IP3 receptor antagonist, 2-APB, attenuates cisplatin induced Ca2+-influx in HeLa-S3 cells and prevents activation of calpain and induction of apoptosis

Background and purpose: Cisplatin drives specific types of tumour cells to apoptosis. In this study we investigate the involvement of intracellular calcium ([Ca2+](i)) in triggering apoptosis in two different cell lines. As cisplatin is used for the treatment of several forms of cancer we choose HeLa-S3 and U2-OS as two examples of tumour cell lines. Experimental approach: Cisplatin (1nM-10 mu M) was applied to HeLa-S3 and U2-OS cells and [Ca2+](i) measured with fluo-4, using laser scanning microscopy. Inositol-1,4,5-trisphosphate (IP3) receptors were visualized with immunostaining. Membrane conductances were measured with patch-clamp techniques. Levels of calpain and caspases were assessed by western blots and apoptotic cells were stained with Hoechst 33342 and counted. Key results: Cisplatin increases [Ca2+](i) concentration-dependently in HeLa-S3 but not in U2-OS cells. This elevation of [Ca2+](i) depended on extracellular Ca2+ but was reduced by the IP3 receptor blocker, 2-APB. This effect was not due to a Ca2+ release triggered by Ca2+ entry. Immunostaining showed IP3-receptors (type1-3) at the cellular membrane of HeLa-S3 cells, but not in U2-OS cells. Electrophysiological experiments showed an increased membrane conductance with cisplatin only when Ca2+ was present extracellularly. Increase of [Ca2+](i) was related to the activation of calpain but not caspase-8 and triggered apoptosis in HeLa-S3 but not in U2-OS cells. Conclusions and implications: Our observations on the activation of IP3-receptors, calcium entry and apoptotic rate by cisplatin in specific carcinogenic cells might open new possibilities in the treatment of some forms of cancer.