Bcl-2 overexpression reduces cisplatin cytotoxicity by decreasing ER-mitochondrial Ca2+ signaling in SKOV3 cells

Abstract

Recent studies have revealed that a small amount of cisplatin can penetrate into the nucleus and induce intranuclear DNA damage. Specifically, most cisplatin accumulates in and stresses different organelles, including mitochondria, endoplasmic reticulum (ER) and the cytosol, where apoptosis signaling is activated and magnified. Bcl-2, which is mainly localized to ER and mitochondria, is identified as a key regulator of survival and apoptosis. Bcl-2 is reported to block cisplatin-induced apoptosis via regulating Ca2+ signaling in a variety of cancer cell lines. However, its target molecule and the mechanism responsible for its inhibitory effect in ovarian cancer are undefined. The present study revealed that Bcl-2 overexpression reduced cisplatin-induced growth inhibition and apoptosis in SKOV3 human ovarian cancer cells. Furthermore, Bcl-2 inhibited cisplatin-induced Ca2+ release from the ER to the cytoplasm and mitochondria, which reduced cisplatin-induced ER stress-mediated apoptosis through the mitochondrial apoptotic pathway. The overexpression of Bcl-2 inhibited the cisplatin-induced increase in the number of ER-mitochondrial contact sites in SKOV3 human ovarian cancer cells. In addition, the present study provided evidence that Bcl-2 reduced the anticancer activity of cisplatin towards ovarian cancer cells in vivo. These results revealed that Bcl-2 attenuates cisplatin cytotoxicity via downregulating ER-mitochondrial Ca2+ signaling transduction. Thus, Bcl-2 which may be a potential therapeutic target for ovarian cancer.

Figure 1.

Bcl-2 overexpression reduces cisplatin-induced growth inhibition and apoptosis in SKOV3 cells. (A) Western blot analysis of the expression of Bcl-2 in pc-SKOV3 and Bcl-2-SKOV3 cells. (B) Pc-SKOV3 and Bcl-2-SKOV3 cells were treated with the indicated doses of cisplatin for 24 h, and then the cell viability was determined by an MTT assay. (C and D) Pc-SKOV3 and Bcl-2-SKOV3 cells were treated with 6 µg/ml cisplatin for 24 h. Subsequently, the cells were stained with Annexin V and 7-AAD and the apoptotic rate was assessed by flow cytometry. The results are the mean ± SD of three independent experiments. *P<0.05.

Figure 2.

Bcl-2 overexpression reduces cisplatin-induced Ca²⁺ release from the ER to the cytoplasm and mitochondria. Pc-SKOV3 and Bcl-2-SKOV3 cells were treated with 6 µg/ml cisplatin for 24 h. Subsequently, the cells were loaded with Fluo-4/AM and Rhod-2/AM for 30 min and fluorescence intensity in the cytoplasm and mitochondria was assessed by confocal microscopy (scale bar, 50 µm). The results are the mean ± SD of three independent experiments. ER, endoplasmic reticulum.

Figure 3.

Bcl-2 overexpression inhibits activation of ER stress-mediated apoptosis by cisplatin in SKOV3 cells. (A) Pc-SKOV3 and Bcl-2-SKOV3 cells were treated with 6 µg/ml cisplatin for 24 h. Calpain-1 expression was observed by confocal microscopy (scale bar, 10 µm). (B and C) Western blot analysis of calpain-1, PDI, Grp78, CHOP and cleaved caspase-4 levels in pc-SKOV3 and Bcl-2-SKOV3 cells after cisplatin treatment. Results are the mean ± SD of three independent experiments. *P<0.05.

Figure 4.

Bcl-2 overexpression inhibits the activation of the mitochondrial apoptosis pathway by cisplatin in SKOV3 cells. (A and B) Pc-SKOV3 and Bcl-2-SKOV3 cells were treated with 6 µg/ml cisplatin for 6 h and then stained with MitoPotential dye and 7-AAD to assess the Δψm by flow cytometry. (C and D) Western blot analysis of cytochrome c, Bcl-2, Bax, cleaved caspase-9 and cleaved caspase-3 levels in pc-SKOV3 and Bcl-2-SKOV3 cells after cisplatin treatment. The results are the mean ± SD of three independent experiments. *P<0.05.

Figure 5.

Bcl-2 overexpression inhibits the induction of ER-mitochondrial interactions by cisplatin in SKOV3 cells. (A) Colocalization of IP3R and VDAC1 in pc-SKOV3 and Bcl-2-SKOV3 cells after cisplatin treatment (bar, 10 µm). (B and C) Representative transmission electron microscopic photomicrographs of pc-SKOV3 and Bcl-2-SKOV3 cells treated with cisplatin for 24 h (scale bar, 200 nm). White arrowheads indicate ER-mitochondrial contacts. Results are the mean ± SD of three independent experiments. *P<0.05. ER, endoplasmic reticulum.

Figure 6.

Bcl-2 attenuates the in vivo antitumor activity of cisplatin in SKOV3 human ovarian cancer xenografts. (A and B) Tumors from different treatment groups were photographed and tumor volumes were assessed on the indicated days. (C) Tumor weights were recorded. (D) The level of cleaved caspase-3 expression was evaluated immunohistochemically (scale bar, 50 µm). The results are the mean ± SD of three independent experiments. *P<0.05.