大蒜素对子宫内膜癌Ishikawa细胞顺铂耐药株增殖和凋亡及侵袭与迁移的影响

Effects of allicin on the proliferation,apoptosis,invasion and metastasis of cisplatin-resistant strains of endometrial carcinoma Ishikawa cells

目的探讨大蒜素对子宫内膜癌顺铂(DDP)耐药细胞株Ishikawa/DDP生物学行为的影响。方法建立子宫内膜癌DDP耐药细胞株,设对照组、DDP处理组、低剂量大蒜素+DDP处理组、中剂量大蒜素+DDP处理组和高剂量大蒜素+DDP处理组,通过细胞计数实验(CCK8)、Transwell实验、划痕实验、流式细胞术检测各组细胞的增殖、侵袭、迁移和凋亡,采用RT-qPCR实验、蛋白质印迹实验分别检测各组细胞中多药耐药基因1(MDR1)mRNA、P-gp蛋白表达水平。结果低剂量大蒜素+DDP处理组、中剂量大蒜素+DDP处理组和高剂量大蒜素+DDP处理组细胞增殖抑制率分别为(20.92±3.28)%、(31.71±2.82)%和(47.34±3.43)%,随着大蒜素浓度增加,细胞增殖抑制率上升,差异有统计学意义,F=346.231,P=0.007。Transwell实验表明,对照组、DDP处理组、低剂量大蒜素+DDP处理组、中剂量大蒜素+DDP处理组和高剂量大蒜素+DDP处理组侵袭细胞数分别为(314.00±7.53)、(278.00±6.52)、(203.00±5.46)、(109.00±5.32)和(65.00±3.41)个,随着大蒜素浓度增加,细胞侵袭力下降,差异有统计学意义,F=437.807,P=0.009。对照组、DDP处理组、低剂量大蒜素+DDP处理组、中剂量大蒜素+DDP处理组和高剂量大蒜素+DDP处理组48 h划痕宽度相对比例分别为(31.48±9.28)%、(33.76±7.43)%、(42.76±5.34)%、(53.56±7.45)%和(64.431±8.32)%,随着大蒜素浓度增加,细胞迁移能力下降,差异有统计学意义,F=345.126,P=0.005。流式细胞术结果表明,对照组、DDP处理组、低剂量大蒜素+DDP处理组、中剂量大蒜素+DDP处理组和高剂量大蒜素+DDP处理组细胞凋亡率分别为(11.32±1.13)%、(13.51±0.83)%、(21.30±1.56)%、(37.64±2.21)%和(51.31±2.42)%,随着大蒜素浓度增加,细胞凋亡率显著提高,组间均值差异有统计学意义,F=423.012,P=0.008。RT-qPCR结果显示,对照组、DDP处理组、低剂量大蒜素+DDP处理组、中剂量大蒜素+DDP处理组和高剂量大蒜素+DDP处理组MDR1 mRNA相对表达量分别为48.16±0.53、42.38±1.52、30.45±0.46、21.34±0.32、12.36±0.41,随着大蒜素浓度增加,MDR1 mRNA表达下降,组间均值差异有统计学意义,F=345.014,P=0.006。蛋白质印迹实验显示,对照组、DDP处理组、低剂量大蒜素+DDP处理组、中剂量大蒜素+DDP处理组和高剂量大蒜素+DDP处理组P-gp蛋白表达水平分别为0.86±0.04、0.67±0.03、0.49±0.05、0.27±0.04、0.07±0.01,随着大蒜素浓度增加,P-gp蛋白表达下降,组间均值差异有统计学意义,F=445.017,P=0.006。结论大蒜素能够抑制子宫内膜癌DDP耐药株Ishikawa/DDP增殖、侵袭和迁移的能力,同时促进肿瘤细胞凋亡,可能通过下调MDR1 mRNA以及P-gp表达逆转Ishikawa/DDP细胞DDP耐药性。

Objective To investigate the effect of allicin on the biological behavior of endometrial cancer cisplatin(DDP)-resistant cell line Ishikawa/DDP.Methods Cisplatin-resistant cell strains cultures of endometrial carcinoma were respectively constituted into control,cisplatin-treated,(low-dose allicin+cisplatin)-treated,(medium-dose allicin+cisplatin)-treated,and(high-dose allicin+cisplatin)-treated groups.The proliferation,invasion,metastasis and apoptosis of the cells in each group were detected by CCK8,transwell assay,scratch assay and flow cytometry,and the expression levels of MDR1 mRNA and P-gp protein in each group were detected by RT-qPCR and western blot assay,respectively.Results The inhibition rates of cell proliferation in the low-dose allicin+DDP-treated group,the medium-dose allicin+DDP-treated group and the high-dose allicin+DDP-treated group were(20.92±3.28)%,(31.71±2.82)%and(47.34±3.43)%,respectively,and the inhibition rates of cell proliferation increased with the increase of allicin concentration,and the difference was significant(F=346.231,P=0.007).Transwell experiments showed that the number of invading cells in the control,DDP-treated,low-dose allicin+DDP-treated,medium-dose allicin+DDP-treated and high-dose allicin+DDP-treatedgroupswere(314.00±7.53),(278.00±6.52),(203.00±5.46),(109.00±5.32)and(65.00±5.32),respectively.The difference was statistically significant(F=437.807,P=0.009).The scratch test showed that the relative proportions of scratch widths after 48 h in the control,DDP-treated,low-dose allicin+DDP-treated,medium-dose allicin+DDP-treated and high-dose allicin+DDP-treated groups were(31.48±9.28)%,(33.76±7.43)%,(42.76±5.34)%,(53.56±7.45)%and(64.43±8.32)%,respectively,and the cell migration capacity decreased with increasing allicin concentration,with statistically significant differences(F=345.126,P=0.005).The flow cytometry results showed that the apoptosis rates in the control,DDP-treated,low-dose allicin+DDP-treated,medium-dose allicin+DDPtreated and high-dose allicin+DDP-treated groups were(11.32±1.13)%,(13.51±0.83)%,(21.30±1.56)%,(37.64±2.21)%and(51.31±2.42)%,the apoptosis rate increased significantly with increasing allicin concentration and the difference was statistically significant(F=423.012,P=0.008).RT-qPCR results showed that the relative expression of MDR1 mRNA in the control,DDP-treated,low-dose allicin+DDP-treated,medium-dose allicin+DDP-treated and highdose allicin+DDP-treated groups were 48.16±0.53,42.38±1.52,30.45±0.46,21.34±0.32,12.36±0.41,respectively,and MDR1 mRNA expression decreased with increasing allicin concentration,with statistically significant differences(F=345.014,P=0.006).Protein blotting experiments showed that the control,DDP-treated,and low dose allicin+DDP treated group,medium dose allicin+DDP treated group and high dose allicin+DDP treated group had P-gp protein expression levels of 0.86±0.04,0.67±0.03,0.49±0.05,0.27±0.04 and 0.07±0.01,respectively,with the increase of allicin concentration,the P-gp protein expression decreased and the difference was statistically significant(F=445.017,P=0.006).Conclusion Allicin inhibits the ability of endometrial cancer DDP-resistant strain Ishikawa/DDP to proliferate,invade and migrate while promoting apoptosis in tumor cells,and may reverse DDP resistance in Ishikawa/DDP cells by downregulating MDR1 as well as P-gp expression.