Chemical activation of prolyl hydroxylase-2 by BBAP-1 down regulates hypoxia inducible factor-1α and fatty acid synthase for mammary gland chemoprevention

Abstract

(4-[7-(Acetyloxy)-2-ethyl-2H-chromen-3-yl] phenyl acetate) (BBAP-1) was identified as a potential prolyl hydroxylase-2 activator and tested for this activity using the 2-oxoglutarate dependent in vitro assay. BBAP-1 was evaluated for its cytotoxic potential against ER + MCF-7 cells, and N-methyl-N-nitrosourea induced estrogen positive mammary gland carcinoma model. The effect of BBAP-1 on cellular morphology was evaluated using in vitro acridine orange/ethidium bromide and JC-1 staining. The morphological symptoms of apoptosis were evident after BBAP-1 treatment when studied through cell staining using acridine orange/ethidium bromide and JC-1 dye. Flow cytometric analysis revealed that BBAP-1 treatment arrested the cell cycle in the G2/M phase. In vivo study revealed the morphological changes of mammary gland tissue when scrutinized using carmine staining, hematoxylin and eosin staining and scanning electron microscopy. BBAP-1 treatment produced a marked effect on histopathological and morphological features when scrutinized against N-methyl-N-nitrosourea induced mammary gland carcinoma. Treatment with BBAP-1 also attenuated the deleterious effects of N-methyl-N-nitrosourea as measured on the basis of oxidative stress markers. Immunoblotting and qRT-PCR analysis revealed the participation of BBAP-1 in the mitochondrial mediated death apoptosis pathway and BBAP-1 also downregulated the hypoxic pathway through activation of prolyl hydroxylase-2. It was concluded that BBAP-1 activated the prolyl hydroxylase-2 enzyme and curtailed the over expression of hypoxia inducible factor-1α and fatty acid synthase along with the mitochondrial mediated death apoptosis pathway.