tram-34 and Endometrial-Neoplasms

The objective of this study was to investigate the effect of intermediate-conductance Ca(2+)-activated K(+) (KCa3.1) channels on the cell proliferation, cell cycle, apoptosis, migration, and invasion in endometrial cancer (EC) cells. Human EC cell lines HEC-1-A and Ishikawa were cultured in vitro and transfected with recombinant plasmid containing KCa3.1-targeting shRNA. RT-qPCR and Western blot were used to examine the mRNA and protein expression levels of KCa3.1 channels in transfected cells. In addition, the specific inhibitor of KCa3.1, TRAM-34, was used to examine the effect of KCa3.1 blockage on migration capacity and invasiveness of EC cells using transwell assay. Proliferation and apoptotic rates of EC cells transfected with KCa3.1 shRNA or treated with TRAM-34 were analyzed using MTT, BrdU incorporation assay, and flow cytometry. Expression of cell cycle proteins and metalloproteinase-2 (MMP-2) was evaluated by RT-qPCR and Western blotting. TRAM-34 treatment and KCa3.1 silencing using shRNA dramatically suppressed both the mRNA and protein expression of KCa3.1 channels (P < 0.01) compared with control groups. Blockage of KCa3.1 by TRAM-34 treatment and KCa3.1 shRNA transfection exerted inhibitory effect on cell growth of both EC cell lines, as demonstrated by increased cell population at G0-G1 phase and decreased cell population at S phase. However, both the treatments did not result in significant changes in the apoptotic rate (P > 0.05) compared to controls. Protein expressions of cyclin D1, cyclin E, and survivin were significantly decreased in the experimental groups comparing to control. We showed that TRAM-34 treatment led to significantly inhibited migration, invasion, and MMP-2 expression in HEC-1-A and Ishikawa cells, compared with the control group (P < 0.01). Blockage of KCa3.1 channel activity or expression inhibits cell proliferation and cell cycle progression without inducing apoptosis in EC cells. Moreover, TRAM-34 could reduce the ability of EC cells to migrate and invade, which might be related to reduced expression of MMP-2.

Topics: Apoptosis; Carcinoma; Cell Line, Tumor; Cell Movement; Cyclin D1; Endometrial Neoplasms; Female; Humans; Inhibitor of Apoptosis Proteins; Intermediate-Conductance Calcium-Activated Potassium Channels; Matrix Metalloproteinase 2; Potassium Channel Blockers; Pyrazoles; Survivin

Potassium (K(+)) channels have been implicated in proliferation of some tumor cells. However, whether K(+) channels are important to the pathogenesis of endometrial cancer (EC) remains unknown. In the present study, we report that intermediate-conductance Ca(2+)-activated K(+) (IKCa1) channels play a critical role in the development of EC. The expression of IKCa1 at both mRNA and protein levels in EC tissues was greatly increased than that in atypical hyperplasia and normal tissues. Treatment of EC cells with clotrimazole and TRAM-34, two agents known to inhibit IKCa1 channels, suppressed the proliferation of EC cells and blocked EC cell cycle at G(0)/G(1) phase. Similarly, downregulation of IKCa1 by siRNA against IKCa1 inhibited EC cell proliferation and arrested its cell cycle at G(0)/G(1) phase. A clotrimazole-sensitive K(+) current was induced in EC cells in response to the increased Ca(2+). The current density induced by Ca(2+) was greatly reduced by clotrimazole, TRAM-34, charybdotoxin or downregulation of IKCa1 by the siRNA against IKCa1. Furthermore, TRAM-34 and clotrimazole slowed the formation in nude mice of tumor generated by injection of EC cells. Our results suggest that increased activity of IKCa1 channel is necessary for the development of EC.

Topics: Animals; Cell Cycle; Cell Proliferation; Charybdotoxin; Clotrimazole; Endometrial Neoplasms; Female; G1 Phase; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice; Mice, Nude; Pyrazoles; Resting Phase, Cell Cycle; RNA, Small Interfering