Synthesis of novel resveralogues

Resveratrol (RSV) has been reported to extend lifespan in various model organisms through activation of the NAD-dependent protein deacetylase, SIRT1 [24], while replenishment of NAD+ improves lifespan and health span in ATM− worms and mice [25]. We therefore set out to rationally design a panel of novel resveratrol-like compounds (Fig. 1a) with the goal of identifying compounds that could restore splicing factor expression to levels comparable with those seen in young cells, but with differing effects on SIRT1 activation and the senescence-associated secretory phenotype (SASP) to allow assessment of molecular mechanism. Synthesis of the backbone was achieved as previously reported [26], with additional functionality and diversity achieved via functional group interconversion (Fig. 1b). Compounds were chosen for further analysis based on (i) structural novelty and low cytotoxicity (ii) differential SIRT1 activation activity (iii) differential effects on the suppression of SASP components and (iv) previously observed increases in the Ki67 positive fraction of MRC5 cultures at 5 μM. We also included the parent compound (resveratrol) and a major metabolite (dihydroresveratrol).

Fig. 1 Synthesis and characterisation of novel resveralogues. a Structures of resveralogues 1–6. Compounds are: 1 resveratrol, 2 resveratrol’s primary metabolite, dihydroresveratrol, 3 (E)-N-(4-(3,5-dimethoxystyryl) phenyl)methanesulfonamide, 4 (E)-N-(4-(3,5-dihydroxystyryl)phenyl)acetamide, 5 (E)-5-(4-(3,5-dimethoxystyryl)phenyl)-1H–tetrazole and 6 (E)-5-(2-(3,5-dimethoxystyryl)phenyl)-1H–tetrazole. b Scheme of synthesis of compounds 3–6 (see Methods for details). c Fluorescence determination of SIRT1 activity in vitro in the presence of 25 μM each compound, normalised against resveratrol (1) and vehicle only control (0). Data are presented as fold change (mean ± SD) in activity normalised to enzyme-only (0) and resveratrol (1), such that 0 represents no activation, and 1.0 indicates activation equivalent to that observed with resveratrol 1. The experiment was carried out in 3 replicates. The numbers on the X axis (1–6) refer to the identity of each resveralogue as indicated above. Uncertainty was calculated by subjecting the standard deviation of the control, Resveratrol and compound data to combination using standard methods for propagation of uncertainty [49] Full size image

SIRT1 activation is significantly altered following side chain modification of resveratrol

Since RSV has been suggested to exert its pro-longevity effects predominantly through activation of SIRT1, we first tested the ability of our novel compounds to activate SIRT1 in an ex vivo enzyme assay (Fig. 1c), with data normalised against activity detected on treatment with resveratrol (RSV, 1). While dihydroresveratrol (Fig. 1a, 2) displayed SIRT1-activation activity equivalent to that of resveratrol, the four novel analogues (3–6) displayed a range of activities from zero (compound 3) to around 75% of control levels (compound 4) (Fig. 1c). These marked differences in SIRT1 activation by the novel resveralogues (compared with RSV and DHRSV) therefore allow us to probe SIRT1-dependence of any biological effects.

Impact of resveralogues on the senescence-associated secretory phenotype

We then set out to determine if treatment with resveratrol or the novel resveralogues had an impact on the senescence-associated secretory phenotype (SASP) in senescent cultures of human fibroblasts (NHDF). The levels of multiple cytokines including key SASP components (IL6, IL8, TNFα, IL2, IL1β, IL-12p70, IL10, INFγ and GMCSF) were determined in senescent NHDF by ELISA (Fig. 2). Although each of the compounds altered cytokine profiles to some extent (Fig. 2, see also Additional file 1: Table S1), there was no consistent pattern with which this occurred. Resveratrol 1 was the only compound to reduce the levels of multiple cytokines including the key SASP mediators IL-6 and IL-8 as well as IL2, TNFα and IFNγ, consistent with previous reports [27]. By contrast, dihydroresveratrol (2) treatment significantly elevated levels of IL-8 and several other inflammatory mediators, whilst 3–6 had variable impact on the expression of the SASP proteins assayed. The only cytokine showing a consistent reduction in level in response to all 6 compounds was IL-10 (Fig. 2, Additional file 1: Table S1).

Fig. 2 Differential effects of resveralogues on the senescence-associated secretory phenotype (SASP). Protein levels of various pro-inflammatory SASP factors was determined using Mesoscale ELISA platform in culture medium of senescent HNDF cultures treated with 5 μM resveralogues 1–6. The heat map indicates fold changes. Con = control (vehicle only). Green indicates up-regulation while red denotes down-regulation. The colour scale refers to percentage change in expression. Experiments were carried out in duplicate a total of 10 times Full size image

Splicing factor expression and splicing patterns of senescence-associated genes are restored in senescent cultures of fibroblasts following treatment with resveralogues

To establish whether RSV and the novel resveralogues could influence splicing regulators, we first measured splicing factor expression by qRT-PCR in senescent cultures of human fibroblasts (NHDF) following 24 h treatment with 5 μM of compounds 1–6. Consistent with previous studies in HEK293 cells [22], we find that resveratrol (1) treatment increased levels of both splicing activators (SRSF transcripts) and inhibitors (HNRNP transcripts) (Fig. 3a). Importantly, novel resveratrol analogues also partially restored levels of both splicing activator and inhibitor transcripts (Fig. 3a, Additional file 2: Table S2). The level of restoration of splicing regulator expression in treated cells was similar to levels previously reported in early passage fibroblasts [2]. This reversal of the age-related decline in splicing factor expression was present for compounds with no discernible SIRT activity (compound 3) as well as those that elevated IL6 and IL8 levels (compounds 2 and 5), indicating that the action of splicing factors is independent of SIRT1 and the SASP.

Fig. 3 Splicing factor regulators are elevated following treatment with resveratrol analogues. Changes to mRNA levels in HNDF cells in response to treatment with 5 μM resveratrol (1) or resveralogues 2–6 determined by quantitative reverse transcription PCR. a Expression of splicing factor regulatory genes (b) Isoform-specific transcripts of genes associated with senescence and/or DNA damage responses. Con = control (vehicle only). Green indicates up-regulated genes, red denotes down-regulated genes. The colour scale refers to fold-change in expression. Data are derived from duplicate testing of 3 biological replicates Full size image

We then asked whether this restoration of a ‘youthful’ complement of splicing factors is biologically relevant. To do this, we examined the alternative splicing profiles of key genes involved in cellular senescence in senescent NHDF cultures treated with each of the compounds (Fig. 3b). In some cases, it was not possible to distinguish an effect on splicing from effects on transcription, since multiple isoforms were affected with the same directionality. For example, both p14ARF and p16INK4A isoforms of the CDKN2A gene, which increases with cellular senescence [28], were down-regulated in response to treatment with most resveralogues. However, in other cases, only one isoform was affected; the expression of the pro-apoptotic p21b isoform, but not the consensus isoform p21a of the CDKN1A gene was altered, demonstrating an effect on splicing. Similarly, increased expression of the CHK1S isoform of the CHK1 gene, which induces mitosis [29], but not the consensus CHK1 isoform which does not, was seen (Additional file 2: Table S2). SIRT1 mRNA expression was upregulated by treatment with the novel resveralogues but not RSV itself. A major regulator of cell proliferation and potential driver of senescence is mTOR: inhibition of mTORC1 by rapamycin increases longevity in animal models [30], while mTORC inhibition can reverse multiple phenotypes of cell senescence [31]. We found elevated expression of both the mTORα and β isoforms, which regulate cell metabolism and cell proliferation respectively [32], on treatment with resveralogues 1–4 (Fig. 3b, Additional file 2: Table S2), though mTORβ was suppressed on exposure of cells to resveralogues 5 and 6. Overall, the changes in alternatively-expressed isoforms following resveralogue treatment are consistent with a shift towards a more proliferation-competent repertoire.

Treatment of senescent cells with resveralogues is associated with reduction in biomarkers of senescence

To assess whether restored splicing factor expression was associated with rescue from cellular senescence, we treated senescent cultures of normal human diploid fibroblasts from three genetically distinct cell strains (NHDF and HF043 dermal fibroblasts and MRC5 lung fibroblasts) for 24 h with compounds 3–6, compared with RSV (1) and DHRSV (2) and measured transcript levels of senescence biomarkers CD248 and CDKN2A (encoding p16INK4/6) by quantitative reverse transcription PCR, normalised against the IDH3B, GUSB and PPIA endogenous control genes, which we have found to be stable in response to senescence and ageing in our previous work [1, 2]. Stability of control genes to resveralogue treatment was verified empirically. While we observed differences between the cell lineages, there was an overall significant decrease in CDKN2A and CD248 molecular markers of senescence compared with vehicle-only control cell populations (Fig. 4a), which was most marked for the foreskin fibroblast line HF043. To further assess senescence, we examined levels of senescence-associated β galactosidase (SA β-Gal). The percentage of NHDF cells staining positive for SA β-Gal decreased from ~75 to ~25%, compared with much lower levels (~7%) in younger cells at PD25 (Fig. 4b), and similar highly significant reductions in SA β-Gal reactivity were seen in senescent cultures of MRC5 and HF043 fibroblasts (Fig. 4b). These reductions in senescence markers were still evident in NHDF cells 4 weeks after initial treatment and larger reductions occurred following repeated treatments at 48 h intervals (Additional file 3: Figure S1). We conclude therefore that senescence markers are markedly diminished upon resveralogue treatment. Given that compound 3 (which does not activate SIRT1) has very similar effects on these senescence biomarkers compared with resveratrol and other resveralogues with variable SIRT-activation activity (4, 5, 6), we can conclude that the decrease in senescence biomarker expression on resveralogue treatment can occur independently of SIRT1 activation.

Fig. 4 Decreased senescence biomarkers on resveralogue treatment (a) Levels of senescence-associated transcripts CDKN2A and CD248 were assessed in senescent populations of NHDF, MRC5 and HF043 fibroblasts by quantitative reverse transcription PCR. Data are expressed relative to stable endogenous control genes GUSB, IDH3B and PPIA, and normalised to the levels of the individual transcripts in untreated controls (c), 1–6 = resveralogues 1–6. Fold change was calculated for in triplicate for three biological replicates (b) Senescence associated β-galactosidase following treatment with resveralogues 1–6 was determined by manually counting the percentage of SA-β gal positive cells (NHDF, MRC5 and HF043) in each treated or control population. n > 300 for each sample. Statistical significance is indicated by * = p < 0.05, ** = p < 0.005, *** = p < 0.0005 (2 way ANOVA) Full size image

Treatment of senescent cells with resveralogues is associated with re-entry of cell cycle

While decreases in senescence biomarkers may be beneficial in alleviating some of the detrimental effects of senescent cells, it is the loss of proliferative capacity of senescent cell populations that is likely to lead to stem cell exhaustion and loss of tissue function/frailty with increasing age [33]. We therefore also assessed cell proliferation and re-entry into the proliferative cell cycle. Initially, using live cell imaging of senescent NHDF cells treated with resveratrol for up to 92 h, we found that some cells within this population showed clear evidence of mitosis within a little as 17.5 h after treatment (Additional file 4: Figure S2). We therefore assessed whether senescent populations of three different fibroblasts lines (NHDF, MRC5 and HF043) could undergo mitosis following treatment with the novel compounds. Remarkably, treatment with even very low doses (5 μM) of the resveralogues led to significant increases (up to 0.6 population doublings) in total cell numbers over only 24 h of drug exposure, while vehicle-only controls remained proliferation-arrested (Fig. 5a). Increases in cell number strongly suggest that a significant proportion of cells in the non-cycling senescent population have been induced to re-enter the mitotic cell cycle.

Fig. 5 Increased proliferation of senescent cell populations following resveralogue treatment. a Cell numbers of NHDF, MRC5 and HF043 fibroblast populations following treatment with resveralogues 1–6. Experiments were carried out in triplicate for three biological replicates and *** represents p < 0.001 (2 way ANOVA). b Proliferation index was assessed for control and treated NHDFs, as well as younger (PD25) cells as assessed by Ki67 immunofluorescence (> 400 nuclei counted per sample, *** p < 0.001 by 2 way ANOVA). c Telomere length was quantified by qPCR relative to the 36B4 endogenous control and normalised to telomere length in vehicle-only controls, younger passage cells (PD25) and in cells treated with compounds 1–6. Experiments were carried out in triplicate for three biological replicates. Statistical significance is indicated by * = p < 0.05, ** = p < 0.005, *** = p < 0.0005 (2 way ANOVA) Full size image

Cell proliferation kinetics are altered in treated cells

To further probe this potential induction of proliferation, the proliferation kinetics of these cultures were determined by immunocytochemical and catalytic histochemical measurement of the levels of the proliferation marker, Ki67, and the senescence marker, SA β-Gal, respectively. Compounds 1–6 induced a consistent increase in the Ki67 positive fraction of cells in senescent NHDF cultures from ~20% of nuclei to ~40%, whereas levels in younger cells at PD25 were > 90% (Fig. 5b), consistent with the findings of increased cell numbers and mitotic figures following drug administration (Fig. 5a, Additional file 4: Figure S2 and data not shown). Since the increased number of cells staining for the proliferation marker Ki67 correlates inversely with the decreased numbers staining for SA-β gal (see Fig. 4b), we suggest that cells have exited senescence to enter the cell cycle.

Treatment of senescent cells with resveralogues is associated with telomere elongation

Telomere shortening is perhaps the best known trigger of cellular senescence. Several splicing factors have been previously demonstrated to unwind telomeres and activate telomerase and could thus potentially lengthen telomeres [13, 14, 34]. We therefore measured telomere length by qPCR in NHDF cells treated with 5 μM resveratrol or resveralogues for 24 h, relative to telomere length in untreated cells. We found that cells treated with resveratrol or any of the novel resveralogues had telomeres that were 1.3–2.4 times longer than vehicle-only controls, compared with younger cells at PD25, which showed telomeres 2.6 times longer than untreated senescent cells (Fig. 5c).

Changes in splicing factor expression and senescence markers are not effects of cell proliferation

To determine whether the changes in splicing factor expression were a cause or consequence of renewed cell proliferation, we measured splicing factor expression and selected senescence markers under low serum conditions, which would induce proliferating cells to enter quiescence. Unsurprisingly, serum-starved cultures demonstrated no increase in cellular proliferation in response to resveralogue treatment, as determined by lack of an observable increase in cell numbers (Fig. 6a) or Ki67 index (Fig. 6b) in treated cells. However effects on both senescence markers (Fig. 6c) and splicing factor expression (Fig. 6d) were still observed, indicating that the effects on senescence and splicing factor expression were independent of proliferation. Uncoupling rescue from proliferation also allows us to quantify more precisely the percentage of cells in which senescence has been reversed from the dilution effect of increased cell number. The number of ‘reverted’ cells is ~15%, which is similar to the levels we had predicted based on the cell proliferation kinetics.

Fig. 6 Effects of resveratrol treatment in cells grown under serum starvation conditions. a Cell numbers of NHDF fibroblasts following treatment with 5 μM resveratrol for 24 h under conditions of serum starvation. Experiments were carried out in triplicate for three biological replicates. (2 way ANOVA). b Proliferation index was assessed for NHDF fibroblasts following treatment with 5 μM resveratrol for 24 h under conditions of serum starvation as assessed by Ki67 immunofluorescence (> 400 nuclei counted per sample). c Senescence associated β-galactosidase following NHDF fibroblasts following treatment with 5 mM resveratrol for 24 h under conditions of serum starvation was determined by manually counting the percentage of SA-β gal positive cells in each treated or control population. n > 300 for each sample. Statistical significance is indicated by *** = p < 0.0005 (2 way ANOVA). d Changes to splicing factor mRNA levels in NHDF fibroblasts following treatment with 5 μM resveratrol for 24 h under conditions of serum starvation determined by qRTPCR. Control = vehicle only. Green indicates up-regulated genes, red denotes down-regulated genes. The colour scale refers to fold-change in expression. Data are derived from duplicate testing of 3 biological replicates Full size image

Decrease in senescent cell fraction is not due to selective death of senescent cells

To exclude the possibility that the decrease in the percentage of senescent cells following treatment resulted from selective cell death of non-proliferating cells, cytotoxicity was assessed using an assay for extracellular lactate dehydrogenase (LDH); this intracellular enzyme is only released into the culture medium upon cell death. In all cases, cells treated with the novel compounds released lower levels of LDH than those treated with RSV (at doses up to 100 μM) in comparison with vehicle only controls (Additional file 5: Figure S3); compound 6 in particular showed very low levels of LDH release. These results demonstrate low cytotoxicity of dihydroresveratrol and all four novel resveralogues.

While necrotic cell death was not detected, it was important to rule out selective loss of senescent cells by apoptosis. Levels of apoptosis in senescent NHDF cultures treated with resveralogues 1–6 were determined by both TUNEL and by Caspase 3 and 7 assays (Additional file 5: Figure S3B and C). No increases in levels of apoptosis were observed in the resveralogue-treated cultures compared with vehicle-only control treatments, suggesting that the increased proliferation on resveralogue treatment was not a consequence of selective death of non-proliferating cells within the population.

ERK agonists and antagonists influence cellular senescence and splicing factor expression

ERK signalling has previously been reported to be influenced by resveratrol [35, 36]. ETS-1, a transcription factor downstream of ERK activation has also been reported to regulate the expression of TRA2B, an important splicing regulator [37]. To investigate the potential interplay between resveratrol and ERK signalling on splicing factor expression and cellular senescence phenotypes, we treated senescent NHDF cells with low dose (1 μM or 10 μM) of trametinib, a well-characterised signalling inhibitor that inhibits the ERK signalling pathway. Treatment of senescent cells with trametinib resulted in a robust decrease in the proportion of senescent cells in the culture, which was apparent at 1 μM and 10 μM, but not at 20 μM. Such dose effects are not uncommon in signalling pathways due to interconnectivity with other signalling pathways and autoregulation (Additional file 6: Figure S4A). Conversely, treatment with the ERK agonist ceramide resulted in a comparable increase in the senescent cell fraction after 24 h. Notably, the effect of ceramide was negated by the addition of 5 μM of any of the novel resveralogues (Additional file 6: Figure S4B). Trametinib also restored splicing factor expression to profiles consistent with earlier passage in a manner similar to that observed with the resveralogues (Additional file 7: Figure S5).