槐耳通过诱导自噬抑制肝癌细胞增殖与迁移

doi:10.3877/cma.j.issn.2095-3232.2019.02.019

中华肝脏外科手术学电子杂志 ›› 2019, Vol. 08 ›› Issue (02) : 169 -174. doi: 10.3877/cma.j.issn.2095-3232.2019.02.019

基础研究

槐耳通过诱导自噬抑制肝癌细胞增殖与迁移

黎文信¹, 王喜城¹, 向美焕², 谭文亮¹, 徐云修修³, 叶义标³, 陈捷³, 陈涛³^,^†(

)

^1. 510120　广州，中山大学孙逸仙纪念医院肝胆外科；510120　广州，中山大学孙逸仙纪念医院　广东省恶性肿瘤表观遗传与基因调控重点实验室
^2. 510120　广州，中山大学孙逸仙纪念医院护理部
^3. 510120　广州，中山大学孙逸仙纪念医院肝胆外科

收稿日期:2018-11-20 出版日期:2019-04-10
通信作者: 陈涛
基金资助:
国家自然基金青年项目(81800560); 广州市科技产学研项目(201604030054)

Huaier inhibits proliferation and metastasis of liver cancer cells by inducing autophagy

Wenxin Li¹, Xicheng Wang¹, Meihuan Xiang², Wenliang Tan¹, Yunxiuxiu Xu³, Yibiao Ye³, Jie Chen³, Tao Chen³^,^†()

^1. Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China; Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
^2. Nursing Department, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
^3. Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China

Received:2018-11-20 Published:2019-04-10
Corresponding author: Tao Chen

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引用本文：

黎文信, 王喜城, 向美焕, 谭文亮, 徐云修修, 叶义标, 陈捷, 陈涛. 槐耳通过诱导自噬抑制肝癌细胞增殖与迁移[J/OL]. 中华肝脏外科手术学电子杂志, 2019, 08(02): 169-174.

Wenxin Li, Xicheng Wang, Meihuan Xiang, Wenliang Tan, Yunxiuxiu Xu, Yibiao Ye, Jie Chen, Tao Chen. Huaier inhibits proliferation and metastasis of liver cancer cells by inducing autophagy[J/OL]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2019, 08(02): 169-174.

目的

探讨槐耳对人肝细胞癌（肝癌）细胞增殖与迁移的影响，并探讨其相关分子机制。

方法

将肝癌细胞株SK-HEP-1和HEP3B分别设为对照组、槐耳处理组和自噬抑制剂组。CCK-8方法检测细胞活性，克隆形成实验检测细胞增殖，流式细胞仪检测细胞凋亡，Transwell实验检测细胞迁移，GFP-LC3荧光融合蛋白检测自噬小体的形成，Western blot检测上皮-间充质转化（EMT）、自噬和Akt/mTOR通路相关蛋白。两组数据比较采用t检验，多组比较采用单因素方差分析和LSD-t检验。

结果

肝癌细胞SK-HEP-1与HEP3B活性随着槐耳浓度升高与时间的增加而降低。6 g/L槐耳处理组肝癌细胞SK-HEP-1和HEP3B的克隆数分别为（77±9）、（84±9）个，明显少于对照组的（231±15）、（257±35）个（t=-8.90，-6.33；P<0.05）。槐耳可明显促进细胞凋亡，具有浓度依赖性（F=120.88，199.59；P<0.05）。8 g/L槐耳处理组肝癌细胞SK-HEP-1和HEP3B的穿膜细胞数分别为（68±13）、（54±13）个，明显少于对照组的（389±28）、（411±15）个（t=-17.98，-31.47；P<0.05）。Western blot检测显示，槐耳处理组N-cadhenin和Vimentin蛋白表达减弱；LC3Ⅱ表达增强，LC3Ⅰ表达减弱；p-Akt、p-mTOR、p62蛋白表达减弱，同时诱导LC3Ⅱ表达增强。自噬抑制剂3-MA可减少槐耳处理组的自噬。

结论

槐耳通过促进肝癌细胞凋亡及抑制细胞增殖和转移而发挥抗肿瘤效应，其机制可能与抑制Akt/mTOR通路诱导自噬和阻止EMT有关。

关键词: 癌，肝细胞, 自噬, 信号通路, 细胞增殖, 细胞运动

Objective

To evaluate the effect of Huaier on the proliferation and metastasis of human hepatocellular carcinoma (HCC) cells, and to explore the molecular mechanism.

Methods

HCC cell lines SK-HEP-1 and HEP3B were divided into the control, Huaier treatment and autophagy inhibitor groups. The cell viability was detected by CCK-8 assay. The cell proliferation was determined by clone formation assay. The cell apoptosis was detected by flow cytometry. The cell metastasis was detected by Transwell assay. The formation of autophagosome was observed by fluorescent GFP-LC3 fusion protein. The related proteins of epithelial-mesenchymal transition (EMT), autophagy and Akt/mTOR signaling pathway were detected by Western blot. Comparison between 2 groups was conducted by t test. Multi-group comparison was carried out by One-way ANOVA and LSD-t test.

Results

The activity of SK-HEP-1 and HEP3B decreased with the increase of Huaier concentration and the prolongation of treatment time. In 6 g/L Huaier treatment group, the number of clone formation of SK-HEP-1 and HEP3B cells was 77±9 and 84±9 respectively, significantly less than 231±15 and 257±35 in control group (t=-8.90, -6.33; P<0.05). Huaier significantly promoted the cell apoptosis in a concentration-dependent manner (F=120.88, 199.59; P<0.05). In 8 g/L Huaier treatment group, the number of trans-membrane SK-HEP-1 and HEP3B cells was 68±13 and 54±13, significantly less than 389±28 and 411±15 in control group (t=-17.98, -31.47; P<0.05). Western blot demonstrated that the expression of N-cadherin and Vimentin proteins was down-regulated, LC3Ⅱ expression was up-regulated and LC3Ⅰ expression was down-regulated in the Huaier treatment group. The expression levels of p-Akt, p-mTOR and p62 proteins were down-regulated and the expression of LC3Ⅱ was up-regulated. Autophagy inhibitor 3-MA could reduce the autophagy in Huaier treatment group.

Conclusions

Huaier exerts anti-tumor effect by promoting the apoptosis and inhibiting the proliferation and metastasis of HCC cells. The mechanism is probably related to suppressing the Akt/mTOR signaling pathway, inducing autophagy and preventing EMT.

Key words: Carcinoma, hepatocellular, Autophagy, Signaling pathway, Cell proliferation, Cell movement

图1 槐耳对肝癌细胞SK-HEP-1和HEP3B细胞增殖和凋亡的影响注：a为生长曲线示随着槐耳浓度与时间的增加细胞活性逐渐减低，b为克隆形成实验示槐耳抑制肝癌细胞增殖，c为流式细胞术检测示槐耳促进细胞凋亡；^*为P<0.05，^**为P<0.01

图2 槐耳对肝癌细胞迁移的抑制作用注：a、b为Transwell实验示槐耳可减少穿膜细胞数，迁移能力抑制作用为浓度依赖性；c为Western blot检测示槐耳可减少N-cadhenin和Vimentin蛋白表达；GAPDH为甘油醛-3-磷酸脱氢酶，^*为P<0.05，^**为P<0.01

图3 槐耳诱导肝癌细胞自噬相关实验注：a为Western blot检测示槐耳可诱导细胞自噬标志蛋白LC3Ⅱ表达增强，自噬抑制剂3-MA可使LC3Ⅱ表达减弱；b为荧光显微镜下示槐耳处理使GFP-LC3标记的自噬小体明显增加，与3-MA共孵育后自噬减少；c为3-MA可抑制槐耳诱导的细胞凋亡；d为3-MA可抑制槐耳对肝癌细胞迁移作用；Huaier为槐耳，^*为P<0.05

图4 Western blot检测自噬与Akt/mTOR相关蛋白注：槐耳可降低p-Akt、p-mTOR、p62蛋白表达，同时诱导LC3Ⅱ表达，自噬抑制剂3-MA可使p-mTOR和p-Akt的水平增加，降低LC3Ⅱ/LC3Ⅰ比值；^*为P<0.05

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