MnSOD表达特性与食管癌细胞放射敏感性的关系

Relationship between characteristics of MnSOD expression and radiosensitivity in esophageal cancer cells

目的:研究锰超氧化物歧化酶(manganese superoxide dismutase,MnSOD)在一氧化氮(mtric oxide,NO)作用下对食管癌TE-1细胞放射敏感性的影响。方法:采用慢病毒转染法将MnSOD重组质粒转染食管癌TE-1细胞,分别建立中、高水平过量表达MnSOD的稳定转染细胞(TE-1Mm、TE-1Mh)及空载体细胞(TE-1Mn)。进一步采用RT-PCR、Western blot检测转染后TE-1细胞、空载体TE-1Mn细胞、稳定转染细胞TE-1Mm及TE-1Mh中MnSOD mRNA和蛋白的表达;四甲基偶氮唑蓝(MTT)比色实验评价NO供体硝普钠(sodium nitroprusside,SNP)、放射线及二者联合对以上4组细胞的抑制;流式细胞术(FCM)及Western blot检测各组细胞凋亡、活性氧(reactive oxygen species,ROS)荧光强度及蛋白表达的变化。结果:成功建立了稳定中、高水平过量表达MnSOD的TE-1细胞株TE-1Mm和TE-1Mh;RT-PCR及Western blot证实上述2种细胞中含有不同表达水平的MnSOD。MTT法及FCM显示,TE-1Mm细胞的存活率下降,细胞凋亡率上调,而TE-1Mh细胞的存活率上调,细胞凋亡率下降,相应组间相比差异具有统计学意义(P<0.05)。0.5～2mmol/L SNP及2、4、6Gy放射线处理48 h,TE-1Mm细胞生长抑制率明显增加,细胞生长明显减慢,放射增敏比增加,细胞凋亡率上调;TE-1Mh细胞抑制率反而降低,细胞生长加速,放射增敏比降低,细胞凋亡率下降,相应组间相比差异均具有统计学意义(P<0.05)。Western blot结果及FCM显示:0.5～2 mmol/L SNP及2、4、6 Gy放射线处理48 h,与TE-1Mm细胞相比,TE-1Mh细胞MnSOD蛋白表达量及细胞内ROS相对降低,相应组间相比差异均具有统计学意义(P<0.05)。结论:中等水平过量表达MnSOD增加食管癌细胞放射敏感性,抑制增殖,诱导凋亡,而高水平过量表达MnSOD则与之相反,为今后通过MnSOD提高放射敏感性来抑制食管癌或通过其抗拒放射来保护正常细胞提供了可能性。

OBJECTIVE：To explore effects of manganese superoxide dismutase（MnSOD） overexpression on radiosensitivity in the esophageal cancer cell line under the action of nitric oxide（NO）.METHODS：Overexpression of MnSOD cDNA was obtained by lentivirus to get two stable transfected TE-1 cell lines of MnSOD with moderate to high expression（TE-1 Mm,TE-1Mh） and empty vector cell（TE-1Mn）.Reverse transcription polymerase chain reaction（RTPCR） and western blot were then introduced to detect the target genes with respect to its expression in esophageal TE-1 cells.Additionally,colorimetric 3-[4,5-dimethy thiazol-2-yl]-2,5-diphenyltetrazolium bromide（MTT） assay,flow cytometry（FCM） and western blot were also used to assess the influence of the moderate to high overexpression of MnSOD that might be found on proliferation of TE-1 cells treated with sodium nitroprusside and irradiation.RESULTS：RT-PCR and western blot showed that TE-1 cells transfected with MnSOD cDNA contained the target genes at different levels,suggesting that esophageal cancer cell of stable transfection had been successfully constructed.MTT and FCM revealed that moderate overexpression of MnSOD decreased growth rates,increased apoptosis;while high overexpression of MnSOD increased growth rates and decreased apoptosis.When combined with radiation or NO,moderate overexpression of MnSOD reduced growth rates,enhanced cell apoptosis and radiosensitivity of esophageal cancer cells,whereas high MnSOD overexpression had the opposite effect.Western blot and FCM demonstrated that TE-1Mh cells decreased MnSOD protein expression and ROS levels treated with radiation or NO compared with TE-1Mm cell.CONCLUSION：Moderate overexpression of MnSOD increased the radiosensitivity of esophageal cancer cells,whereas high MnSOD overexpression had the opposite effect on cell radiosensitivity.This finding suggests a potential new method to kill certain radioresistant tumors and to provide radioresistance to normal cells.