Nifedipine induced autophagy through Beclin1 and mTOR pathway in endometrial carcinoma cells
Nifedipine induced autophagy through Beclin1 and mTOR pathway in endometrial carcinoma cells
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硝苯地平通过 Beclin1 和 mTOR 通路诱导子宫内膜癌细胞自噬
DOI：
10.3760/cma.j.issn.0366-6999.2012.17.028
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发表时间：
2012-09-05
影响因子：
6.1
通讯作者：
Wang Jian-liu
中科院分区：
文献类型：
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作者：
Bao Xiao-xia;Xie Bu-shan;Wang Jian-liu
研究背景子宫内膜癌是最常见的女性生殖道恶性肿瘤之一。L类钙通道拮抗剂硝苯地平可抑制肿瘤细胞增殖。最近的研究表明，细胞内游离钙([Ca~(2+)](C))的升高是自噬的有力诱因。为探讨硝苯地平与Hec-1A细胞自噬的关系。方法用硝苯地平(10MU/L)培养Hec-1A细胞，不同时间收获细胞计数。用四甲基偶氮唑盐比色法检测细胞活力，Transwell法检测细胞迁移情况。Annexin V/PI法检测细胞凋亡率。3-甲基腺嘌呤(3-MA)(2.5 mm o l/L)作用0、5、15、30、60、120 m in，检测细胞内L型钙通道α1D(Cav1.3)蛋白的表达。将培养的细胞分为4组：未处理组(对照组)、10mU/L硝苯地平(N组)、2.5 mU/L 3-MA组(3-MA组)、10 mU/L硝苯地平+2.5 mmol/L 3-MA组(N+3 MA组)。结果硝苯地平作用24、48、96h后，Hec-1a细胞增殖明显受到抑制(P=0.000)。硝苯地平对细胞迁移能力的抑制作用(94.00/-8.2%)与未处理细胞(160.00/-9.5%)相比差异有统计学意义(P=0.021)。硝苯地平诱导的早期细胞凋亡率为(2.21±0.19)%，对照组为(2.9±0.13)%(P=0.052)，晚期细胞凋亡率分别为(10.38±0.96)%和(4.40±0.60)%(P=0.020)。3-MA组在15分钟内Cav1.3水平略有升高，但在30分钟后显著降低。N组MDC标记的自噬空泡(20.63+/-3.36)明显多于对照组(6.29+/-0.16，P=0.015)。GFP-LC3定位显示，3-MA组、N+3MA组和3-MA组细胞的LC3水平分别为2.80+/-0.29、2.30+/-0.17和1.80+/-0.21。N组细胞自噬明显增强(P
Background Endometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated that a rise in the free cytosolic calcium ([Ca2+](c)) was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-1A cells.Methods Cells were cultured with nifedipine (10 mu mol/L) and harvested at different times for counting cell number. MTT assay was applied to evaluate the cell viability and transwell assay to reveal cell migration. Apoptotic cells were detected with annexin V/PI assay. Then cells were treated with 3-methyladenine (3-MA) (2.5 mmol/L) for 0, 5, 15, 30, 60, and 120 minutes and the expression of the L-type calcium channel alpha1D (Cav1.3) protein was detected. At last, cells were cultured and assigned to four groups with different treatment: untreated (control group), 10 mu mol/L nifedipine (N group), 2.5 mmol/L 3-MA (3-MA group), and 10 mu mol/L nifedipine plus 2.5 mmol/L 3-MA (N+3MA group). Autophagy was detected with GFP-LC3 modulation by fluorescent microscopy, and expression of the autophagy-associated proteins (LC3, Beclin1 and P70s6K) by Western blotting and monodansylcadaverine (MDC) labeled visualization.Results Proliferation of Hec-1A cells was obviously suppressed by nifedipine compared with that of the untreated cells for 24, 48, and 96 hours (P=0.000 for each day). The suppression of migration ability of the nifedipine-treated cells (94.0 +/- 8.2) was significantly different from that of the untreated cells (160.00 +/- 9.50, P=0.021). The level of early period cell apoptosis induced by nifedipine was (2.21 +/- 0.19)%, which was (2.90 +/- 0.13)% in control group (P=0.052), whereas the late period apoptosis level reached (10.38 +/- 0.96)% and (4.40 +/- 0.60)% (P=0.020), respectively. The 3-MA group induced a slight increase in the Cav1.3 levels within 15 minutes, but significantly attenuated the Cav1.3 levels after 30 minutes. There were more autophagic vacuoles labeled by MDC in the N group (20.63 +/- 3.36) than the control group (6.29 +/- 0.16, P=0.015). GFP-LC3 localization revealed that the LC3 levels of cells in 3-MA group, N+3MA group, 3-MA group were 2.80 +/- 0.29, 2.30 +/- 0.17, and 1.80 +/- 0.21, respectively. Cells in the N group showed significant augmentation of autophagy (P