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...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-IA cells. Methods 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 alphalD （Cavl.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, Beclinl and P70s6K） by Western blotting and monodansylcadaverine （MDC） labeled visualization. Results Proliferation of Hec-lA 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 Cavl.3 levels within 15 minutes, but significantly attenuated the Cavl.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 Beclinl revealed significant differences between the N＋3-MA group and control group （P=0.025; Beclinl： P=-0.015）. Conclusions Proliferation and migration in vitro of endometrial carcinoma Hec-lA cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-lA cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cavl.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclinl and mTOR pathways.展开更多
文摘目的高通量筛选技术筛选抑制肝癌(PLC)细胞生长的新型小分子化合物。方法将PLC HepG2、Huh7细胞进行培养,培养后用于实验。选择3个小分子化合物数据库(合计3272个小分子化合物),分别为Prestwick、Tocriscreen、Selleck Chem。以及进行高通量筛选和靶化合物的筛选。结果本研究从3272个小分子化合物中利用高通量筛选技术选出抑制PLC细胞生长的目的化合物(hits)。初筛出的hits为z-score<-1.5,差异有统计学意义( P <0.05)。本研究通过比较9块化合物筛选板2种PLC细胞系(HepG2、Huh7)的重叠区域,共筛选出31种能够同时抑制这2个细胞系生长,且z-score<-1.5的化合物。最终筛选出36种hits化合物,包括5种临床治疗PLC的常用化疗药物和分子式。阿霉素作为hits被2次筛选出来,故最终初筛成功的新型化合物共有31种。结论通过高通量筛选技术筛选出31种可能具有抑制PLC细胞生长作用的新型小分子化合物,且有可能成为PLC治疗中的新型靶向药物。
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 30973182）.
文摘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-IA cells. Methods 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 alphalD （Cavl.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, Beclinl and P70s6K） by Western blotting and monodansylcadaverine （MDC） labeled visualization. Results Proliferation of Hec-lA 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 Cavl.3 levels within 15 minutes, but significantly attenuated the Cavl.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 Beclinl revealed significant differences between the N＋3-MA group and control group （P=0.025; Beclinl： P=-0.015）. Conclusions Proliferation and migration in vitro of endometrial carcinoma Hec-lA cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-lA cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cavl.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclinl and mTOR pathways.
