METHODS: Cell viability and colony formation assays were conducted to evaluate the sensitivity of cells to lenvatinib and dicoumarol. RNA-Seq was used to determine the differences in transcriptome between parental cells and lenvatinib-resistant (LR) cells. The upregulated genes were analyzed by GO and KEGG analyses. Then, qPCR and Western blotting were employed to determine the relative gene expression levels. Afterwards, the intracellular reactive oxygen species (ROS) and apoptosis were detected by flow cytometry.
RESULTS: PLC-LR and Hep3B-LR were established. There was a total of 116 significantly upregulated genes common to both LR cell lines. The GO and KEGG analyses indicated that these genes were involved in oxidoreductase and dehydrogenase activities, and reactive oxygen species pathways. Notably, NAD(P)H:quinone oxidoreductase 1 (NQO1) was highly expressed in LR cells, and was involved in the lenvatinib resistance. The high expression of NQO1 decreased the production of ROS induced by lenvatinib, and subsequently suppressed the apoptosis. The combination of lenvatinib and NQO1 inhibitor, dicoumarol, reversed the resistance of LR cells.
CONCLUSIONS: The high NQO1 expression in HCC cells impedes the lenvatinib-induced apoptosis by regulating the ROS levels, thereby promoting lenvatinib resistance in HCC cells.
方法:进行细胞活力和集落形成测定以评估细胞对乐伐替尼和双香豆醇的敏感性。RNA-Seq用于确定亲本细胞和lenvatinib抗性(LR)细胞之间的转录组差异。通过GO和KEGG分析来分析上调的基因。然后,使用qPCR和Western印迹来确定相对基因表达水平。之后,流式细胞术检测细胞内活性氧(ROS)和细胞凋亡。
结果:建立了PLC-LR和Hep3B-LR。两种LR细胞系共有总共116个显著上调的基因。GO和KEGG分析表明,这些基因参与氧化还原酶和脱氢酶活性,和活性氧途径。值得注意的是,NAD(P)H:醌氧化还原酶1(NQO1)在LR细胞中高表达,并参与了lenvatinib耐药性。NQO1的高表达降低了乐伐替尼诱导的ROS的产生,随后抑制了细胞凋亡。lenvatinib和NQO1抑制剂的组合,双香丸,逆转LR细胞的抗性。
结论:肝癌细胞中NQO1的高表达通过调节ROS水平阻碍来伐替尼诱导的细胞凋亡,从而促进肝癌细胞对乐伐替尼的耐药性。