Inhibitor of Nicotinamide Phosphoribosyltransferase Sensitizes Glioblastoma Cells to Temozolomide via Activating ROS/JNK Signaling Pathway

Abstract

Overcoming temozolomide (TMZ) resistance is a great challenge in glioblastoma (GBM) treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide and has a crucial role in cancer cell metabolism. In this study, we investigated whether FK866 and CHS828, two specific NAMPT inhibitors, could sensitize GBM cells to TMZ. Low doses of FK866 and CHS828 (5 nM and 10 nM, resp.) alone did not significantly decrease cell viability in U251-MG and T98 GBM cells. However, they significantly increased the antitumor action of TMZ in these cells. In U251-MG cells, administration of NAMPT inhibitors increased the TMZ (100 μM)-induced apoptosis and LDH release from GBM cells. NAMPT inhibitors remarkably enhanced the activities of caspase-1, caspase-3, and caspase-9. Moreover, NAMPT inhibitors increased reactive oxygen species (ROS) production and superoxide anion level but reduced the SOD activity and total antioxidative capacity in GBM cells. Treatment of NAMPT inhibitors increased phosphorylation of c-Jun and JNK. Administration of JNK inhibitor SP600125 or ROS scavenger tocopherol with TMZ and NAMPT inhibitors substantially attenuated the sensitization of NAMPT inhibitor on TMZ antitumor action. Our data indicate a potential value of NAMPT inhibitors in combined use with TMZ for GBM treatment.

Figure 1

NAMPT inhibitor sensitizes glioblastoma cells to TMZ treatment. (a) Upregulation of MGMT in U251-MG and T89-MG cells compared with normal human astrocyte (NHA) cells. (b) Confirmation of inhibitory effect of FK866 and CHS828 on NAD concentration in U251-MG and T89-MG cells. (c) Effects of low (5 nM) and high (100 nM) doses of FK866 on cell viability in U251-MG GBM cells. ^∗∗ P < 0.01 versus control. ^# P < 0.05 versus FK866 (5 nM). N = 8. (d) Effects of low (10 nM) and high (200 nM) doses of CHS828 on cell viability in U251-MG GBM cells. ^∗ P < 0.05 versus control. ^# P < 0.05 versus CHS828 (10 nM). N = 8. (e) Effects of low (5 nM) and high (100 nM) doses of FK866 on cell viability in T89 GBM cells. ^# P < 0.05 versus FK866 (5 nM). N = 8. (f) Effects of low (10 nM) and high (200 nM) doses of CHS828 on cell viability in T89 GBM cells.

Figure 2

NAMPT inhibitor increases the TMZ-induced apoptosis and necrosis in glioblastoma cells. (a) Fluorescent images of TUNEL assay and quantitative analysis showing the effect of low doses of FK866 and CHS828 (10 nM and 20 nM, resp.) on the apoptosis in U251-MG GBM cells. DAPI was used to stain nuclei. ^∗ P < 0.05 versus TMZ alone. N = 8. At least 20 visual fields were included for analysis. (b-c) LDH assay showing the LDH content in culture medium of TMZ alone, TMZ plus FK866, and TMZ plus CHS828 treated U251-MG GBM cells. ^∗ P < 0.05 and ^∗∗ P < 0.01 versus TMZ alone. N = 8.

Figure 3

NAMPT inhibitor enhances the caspase-1, caspase-3, and caspase-9 activities in glioblastoma cells. (a–c) Effects of NAMPT inhibitors FK866 and CHS828 on the activities of caspase-1, caspase-3, and caspase-9 in U251-MG GMB cells. ^∗∗ P < 0.01 versus TMZ alone. N = 8.

Figure 4

NAMPT inhibitor augments the TMZ-induced ROS production in glioblastoma cells. Relative ROS level (a), superoxide anion level (b), total SOD activity (c), and total-oxidant capacity (d) in U251-MG glioma cells. ^∗ P < 0.05 versus TMZ alone. N = 8.

Figure 5

NAMPT inhibitor activates c-Jun/JNK signaling pathway in glioblastoma cells. (a) Representative images of phosphorylation of c-Jun and JNK in TMZ alone, TMZ plus FK866, and TMZ plus CHS828 treated U251-MG GBM cells. (b-c) Quantitative analysis on the phosphorylation of c-Jun and JNK in TMZ alone, TMZ plus FK866, and TMZ plus CHS828 treated U251-MG GBM cells. ^∗ P < 0.05 versus TMZ alone. N = 5.

Figure 6

The c-Jun/JNK pathway and ROS production contribute to the sensitization of NAMPT inhibitor on TMZ antitumor action in glioblastoma cells. (a-b) Effects of SP600125, a specific inhibitor of JNK pathway, on the antitumor action of FK866 (a) and CHS828 (b). ^∗ P < 0.05 versus FK866 (5 nM) or CHS828 (10 nM). N = 8. (c-d) Effects of tocopherol, a ROS scavenger, on the antitumor action of FK866 (c) and CHS828 (d). ^∗ P < 0.05 versus FK866 (5 nM) or CHS828 (10 nM). N = 8. ^# P < 0.05 versus control. ^∗∗ P < 0.01 versus FK866 (5 nM) or CHS828 (10 nM) alone.