3协同作用以降低其毒副作用。近期研究结果发现，As2O3治疗HRPCa与MAPK通路JNK蛋白磷酸化有关，并且在VK3存在下其治疗效果明显增强。本项目将研究As2O3作用下的MAPK通路蛋白磷酸化和下游相关蛋白变化，以及Vk3和As2O3联合作用下上述蛋白及氧化应激酶的变化，旨在阐明As2O3治疗HRPCa机制并探索Vk3与其协同作用的机理，打破As2O3剂量瓶颈，为这个古老的小分子化合物治疗HRPCa以及众多实体瘤临床推广应用奠定基础。

申请人于2004年在国内率先报导As2O3可以诱导前列腺癌(PCa)凋亡,但作用机制尚不清楚。本项目通过研究As2O3对PC-3、DU145细胞株的影响、As2O3 作用下 HRPCa 的 MAPK 信号通路变化、利用裸鼠 HRPCa 模型来验证 As2O3的体内疗效和机制和通过免疫组化检测前列腺癌和前列腺增生组织蜡块中JNK、p-JNK、p-ERK及ERK蛋白表达情况，最后发现JNK和ERK的磷酸化对诱导前列腺癌细胞凋亡及预测前列腺癌患者预后有重要作用，可能成为临床中治疗前列腺癌的新靶点。本课题组临床研究发现Wnt通路拮抗剂SFPR-1为判定PCa预后的独立因素,通过BSP方法检测发现PCa细胞系中SFPR1启动子高甲基化,As2O3能逆转其甲基化,并以剂量依赖的方式上调SFRP1和下调Wnt通路关键蛋白β-catenin的表达,影响其下游靶基因表达,诱导PCa凋亡。故提出“As2O3抑制PCa与逆转SFRP1启动子的高甲基化,恢复沉默的SFRP-1的表达,关闭Wnt信号通路有关”的假说。本项目将通过蛋白质免疫印迹、Rt-PCR,RNAi等方法验证SFRP1为As2O3诱导 PCa凋亡的靶标蛋白;阐明 As2O3逆转SFRP-1高甲基化的机制;并通过动物模型探索通过调控S FRP-1启动子甲基化,治疗PCa的可行性,为PCa治疗提供新的思路和靶点;为As2O3临床推广及应用奠定基础。