目的 探究葫芦素B对肾癌细胞增殖、侵袭、转移和自噬的影响及其经由的细胞通路.方法 培养人肾癌768-O细胞,利用MTT细胞增殖实验,得到最佳作用浓度.即利用培养基饥饿过夜处理后加入不同浓度的(20、40、60、80 nmol/L)葫芦素B处理48 h,通过不同处理方法分为对照组、葫芦素B组、Akt通路抑制剂LY294002组、葫芦素B+LY294002组,分别通过MTT法、划痕实验、Transwell实验检测细胞增殖、迁移及侵袭能力4种处理方法分为对照组、葫芦素 B 组、Akt 通路抑制剂 LY294002 组、葫芦素 B+LY294002 组;Western Blot 实验检测 t-Akt、p-Akt、N-cadherin、EG-FR、LC3-Ⅱ蛋白的表达情况;免疫荧光实验检测细胞自噬情况.结果 葫芦素B呈浓度梯度抑制细胞增殖;葫芦素B组细胞增殖、迁移、侵袭明显低于对照组,自噬增加(P＜0.01);Western Blot结果显示,与对照组相比,加入葫芦素B后,细胞的p-Akt、N-cadherin及EGFR蛋白表达减少,LC3-Ⅱ表达增加(P＜0.01).结论 葫芦素B在768-O肾癌细胞中通过下调EGFR、p-Akt、N-cadherin的表达,抑制肾癌细胞增殖、侵袭、转移,促进自噬.
The Effect of Cucurbitacin B on the Biological Behavior of Renal Cancer 768-O Cells
Objective To investigate the effects of cucurbitacin B on proliferation,invasion,metastasis,and autophagy of renal cancer cells,as well as possible cellular pathways.Methods Human renal cancer 768-O cells were cultured and subjected to MTT assay.Firstly,the culture medium was starved overnight and treated with different concentrations of(20,40,60,80 nmol/L)cu-curbitacin B for 48 hours to obtain the optimal concentration of action.Then,different treatment methods were used to divide the cells into control group,cucurbitacin B group,LY294002 group,and cucurbitacin B+LY294002 group.Four treatment methods were used to detect cell proliferation,migration,and invasion ability,including MTT assay,scratch assay,and Transwell assay.The cells were divided into control group,cucurbitacin B group,Akt pathway inhibitor LY294002 group,and cucurbitacin B+LY294002 group;Western Blot assay was used to detect the expression of t-Akt,p-Akt,N-cadherin,EGFR,and LC3-Ⅱ proteins;immu-nofluorescence assay was used to detect cellular autophagy.Results Cucurbitacin B inhibited cell proliferation in a concentration gra-dient;the proliferation,migration,and invasion of cells in the cucurbitacin B group were significantly lower than those in the control group,and autophagy increased(P＜0.01);Western Blot results showed that compared with the control group,the addition of cu-curbitacin B resulted in a decrease in the expression of p-Akt,N-cadherin,and EGFR proteins in the cells,while the expression of LC3-Ⅱ increased(P＜0.01).Conclusion Cucurbitacin B inhibits the proliferation,invasion,and metastasis of renal cancer cells and promotes autophagy by downregulating the expression of EGFR,p-Akt,and N-cadherin in 768-O renal cancer cells.
cucurbitacin Brenal cancerproliferation and invasionautophagy
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