Combination of Metformin, Sodium Oxamate and Doxorubicin Induces Apoptosis and Autophagy in Colorectal Cancer Cells via Downregulation HIF-1α

Abstract

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.

Gut metabolite L-lactate supportsCampylobacter jejunipopulation expansion during acute infection

“…To further explore the role of lactate during C. jejuni infection of colonocytes, we treated HCT116 cells with two inhibitors i) sodium oxamate, a competitive inhibitor of lactate dehydrogenase and ii) 2-deoxyglucose, a non-competitive hexokinase inhibitor that reduces lactate production by inhibiting the glycolysis pathway (25, 26). Both inhibitors were confirmed to significantly reduce lactate levels when compared to untreated cells, without reducing cell viability ( Fig 5E ).…”

Metformin Downregulates the Expression of Epidermal Growth Factor Receptor Independent of Lowering Blood Glucose in Oral Squamous Cell Carcinoma

“…In breast and other cancers, metformin activates the AMPK signaling pathway and inhibits the mTOR pathway to trigger its anti-cancer effects (23,24). In addition to the AMPK pathway, several non-AMPK pathways, such as RAS, AKT, and HIF-1a, may contribute to the anti-cancer effect of metformin (25)(26)(27). Moreover, metformin acts directly on mitochondria by reducing mitochondrial respiration and overall energy efficiency (28).…”

Transcriptome Analysis Reveal Candidate Genes and Pathways Responses to Lactate Dehydrogenase Inhibition (Oxamate) in Hyperglycemic Human Renal Proximal Epithelial Tubular Cells

“…The HIF inhibiting properties were removed using oxamate in the hyperglycemic rat proximal tubular cells ( 6 ). Studies have been reported that oxamate could induce autophagy via downregulation HIF-1α through inhibiting the Akt-mTOR signaling pathway in cancer cells ( 35 , 36 ). BECN1 is a haploinsufficient tumor suppressor gene as a core component of the class III phosphatidylinositol 3-kinase that is involved in autophagosome formation and vesicular trafficking ( 37 ).…”