Chemopotentiating effects of a novel NAD biosynthesis inhibitor, FK866, in combination with antineoplastic agents.

FK866 is a novel anticancer agent that was previously shown to interfere with NAD superset+ biosynthesis by inhibition of nicotinamide phosphoribosyltransferase and to initiate apoptosis in cancer cells. As NAD superset+ is involved in cellular DNA repair processes, the present in vitro study on THP-1 and K562 leukemia cells was conducted to investigate the cytotoxicity of FK866 combination treatment with various cytotoxic agents: the antimetabolite Ara-C, the DNA-intercalating agent daunorubicin and the alkylating compounds 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG) and melphalan. Cell viability after drug exposure was assessed by propidium iodide (PI) staining. Non-cytotoxic concentrations of FK866 (10 superset-9 M or less), applied simultaneously or 24 hours before adding cytotoxic agents, caused a depletion in the intracellular NAD superset+ and--to a lesser extent-- NADH levels in THP-1 cells. After 48 and 72 hours treatment with daunorubicin and Ara-C, respectively, increased cell death was observed in THP-1 cells that were pretreated with FK866, as compared to cells exposed to antineoplastic drugs alone. However, this effect was transient, and there was no difference in cell survival after 72 hours incubation with daunorubicin or 96 hours with Ara-C. - Non-toxic concentrations of FK866 added 8, 16, or 24 hours before starting treatment with the PARP-activating agent MNNG synergistically decreased intracellular NAD superset+ contents, and increased MNNG-induced cytotoxicity both in THP-1 and K562 cells for at least 72 hours. This effect was less pronounced when FK866 was used in combination with another alkylating agent, melphalan. The PARP inhibitor 3-aminobenzamide delayed MNNG-induced cytotoxicity by 24 hours both in cells that were pretreated with FK866 and in non-pretreated cells. 48 hours later, the protective effect of 3-aminobenzamide could no longer be observed, but FK866-pretreated cells retained increased sensitivity to MNNG. - In conclusion, the chemosensitizing effect of FK866 on cell death induced by antineoplastic drugs was particularly obvious in combination with substances like MNNG that cause NAD superset+ depletion per se. It was less pronounced and only transiently measurable in combination with daunorubicin, Ara-C, and melphalan, respectively. These results may indicate different levels of DNA damage implicated in the action of the cytotoxic agents used.

Cytoprotective features of selenazofurin in hematopoietic cells.

OBJECTIVES: Antineoplastic activity of tiazofurin (Tz) and selenazofurin (Se) depends on their conversion to substances which are analogs of NAD. NAD performs pleiotropic and essential cellular functions, both as a cofactor in oxidation-reduction reactions and as a substrate for poly- and mono-ADP-ribosylation reactions. The therapeutic potential of modulating intracellular NAD levels and activity of NAD-dependent enzymes by concomitant administration of conventional anticancer agents merits further research. Our aim was to investigate the cytotoxic effects of Tz and Se in hematopoietic cells and to test their ability to potentiate the effects of DNA strand-disrupting agents. MATERIAL: THP-1, a cell line, derived from human acute monoblastic leukemia, was used. CLL lymphocytes were obtained from 8 patients with CLL. METHODS: The WST-l test was used to detect the function of NAD(P)-dependent dehydrogenases after exposure of THP-1 cells to Tz or Se. Cytotoxicity of Tz, Se, MNNG and chlorambucil was assessed using the membrane permeability assay (PI test). RESULTS: THP-1 cells were sensitive to cytotoxic effects of Tz and Se, with IC50 values of 2.5 x 10(-5) M for Tz and 2 x 10(-6) M for Se, as determined with the WST-1 test; 10 microM Se induced cell membrane disruption in more than 20% of THP-1 cells 48 hours after commencement of treatment, whereas the same concentration of Tz failed to increase membrane permeability. Pretreatment of THP-1 cells with 0.5 - 1.5 microM Se had no effect on the time course of cell death, induced by treatment with the DNA-damaging agent 1-methyl-3-nitro-1 - nitrosoguanidinium (MNNG) for 36 hours. However, when incubation of THP-1 cells with MNNG was prolonged (72 hours) without changing the incubation medium, pretreatment with Se had the following effects: the relative number of cells that died spontaneously decreased, and the cytotoxicity of MNNG was diminished. This effect was also demonstrated ex vivo in 6 of 8 cases of CLL, treated with MNNG and chlorambucil. CONCLUSIONS: Contrary to other investigations, we here demonstrate that preincubation with Se may partially protect cells from cell death induced by the alkylating agents MNNG and chlorambucil in the THP-1 cell line and in CLL lymphocytes presumably by affecting spontaneous cell death.

Poly ADP-ribose polymerase (PARP) inhibitors transiently protect leukemia cells from alkylating agent induced cell death by three different effects.

Polyadenosylation of nuclear enzymes is well known to regulate the cellular repair capacity after DNA damage. PARP mediates the transfer of poly-ADP-ribose moieties on itself and other nuclear proteins by the breakdown of NAD+. The present study investigated how modulation of PARP activity interferes with cell death induced by two different alkylating agents used in cancer chemotherapy. 1-methyl-3-nitro-1-nitrosoguanidinium (MNNG) decreased cellular reduction capacity (WST-1 assay) in HL60 and CCRF-CEM cells, accompanied by increased activity of PARP and depletion of intracellular NAD+ and ATP. Pretreatment with the PARP inhibitors 3-AB or 4-AN resulted in transient cell protection, which was associated with a switch from necrosis to apoptosis in CCRF-CEM cells and enhanced apoptosis in HL60 cells. Both PARP inhibitors delayed the drop in WST-1 reduction and retained NAD+ and ATP levels required for apoptosis. Furthermore, 3-AB or 4-AN prevented progressive DNA degradation in MNNG-treated CCRF-CEM cells. In contrast to MNNG, we did not observe early activation of PARP, decrease in WST-1 reduction, or wasteful consumption of NAD+ and ATP after treatment with melphalan. However, preincubation with 3-AB or 4-AN resulted in decreased HL60 cell membrane blebbing and reduced formation of apoptotic bodies. In conclusion, the cell death preventing effects of PARP inhibitors are mediated by their ability to maintain cellular energy metabolism, to inhibit the activation of endonucleolytic DNA degradation and to prevent cell blebbing. Surprisingly, these protective effects of PARP inhibitors on different cell functions seem to be independent of each other and are rather determined by the respective cytotoxic mechanisms implicated by different drugs. Our results support the hypothesis, that PARP activation and/or cleavage plays a regulatory role in the induction of apoptosis.