IP(3) receptor antagonist, 2-APB, attenuates cisplatin induced Ca2+-influx in HeLa-S3 cells and prevents activation of calpain and induction of apoptosis.
OBJECTIVE
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.


METHODS
Cisplatin (1 nM-10 microM) 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.


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 (type 1-3) 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
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.
Splettstoesser
F
F
Institut für Physiologie, Universitätsklinikum, Universität Duisburg-Essen, Essen, Germany.
Florea
A-M
AM
Büsselberg
D
D
D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002154:Calpain / Q000187:drug effects / Q000378:metabolism; D053181:Caspase 8 / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000008:administration & dosage / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006367:HeLa Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000187:drug effects*; D018613:Microscopy, Confocal
D000970:Antineoplastic Agents; D001896:Boron Compounds; D053496:Inositol 1,4,5-Trisphosphate Receptors; C109986:2-aminoethoxydiphenyl borate; D002154:Calpain; D053181:Caspase 8; D002945:Cisplatin; D002118:Calcium