Calcium Efflux From The ER Regulates Cisplatin-induced Apoptosis In Human Cervical Cancer Hela Cells

Backgroud:Cisplatin (cis-diamminedichloroplatinum II, CDDP) is one of thetraditionalanti-tumoragents, it is also one of the common chemotherapy drugs againstgynecological tumors, including cervical cancer. However, relapse following cisplatintherapy is inevitable. Thus, it is particularly important to elucidate the cell-killingmechanism of cisplatin. Cisplatin is generally considered to kill cancer cells bydamaging DNA and inhibiting DNA synthesis, which induces mitochondrial-mediatedapoptosis and consequently cell death. Previous research suggests thatchemotherapeutic agents target reactive oxygen species (ROS) metabolism and ATPproduction in mitochondria, activate mitochondrial-mediated cell apoptosis andinhibit cell growth and proliferation via an increase in cellular calciummobilization.Meanwhile, increasing evidences reveal that cisplatin triggers apoptoticevents via endoplasmic reticulum (ER) stress.However, ER is an intracellular store ofcalcium, but the function of calcium signaling in ER-derived apoptosis induced bychemotherapy drugs is not clear.Objective:To illustrate the impacton calcium level by cisplatin, investigate the role ofcalcium efflux from ER in apoptosis induced by cisplatin in HeLa cells and furthermechanistic insight on the tumor cell-killing effect of cisplatin.Methods:1. Cells were treated with cisplatin (2.5μg/mL,5μg/mL and10μg/mL) for0h,6h,12h and24h, and incubated with the fluorescentcalcium indicator Fluo-4/AM orRhod-2/AM. Calcium concentrations in the cytosol or mitochondriawere observed by confocal microscopy;2. HeLa cells were treated with cisplatin (5μg/mL) with or without BAPTA/AM (2.5μM) and2-APB (100μM) for12h and24h. Cells were incubated with thefluorescent calcium indicator Fluo-4/AM. Calcium concentrations in the cytosolwere observed by confocal microscopy;3. HeLa cells were treated with cisplatin (5μg/mL) with or without BAPTA/AM (2.5μM) and2-APB (100μM) for24h. Cell viability was determined using the MTTassay4. HeLa cells were treated with cisplatin (5μg/mL) with or without BAPTA/AM (2.5μM) and2-APB (100μM) for24h and then weused flow cytometry to detect theratio of cell apoptosis in HeLa cells;5. HeLa cells were treated with cisplatin (5μg/mL) with or without BAPTA/AM (2.5μM) and2-APB (100μM) for24h, and stained with Hoechst33342. We examinedapoptotic chromatin condensation by confocal microscopyfollowing Hoechst33342staining;6. The expression of mitochondrial-mediated apoptosis associated proteins and ERstress proteins in HeLa cells treated with cisplatin (5μg/mL) with or withoutBAPTA/AM (2.5μM) and2-APB (100μM) for12h were detectedby westernblotting;7. The expression of cleaved caspase-3, GRP78and CHOP in HeLa cells treated withcisplatin (5μg/mL) with or without BAPTA/AM (2.5μM) and2-APB (100μM) for12h wereexaminedby confocal microscopy.Results:1. Free Ca2+levels in the cytosol and mitochondria increased in both a dose-andtime-dependent manner in HeLa cells.The alteration of free Ca2+levels induced bycisplatin in the cytosol and mitochondria substantially decreased in groups treatedwith cisplatin combined with BAPTA/AM or2-APB;2. Cisplatin inhibited cell viability and induced apoptosis in HeLa cells. Treatmentcombined with BAPTA/AM or2-APB decreased the cytotoxic effects of cisplatin; 3. Cisplatin enhanced the expression of CAPN1and cleaved caspase-3. Treatmentwith cisplatin combined with BAPTA/AM or2-APB decreased the level of CAPN1and cleaved caspase-3induced by cisplatin;4. Cisplatin enhanced the expression of GRP78, CHOP and cleaved caspase-4.Treatment with cisplatin combined with BAPTA/AM or2-APB decreased the levelofGRP78, CHOP and cleaved caspase-4induced by cisplatin;Conclusions:Cisplatin induces ER stress and results in the dysfunction of ER calciumhomestasis in human cervical cancer HeLa cells, which triggers calcium effluxfromER. Calcium signaling from ER regulates mitochondrial-mediated andER-associated apoptosis induced by cisplatin.