Troglitazone (TRO) is a thiazoladinedione that exhibits both anti-hyperglycemic and anti-proliferative effects. The anti-proliferative action of TRO is exerted either through a peroxisome proliferators activated receptor γ (PPARγ) dependent or through PPARγ independent biochemical pathways. We describe in this study a novel action of TRO that induces prolonged cellular acidosis through inhibition of the Na+/H+ exchanger (NHE) in breast carcinoma derived cell lines MCF7 and MDA-MD-231. Cells incubated in Krebs-Henseleit Hepes (KHH) media plus 20μM TRO showed an acute decrease in the cellular pH (pHi) from an average of 7.54 ± 0.09 to 6.76 ± 0.03 and from 7.38 ± 0.18 to 6.89 ± 0.25 after 12minutes (p<0.0001 vs. initial time) in MCF7 and MDA-MD-231 cells, respectively, as determined spectrofluorimetrically with (2’,7’)-biscarboxyethyl-5-(6)-carboxyfluorescein) (BCECF). Noteworthy, this profound cellular acidosis was not reversed by removing TRO from the media suggesting that the effect involves, at least in part, some site(s) other than the external sodium binding site. Both cell lines exhibited a robust response to an NH4Cl acid load that was reduced by 64%(both p<0.01,N=5) in the presence of 20uM TRO consistent with inhibition of sodium-hydrogen ion exchange activity. The hallmark of sustained cellular acidosis is enhanced ammoniagenesis from glutamine as previously shown. Consistent with a sustained cellular acidosis ammonium production was elevated in both cell lines as compared to controls (p<0.05) after 18 h of incubation with TRO. At this time, the level of DNA synthesis as determined by [3H]-thymidine incorporation was reduced by 25% and 50% in MCF7 and MDA-MD-231 cells, respectively (p<0.05). The TRO-mediated reduction in [3H]-thymidine incorporation was not reversed by the presence of 5 μM of the PPARγ specific inhibitor GW9662 consistent with a PPARγ -independent mechanism. Furthermore, TRO(20uM) mediates a similar degree of acidosis in PPARγ −/− fibroblasts (NIH-3T3) and inhibits DNA synthesis by 98% in these cells. Our data support a NHE-mediated action of TRO that exerts its anti-proliferative effect independent of PPARγ and suggest that NHE and PPARγ may interact perhaps via MEK in modulating this effect in tumorigenic cell lines.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]