胃癌细胞的分化程度对凋亡素选择性抗肿瘤效应的影响及机制研究

Gastric cancer is one of the most common global cancers, and the main technologies used for treatment is chemotherapy. Due to the low survival rate at present, it's necessary to develop highly efficiency anti-cancer agents with lower toxicity. Apoptin can induce the tumor cell apoptosis selectively, but has no influence on normal cells. Thus, it has been regarded as an anti-cancer agent with good prospect. However, the mechanism of apoptin is not well explained. We found that, apoptin had different apoptosis-inducing effect on different types of cancer, and to different differentiated gastric cancer cells, its apoptosis also had different apoptosis-inducing effects. In respect that autophagy is closely related with cell differentiation and apoptosis, we did further research and found that, apoptin shown different autophagy regulation effects on different differentiated gastric cancers, which suggested that the apoptosis- inducing effect of apoptin might be related with the autophagy regulation. In this project, we plan to carry out experiments in vitro and in vivo, confirm variations in autophagy level of different differentiated gastric cancers, investigate the interaction and mechanism of the apoptosis-inducing and autophagy regulation effects by apoptin in different differentiated gastric cancers, and improve the apoptosis-inducing effect of apoptin by intervening autophagy. This study not only further clarify the mechanism of apoptin, identify its efficient anti-tumor target, and contribute to the development of new anti-tumor agents, but also is able to reveal the relationship of autophagy and tumorigenesis of different differentiated gastric cancer, and improve the anti-tumor treatment strategy by regulating autophagy. This project has important theoretical significance and clinical value.

胃癌是全球高发肿瘤，化疗是重要治疗手段，研发高效低毒的抗肿瘤药物很有必要。凋亡素可选择性地诱导肿瘤细胞凋亡，被认为是很有前景的抗肿瘤制剂，但其作用机制尚未完全阐明。我们研究发现：对于不同肿瘤，凋亡素的凋亡诱导效率不同；分化程度不同的胃癌细胞，其凋亡诱导效率也不同。由于自噬与分化、凋亡均有密切联系，我们进一步研究发现，凋亡素对不同分化程度胃癌细胞自噬的影响是不同的，提示其凋亡诱导效应可能与调节自噬有关。本课题拟通过体内外实验，明确不同分化阶段胃癌细胞的自噬水平，研究凋亡素和不同分化程度胃癌细胞自噬的相互作用机制及与其凋亡诱导效应的关系，并通过干预自噬以提高凋亡素的诱导凋亡效应。本项目不仅可进一步明确凋亡素的作用机制，确定高敏感的肿瘤靶标，有助于抗肿瘤制剂的开发；还能揭示不同分化程度胃癌的自噬与肿瘤发生的关系，完善调节自噬的抗肿瘤治疗策略，具有重要的理论和临床应用意义。

凋亡素能选择性诱导肿瘤细胞凋亡，而对正常细胞无作用，使得它成为潜在抗癌治疗的候选者。在实验室前期实验中，通过透膜肽PTD4与Apoptin 融合，获得PTD4-Apoptin融合蛋白，该蛋白可穿透细胞膜，进而发挥凋亡素的凋亡诱导效应。本课题拟以不同分化程度的人源胃癌细胞系MKN45（低分化）、SGC7901（中低分化）、BGC823(中分化)、MKN28（高分化）、化学诱导的高分化WD-MKN45、低分化LD-SGC7901、高分化WD-SGC7901为研究对象，并构建裸鼠皮下及原位胃癌模型，观察各细胞3组织及自噬水平的差异，探究凋亡素对胃癌的生长抑制效应及自噬水平的影响，并结合自噬抑制剂氯喹（Chloroquine，CQ）和自噬诱导剂雷帕霉素（Rapamycin）探索干预细胞自噬对凋亡素的诱导凋亡效应的影响。.结果表明低分化的细胞（MKN45）及肿瘤组织的LC3-II表达水平较低，而高分化的细胞（MKN28）及肿瘤组织自噬水平较高。PTD4-Apoptin可显著抑制不同分化程度胃癌细胞的增殖活性，并诱导胃癌细胞发生凋亡和自噬，并且对低分化的细胞（MKN45、LD-SGC7901）抑制作用最强。当PTD4-apoptin蛋白与自噬抑制剂氯喹、自噬诱导剂雷帕霉素联合作用后，发现抑制自噬可增强凋亡素对胃癌细胞的凋亡诱导效应，促进自噬的细胞可抑制凋亡素的凋亡诱导效应。同时细胞孵育PTD4-apoptin蛋白后，mTOR信号通路关键蛋白mTOR和p-mTOR的表达水平显著下降，表明凋亡素可能通过抑制mTOR信号通路从而促进细胞自噬和凋亡。建立不同分化程度胃癌细胞的裸鼠皮下及原位胃癌模型，凋亡素可以明显的抑制胃癌细胞SGC7901移植瘤和低分化的胃癌细胞LD-SGC7901移植瘤的生长，自噬抑制剂氯喹可以增强凋亡素的抑瘤效应。.因此，这些发现表明凋亡素能够作为一个潜在的胃癌抗癌制剂，且完善了调节自噬的抗肿瘤治疗策略，将有助于更好的利用凋亡素的抗肿瘤特性，从而为开发新的抗肿瘤制剂以及联合用药提供理论依据。