基于 CRT迁移信号通路研究Rh2-O诱导癌细胞免疫原性死亡的分子机制

Cancer cells escape immune surveillance and recognition due to its weak immunogenicity and immune tolerance. It is considerable realistic significance to identify new functional ingredients which could effectively kill cancer cells and make the body produce antitumor immune response to cancer cells. It is well known that Rh2 has a significant anti-cancer action, however, it was confirmed to have a low bioavailability in vivo and difficult to be used for clinic. Previous, we have synthesized Rh2 octyl ester (Rh2-O) and reported that Rh2-O had a better bioavailability and a higher anti-cancer activity than Rh2. Meanwhile, we found by accident that Rh2-O might improve the immune function in H22 tumor-bearing mice, and induce the exposure of calreticulin (CRT) on the cell surface. It was reported that endoplasmic reticulum stress plays an important role in the exposure of CRT. Therefore, we speculate that Rh2-O may induce the exposure of CRT by Fas/CXCL8-mediated endoplasmic reticulum stress, and then lead to immunogenic cell death (ICD). In order to confirm our scientific hypothesis，the cultivation of human hepatocellular carcinoma cell line HepG2 cells in vitro and H22 tumor-bearing mice model were used to further study of Rh2-O- induced ICD and its role in the antitumor immune regulation by RNA interference, immunofluorescence, anti-cancer vaccine in mice experiment and flow cytometry technology. The results of this study could provide scientific data for the function exploration and application of ginsenoside.

癌细胞的弱免疫原性和机体对癌细胞的免疫耐受导致癌细胞逃脱免疫监视，因此挖掘既可杀死癌细胞又能使机体产生免疫应答的活性成分具有重要意义。人参皂苷Rh2抗癌活性显著，然而其在体内利用率低，难以发挥显著功效。我们前期对Rh2结构进行修饰，合成的Rh2辛酸酯(Rh2-O) 可显著提高Rh2生物利用率及诱导癌细胞凋亡率，且意外发现Rh2-O可调节小鼠免疫功能，诱导免疫原性死亡标志蛋白CRT迁移至细胞膜表面。因此，基于内质网应激在CRT迁移中的关键作用，我们推测Rh2-O可能通过Fas/CXCL8介导的内质网应激诱导CRT迁移从而导致细胞发生免疫原性死亡。为了证实这一科学假说，本项目以HepG2细胞和H22荷瘤小鼠为研究对象，应用RNAi技术、免疫荧光、小鼠抗癌疫苗实验及流式细胞术等，从细胞到动物，深入研究Rh2-O诱导细胞发生免疫原性死亡及其在抗癌中的作用，为人参皂苷的功能发掘与应用提供科学数据。

癌细胞的弱免疫原性和机体对癌细胞的免疫耐受导致癌细胞逃脱免疫监视，因此挖掘既可杀死癌细胞又能使机体产生免疫应答的活性成分具有重要意义。人参皂苷Rh2抗癌活性显著，然而其在体内利用率低，难以发挥显著功效。我们前期对Rh2结构进行修饰，合成的Rh2辛酸酯(Rh2-O) 可显著提高Rh2生物利用率及诱导癌细胞凋亡率，且意外发现Rh2-O可调节小鼠免疫功能，诱导免疫原性死亡标志蛋白CRT迁移至细胞膜表面。本项目通过体外细胞实验及体内荷瘤小鼠实验研究Rh2-O诱导Huh-7肝癌细胞免疫原性细胞死亡的作用及其可能的机制。结果发现：①通过体外细胞实验，证实Rh2-O处理显著增强肝癌细胞HMGB1的分泌、ATP的释放及CRT的膜外翻（P <0.05)；②Rh2-O通过诱导内质网应激引起CRT 膜外翻，并通过诱导细胞线粒体自噬引起ATP释放的增加；③通过体内疫苗试验，发现Rh2-O处理的Hepa1-6细胞能显著抑制肿瘤的生长，进一步证实Rh2-O可诱导肝癌细胞免疫原性细胞死亡；④通过构建Hepa1-6小鼠肝癌模型，发现Rh2-O能增强脾淋巴细胞的增殖能力和细胞毒性，并诱导肿瘤相关M2型巨噬细胞重编程，从而改善荷瘤小鼠的免疫功能。本项目的研究成果可为人参皂苷的功能发掘与应用提供科学数据。