内质网降解系统在rLZ-8触发胃癌细胞自噬依赖性死亡过程中机制研究

Endoplasmic Reticulum Associated Degradation (ERAD) and endoplasmic reticulum stress-induced autophagy are increasingly hot topics in the study of cell death.The previous studies have proved that ERAD mainly comprises the autophagic lysosomal pathway and the ubiquitin-proteasome pathway; the former is used primarily in clearing up insoluble or massive accumulation of protein aggregates, and the latter plays an important role for the degradation of misfolding or overexpression protein. The preliminary work shows that the recombinant Ganoderma Lucidum Immunomodulatory Protein (rLZ-8) entered into the Gastric Cancer Cell Line SGC-7901 via caveolae-mediated endocytosis, and then massively accumulated into the endoplasmic reticulum, thereby triggered autophagic response by causing endoplasmic reticulum stress. As a result, it induced an autophage-dependent cell death process in tumor cells without Caspase involving.The initial findings suggested that the aggregated rLZ-8 in the endoplasmic reticulum can be removed through autophagic-lysosomal degradation pathway in SGC-7901 cells, but the over-reaction of the degradation pathway can eventually lead to cell death. However, the specific mechanism of the procedure and form characteristics has still been an enigma.The project will attempt to use cell biology and molecular biology methods to investigate the role of ERAD in the autophagy-dependent cell death triggered by rLZ-8.

内质网降解系统与内质网应激引起自噬是国内外细胞死亡学科中研究的热点。已有文献表明内质网降解系统主要包括自噬溶酶体通路和泛素蛋白酶体通路，前者主要清除不溶性或大量积累的蛋白凝集物，后者主要负责降解错折叠或过表达的蛋白。我们的前期工作证明重组灵芝免疫调节蛋白（rLZ-8）可通过小窝介导的胞吞作用进入胃癌细胞SGC-7901中，并大量积累在内质网，进而引起内质网应激触发自噬，其结果会诱导肿瘤细胞发生非Caspase途径的自噬依赖性细胞死亡过程。初步研究结果提示：胃癌细胞SGC-7901可通过自噬溶酶体降解通路来清除滞留在内质网中的rLZ-8,而该降解通路的过度反应最终导致了细胞死亡，但具体作用机制和形态学特征尚未被阐明。本项目拟采用细胞生物学、分子生物学手段研究内质网降解通路在自噬依赖细胞死亡方式中的作用。

本项目分别评价了rLZ-8在体内和体外的抗肿瘤活性，发现rLZ-8不仅可在体外杀伤肝癌Hep G2细胞，而且在实验动物体内也对肝肿瘤有显著的抑制效果。我们进一步rLZ-8内化和抗肿瘤机制进行了深入研究，借助3D-SIM超高分辨率显微镜我们观察到了rLZ-8的内化过程，并建立起了一种可以实时动态观察细胞巨胞饮的成像方法。我们研究发现，EGFR是rLZ-8在肿瘤细胞中的主要靶标，rLZ-8与EGFR结合通过巨胞饮强烈内化引发了一种新的细胞死亡方式，这种肿瘤细胞死亡既与细胞凋亡、坏死和胀亡等死亡方式均有较大区别，也与巨泡化死亡方式不同。更重要的是，rLZ-8与EGFR结合后没有导致EGFR二聚化和磷酸化，所形成的晚期内吞体不发生自身融合，也不与溶酶体融合，而是停止在细胞质中，由于膜结构无法循环回膜重组，导致原细胞膜表面积越来越少，胞质内吞物大量堆积，最终胞膜崩解而死亡。最终我们揭示了rLZ-8在肝癌细胞中的内化和抗肿瘤机制，为rLZ-8抗肿瘤新药的研发提供了重要的生物学依据。