Abstract
Previously, we demonstrated that malignant glioma cell lines have increased intracellular pH (pHi) as a result of increased activities of the type I sodium/hydrogen exchanger (NHE1). This alkalotic pHi of 7.2 to 7.4 is favorable for augmented glycolysis, DNA synthesis, and cell cycle progression. Conversely, reductions in pHi have been associated with reduced rates of proliferation in transformed cell types. The effects of reducing pHi directly and by NHE1 inhibition on human malignant glioma cells were systematically compared with those on primary rat astrocytes. Neither cariporide, nor direct acidification to pHi 6.9 altered the proliferative rates or viabilities of human U87 or U118 malignant glioma cell lines. However, amiloride significantly impaired glioma cell proliferation and viability while not affecting astrocytes at concentrations (500 μM) that exceeded its inhibition of NHE1 in glioma cells (IC50 = 17 μM). Preventing a reduction of pHi did not alter the drug's antiproliferative and cytotoxic effects on glioma cells. These findings indicated that amiloride's cytotoxic effects on glioma cells are independent of its ability to inhibit NHE1 or to reduce intracellular pHi. The amiloride derivative 2,4 dichlorobenzamil (DCB) inhibits the sodium-calcium exchanger (NCX) and was both antiproliferative and cytotoxic to glioma cells at low doses (20 μM). By contrast, KB-R7943 [(2-[2-[4-nitrobenzyloxy]phenyl]ethyl)-isothioureamethanesulfonate] preferentially blocks sodium-dependent calcium influx by NCX (reverse mode) and was nontoxic to glioma cells. It is proposed that DCB (20 μM) and amiloride (500 μM) impair calcium efflux by NCX, leading to elevations of intracellular calcium that initiate a morphologically necrotic, predominantly caspase-independent glioma cell death.
Footnotes
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This work was supported by funding from the National Institutes of Health Grants NS40489 and HL31179 and the University of California Cancer Research Coordinating Committee. This work will be submitted for partial fulfillment of the dissertation requirements for Manu Hegde, Ph.D. candidate in the Neuroscience Graduate Group, University of California, Davis.
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DOI: 10.1124/jpet.103.065029.
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ABBREVIATIONS: pHi, intracellular pH; NHE1, sodium hydrogen exchanger type 1; NCX, sodium calcium exchanger; DCB, 2,4 dichlorobenzamil; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PBS-CMF, phosphate-buffered saline-Ca2+ and Mg2+ free; TBS, Tris-buffered saline; BCECF, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM; HR, HEPES Ringer; pHe, extracellular (buffer) pH; [Ca2+]i, intracellular calcium concentration.
- Received December 30, 2003.
- Accepted March 9, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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