Role of Epidermal Growth Factor Receptor (EGFR)-Signaling Versus Cellular Acidosis Via Na+/H+ Exchanger1(NHE1)-Inhibition in Troglitazone-Induced Growth Arrest of Breast Cancer-Derived Cells MCF-7

Purpose:We previously showed that troglitazone (TRO) induces a profound cellular acidosis in MCF-7 cells as a result of inhibiting Na + /H + exchanger (NHE)1-mediated acid extrusion and this was associated with a marked reduction in cellular proliferation. The present study focuses on TRO-activated signaling pathways versus TRO-mediated NHE1-inhibition in reducing DNA synthesis. Experimental Design: TRO activation of the signaling pathway involving epidermal growth factor receptor (EGFR)/MAPK/ERK kinase (MEK) 1/2/extracellular signal-regulated kinase (ERK) 1/2 was studied by Western blotting and phospho-specific antibodies. TRO induction of cellular acidosis and inhibition of NHE1 activity were measured using (2, 7)-biscarboxyethyl-5 (6)-carboxyfluorescein (BCECF) assay and NH4 + /NH 3 pulsing. Cellular proliferation was assessed as DNA synthesis by 3 H-thymidine incorporation. Results: TRO simultaneously reduces pH i and elevates phosphorylated-extracellular signal-regulated kinase (p-ERK). These responses reflected inhibition of acid extrusion and EGFR activation respectively and were sustained over 18h associated with a large decrease in DNA synthesis. Preventing TRO-induced ERK activation did not restore DNA synthesis or cellular pH. Conclusions: TRO activates two parallel pathways: I] EGFR/MEK1/2/ERK1/2 and II] NHE1 inhibition/cellular acidosis. Elimination of I] did not prevent the inhibition of DNA synthesis consistent with TRO-induced growth arrest dependent upon II] in tumorigenic non-metastatic breast cancer derived MCF-7 cells.