Cdc25c是调节细胞周期的关键因子，研究显示过氧化氢使得Cdc25c的半胱氨酸Cys377和Cys330形成分子内二硫键，但活性氧引起的Cdc25c的翻译后修饰与它的降解以及与细胞周期的关系并不明确。我们发现新型天然产物PP31J能够诱导肿瘤细胞G2/M周期阻滞从而发挥良好的体内外抗肿瘤活性，该过程中伴随着周期调节蛋白Cdc25c的下调且与活性氧密切相关。本项目以化合物PP31J为研究对象，检测 PP31J通过活性氧对细胞周期通路中周期调节蛋白Cdc25c的翻译后修饰的影响，对PP31J调节细胞周期发挥抗肿瘤活性的机制进行深入研究，有望丰富活性氧介导的蛋白质翻译后修饰调控理论，有助于发现全新的抗肿瘤药物靶点，推动新型抗肿瘤治疗药物的研发。

Cdc25c protein is a key factor in cell cycle regulation. Studies showed that H2O2 induced a disulfide bond in Cdc25c between the active-site cysteine at position 377 and an invariant cysteine at position 330, but the relationship between post-translational modification of Cdc25c which induced by reactive oxygen species, its degradation and cell cycle regulation is not clear. We found a new natural product PP31J could induce G2/M cell cycle arrest and showed antitumor activity in vitro and in vivo. The cell cycle arrest was accompanied by a decrease in Cdc25c protein levels which was closely related to reactive oxygen species. The project will investigate the post-translational modification Cdc25c driven by ROS to clarify the mechanism of antitumor effect of PP31J through cell cycle regulation. The project is expected to enrich the theory of post-transaltional modification of protein mediated by reactive oxygen species, help to identify new targets for anticancer drugs and promote research and development of novel anticancer drug.

实验目的：PP31J是从茄科 （Solanaceae）酸浆属（Physalis）植物毛酸浆（Physalis pubescens L.）中提取出来的新型迈克尔反应受体分子化合物。近年来的研究表明多种该类化合物具有抗肿瘤作用。本课题对PP31J体内外抗肿瘤作用进行系统的评价，并探索其抗肿瘤作用 靶点和分子机制。方法与结果：采用MTT法测定PP31J体外抑制肿瘤细胞增殖的能力，结果显示，PP31J对人乳腺癌细胞株MDA-MB-231，前列腺癌细胞株PC3，Du145，肝癌细胞株HepG2，卵巢癌细胞株SKOV3有不同程度的抑制作用，其中在人前列腺癌细胞株PC3，Du145中具有较好的抑制细胞增殖活性,IC50分别为1.85 μM和5.1 μM。我们选取人前列腺癌PC3裸小鼠皮下移植瘤模型评价该化合物体内抗肿瘤作用，结果显示50mg/kg PP31J,每3天给药1次，能显著抑制移植瘤的生长，第30天抑瘤率为63.8%。PI染色法检测细胞周期结果显示, PP31J剂量依赖和时间依赖的诱导肿瘤细胞的细胞周期阻滞于G2/M期。Western Blot 结果显示PP31J能够引起周期蛋白CyclinB1的上调，Cdc25C蛋白水平的下调。采用DCFDA荧光探针结合流式细胞术测定PP31J作用PC3细胞内活性氧的水平，结果显示PP31J引起细胞内活性氧的升高。抗氧化剂GSH，NAC逆转了PP31J引起的Cdc25蛋白水平的降低；此外采用流式细胞术也证实抗氧化剂GSH和NAC同样能逆转PP31J引起的细胞周期阻滞。结论：本研究证实PP31J具有显著的体内外抗肿瘤作用。该化合物是通过引起肿瘤细胞周期阻滞发挥抗肿瘤作用的，细胞周期阻滞和PP31J引起细胞活性氧升高密切相关，我们的研究为PP31J的应用提供实验基础。