新型吲哚酮类衍生物诱导肿瘤细胞凋亡的机制研究

Abnormal of the tumor suppressor p53 is well assosiated with chemotherapy resistance of tumor cells. In our previous works, we designed and synthesized a series of isatin derivatives .The HKL-3e exhibited the highest cytotoxic activityaganist K562 cell lines (IC50=4 nM) . The observed cytotoxic effect in K562 cells was associated with a decrease of cell viability, leading to apoptotic cell death characterized DNA fragmentation. Pretreatment of K562 cells with pan caspase inhibitor, z-VAD did not inhibit HKL-3e induced apoptosis. Furthermore, HKL-3e induced the translocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. Taken together, Our findings demonstrated that HKL-3e may trigger caspase-independent and AIF-mediated apoptotic cell death in K562 cells. Our study also revealed that human myeloblastic leukemia HL-60 and colon carcinoma HT-29 cells underwent morphological changes characteristic of apoptosis after HKL-3e treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. These data suggest that a wider understanding of HKL-3e-induced apoptosis is could theoretically pave the way for the development of new drugs that modulate apoptosis. The aim of this study is to understand the antitumor mechanism of HKL-3e in drug resistance cancer cell lines and set up the foundation for a rational therapeutic approach in the treatment of resistant abnormal p53 cancers. In order to clarify the exact antitumor mechanism of HKL-3e, this project will focus on following three aspects: 1) Whether the tumor cell apoptosis induced by the HKL-3e is an important mechanism of its antitumor effect? 2) Which molecular in p53 downstream pathway is a key protein in HKL-3e-induced tumor cell apoptosis? 3) What are the important regulatory factors for AIF translocation in HKL-3e-induced apoptosis process

p53基因缺失/变异肿瘤细胞的耐药性机制及其对策是目前肿瘤生物学的研究热点和难点。我们发现新型吲哚酮类衍生物HKL-3e能高效高选择性杀伤p53异常的K562细胞（IC50=4 nM），并诱导该细胞发生Caspase不依赖性凋亡、促进凋亡诱导因子AIF转位。HKL-3e诱导的凋亡不依赖于p53信号通路，也没有Caspase的切割活化，表明其抗肿瘤机制与常规化药不同，很可能与其高效高选择性杀伤p53异常肿瘤细胞的特性有关，尚需深入研究。本项目拟利用HKL-3e选择性杀伤53异常耐药性肿瘤细胞模型，研究以下问题：（1）阐明HKL-3e引发肿瘤细胞凋亡的机理，明确其诱导的凋亡是否是其抗肿瘤的重要机制。（2）探究HKL-3e调控p53下游通路使耐药肿瘤细胞发生凋亡的具体机制。（3）明确HKL-3e是否是通过影响Bcl-2家族或calpains/Cathepsins家族促进AIF转位引发凋亡的。

p53基因缺失/变异肿瘤细胞的耐药性机制及其对策是目前肿瘤生物学的研究热点和难点。.本课题组在前期工作中合成了一系列新型吲哚酮类衍生物，并优化得到了若干个抗肿瘤活性较好的先导化合物。我们发现新型吲哚酮类衍生物HKL-3e能高效高选择性杀伤p53异常的K562细胞，并诱导该细胞发生Caspase不依赖性凋亡、促进凋亡诱导因子AIF转位。HKL-3e诱导的凋亡不依赖于p53信号通路，也没有Caspase的切割活化，表明其抗肿瘤机制与常规化药不同，很可能与其高效高选择性杀伤p53异常肿瘤细胞的特性有关，尚需深入研究。本课题的研究重点是阐明新型吲哚酮类的抗肿瘤活性特点及作用机制，为开发新型高效低毒、具有自主知识产权的的抗肿瘤药物奠定基础。.首先，研究了化合物HKL-3e对肿瘤细胞增殖的影响。发现化合物HKL-3e对K562细胞的增殖均有明显的抑制作用，对K562细胞半数抑制浓度0.004 μM。化合物HKL-3e在实验浓度范围内对18种人源的肿瘤细胞增殖有明显抑制作用，其中对白血病细胞K562，Jurkat，HL-60以及胃癌细胞MGC-803的细胞增殖有十分显著的抑制作用，IC50均达到nM级别。化合物HKL-3e对正常细胞HUV-EC，MRC-5，L-02，HEK-293与其对K562细胞增殖的抑制作用相比，其IC50大至少一个数量级。初步认定在抑制肿瘤生长的有效给药浓度下，对正常细胞增殖的影响较小。.接下来，研究了化合物HKL-3e对K562细胞凋亡和细胞周期的影响。发现30 nM HKL-3e处理K562细胞一定时间后能诱导K562细胞发生DNA损伤。化合物HKL-3e诱导K562细胞经历的凋亡途径为caspase途径和程序性坏死样凋亡途径共同作用。证明线粒体途径参与了HKL-3e诱导的细胞凋亡。发现30 nM HKL-3e处理K562细胞后G2/M期细胞比DMSO对照组出现明显增多，并且在化合物处理12 h时出现峰值，所占比例为43.22%。 .最后，HKL-3e（10nM）处理K562细胞， 48 h后收收集细胞，western blot 法检测凋亡诱导因子(apoptosis-inducing factor, AIF)，发现细胞核内AIF水平显著上调，并且线粒体内AIF减少，表明HKL-3e 化合物在K562细胞中AIF被激活，并参与诱导凋亡过程。