缺氧条件下白花丹醌对肝癌HepG2细胞增殖、凋亡与侵袭及HIF-1α表达的影响

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

关键词:  白花丹醌  肝癌  细胞凋亡  肿瘤侵袭  氯化钴  低氧诱导因子1α  缺氧

Key words:  plumbagin  hepatocellular carcinoma  apoptosis  tumor invasion  cobalt chloride  hypoxia induced factor 1α (HIF-1α)  hypoxia

摘要:

目的 研究白花丹醌在缺氧条件下对人肝癌细胞HepG2增殖、凋亡、侵袭和低氧诱导因子1α(hypoxia induced factor1α，HIF-1α)及其下游基因表达的影响。方法 采用二氯化钴(CoCl₂)化学诱导法建立HepG2细胞体外缺氧模型，经不同浓度(2，4，8 μmol·L^–1)白花丹醌处理24 h后，MTT、平板克隆形成试验观察HepG2细胞增殖水平变化；使用Annexin V/PI双染流式细胞术检测HepG2细胞凋亡情况；使用Transwell试验观察HepG2细胞侵袭能力的变化；使用qRT-PCR检测HepG2细胞内HIF-1A mRNA转录水平变化；使用Western blotting检测细胞内HIF-1α及其下游基因c-Myc、VEGFA、MMP9和TWIST1的蛋白表达水平。结果 当CoCl₂处理浓度为150 μmol·L^–1时，HepG2细胞增殖水平未见显著变化，而HIF-1α蛋白表达水平显著升高(P<0.01)，提示体外缺氧模型建立成功。与常氧对照组相比，MTT、平板克隆形成试验结果显示不同浓度白花丹醌作用HepG2细胞后，其增殖水平受到显著抑制(P<0.05或P<0.01)； Transwell试验结果显示白花丹醌可显著抑制HepG2细胞侵袭(P<0.05或P<0.01)；流式细胞术结果表明白花丹醌能显著诱导HepG2细胞凋亡(P<0.05或P<0.01)；Western blotting及qRT-PCR检测结果显示，白花丹醌能显著下调HIF-1α蛋白及HIF-1A mRNA表达水平(P<0.05或P<0.01)。Western blotting检测结果显示HIF-1α及其下游基因c-Myc、VEGFA、MMP9和TWIST1的蛋白表达水平均显著下调(P<0.05或P<0.01)。结论 CoCl₂体外模拟肝癌HepG2细胞缺氧的适宜浓度为150 μmol·L^–1；在缺氧条件下，白花丹醌可显著抑制肝癌HepG2细胞的增殖与侵袭，同时诱导细胞凋亡，其作用机制可能与下调HIF-1α蛋白及其下游靶基因蛋白表达有关。

Abstract:

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.