Lanatoside C induces autophagy in Huh-7 cells and promotes the combination of Beclin1 with VPS34 and VPS15
10.3781/j.issn.1000-7431.2024.2310-0548
- VernacularTitle:毛花苷C诱导肝癌Huh-7细胞自噬并促进Beclin1与VPS34和VPS15的相互结合
- Author:
Mengling WANG
1
;
Dan WU
;
Shengqiang TAN
;
Xiong TAN
;
Changqi GUO
;
Hongwen LI
;
Ning TAN
Author Information
1. 桂林医学院基础医学院,广西壮族自治区 桂林 541199
- Publication Type:Journal Article
- Keywords:
Hepatocellular carcinoma;
Lanatoside C;
Autophagy;
Beclin1;
LC3-Ⅱ
- From:
Tumor
2024;44(4):334-345
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of lanatoside C on autophagy in hepatocellular carcinoma Huh-7 cells,and the possible mechanism of action.Methods:After Huh-7 cells were treated with gradient concentration of lanatoside C for 24 h,the IC50 value of lanatoside C on Huh-7 cells was detected by CCK-8 assay,and the role of intervention of gradient concentration of lanatoside C in the proliferation and clone formation of Huh-7 cells was analyzed by CCK-8 assay and clone formation assay.Western blotting was used to detect the expression levels of autophagy markers in Huh-7 cells,such as microtubule-associated protein light chain 3(LC3)-II,LC3-Ⅰ,p62,Beclin1,vacuolar protein sorting 34(VPS30),VPS15 and autophagy-related protein 3(ATG3)in Huh-7 cells,and the changes of LC3-II/LC3-Ⅰ ratio were analyzed.The effects of lanatoside C on the formation of autophagic vesicles in Huh-7 cells were observed by laser confocal microscopy,and the effects of lanatoside C on the formation of autophagic vesicles in Huh-7 cells were confirmed by artificial intelligence image analysis.Finally,the effects of lanatoside C on the interaction between Beclin1,VPS34 and VPS15 on the formation of autophagic vesicles in Huh-7 cells were detected by co-immunoprecipitation.Results:The IC50 value of lanatoside C on Huh-7 cells at 24 h was(0.49±0.07)μg/mL.The results of the CCK-8 assay and clone formation assay suggested that lanatoside C had a significant inhibitory effect on the growth of Huh-7 cells in a concentration-dependent manner(both P<0.01).The results of Western blotting assay showed that the LC3-II/LC3-Ⅰ ratio was significantly increased with the increase in the concentration of lanatoside C(P<0.01),while the expression level of Beclin1 protein was up-regulated(P<0.05),and the expression level of p62 protein was significantly down-regulated(P<0.01).The results of laser confocal microscopy showed that lanatoside C could significantly induce autophagic vesicle formation in Huh-7 cells(P<0.01).The results of artificial intelligence image analysis confirmed this effect of lanatoside C(all P<0.01).The results of co-immunoprecipitation confirmed that lanatoside C was able to promote the binding of Beclin1 to VPS34 and VPS15 with each other(both P<0.01).Conclusion:Lanatoside C significantly inhibits the proliferation of Huh-7 cells and and induces Huh-7 cells autophagy,the mechanism of this effect may be related to up-regulating Beclin1 expression and promoting the combination of Beclin1 protein with VPS34 and VPS15 proteins.
