Guanosine supplementation reduces the antiproliferative and apoptotic effects of the IMPDH inhibitor gnidilatimonoein in K562 cells

Abstract

IMPDH (inosine 5'-monophosphate dehydrogenase) is the rate-limiting enzyme in the de novo biosynthetic pathway of guanine nucleotides, which is usually up-regulated in human leukaemia cell lines. Our previous studies have classified gnidilatimonoein, isolated from Daphne mucronata, as an IMPDH inhibitor and a strong antiproliferative agent among several types of leukaemia cells. In the present study, we investigated the effects of gnidilatimonoein on intracellular GTP pool size and its link to differentiation and apoptosis of K562 cells. It was found that gnidilatimonoein inhibited cell proliferation and induced G0/G1 cell cycle arrest in K562 cells after 24 h exposure to a single dose of gnidilatimonoein (1.5 μM), while no significant effects were observed on unstimulated and phytohaemagglutinin-stimulated peripheral blood lymphocyte cells at the gnidilatimonoein dose (1.5 μM) used. Based on the morphological changes, Wright-Giemsa staining, benzidine assay and the expression of cell surface markers [GPIIb (glycoprotein IIb) and glycophorin A], as analysed by flow cytometry, we found that K562 cells had differentiated towards megakaryocytic lineage. In addition, gnidilatimonoein induced apoptosis among K562 cells based on Acridine Orange/ethidium bromide and annexin V/propidium iodide double-staining observations. These changes, which were abrogated by the addition of guanosine, became evident when the intracellular GTP level decreased to approx. 20-35% of the untreated control level. Based on these findings, it can be concluded that gnidilatimonoein induces differentiation and apoptosis in K562 cells through perturbation of GTP metabolism, as one of its routes of action.