Activation of p53 by costunolide blocks glutaminolysis and inhibits proliferation in human colorectal cancer cells

Colorectal Cancer is a leading cause of cancer-related death. Glutaminolysis has been suggested as a therapeutic target for Cancer. Costunolide is a natural sesquiterpene lactone showing potent antitumor activity. Our studies were aimed at evaluating how costunolide affected glutaminolysis leading to proliferation inhibition in human colorectal Cancer cells. Costunolide suppressed viability and proliferation of HCT116 cells concentration-dependently, but did not apparently affect human intestinal epithelial cells. Costunolide at 20 μM reduced viability and proliferation of HCT116 cells time-dependently. Costunolide also repressed phosphorylation of mTOR and its downstream kinases p70S6K and 4E-BP1. Examinations of glutaminolysis metabolites showed that costunolide increased intracellular glutamine levels, but decreased intracellular levels of glutamate, α-ketoglutarate (α-KG), and ATP in HCT116 cells, suggesting costunolide blockade of glutaminolysis. Furthermore, costunolide inhibited promoter activity of Glutaminase 1 (GLS1), the first rate-limiting Enzyme in glutaminolysis, and reduced mRNA and protein expression of GLS1 in HCT116 cells, The GLS1 inhibitor BPTES, similar to costunolide, significantly reduced intracellular levels of α-KG and ATP and inhibited proliferation in HCT116 cells. Finally, costunolide increased phosphorylation and nuclear translocation of p53 in HCT116 cells. Both p53 inhibitor pifithrin-α and p53 siRNA significantly rescued costunolide suppression of GLS1 promoter activity and expression in HCT116 cells. These data in aggregate suggested that activation of p53 was required for costunolide inhibition of GLS1 resulting in blockade of glutaminolysis and inhibition of proliferation in colorectal Cancer cells, which was a novel mechanism underlying the antitumor activity of costunolide against colorectal Cancer.