Benzamide riboside induces apoptosis independent of Cdc25A expression in human ovarian carcinoma N.1 cells

Abstract

One of the mechanisms of action of a new oncolytic agent, benzamide riboside (BR) is by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH) which catalyzes the formation of xanthine 5'-monophosphate from inosine 5'-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. In the present study BR (10 ± 20 mM) induced apoptosis in a human ovarian carcinoma N.1 cell line (a monoclonal derivative of its heterogenous parent line HOC-7). This was ascertained by DNA fragmentation, TUNEL assay, [poly(ADP)ribose polymerase]-cleavage and alteration in cell morphology. Apoptosis was accompanied by sustained c-Myc expression, concurrent down-regulation of cdc25A mRNA and protein, and by inhibition of Cdk2 activity. Both Cdk2 and cdc25A are G 1 phase specific genes and Cdk2 is the target of Cdc25A. These studies demonstrate that BR exhibits dual mechanisms of action, first by inhibiting IMPDH, and second by inducing apoptosis, which is associated with repression of components of the cell cycle that are downstream of constitutive cMyc expression.

Subcellular localisation of Cdc25A determines cell fate

“…In N.1 cells, it was demonstrated that the induction of apoptosis by TNFa and benzamide riboside repressed endogenous Cdc25A. 30,31 Hence, we analysed the expression of Cdc25A in the presence of TNFa by Western blotting to confirm that ectopic Cdc25A remained expressed ( Figure 4a). This was essential for the performance of subsequent experiments.…”

Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside

“…It was previously demonstrated that BR exhibits strong anti neoplastic activity in a panel of human tumour cell lines 3 and was most effective in leukaemia cells by inducing apoptosis. 13,14,40 The oncolytic activity of BR 3 was assumed to be due to its IMPDH-inhibitory and, therefore, dGTP-limiting action. 2,12 This hypothesis was strongly encouraged by the observation, that guanosine, a precursor of dGTP, prevented the oncolytic activity of BR.…”

“…Thus, ATP and/or dATP levels seem to determine cell death modes as it was previously suggested by others. 15,24,25,39,42 In earlier investigations it was demonstrated that BR suppressed survival pathways, induced apoptosis-relevant genes 13,14 and activated Caspase 8 but not Caspase 9 (Polgar et al, submitted). However, only low doses of BR (5 and 10 mM), but not high doses (20 mM and more) induced apoptosis by a pathway that culminated in Caspase 3 activation.…”

“…13,14 Higher BR-concentrations however, provoked necrosis, which is a common phenomenon of pro-apoptotic drugs 15 ± 17 and limits chemotherapy because of non-specific drug toxicity. Overdosing results in necrosis and spilling of intracellular fluids into the peri-cellular space, leading to inflammatory responses with wide ranging destructions of surrounding tissues.…”

Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside

“…9 ± 11 BR, exhibited stronger antiproliferative activity in the K562 cells than TR 12 and was shown to induce apoptosis in HL-60 13 and N.1 ovarian carcinoma cells. 13,14 Higher BR-concentrations however, provoked necrosis, which is a common phenomenon of pro-apoptotic drugs 15 ± 17 and limits chemotherapy because of non-specific drug toxicity. Overdosing results in necrosis and spilling of intracellular fluids into the peri-cellular space, leading to inflammatory responses with wide ranging destructions of surrounding tissues.…”