Independent [Ca²⁺]_i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells

The effect of the carcinogen safrole on intracellular Ca²⁺ movement and cell proliferation has not been explored previously. The present study examined whether safrole could alter Ca²⁺ handling and growth in human oral cancer OC2 cells. Cytosolic free Ca²⁺ levels ([Ca ²⁺]_i) in populations of cells were measured using fura-2 as a fluorescent Ca²⁺ probe. Safrole at a concentration of 325 μM started to increase [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced by 40% by removing extracellular Ca²⁺, and was decreased by 39% by nifedipine but not by verapamil or diltiazem. In Ca²⁺-free medium, after pretreatment with 650 μM safrole, 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) barely induced a [Ca²⁺]_i rise; in contrast, addition of safrole after thapsigargin treatment induced a small [Ca ²⁺]_i rise. Neither inhibition of phospholipase C with 2 μM U73122 nor modulation of protein kinase C activity affected safrole-induced Ca²⁺ release. Overnight incubation with 1 μM safrole did not alter cell proliferation, but incubation with 10-1000 μM safrole increased cell proliferation by 60±10%. This increase was not reversed by pre-chelating Ca²⁺ with 10 μM of the Ca²⁺ chelator BAPTA. Collectively, the data suggest that in human oral cancer cells, safrole induced a [Ca²⁺]_i rise by causing release of stored Ca²⁺ from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca²⁺ influx via nifedipine-sensitive Ca²⁺ entry. Furthermore, safrole can enhance cell growth in a Ca²⁺-independent manner.