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, 83 (2), 339-53

Enhanced Degradation of Dihydrofolate Reductase Through Inhibition of NAD Kinase by Nicotinamide Analogs

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Enhanced Degradation of Dihydrofolate Reductase Through Inhibition of NAD Kinase by Nicotinamide Analogs

Yi-Ching Hsieh et al. Mol Pharmacol.

Abstract

Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin's lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment.

Figures

Fig. 1.
Fig. 1.
Screening of NAD/NADP analogs for cytotoxicity. (A) Two thousand DG44-(DGFR-EGFP) cells per well were plated and grown in Ham’s F12 medium supplemented with 10% dialyzed FBS containing no glycine, hypoxanthine, or thymidine. Concentrations of drugs tested ranged from 200 μM concentration. After incubation with NAD/NADP analogs for 96 hours, the viability of the transfectants was assessed using the MTS proliferation assay (see Materials and Methods for detail). (B) NADS and NADPS were tested in C85 and CCRF-CEM cells in RPMI 1640 medium for 96 hours. Although cell toxicity in C85 cells was determined using MTS assay, Trypan blue exclusion assay was used to determine cell viability in the presence of NADS and NADPS in CCRF-CEM cells. (C) Cytotoxicity of NADS and NADPS was tested for DG44-(DHFR-EGFP) cells in RPMI-1640 and Ham’s F12 using the MTS assay. (D) Seventy-five thousand C85 cells and mesenchymal stem cells (MSCs) were plated into 24-well tissue culture plates in triplicate in RPMI-1640 (C85) or Dulbecco's modified Eagle medium (DMEM) (mesenchymal stem cells) supplemented with 10% FBS. The following day, the medium was removed and replaced with fresh medium containing NADPS. After 96 hours of incubation, the cell viability was determined. The percentage cell survival was determined by Softmax Pro software, and GraphPad Prism 4 software was used to determine ED50 values using a sigmoidal dose-response curve fit.
Fig. 2.
Fig. 2.
NADS or NADPS treatment decreased steady-state levels of DHFR. (A) Western blot analysis of total lysates from DG44-(DHFR-EGFP) cells was performed after treatment with 0, 1, and 10 μM NADS for 48 hours. DHFR-EGFP fusion protein was detected using an antibody against EGFP. (B) CCRF-CEM and CCRF-CEM/R cells were exposed to 0, 1, and 10 μM NADS and NADPS for 0, 24, and 48 hours. The DHFR levels were detected using an anti-DHFR antibody. Equal loading was determined with GAPDH or α-tubulin, respectively.
Fig. 3.
Fig. 3.
Cytotoxic effect of NADPS is primarily attributable to targeting DHFR. (A) Western blot analysis of DHFR and other dehydrogenases (ALDH1a, GAPDH, and IMPDH). Protein levels were determined using antibodies specific to each dehydrogenase after treating C85 cells with 10 μM NADP-S for 0, 4, 6, 15, and 24 hours; α-tubulin was used as a loading control. (B) Trypan blue exclusion assay was used to determine cell viability of CCRF-CEM/R cells, containing a 5-fold increase of DHFR levels and CCRF-CEM parenteral cells after NADPS treatment for 96 hours. The cells were grown in RPMI-1640 medium supplemented with 10% dialyzed FBS. After harvesting cells, cell viability was determined using the Vi-CELL Series Cell Viability Analyzer. (C) MTS assay, as described in Materials and Methods, was performed to determine cytotoxic effect of NADPS in parental DG44 cells, a hamster ovary cell line with no DHFR expression and in DG44- (DHFR-EGFP), a subline of DG44 cells stably transfected with a DHFR-EGFP fusion construct. Parental DG44 cells were grown in RPMI 1640 medium that was supplemented with hypoxanthine and thymidine to allow these cells to grow in the absence of DHFR (▪, red). DG44-(DHFR-EGFP) cells were grown in RPMI 1640 media with (▴, blue) and without (▾, black) hypoxanthine and thymidine supplementation. Two thousand cells per well were plated, and cells were incubated with NADPS for 96 hours. The percentage cell survival was determined using Softmax Pro software, and GraphPad Prism 4 software was used to determine ED50 values using a sigmoidal dose-response curve fit.
Fig. 4.
Fig. 4.
Transcriptional downregulation of DHFR mRNA through G1/S block cannot explain the decrease in DHFR levels of NADPS-treated cells. (A–C) Nonsynchronized C85 cells were treated with 0, 10, and 40 μM NADPS for 24 hours and processed for propidium iodide labeling and flow cytometry. (D) Nonsynchronized C85 cells were treated with 10 nM MTX for 24 hours and processed for PI-labeling and flow cytometry. (E–G) Recovery G1 block by removing NADPS in C85 cells. Nonsynchronized C85 cells were treated with 0 or 10 μM NADPS for 48hours (E and F). (G) C85 cells were incubated with 10 μM NADPS for 24 hours and then replaced with new medium in the absence of NADPS for 24 hours. Cells were harvested and processed for flow cytometry. (H) After treating CCRF-CEM cells with 10 μM NADPS and 10 nM MTX for 24 hours, total cytoplasmic RNA was extracted for quantitative RT-PCR analysis. DHFR mRNA levels were normalized to β-actin mRNA levels. The expression in the untreated sample was set to one. The y-axis is the mean of the fold changes in DHFR mRNA expression level; bars, standard deviation. The results were averaged from three independent experiments, and each experiment was performed in triplicate.
Fig. 5.
Fig. 5.
A decreased DHFR level by NAD/NADP analogs is attributable to neither inhibition of MTX-mediated translational regulation of DHFR protein nor inhibition of ubiquitin proteasome pathway. DHFR protein levels were determined by Western blotting in CCRF-CEM cells. (A) CCRF-CEM cells were treated with 10 nM MTX alone, 10 μM NADPS alone, and simultaneously with 10 nM MTX and 10 μM NAPDS for 24 and 48 hours. (B) CCRF-CEM cells were treated with 10 nM MTX alone, 10 μM NADS alone, and simultaneously with 10 nM MTX and 10 μM NAPS for 24 and 48 hours. DHFR protein was detected by polyclonal anti-DHFR antibodies, and GAPDH was used as a loading control (A and B). Quantification of DHFR level was shown at the bottom of gels and measured by Image J program (provided by National Institutes of Health). (C) MCF-7 cells treated with 0.5 μM MG132, a proteasome inhibitor, in the presence and absence of 10 μM NADPS for 48 hours. GAPDH was used as the loading control, and Cyclin D1 was used as positive control for the activity of MG132. MCF-7 cells were also treated with MTX in the presence and absence of NADPS and MG132.
Fig. 6.
Fig. 6.
NAD kinase is inhibited by NADPS, leading to decreased half-life of DHFR. (A) Inhibition of NAD kinase was determined spectrophotometrically by measuring the increase in absorbance at 340 nm caused by the reduction of NADP to NADPH by G6PDH using a coupled assay (see details in Materials and Methods). (B) Effect of NADK suppression on DHFR protein levels was determined by Western blotting in C85 cells after transfection for 48 hours with siRNA targeting NADK. DHFR protein levels were compared with untreated cells and with NADPS-treated cells. (C) The half-life of DHFR was measured by a 35S methionine radiolabeled pulse-chase experiment. CCRF-CEM/R cells were treated with 10 μM NADPS, and at the indicated times, samples were subjected to immunoprecipitation with an anti-DHFR antibody, followed by SDS-PAGE and autoradiography. The lower panel shows the quantification of results in the presence and absence of NADPS. Values shown are the means ± S.D. for three independent pulse-chase experiments. For statistical analysis, Student's t test was used; paired comparisons of cellular DHFR levels between NADPS treated and untreated were made. P values were <0.05 both at 16 and 23 hours, suggesting that there was a statistical difference in DHFR levels between treated and untreated cells.
Fig. 7.
Fig. 7.
NADPS+ has a more favorable binding to NAD kinase, compared with NADP+. (A) Human NAD kinase (cartoon) complexed with NAD+ (space-filling model). Chain A is colored deep blue, and chain B is colored forest green (last snapshot from 2 ns of dynamics in TIP3P water box). Built from Protein Data Bank codes 3PFN (Human) and 1Z0Z (Arch. fulgidus). (B) Close-up of binding region with NAD+ and key interacting residues depicted as stick models where NAD+ (ball and stick model) carbon atoms are colored gray and chain A protein residue carbon atoms are colored navy blue, with chain B residue carbon atoms colored teal. (C) Close-up of binding region with NADP+ and key interacting residues depicted as stick models where NADP+ (ball and stick model) carbon atoms are colored gray and chain A protein residue carbon atoms are colored navy blue, with chain B residue carbon atoms colored teal. (D) Close-up of binding region with NADPS+ and key interacting residues depicted as stick models where NADPS+ (ball and stick model) carbon atoms are colored gray and chain A protein residue carbon atoms are colored navy blue, with chain B residue carbon atoms colored teal.
Fig. 8.
Fig. 8.
MTX protects cells from DHFR degradation by NADPS. DHFR protein levels were determined by Western blotting in DG44-(DHFR-EGFP) cells. (A and B) DG44 DHFR-EGFP cells or DG44 DHFRS118AEGFP cells were treated with 10 nM MTX alone, 10 μM NADPS alone, and the simultaneously addition of 10 nM MTX and 10 μM NAPDS for 24 and 48 hours. DHFR-EGFP and DHFRS118AEGFP protein were detected by a monoclonal anti-EGFP antibody. GAPDH was used as a loading control. Quantification of DHFR levels (shown at the bottom of gels) was measured by the Image J program (provided by NIH).
Fig. 9.
Fig. 9.
NADPS has a synergetic cytotoxic effect with MTX. DHFR levels were determined by Western blotting after sequential addition of NADPS and MTX. C85 (A) cells were treated with 10 nM MTX alone for 24 or 48 hours, pretreated with 10 mM NADPS for 4 or 6 hours, and followed by treatment with 10 nM MTX for 24 or 48 hours. DHFR protein was detected by an anti-DHFR antibody, and GAPDH was used as a loading control. (B) A clonogenic assay was performed to determine the cytotoxic effect of the combination of NADPS and MTX in C85 cells. Values were given as means ± SD for three independent experiments. (C) Combination index plots for C85 cells exposed to NADPS and MTX (see details in Materials and Methods).
Fig. 10.
Fig. 10.
NADPS is still effective in MTX transport–resistant cells. Parental CCRF-CEM (A) and CCRF- CEM /T cells, resistant to classic antifolates, such MTX and D1694 (raltitrexed; Tomudex) because of impaired transport (Mini et al., 1985) (B) were treated with MTX, D1694, and the lipophilic antifolate TMTX. The cells were grown in RPMI-1640 medium supplemented with 10% dialyzed FBS. After incubation with NADPS for 96 hours, cells were collected and viability was determined using Trypan blue exclusion assay with the Vi-CELL Series Cell Viability Analyzer. The percentage cell survival was determined by Softmax Pro software, and sigmoidal dose-response curve fit of the graphs drawn by GraphPad Prism 4 software was used to determine ED50 values. Although the concentrations of NADPS used in the experiment were micromolar, the antifolate concentrations were nanomolar.

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