神经酰胺调控Ca2+-ERS通路诱导涎腺腺样囊性癌细胞凋亡及其分子机制研究

The function of endoplasmic reticulum (ER) is the key to rapid proliferation of tumor cells and delivery of malignant substances. Strong external stimuli interrupt the function of endoplasmic reticulum, triggering the endoplasmic reticulum stress (ERS), and activating vast cascades of pro-apoptosis signaling pathways. It represents a new anti-tumor therapeutic strategy to target the ER and to trigger downstream pro-apoptosis signaling cascades. The Ca2+ signal in the ER synergize with ERS to promote apoptosis, but little is known about the mechanism of cross-talk between the two signaling pathways in the tumor cells. We had discovered in our previous study that ceramide could initiate ERS in the salivary adenoid cystic carcinoma cell lines, and that ceramide enhanced the Ca2+ release from the ER. Basing on our discovery, we planned in this project to adopt mouse model with genetic point mutation, and to establish tumor-bearing mouse model. Biological effects of ceramide on salivary gland tissue and tumor tissue were examined. Lentiviral transfection was performed to up-regulate or down-regulate cell signaling molecule in the cascades investigated, thus to explore the molecular mechanism of ceramide-midiated regulation of salivary adenoid cystic carcinoma cell apoptosis by crosstalk between Ca2+ signaling and ERS depended apoptotic pathways. Project results will disclose the molecular mechanism of ceramide-induced apoptotic effects on endoplasmic reticulum level, thus laying theoretical and experimental foundation for new anti-tumor therapies.

内质网功能在肿瘤细胞快速增殖和恶性物质分泌过程中起关键作用，持续强烈的外界刺激可干扰内质网功能，启动内质网应激(ERS)，激活下游广泛的促凋亡途径。靶向干预内质网并启动多重放大的凋亡信号，代表着肿瘤治疗的新策略。内质网Ca2+信号与ERS可协同诱导凋亡，然而其在肿瘤细胞中的级联串话机制不明。申请者前期研究发现神经酰胺可激活涎腺腺样囊性癌细胞的ERS反应，并促进内质网Ca2+释放。本课题拟在此基础上采用基因突变小鼠模型和荷瘤裸鼠模型，检测神经酰胺对正常涎腺组织和肿瘤组织的生物学效应；通过慢病毒转染对细胞信号通路各级分子采用增效或失效设计，研究通过内质网Ca2+信号介导，神经酰胺调控的ERS促凋亡途径与Ca2+释放凋亡途径级联串话，进而诱导细胞凋亡的分子机制。研究结果将在亚细胞器内质网水平上揭示神经酰胺诱导凋亡作用的分子机制，为开发新的肿瘤治疗方法提供理论和实验基础。

内质网不仅是细胞内主要的钙库，也是蛋白质翻译合成的场所。干扰内质网的正常功能，激活内质网应激(ERS)及其下游的凋亡通路，代表着肿瘤治疗的新策略。神经酰胺是近年来发现的具有诱导肿瘤细胞凋亡能力的脂质分子，然而其具体的作用机制仍不明确。在本项目中，我们发现外源性神经酰胺可激活涎腺腺样囊性癌细胞的内质网应激，神经酰胺对内质网应激三条信号通路PERK/eIF2α通路、ATF6通路和IRE1/XBP1通路的激活效应具有时间—剂量效应关系。通过转染神经酰胺合酶同样可诱导多个内质网应激标志物的表达，而靶向干扰内质网应激关键因子Grp78显著抑制神经酰胺的激活作用。此外，神经酰胺可干扰细胞内钙稳态，抑制钙离子通道蛋白SERCA2α、SERCA2β和SERCA3的表达。通过对内质网功能的双重影响，神经酰胺同时激活内质网应激下游凋亡通路和钙离子依赖的凋亡通路，诱导CHOP、JNK、MAPK等多个凋亡因子的表达。最后，我们通过细胞实验和荷瘤裸鼠模型证实了神经酰胺显著抑制涎腺腺样囊性癌的增殖，并诱导肿瘤发生凋亡。通过本项目，我们明确了神经酰胺通过激活内质网应激和干扰钙稳态，诱导涎腺腺样囊性癌细胞凋亡的分子机制，为其临床应用提供了理论基础。