低糖联合棕榈酸通过诱导活性氧产生和DNA损伤促进结直肠癌细胞放射增敏

Low-glucose combined with palmitic acid enhances the radiosensitivity of colorectal cancer through inducing reactive oxygen species generation and DNA damage

目的探讨低糖联合棕榈酸的代谢环境对人结直肠癌细胞SW480的抑制作用及其促进放射增敏的机制研究。方法在低糖、棕榈酸、低糖联合棕榈酸的处理下CCK-8筛选明显抑制SW480细胞增殖的处理条件。通过流式细胞术检测细胞活性氧(ROS)水平、线粒体膜电位及凋亡变化;免疫荧光γ-H2AX染色及克隆形成实验检测放射敏感性变化;蛋白印迹法检测蛋白质水平变化。结果与对照组相比,低糖联合120μmol/L棕榈酸处理显著抑制SW480细胞增殖(P<0.01);CPT1a、PFKFB3、PKM表达增加,NDUFV1、NDUFV2、NDUFS1表达减少;ROS水平增加(P<0.01);ATP水平降低(P<0.01)。射线处理后低糖联合120μmol/L棕榈酸处理组γ-H2AX焦点数量增多(P<0.05);D0值降低(P<0.01);ROS水平增加(P<0.001);细胞凋亡增加(P<0.001);γ-H2AX蛋白表达增加(P<0.01)。NAC预处理能够逆转ROS、凋亡和γ-H2AX蛋白表达的变化。结论低糖联合棕榈酸诱导SW480细胞代谢应激,抑制肿瘤增殖,在联合射线照射时通过诱导ROS产生和DNA损伤促进SW480细胞放射增敏。

Objective To investigate the mechanism underlying the inhibiting effect of low-glucose combined with palmitic acid on human colon cancer cells and its influence on the radiosensitivity.Methods Under the treatment of low-glucose,palmitic acid and low-glucose combined with palmitic acid,the treatment condition that significantly inhibited the proliferation of SW480 was screened by CCK-8 assay.The reactive oxygen species(ROS)level,mitochondrial membrane potential and apoptosis rate were detected by flow cytometry.The changes in the radiosensitivity were detected by immunofluorescence-based γ-H2AX quantification and colony formation assay.The protein expression level was detected by Western blot.Results Compared with the control group,the condition of low-glucose combined with 120μmol/L palmitic acid significantly inhibited the proliferation of SW480 cells(P<0.01).The expression levels of CPT1a,PFKFB3 and PKM were significantly up-regulated,the expression levels of NDUFV1,NDUFV2 and NDUFS1 were remarkably down-regulated,the ROS level was significantly increased and the ATP level was considerably reduced in the cells under metabolic stress(all P<0.01).After irradiation,the number ofγ-H2AX foci was significantly increased(P<0.05),and the D0 value was significantly reduced(P<0.01),the ROS level was considerably increased(P<0.001),the apoptosis rate was significantly increased(P<0.001)and the expression level ofγ-H2AX protein was remarkably up-regulated(P<0.01)in the low-glucose combined with 120μmol/L palmitic acid group.Pretreatment with NAC could reverse the changes of ROS,apoptosis andγ-H2AX protein expression.Conclusions The combination of low-glucose and palmitic acid can induce metabolic stress in SW480 cells,inhibit tumor proliferation and increase the radiosensitization when combined with radiotherapy by inducing the generation of ROS and DNA damage.