NDGA inhibits p300 in vitro

To test the hypothesis that NDGA is a p300 inhibitor, we used two different in vitro enzymatic assays that measure the acetyltransferase activities of p300 and PCAF (p300/CBP-associated factor). In the first assay, we used purified human recombinant p300 BHC protein29 (a construct containing the bromodomain, catalytic acetyltransferase domain, and C/H3 domain, amino acids 965–1810) that represents the acetyltransferase activity of full length protein,30 histone H3 (amino acids 1–21) peptide as a substrate, and acetyl-CoA as an acetyl-group donor, to measure the activity of p300 on histone H3 in the presence of NDGA. In this fluorimetric assay, NDGA inhibited p300 BHC with an IC 50 of 10.3 μM, compared with 75 μM for the known p300 inhibitor anacardic acid31 (Fig. 1a). When we evaluated NDGA for the inhibition of human recombinant PCAF HAT (histone acetyltransferase domain), we observed no inhibition even at high concentrations of NDGA (Fig. 1b), ruling out a nonspecific mechanism of action. In the second assay, we used purified human recombinant p300 HAT protein consisting of the core HAT only (amino acids 1284–1673), histone H3 peptide (amino acids 1–21), and acetyl–CoA mixture in reaction buffer containing increasing concentrations of NDGA. Consistent with Fig. 1a, b, NDGA inhibited p300 HAT activity (Fig. 1c) but failed to inhibit purified human PCAF HAT activity (Fig. 1d). To further understand the mechanism of p300 inhibition, we determined inhibition kinetics of p300 HAT activity by NDGA for both substrates acetyl-CoA and H3 peptide. Double-reciprocal plots were constructed to find the nature of inhibition. Inhibition kinetics studies clearly showed that NDGA is a noncompetitive inhibitor for both acetyl-CoA (Fig. 1e) and histone H3 binding sites (Fig. 1f). These results indicate that NDGA is a selective inhibitor of p300 acetyltransferase activity in vitro.

Fig. 1 NDGA inhibits histone acetyltransferase activity of p300 in vitro. a The acetyltransferase activity of p300 on histone H3 peptide (aa 1–21) was measured in the presence of NDGA or anacardic acid. Results are expressed as percentage activity in the absence of NDGA. A paired t-test was performed for statistical significance, p = 0.0013. b Effect of NDGA on PCAF HAT activity expressed as percentage activity in the absence of NDGA. Inhibitory effects of NDGA on p300 (c) and PCAF (d) using another enzymatic assay were measured and are expressed as percentage inhibition relative to control DMSO. Effects of varying concentrations of acetyl-CoA (e) and substrate peptide histone H3 in the presence of varying concentrations of NDGA were measured (f). Data represent the results from three independent experiments (mean ± S.D.), and curves were generated using nonlinear regression fit Full size image

NDGA inhibits p300-mediated histone acetylation in human, mouse and fruit fly cells

As an epigenetic regulator, p300 orchestrates gene expression via acetylation of specific lysine residues in specific cellular histone proteins in cells.32 To determine whether NDGA influences histone acetyl marks in cells, we monitored the acetylation status of different lysine residues in HEK293T cells treated with NDGA. Specifically, we extracted the histones and conducted western blot analysis using antibodies selective for unique acetylated histone residues and determined IC 50 values for each residue (Fig. 2a, Supplementary Fig. 1). Previous genetic studies reported that deletion of p300 specifically reduces acetylation on H3K27 but not H3K9.33 Consistent with our hypothesis that NDGA selectively inhibits p300 in cells, we found that histone H3K27 acetylation was the most suppressed in cells treated with NDGA with an IC 50 of 8.8 μM (Fig. 2a–c). The acetylation of other histone residues was also suppressed but in most cases with IC 50 > 25 μM, except for H3K14 (Fig. 2a). As expected, residues that are not p300 targets, such as H3K9, were unchanged by NDGA (Fig. 2a–c).

Fig. 2 NDGA inhibits p300-driven histone acetylation in HEK293T cells. a Median inhibitory concentration (IC 50 ) values for NDGA on different acetylated histone residues and median effective concentration (EC 50 ) value on H3K27 tri-methylation were determined by immunoblotting of histones extracted from cells after 24-h treatments with varying concentrations of NDGA (ND, not determined). b Differences in acetylation and tri-methylation of p300 target (H3K14, H3K18, and H3K27) and non-target (H3K9) histones were monitored by immunoblotting after indicated concentrations of NDGA treatment for 24 h and histone extraction. c Dose-dependent changes in acetylation and tri-methylation on H3K27 after 24-h NDGA treatment were analyzed by immunoblotting and are represented as percentage change in histone modification relative to DMSO treatment. d Decreases in H3K27 acetylation upon 30 µM of NDGA treatment for 24 h in human (HEK293T), mouse (MEF) and fruit fly (S2 Schneider) cells were monitored by immunoblotting after histone extraction. e. Effects of wildtype p300 overexpression on NDGA-dependent H3K27 hypoacetylation were determined by densitometric analysis of immunoblotting data. Cells were transfected with different amounts of human wildtype EP300 encoding expression plasmid pCI-p300(WT) and/or empty vector (EV) and treated with varying NDGA concentrations for 24 h. The data represent percentage change in H3K27 acetylation relative to DMSO treatment and the change in IC 50 values. f The rescue of p300 overexpression on H3K27 hypoacetylation after 30 µM NDGA treatment was performed as in panel E. g Effects of overexpression of wildtype and catalytically inactive p300 mutants (Y1503A and F1504A) on NDGA-induced H3K27 hypoacetylation were determined by immunoblotting and densitometry. Cells were transfected by the same amount of expression plasmids or empty vector (EV) and treated with varying NDGA concentrations. Histones were extracted and analyzed. h The effects of wildtype and mutant (Y1503A or F1504A) p300 overexpression on NDGA-induced histone hypoacetylation and hypermethylation were determined by immunoblotting after 30 µM NDGA treatment for 24 h. i Effects of wildtype and mutant p300 overexpression on NDGA-induced histone hypoacetylation were analyzed by immunoblotting and densitometry. j Decreased H3K27 tri-methylation upon wildtype p300 overexpression was detected by immunoblotting and densitometry. Total histone antibodies were used as loading controls in all experiments. Data represent the results from two independent experiments (mean ± S.D.) and curves were generated using nonlinear regression fit Full size image

To test the effect of NDGA on H3K27 acetylation in different organisms, we also treated mouse (MEF) and fruit fly (S2 Schneider) cell lines with NDGA (30 μM) for 24 h, and analyzed extracted histones by immunoblotting. As with HEK293T, H3K27 acetylation was also found to be decreased also in these cell lines upon NDGA treatment (Fig. 2d, Supplementary Fig. 2), demonstrating the suppressive effect of NDGA on H3K27 acetylation in evolutionarily distant organisms.

NDGA-induced suppression of H3K27ac is associated with increased tri-methylation of the same residue in HEK293T cells

Decreased histone H3 K27 tri-methylation (H3K27me3) is a characteristic hallmark of aging in both invertebrates and vertebrates. Worms and fruit flies exhibit an age-associated decrease in H3K27me3 marks,34 but in humans, a strong decrease in this modification has been observed in primary fibroblasts from patients with Hutchinson–Gilford progeria syndrome, a premature aging disease.35 Conversely, the naked mole rat, which is the longest-living rodent with a lifespan of more than 28 years,36 bears higher levels of H3K27 tri-methylation compared to mice.37 Since acetylation and methylation are mutually exclusive on a single lysine, decreased acetylation can result in an increase in methylation on the same residue.38 Therefore, we treated HEK293T cells with increasing concentrations of NDGA, extracted histones, and determined the methylation status of H3K27 by immunoblotting. As predicted and in agreement with the decrease in H3K27 acetylation, cells treated with NDGA showed a significant increase in H3K27 tri-methylation with a half maximal effective concentration (EC 50 ) of 9.80 μM (Fig. 2a–c).

Overexpression of p300 reverses NDGA-induced loss of H3K27 acetylation

To further assess p300 as a relevant target of NDGA, we overexpressed different amounts of full-length p300 in HEK293T cells and treated them with NDGA. In support of a role for p300 in sustaining H3K27 acetylation, we found that overexpression of p300 suppressed the effect of NDGA in a dose-dependent manner and increased H3K27 acetylation (Fig. 2e, Supplementary Fig. 3). The IC 50 of NDGA in rescue experiments was strongly correlated with the amount of p300-expressing plasmid transfected (Fig. 2f, Supplementary Table 1). Furthermore, addition of catalytically inactive p300 mutants (p300Y1503A and F1504A)39 failed to revert NDGA-mediated inhibition (Fig. 2g, Supplementary Fig. 3). In addition to H3K27 acetylation, we also evaluated the acetylation of other histone residues with an IC 50 lower than 50 μM (H3K4, H3K14, H3K18, H2AK13). While we observed a significant rescue effect on H3K27 and a slight rescue in H3K4 acetylation with wildtype p300 overexpression, changes in other acetylated residues were insignificant (Fig. 2h, i, Supplementary Fig. 4). In agreement with our previous result, we observed a significant decrease in H3K27 tri-methylation when wildtype p300 was overexpressed; whereas overexpression of mutant p300 was unable to change the levels of H3K27me3 (Fig. 2j, Supplementary Fig. 4). These findings support the hypothesis that NDGA-mediated H3K27 hypoacetylation and elevated H3K27 tri-methylation are specifically due to inhibition of p300 acetyltransferase activity.

p300 is a direct target of NDGA in HEK293T cells

To provide additional evidence that NDGA directly binds to p300 in cells, we used a cellular thermal shift assay (CETSA) that uniquely allows monitoring of drug engagement inside cells.40 Based on the principle of ligand-induced protein stabilization, compound-bound proteins precipitate at higher temperatures than unbound proteins and can be detected at higher levels in the soluble fraction. To identify direct targets of NDGA, we tested different acetyltransferases. We exogenously expressed p300 HAT (p300/CBP family), GCN5 (general control of amino acid synthesis protein 5-like 2) and PCAF (GNAT family), and TIP60 (60-kDa Tat-interactive protein, MYST family) in HEK293T cells and treated the cells with NDGA. Intact cells were heated at different temperatures for 3 min to denature and precipitate proteins. These cells were then subjected to three freeze-thaw cycles to lyse them, isolation of a soluble fraction after centrifugation (20,000 × g) and analysis by immunoblotting. We observed an increase in the thermostability of p300 HAT domain by 10.8 °C upon NDGA treatment (Fig. 3a, Supplementary Fig. 5). To confirm this thermostabilization in a dose-dependent manner, we treated cells with different concentrations of NDGA and heated them at a single temperature (55 °C) where we previously detected 35% of the total amount of p300 in the soluble fraction. Maximal stabilization (100%) occurred at 30 μM NDGA (Fig. 3b, Supplementary Fig. 5). Importantly, CETSA analysis of GCN5, PCAF, and TIP6 (Fig. 3c–e, Supplementary Fig. 5) showed no significant change in thermostability upon NDGA treatment. The difference in melting temperatures (T m ) of proteins tested are shown in Fig. 3f. These results further support the model that NDGA specifically binds to p300 HAT at a concentration consistent with its inhibitory activity on histone acetylation (Fig. 2).

Fig. 3 NDGA is a target of NDGA in HEK293T cells. a Melting curves of the p300 HAT domain were generated from a CETSA experiment performed in intact cells expressing HA tagged p300-HAT construct following 3-h DMSO or NDGA treatment. b Isothermal dose-response fingerprint-CETSA (ITDRF CETSA ) graph was generated from cells expressing the p300-HAT construct. Intact cells were treated with different concentrations of NDGA for 3 h and subjected to heating at 55 °C for 3 min to monitor thermostabilization. Melting curves of different acetyltransferases were generated from intact cells expressing HA-GCN5 (c), HA-PCAF (d) or HA-TIP60 (e) after 3-h DMSO or 30 µM NDGA treatment and CETSA. f Change in melting temperatures (T m ) of tested proteins upon NDGA treatment. Data represent the results from two independent experiments (mean ± S.D.), and curves were generated using nonlinear regression fit Full size image

NDGA induces autophagy in HEK293T and HeLa cells

While we demonstrated a potential molecular mechanism for the role of NDGA in aging cells above, the cellular mechanism by which NDGA elicits these effects remains unexplored. p300, the molecular target of NDGA, is a regulator of cellular autophagy, an evolutionarily conserved process for efficient removal of damaged and potentially harmful cellular contents, including long-lived proteins and cellular organelles.41 To accomplish this cellular cleansing effort, the coordinated actions of various autophagy-related (Atg) proteins are required. Atg proteins provide the main molecular machinery essential for initiating autophagosome formation via an ubiquitin-like conjugation system.42 Interestingly, p300 modulates the acetylation status of several Atg proteins. Silencing of p300 expression reduces the acetylation of Atg5, Atg7, LC3, and Atg12, and increases their stability and cellular level, resulting in autophagy pathway activation. Overexpression of p300, on the other hand, causes a significant increase in acetylation, decreases the stability of mentioned key proteins, and consequently blocks autophagy by lowering their cellular level. These previous results predict that inhibiting p300 by NDGA could act through activating autophagy. To test this hypothesis, we measured the level of autophagy after NDGA treatment and in response to serum-free media as a positive control. Adding NDGA to HEK293T cells induced several molecular markers of autophagy, including AMPK phospho-activation and upregulation of Beclin-1 (Fig. 4a, Supplementary Fig. 6).

Fig. 4 NDGA induces autophagy in HEK293T and HeLa cells. a Activation of the autophagy signaling pathway was monitored by immunoblotting of total proteins isolated from HEK293T cells after a 24-hour treatment with DMSO or varying concentrations of NDGA. Serum-free medium (SFM) was used as a positive control of autophagy. b Autophagosome formation in HeLa-GFP-LC3 cells was monitored by counting GFP-LC3 puncta after a 24-h treatment with DMSO or NDGA, with or without Bafilomycin A1 (BafA, 100 nM) or Chloroquine (Chq, 100 µM) treatment 3 h before sample collection. c The percentage of autophagy was measured in HeLa-GFP-LC3 cells treated with DMSO or NDGA, with or without Bafilomycin A1 or Chloroquine treatment for 3 h prior to sample collection. The threshold value to determine autophagy-positive cells was based on previous measurements and DMSO-treated cells; d. Autophagy induction in HeLa-GFP-LC3 cells was analyzed by FACS after a 24-h treatment with varying NDGA concentrations. e GFP-LC3 puncta (green) in HeLa-GFP-LC3 cells treated with DMSO or NDGA, with or without Bafilomycin A1 or Chloroquine were captured by confocal microscopy. Cells were fixed after 24-h treatment, stained with DAPI (blue) and analyzed. f Effects of Bafilomycin A and Chloroquine treatment for 3 h on autophagy marker proteins were quantitated by immunoblotting following total protein extraction. g Effects of wildtype and mutant (Y1503A and F1504A) p300 overexpression on NDGA-induced autophagy were monitored by immunoblotting. h Effects of p300 inhibitors on H3K27 acetylation and autophagy induction were analyzed by immunoblotting. HEK293T cells treated with NDGA (100 µM), A485 (100 nM) or C646 (100 µM) for 24 h, and total protein or histones were extracted. Numbers in immunoblotting images (f–h) indicate the fold change determined by densitometry analysis. β-Actin and total histone H3 were used as loading controls. Data represent the results from two independent experiments (mean ± S.D.), and statistical significance was determined by student t-test (*p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001) Full size image

Atg proteins together regulate the formation of a double membrane structure (autophagosome) that engulfs the cellular cargo targeted for degradation.43 Briefly, Atg7 induces Atg12-Atg5 conjugation and this conjugate specifies localization of LC3 on the lipid membrane. Our results showed a dose-dependent increase in Atg7 levels, and an increase in Atg5/Atg12 dimerization (Fig. 4a, Supplementary Fig. 6), consistent with induction of autophagy. In addition, we observed upregulation of LC3-II, which is associated with autophagosome formation as well as upregulation of the autophagy cargo receptors NBR1 (Neighbor of BRCA1 gene 1) and p62 (SQSTM1, Sequestosome-1) upon treatment with NDGA (Fig. 4a, Supplementary Fig. 6). To confirm the induction of autophagy in cells, we visualized the process by imaging HeLa cells stably expressing GFP-LC3. Following a 24-h treatment with NDGA (30 μM) or DMSO, we analyzed LC3 lipidation. LC3-positive puncta were significantly increased with NDGA treatment. Moreover, treatment with Bafilomycin A1, an V-ATPase inhibitor that prevents the fusion of autophagosomes and lysosomes,44 or with the lysosomal pH-neutralizing compound Chloroquine45 resulted in an additional increase in GFP-LC3 puncta in treated cells (Fig. 4b, e). These results indicate the accumulation of autophagosomes demonstrating that HeLA-GFP-LC3 cells have a functional autophagy pathway. An increase in autophagosome number upon NDGA treatment caused a rise in the number of cells undergoing autophagy (Fig. 4c, e). We also observed that the pro-autophagic effect of NDGA was dose-dependent (Fig. 4d, Supplementary Fig. 7). NDGA-treated HEK293T cells showed increased levels of endogenous NBR1, p62, and LC3-II upon NDGA treatment; Bafilomycin A or Chloroquine treatment further enhanced this effect (Fig. 4f, Supplementary Fig. 6). Since the overexpression of p300 rescues H3K27 hypoacetylation (Fig. 2f), we next evaluated p62 and LC3-II levels in wildtype versus mutant p300 overexpressing HEK293T cells upon NDGA treatment. Figure 4g shows the rescue effect of excess p300 expression by downregulation of these autophagy markers (Supplementary Fig. 6). When we compared NDGA with other p300 inhibitors C646 and A485 for autophagy induction, we discovered that these inhibitors also increased the levels of autophagy markers NBR1, p62, and LC3-II in HEK293T cells (Fig. 4h, Supplementary Fig. 6). These data indicate that NDGA effectively increases autophagic flux in HEK293T and GFP-LC3 HeLa cells, and that p300 activity is a key element in NDGA-induced autophagy.

NDGA increases median lifespan, decreases histone acetylation, and induces autophagy in worms

To determine the prolongevity property of NDGA in vivo, we treated wildtype C. elegans (N2 Bristol) with 100 μM NDGA. The median lifespan of worms exposed to NDGA was 21.4% greater than that of worms exposed to the vehicle alone (DMSO) (Fig. 5a). To rule out potential variations in food availability caused by NDGA, we examined the effect of NDGA on feeding bacteria (OP50) and found that OP50 growth was unaffected by NDGA (Fig. 5b). These findings indicate that NDGA has a beneficial effect on C. elegans lifespan that is independent of the feeding bacteria growth.

Fig. 5 NDGA increases median lifespan and induces autophagy in worms. a The effect of NDGA on worm lifespan was measured by exposing N2 worms to DMSO or NDGA (100 µM) on NGM plates. Average values from three independent experiments were plotted as a lifespan curve. Lower panel indicates the median lifespan of each group as days and percentage; b The effect of NDGA on feeding bacteria (E. coli OP50) growth was measured spectrophotometrically (A 595 ) every hour for 8 h in LB medium with 100 µM of NDGA or DMSO. c, d Total histone H3 acetylation in worms was measured by immunoblotting after 24-h NDGA (100 µM) or DMSO exposure and total protein extraction. e Changes in mCherry puncta number in MAH215 worms were monitored by fluorescence microscopy after a 24-h NDGA or DMSO treatment. f Effects of NDGA treatment on GFP by time were measured by fluorescence microscopy after NDGA or DMSO (Control) treatment for 2 and 24 h and represented relative to mCherry intensity. g Monitoring of tandem reporter lgg-1p::mCherry::GFP::lgg-1 expressing worms was performed by fluorescence microscopy. h, i Monitoring Lysotracker Red staining in N2 worms after 24-h treatment with NDGA (100 µM) or DMSO was performed by fluorescence microscopy, and fluorescence intensity was measured for individual animals in each group. Data represent the results from three independent experiments (mean ± S.D.), and statistical significance was determined by student t-test (*p < 0.05; **p < 0.01, ***p < 0.001) Full size image

CBP-1, the nematode orthologue of p300/CBP, is responsible for histone acetylation in C. elegans, and animals with catalytically inactive cbp-1 have decreased global histone acetylation.46 With this knowledge, we evaluated the possible suppressive effect of NDGA on histone H3 acetylation. We treated worms with 100 μM NDGA for 24 h, analyzed histone H3 acetylation by immunoblotting and observed a significant decrease in histone H3 acetylation in treated animals (Fig. 5c, d, Supplementary Fig. 8).

To analyze autophagy induction in worms in depth, we used described reporter strain (MAH215) expressing pH sensitive GFP in a tandem lgg-1p::mCherry::GFP::lgg-1 in which mCherry puncta indicates autophagosomes and autolysosomes.47 Worms exposed to NDGA had an increase in mCherry-only puncta, indicating an elevated autophagic activity as assessed by increased fusion of autophagosomes to lysosomes (Fig. 5e, g). MAH215 worms with this tandem reporter allow us to analyze the autophagosome maturation process by monitoring the GFP and mCherry fluorescence intensity over time. Increased autophagic flux and lysosomal activity following NDGA treatment is also evident from observed decreases in pH-sensitive GFP fluorescence over time resulting from the quenching of GFP within the acidic lysosome. We examined the NDGA-treated worms (2–24 h) by measuring the ratio of GFP relative to mCherry fluorescence. We observed that GFP signal was significantly less in animals treated for 2 h than in the vehicle-treated animals (DMSO), and those levels continued to decrease at 24 h (Fig. 5f). In agreement with these results, worms treated with NDGA also show increased lysotracker staining, suggesting an increased number of lysosomes/late endosomes most likely as a result of increased autophagic activity (Fig. 5h, i). These data provide evidence that NDGA treatment causes an increase in autophagic flux in C. elegans.