Troglitazone acts on cellular pH and DNA synthesis through a peroxisome proliferator-activated receptor gamma-independent mechanism in breast cancer-derived cell lines

Abstract

Purpose: The purpose of this study was to assess whether troglitazone (TRO) would induce cellular acidosis by inhibiting Na(+)/H(+) exchanger (NHE) 1 in breast carcinoma-derived cell lines and, if so, whether cellular acidosis would be associated with a reduction in proliferation.

Experimental design: Intracellular pH (pH(i)) and acid extrusion capacity after an exogenous acid load were assayed using (2, 7)-biscarboxyethyl-5(6)-carboxyfluorescein in MCF-7 and MDA-MB-231 cells treated with TRO. Radiolabeled thymidine incorporation was used to assess DNA synthesis. Peroxisome proliferator-activated receptor (PPAR) gamma involvement was assessed using an antagonist and PPARgamma(-/-) NIH3T3 cells.

Results: TRO induced a prompt (<4 minute) and severe cellular acidosis in both MCF-7 (7.54 +/- 0.23 to 6.77 +/- 0.06; P < 0.001) and MDA-MB-231 cells (7.38 +/- 0.18 to 6.89 +/- 0.25; P < 0.05) after 12 minutes, without increasing acid production. Acid extrusion as assessed by the response to an exogenous acid load (NH(4)Cl pulse) was markedly blunted (MDA-MB-231, P < 0.01) or eliminated (MCF-7, P < 0.001). Chronic exposure to TRO resulted in NHE1 activity reduction (P < 0.05) and a dose-dependent decrease in DNA synthesis (<75% inhibition at 100 micromol/L; P < 0.001 and P < 0.01 for MCF-7 and MDA-MB-231, respectively) associated with a decreased number of viable cells. TRO-mediated inhibition of proliferation was not reversed by the presence of the PPARgamma inhibitor GW9662 and was demonstrable in PPARgamma(-/-) NIH3T3 cells, consistent with a PPARgamma-independent mechanism.

Conclusions: TRO induces marked cellular acidosis in MCF-7 and MDA-MD-231 cells. Sustained acidosis is consonant with decreased proliferation and growth that is not reversed by a PPARgamma antagonist. Our results support a NHE-mediated action of TRO that exerts its effect independent of PPARgamma.