An Inhibitor of Na+/H+ Exchanger (NHE), Ethyl-Isopropyl Amiloride (EIPA), Diminishes Proliferation of MKN28 Human Gastric Cancer Cells by Decreasing the Cytosolic Cl- Concentration via DIDS-Sensitive Pathways

Abstract

Background/Aims: Tumor cells produce a large amount of acidic metabolites due to their high metabolic condition. However, cytosolic pH (pH_c) of tumor cells is identical to or even slightly higher than that of normal cells. To maintain pH_c at a normal or higher level, tumor cells would have to have higher expression and/or activity of H⁺ transporting systems than normal cells. The purpose of the present study was to identify effects of ethyl-isopropyl amiloride (EIPA, an inhibitor of Na⁺/H⁺ exchanger (NHE)) on proliferation of human gastric cancer MKN28 cells. Methods: Effects of EIPA on proliferation, pH_c, [Cl^-]_c and expression of proteins regulating cell cycle and MAPKs were studied in MKN28 expressing NHE exposed to EIPA for 48 h. Results: EIPA suppressed proliferation of MKN28 cells by causing G₀/G₁ arrest without any significant effects on pH_c, but associated with reduction of [Cl^-]_c. Although EIPA alone had no effects on pH_c, EIPA co-applied with DIDS (an inhibitor of Cl^-/HCO₃^- exchangers; i.e., anion exchanger (AE) and Na+-driven Cl^-/HCO₃^- exchanger (NDCBE)) reduced pH_c, suggesting that DIDS-sensitive Cl^-/HCO₃^- transporters such as AE and/or NDCBE keep pH_c normal by stimulating HCO₃^- uptake coupled with Cl^- release under an NHE-inhibited condition. EIPA-induced lowered [Cl^-]_c up-regulated expression of p21associated with phosphorylation of MAPKs, suppressing proliferation associated with G₀/G₁ arrest. Conclusions: EIPA suppressed proliferation of MKN28 cells through up-regulation of p21 expression via reduction of [Cl^-]_c as a result from DIDS-sensitive Cl^-/HCO₃^- exchanger-mediated compensation for keeping pH_c normal under an NHE-inhibited condition. This is the first study revealing that an NHE inhibitor suppressed the proliferation of cancer cells by reducing [Cl^-]_c but not pH_c.