Effects of Plumbagin on Proliferation, Apoptosis, Invasion and Expression of HIF-1α in Hepatocellular HepG2 Cells Under Hypoxia Condition

OBJECTIVE To investigate the effects of plumbagin on proliferation, apoptosis, invasion and expression of hypoxia induced factor 1α(HIF-1α) as well as its target genes in HepG2 cells under hypoxia condition. METHODS Cobalt chloride(CoCl₂) was used to induce a chemical hypoxia condition for HepG2 cells. Under this hypoxia condition, HepG2 cells were treated with plumbagin at 2, 4, 8 μmol·L^–1 for 24 h respectively. The proliferation of HepG2 cells were measured by MTT and plate clone formation assay. The apoptotic of HepG2 cells were detected by flow cytometry to detect labeled Annexin V/PI. Transwell experiment was conducted to analyze the invasion ability of HepG2 cells after plumbagin treatment. qRT-PCR were used to detect the transcription level of its coding gene, HIF-1A. Western blotting analysis was further used to detect the protein expression level of HIF-1α and its downstream target genes such as c-Myc, VEGFA, MMP9 and TWIST1 in cells. RESULTS The concentration of CoCl₂ were optimized to 150 μmol·L^–1 without interference with cell proliferation and the expression level of HIF-1α was significantly increased(P<0.01), indicating successfully establishment of the hypoxia model by CoCl₂ treatment. Compared to the normoxia control group, MTT and plate clone formation assay results showed that proliferation of HepG2 cells was significantly inhibited by treatment with different concentration of plumbagin(P<0.05 or P<0.01). In addition, Transwell experiment result showed that the invasion ability of HepG2 cells was also decreased by plumbagin(P<0.05 or P<0.01). Flow cytometry results indicated that plumbagin could significantly induce the apoptosis of HepG2 cells(P<0.05 or P<0.01). The results of Western blotting and qRT-PCR showed that plumbagin could significantly down-regulate the expression levels of HIF-1α protein and HIF-1A mRNA(P<0.05 or P<0.01). Western blotting analysis results further showed that the protein expression levels of HIF-1α and its downstream genes c-Myc, VEGFA, MMP9 and TWIST1 were significantly down-regulated(P<0.05 or P<0.01). CONCLUSION The optimal modeling concentration of CoCl₂ is measured as 150 μmol·L^–1. Under hypoxia conditions, plumbagin can also significantly inhibit the proliferation and invasion of HepG2 cells. The apoptosis-inducing ability of plumbagin is also strong under hypoxia condition, which may be related to the significantly down-regulated expression of HIF-1α and its target genes.