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Nifedipine induced autophagy through Beclinl and mTOR pathway in endometrial carcinoma cells 被引量:11

Nifedipine induced autophagy through Beclinl and mTOR pathway in endometrial carcinoma cells
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摘要 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. 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.
出处 《Chinese Medical Journal》 SCIE CAS CSCD 2012年第17期3120-3126,共7页 中华医学杂志(英文版)
基金 This study was supported by a grant from the National Natural Science Foundation of China (No. 30973182).
关键词 NIFEDIPINE L-TYPE AUTOPHAGY endometrial carcinoma nifedipine L-type autophagy endometrial carcinoma
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  • 1Zhang L, Li X, Zhao L, Zhang G, Wang J, Wei L. Nongenomic effect of estrogen on the MAPK signaling pathway and calcium influx in endometrial carcinoma cells. J Cell Biochem 2009; 106: 553-562.
  • 2Yu L, McPhee CK, Zheng L, Mardones GA, Rong Y, Peng J, et al. Termination of autophagy and reformation of lysosomes regulated by roTOR. Nature 2010; 465: 942-946.
  • 3Chen N, Karantza V. Autophagy as a therapeutic target in cancer. Cancer Biol Ther 2011 ; 11 : 157-168.
  • 4Janku F, McConkey D J, Hong DS, Kurzrock R. Autophagy as a target for anticancer therapy. Nat Rev Clin Oncol 2011; 8: 528-539.
  • 5Zhang J, Ren C, Chen L, Navedo MF, Antos LK, Kinsey SP, et al. Knockout of Na^+/Ca^2+exchanger in smooth muscle attenuates vasoconstriction and L-type Ca^2+ channel current and lowers blood pressure. Am J Physiol Cric Physiol 2010; 298: H472-H483.
  • 6Hφyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, et al. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell 2007; 25: 193-205.
  • 7Decuypere JP, Monaco G, Bultynck G, Missiaen L, De Smedt H, Parys JB. The 1P3 receptor-mitochondria connection in apoptosis and autophagy. Biochimica et Biophysica Acta 2011; 1813: 1003-1013.
  • 8Decuypere JP, Bultynck G, Parys JB. A dual role for Ca^2+ in autophagy regulation. Cell Calcium 2011, 50: 242-250.
  • 9Criollo A, Maiuri MC, Tasdemir E, Vitale I, Fiebig AA, Andrews D, et al. Regulation of autophagy by the inositol trisphosphate receptor. Cell Death Differ 2007; 14: 1029-1039.
  • 10Liu G, Hu X, Premkumar L, Chakrabarty S. Nifedipine synergizes with calcium in activating the calcium sensing receptor, suppressing the expression of thymidylate synthase and surviving and promoting sensitivity to fluorouraci! in human colon carcinoma cells. Mol Carcinog 2011; 50: 922-930.

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