4-氨基吡啶对卵巢上皮性癌细胞增殖的影响

Influence of 4-aminopyridine on cell proliferation through inhibiting voltage-gated K^+ channel in ovarian cancer

目的探讨4-氨基吡啶(4-AP)对卵巢上皮性癌细胞株SKOV3细胞增殖的抑制作用。方法应用RT-PCR技术分析SKOV3细胞膜上电压门控式钾离子通道(Kv)mRNA的表达;应用膜片钳记录4-AP对SKOV3细胞膜上电压门控式钾离子电流(IK)的抑制作用;应用流式细胞仪检测4-AP对SKOV3细胞增殖周期的影响;采用四甲基偶氮唑蓝(MTT)比色法证实4-AP对SKOV3细胞增殖的抑制作用。以未加4-AP的SKOV3细胞为对照。结果RT-PCR技术检测显示,SKOV3细胞表达KvmRNA;膜片钳记录显示,5mmol/L的4-AP可以降低IK值52·5%;流式细胞仪检测结果显示,应用5mmol/L的4-AP作用72h后,SKOV3细胞周期分布与对照相比,G0/G1期细胞由39·7%增加到62·3%;S期和G2/M期细胞的比例下降,分别由57·3%、3·0%下降到36·2%、1·4%(P<0·05);MTT比色法显示,0·1、1、5、10、15、20mmol/L的4-AP均能抑制SKOV3细胞的增殖,抑制率分别为17·5%、35·0%、54·6%、69·1%、71·2%、72·8%,各浓度分别与对照比较,差异均有统计学意义(P<0·01),且抑制程度与药物浓度呈正相关关系。结论4-AP能够抑制SKOV3细胞的增殖,Kv在卵巢上皮性癌细胞株SKOV3细胞的增殖过程中起着重要作用。

Objective To study the inhibition of cell proliferation by 4-aminopyridine（4-AP） in the human ovarian cancer cell SKOV3. Methods The expression of voltage-gated K^＋ channel on the SKOV3 cell line was detected by reverse transcription polymerase chain reaction. Inhibition of voltage-gated K^＋ current by 4-AP through the whole-cell patch-clamp technique on SKOV3 cell line was recorded. The influence on the cell-cycle of the SKOV3 cell line by 4-AP was observed by flow cytometry and the inhibition of the cell proliferation was studied using methyl thiazolyl tetrazolium （MTT） method. Results Voltagegated K^＋ channel was expressed in SKOV3 cell. Exposure of the SKOV3 cell to 5 mmol/L 4-AP reduced K^＋ current by 52. 5%. The 4-AP at 5 mmol/L significantly effected the progression of cell cycle with a 72 h treatment of SKOV3 cell. Exponentially growing cells without inhibitors had a distribution of 39.7% in G0/G1 phase, 57.3% in S phase, and 3.0% in G2/M phase. In 4-AP-treated cells, the proportion of G0/G1 cells increased significantly to 62. 3% （P 〈0. 05）, while there was a significant decrease in S phase cells （36. 2%, P 〈 0. 05 ） and G2/M phase （ 1.4%, P 〈 0. 05 ）. Incubation of the SKOV3 cells with 0. 1, 1, 5, 10, 15, 20 mmol/L 4-AP resulted in a concentration-dependent reduction in the number of viable cells as compared with the control, and the inhibition rate was 17.5% , 35.0% , 54.6% , 69.1% , 71.2% , 72. 8% respectively（vs control, P 〈 0.01 ）. Conclusion The voltage-gated K^＋ channels expressed by SKOV3 play an important role in SKOV3 cell proliferation.