摘要
目的 探究槲皮素与去铁胺联合使用对于宫颈癌细胞增殖、细胞周期及细胞凋亡的影响.方法 人正常宫颈上皮细胞End Ⅰ,人宫颈癌细胞HeLa、C33A购自美国典型培养物保藏中心.采用总铁离子比色法检测End Ⅰ、HeLa和C33A中铁离子水平.将宫颈癌细胞分为空白对照组、溶剂对照组、去铁胺处理组、槲皮素处理组、槲皮素联合去铁胺处理组,采用总铁离子比色法比较各组铁离子浓度,细胞计数试剂盒检测细胞增殖、计算药物联合指数,流式细胞术检测细胞周期及凋亡,蛋白质免疫印记实验检测周期及凋亡相关蛋白表达水平.转录组测序探讨槲皮素联合去铁胺对宫颈癌细胞作用的可能机制.两组样本采用独立样本t检验进行显著性分析,多组样本之间的显著性分析采用单因素方差分析.结果 HeLa和C33A细胞中铁离子浓度均高于End Ⅰ细胞[(40.67±1.93)μmol/L、(36.93±2.40)µmol/L 比(18.40±1.00)µmol/L,t=-17.718、-12.337,P＜0.05].HeLa、C33A细胞中槲皮素联合去铁胺组铁离子水平低于对照组及单药处理组[(20.80±1.08)µmol/L 比(40.63±3.65)µmol/L、(25.73±2.11)μmol/L、(31.90±3.44)µmol/L,(20.00±1.10)μmol/L 比(37.23±1.17)μmol/L、(26.17±1.26)µmol/L、(30.50±1.50)µmol/L,F=35.929、108.954,P＜0.05],细胞吸光度低于对照组及单药处理组[1.35±0.03 比 2.56±0.01、2.32±0.09、2.19±0.11,0.89±0.05 比 1.77±0.05、1.47±0.03、1.21±0.06,F=168.152、184.352,P＜0.05],并且具有协同作用[联合指数(CI)＜1].槲皮素联合去铁胺组G1/S期的HeLa细胞比例高于对照组及单药处理组[(86.10±1.65)％比(71.90±0.66)％、(75.1±0.87)％、(81.93±1.42)％,F=95.119,P＜0.05],p21 蛋白表达水平高于对照组(2.48±0.15比1.00±0.18,t=-10.911,P＜0.05),细胞周期蛋白依赖性激酶4(CDK4)、细胞周期蛋白依赖性激酶6(CDK6)和细胞周期蛋白D1(CCND1)的表达低于对照组(0.19±0.07比1.00±0.02、0.52±0.01 比 1.00±0.08、0.22±0.11 比 1.00±0.30,t=19.071、10.654、4.201,P＜0.05).槲皮素联合去铁胺组HeLa细胞凋亡比例高于对照组及槲皮素处理组[(23.43±0.41)％比(6.31±0.26)％、(19.60±1.73)％,F=308.920,P＜0.05],半胱氨酰天冬氨酸特异性蛋白酶-3前体(pro Caspase-3)、B细胞淋巴瘤/白血病-2(bcl-2)蛋白的表达量低于对照组(0.48±0.05比1.00±0.14、0.25±0.11 比 1.00±0.12,t=6.181、8.239,P＜0.05),活化半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved Caspase-3)和bcl-2关联X蛋白(bax)蛋白的表达高于对照组(3.99±0.28比1.00±0.11、1.78±0.28 比 0.98±0.04,t=-17.079、-4.930,P＜0.05).京都基因与基因组百科全书数据库(KEGG)富集分析显示,槲皮素联合去铁胺处理组差异基因IGFBP3、CCND1、CASP3、CDK4、BAX等在p53信号通路有显著富集(P＜0.05),p53蛋白表达水平高于对照组(3.39±0.47比1.00±0.08,t=-8.728,P＜0.05).结论 槲皮素联合去铁胺能够协同降低宫颈癌细胞内铁离子水平、抑制细胞增殖,通过p53信号通路诱导细胞周期阻滞及细胞凋亡.
Abstract
Objective To investigate the effects of the combination of quercetin and deferoxamine on cell proliferation,cell cycle and apoptosis in cervical cancer cells.Methods The human normal cervi-cal epithelial cells(End Ⅰ)and the human cervical cancer cells(HeLa and C33A)were obtained from the american type culture collection(ATCC).The iron ion levels in End Ⅰ,HeLa and C33A were quantified using the total iron ion colourimetric assay.The cervical cancer cells were subdivided into the following ex-perimental groups:blank control group,solvent control group,deferoxamine-treated group,quercetin-trea-ted group and quercetin-combined deferoxamine-treated group.A total iron ion colourimetric assay was em-ployed to compare the iron ion concentration of each experimental group.Cell counting kit-8 was utilised to detect the impact of the drug combination on cell proliferation and calculate the drug combination index.Flow cytometry was used to detect cell cycle and apoptosis,while western blotting was employed to detect the expression levels of proteins associated with cell cycle and apoptosis.RNA-seq was used to investigate the possible mechanisms of action of quercetin combined with deferoxamine on cervical cancer cells.Signif-icance analyses were conducted using independent samples t-test for two groups of samples and one-way ANOVA for multiple groups of samples.Results Iron ion concentrations were higher in HeLa and C33A cells than in End Ⅰ cells[(40.67±1.93)μmol/L,(36.93±2.40)μmol/L vs.(18.40±1.00)μmol/L,t=-17.718,-12.337,P＜0.05].In HeLa and C33A cells,the quercetin-combined deferoxamine-treated group exhibited lower levels of iron ions[(20.80±1.08)μmol/L vs.(40.63±3.65)μmol/L,(25.73±2.11)μmol/L,(31.90±3.44)μmol/L,(20.00±1.10)μmol/L vs.(37.23±1.17)μmol/L,(26.17±1.26)μmol/L,(30.50±1.50)μmol/L,F=35.929,108.954,P＜0.05]and reduced cellular absorbance[1.35±0.03 vs.2.56±0.01,2.32±0.09,2.19±0.11,0.89±0.05vs.1.77±0.05,1.47±0.03,1.21±0.06,F=168.152,184.352,P＜0.05]relative to the control and mono-treatment groups,demonstrating a synergistic effect[combination index(CI)＜1].In the quercetin-combined def-eroxamine-treated group,a higher proportion of HeLa cells was observed in the Gt/S phase in comparison to the control and single agent treatment groups[(86.10±1.65)％vs.(71.90±0.66)％,(75.1±0.87)％,(81.93±1.42)％,F=95.119,P＜0.05],while the p21 protein was more highly expressed(2.48±0.15 vs.1.00±0.18,t=-10.911,P＜0.05),and the expression of cyclin-dependent kinase 4(CDK4),cyclin-dependent kinase 6(CDK6)and cyclin D1(CCND1)was lower than in the control group(0.19±0.07 vs.1.00±0.02,0.52±0.01 vs.1.00±0.08,0.22±0.11 vs.1.00±0.30,t=19.071,10.654,4.201,P＜0.05).The combined treatment with quercetin and deferoxamine resulted in a higher percentage of HeLa cell apoptosis compared to the control and quercetin-treated groups[(23.43±0.41)％vs.(6.31±0.26)％,(19.60±1.73)％,F=308.920,P＜0.05],while the expression of precursor cysteinyl aspartate-specific protease-3(pro-Caspase-3)and B cell lymphoma/leukemia-2(bcl-2)was lower than the control(0.48±0.05 vs.1.00±0.14,0.25±0.11 vs.1.00±0.12,t=6.181,8.239,P＜0.05)and the expression of cleaved Caspase-3 and bcl-2 Associated X protein(bax)was higher(3.99±0.28 vs.1.00±0.11,1.78±0.28 vs.0.98±0.04,t=-17.079,-4.930,P＜0.05).Kyoto Encyclopedia of Genes and Genomes database(KEGG)enrichment analysis showed that the differential genes in the quercetin-combined deferoxamine-treated group including IGFBP3,CCND1,CASP3,CDK4,BAX were significantly enriched in the p53 pathway,and the p53 protein expression level was higher than that in the control group(3.39±0.47 vs.1.00±0.08,t=-8.728,P＜0.05).Conclusion The combination of quercetin and deferoxamine demonstrated a synergistic effect in reducing intracellular iron ion levels and in-hibiting cell proliferation in cervical cancer cells.Furthermore,this combination induced cell cycle arrest and apoptosis through the p53 signaling pathway.
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