【作者】 龚克；

【导师】 舒红兵； 李文化；

【作者基本信息】 武汉大学 ， 细胞生物学， 2013， 博士

【摘要】 癌症是目前人类面临的重大健康问题。其中,肝癌死亡率高、预后差,中国是肝癌患病的高发区。肝癌的治疗手段主要包括手术切除、放射疗法、化学药物治疗等,现今临床上用于肝癌治疗的药物十分有限,发现新型抗肝癌药物意义重大。传统中药是抗癌药物的潜在宝库,从紫草中提取的一种萘醌类物质——紫草素,被证明具有抗肿瘤活性,但其抗癌机制一直不明。本研究对紫草素在体内、体外的抗肝癌活性及其机制进行了初步研究。细胞程序性死亡是化疗药物有效杀伤肿瘤细胞的重要机制,主要包括细胞凋亡和细胞自噬。本研究结果显示：较低浓度紫草素(≤2.5μM)处理肝癌细胞和正常肝细胞12小时诱导细胞自噬产生。LC3转换检测、GFP-LC3荧光点聚集和吖啶橙染色自噬泡证明了紫草素促进细胞自噬的产生；细胞自噬早期抑制剂3-MA能有效阻断紫草素诱导的细胞自噬；LC3周转和p62降解则从动态上证明了自噬潮的产生。另一方面,Western blot检测细胞凋亡标志蛋白以及Annexin-V FITC/PI双染检测结果表明,较高浓度紫草素(6-8μM)处理24小时可有效诱导肝癌细胞凋亡,但不影响非恶化的肝细胞存活。CASPASE广‘谱抑制剂z-VAD预处理细胞可阻止紫草素诱导的肝癌细胞凋亡,紫草素诱导的细胞凋亡依赖于CASPASE蛋白的剪切。以上实验结果初步表明紫草素能在体外能诱导肝癌细胞发生细胞凋亡和细胞自噬。诱导活性氧自由基ROS是一些化疗药物诱导肿瘤细胞凋亡的重要分子机制。对紫草素诱导肝癌细胞凋亡和自噬的分子机制研究发现,紫草素处理可导致细胞内ROS积累,并参与细胞凋亡与细胞自噬的调控过程,ROS的清除剂能拯救其诱导的细胞凋亡和细胞自噬。Akt、RIP/NF-κB等信号分子是影响细胞死活命运的重要蛋白。对紫草素诱导肝癌细胞凋亡的分子机制研究发现,较高浓度(≥4μM)紫草素可抑制Akt、RIP/NF-κB活性,并参与调节紫草素诱导的细胞凋亡。外源过表达这些蛋白能部分拯救紫草素诱导的细胞凋亡,其抑制剂则能与紫草素协同诱导细胞凋亡。此外,我们研究还发现ROS的积累是这些信号通路的上游事件。Akt/mTOR、MAPK、ATG家族蛋白等是调节细胞自噬的重要通路。对紫草素诱导的肝癌细胞自噬分子机制研究发现,ERK、ATG7参与了紫草素诱导的细胞自噬过程,较低浓度(≤2.5μM)紫草素处理可激活ERK与ATG7,降低其活性可抑制细胞自噬。另外,RIP通路也部分参与了紫草素诱导的细胞自噬过程,过表达RIP可抑制细胞自噬的发生,RIP抑制剂Nec-1能与紫草素协同诱导细胞自噬。在紫草素诱导细胞自噬的过程中,ROS的积累也发生在RIP和ERK的上游。除体外细胞培养实验外,我们还通过建立Huh7肝癌细胞裸鼠异种移植瘤模型,研究紫草素在体内对肝癌细胞的影响。通过比较肿瘤生长曲线、动物存活率、肿瘤重量,我们发现紫草素在体内也有很好的抑癌效果。动物体重变化曲线和血清谷丙转氨酶活性分析表明紫草素对动物的毒性较低。细胞自噬相关蛋白表达分析和电子显微镜观察结果表明,较低浓度紫草素(每千克裸鼠体重灌胃2.5-5mg)可诱导体内肿瘤发生细胞自噬；细胞凋亡相关蛋白表达分析和TUNEL染色结果表明,在较高浓度紫草素(每千克裸鼠体重灌胃5-10mg)作用下,肿瘤细胞发生细胞凋亡。此外,体内信号通路变化,如细胞凋亡标志蛋白的剪切、LC3的转换、Akt、RIP在细胞凋亡过程中的下调,以及ERK、ATG7在细胞自噬过程中的上调等,也与体外细胞培养的实验结果保持一致。综上所述,我们研究发现,紫草素既可可通过ROS/Akt、ROS/RIP/NF-κB通路诱导CASPASE依赖的细胞凋亡,也可通过ROS/ERK、ROS/RIP以及ATG7通路诱导细胞自噬发生。因此,本研究表明紫草素可作为细胞凋亡与细胞自噬的诱导剂,在肝癌化学治疗中具有潜在的应用前景。更多还原

【Abstract】 Cancer is one of the biggest health issues around the world. Most cases of hepatocellular carcinoma (HCC) occur in China, with high mortality rate and bad prognosis. The treatments of hepatocellular carcinoma contain majorly surgical resection, radiotherapy, chemotherapy, and so on. As there are limited types of drugs on clinical treatment for HCC now, it is very important to develop novel anti-HCC drugs. The traditional Chinese medicine (TCM) is a great source for anti-tumor agents. Shikonin a naphthoquinone derived from the Chinese medicinal plant Lithospermum erythrorhizon, shows anti-cancer capability. However, the detailed mechanism is still unclear. In the presented research, we investigated the inhibition effect of shikonin on HCC in vivo and in vitro.Programmed cell death (PCD) is an essential mechanism for chemotherapeutics killing tumor cells effectively, which mainly contains apoptosis and autophagy. Our results indicated that a low dose of shikonin (2-2.5μM) treatment for a short time (12hours) induced HCC and normal liver cells autophagy, which was determined by LC3conversion assay, GFP-LC3puncta formation, and AV staining.3-MA could block early autophagosome formation induced by shikonin. Autophagic flux was confirmed by LC3turnover assay and degradation of p62. On the other hand, results from Western blot analysis of apoptosis-related proteins and Annexin-V FITC/PI staining plus FACS analysis referred that shikonin treatment at a high dose (6-8μM) and for a long time (24hours) might induce HCC cells but not normal cells apoptotic cell death. Shikonin-induced apoptosis could be blocked by z-VAD, a CASPASE pan-inhibitor, thus it was CASPASE-dependent. Our results showed that shikonin could trigger HCC cell lines apoptosis and autophagy in vitro.As ROS was reported to be involved in mechanisms of some anti-cancer drugs inducing apoptosis, we next investigated the mechanism and found ROS accumulation after shikonin treatment. The ROS scavengers NAC and Tiron completely blocked apoptosis and autophagy. Therefore, the ROS accumulation played an essential role in shikonin-induced apoptosis and autophagy.Akt and RIP/NF-κB are important in cell fate decision. In our studies on the mechanism of shikonin-induced apoptosis, downregulation of Akt and RIP/NF-κB activity was found to be involved in shikonin-induced apoptosis. Because ectopic expression of Akt or RIP partly abrogated the effects of shikonin, and downregulation by Akt inhibitor and RIP inhibitor synergistically induced apoptosis in conjunction with shikonin treatment. Moreover, ROS scavengers blocked shikonin-induced inactivation of Akt and RIP/NF-κB, but Akt or RIP/NF-κB did not regulate ROS generation, suggesting that Akt and RIP/NF-κB signals are downstream of ROS generation.On the other hand, we found that activated the ERK and ATG7are connected to shikonin-induced autophagy, since downregulation of these two pathways led to block of autophagy, and ERK inhibitor enhanced shikonin-induced autophagy. Meanwhile, RIP pathway was involved in autophagy as well, as RIP inhibitor plus shikonin cotreatment triggered enhanced autophagy, and RIP overexpression inhibited autophagy. ROS accumulation was found to be an upper stream event of RIP and ERK pathways as well.Moreover, experiments in vivo revealed similar results: shikonin treatment in Huh7tumor xenograft model of nude mice caused suppression of tumor growth with a low toxicity to animals. Shikonin induced tumor cells autophagy at a low dose (2.5-5mg/kg body weight) and apoptosis at a high dose (5-10mg/kg body weight in vivo, via the similar pathways as in vitro.Taken together, our findings suggest that shikonin is an inducer of autophagy and apoptosis, and may be a promising agent in the treatment of liver cancer. The molecular mechanisms of shikonin are induction of apoptosis through ROS/Akt and ROS/RIP/NF-κB pathways, and triggering autophagy via. ROS/ERK、ROS/RIP、 ATG7pathways.更多还原