| 柴效科,周聪,宋爱琳.双硫仑螯合铜对耐紫杉醇乳腺癌细胞增殖、迁移、凋亡及自噬的影响[J].安徽医药,2022,26(12):2383-2389. |
| 双硫仑螯合铜对耐紫杉醇乳腺癌细胞增殖、迁移、凋亡及自噬的影响 |
| Effect of DSF-Cu on the proliferation, migration, apoptosis and autophagy of paclitaxel-resistant breast cancer cells |
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| DOI：10.3969/j.issn.1009-6469.2022.12.011 |
| 中文关键词: 乳腺肿瘤 双硫仑 紫杉醇 抗药性，肿瘤 bcl-2相关 X蛋白质 自噬相关蛋白质类 |
| 英文关键词: Breast neoplasms Disulfiram Paclitaxel Drug resistance, neoplasm Bcl-2-associated X protein Autophagy-related protein |
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| 中文摘要: |
| 目的探讨双硫仑螯合铜( DSF-Cu)对耐紫杉醇乳腺癌细胞增殖、迁移、凋亡及自噬的影响。方法 2018年 1月至 2019年 10月，培养 MCF-7人乳腺癌细胞和耐紫杉醇乳腺癌 MCF-7/紫杉醇细胞，用定量紫杉醇持续诱导 MCF-7/紫杉醇细胞维持耐药性。通过 CCK-8法检测 MCF-7/紫杉醇细胞耐药指数；通过 CCK-8法检测 MCF-7/紫杉醇细胞在不同浓度 DSF-Cu(5、10、 15、20、25 μmol/L)干预不同时间( 24 h和 48 h)后的抑制率，并分析细胞抑制率与 DSF-Cu浓度和作用时间的关系；通过划痕实验检测 DSF-Cu对 MCF-7/紫杉醇细胞迁移能力的影响；通过实时荧光定量逆转录聚合酶链反应( qRT-PCR)和蛋白质印迹法检测 MCF-7和 MCF-7/紫杉醇细胞中 B细胞淋巴瘤 -2(Bcl-2)、 Bcl-2相关 X蛋白( Bax)和自噬效应蛋白 Beclin-1基因和蛋白的表达，并在紫杉醇和 DSF-Cu单药及联合干预 MCF-7/紫杉醇细胞中后，检测 Bcl-2、Bax和 Beclin-1蛋白的表达；通过流式细胞计数技术检测不同浓度 DSF-Cu(5、10、15、20 μmol/L)干预 MCF-7/紫杉醇细胞不同时间( 12 h、24 h和 48 h)后细胞凋亡情况。结果 MCF-7和 MCF-7/紫杉醇细胞在紫杉醇干预 24 h后 IC50值分别为 1 394.79 μmol/L和 321.42 μmol/L，MCF-7/紫杉醇细胞耐药指数为 4.34；在不同浓度 DSF-Cu(5、10、15、20、25 μmol/L)DSF-Cu干预 MCF-7/紫杉醇细胞 24 h后细胞抑制率分别为( 27.42±3.38)%、(42.71±0.77)%、(57.52±7.10)%、(73.83±1.76)%、(85.55±2.25)%，48 h后细胞抑制率分别为( 51.27±4.59)%、(57.34±5.94)%、(71.50±1.80)%、(81.54±4.36)%、(90.96±2.13)%，DSF-Cu以浓度与时间依赖性抑制乳腺癌 MCF-7/紫杉醇细胞增殖( P<0.05)；划痕实验证实 DSF-Cu能抑制乳腺癌 MCF-7/紫杉醇细胞迁移； qRT-PCR结果显示，与 MCF-7细胞相比， MCF-7/紫杉醇细胞中 Bcl-2和 Beclin-1基因表达升高， Bax基因表达降低( P<0.05)；蛋白质印迹法结果显示，与 MCF-7细胞比较， MCF-7/紫杉醇细胞中 Bcl-2和 Beclin-1蛋白表达升高， Bax蛋白表达降低( P<0.05)。 MCF-7/紫杉醇细胞在紫杉醇和 DSF-Cu单药及联合干预后 Bcl-2表达分别为 1.81±0.28、1.25±0.09、0.87±0.10，Bax表达分别为 0.65±0.15、1.07±0.15、1.34±0.04，Beclin-1表达分别为 1.53±0.22、0.85±0.18、0.53±0.08，MCF-7/紫杉醇细胞在 DSF-Cu干预后 Bax蛋白表达升高， Bcl-2和 Beclin-1蛋白表达降低( P<0.05)；式结果显示不同浓度 DSF-Cu(0、5、10、15、20 μmol/L)干预 MCF-7/紫杉醇细胞 24 h后，细胞凋亡率分别为( 5.50±1.01)%、流(11.03±1.04)%、(55.12±5.06)%、(68.86±3.36)%、(80.07±4.30)%，在 IC50浓度的 DSF-Cu干预 MCF-7/紫杉醇细胞不同时间( 0h、 12 h、24 h、48 h)后，细胞凋亡率分别为( 4.47±0.57)%、(15.92±2.63)%、(34.85±2.70)%、(74.94±1.13)%，MCF-7/紫杉醇在 DSF-Cu干预后细胞凋亡情况与 DSF-Cu呈浓度和时间依赖性( P<0.05)。结论 DSF-Cu可通过诱导凋亡、抑制自噬来抑制乳腺癌 MCF-7/紫杉醇细胞增殖和迁移，逆转乳腺癌 MCF-7细胞对紫杉醇耐药性。 |
| 英文摘要: |
| Objective To investigate the effect of disulfiram chelated with Cu2+ (DSF-Cu) on the proliferation, migration, apoptosis and autophagy of paclitaxel-resistant breast cancer cells.Methods From January 2018 to October 2019, MCF-7 human breast cancer cells and paclitaxel-resistant breast cancer MCF-7/paclitaxel cells were cultured, and MCF-7/paclitaxel cells were continuously in.duced to maintain drug resistance with quantitative paclitaxel. The drug resistance index of MCF-7/paclitaxel cells was detected by CCK-8 assay; the inhibition rate of MCF-7/paclitaxel cells after different concentrations of DSF-Cu (5, 10, 15, 20, 25 μmol/L) interven. tions for different times (24 h and 48 h) was detected by CCK-8 assay, and the relationship between cell inhibition rate and DSF-Cu concentration and duration of action was analyzed; the inhibition rate of DSF-Cu on the migration ability of MCF-7/paclitaxel cells was examined by scratch assay. Real-time quantitative polymerase chain reaction (qRT.PCR) and Western blotting were used to detect the expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and Beclin gene and protein in MCF-7 and MCF-7/paclitax. el cells after combined intervention in MCF-7/paclitaxel, and Bcl-2, Bax and Beclin-1 protein expression in MCF-7/paclitaxel cells af. ter single and combined intervention with paclitaxel and DSF-Cu. Apoptosis was detected by flow cytometry after different concentra. tions of DSF-Cu (5, 10, 15 and 20 μmol/L) interventions on MCF-7/paclitaxel cells at different times (12 h, 24 h and 48 h).Results The IC50 values of MCF-7 and MCF-7/paclitaxel cells were 1 394.79 μmol/L and 321.42 μmol/L, respectively. After 24 h of paclitaxel intervention, the MCF-7/paclitaxel cell resistance index was 4.34. The inhibition rates of MCF-7/paclitaxel cells were (27.42±3.38)%,(42.71±0.77)%, (57.52±7.10)%, (73.83±1.76)%, and (85.55±2.25)% after 24 h of intervention with different concentrations of DSF-Cu (5, 10, 15, 20, 25 μmol/L), and were (51.27±4.59)%, (57.34±5.94)%, (71.50±1.80)%, (81.54±4.36)%, and (90.96±2.13)% after 48 h.DSF-Cu inhibited the proliferation of breast cancer MCF-7/paclitaxel cells in a concentration-and time-dependent manner (P<0.05). Scratch assays confirmed that DSF-Cu inhibited breast cancer MCF-7/paclitaxel cell migration; qRT.PCR results showed that Bcl-2 and Beclin-1 gene expression was elevated and Bax gene expression was decreased in MCF-7/paclitaxel cells compared with MCF-7 cells (P<0.05). The Western blotting results showed elevated Bcl-2 and Beclin-1 protein expression and decreased Bax protein expres. sion in MCF-7/paclitaxel cells compared with MCF-7 cells (P<0.05). In MCF-7/paclitaxel cells, after DSF-Cu and Taxol single drug and combined intervention, the expression of Bcl-2 was 07±0.15 and 1.34±0.04, and the expression of Beclin-1 was 1.53±0.22, 0.85±0.18, and 0.53±0.08 after paclitaxel and DSF-Cu single and combined interventions, respectively. MCF-7/paclitaxel cells showed elevated Bax protein expression and decreased Bcl-2 and Beclin-1 protein expression after DSF-Cu intervention (P<0.05). Flow cytometry showed that after 24 h of intervention with different concentrations of DSF-Cu (0, 5, 10, 15, 20 μmol/L) in MCF-7/paclitaxel cells, theapoptosis rates were (5.50±1.01)%, (11.03±1.04)%, (55.12±5.06)%, (68.86±3.36)%, and (80.07±4.30)%, respectively. The apoptosisrates were (4.47±0.57)%, (15.92±2.63)%, (34.85±2.70)% and (74.94±1.13)%, respectively, after DSF-Cu intervention of MCF-7/pacli. taxel cells at different times (0 h, 12 h, 24 h, 48 h) at the IC50 concentration. The apoptosis of MCF-7/paclitaxel cells after DSF-Cu inter. vention was concentration-and time-dependent with DSF-Cu (P<0.05).Conclusion DSF-Cu can inhibit the proliferation and migra. tion of breast cancer MCF-7/paclitaxel cells by inducing apoptosis and inhibiting autophagy and can reverse paclitaxel resistance in breast cancer MCF-7 cells |
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