JNK/P38MAPK在脱氧鬼臼毒素诱导胶质瘤细胞Parthanatos（PARP-1依赖性死亡）中的作用及机制研究

Gliomas are the common malignant intracranial primary tumors with higher mortality, higher recurrence and no effective treatment. Parthanatos is a type of programmed cell death characterized with PARP-1 hyper-activation and cytoplasm accumulation of PAR. Different with improving the sensitivities of glioma cells to current chemotherapy and radiotherapy by inhibition of PARP-1 activation, induction of parthanatos might become a new strategy to eliminate glioma cells in further. We found that deoxypodophyllotoxin (DPT) could induce glioma cell parthanatos in vitro and in vivo, in which JNK/P38MAPK might play a crucial role. Thus, we hypothesize that JNK/P38 MAPK might participate in the regulation glioma cell parthanatos induced by DPT. In this research project, we will use various glioma cell lines and naked mice model of xenograft glioma, and molecular biology technologies such as western blotting analysis and immunoprecipitation to investigate the following issues: (1) The effect of JNK/P38 MAPK on the glioma cell parthanatos triggered by DPT; (2) The mechanism underlying the activation of JNK/P38 MAPK caused by DPT; (3) The mechanism via which JNK/P38MAPK regulate PARP-1 hyper-activation; (4) The effect of JNK/P38 MAPK on PARG and its underlying mechanism. We hope to clarify the role of JNK/P38 MAPK in glioma cell parthanatos and its potential mechanism, and present theoretical basis for developing corresponding treatment targeting parthanatos induction.

脑胶质瘤是颅内最常见的具有高复发率和高死亡率特点的恶性肿瘤，目前缺乏有效的治疗方法。Parthanatos是以PARP-1过度激活和细胞中PAR积聚为特征的程序性死亡方式。我们发现，脱氧鬼臼毒素（DPT）在体外和体内均能诱导胶质瘤细胞Parthanatos，抑制JNK或P38均可以减少DPT诱导的胶质瘤细胞死亡，进而提出科学假说“JNK/P38MAPK调节了胶质瘤细胞的Parthanatos”。在本项目中，我们采用体外和体内研究，应用免疫沉降、蛋白印迹等方法探讨：（1）DPT激活JNK/P38的机制；（2）JNK/P38在DPT诱导胶质瘤细胞Parthanatos中的作用；（3）JNK/P38调节PARP-1过度激活的机制；（4）JNK/P38对PARG（PAR降解酶）的影响及机制。我们期望通过本研究阐明JNK/P38MAPK对胶质瘤细胞Parthanatos的调节作用和机制。

Parthanatos是一种以PARP1过渡激活、细胞内PAR多度聚集为特征的新型程序性细胞死亡方式，但是其调节机制尚不清楚。在本项目中，我们在发现天然小分子化合物脱氧鬼臼毒素（Deoxypodophyllotoxin，DPT）能够诱导脑胶质瘤细胞Parthanatos死亡的基础上，通过体外细胞培养以及建立裸鼠皮下荷胶质瘤模型， 采用细胞生物学以及分子生物学研究方法探讨并阐明了JNK/P38MAPK信号通路对脑胶质瘤细胞Parthanatos死亡的调节作用及相关机制。我们发现：（1）JNK/P38MAPK信号通路促进了胶质瘤细胞Parthanatos死亡；JNK/P38MAPK信号通路的激活，提高了细胞中的活性氧，增强了DPT所诱导的活性氧依赖性DNA双链断裂，促进了PARP1的过度激活以及胞浆和胞核中细胞毒性物质PAR的积聚和胶质瘤细胞死亡。（2）JNK/P38MAPK通过激活NADPH氧化酶1（NOX1）、抑制catalase提高了细胞中活性氧水平，同时还通过抑制xCT抑制了细胞内抗氧化剂GSH的含量，进而促进了胶质瘤细胞PARP1的过度激活、PAR的积聚以及胶质瘤细胞死亡；激活的NOX1则进一步激活了JNK/P38MAPK信号通路，形成了正反馈调节。（3）JNK/P38MAPK通过激活Sirt1，提高了细胞内活性氧的含量，增强了DNA的双链断裂，促进了胶质瘤细胞PARP1的过度激活、PAR的积聚以及胶质瘤细胞死亡; 激活的Sirt1则进一步促进了JNK/P38MAPK的激活，形成了正反馈调节；（4）发现了DPT激活通过上调ASK1在Thr845位点的磷酸化，进而促进了JNK和P38的激活；而且NOX1和Sirt1均通过上调ASK1在Thr845位点的磷酸化，激活了JNK和P38信号通路。通过本项目，我们明确了JNK/P38MAPK在脑胶质瘤细胞Parthanatos中的作用和机制，这不仅有助于我们深入地理解脑胶质瘤细胞中Parthanatos激活机制及途径，还有利于以JNK/P38MAPK为靶点探讨脑胶质瘤治疗的新方案和新药物。同时，我们还阐明了小分子化合物脱氧鬼臼毒素诱导脑胶质瘤细胞Parthanatos死亡的机制，有利于以脱氧鬼臼毒素为基础设计新型的小分子药物提供了理论依据。