Exploration of the effect of the alkaloid colchicine on Ca2+ handling and its related physiology in human oral cancer cells

Abstract

Objective: Colchicine, extracted from plants of the genus Colchicum, is a commonly prescribed drug for inflammatory diseases. It has been shown that colchicine affected various physiological responses in different models. However, the effect of colchicine on cytosolic free Ca2+ levels ([Ca2+]i) and its related physiology in human oral cancer cells is unknown. This study examined whether colchicine altered Ca2+ homeostasis and caused cytotoxicity in OC2 human oral cancer cells.Methods: The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca2+]i. Cell viability was measured by the fluorescent reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay.Results: Colchicine at concentrations of 250–650 μM induced [Ca2+]i rises concentration-dependently. The response was reduced by approximately 40% by removing extracellular Ca2+. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited colchicine-evoked [Ca2+]i rises. Conversely, treatment with colchicine inhibited thapsigargin-evoked [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished colchicine-induced Ca2+ release. In Ca2+-containing medium, colchicine-induced Ca2+ entry was supported by Mn2+-caused quenching of fura-2 fluorescence and the entry was partly inhibited by protein kinase C (PKC) modulators (phorbol 12-myristate 13 acetate, PMA; and GF109203X) and by three modulators of store-operated Ca2+ channels (nifedipine, econazole and SKF96365). Colchicine at 250–650 μM decreased cell viability, which was not reversed by pretreatment with the Ca2+chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM).Conclusions: In OC2 cells, colchicine induced [Ca2+]i rises by evoking PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ entry. Furthermore, colchicine caused cell death that was not triggered by preceding [Ca2+]i rises.