Combination of Metformin, Sodium Oxamate and Doxorubicin Induces Apoptosis and Autophagy in Colorectal Cancer Cells via Downregulation HIF-1α.
Combination of Metformin, Sodium Oxamate and Doxorubicin Induces Apoptosis and Autophagy in Colorectal Cancer Cells via Downregulation HIF-1α.
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DOI：
10.3389/fonc.2021.594200
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发表时间：
2021
影响因子：
4.7
通讯作者：
Pérez-Plasencia C
中科院分区：
文献类型：
--
作者：
Coronel-Hernández J;Salgado-García R;Cantú-De León D;Jacobo-Herrera N;Millan-Catalan O;Delgado-Waldo I;Campos-Parra AD;Rodríguez-Morales M;Delgado-Buenrostro NL;Pérez-Plasencia C
结直肠癌(CRC)是全球癌症相关死亡的第三大原因，无论男女。目前的治疗方法包括手术、化疗和靶向治疗；然而，长期暴露在化学制剂中会导致患者的毒性和耐药性。因此，我们实施了一种基于阿霉素、二甲双胍和草酸钠的联合治疗策略，称为三联疗法(TT)。我们发现，与阿霉素相比，TT通过抑制mTOR/AKT途径显著抑制了结直肠癌细胞的增殖，促进了细胞的凋亡和自噬。Western印迹分析了几个自噬基因：TT过表达ULK1、ATG4和LC3II基因。有趣的是，ULK1是唯一一个在TT给药过程中逐渐过度表达的自噬相关蛋白。因此，我们假设在自噬激活过程中，存在一种由microRNAs调节UKL1表达的转录后机制。通过生物信息学方法，我们确定ULK1可能成为mir-26a的靶点，mir-26a在结直肠癌晚期过度表达。体外实验表明，mir-26a的过表达显著降低了ULK1、mRNA和蛋白的表达。相反，TT通过mir-26a下调恢复了ULK1的表达。由于三联疗法抑制了mir-26a的表达，我们推测这种药物组合可能参与了mir-26a的转录调控。因此，我们分析了Mir-26a启动子序列，发现了两个HIF-1α转录因子识别位点。我们建立了两种不同的HIF-1α稳定模型。两者在低氧条件下均表现为mir-26a高表达和ULK1降低。进行免疫沉淀实验，观察到HIF-1α在mir-26a启动子中的丰度。令人惊讶的是，TT降低了HIF-1mRNA的表达，并恢复了ULK1mRNA的表达。这些结果揭示了一个重要的调控机制，该机制由激活HIF-1α的信号控制，进而调节mir-26a的转录。
Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide in both sexes. Current therapies include surgery, chemotherapy, and targeted therapy; however, prolonged exposure to chemical agents induces toxicity in patients and drug resistance. So, we implemented a therapeutic strategy based on the combination of doxorubicin, metformin, and sodium oxamate called triple therapy (Tt). We found that Tt significantly reduced proliferation by inhibiting the mTOR/AKT pathway and promoted apoptosis and autophagy in CRC derived cells compared with doxorubicin. Several autophagy genes were assessed by western blot; ULK1, ATG4, and LC3 II were overexpressed by Tt. Interestingly, ULK1 was the only one autophagy-related protein gradually overexpressed during Tt administration. Thus, we assumed that there was a post-transcriptional mechanism mediating by microRNAs that regulate UKL1 expression during autophagy activation. Through bioinformatics approaches, we ascertained that ULK1 could be targeted by mir-26a, which is overexpressed in advanced stages of CRC. In vitro experiments revealed that overexpression of mir-26a decreased significantly ULK1, mRNA, and protein expression. Contrariwise, the Tt recovered ULK1 expression by mir-26a decrease. Due to triple therapy repressed mir-26a expression, we hypothesized this drug combination could be involved in mir-26a transcription regulation. Consequently, we analyzed the mir-26a promoter sequence and found two HIF-1α transcription factor recognition sites. We developed two different HIF-1α stabilization models. Both showed mir-26a overexpression and ULK1 reduction in hypoxic conditions. Immunoprecipitation experiments were performed and HIF-1α enrichment was observed in mir-26a promoter. Surprisingly, Tt diminished HIF-1α detection and restored ULK1 mRNA expression. These results reveal an important regulation mechanism controlled by the signaling that activates HIF-1α and that in turn regulates mir-26a transcription.
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