Fig 1: NAMPT expression is significantly increased in PCa with extraprostatic invasion. Immunohistochemistry revealed minimal NAMPT detection in normal (A) and benign hyperplastic (B) prostate tissues. Antibody negative controls (C) showed no staining in PCa tissue. NAMPT immunostaining (arrows) was weak to moderate in histologic Group 1 (D, E) or Group 3 (F) organ-confined PCa (Gleason scores 3 + 3 = 6 or 4 + 3 = 7) but was markedly increased in extraprostatic invasive PCa with Gleason score 3 + 3 = 6, Group 1 (G), 3 + 4 = 7, Group 2 (H) or 5 + 5 = 10, Group 5 (I) and invasion into periprostatic fat (*) and nerve (n). ImageJ software semi-quantitation of NAMPT immunostaining intensity (J) represented as mean Gray Value confirmed significantly higher NAMPT staining in PCa tissues compared to benign prostate tissues (n = 10) (* p<0.05) with further significant increased expression in extraprostatic-invasive PCa (n = 13) compared to organ-confined PCa (n = 10) (** p<0.05). A-I, x 200.
Fig 2: Plasma eNAMPT levels track with human PCa and its invasion. A. Compared to healthy male controls (Controls), age-matched healthy male controls (M-Controls) and subjects with high PSA levels but without PCa (High risk), plasma eNAMPT levels were significantly higher in subjects developed PCa (PCa) (*, **, ***p<0.0001). B. Plasma eNAMPT levels were measured using the NAMPT ELISA detection kit from Thermo Fisher Scientific. This confirmed the significant increase in plasma eNAMPT levels in PCa (n = 70) compared to subjects without PCa (n = 50) (* p<0.05). C. Plasma eNAMPT levels were significantly higher in extraprostatic invasive PCa compared to organ-confined PCa (*p<0.05). D. Calculated by MedCal, the plasma eNAMPT levels (n = 34 PCa, 130 high risk, and 105 healthy male control) had significant (AUC 0.79, p<0.0001) predictive value for diagnosing PCa. Calculated by MedCal, the plasma eNAMPT levels (n = 10 organ-confined PCa, 13 extraprostatic-invasive PCa) had significant (AUC 0.76, p<0.05) predictive value for diagnosing invasive PCa. E. In contrast, there was no significant difference in presurgical blood PSA levels of subjects with organ-confined versus extraprostatic-invasive PCa (p>0.05). F. There was no significant correlation among the PSA levels and eNAMPT levels (n = 164, p>0.05).
Fig 3: Influence of PCa-relevant transcription regulators on NAMPT promoter and eNAMPT secretion. PC3 and Du145 cell cultures were treated with FG-4592 100 uM, or EGF 100 ng/ml, or testosterone 100 nM for 4 or 24 h. The NAMPT promoter activities (using dual-luciferase assay) were significantly increased in response to FG-4592 and EGF (p<0.05) as early as 4 h and were sustained for up to 24 h (A, B), results confirmed by western blots of NAMPT protein levels in cells at 24 h (C, D).
Fig 4: Effect of eNAMPT on in vivo PCa invasion assays. PC3 cells invaded through diaphragm muscle layer in untreated SICD mice (n = 5) (A); intraperitoneal injection with eNAMPT neutralizing antibody significantly inhibited the tumor invasion (n = 5) (B, G, *p<0.05). Immunohistochemistry showed abundant Ki67-positive PC3 cells in untreated group (C) compared to fewer Ki67-positive cells in eNAMPT antibody-treated PC3 cells (D). The percentages of Ki67-positive cells were significantly higher in untreated group (Veh) compared to eNAMPT pAb-treated group (eNAMPT ab) (H, *p<0.05). PC3 cells highly expressed NAMPT in vivo detected by immunohistochemistry (E) and the plasma eNAMPT levels were significantly higher in untreated group (Veh) compared to normal mice (Ctr) and eNAMPT pAb treated group (Ab) (F, *p<0.05). A-D x200; E x100.
Supplier Page from Thermo Fisher Scientific for Human Visfatin ELISA Kit