Hypoxia regulates glycolysis through the HIF-1α/BMAL1/ALDOC axis to reduce oxaliplatin sensitivity in colorectal cancer

Abstract

Background: Oxaliplatin (L-OHP) is a first-line chemotherapy agent for advanced colorectal cancer (CRC), but the development of resistance often compromises its efficacy. Tumor hypoxia and metabolic reprogramming are known to influence chemotherapy sensitivity, yet their interrelationship remains inadequately explored. Methods: In vitro assays were conducted using human colorectal cancer cell lines (DLD1 and LoVo) under hypoxic conditions induced by cobalt chloride (CoCl2). The expression levels of key proteins involved in the HIF-1α/BMAL1/ALDOC pathway were assessed through Western blotting and quantitative real-time PCR (qPCR). Cell viability, apoptosis, and glycolytic activity were evaluated using CCK-8 assays, flow cytometry, and lactate/ATP measurements. Results: Hypoxia significantly enhanced glycolysis in CRC cells, decreasing sensitivity to L-OHP. The HIF-1α/BMAL1/ALDOC axis was identified as a crucial mediator in this process, with HIF-1α upregulating BMAL1, which increased ALDOC expression. This cascade promoted glycolytic activity and reduced apoptosis in hypoxic conditions. Notably, a positive correlation between HIF-1α and ALDOC expression was confirmed in clinical CRC samples. Conclusion: The findings reveal a novel mechanism by which hypoxia diminishes L-OHP sensitivity in CRC through the HIF-1α/BMAL1/ALDOC pathway. These insights provide potential biomarkers for predicting treatment outcomes and suggest new therapeutic strategies to enhance chemosensitivity in colorectal cancer.

Keywords: ALDOC; HIF-1α; chemotherapy; glycolysis; hypoxia.

Figure 1

CoCl2-induced hypoxia reduces the sensitivity of CRC cells to L-OHP and enhances glycolysis. (A) Western blot analysis of HIF-1α protein expression under different concentrations of CoCl2. (B) Cell viability assay under normoxic and hypoxic conditions with varying L-OHP concentrations. IC50 values were determined using GraphPad Prism 6.0 software by fitting dose-response curves to a four-parameter logistic model. (C) Clone formation assay results under normoxic and hypoxic conditions with L-OHP treatment. (D-G) Additional assays demonstrating apoptosis, lactate production, and ATP levels. *p < 0.05; **p < 0.01 and * * * p < 0.001).

Figure 2

CoCl2-induced hypoxia reduces L-OHP sensitivity by modulating the glycolytic capacity of CRC cells. (A) Cell viability assay detects the cell viability of DLD1 and LoVo cells under normoxia versus hypoxia conditions with L-OHP and 2-DG treatment. (B) Clone formation assay detects the colony numbers of DLD1 and LoVo cells under normoxia or hypoxia conditions with L-OHP and 2-DG treatment. (C) Flow cytometry detects the apoptosis of DLD1 and LoVo cells under normoxia versus hypoxia conditions with L-OHP and 2-DG treatment. (D) Western blot detects the apoptosis-related protein expression in DLD1 and LoVo cells under normoxia versus hypoxia conditions with L-OHP and 2-DG treatment. (E) Lactate measurement assay detects the lactate concentration in DLD1 and LoVo cells under normoxia versus hypoxia conditions with L-OHP and 2-DG treatment. (F) Adenosine triphosphate (ATP) assay detects the ATP production in DLD1 and LoVo cells as indicated. (*p < 0.05, * * p < 0.01, and * * * p < 0.001) (L-OHP (N) represents L-OHP-treated normoxic tumor cells and L-OHP (H) represents L-OHP-treated hypoxic tumor cells).

Figure 3

Hypoxia reduces L-OHP sensitivity of CRC cells by regulating ALDOC-mediated glycolysis. (A) PCR detects the mRNA expression in DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP treatment. (B) Western blot analyses of the expression of ALDOC protein in DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP treatment. (C) PCR detects the ALDOC mRNA expression in DLD1 and LoVo cells under normoxia versus hypoxia after L-OHP and siHIF-1α treatment. (D) Western blot analyses of the expression of ALDOC protein of DLD1 and LoVo cells under normoxia versus hypoxia after L-OHP and siHIF-1α treatment. (E)Lactate measurement assay detects the lactate concentrations in DLD1 and LoVo cells under normoxia versus hypoxia after L-OHP and siALDOC treatment. (F) Adenosine triphosphate (ATP) assay detects the ATP production of DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP and siALDOC treatment. (G) Cell viability assay detects the cell viability of DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP and siALDOC treatment. (H) Clone formation assay detects the colony numbers of DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP and siALDOC treatment. (I) Flow cytometry detects the apoptosis of DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP and siALDOC treatment. (J) Western blot detects the apoptosis-related protein expression in DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP and siALDOC treatment. (*p < 0.05, * * p < 0.01, and * * * p < 0.001) (L-OHP (N) represents L-OHP-stimulated normoxic tumor cells and L-OHP (H) represents L-OHP-stimulated hypoxic tumor cells).

Figure 4

CoCl₂-induced hypoxia mediates glycolysis through the HIF-1α/BMAL1/ALDOC pathway, thus reducing the sensitivity to L-OHP in colorectal cancer. (A) UCSC and Jaspar online tools predict the binding sites of HIF-1α to BMAL1 promotor and BMAL1 to ALDOC promotor. (B) PCR detects BMAL1 mRNA expression in DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP treatment. (C) Western blot analyses of the expression of BMAL1 protein in DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP treatment. (D) PCR detects BMAL1 mRNA expression in DLD1 and LoVo cells under normoxia versus hypoxia conditions with L-OHP and siHIF-1α treatment. (E)Western blot analyses of the expression of BMAL1 protein in DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP and siHIF-1α treatment. (F) PCR detects ALDOC mRNA expression in DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP and siBMAL1 treatment. (G) Western blot analyses of the expression of ALDOC protein of DLD1 and LoVo cells under normoxia versus hypoxia with L-OHP and siBMAL1 treatment. (*p < 0.05, * * p < 0.01, and * * * p < 0.001) (L-OHP (N) represents L-OHP-treated normoxic tumor cells and L-OHP (H) represents L-OHP-treated hypoxic tumor cells).

Figure 5

The positive correlation between HIF-1α and ALDOC expression was verified in samples of CRC patients (A) IHC was used to detect the expression of HIF-1α and ALDOC in patients with SD or PR (n=16) and PD (n=17). (B) The correlation between HIF-1α and ALDOC expression was analyzed by IHC. (*p < 0.05, * * p < 0.01, and * * * p < 0.001).