Molecular basis of the therapeutic effect of butyrate in glioblastoma revealed by in vitro and in silico approach

Abstract

Glioblastoma (GBM) remains among the most difficult to treat cancers globally. Complexity of this disease and the existing inadequacies of modern therapy put forward the need for ongoing research into new therapies. Short Chain Fatty Acids (SCFAs) are among the major metabolites secreted by the gut microbiome, and are thought to play an important role in gut microbiome-brain interactions. While earlier experimental studies have hinted towards a therapeutic potential of butyrate in glioblastoma, we demonstrate here the molecular mechanism behind this effect through a combination of in vitro and in silico approaches. LN229 and U87 GBM cells were treated with butyrate, and anti-proliferative potential was evaluated through colony formation assay, flow cytometry and qRT-PCR experiments. Molecular mechanism was mapped through butyrate target prediction, molecular docking, molecular dynamics simulations and analyses of binding free energy calculations. Butyrate inhibited the proliferation, and colony formation in U87 and LN229 GBM cell lines. We also found that it arrested the cell cycle progression at the G0/G1 phase in U87 cells and the G2/M Phase in LN229 cell lines, along with initiation of apoptosis in both the cells. Similarly, butyrate treatment also reduced the expression of Epidermal Growth Factor Receptor (EGFR) in a dose-dependent manner. The molecular mechanism behind the anti-cancer effect of butyrate was demonstrated through molecular docking and simulation studies. Butyrate bound to Histone Deacetylase-3 (HDAC3)-Nuclear CoRepressor2 complex, and induced allosteric dynamics that further blocked HDAC3 activation. The observations in this study demonstrate the potential of butyrate as a viable therapeutic agent for glioblastoma.