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.
OBJECTIVE
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 G(0)/G(1) 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(-)/HCO(3)(-) exchangers; i.e., anion exchanger (AE) and Na+-driven Cl(-)/HCO(3)(-) exchanger (NDCBE)) reduced pH(c), suggesting that DIDS-sensitive Cl(-)/HCO(3)(-) transporters such as AE and/or NDCBE keep pH(c) normal by stimulating HCO(3)(-) 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 G(0)/G(1) 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(-)/HCO(3)(-) 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).
Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan. hosogi@koto.kpu-m.ac.jp
Shigekuni
S
Hosogi
Hiroaki
H
Miyazaki
Ken-Ichi
K
Nakajima
Eishi
E
Ashihara
Naomi
N
Niisato
Katsuyuki
K
Kusuzaki
Yoshinori
Y
Marunaka
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D000584:Amiloride / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D010750:Phosphoproteins / Q000037:antagonists & inhibitors*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000473:pathology*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D018118:Chloride Channels; D002712:Chlorides; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D000584:Amiloride; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C039614:ethylisopropylamiloride