Adjuvant chemotherapy with 5-fluorouracil (5-FU) does not improve survival of patients suffering from a form of colorectal cancer (CRC) characterized by high level of microsatellite instability (MSI-H). Given the importance of autophagy and multi-drug-resistant (MDR) proteins in chemotherapy resistance, as well as the role of casein kinase 1-alpha (CK1α) in the regulation of autophagy, we tested the combined effect of 5-FU and CK1α inhibitor (D4476) on HCT116 cells as a model of MSI-H colorectal cancer. To achieve this goal, the gene expression of Beclin1 and MDR genes, ABCG2 and ABCC3 were analyzed using quantitative real-time polymerase chain reaction. We used immunoblotting to measure autophagy flux (LC3, p62) and flow cytometry to detect apoptosis. Our findings showed that combination treatment with 5-FU and D4476 inhibited autophagy flux. Moreover, 5-FU and D4476 combination therapy induced G2, S and G1 phase arrests and it depleted mRNA of both cell proliferation-related genes and MDR-related genes (ABCG2, cyclin D1 and c-myc). Hence, our data indicates that targeting of CK1α may increase the sensitivity of HCT116 cells to 5-FU. To our knowledge, this is the first description of sensitization of CRC cells to 5-FU chemotherapy by CK1α inhibitor.

Graphic abstract

中文翻译：

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酪蛋白激酶-1-α 抑制剂 (D4476) 通过自噬通量抑制使微卫星不稳定的结肠直肠癌细胞对 5-氟尿嘧啶敏感

用 5-氟尿嘧啶 (5-FU) 进行辅助化疗不会提高患有以高水平微卫星不稳定性 (MSI-H) 为特征的结直肠癌 (CRC) 患者的生存率。鉴于自噬和多药耐药 (MDR) 蛋白在化疗耐药中的重要性，以及酪蛋白激酶 1-α (CK1α) 在调节自噬中的作用，我们测试了 5-FU 和 CK1α 的联合作用HCT116 细胞上的抑制剂 (D4476) 作为 MSI-H 结直肠癌的模型。为了实现这一目标，Beclin1和 MDR 基因ABCG2和ABCC3的基因表达使用定量实时聚合酶链反应进行分析。我们使用免疫印迹来测量自噬通量（LC3，p62）和流式细胞术来检测细胞凋亡。我们的研究结果表明，5-FU 和 D4476 的联合治疗抑制了自噬通量。此外，5-FU 和 D4476 联合治疗可诱导 G2、S 和 G1 期阻滞，并耗尽细胞增殖相关基因和 MDR 相关基因（ABCG2、细胞周期蛋白 D1和c-myc）的 mRNA 。因此，我们的数据表明靶向 CK1α 可能会增加 HCT116 细胞对 5-FU 的敏感性。据我们所知，这是第一次描述 CRC 细胞对 CK1α 抑制剂对 5-FU 化疗的敏感性。

图形摘要