Ca2+在顺铂诱导人卵巢癌SKOV3细胞自噬反应中的作用

摘要

Background and purpose:Ca2+plays a very important role in the maintenance of cell biological functions. The storage, release and uptake capacity of Ca2+ is controlled by endoplasmic reticulum (ER). Ca2+ homeo-stasis is essential for cellular energy metabolism and proper protein folding. This study aimed to investigate the effect of cytoplasmic Ca2+ on cisplatin induced ER stress-mediated autophagy in ovarian carcinoma SKOV3 and its underlying mechanism.Methods:The ovarian cancer SKOV3 was used as a study object. The experiment consisted of three parts:① To explore the possible relationship between cisplatin-induced ER stress and autophagy, SKOV3 cells were treated with cisplatin for 0, 6, 12 and 24 h, respectively;② To explore the possible relationship between ER stress induced Ca2+ effux and autophagy, SKOV3 cells were treated with cisplatin for 0, 9 and 12 h, respectively, and TG was used as a positive control;③ To explore the effects of blocking calcium effux on autophagy, SKOV3 cells were divided into control group, cisplatin group, TG group, BAPTA-AM group, cisplatin combined with BAPTA-AM group and TG com-bined with BAPTA-AM group. Western blot was used to detect the protein levels of GRP78 and LC3. Fluo-4 calcium lfuorescent probe was used to examine cytoplasmic Ca2+ levels. Confocal microscopy was used to detect LC3 level by immunolfurescence staining.Results:Compared to control group (0.679±0.011), GRP78 was signiifcantly accumulated at 6, 12 and 24 h after cisplatin treatment and reached the maximum value at 6 h (1.393±0.004,P=0.000). Similarly, compared to control group (0.038±0.000), LC3 puncta were clearly seen after cisplatin treatment and reached the maxi-mum value at 12 h (0.072±0.002,P=0.000). Using confocal microscopy, we found that cisplatin and TG increased LC3 punctate accumulation and cytoplasmic Ca2+ levels in a time-dependent manner. Immunolfuorescent method showed that treatment with cisplatin combined with BAPTA-AM or TG combined with BAPTA-AM increased LC3 punctate accumulation induced by cisplatin or TG. The results of Western blot showed that cisplatin combined with BAPTA-AM (0.071±0.001) or TG combined with BAPTA-AM (0.065±0.001) signiifcantly increased LC3Ⅱ/LC3Ⅰ ratio induced by cisplatin (0.039±0.000,P=0.000) or TG (0.035±0.001,P=0.000).Conclusion:Cisplatin induces intracellular ER stress and autophagy in SKOV3 cells, accompanied by increased cytoplasmic Ca2+ levels. Chelating cytoplasmic Ca2+enhanc-es cisplatin-induced autophagy.%背景与目的：Ca2+在维持细胞生物活性方面扮演着很重要的角色，其在细胞内的储存、释放和摄取主要受内质网调节，细胞内Ca2+浓度的稳态是维持细胞生物能量代谢、蛋白质折叠和分泌的基础条件。本研究探讨Ca2+在顺铂诱导SKOV3细胞内质网应激-自噬反应中的作用机制。方法：取人卵巢癌SKOV3细胞系为研究对象，按以下步骤分组：①探讨顺铂诱导内质网应激与自噬反应，用6μg/mL的顺铂处理SKOV3细胞0、6、12和24 h；②了解顺铂和毒胡萝卜内酯(thapsigargin, TG)诱导内质网应激释放的Ca2+与自噬的关系，分别用TG和顺铂处理SKOV3细胞0、9和12 h；③探究Ca2+对自噬的作用机制，分成对照组、顺铂组、TG组、BAPTA-AM组、顺铂联合BAPTA-AM组和TG联合BAPTA-AM组。用蛋白[质]印迹法(Western blot)检测内质网应激相关蛋白GRP78和自噬标志性蛋白LC3蛋白的表达水平；用Fluo-4钙离子荧光探针检测细胞质中的Ca2+浓度变化；间接免疫荧光染色后，用共聚焦显微镜检测LC3蛋白的表达情况。结果：SKOV3细胞经6μg/mL顺铂作用6 h时GRP78灰度值(1.393±0.004)与其对照组(0.679±0.011)相比显著提高(t=113.2，P=0.000)，在12 h时LC3灰度值(0.072±0.002)与其对照组(0.038±0.000)相比显著提高(t=25.5，P=0.000)。间接免疫荧光结果显示，顺铂(6μg/mL)组和TG(3μmol/L)组随作用时间的延长，细胞内LC3荧光斑点会逐渐增多，并伴随着细胞质Ca2+浓度上升。后经钙离子络合剂BAPTA-AM干预后，细胞内LC3荧光强度进一步增强。Westren blot结果显示，顺铂组LC3灰度值(0.039±0.000)小于顺铂联合BAPTA-AM组(0.071±0.001)，TG组(0.035±0.001)小于TG联合BAPTA-AM组(0.065±0.001)，差异有统计学意义(P=0.000)。结论：顺铂诱导SKOV3细胞内质网应激和自噬的发生，并伴随着细胞质内Ca2+浓度的上升。络合细胞质内Ca2+能增强顺铂诱导的自噬反应。