Effect of diindolylmethane on Ca²⁺ homeostasis and viability in MDCK renal tubular cells

Abstract

The effect of the natural product diindolylmethane (DIM) on cytosolic Ca(2+) concentrations ([Ca(2+)]i) and viability in MDCK renal tubular cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)]i. DIM at concentrations 1-50 μM induced a [Ca(2+)]i rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). DIM induced Mn(2+) influx leading to quenching of fura-2 fluorescence. 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 inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) greatly inhibited DIM-induced [Ca(2+)]i rise. Incubation with DIM abolished TG or BHQ-induced [Ca(2+)]i rise. Inhibition of phospholipase C with U73122 reduced DIM-induced [Ca(2+)]i rise by 50%. At 1, 10, 40 and 50 μM, DIM slightly enhanced cell proliferation. The effect of 50 μM DIM was reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In sum, in MDCK 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 did not induce cell death.

Effect of Protriptyline on [Ca^(2+)]i and Viability in MDCK Renal Tubular Cells

“…The [Ca 2+ ] i was measured as previously described (6,8,11,23,32). Confluent cells grown on 6 cm dishes were trypsinized and suspended in culture medium at a density of 10 6 /ml.…”

“…Viability was assessed as previously described (6,8,11). The measurement of viability was based on the ability of cells to cleave tetrazolium salts by dehydrogenases.…”

“…The MDCK cell is commonly applied for renal studies. It has been shown that in this cell, [Ca 2+ ] i rises can be induced in response to the stimulation of various compounds such as thymol (6), diindolylmethane (11) and angiotensin II (23).…”

Anticancer Activity of 3,3′‐Diindolylmethane and the Molecular Mechanism Involved in Various Cancer Cell Lines

“…In the absence of extracellular Ca 2+ , the addition of the ER Ca 2+ pump inhibitor 2,5‐di‐ tert ‐butylhydroquinone, and suppression of phospholipase C abolished DIM‐induced cytosolic Ca 2+ rise. DIM has been reported to induce Ca 2+ release from the ER of human hepatoma, prostate, and renal tubular cancer cell . Staining with Annexin V/propidium iodide showed that DIM induced apoptosis in a concentration‐dependent manner.…”