5α-双氢睾酮对前列腺癌LNCaP细胞钙离子移动和细胞生长的影响

Effects of 5α-dihydrostestosterone on calcium mobilization and growth of prostate cancer cell line LNCaP

目的:探讨5α-双氢睾酮(DHT)对前列腺癌LNCaP细胞钙离子移动的影响及其机制。方法:应用Fura-2/AM Ca2+荧光探针法结合MiraCal荧光成像系统动态检测DHT刺激以及Ca2+通道阻滞剂干预后LNCaP细胞内钙离子浓度([Ca2+]i)的变化。应用MTT法观察细胞活力,流式细胞仪观察早期细胞凋亡率。结果:DHT浓度为1、10、100和1000nmol/L时,能快速诱导[Ca2+]i升高,在20s～3min升至峰值。细胞外液无Ca2+时,1000nmol/L DHT未能诱导[Ca2+]i升高。细胞膜L-型电压门控Ca2+通道阻滞剂维拉帕米(50μmol/L)、地尔硫(100μmol/L)或硝苯地平(5mmol/L)37℃孵育细胞5min后,能完全抑制1000nmol/L DHT诱导的[Ca2+]i升高。磷脂酶C抑制剂新霉素(1mmol/L)37℃孵育细胞5min或兰尼定受体阻滞剂普鲁卡因(50mmol/L)37℃孵育细胞3min后,对1000nmol/L DHT诱导的[Ca2+]i升高没有影响。1000nmol/LDHT作用细胞48h后,与1000nmol/L DHT作用前用维拉帕米预孵细胞相比,细胞光密度(D)值[(0.67±0.10)vs(2.13±0.16))和早期细胞凋亡率[(14.31±2.29)%vs(1.07±0.19)%]差异有统计学意义(P<0.01)。结论:DHT可快速地、剂量依赖性地诱导LNCaP细胞[Ca2+]i升高;DHT诱导的LNCaP细胞[Ca2+]i的升高是通过细胞外Ca2+经细胞膜L-型电压门控Ca2+通道流入细胞内实现的,细胞内贮钙库未释放Ca2+。DHT诱导的LNCaP细胞[Ca2+]i升高促进细胞凋亡、抑制细胞生长。

Objective：To investigate the effects of 5α- dihydrostestosterone （DHT） on calcium mobilization and growth of prostate cancer cell line LNCaP. Methods： Intracellutar calcium concentration （[Ca2＋ ） was assayed by MiraCal Image System using Fura-2/AM as Ca2＋ fluorescence probe. Cell viability was observed by MTT assay and apoptosis by flow cytometry. Results： The calcium levels rapidly increased following addition of DHT, with the latency of response only in seconds. DHT at the concentrations of 1, 10, 100 and 1 000 nmol/L increased [Ca2＋ ]i from （28±5）, （29±5）, （28±4） and （28±9） nmol/L to （31±3） （P〉0.05,65±9） （P〈0.01）, （193±33） （P〈0. 001） and （208±42） nmol/L （P〈0.001）, respectively. The response induced by 1 000 nmol/L DHT was similar to that induced by 100 nmol/L DTH. DHT 1 000 nmol/L did not increase [Ca2＋ ]i under extracellular Ca2＋ free condition. Blockers of I. type voltage-gated calcium channels, including verapamil （50 μmol/L）, dihiazem （100μmol/L） or nifedipine （5 mmol/L） at 37℃ for 5 min prior to stimulation with 1 000 nmol/L DHT, complctely inhibited DHT-induced [Ca2＋]i rise. Pre-treatment with inhibitor of phospholipase C such as neomycin sulfate （1 mmol/L） at 37 C for 3 min or inhibitor of ryanodine receptor such as procaine （50 mmol/L） at 37℃ for 3 min had no influence on [Ca2＋] rise induced by 1 000 nmol/L DHT. The optical density （D） values and early apoptosis rates of the cells stimulated with 1 000 nmol/L DHT for 48 h were significantly different from those of cells pre-treated with verapamil prior to stimulation with 1 000 nmol/L DHT （[0. 67±0.10] %0 vs [2. 13±0.16] %and [14. 31±2. 29]% vs [1. 07±0. 19]% ,P〈0.01）.Conclusion： DHT can induce rapid [Ca2＋ ]i rise in LNCaP cells in a concentration-dependent manner. The increase of [Ca2＋]i induced by DHT involves L-type voltagc-gated calcium channels, but does not involve release of intracellular Ca2＋ stores. The increase of [Ca2＋]i induced by DHT increases apoptosis and inhibits growth of LNCaP cells.