Briefly, THP-1 cells were pretreated by plating at in 24-well plates. Compounds were then incubated with an NAMPT inhibitor for 5 h to evaluate decreases in NAD. Cells were collected, and NAD was quantified using a slightly modified version of a previously de- scribed enzymatic cycling procedure . After washing with chilled PBS (4 °C), cells were treated with 50 μL of 1 N HClO4 for 15 min on ice and then neutralized with an equal volume of 1 N KOH and 100 μl of Bicine (200 mM, pH 8.0). A 50- μl aliquot of cell extract was mixed with an equal volume of the Bicine buffer containing 23 μL/mL of ethanol, 0.17 mg/ml of MTT, 0.57 mg/ml of phenazine ethosulfate, and 10 μg of alcohol dehydrogenase. The mixture was maintained at room temperature for 30 min, and absorbance was measured at 570 nm using an Infinite M1000 microplate reader (Tecan, Männedorf, Switzerland). A standard curve allowed for quantification of NAD.
Fig. 3. KN-93 and KN-92 do not affect decreases in NAD induced by NAMPT in- hibitors. THP-1 cells were preincubated with 10 μM KN-93 or KN-92 for 24 h, fol- lowing the addition of 10 μM AS1604498 or 1 μM FK866. After 5 h, cells were harvested, and NAD was measured. Values are expressed as mean7S.E.M from independent experiments. n.s., not significant in the samples evaluated (one-way ANOVA). npo0.05 vs. control.
2.6. Real-time quantitative PCR
THP-1 cells were plated at 6 105/3 mL with compounds in 6-well plates and incubated for 40 h. Total cellular RNA was iso- lated using an RNeasy mini kit (Qiagen, Valencia, CA, USA). Total RNA was reverse-transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN, USA) in accordance with the manufacturer’s instructions. The following PCR primer sets were used: 5’-GCAGAGCTGGAAGTCGAGTG-3’ (F), 5’-GAGCAGAAGAGTTTGGATATCAG-3’ (R) for Noxa; 5’-CGGAGGAT- GAGTGACGAGTT-3’ (F), 5’-CCACCAGGACTGGAAGACTC-3’ (R) for Bad; 5’-GACCATGGAGGTTCTTGGCA-3’ (F), 5’-AGGCTCACGTC- CATCTCGTC-3’ (R) for Bik; 5’-CCTTTTCTACTTTGCCAGCAAAC-3’ (F), 5’-GAGGCCGTCCCAACCAC-3’ (R) for Bax; 5’-GAAGCATTGGGGAT- CAAGAA-3’ (F),5’-AGCAGATGGCTCGAGAATACA-3’ (R) for YWHAZ; 5’-GACGACCTCAACGCACAGTA-3’ (F), 5’-AGGAGTCCCATGATGA- GATTGT-3’ (R) for Puma; 5’-CAACACAAACCCCAAGTCCT-3’ (F), 5’- GCATATCTGCAGGTTCAGCC-3’ (R) for Bim; 5’-CTGAGTACCT- GAACCGGCA-3’ (F), 5’-GAGAAATCAAACAGAGGCCG-3’ (R) for Bcl-2; 5’-GCGTGGAAAGCGTAGACAAG-3’ (F), 5’-TGCTGCATTGTTCCCATA- GA-3’ (R) for Bcl-xl; 5’-CAGTGCATTGCAGACCAGTT-3’ (F), 5’- TTCAAAGCAAGGTTGTGCAG-3’ (R) for Bmf. YWHAZ was used as an independent experiments. n.s., not significant and nnnpo0.001 vs. AS only.
2.7. Statistical analysis
Significance was assessed using Student’s t-test or one-way analysis of variance followed by Dunnett’s multiple comparison test unless otherwise noted in the. p o 0.05 was considered significant.
3.1. KN-93 inhibits apoptosis induced by NAMPT inhibitors
In the evaluation of compounds with physiological activities to identify the signaling pathway by which apoptosis is induced by NAMPT inhibitors, the CaMKII inhibitor KN-93 was found to ef- fectively reduce apoptosis. Time-lapse imaging was used to de- termine the time course of apoptosis induced by the NAMPT in- hibitor AS1604498. Following exposure to AS1604498, apoptosis occurred at 30 h and peaked at 48 h. KN-93 inhibited and delayed apoptosis (Fig. 1A) and dose-dependently inhibited caspase 3/7 activation at 40 h (Fig. 1B). Inhibition of apoptosis was also con- firmed by trypan blue exclusion assay (Fig. 1C).
3.2. Inhibition of apoptosis by KN-93 is independent of CaMKII
The role of CaMKII activity in the inhibition of apoptosis was in- vestigated using Autocamtide-2-related Inhibitory Peptide II, Cell- permeable (Ant-AIP-II), a peptide inhibitor reported to be effective in cell-based assays at 10 μM . Ant-AIP-II did not inhibit AS1604498- induced apoptosis (Fig. 2). The effect of KN-92, the inactive analog of KN-93 that exerts no inhibition of CaMKII , on apoptosis was then investigated. As shown in Fig. 2, KN-92 inhibited apoptosis, indicating that a mechanism other than CaMKII activity is involved. Buy NMN
Fig. 5. NAMPT inhibitors upregulate Bim and KN-92 does not affect this induction. AS1604498 was added at 10 μM to THP-1 cells with or without KN-92 at 10 μM. Cells were harvested after 40 h, and the expression of mRNAs was evaluated by quantitative real-time PCR. Fold index was calculated with the expression in con- trol sample without a standard. Data are shown as mean of two independent ex- periments. n.s., not significant.
Fig. 6. L-type Ca2 þ channel blockade partially inhibits apoptosis induced by NAMPT inhibitors. Effects of L-type Ca2 þ channel blocker verapamil and nimodi- pine were evaluated. AS1604498 was added at 10 μM, and concentrations of ver- apamil and nimodipine are presented in micromolar values. Caspase 3/7 activity was evaluated after 40 h. Values are expressed as mean7S.E.M from three in- dependent experiments. nnpo0.01, nnnpo0.001 vs. AS only.