Two newly synthesized 5-methyltetrahydrofolate-like compounds inhibit methionine synthase activity accompanied by cell cycle arrest in G1/S phase and apoptosis in vitro

Abstract

Cobalamin-dependent methionine synthase, with a cofactor of vitamin B12, catalyzes the reaction of 5-methyltetrahydrofolate and homocysteine to form methionine and tetrahydrofolate, which takes a core position in folate cycle, one-carbon-unit transfer, and sulfur amino acid pathways. The 'methyl folate trap' hypothesis suggests that methionine synthase is a potential target for anticancer drug development. ZL031 and ZL033 are 5-methyltetrahydrofolate-like compounds that have been newly synthesized as potential inhibitors of the enzyme. To identify the effect of these two compounds on methionine synthase activity, a spectrophotometric assay was used and the results proved that ZL031 and ZL033 inactivated methionine synthase in HL-60 cells with an IC50 dose of 10.0 and 1.4 mumol/l, respectively. Moreover, obvious inhibitory effect on proliferation of HL-60 cells was observed, leading to our further investigation of the underlying anticancer mechanism. Under the circumstances of methionine synthase deficiency and subsequent folate depletion, cell cycle was arrested in G1/S phase and apoptosis was also observed. Analysis of cell cycle regulatory proteins demonstrated that cyclin E and cyclin-dependent kinase 2 were both increased. Furthermore, reduction of caspase-3, poly (ADP-ribose) polymerase, caspase-8, and caspase-9 protein levels were observed. In all the biological experiments we have performed, ZL033 has shown a better efficacy compared with ZL031. These results suggest that ZL031 and ZL033, as novel methionine synthase inhibitors, caused G1/S phase delay and apoptosis and eventually inhibit the proliferation of HL-60 cells in vitro. ZL033, with a carboxylic acid substituent, might have a better potential for drug development than ZL031 with an ester substituent.