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Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes With Chemotherapy

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Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes With Chemotherapy

Philip M Tedeschi et al. Mol Pharmacol.

Abstract

NAD(+) kinase (NADK) is the only known cytosolic enzyme that converts NAD(+) to NADP(+), which is subsequently reduced to NADPH. The demand for NADPH in cancer cells is elevated as reducing equivalents are required for the high levels of nucleotide, protein, and fatty acid synthesis found in proliferating cells as well as for neutralizing high levels of reactive oxygen species (ROS). We determined whether inhibition of NADK activity is a valid anticancer strategy alone and in combination with chemotherapeutic drugs known to induce ROS. In vitro and in vivo inhibition of NADK with either small-hairpin RNA or thionicotinamide inhibited proliferation. Thionicotinamide enhanced the ROS produced by several chemotherapeutic drugs and produced synergistic cell kill. NADK inhibitors alone or in combination with drugs that increase ROS-mediated stress may represent an efficacious antitumor combination and should be explored further.

Figures

Fig. 1.
Fig. 1.
Thionicotinamide (ThioNa) is a prodrug of NADS and NADPS. (A) All three compounds result in the destabilization of dihydrofolate reductase (DHFR), an indication of NADK inhibition. Methotrexate (MTX) causes a stabilization of DHFR and results in an increase of detectable protein. (B) These compounds have similar toxicity profiles in C85 colorectal cancer cells. (C) NADK shRNA knockdown and thionicotinamide toxicity result in similar colony growth in C85 cells. Con., control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Fig. 2.
Fig. 2.
NADPS is both a substrate and inhibitor of human G6PD. (A) NADPS, derived from NADS phosphorylated by NADK in this reaction, can be reduced by G6PD to NADPSH, which absorbs at 405 nm. (B) NADPS inhibits NADP reduction. (C) Using a Dixon plot, the Ki of NADPS for human G6P D is 1 µM, as opposed to the NADP Km of 7.1 µM (Wang and Engel, 2009).
Fig. 3.
Fig. 3.
Exogenous nicotinamide in culture media can abrogate thionicotinamide (ThioNa) toxicity. (A) Thionicotinamide toxicity is inversely correlated with nicotinamide levels. Untreated C85 cells and C85 cells stably knocking down NADK are unaffected. Representative wells are shown for each condition. (B) Average colony increases in thionicotinamide-treated cells as nicotinamide levels increase. (C) Average colony number increases as nicotinamide levels increase. (D) The proposed intracellular biosynthetic pathway from thionicotinamide to NADPSH. Nam, nicotinamide; n.s., not significant.
Fig. 4.
Fig. 4.
Treatment with thionicotinamide (ThioNa) reduces cellular pools of NADP/NADPH and inhibits biosynthetic pathways. (A) NADP and (B) NADPH cellular pools decrease in C85 cells with 100 µM thionicotinamide treatment. (C) The protein synthesis rate, measured by [3H]4,5-leucine incorporation, is reduced with thionicotinamide treatment. (D) Neutral fatty acid levels in cells treated with thionicotinamide are reduced, as measured by Oil Red O staining. CPM, counts per minute; n.s., not significant.
Fig. 5.
Fig. 5.
Thionicotinamide (ThioNa) causes a rise in cellular ROS levels and synergizes with chemotherapy. (A) Treatment of C85 cells with 100 µM of thionicotinamide, NADS, or NADPS causes an increase in steady-state ROS levels. (B) C85 cells under oxidative stress from 1 mM hydrogen peroxide (H2O2) are more sensitive when treated with 100 µM thionicotinamide. (C) C85 cells treated with thionicotinamide or containing a knockdown of NADK are more sensitive to menadione, a generator of ROS. (D) Thionicotinamide synergizes with ROS-inducing chemotherapy gemcitabine, docetaxel, and irinotecan. Confidence interval values (Chou and Talalay, 1984) and ROS levels after 24 hours of treatment are described. *P ≤ 0.05 when compared with untreated cells.
Fig. 6.
Fig. 6.
Combination of thionicotinamide (ThioNa) and irinotecan results in DNA damage and induction of apoptosis. (A) An increase in γ-H2AX, an indication of DNA double-strand breaks, is markedly increased in C85 cells treated with thionicotinamide and irinotecan. (B) The presence of cleaved caspase 3 and poly(ADP-ribose) polymerase (PARP) in C85 cells treated with thionicotinamide and irinotecan is indicative of apoptosis. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Fig. 7.
Fig. 7.
NADK inhibition is effective in xenograft models of colon cancer and lymphoma. (A) Stable knockdown of NADK in C85 cells caused slow growth in xenografts. NOD/SCID mice bearing C85 xenografts treated with 100 mg/kg thionicotinamide (ThioNa) displayed inhibited tumor growth for the duration of treatment (inset) with little low general toxicity. (B) Moderate tumor regression was observed in a second xenograft study using the diffuse large B-cell lymphoma cell line RL using a dose of 100 mg/kg of thionicotinamide. Arrows indicate treatment. *P < 0.05; **P < 0.01; ***P < 0.001.

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