C-MYC and HIF1α promoter G-quadruplexes dependent metabolic regulation mechanism of berberine in colon cancer.

Abstract

G-quadruplexes are molecular switches regulating gene transcription. c-MYC and hypoxia-inducible factor 1-alpha (HIF1α) play important roles in cell proliferation, apoptosis, and metabolic regulation in colon cancer. Whether berberine can regulate metabolism by interacting with c-MYC and HIF1α G-quadruplexes in colon cancer needs to be explored. The binding mode of berberine with c-MYC and HIF1α G-quadruplexes were explored by ultraviolet and visible absorption spectroscopy and fluorescence spectroscopy. Circular dichroism (CD) spectroscopy was performed to evaluate the effects of berberine on the stability of c-MYC and HIF1α G-quadruplexes. After different concentrations of berberine acting on HCT116 cells for 24 h, cell proliferation and apoptosis were detected by MTT assay and flow cytometry; quantitative real-time polymerase chain reaction and western blot were performed to detect mRNA and protein expression of c-MYC and HIF1α; transcriptome sequencing was used to analyze the metabolic pathways. For the effects of berberine on colon cancer mouse model with dose of 50 mg·kg-1 for 14 days, tumor growth were monitored, hematoxylin and eosin staining and immunofluorescence staining were performed to analyze histopathology and protein expression of c-MYC and HIF1α, central carbon metabolism was detected in tumor tissues. The binding ability of berberine with c-MYC G-quadruplex was different to that of berberine with HIF1α G-quadruplex. Both binding modes involved π-π stacking. The stoichiometric ratios were 1:1, 1:3, and 3:1 for berberine with c-MYC G-quadruplex and only 1:1 for berberine with HIF1α G-quadruplex. Temperature had a greater effect on the binding of berberine to c-MYC G-quadruplex. Berberine could improve the thermal stability of both c-MYC and HIF1α G-quadruplexes. Berberine inhibited the gene transcription and protein expression of c-MYC and HIF1α in colon cancer HCT116 cells. In vivo, berberine delayed tumor progression and inhibited the protein expression of c-MYC and HIF1α. Twelve differential metabolites such as decreased adenosine triphosphate were obtained, indicating that berberine could regulate the metabolic pathways of the tricarboxylic acid (TCA) cycle and glycolysis/gluconeogenesis, among others. Berberine may inhibit colon cancer by regulating the TCA cycle and glycolysis/gluconeogenesis based on the interaction with c-MYC and HIF1α G-quadruplexes.