Effect of diindolylmethane on Ca ²⁺ movement and viability in HA59T human hepatoma cells

The effect of diindolylmethane, a natural compound derived from indole-3-carbinol in cruciferous vegetables, on cytosolic Ca ²⁺ concentrations ([Ca ²⁺] _i) and viability in HA59T human hepatoma cells is unclear. This study explored whether diindolylmethane changed [Ca ²⁺] _i in HA59T cells. The Ca ²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca ²⁺] _i. Diindolylmethane at concentrations of 1-50 μM evoked a [Ca ²⁺] _i rise in a concentration-dependent manner. The signal was reduced by removing Ca ²⁺. Diindolylmethane-induced Ca ²⁺ influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators but was inhibited by aristolochic acid. In Ca ²⁺-free medium, treatment with the endoplasmic reticulum Ca ²⁺ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca ²⁺] _i rise. Incubation with diindolylmethane inhibited thapsigargin or BHQ-induced [Ca ²⁺] _i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca ²⁺] _i rise. At concentrations of 10-75 μM, diindolylmethane killed cells in a concentration-dependent manner. The cytotoxic effect of diindolylmethane was not reversed by chelating cytosolic Ca ²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid. Propidium iodide staining data suggest that diindolylmethane (25-50 μM) induced apoptosis in a concentration-dependent manner. Collectively, in HA59T cells, diindolylmethane induced a [Ca ²⁺] _i rise by causing phospholipase C-dependent Ca ²⁺ release from the endoplasmic reticulum and Ca ²⁺ influx via phospholipase A ₂-sensitive channels. Diindolylmethane induced cell death that may involve apoptosis.