3,3'-Diindolylmethane Alters Ca ²⁺ Homeostasis and Viability in MG63 Human Osteosarcoma Cells

The effect of the natural product 3,3'-diindolylmethane (DIM) on cytosolic Ca ²⁺ concentrations ([Ca ²⁺] _i) and viability in MG63 human osteosarcoma cells was explored. The Ca ²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca ²⁺] _i. DIM at concentrations of 40-80μM induced a [Ca ²⁺] _i rise in a concentration-dependent manner. The response was reduced partly by removing Ca ²⁺. DIM-evoked Ca ²⁺ entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca ²⁺, incubation with the endoplasmic reticulum Ca ²⁺ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished DIM-induced [Ca ²⁺] _i rise. Incubation with DIM also inhibited thapsigargin or BHQ-induced [Ca ²⁺] _i rise. Inhibition of phospholipase C with U73122 abolished DIM-induced [Ca ²⁺] _i rise. At concentrations of 10-50μM, DIM killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca ²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40μM) induced apoptosis in a concentration-dependent manner. In sum, in MG63 cells, DIM induced a [Ca ²⁺] _i rise by evoking phospholipase C-dependent Ca ²⁺ release from the endoplasmic reticulum and Ca ²⁺ entry via protein kinase C-sensitive store-operated Ca ²⁺ channels. DIM caused cell death that may involve apoptosis.