The Role Of KCa3.1 Channels In Endometrial Cancer Cells Proliferation

Worldwide, endometrial cancer (EC) is the seventh most common malignant disorder; the incidence is at 15-20 per 100000 women per year, which serious threats the health and life of women. Owing to the development technology and medical treatment, the curability of EC raised, but the tumors with particular morphological variants, adverse histopathological features and /or advanced stage, the five years survival rate is very low. Despite extensive etiopathogenisis and pathogenetic studies, the molecular mechanisms of EC remain elusive.The intermediate-conductance Ca2 +-activated K+ (IKCa, KCa3.1) channels (single channel conductance ) belong to the Ca2 +-activated K+ channel superfamily that includes large-conductance (BKCa), intermediate-conductance Ca2 +-activated K+ (IKCa) channels and small-conductance (SKCa) Ca2 +-activated K+ channels. The activation of KCa3.1 is Ca²⁺-dependent with little or no voltage dependency. Recently, it has been demonstrated that KCa3.1 channels play a vital role in sustaining the intracellular Ca²⁺ levels for lymphocyte activation and proliferation, and oncogenesis. But little is known about the relationship between KCa3.1 channels and gynaecological malignancy, tumor cell biological behaviour. Our experiments began with an investigation of the expression of KCa3.1 channels in normal endometrial tissues and endometrial cancer tissues, RNA interference knocked down KCa3.1 channels expression in endometrial cancer HEC-1-A cells, and subsequently, western blot, RT-PCR, Real-Time PCR, [³H]thymidine incorporation, flow cytometric assessment, patch clamp technique, inhibitors of KCa3.1 channel were applied to explore the effect of KCa3.1 channel on HEC-1-A cells proliferation, cell cycle and electrophysiological characteristics.These experiments are divided into 3 parts,as following: â‘ Expression of KCa3.1 channels in normal endometrial tissues and endometrial cancer tissues; â‘¡The study on electrophysiological characteristics of HEC-1-A cells and the effects of KCa3.1 channels on endometrial cancer cell growth; â‘¢Inhibitor of KCa3.1 channel inhibit the development of transplanted tumor in nude mice.Section D Expression of KCa3.1 channels in normal endometrial tissues and endometrial cancer tissuesObjective To examine the expression of KCa3.1 channels in human normal endometrial tissues and endometrial cancer tissues and explore the relationship between the abberant expression of this gene and tumorigenesis of EC.Methods RT-PCR , Real-Time PCR and western bloting were used to examine the expression of KCal channels in 13 normal endometrial specimens and 25 endometrial cancer specimens.Results Only one of 13 normal human endometrial specimens has moderate expression of KCa3.1 channels mRNA , the others have weak or no expression, but almost of 25 endometrial cancer specimens have moderate and even serious expression of KCa3.1 channels mRNA(The result of Real-Time PCR nearly consisted with the result of RT-PCR) ; the expression of KCa3.1 channels protein of all specimens nearly consisted with KCa3.1 channels mRNA of all specimens, two of 13 normal human endometrial specimens have moderate expression of KCa3.1 channels protein, the others have weak or no expression, but almost of 25(only 4 specimens have weak expression) endometrial cancer specimens have moderate and even serious expression of KCa3.1 channels protein, the intensities of KCa3.1 channels mRNA and protein in endometrial cancer specimens were significantly higher than that in normal endometrial specimens (P<0.01).Conclusions The aberrant expression of KCa3.1 channels may be closely related to malignant transformation and development of endometrial cancers.Section DThe study on electrophysiological characteristics of HEC-1-A cells and the effects of KCa3.1 channels on endometrial cancer cell growthObjective To study on electrophysiological characteristics of HEC-1-A cells and the effects of KCa3.1 channels on endometrial cancer cell growthMethods We chose HEC-1-A cells, which has higher KCa3.1 channels expression and KLE cells which has moderate KCa3.1 channels expression to be targeted cells. CD Standard whole-cell patch-clamp recording was applied in HEC-1-A cells. In the experiments, different concentration , different kinds of inhibitors, differentconcentration Ca²⁺ and small interfering RNA against KCa3.1 channels were used to explore the electrophysiological characteristics of HEC-1-A cells KCa3.1 channels. The recording of resting membrane potential was corrected with liquid junction potential. The membrane potential was clamped at -70 mV during recording and the membrane currents were recorded with a MultiClamp 700A amplifier in the whole-cell configuration digitized by pClamp9.0/Digidata 1322A (Axon Instruments, CA). Software Clampfit 9.0 (Axon Instruments, CA) was used for off-line analysis. (2)a pair specific siRNAs against KCa3.1 channels and a pair nonsilence siRNAs as control were designed, The siRNAs were transfected into HEC-1-A cells and RNA interference was used to knockdown KCa3.1 channel expression. The efficacy of siRNA transfection was assessed by western blot and efficacy of FITC-siRNA transfection.(3)The effect of the knockdown of KCa3.1 channels and the ihibition of KCa3.1 channels by inhibitors on HEC-1-A and KLE cells proliferation and cell cycle were determined by [³H] thymidine incorporation, cell number and flow cytometry. â‘£Moreover, we investigated the expression of cyclinD1 closely related with cell cycle after knock-down of KCa3.1 channels or inhibition of KCa3.1 channels with inhibitor.Result â‘ siRNA against KCa3.1 knocked down the expression of KCa3.1 channel specifically and effectively. â‘¡The membrane potential hyperpolarized with 8.7 mM Ca²⁺ pipette solution was -62.4±1.9mV against 12.5±1.4mV, the membrane potential with 1 mM Ca²⁺ pipette solution. Ramp currents were elicited by 280 ms voltage ramps from -120 mV to +80 mv, the current density was 42.8±5.1 pA/pF (n=15) with 8.7 mM Ca²⁺ pipette solution, whereas, 5.5±1.4 pA/pF (n=9) with 1 mM Ca²⁺ pipette solution when membrane potential was +70 mV. The current density under 8.7 mM Ca²⁺ pipette solution was greatly reduced in a dose-dependent manner by clotrimazole, Charybdotoxin and Tram-34. Moreover, siRNA against KCa3.1 channel markedly reduces the current density of KCa3.1 channels, â‘¢small interfering RNA inhibited EC cell proliferation (P<0.01), reduced [³H]thymidine incorporation , retard cell cycle at G0-G1 phase and reduce S phase in HEC-1-A cells in comparison with that found in the cells transfected with non-silencing RNA(P<0.01). â‘£ clotrimazole, an inhibitor of KCa3.1 channels known to suppress the function of the channels, caused time-dependent decrease in cell number of HEC-l-A cells; in a dose-dependent manner inhibite [³H]thymidine incorporation; clotrimazole and TRAM-34 in a dose-dependent manner decrease HEC-1-A and KLE cells number (5). Clotrimazoleand TRAM-34 in a dose-dependent manner retard cell cycle at G0-G1 phase, and reduce S phase in HEC-1-A and KLE cells.â‘¥The expression of cyclinDl was decreased after knock-down of KCa3.1 channels or inhibition of KCa3.1 channels.Conclusion â‘ the existence of functional KCa3.1 channels in HEC-1-A cells, the functional KCa3.1 currents in HEC-1-A cells were indeed induced with a high Ca²⁺ intracellular solution not voltage; â‘¡KCa3.1 channels involved in regulation in HEC-1-A and KLE cells proliferation and cell cycle, and the mechanisms may be due to transcriptional regulation on cyclinD1, perhaps others; the expression of KCa3.1 channels may be closely related to malignant transformation of endometrial cancers and regarded as a new target for anticancer treatment.SectionIII Inhibitor of KCa3.1 channel inhibits the growth of transplanted tumor in nude miceObjective To provide direct evidence that KCa3.1 channels are responsible for tumor development.Methods A total of 18 female nude mice were randomly divided into control and treated groups. Treated groups were injected with 8×10⁶ HEC-l-A cells with 20μM clotrimazole into the flank of the mice, and control group injected with 8×10⁶ HEC-1-A cells incorporating 0.4ul dimethyl sulfoxide (DMSO), which was to use for dissolving clotrimazole in the treated group. General states of health were observed; the tumor size in volume was measured with calipers at every six days, seventeen days after injection, the mice were sacrificed by aether gas environment. The tumor size was measured in volume with caliper and in weigh with scale.Result There was no difference in general states of health between two groups, but there were markedly differences in tumor volume (mm³) between two groups at six days, at twelve days , at eighteen days:: control group versus treated group, 189.3±59.6 versus 127.4±50.7 (P<0.05) ; 388.8±116.4 versus 233.8±103.9 (P <0.01) ; 788.9±131.8 versus 483.3±180.3 (P<0.01) .There was markedly difference in tumor weigh (g) between two groups: control group versus treated group, 0.72±0.12g versus 0.41±0.19g (P<0.01) .Conclusion KCa3.1 channel play a vital role in EC cell growth and can beconsidered as a new target for anticancer treatment.Our research demonstrates that KCa3.1 channels is over-expressed in endometrial cancer specimens, and regulate cell proliferation and cell cycle. In the present study, we also examined electrophysiological characteristics of HEC-1-A cells KCa3.1 channels and the change of gene expression of cyclinDl and primarily explained the mechanisms that KCa3.1 channels regulated cell proliferation and cell cycle. Our research provides a new path for further studying the oncogenesis and progression of EC. Moreover, by using RNAi technology, our study provides a new strategy for gene therapy of refractory EC.