Furosemide, a Blocker of Na+/K+/2Cl− Cotransporter, Diminishes Proliferation of Poorly Differentiated Human Gastric Cancer Cells by Affecting G0/G1 State

Abstract

Furosemide, a blocker of Na + /K + /2Cl -cotransporter (NKCC), is often used as a diuretic to improve edema, ascites, and pleural effusion of patients with cancers. The aim of the present study was to investigate whether an NKCC blocker affects cancer cell growth. If so, we would clarify the mechanism of this action. We found that poorly differentiated gastric adenocarcinoma cells (MKN45) expressed the mRNA of NKCC1 three times higher than moderately differentiated ones (MKN28) and that the NKCC in MKN45 showed higher activity than that in MKN28. A cell proliferation assay indicates that furosemide significantly inhibited cell growth in MKN45 cells, but not in MKN28 cells. Using flow cytometrical analysis, we found that the exposure to furosemide brought MKN45 cells to spend more time at the G 0 /G 1 phase, but not MKN28 cells. Based on these observations, we indicate that furosemide diminishes cell growth by delaying the G 1 -S phase progression in poorly differentiated gastric adenocarcinoma cells, which show high expression and activity of NKCC, but not in moderately differentiated gastric adenocarcinoma cells with low expression and NKCC activity.Key words: gastric cancer cell, Na + /K + /2Cl -cotransporter (NKCC), furosemide, cell cycle, cell proliferation.In the past two decades, many researchers have reported the characteristics of ion channels/transporters in various types of cells, including cancer cells. Several types of K + , Ca 2+ , and Cl -channels have been identified in colorectal, breast, lung, and prostate cancer cells [1,2]. Some studies indicate that the transepithelial ion transport plays an important role in cell apoptosis [3,4]. Particularly, the modulation of Cl -transport via Cl -channel, K + /Cl -cotransporter (KCC), and Na + /K + /2Cl -cotransporter (NKCC) occurs in cell proliferation [5][6][7]. However, it is unknown whether NKCC affects the proliferation of cancer cells. In the treatment of patients with terminal stage cancers, however, furosemide (an NKCC blocker, a loop diuretic) is often used as a diuretic to keep urine output and to improve edema, ascites, or pleural effusion. Therefore we performed the present study to clarify the effect of furosemide on the proliferation of cancer cells.In the present study, we used two human gastric cancer cell lines, MKN28 and MKN45, which were established from moderately and poorly differentiated adenocarcinoma, respectively [8]. We determined the mRNA and functional expression levels of the NKCC and investigated the inhibitory effect of an NKCC blocker, furosemide, on the proliferation in these two cancer cell lines. We found that mRNA and the functional expression levels of NKCC were both higher in MKN45 than in MKN28 cells. Furthermore, furosemide diminished the cell proliferation, delaying the G 1 -S phase progression in MKN45 cells, but not in MKN28 cells. MATERIALS AND METHODSCell culture. The moderately differentiated human gastric adenocarcinoma cell line, MKN28, and the poorly differentiated human gastric adenocarcinoma cel...

Enhanced insulin secretion and improved glucose tolerance in mice with homozygous inactivation of the Na+K+2Cl− co-transporter 1

“…Although the mechanisms involved in proliferation of b-cells lacking NKCC1 remain unknown, our findings are in contrast to the direct positive linear relationship that exists between b-cell mass and body weight (Montanya et al 2000, Herbach et al 2011 and to the known negative impact that pharmacological inhibition of NKCC1 or depletion of [Cl K ] i have on normal cell growth and proliferation (O'Brien et al 1988, Panet et al 1994, 2002, Lang et al 1998, Iwamoto et al 2004, Shiozaki et al 2006. Indeed, NKCC1 KO mice are half the weight of NKCC1…”

Intracellular chloride regulates the G1/S cell cycle progression in gastric cancer cells

“…These reports suggest that the changes in the [Cl -]i might act as a regulator of various types of intracellular enzymes. We previously showed that furosemide, a blocker of NKCC, diminished cell growth by delaying the G1-S phase progression in gastric cancer cells with high expression and activity of NKCC [12] (Figure 1 [17,18] . These findings indicated that our experimental system using the low Cl -medium can be used as a model of the [Cl -]i regulation (Figure 2).…”

“…-cotransporter (NKCC) were higher in poorly-differentiated type gastric adenocarcinoma cells than in differentiated cells [12] . These reports indicate that the transepithelial Cl -transport plays an important role in cell proliferation of gastrointestinal cancer cells.…”

Pleural lavage with distilled water during surgery for esophageal squamous cell carcinoma

“…These findings clearly support the efficacy of pleural lavage with distilled water during surgery for ESCC. The coverslip with the cells was set in a perfusion chamber that was mounted on the stage of a DIC microscope (BX50Wi, Olympus, Tokyo, Japan) connected to a video-enhanced contrast (VEC) system (ARGUS-20, Hamamatsu Photonics, Hamamatsu, Japan), and images were continuously recorded by a video recorder according to the procedure described previously (9). The experiments were performed at 37˚C.…”