目的 研究海洋真菌Aspergillus sp. XBB-4中生物碱类代谢产物3-羟基-β-咔啉诱导人肝细胞癌细胞凋亡及其作用机制。方法 不同浓度的3-羟基-β-咔啉处理肝细胞癌细胞,MTT法和流式细胞术分别检测3-羟基-β-咔啉对细胞增殖和凋亡的影响,Western blot检测DNA修复酶(PARP)切割,cleaved-caspase3和Bcl2等凋亡相关蛋白和相关通路蛋白ERK、AKT、JNK等磷酸化水平的变化。流式细胞术检测3-羟基-β-咔啉对胞内活性氧(ROS)水平的影响,并检测ROS清除剂对3-羟基-β-咔啉介导的细胞凋亡的影响。结果 3-羟基-β-咔啉显著抑制肝细胞癌细胞HepG2、QGY7701和SMMC7721的增殖,PARP断裂条带明显增加,Cleaved-caspase3蛋白表达上调,抗凋亡蛋白Bcl2表达下调,ERK1/2蛋白磷酸化水平降低。流式细胞术检测发现3-羟基-β-咔啉诱导肝癌细胞凋亡。3-羟基-β-咔啉处理肝癌细胞后胞内ROS水平升高,ROS抑制剂NAC或脂质ROS抑制剂Liproxstatin-1可拮抗3-羟基-β-咔啉诱导的SMMC7721或HepG2细胞的凋亡。结论 3-羟基-β-咔啉能够抑制肝癌细胞增殖,升高细胞ROS水平,诱导细胞凋亡。
Abstract
OBJECTIVE To study the effect of 3-hydroxy-β-carboline, an alkaloid metabolite in the marine fungus Aspergillus sp. XBB-4, on apoptosis induction in human hepatocellular carcinoma (HCC) cells. METHODS HCC cells were treated with different concentrations of 3-hydroxy-β-carboline for 48 h with 0.1% DMSO as the control group. MTT assay was used to detect the inhibitory effect of 3-hydroxy-β-carboline on proliferation of HCC cells. Flow cytometry was used to detect the effect of 3-hydroxy-β-carboline on apoptosis of HCC cells, and the expression of apoptosis related proteins PARP, cleaved caspase-3 and anti-apoptotic protein Bcl2 was detected by Western blot. Then changes of intracellular ROS levels and phosphorylation levels of ERK, AKT and SARP/JNK were respectively detected by flow cytometry and Western blot. The effects of ROS blockers on 3-hydroxy-β-carboline mediated apoptosis were also detected. RESULTS 3-Hydroxy-β-carboline significantly inhibited proliferation of HCC cells, up-regulated expression of protein PARP cleavage and cleaved caspase-3, down-regulated expression of anti-apoptotic protein Bcl2, and induced apoptosis using flow cytometry. 3-Hydroxy-β-carboline induced intracellular ROS level in HCC cells, while phosphorylation level of signaling pathways protein ERK1/2 was reduced. Meanwhile, liproxstatin-1, a lipid ROS inhibitor or NAC could antagonize apoptosis of HepG2 cells or SMMC7721 cells induced by 3-hydroxy-β-carboline. CONCLUSION 3-Hydroxy-β-carboline can inhibit the proliferation of HCC cells, increase intracellular ROS level and induce apoptosis.
关键词
3-羟基-β-咔啉 /
肝细胞癌 /
活性氧 /
细胞凋亡
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Key words
3-hydroxy-β-carboline /
hepatocellular carcinoma /
active oxygen species /
apoptosis
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中图分类号：
R965.1
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参考文献
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脚注
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基金
安徽高校自然科学研究重点项目资助(KJ2019A1250)
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