BH3模拟物ABT737诱导人卵巢癌SKOV3/DDP细胞凋亡与自噬
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摘要
目的观察ABT737作用于人卵巢癌顺铂耐药SKOV3/DDP细胞后,细胞凋亡和自噬的发生,探讨抑制自噬后对凋亡的影响。方法体外培养SKOV3/DDP细胞,按ABT737药物终浓度0、2.5、5.0和10.0μmol/L分为4组,分别作用12和24 h。流式细胞术检测各组细胞凋亡率,分别采用免疫荧光和免疫印迹法(Western blot)检测各组细胞自噬相关蛋白LC3和Beclin-1表达水平。将细胞分为对照组、ABT737组、3-Ma组和联合用药组,分别以细胞培养基、ABT737(5.0μmol/L)、3-Ma(1.0 mmol/L)和ABT737联合3-Ma作用于SKOV3/DDP细胞,Western blot法检测12和24 h后凋亡相关蛋白active Caspase-3和cyto C的表达水平。结果与对照组比较,12和24 h各剂量ABT737组细胞凋亡率均明显增高,差异有统计学意义(P<0.01)。间接免疫荧光法检测LC3和Beclin-1蛋白表达,结果显示随着ABT737药物终浓度增加和作用时间的延长,两蛋白阳性表达亮度均越来越强,提示蛋白表达增强。Western blot检测LC3和Beclin-1蛋白表达水平,结果显示随着ABT737药物终浓度增加和作用时间的延长,细胞中LC3(F=10.878,F=13.256,P<0.05)和Beclin-1(F=6.589,F=7.365,P<0.05)蛋白表达水平均逐渐升高。3-Ma抑制自噬后,与ABT737组相比,联合用药组active Caspase-3 12和24 h表达水平明显增强,12和24 h cyto C表达水平也明显增强,差异有统计学意义(P<0.05)。结论 ABT737诱导人卵巢癌顺铂耐药SKOV3/DDP细胞凋亡的同时伴随着自噬的发生,抑制自噬能够促进凋亡。
Objective To observe the effect of ABT737 on apoptosis and autophagy in human ovarian cancer SKOV3/DDP cells,and to explore the effect of inhibiting autophagy on apoptosis. Methods The SKOV3/DDP cells were cultured with various concentrations ABT737 of 0,2.5,5.0,10.0 μmol/L for 12 and 24 h. The changes of apoptosis rates were detected by flow cytometry;the expressions of LC3 and Beclin-1 in various groups were detected by both indirect immunofluorescence method and Western blot. Then SKOV3/DDP cells were divided into control group,ABT737 group,3-Ma group and combined group,which were applied by cell culture medium, ABT737(5.0 μmol/L), 3-Ma(1.0 mmol/L)and ABT737 combined with 3-Ma.The expression levels of apoptosis related protein active Caspase-3 and cyto C were detected by Western blot. Result Compared with control group, the rates of apoptosis in 12 and 24 h groups were significantly increased. The immunofluorescence result showed that the expressions of LC3 and Beclin-1 were significantly increased in different doses of ABT737 groups with the increase of dose and time. The expression levels of LC3(F=10.878,F=13.256,P<0.05) and Beclin-1(F=6.589,F=7.365,P<0.05)were significantly increased in different doses of ABT737 groups with the increase of dose and time. Following the inhibition of autophagy by 3-Ma, compared with ABT737 group, the expression levels of active Caspase-3 12 and 24 h were significantly increased in combined group, and the expression levels of cyto C 12 and 24 h were also increased. Conclusion ABT737 can induce both apoptosis and autophagy in SKOV3/DDP cells, and the inhibition of autophagy promotes apoptosis.
Objective To observe the effect of ABT737 on apoptosis and autophagy in human ovarian cancer SKOV3/DDP cells,and to explore the effect of inhibiting autophagy on apoptosis. Methods The SKOV3/DDP cells were cultured with various concentrations ABT737 of 0,2.5,5.0,10.0 μmol/L for 12 and 24 h. The changes of apoptosis rates were detected by flow cytometry;the expressions of LC3 and Beclin-1 in various groups were detected by both indirect immunofluorescence method and Western blot. Then SKOV3/DDP cells were divided into control group,ABT737 group,3-Ma group and combined group,which were applied by cell culture medium, ABT737(5.0 μmol/L), 3-Ma(1.0 mmol/L)and ABT737 combined with 3-Ma.The expression levels of apoptosis related protein active Caspase-3 and cyto C were detected by Western blot. Result Compared with control group, the rates of apoptosis in 12 and 24 h groups were significantly increased. The immunofluorescence result showed that the expressions of LC3 and Beclin-1 were significantly increased in different doses of ABT737 groups with the increase of dose and time. The expression levels of LC3(F=10.878,F=13.256,P<0.05) and Beclin-1(F=6.589,F=7.365,P<0.05)were significantly increased in different doses of ABT737 groups with the increase of dose and time. Following the inhibition of autophagy by 3-Ma, compared with ABT737 group, the expression levels of active Caspase-3 12 and 24 h were significantly increased in combined group, and the expression levels of cyto C 12 and 24 h were also increased. Conclusion ABT737 can induce both apoptosis and autophagy in SKOV3/DDP cells, and the inhibition of autophagy promotes apoptosis.
引文
[1] Tanda ET, Budroni M, Cesaraccio R, et al. Epidemiology of ovarian cancer in North Sardinia, Italy, during the period 1992-2010[J]. Eur J Gynaecol Oncol, 2015, 36(1):69-72.
[2] Song H, Wei M, Liu W, et al. Cisplatin induced apoptosis of ovarian cancer A2780s cells by activation of ERK/p53/PUMA signals[J].Histol Histopathol, 2018, 33(1):73-79.
[3] Pannuru P, Dontula R, Khan AA, et al. miR-let-7f-1 regulates SPARC mediated cisplatin resistance in medulloblastoma cells[J].Cell Signal, 2014, 26(10):2193-2201.
[4] Kasaian J, Mosaffa F, Behravan J, et al. Modulation of multidrug resistance protein 2 efflux in the cisplatin resistance human ovarian carcinoma cells A2780/RCIS by sesquiterpene coumarins[J]. Phytother Res, 2016, 30(1):84-89.
[5] Bhatt M, Ivan C, Xie X, et al. Drug-dependent functionalization of wild-type and mutant p53 in cisplatin-resistant human ovarian tumor cells[J]. Oncotarget, 2017, 8(7):10905-10918
[6] Yasui K, Mihara S, Zhao C, et al. Alteration in copy numbers of genes as a mechanism for acquired drug resistance[J]. Cancer Res, 2004, 64(4):1403-1410.
[7] Ganesh S, Iyer AK, Weiler J, et al. Combination of siRNA-directed gene silencing with cisplatin reverses drug resistance in human non-small cell lung cancer[J]. Mol Ther Nucleic Acids, 2013, 2(7):e110.
[8] Sample A, He YY. Autophagy in UV damage response[J]. Photochem Photobiol, 2017, 93(4):943-955.
[9] Costa L, Amaral C, Teixeira N, et al. Cannabinoid-induced autophagy: Protective or death role? [J]. Prostaglandins Other Lipid Mediat, 2016,122(1):54-63.
[10] Tsujimoto Y, Finger LR, Yunis J, et al. Loning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation[J]. Science, 1984, 226(4678):1097-1099.
[11] Tsujimoto Y, Croce CM. Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma[J]. Proc Natl Acad Sci USA, 1986, 83(14):5214-5218.
[12] Huang K, Zhang J, O′Neill KL, et al. Cleavage by Caspase 8 and mitochondrial membrane association activate the BH3-only protein bid during TRAIL-induced apoptosis[J]. J Biol Chem, 2016, 291(22):11843-11851.
[13] Tomicic MT, Meise R, Aasland D, et al. Apoptosis induced by temozolomide and nimustine in glioblastoma cells is supported by JNK/c-Jun-mediated induction of the BH3-only protein BIM[J].Oncotarget, 2015, 6(32):33755-33768.
[14] Ebrahim AS, Sabbagh H, Liddane A, et al.Hematologic malignancies: newer strategies to counter the BCL-2 protein[J].J Cancer Res Clin Oncol, 2016, 142(9):2013-2022.
[15] Martina BP, Nina BB, Stefan M, et al. The BH3 mimetic drug ABT-737 induces apoptosis and acts synergistically with chemotherapeutic drugs in thyroid carcinoma cells [J]. Cancer Cell Int, 2016, 16(1):1-12.
[16] Wang S, Mao Y, Xi S, et al. Nutrient starvation sensitizes human ovarian cancer SKOV3 cells to BH3 mimetic via modulation of mitochondrial dynamics[J]. Anat Rec (Hoboken), 2017, 300(2):326-339.
[17] Swart C, Du Toit A, Loos B. Autophagy and the invisible line between life and death[J].Eur J Cell Biol, 2016, 95(12):598-610.
[18] Mowers EE, Sharifi MN, Macleod KF. Novel insights into how autophagy regulates tumor cell motility[J]. Autophagy, 2016, 12(9):1679-1680.
[2] Song H, Wei M, Liu W, et al. Cisplatin induced apoptosis of ovarian cancer A2780s cells by activation of ERK/p53/PUMA signals[J].Histol Histopathol, 2018, 33(1):73-79.
[3] Pannuru P, Dontula R, Khan AA, et al. miR-let-7f-1 regulates SPARC mediated cisplatin resistance in medulloblastoma cells[J].Cell Signal, 2014, 26(10):2193-2201.
[4] Kasaian J, Mosaffa F, Behravan J, et al. Modulation of multidrug resistance protein 2 efflux in the cisplatin resistance human ovarian carcinoma cells A2780/RCIS by sesquiterpene coumarins[J]. Phytother Res, 2016, 30(1):84-89.
[5] Bhatt M, Ivan C, Xie X, et al. Drug-dependent functionalization of wild-type and mutant p53 in cisplatin-resistant human ovarian tumor cells[J]. Oncotarget, 2017, 8(7):10905-10918
[6] Yasui K, Mihara S, Zhao C, et al. Alteration in copy numbers of genes as a mechanism for acquired drug resistance[J]. Cancer Res, 2004, 64(4):1403-1410.
[7] Ganesh S, Iyer AK, Weiler J, et al. Combination of siRNA-directed gene silencing with cisplatin reverses drug resistance in human non-small cell lung cancer[J]. Mol Ther Nucleic Acids, 2013, 2(7):e110.
[8] Sample A, He YY. Autophagy in UV damage response[J]. Photochem Photobiol, 2017, 93(4):943-955.
[9] Costa L, Amaral C, Teixeira N, et al. Cannabinoid-induced autophagy: Protective or death role? [J]. Prostaglandins Other Lipid Mediat, 2016,122(1):54-63.
[10] Tsujimoto Y, Finger LR, Yunis J, et al. Loning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation[J]. Science, 1984, 226(4678):1097-1099.
[11] Tsujimoto Y, Croce CM. Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma[J]. Proc Natl Acad Sci USA, 1986, 83(14):5214-5218.
[12] Huang K, Zhang J, O′Neill KL, et al. Cleavage by Caspase 8 and mitochondrial membrane association activate the BH3-only protein bid during TRAIL-induced apoptosis[J]. J Biol Chem, 2016, 291(22):11843-11851.
[13] Tomicic MT, Meise R, Aasland D, et al. Apoptosis induced by temozolomide and nimustine in glioblastoma cells is supported by JNK/c-Jun-mediated induction of the BH3-only protein BIM[J].Oncotarget, 2015, 6(32):33755-33768.
[14] Ebrahim AS, Sabbagh H, Liddane A, et al.Hematologic malignancies: newer strategies to counter the BCL-2 protein[J].J Cancer Res Clin Oncol, 2016, 142(9):2013-2022.
[15] Martina BP, Nina BB, Stefan M, et al. The BH3 mimetic drug ABT-737 induces apoptosis and acts synergistically with chemotherapeutic drugs in thyroid carcinoma cells [J]. Cancer Cell Int, 2016, 16(1):1-12.
[16] Wang S, Mao Y, Xi S, et al. Nutrient starvation sensitizes human ovarian cancer SKOV3 cells to BH3 mimetic via modulation of mitochondrial dynamics[J]. Anat Rec (Hoboken), 2017, 300(2):326-339.
[17] Swart C, Du Toit A, Loos B. Autophagy and the invisible line between life and death[J].Eur J Cell Biol, 2016, 95(12):598-610.
[18] Mowers EE, Sharifi MN, Macleod KF. Novel insights into how autophagy regulates tumor cell motility[J]. Autophagy, 2016, 12(9):1679-1680.
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