Article Figures & Data
Figures
- Fig. 1.
Inhibition of NAMPT sensitizes primary human tissue to the growth inhibitory effects of MTX. Primary human fibroblasts were transfected with either control scrambled siRNA or with NAMPT-specific siRNA. (A) Twenty-four hours after transfection, cells were treated with MTX at concentrations up to 10 µM for 96 hours. (B) Isolated human lymphocytes were left unstimulated or were stimulated with 2% phytohemagglutinin and treated with either 10 nM MTX alone, 0.1 nM FK-866 alone, or 10 nM MTX in combination with 0.1 nM FK-866. Cell viability was measured by fluorescence spectroscopy using the resazurin reduction assay. Experiments were conducted in triplicate and the resulting mean ± S.D. for each experiment is presented. Statistical testing was conducted using t test analysis. Scr, scrambled.
- Fig. 2.
MTX activity in NAMPT-deficient cells is folate dependent. A549 cells were transfected with either control scrambled siRNA or with siRNA directed toward NAMPT. Twenty-four hours after transfection, cells were treated with different concentrations of MTX alone or in combination with 10 μM folinic acid for 96 hours. (A–C) Knockdown of NAMPT by siRNA was confirmed by Western blot analysis (A), quantified by densitometry analysis (B), and verified by real-time-PCR (C). Cell viability was measured by fluorescence spectroscopy using the resazurin reduction assay. (D) The effect of MTX toxicity alone or in combination with 10 μM folinic acid was determined as percent viability based on untreated control cells. Experiments were conducted in triplicate and the resulting mean ± S.D. for each experiment is presented. Concentrations of MTX required for half-maximal inhibition of 50% inhibition of cell viability were determined and compared by t test analysis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MW, molecular weight; Scr, scrambled.
- Fig. 3.
NAD supplementation reverses enhanced MTX sensitivity in NAMPT-deficient cells. (A) A549 cells were transfected with either control scrambled siRNA or with siRNA directed toward NAMPT and cellular NAD levels were measured 96 hours after transfection. (B) Cellular viability was measured in NAMPT-deficient cells and treated with various concentrations of MTX with or without supplementation of 200 μM NAD. Experiments were conducted in triplicate and the resulting mean ± S.D. for each experiment is presented. NAD levels were compared by t test analysis. Concentrations of MTX required for half-maximal inhibition of 50% inhibition of cell viability were determined and compared by t test analysis. Scr, scrambled.
- Fig. 4.
Inhibition of NAMPT does not affect DHFR expression or intracellular levels of folate or MTX. (A) A549 cells were transfected with either control scrambled siRNA or with siRNA directed toward NAMPT and protein levels of DHFR were monitored 96 hours after transfection. (B) For densitometry analysis, DHFR protein levels were normalized to GAPDH. (C and D) Cellular concentrations of folate (C) and MTX levels (D) were analyzed in control and NAMPT-deficient cells treated for 24 hours with 1000 nM MTX. Experiments were conducted in triplicate and the resulting mean ± S.D. for each experiment is presented and compared by t test analysis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, not significant; Scr, scrambled.
- Fig. 5.
MTX does not cause synergistic depletion of cellular NAD in NAMPT-deficient cells. (A) A549 cells were transfected with either control scrambled siRNA or with siRNA directed toward NAMPT; 24 hours after transfection, cells were treated with 1000 nM MTX for 96 hours or 50 mM etoposide for 24 hours to determine activation of PARP by measuring PAR formation by Western blot analysis. (B) Control and NAMPT-deficient cells were treated with different concentrations of MTX for 96 hours and total DNA was separated on 1% agarose gel to evaluate DNA damage. (C) Total NAD levels were monitored in control and NAMPT-deficient cells treated with different concentrations of MTX for 96 hours. Experiments were conducted in triplicate and the resulting mean ± S.D. for each experiment is presented and compared by t test analysis. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MW, molecular weight; NS, not significant; PAR, poly(ADP-ribose); Scr, scrambled.
- Fig. 6.
Increased MTX activity does not occur through activation of PARP. A549 cells transfected with either control scrambled siRNA or with siRNA directed toward NAMPT were treated with different concentrations of MTX with or without 10 μM olaparib. (A and B) The effect of inhibition of PARP-1 by olaparib on MTX toxicity in control (A) and NAMPT-deficient (B) cells was determined by normalizing cell growth inhibition with MTX against control cells. Experiments were conducted in triplicate and the resulting mean ± S.D. for each experiment is presented. Concentrations of MTX required for half-maximal inhibition of 50% inhibition of cell viability were determined and compared by t test analysis. Scr, scrambled.
- Fig. 7.
NAMPT inhibition results in the synergistic depletion of cellular ATP by MTX. (A and B) A549 cells transfected with either control scrambled siRNA or with siRNA directed toward NAMPT were treated with different concentrations of MTX and cellular ATP levels were measured 96 hours after transfection (A) and in response to treatment with different concentrations of MTX over 96 hours (B). The effect of MTX on cellular ATP was determined by normalizing cellular ATP levels to the untreated control cells. Experiments were conducted in triplicate and the resulting mean ± S.D. for each experiment is presented and compared by t test analysis. Scr, scrambled.
- Fig. 8.
NAMPT inhibition does not affect AICART activity but is associated with an increase in intracellular intermediates of de novo purine biosynthesis. A549 cells were transfected with either control scrambled siRNA or with NAMPT-specific siRNA. (A–C) After 72 hours under normal culture conditions, cells were subsequently harvested and intracellular concentrations of ZMP (A) and IMP (B) and the ratio of IMP/ZMP (C) were determined. Experiments were conducted in triplicate and the resulting mean ± S.D. for each experiment is presented and compared by t test analysis. NS, not significant; Scr, scrambled.
- Fig. 9.
Supplementation of nucleotide biosynthesis rescues cell viability and ATP levels, but not NAD levels. (A–C) A549 cells transfected with either control scrambled siRNA or with siRNA directed toward NAMPT were treated with 1000 nM MTX alone or together with a combination of 100 μM hypoxanthine and 100 μM thymidine for 96 hours and measured for cellular viability (A), cellular NAD levels (B), and cellular ATP levels (C). Experiments were conducted in triplicate and the resulting mean ± S.D. for each experiment is presented and compared by t test analysis. NS, not significant; Scr, scrambled.
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NAMPT Deficiency Potentiates ATP Depletion by Methotrexate
