吉西他滨联合热疗对胰腺癌ASPC-1细胞增殖、迁移、侵袭和凋亡的影响及机制研究

Effects of gemcitabine combined with hyperthermia on proliferation,migration,invasion and apoptosis of pancreatic cancer ASPC-1 cells and its mechanism

目的:探讨吉西他滨(GEM)联合热疗对胰腺癌ASPC-1细胞增殖、迁移、侵袭和凋亡的影响及可能的机制。方法:将ASPC-1细胞随机分为对照组(无任何处理)、GEM组(给予30μmol/L GEM)和热化疗组(给予热疗联合30μmol/L GEM)。采用MTT法检测细胞增殖及活力情况。采用划痕实验和Transwell实验检测细胞迁移和侵袭能力。流式细胞仪检测细胞凋亡情况。采用Westerm blot和实时荧光定量PCR(RT-qPCR)检测特异性蛋白1(Sp1)、细胞周期蛋白D1(Cyclin D1)、环氧合酶-2(COX-2)、B细胞淋巴瘤-2(Bcl-2)蛋白及mRNA表达情况。结果:30μmol/L GEM对ASPC-1细胞增殖有抑制作用,热化疗组抑制作用更明显(均P<0.05)。GEM前24 h给予43℃热疗1 h对细胞抑制作用更明显(均P<0.05)。与对照组比较,GEM组和热化疗组细胞迁移能力降低,且热化疗组降低更加明显(均P<0.05)。GEM组和热化疗组细胞侵袭能力较对照组降低,且热化疗组更明显(均P<0.05)。与对照组比较,GEM组和热化疗组细胞凋亡率升高,且热化疗组高于GEM组(均P<0.05)。与对照组比较,GEM组细胞Sp1、COX-2、Bcl-2蛋白水平降低(均P<0.05)。与GEM组比较,热化疗组Sp1、COX-2蛋白水平降低(均P<0.05)。与对照组比较,GEM组细胞Sp1、COX-2 mRNA表达降低(均P<0.05)。与GEM组比较,热化疗组Sp1、COX-2 mRNA表达降低(均P<0.05)。结论:GEM联合热疗可抑制胰腺癌ASPC-1细胞增殖、迁移和侵袭,并诱导凋亡,其机制可能与抑制Sp1及其下游基因COX-2有关。

Objective:To investigate the effects of gemcitabine(GEM)combined with hyperthermia on the proliferation,migration,invasion and apoptosis of pancreatic cancer ASPC-1 cells and its possible mechanism.Methods:ASPC-1 cells were randomly divided into control group(without any treatment),GEM group(given 30μmol/L GEM)and thermochemotherapy group(given hyperthermia combined with 30μmol/L GEM).Cell proliferation and viability were detected by MTT assay.Wound healing assay and Transwell assay were used to detect cell migration and invasion.Apoptosis was assessed by flow cytometry.Western blot and RT-qPCR were used to detect the protein and mRNA expressions of Sp1,Cyclin D1,COX-2,and Bcl-2.Results:GEM(30μmol/L)inhibited the proliferation of ASPC-1 cells,especially in the thermochemotherapy group(all P<0.05).Hyperthermia at 43℃for 1 hour at 24 hours before GEM had a more significant inhibitory effect on the cells(all P<0.05).Compared with the control group,the cell migration ability was decreased in GEM group and thermochemotherapy group,and the decrease was more obvious in thermochemotherapy group(all P<0.05).The invasive ability of GEM group and thermochemotherapy group was lower than that of the control group,and the thermochemotherapy group was more obvious(all P<0.05).Compared with the control group,the apoptosis rate of GEM group and thermochemotherapy group was increased,and the thermochemotherapy group was higher than GEM group(all P<0.05).Compared with the control group,the protein levels of Sp1,COX-2 and Bcl-2 in the GEM group were decreased(all P<0.05).Compared with the GEM group,the protein levels of Sp1 and COX-2 in the thermochemotherapy group were decreased(all P<0.05).Compared with the control group,the mRNA expression of Sp1 and COX-2 in the GEM group decreased(both P<0.05).Compared with the GEM group,the mRNA expression of Sp1 and COX-2 in the thermochemotherapy group decreased(all P<0.05).Conclusion:GEM combined with hyperthermia can inhibit the proliferation,migration and invasion and induce apoptosis of pancreatic cancer ASPC-1 cells possibly by inhibiting Sp1 and its downstream gene COX-2.