Effect of clotrimazole on cytosolic Ca²⁺ rise and viability in HA59T human hepatoma cells

Clotrimazole is an antimycotic imidazole derivative that interferes with cellular Ca²⁺ homeostasis. This study examined the effect of clotrimazole on cytosolic Ca²⁺ concentrations ([Ca²⁺]i) and viability in HA59T human hepatoma cells. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]i. Clotrimazole induced [Ca²⁺]i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca²⁺. Clotrimazole-evoked Ca²⁺ entry was suppressed by store-operated channel inhibitors (nifedipine, econazole and SK&F96365) and protein kinase C modulators (GF109203X and phorbol, 12-myristate, 13-acetate). In Ca ²⁺-free medium, incubation with the endoplasmic reticulum Ca ²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone abolished clotrimazole-induced [Ca²⁺]i rise. Inhibition of phospholipase C with U73122 abolished clotrimazole-induced [Ca²⁺]i rise. At 10-40 M, clotrimazole inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺. Clotrimazole at 10 and 30 M also induced apoptosis. Collectively, in HA59T cells, clotrimazole-induced [Ca²⁺]i rises by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via store-operated Ca²⁺ channels. Clotrimazole also caused apoptosis.