人端粒酶逆转录酶线粒体转位在蟾蜍灵诱导的人肿瘤细胞氧化应激中的作用与机制

Bufalin，a major immunoreactive component of the Chinese traditional medicine Chan'su, has been shown to induce apoptotic cell death in many types of cancer cell lines.However, the detailed molecular mechanisms of its induction of apoptosis are still unclear. Our previous study has demonstrated that bufalin induced apoptosis in cancer cells by reactive oxygen species(ROS)-dependent mitochondrial pathway.Mitochondria is the critical intracellular source of (ROS),and apoptotic insults including various chemotherapeutic agents induce apoptosis in cancer cells by oxidative stress-mediated mitochondrial pathway.The telomerase holoenzyme is a multi component ribonucleoprotein that plays a crucial role in telomere maintenance via the addition of telomeric repeats at the 3'ends of the DNA, thus helping overcome the end replication problem and protecting genome DNA integrity.The human telomerase reverse transcriptase (hTERT),the catalytic subunit of the telomerase holoenzyme has been proposed as the rate-limiting factor for telomerase activity.Evidence is accumulating to support non-canonical activity of hTERT in cancer cells.In addition to the non-telomere-related functions for nuclear hTERT,an increasing body of evidence shows that hTERT accumulates in mitochondria under oxidative stress.The N terminal of hTERT has a mitochondrial targeting sequence(MTS) that is sufficient and required for mitochondrial localization.But the exact reason for hTERT accumulation in mitochondria under oxidative stress is still unknown.Despite some descriptive work about mitochondrial hTERT, fundamental questions about its biology and function in the organelle remain unanswered.Our preceding results showed that hTERT translocated from nucleus to mitochondria in oxidative stress damage induced by bufalin in cancer cells. The present study aimed to investigate the effect of hTERT mitochondrial translocation in bufalin-induced oxidative stress-dependent apoptosis.We intended to inhibit hTERT mitochondrial targeting by constructing truncated MTS deleting vector to transfect human cancer cells,and interrupte classical protein nuclear export and mitochondrial transmembrane import pathways by leptomycin B and RNA interference methods repectively to determine the functions and mechanisms of the hTERT nuclear-mitochondrial shuttling under oxidative stress.Our study would provide theoretical basis on improving anticancer mechanisms of chinese traditional medicine.

蟾蜍灵是传统中药蟾酥的单体成分之一，在体外可抑制多种肿瘤细胞增殖，但确切的机制尚不清楚。我们的研究表明，蟾蜍灵以活性氧依赖的线粒体途径诱导肿瘤细胞凋亡。线粒体是氧化应激的中心，多种药物以氧化应激性损伤的方式诱导肿瘤细胞凋亡。人端粒酶逆转录酶(hTERT)除合成端粒外,还定位于线粒体参与氧化应激，但其核-线粒体转位的机制与在氧化应激性损伤中的作用尚不明确。本课题组前期工作证实，在蟾蜍灵诱导的肿瘤细胞氧化应激性损伤中hTERT由细胞核向线粒体转位。本研究拟通过构建线粒体定位序列缺失的截短型hTERT载体转染肿瘤细胞，阻断hTERT线粒体定位，进一步明确hTERT线粒体转位在蟾蜍灵诱导的肿瘤细胞氧化应激中的作用；并利用leptomycin B和RNAi技术阻断经典的蛋白质核输出及线粒体输入通路，探讨hTERT核浆穿梭的机制。本研究将为完善传统中药以线粒体hTERT为靶点的抗肿瘤机制奠定理论基础。

蟾蜍灵（Bufalin）是传统中药蟾酥的主要活性成分之一,具有很强的抑瘤作用，诱导肿瘤细胞发生线粒体依赖的凋亡，但具体的分子机制尚不清楚。本课题组发现蟾蜍灵诱导人胰腺癌 CAPAN-2 及舌癌 CAL-27细胞凋亡，促进线粒体氧化应激并下调人端粒酶逆转录酶（hTERT）表达；利用 siRNA干扰 hTERT基因表达，促进肿瘤细胞线粒体相关途径凋亡。我们还发现，蟾蜍灵在诱导人胰腺癌 CAPAN-2 及舌癌 CAL-27细胞凋亡过程中增强JNK 磷酸化及 p38-MAPK，利用JNK抑制剂 SP600125 或 p38-MAPK 抑制剂 SB203580阻断 JNK/p38-MAPK 通路能够逆转蟾蜍灵诱导的hTERT下调，因此JNK/p38通路可能参与蟾蜍灵诱导的 hTERT下调以及相关的线粒体凋亡途径。另外，针对目前阻碍蟾蜍灵临床应用的缺乏肿瘤细胞选择性的缺点，本课题组在高通透高滞留效应（Enhanced Permeability and Retention;EPR)以及肿瘤细胞高表达生物素受体，生物素化纳米粒快速胞内摄取的理论基础上，设计并合成了肿瘤靶向药物输送系统——装载蟾蜍灵的生物素化壳聚糖纳米粒( Bu-BCS-NPs )。体内外实验表明，与天然的蟾蜍灵相比 Bu-BCS-NPs 对乳腺癌表现出更显著的细胞毒性，诱导 MCF-7 细胞内活性氧产生并降低线粒体膜电位。我们的研究结果提示，Bu-BCS-NPs有望成为有效的具有肿瘤靶向作用的蟾蜍灵制剂。另外，我们还对线粒体相关蛋白 Tumor necrosis factor Receptor-Associated Protein 1 (TRAP1)在肿瘤发生发展中的作用进行了研究。结果显示，TRAP1在食管癌（尤其是低分化）中高表达，降低食管癌 ECA109及 EC9706 细胞 TRAP1基因表达，促进活性氧产生、线粒体去极化，诱导细胞周期阻滞；重述表达 TRAP1 能够逆转细胞凋亡及增殖抑制。皮下移植瘤生物发光结果显示，TRAP1敲减能够明显抑制体内食管癌细胞的生长。我们的研究结果显示，线粒体相关蛋白 TRAP1 在绝大多数食管癌中高表达，增强肿瘤细胞的抗凋亡能力，有望成为以线粒体为靶向的肿瘤治疗的新靶点。本研究为以线粒体氧化应激为靶向的肿瘤治疗策略提供了理论依据及新思路。