Department of Obstetrics and Gynecology, Peking University People's Hospital, Beijing 100044, China.
Chin Med J (Engl). 2012 Sep;125(17):3120-6.
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 (Ca(2+)) was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-1A cells.
Cells were cultured with nifedipine (10 µmol/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 µmol/L nifedipine (N group), 2.5 mmol/L 3-MA (3-MA group), and 10 µmol/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.
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 < 0.05). Western blotting analysis confirmed the down-regulation of LC3 levels in 3-MA group (0.85 ± 0.21) and N+3MA group (1.21 ± 0.12) compared with nifedipine treatment (2.64 ± 0.15, P < 0.05). The annexin-V-FITC/PI assay showed that the level of early period cell apoptosis induced in the N+3-MA group ((11.22 ± 0.91)%) differed significantly from that of the control group ((2.51 ± 0.70)%) and N group ((3.47 ± 0.39)%). Similarly, the late period level of the N+3-MA group ((55.19 ± 2.51)%) differed significantly from that of the control group ((15.81 ± 1.36)%) and the N group ((22.09 ± 2.48)%, P < 0.05). The down-regulated expression of P70s6k and up-regulated expression of the Beclin1 revealed significant differences between the N+3-MA group and control group (P = 0.025; Beclin1: P = 0.015).
Proliferation and migration in vitro of endometrial carcinoma Hec-1A cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-1A cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cav1.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclin1 and mTOR pathways.
子宫内膜癌是女性生殖道最常见的恶性肿瘤之一。硝苯地平是一种 L 型钙通道拮抗剂，能够抑制癌细胞增殖。最近的研究表明，细胞内游离钙浓度的升高(Ca(2+))是诱导自噬的有效因素。本研究旨在探讨硝苯地平与 Hec-1A 细胞自噬之间的关系。
用硝苯地平（10 μmol/L）处理细胞，在不同时间点计数细胞数量。MTT 法评估细胞活力，Transwell 法检测细胞迁移。用 annexin V/PI 法检测凋亡细胞。然后用 3-甲基腺嘌呤（3-MA）（2.5 mmol/L）处理细胞 0、5、15、30、60 和 120 分钟，检测 L 型钙通道 α1D 亚基（Cav1.3）蛋白的表达。最后，细胞培养并分为 4 组进行不同处理：未处理（对照组）、10 μmol/L 硝苯地平（N 组）、2.5 mmol/L 3-MA（3-MA 组）和 10 μmol/L 硝苯地平加 2.5 mmol/L 3-MA（N+3MA 组）。用 GFP-LC3 调制荧光显微镜检测自噬，用 Western blot 和单丹磺酰尸胺（MDC）标记可视化检测自噬相关蛋白（LC3、Beclin1 和 P70s6K）的表达。
与未处理组相比，硝苯地平处理 24、48 和 96 小时后 Hec-1A 细胞的增殖明显受到抑制（P = 0.000 各日）。硝苯地平处理组细胞迁移能力的抑制（94.0 ± 8.2）与未处理组（160.00 ± 9.50，P = 0.021）有显著差异。硝苯地平诱导的早期细胞凋亡水平为（2.21 ± 0.19）%，对照组为（2.90 ± 0.13）%（P = 0.052），而晚期凋亡水平分别达到（10.38 ± 0.96）%和（4.40 ± 0.60）%（P = 0.020）。3-MA 组在 15 分钟内引起 Cav1.3 水平轻微升高，但在 30 分钟后显著降低 Cav1.3 水平。N 组自噬小体标记物 MDC 的阳性细胞数（20.63 ± 3.36）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 < 0.05）。Western blot 分析证实 3-MA 组（0.85 ± 0.21）和 N+3MA 组（1.21 ± 0.12）LC3 水平下调与硝苯地平处理组（2.64 ± 0.15，P < 0.05）相比。Annexin-V-FITC/PI 检测显示 N+3-MA 组诱导的早期细胞凋亡水平（（11.22 ± 0.91）%）与对照组（（2.51 ± 0.70）%）和 N 组（（3.47 ± 0.39）%）有显著差异。同样，N+3-MA 组晚期细胞凋亡水平（（55.19 ± 2.51）%）与对照组（（15.81 ± 1.36）%）和 N 组（（22.09 ± 2.48）%）有显著差异（P < 0.05）。N+3-MA 组 P70s6k 表达下调和 Beclin1 表达上调与对照组相比有显著差异（P = 0.025；Beclin1：P = 0.015）。
硝苯地平显著抑制子宫内膜癌 Hec-1A 细胞的体外增殖和迁移。硝苯地平诱导自噬以拮抗 Hec-1A 细胞凋亡。3-MA 抑制自噬导致 Cav1.3 下调，并增强硝苯地平诱导的细胞死亡。硝苯地平诱导的自噬与 Beclin1 和 mTOR 通路有关。
