Shikonin induces hepatocellular carcinoma cell apoptosis by suppressing PKM2/PHD3/HIF-1α signaling pathway.
10.12122/j.issn.1673-4254.2023.01.12
- Author:
Huan Huan ZHANG
1
;
Zhuo CHEN
1
;
Xiang Di ZHAO
1
;
Qiang HUO
1
;
Xiu CHENG
1
Author Information
1. Anhui Provincial Engineering Technology Research Center of Biochemical Drugs, Bengbu 233030, China.
- Publication Type:Journal Article
- Keywords:
apoptosis;
hepatocellular carcinoma;
hypoxia-inducible factor-1α;
prolyl hydroxylase 3;
pyruvate kinase M2;
shikonin
- MeSH:
Humans;
Prolyl Hydroxylases;
Carcinoma, Hepatocellular;
Caspase 3;
bcl-2-Associated X Protein;
Liver Neoplasms;
Signal Transduction;
Apoptosis;
Adenosine Triphosphate
- From:
Journal of Southern Medical University
2023;43(1):92-98
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To investigate the mechanism of shikonin-induced death of human hepatocellular carcinoma SMMC-7721 cells.
METHODS:Cultured SMMC-7721 cells and normal hepatocytes (L-02 cells) were treated with 4, 8, or 16 μmol/L shikonin, and the changes in cell viability was assessed using MTT assay. The levels of ATP and lactic acid in the cell cultures were detected using commercial kits. Co-immunoprecipitation and immunofluorescence staining were used to determine the relationship among pyruvate kinase M2 (PKM2), prolyl hydroxylase 3 (PHD3), and hypoxia-inducible factor-1α (HIF-1α). The expressions of PHD3, PKM2, HIF-1α, Bax, cleaved caspase-3, and Bcl-2 in SMMC-7721 cells were detected with Western blotting, and cell apoptosis was analyzed with annexin V-FITC/PI staining. The effects of RNA interference of PKM2 on PHD3 and HIF-1α expressions in SMMC-7721 cells were detected using Western blotting.
RESULTS:The IC50 of shikonin against SMMC-7721 and L-02 cells was 8.041 μmol/L and 31.75 μmol/L, respectively. Treatment with shikonin significantly inhibited the protein expressions of PKM2, HIF-1α and PHD3 and nuclear translocation of PKM2 and HIF-1α in SMMC-7721 cells. Coimmunoprecipitation and immunofluorescence staining confirmed that shikonin inhibited the formation of PKM2/PHD3/HIF-1α complex and significantly reduced the contents of lactic acid and ATP in SMMC-7721 cells (P < 0.05). The expressions of PHD3 and HIF-1α decreased significantly after PKM2 knockdown (P < 0.05). Shikonin treatment significantly increased the apoptosis rate, enhanced the expressions of Bax and cleaved caspase-3, and decreased Bcl-2 expression in SMMC-7721 cells (P < 0.05).
CONCLUSIONS:Shikonin induces apoptosis of SMMC-7721 cells possibly by inhibiting aerobic glycolysis through the PKM2/PHD3/HIF-1α signaling pathway to cause energy supply dysfunction in the cells.
