IP 3 receptor antagonist, 2-APB, attenuates cisplatin induced Ca 2+-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 ([Ca ²⁺] _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-10μM) was applied to HeLa-S3 and U2-OS cells and [Ca ²⁺] _i measured with fluo-4, using laser scanning microscopy. Inositol-1,4,5-trisphosphate (IP ₃) 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 [Ca ²⁺] _i concentration- dependently in HeLa-S3 but not in U2-OS cells. This elevation of [Ca ²⁺] _i depended on extracellular Ca ²⁺ but was reduced by the IP ₃ receptor blocker, 2-APB. This effect was not due to a Ca ²⁺ release triggered by Ca ²⁺ entry. Immunostaining showed IP ₃-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 Ca ²⁺ was present extracellularly. Increase of [Ca ²⁺] _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 IP ₃-receptors, calcium entry and apoptotic rate by cisplatin in specific carcinogenic cells might open new possibilities in the treatment of some forms of cancer.