白藜芦醇降低缺氧诱导因子1α蛋白SUMO化抑制皮肤鳞状细胞癌新生血管形成

Inhibitory effect of resveratrol on neovascularization in cutaneous squamous cell carcinoma by suppressing the SUMOylation of hypoxia-inducible factor 1α protein

目的从蛋白质泛素/类泛素化修饰平衡角度探讨白藜芦醇抑制皮肤鳞状细胞癌新生血管形成的内在机制。方法以人皮肤鳞状细胞癌A431细胞株为研究对象,分别给予培养基中加入50μmol/L和100μmol/L白藜芦醇处理对数生长期A431细胞作为实验组,以不加白藜芦醇培养基处理为对照组。按照上述实验分组,培养48 h采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)实验检测各组细胞增殖活性;采用血管模拟形成实验检测白藜芦醇处理12 h对A431细胞血管拟态形成能力的影响;采用Western印迹检测白藜芦醇处理48 h各组细胞泛素(ubiquitin)、小泛素相关修饰蛋白1(SUMO1)、缺氧诱导因子1α(HIF-1α)、血管内皮生长因子受体(VEGFR)相对表达水平。将24只8周龄BALB/c雄性去胸腺小鼠随机均分3组,于腹股沟皮下接种A431细胞,治疗组予1 mg/kg或2 mg/kg白藜芦醇腹腔注射给药,对照组注射相同体积的生理NaCl溶液,每3天注射1次,第21天处死小鼠,解剖肿瘤称重;采用免疫组化检测各组肿瘤组织中CD31的表达。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果对照组、50μmol/L组、100μmol/L组A431细胞增殖率差异有统计学意义(F=17.75,P=0.017),50μmol/L组、100μmol/L组低于对照组(66.53%±5.09%、35.88%±4.28%比100%,LSD-t=21.17、29.04,P=0.011、0.004);3组A431细胞血管管壁样细胞嵴的总长度差异有统计学意义(F=21.37,P=0.004),50μmol/L组、100μmol/L组低于对照组[(102.73±11.36)μm、(37.83±4.19)μm比(185.26±8.02)μm,均P<0.05];3组细胞泛素、SUMO1、HIF-1α、VEGFR相对表达差异均有统计学意义,50μmol/L组、100μmol/L组泛素相对表达均高于对照组(2.09±0.13、3.53±0.16比0.68±0.11,均P<0.05),SUMO1、HIF-1α、VEGFR的相对表达均低于对照组(SUMO1:1.87±0.13、1.02±0.11比3.10±0.11,HIF-1α:0.81±0.06、0.45±0.06比0.97±0.08,VEGFR:0.73±0.09、0.39±0.05比0.98±0.07,均P<0.05)。动物试验中,对照组、1 mg/kg组、2 mg/kg组小鼠皮下肿瘤质量[(3.29±0.57)g、(2.91±0.49)g、(2.55±0.52)g]、肿瘤组织CD31细胞阳性率(76.24%±5.51%、39.45%±5.48%、12.07%±3.54%)差异均有统计学意义(F=14.33、15.34,P=0.019、0.021),1 mg/kg组、2 mg/kg组均低于对照组(均P<0.05)。结论白藜芦醇能够抑制肿瘤生长与肿瘤组织新生血管形成,可能与通过降低HIF-1α蛋白泛素/类泛素化修饰途径抑制皮肤鳞癌新生血管形成有关。

Objective To explore intrinsic mechanisms underlying the inhibitory effect of resveratrol on neovascularization in cutaneous squamous cell carcinoma from the perspective of ubiquitin/ubiquitin-like protein modification balance.Methods The human cutaneous squamous cell carcinoma cell line A431 was used as the research object.Cultured A431 cells at exponential growth phase were divided into 3 groups(control group,50μmol/L resveratrol group,and 100μmol/L resveratrol group)to be cultured with mediums containing 0,50,and 100μmol/L resveratrol,respectively.Cell proliferation activity was assessed by the 3-(4,5)-dimethylthiazol(-z-y1)-2,5-di-phenytetrazoliumromide(MTT)assay after 48-hour culture;the vasculogenic mimicry formation assay was performed to evaluate the vasculogenic mimicry formation ability of A431 cells after 12-hour treatment with resveratrol;Western blot analysis was conducted to detect the relative protein expression levels of ubiquitin,small ubiquitin-related modifier-1(SUMO1),hypoxia-inducible factor 1α(HIF-1α),and vascular endothelial growth factor receptor(VEGFR)in different groups after 48-hour treatment with resveratrol.Then,248-week-old BALB/c male thymectomized mice were randomly and equally divided into 3 groups to be subcutaneously inoculated with A431 cells in the inguinal region,followed by intraperitoneal injections of 1 mg/kg or 2 mg/kg resveratrol(1 mg/kg or 2 mg/kg resveratrol group),or the same volume of physiological sodium chloride solutions(control group);the intraperitoneal injections were done once every 3 days in all groups;all the above mice were sacrificed on the 21st day,and the tumors were resected and weighed.Immunohistochemistry assay was performed to determine the CD31 expression in tumor tissues.One-way analysis of variance was used for comparisons among multiple groups,and least significant difference(LSD)-t test was used for multiple comparisons.Results The proliferation rate of A431 cells significantly differed among the control group,50μmol/L resveratrol group,and 100μmol/L resveratrol group(F=17.75,P=0.017),and was significantly lower in the 50μmol/L resveratrol group(66.53%±5.09%)and the 100μmol/L resveratrol group(35.88%±4.28%)than in the control group(100%,LSD-t=21.17,29.04,P=0.011,0.004,respectively);the total length of vessel wall-like structures formed by A431 cells significantly differed among the 3 groups(F=21.37,P=0.004),and was significantly lower in the 50μmol/L resveratrol group(102.73±11.36μm)and the 100μmol/L resveratrol group(37.83±4.19μm)than in the control group(185.26±8.02μm,both P<0.05);the relative protein expression levels of ubiquitin,SUMO1,HIF-1α,and VEGFR also significantly differed among the 3 groups,the ubiquitin protein expression was significantly higher in the 50μmol/L resveratrol group(2.09±0.13)and the 100μmol/L resveratrol group(3.53±0.16)than in the control group(0.68±0.11,both P<0.05),while the protein expression of SUMO1,HIF-1α,and VEGFR was significantly lower in the 50μmol/L resveratrol group(1.87±0.13,0.81±0.06,0.73±0.09,respectively)and the 100μmol/L resveratrol group(1.02±0.11,0.45±0.06,0.39±0.05,respectively)than in the control group(3.10±0.11,0.97±0.08,0.98±0.07,respectively,all P<0.05).In the mice experiment,the weight of subcutaneous tumors and the proportion of CD31-positive cells in tumor tissues significantly differed among the control group,1 mg/kg resveratrol group,and 2 mg/kg resveratrol group(weight:3.29±0.57 g,2.91±0.49 g,2.55±0.52 g;proportion:76.24%±5.51%,39.45%±5.48%,12.07%±3.54%;F=14.33,15.34,P=0.019,0.021,respectively),and were significantly lower in the 1 mg/kg resveratrol group and 2 mg/kg resveratrol group than in the control group(all P<0.05).Conclusion Resveratrol could inhibit tumor growth and neovascularization in tumor tissues,which were possibly associated with the inhibitory effect of resveratrol on neovascularization in cutaneous squamous cell carcinoma by suppressing the SUMOylation of HIF-1αprotein via ubiquitin/ubiquitin-like protein modification pathways.