关键词: 　顺铂, Ca²⁺, 内质网应激, 自噬

Abstract: 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⁺ homeostasis 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⁺ efflux 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 efflux on autophagy, SKOV3 cells were divided into control group, cisplatin group, TG group, BAPTA-AM group, cisplatin combined with BAPTA-AM group and TG combined with BAPTA-AM group. Western blot was used to detect the protein levels of GRP78 and LC3. Fluo-4 calcium fluorescent probe was used to examine cytoplasmic Ca²⁺ levels. Confocal microscopy was used to detect LC3 level by immunoflurescence staining. Results: Compared to control group (0.679±0.011), GRP78 was significantly 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 maximum 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. Immunofluorescent 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) significantly 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⁺ enhances cisplatin-induced autophagy.

Key words: Cisplatin, Ca²⁺, ER Stress, Autophagy