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–10μM) 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.

British Journal of Pharmacology (2007) 151, 1176–1186; doi:10.1038/sj.bjp.0707335