Resveratrol Reactivates Latent HIV through Increasing Histone

May 4, 2017 - Although the application of combination antiretroviral therapy (cART) can successfully reduce HIV replication to undetectable levels, th...
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Resveratrol Reactivates Latent HIV through Increasing Histone Acetylation and Activating Heat Shock Factor 1 Xiaoyun Zeng,† Xiaoyan Pan,†,§ Xinfeng Xu,† Jian Lin,† Fuchang Que,† Yuanxin Tian,† Lin Li,† and Shuwen Liu*,†,‡ †

Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China § State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China ‡ State Key Laboratory of Organ Failure Research, Guangdong Provincial Institute of Nephrology, Southern Medical University, Guangzhou 510515, China S Supporting Information *

ABSTRACT: The persistence of latent HIV reservoirs presents a significant challenge to viral eradication. Effective latency reversing agents (LRAs) based on “shock and kill” strategy are urgently needed. The natural phytoalexin resveratrol has been demonstrated to enhance HIV gene expression, although its mechanism remains unclear. In this study, we demonstrated that resveratrol was able to reactivate latent HIV without global T cell activation in vitro. Mode of action studies showed resveratrolmediated reactivation from latency did not involve the activation of silent mating type information regulation 2 homologue 1 (SIRT1), which belonged to class-3 histone deacetylase (HDAC). However, latent HIV was reactivated by resveratrol mediated through increasing histone acetylation and activation of heat shock factor 1 (HSF1). Additionally, synergistic activation of the latent HIV reservoirs was observed under cotreatment with resveratrol and conventional LRAs. Collectively, this research reveals that resveratrol is a natural LRA and shows promise for HIV therapy. KEYWORDS: resveratrol, latent HIV, histone acetylation, heat shock factor 1, synergistic reactivation

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INTRODUCTION Although the application of combination antiretroviral therapy (cART) can successfully reduce HIV replication to undetectable levels, the latent HIV reservoirs constitute the primary hurdle for viral eradication.1,2 The latent HIV reservoirs, which are primarily composed of latently infected resting memory CD4+ T cells, contain integrated but transcriptionally silent provirus, thus evading immune surveillance. However, the integrated provirus is still replication-competent so that the cessation of therapy would lead to a sudden rebound of viremia, thus imposing the requirement for lifelong cART on HIVinfected individuals.3 Long-term toxicity and drug resistance of cART necessitate a more urgent need for more pharmacological strategies.4,5 In recent years, the “shock and kill” strategy has come to public attention. The strategy aims at purging latent reservoirs by awakening latent infected cells while preventing the spread of infection by implementing cART, ultimately resulting in elimination of the activated cells due to viral cytopathic effects or the host immune response.6 Much progress has been made in understanding the events involved in the establishment and maintenance of HIV latency, which comprise deleterious mutations in the viral genome, transcriptional interference, changes in chromatin structure, epigenetic silencing, the presence of negative transcription factors, the absence of positive transcription factors, and some other interferences.7 The latency reversing agents (LRAs) against the events described above can be classified as follows: histone deacetylase (HDAC) inhibitors, histone methyltrans© 2017 American Chemical Society

ferase (HMT) inhibitors, DNA methyltransferase inhibitors, protein kinase C (PKC) activators, and positive transcription elongation factor b (P-TEFb) activators.8 Particularly, HDAC inhibitors, such as vorinostat (also known as SAHA),9 romidepsin,10,11 and panobinostat,12 hold promise as good candidates for viral purging due to their ability to induce viral replication in resting CD4+ T cells from patients on cART. However, their use individually failed to reduce the size of reservoirs from the infected individuals. Natural products appear to be a source of LRA discovery and offer new prospects for HIV cure.13−16 Resveratrol (3,5,4′-trihydroxystilbene) is a well-known natural phytoalexin existing in many plant species such as red grapes and peanuts, especially abundant in red wine. There has been increasing interest in resveratrol since an inverse correlation between red wine consumption, and thus uptake of resveratrol, and the incidence of cardiovascular diseases was reported.17 Since then, its therapeutic potentials against multiple diseases including cancer, cardiovascular diseases, and nervous system diseases have been widely reported.18 Recently, resveratrol has been screened by our group to be capable to reactivate latent HIV.19 However, the underlying mechanism involved remains poorly defined. This study aimed to investigate the ability of resveratrol in the reactivation of Received: Revised: Accepted: Published: 4384

January 26, 2017 May 3, 2017 May 4, 2017 May 4, 2017 DOI: 10.1021/acs.jafc.7b00418 J. Agric. Food Chem. 2017, 65, 4384−4394

Article

Journal of Agricultural and Food Chemistry

centrifugation using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA) and were cultured in RPMI1640 medium with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere. Cytotoxicity Assays. Cytotoxicity of resveratrol was evaluated in J-Lat clones (A2 and 10.6), ACH2 cells, Jurkat T cells, and human PBMCs. Cells were plated in 96-well plates and treated with various concentrations of resveratrol for 24, 48, or 72 h. After drug treatment, the cells were precipitated by centrifugation and the cultural supernatant was aspirated. One hundred and twenty-five microliters of XTT solution dissolved in the culture medium at a final concentration of 200 μg/mL containing 50 μM PMS was added. Then the mixture was incubated for an additional 4 h at 37 °C. After 4 h, the absorbance was measured at 450 nm and the background was determined at 650 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The 50% cytotoxicity concentration (CC50) was calculated by the CalcuSyn software. The influence of EX527, SRT1720, or KRIBB11 alone or in cotreatment with resveratrol or carfilzomib (CFZ) on the cell viability of J-Lat A2 cells is shown in Figure S1. Quantitative Analysis of Synergy of Latency Reversing Agent Combinations. The Bliss independence model was adapted to calculate synergy when resveratrol was combined with other conventional LRAs.21 For drug x and drug y, faxy, P is the predicted fraction affected by a combination of drug x and drug y based on the experimentally observed fraction affected by drug x ( fax) and drug y ( fay) individually and is calculated from the equation faxy, P = fax + fay − ( fax)(fay). faxy, O is the experimentally observed fraction affected by a combination of drug x and drug y. According to the Bliss model, faxy, O was compared with faxy, P with the following computation: Δfaxy = faxy, O − faxy, P. If Δfaxy > 0 with statistical significance, the combination effect of two drugs exceeds that predicted by the Bliss model and the drug combination displays synergy. If Δfaxy < 0 with statistical significance, the combination effect of two drugs is less than that predicted by the Bliss model and the drug combination displays antagonism. In our analysis, fax = % GFP-positive cells after treatment with drug x − % GFP-positive cells of untreated group. Detection of T Cell Activation Markers by Flow Cytometry. Isolated PBMCs were incubated with resveratrol (40 or 60 μM) or 1 μM prostratin. After 24 h of treatment, the cells were collected, washed with phosphate buffer saline (PBS), and then immune-stained with CD25-PE and CD69-APC conjugated antibodies at 4 °C for 30 min. The T cell activation markers (CD25 and CD69) were evaluated by flow cytometry in BD FACSCanto II Flow Cytometer, and the data were analyzed using FlowJo software. Protein Isolation and Western Blot Analysis. After drug treatment, the cells were collected, washed with PBS, and then lysed in the prechilled RIPA lysis buffer with proteinase and phosphatase inhibitors for 15 min as previously described.19 The lysates were centrifuged at 12,000g for 15 min at 4 °C to pellet cellular debris, and the supernatant were transferred to a fresh prechilled tube. Equal amounts of protein were subjected to SDS−polyacrylamide gel electrophoresis. Separated proteins were transferred to polyvinylidene difluoride membranes (Roche, Indianapolis, IN, USA). Then the membranes were blocked with 5% nonfatty milk solution for 1 h, probed with primary antibodies overnight at 4 °C, and incubated with secondary peroxidase-conjugated antibodies at room temperature for 1 h. The chemiluminescent signals were visualized using Lumiglo reagent (Cell Signaling Technology) and then exposed to X-ray film (Fujifilm Europe GmbH, Dusseldorf, Germany). HDAC Activity Assays. An HDAC activity fluorometric assay kit (Biovision, Santa Clara, CA, USA) was used to examine the enzymatic activity of HDACs present in the nuclear extracts isolated from J-Lat A2 cells according to manufacturer’s instruction. Nuclear extracts were isolated using the Nuclear Extraction Kit (Cayman, Ann Arbor, MN, USA) according to the manufacturer’s protocol. The extracts were diluted with distillation−distillation H2O to achieve a final amount of 15 μg/well and added to a 96-well clear-bottom black plate. Resveratrol at different concentrations or SAHA (1 μM) was added to the samples. Then HDAC assay buffer and the substrate were added

latent HIV and the molecular mechanism involved in resveratrol-mediated reactivation.

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MATERIALS AND METHODS

Cell Lines and Culture. J-Lat cells (clones A2 and 10.6) (encoding the green fluorescent protein (GFP) to monitor HIV long terminal repeat (LTR) expression), ACH2 cells (harboring HIV provirus), and Jurkat T cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. Cells were cultured in RPMI1640 medium (Gibco, Grand Island, NY, USA) with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and maintained in a humidified 5% CO2 incubator at 37 °C. Reagents. Antibodies specific to SIRT1 and p-HSF1(Ser320) and acetyl-H4K8 used for ChIP assay were obtained from abcam Inc. (Cambridge, MA, USA). Antibodies specific for acetyl-H3K9, acetylH3K14, acetyl-H4K8, and β-actin were from Cell Signaling Technology (Beverly, MA, USA). Antibody specific to HSF1 and dihydroresveratrol were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody specific to p24 (183-12H-5C) was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. Human PE conjugated anti-CD25 and APC conjugated anti-CD69 antibodies were from BD Biosciences (San Jose, CA, USA). Resveratrol, JQ1, carfilzomib, piceatannol, and pterostilbene were obtained from MedChem Express (Monmouth Junction, NJ, USA). EX527 and SRT1720 were purchased from Selleck Chemicals (Houston, TX, USA). SAHA, prostratin, 2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino)carbonyl-2H-tetrazolium hydroxide (XTT), phenazine methosulfate (PMS), KRIBB11, triacetyl resveratrol, and resveratrol trimethyl ether were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polydatin was kindly provided by Dr. Heqing Huang (Sun Yat-Sen Univesity, China). HIV Reactivation Detection by Flow Cytometry. J-Lat clones (A2 and 10.6) were stimulated with the indicated compounds for 48 h, and the GFP fluorescence pattern was analyzed by flow cytometry. The percentage of GFP positive cells was used as a measure of HIV reactivation and was evaluated on a BD FACSCanto II Flow Cytometer (San Jose, CA, USA). The data were analyzed using FlowJo software (Treestar, San Carlos, CA, USA). Real-Time Quantitative PCR Analysis of HIV Expression. Total RNA was isolated from ACH2 cells using Trizol (Invitrogen) followed by reverse transcription using a PrimeScript RT reagent kit (Takara, Japan). Real-time PCR (Takara, Japan) was performed on a 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer’s protocol using the following primers: HIV LTR forward primer, 5′-GCCTCCTAGCATTTCGTCACAT-3′, reverse primer, 5′-GCTGCTTATATGTAGCATCTGAGG-3′; HIV Gag forward primer, 5′-GTCCAGAATGCGAACCCAGA-3′, reverse primer, 5′-GTTACGTGCTGGCTCATTGC-3′. The mRNA level of the endogenous control GAPDH was used as a reference for normalization. The primers for GAPDH were as follows: forward primer, 5′-CTCTGCTCCTCCTGTTCGAC-3′; reverse primer, 5′AGTTAAAAGCAGCCCTGGTGA-3′. Cycling conditions were as follows: 95 °C for 15 s for an initial denaturation, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The comparative 2−ΔΔCT method was adopted to analyze a fold-increase versus untreated controls. Measurement of HIV p24 Antigen in the Supernatant of ACH2 Cells. ACH2 cells were treated with resveratrol at indicated concentrations for 48 h. Prostratin (1 μM) was used as a positive control. After incubation, the cells were centrifuged and the supernatant was collected and mixed with an equal volume of 5% Triton X-100. Then the virus lysates were measured for p24 antigen by ELISA as previously described.20 Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs). Whole peripheral blood was collected from healthy laboratory members with written informed consent. The experiment was approved by the Human Ethics Committee of Southern Medical University, China. PBMCs were isolated by density gradient 4385

DOI: 10.1021/acs.jafc.7b00418 J. Agric. Food Chem. 2017, 65, 4384−4394

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Figure 1. Resveratrol reactivates HIV expression in latent cell models. (A) Chemical structure of resveratrol. (B, C) J-Lat clone A2 (B) and clone 10.6 (C) were exposed to the indicated concentrations of resveratrol or 1 μM prostratin for 48 h. The percentage of GFP-positive cells was examined by flow cytometry analysis. (D) ACH2 cells were treated with resveratrol (40 and 60 μM) or 1 μM prostratin for the indicated time points, and HIV transcription was measured by real-time quantitative RT-PCR using primers specific for HIV LTR and Gag. (E, F) ACH2 cells were exposed to the indicated concentrations of resveratrol or 1 μM prostratin. After 48 h of treatment, the cells and the supernatant were collected and used for further analysis. (E) The expression level of p24 antigen in the cell extracts was examined by Western blot. The expression level of β-actin was used as a loading control. (F) HIV p24 antigen in the supernatant of ACH2 cells was evaluated by ELISA. (G) J-Lat A2, J-Lat 10.6, ACH2, Jurkat T cells, and human PBMCs were treated with resveratrol at different concentrations for 24, 48, and 72 h, and then the cell viability of the cells was evaluated by XTT assays. Data represent the mean ± SD of three independent experiments. (∗) p < 0.05 versus control group; (∗∗) p < 0.01 versus control group. to each well sequentially. After incubation, the lysine developer was added to cleave the deacetylated substrates and fluorophores were released. Finally, the plate was read under a fluorometer (excitation, 350 nm; emission, 480 nm). To exclude possible interferences from the green fluorescence in the cells and the autofluorescence of resveratrol, additional background controls with nuclear extracts of the untreated cells and 80 μM resveratrol were performed. There was no disturbance detected in the experiment condition conducted. Chromatin Immunoprecipation (ChIP). Chromatin immunoprecipation was performed using a ChIP assay kit (Cell Signaling Technology, Beverly, MA, USA) and following the instructions from the manufacturer. Briefly, J-Lat A2 cells (5 × 107) were treated with or without resveratrol (60 μM) or SAHA (1 μM) for 24 h. After the treatment, the cells were collected and cross-linked with 1% formaldehyde for 10 min at room temperature. The cross-link reaction was quenched by adding glycine solution. The cross-linked cells were then lysed and sonicated into chromatin fragments. The chromatin fragments were immunoprecipated with antibodies against acetylH3K9, acetyl-H3K14, acetyl-H4K8, or normal rabbit IgG (negative control) at 4 °C overnight. Protein A/G magnetic beads were subsequently added to collect immune complexes. Following washing,

the chromatin was eluted from the protein A/G magnetic beads. DNA was recovered and purified according to the kit instruction. Finally, real-time quantitative PCR was used to quantify the level of HIV LTR with the following primers: forward, 5′-AGCTTGCTACAAGGGACTTTCC-3′; and reverse, 5′-ACCCAGTACAGGCAAAAAGCAG3′. The signals obtained by real-time quantitative PCR were normalized to the input DNA. Statistical Analysis. Experimental data were represented as the mean ± SD of at least three independent experiments. Statistical analysis for comparison between groups was performed with a oneway ANOVA test followed by Tukey’s t test using GraphPad Prism 5.0 (San Diego, CA, USA). p values of 0. Statistical significance was calculated using a t test comparing the experimentally observed and predicted fractional effects. (∗) p < 0.05; (∗∗) p < 0.01.

Figure 3. Resveratrol does not cause global T cell activation. Human PBMCs were treated with either resveratrol (40, 60 μM) or 1 μM prostratin for 24 h. The expressions of activation marker CD69 and CD25 were detected by flow cytometry using specific antibodies.

cells expressing Tat and GFP under the control of HIV LTR (LTR-Tat-IRES-GFP),22 were used in our study. After J-Lat A2 cells were treated with increasing concentrations of resveratrol for 48 h, the percentage of GFP-positive cells, which reflected latent HIV reactivation, was analyzed by flow cytometry. As shown in Figure 1B, resveratrol induced HIV LTR-driven expression of GFP in a dose-dependent manner and yielded up to 17% GFP-positive cells at 60 μM. To further determine whether the reactivation effects on latent HIV could be obtained in other HIV latency cell models, J-Lat clone 10.6,23 which is also a J-Lat cell line but exhibits a different activation profile from J-Lat A2 cells, and ACH2 cells, which are derived from A3.01 infected chronically with HIV,24 were used. The results from flow cytometry showed that resveratrol could also induce latent HIV transcription in a dosedependent manner in J-Lat clone 10.6 (Figure 1C). The effect of resveratrol on latent HIV in ACH2 cells was also stimulatory. As shown in Figure 1D, ACH2 cells exposed to resveratrol showed increasing transcription of HIV LTR and Gag. The transcription of the genes after resveratrol treatment for 48 and 72 h was elevated more apparently compared with that after treatment for 24 h. Consistent with the results of real-time quantitative PCR, the expression levels of HIV capsid protein p24 in the cell extracts and virions released from the cells were increased after incubation with resveratrol for 48 h (Figure 1E,F). To determine whether resveratrol caused any cytotoxic effects on cells, we assessed the cytotoxicity of resveratrol to J-

Lat A2 cells, J-Lat 10.6 cells, ACH2 cells, Jurkat T cells, and human PBMCs. On the basis of the XTT assay results, resveratrol exhibited minimal cytotoxicity to these cell lines at 24 and 48 h. Although resveratrol showed mild cytotoxicity to J-Lat 10.6, ACH2, and Jurkat T cells at 72 h, the CC50 of resveratrol in human PBMCs at 72 h was 230.73 μM, which was much higher than the effective concentration (Figure 1G). Taken together, these data showed that resveratrol was potent in reactivating latent HIV in vitro. Resveratrol Synergistically Reactivates Latent HIV Production. Combinational LRAs strategies are widely assumed to be able to more efficiently reactivate the latent HIV reservoirs.7,21,25 Prostratin, JQ1, and SAHA are potential and conventional LRAs with different mechanisms. Prostratin is a phorbol ester with an ability to induce HIV transcription via NF-κB and AP-1 pathways, and JQ1 is a BET inhibitor that could block the interaction of BRD4 and P-TEFb, whereas SAHA is a pan-HDAC inhibitor to create a less repressive chromatin environment for HIV transcription.9,22,26 To assess whether resveratrol synergistically reactivates latent HIV in JLat A2 cells when combined with these three conventional LRAs, we treated J-Lat A2 cells with resveratrol alone (20 μM), prostratin alone (0.5 μM), JQ1 alone (0.5 μM), SAHA alone (1 μM), or resveratrol (20 μM)/prostratin (0.5 μM), resveratrol (20 μM)/JQ1 (0.5 μM), resveratrol (20 μM)/SAHA (1 μM) for 48 h, respectively. As shown in Figure 2A, mock treatment of J-Lat A2 cells induced GFP expression of 0.68%. Stimulation of J-Lat A2 cells with resveratrol, prostratin, JQ1, or SAHA 4387

DOI: 10.1021/acs.jafc.7b00418 J. Agric. Food Chem. 2017, 65, 4384−4394

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Figure 4. The reactivation effect of resveratrol on latent HIV is independent of SIRT1 activation. (A) Western blot detection of SIRT1 after J-Lat A2 cells were treated with resveratrol at the indicated concentrations for 48 h. The expression level of β-actin was used as a loading control. (B, D) J-Lat A2 cells were treated with 60 μM resveratrol alone or in combination with Ex527 (B) or SRT1720 (D) at the indicated concentrations for 48 h. The percentage of GFP-positive cells was measured by flow cytometry. Data represent the mean ± SD of three independent experiments. (C, E) The expression levels of SIRT1 and the putative target of SIRT1 acetyl-H3K9 were analyzed by Western blot after J-Lat A2 cells were treated with resveratrol (60 μM) alone or in combination with EX527 (10 μM) (C) or SRT1720 (5 μM) (E). Additionally, the effects of SAHA (1 μM), known to block HDAC activity, are shown. The expression level of β-actin was used as a loading control. (*) p < 0.05 versus resveratrol treatment alone; (**) p < 0.01 versus resveratrol treatment alone.

with resveratrol (40, 60 μM) or prostratin (1 μM). Consistent with previous findings,29 prostratin stimulated the expression of both T cell activation markers robustly. In contrast to prostratin, there was no significant change in the expression of CD25 and CD69 under the treatment with resveratrol compared with untreated cells (Figure 3). Consequently, resveratrol is a potential LRA without global T cell activation. Silent Mating Type Information Regulation 2 Homologue 1 (SIRT1) Is Not Involved in Resveratrol-Mediated HIV Reactivation. It has been previously identified that SIRT1, which belongs to class 3 HDAC, serves as a regulator of HIV transcription.30,31 Additionally, resveratrol is a well-known activator of SIRT1 and is found to up-regulate the transcription and deacetylase activity of SIRT1.32,33 We therefore wanted to determine the requirement of SIRT1 in resveratrol-mediated reactivation in J-Lat A2 cells. To this end, we examined the protein expression level of SIRT1 in J-Lat A2 cells treated with resveratrol. However, there was no significant induction of SIRT1 when cells were treated for 48 h (Figure 4A). Besides, the addition of the SIRT1 inhibitor, EX527, to J-Lat A2 cells did not demonstrate any inhibition in resveratrol-induced

alone induced GFP expressions of 5.14, 11.97, 15.7, or 19.03%, respectively. The cells expressing GFP were significantly increased by resveratrol/prostratin (35.3%), resveratrol/JQ1 (64.43%), or resveratrol/SAHA (53.67%). To further evaluate whether the drug combination effects meet the metric of drug synergy, the Bliss independence model was adapted.21 On the basis of the Bliss independence model, we found that the combined effects of resveratrol and prostratin, JQ1, or SAHA were greater than the idealized Bliss independence prediction (Δfaxy > 0) (Figure 2B), implying that resveratrol in combination with prostratin, JQ1, and SAHA resulted in synergistic reactivation of the latent HIV production. Resveratrol Does Not Induce Global T Cell Activation. Global T cell activation would potentially cause release of proinflammatory cytokines, thus resulting in undesirable cell toxicity.27,28 Thus, the LRAs, which do not nonspecifically induce global T cell activation, are more worthwhile being investigated and developed.8 To investigate the effect of resveratrol on T cell activation, the expressions of T cell activation biomarkers CD25 and CD69 on the isolated human PBMCs surface were analyzed following 24 h of stimulation 4388

DOI: 10.1021/acs.jafc.7b00418 J. Agric. Food Chem. 2017, 65, 4384−4394

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Journal of Agricultural and Food Chemistry

Figure 5. Resveratrol-mediated HIV reactivation is associated with increasing histone acetylation. (A) Overall HDAC inhibition in the nuclear extracts of J-Lat A2 cells by increasing concentrations of resveratrol (10, 20, 40, 60, and 80 μM) or solvent as control. For positive reference, SAHA (1 μM) was used. (B, C) After J-Lat A2 cells were treated with resveratrol at the indicated concentrations or SAHA (1 μM) for 24 h (B) or with 60 μM resveratrol for the indicated time points (C), acetylated histone marks acetyl-H3K9, acetyl-H3K14, and acetyl-H4K8 were detected by Western blot. The expression level of β-actin was used as a loading control. (D) J-Lat A2 cells were stimulated with resveratrol (60 μM) or SAHA (1 μM) for 24 h, and the relative change of acetyl-H3K9, acetyl-H3K14, and acetyl-H4K8 bound to HIV LTR relative to untreated control was determined using ChIP-qPCR. Data represent the mean ± SD of three independent experiments. (∗) p < 0.05 versus control group; (∗∗) p < 0.01 versus control group.

disruption of HIV latency or the expression of SIRT1 (Figure 4B,C). Nevertheless, acetylation of the histone residue H3K9, a putative target of SIRT1, was found to be increased when cells were treated with resveratrol, which was not influenced after cotreatment with EX527 (Figure 4C). On the contrary, selective SIRT1 agonist SRT1720 attenuated the GFP expression induced by resveratrol (Figure 4D). However, the expression level of SIRT1 as well as acetyl-H3K9 remained unchanged under treatment with SRT1720 (Figure 4E). Hence, our findings indicated that other targets rather than SIRT1 might contribute to resveratrol-induced reactivation of latent HIV. Resveratrol Increases Histone Acetylation in HIV LTR in J-Lat A2 Cells. The presence of HDACs in HIV LTR would induce a repressive effect on chromatin structure, thereby blocking HIV transcription.7,34 It has been previously published that resveratrol has a moderate inhibitory effect on the activity of human HDACs of classes 1, 2, and 4 via in vitro analysis.35 In light of the aforementioned observation that resveratrol upregulated acetyl-H3K9, we further determined whether resveratrol affected HDAC activity. To this end, a HDAC activity assay was performed. As shown in Figure 5A, resveratrol showed moderate inhibitory activity against HDACs expressed in nuclear extracts of J-Lat A2 cells at high concentrations of 40, 60, and 80 μM. To further substantiate

the mechanism of action of resveratrol, we investigated the effect of resveratrol in the levels of other histone acetylation marks including acetyl-H3K14 and acetyl-H4K8. After J-Lat A2 cells were treated with resveratrol for 24 h, the expression levels of acetyl-H3K9, acetyl-H3K14, and acetyl-H4K8 were increased significantly, which was also detected upon treatment with HDAC inhibitor SAHA (Figure 5B). In addition, acetyl-H3K9 and acetyl-H3K14 dramatically increased from 3 h compared with control (0 h), whereas acetyl-H4K8 was up-regulated from 12 h (Figure 5C). To determine if the reactivation effect of resveratrol on latent HIV was due to hyperacetylation of HIV LTR promoter region, a ChIP assay was performed. We found that the levels of histone acetylation marks acetyl-H3K9, acetylH3K14, and acetyl-H4K8 were increased in HIV LTR upon stimulation with resveratrol (Figure 5D). These findings suggested resveratrol might induce levels of histone acetylation in HIV LTR to promote latent HIV transactivation. Resveratrol Promotes HIV Transcription through Activating Heat Shock Factor 1 (HSF1). HSF1, a conserved heat shock factor, has been reported by our group as a key transcription factor regulating HIV transcription.19,36 To test the possibility that resveratrol reactivated HIV by affecting HSF1 signaling pathway, the effect of resveratrol on HSF1 Ser320 phosphorylation was further determined. The proteasome carfilzomib, which was reported by our group to activate 4389

DOI: 10.1021/acs.jafc.7b00418 J. Agric. Food Chem. 2017, 65, 4384−4394

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Journal of Agricultural and Food Chemistry

Figure 6. Resveratrol activates the HSF1 signaling pathway. (A) J-Lat A2 cells were treated with various concentrations of resveratrol or carfilzomib (50 nM) for 48 h. Then the cells were lysed, and Ser320 phosphorylated HSF1 and total HSF1 were detected by Western blot with corresponding antibodies. (B) After J-Lat A2 cells were treated with resveratrol (60 μM) or carfilzomib (50 nM) alone with or without HSF1 inhibitor KRIBB11 (1.25, 2.5, 5 μM) for 48 h, GFP expression was measured by flow cytometry. (*) p < 0.05 versus resveratrol or carfilzomib treatment alone; (**) p < 0.01 versus resveratrol or carfilzomib treatment alone.

the HSF1 pathway,36 was used as the positive control. In this respect, the treatment of resveratrol or carfilzomib stimulated the phosphorylation of HSF1 in J-Lat A2 cells (Figure 6A), indicating that resveratrol treatment facilitated the phosphorylation of HSF1, which might contribute to its increased transcription signaling in J-Lat A2 cells. To further examine whether activation of HSF1 was involved in modulating resveratrol-mediated HIV transcription, KRIBB11, which was found to selectively inhibit the transcriptional activity of HSF1,37 was used. Treatment of J-Lat A2 cells with KRIBB11 significantly reversed HIV LTR-driven expression of GFP induced by resveratrol or carfilzomib (Figure 6B). These data suggested that HSF1 signaling pathway might be involved in resveratrol-mediated reactivation of latent HIV expression. Resveratrol Derivatives in the Reactivation of Latent HIV. Once resveratrol was demonstrated to be a potent LRA, we addressed whether its synthetic or natural derivatives could have reactivation effects on latent HIV. Therefore, potential HIV latency reactivation effects of synthetic resveratrol derivatives (triacetyl resveratrol (triacetyl-Rsv), dihydroresveratrol, and resveratrol trimethyl ether) and natural resveratrol derivatives (piceatannol, pterostilbene, and polydatin) were evaluated in J-Lat A2 cells (Figure 7A). Unexpectedly, we observed that triacetyl resveratrol induced a similar reactivation effect on latent HIV with resveratrol, whereas other derivatives slightly influenced HIV transcription (Figure 7B). Similarly, the combination treatment of triacetyl resveratrol and prostratin, JQ1, or SAHA produced a greater activation level than the effect produced by resveratrol in J-Lat A2 cells (Figure 7C). The combined effects of triacetyl resveratrol and prostratin, JQ1, or SAHA were greater than the idealized Bliss independence prediction under the Bliss independence model (Figure 7D). The results indicated that triacetyl resveratrol in combination with prostratin, JQ1, or SAHA had synergistic effects on the reactivation of HIV from latency.

system diseases, and cancer.38 Multiple clinical studies have indicated that daily oral administration of 1 g of resveratrol is generally well tolerated in healthy participants, even in older adults.39−41 In this study, we provided multiple lines of evidence for the potential impact of resveratrol on latent HIV. First, resveratrol potently reactivated latent HIV in three widely used latency cell models with lower toxicity. Second, resveratrol did not evoke global T cell activation while reactivating latent HIV. Third, resveratrol in combination with conventional LRAs (prostratin, JQ1, and SAHA) had synergistic effects on the reactivation of HIV. The synergism that results from combining resveratrol and conventional LRAs can augment the potency in latency reversal and thus is beneficial for reducing the size of the reservoirs. On the other hand, resveratrol also possesses antiHIV activity against wild-type HIV strains as well as drugresistant HIV strains, including lamivudine and emtricitabineresistant HIV, and resveratrol still exhibits synergistic anti-HIV effects with nucleoside derivatives,42−44 which may be additional beneficial effects when applying a “shock and kill” strategy to purge the latent reservoirs. Although resveratrol was thought to be an activator of SIRT1,33 emerging evidence has shown that resveratrol serves as a synergistic factor of endogenous SIRT1 deacetylase activator but not a direct activator of SIRT1.45−47 Consistent with these findings, our present study showed that inhibition of SIRT1 activation failed to influence resveratrol-mediated reactivation and that the acetylation of H3K9, a putative substrate of SIRT1, was up-regulated under treatment with resveratrol. Moreover, we also found that the specific SIRT1 activator SRT1720 could antagonize resveratrol-mediated reactivation of latent HIV. This might be because SIRT1 could negatively regulate HIV transcription through deacetylating the p65 subunit of nuclear factor-κB (NF-κB).30 Thus, we favored the hypothesis that resveratrol reactivated latent HIV in a manner which was not related to SIRT1. The transcription of integrated HIV, similar to the transcription of host genes, could be epigenetically modified. Acetylation states of the core histones in the HIV LTR, which are associated with HIV transcription, are balanced by two competing enzyme complexes (histone acetyltransferases (HATs) and HDACs).9,48 HDAC activity assays showed that resveratrol was able to inhibit HDAC activity, and Western blot analysis and ChIP assay further indicated that resveratrol treatment would lead to increasing histone acetylation in HIV LTR, which would create a less repressive chromatin state and allow for HIV transcription.

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DISCUSSION Resveratrol is a natural product widely found in many plants as well as red wine, so it is possible to supplement with resveratrol in our daily lives. Ever since resveratrol was discovered in preventing cardiovascular diseases, it has been paid great attention for its more pharmacological activity.17 A review of the database http://clinicaltrials.gov/ reveals a total of 118 clinical trials involving resveratrol, including 6 trials in phase 4 that are completed or recruiting. These trials aim at investigating the potential role of resveratrol in the management of multiple diseases, such as diabetes, obesity, nervous 4390

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Figure 7. Resveratrol derivatives in the reactivation of latent HIV. (A) Chemical structures of resveratrol derivatives. (B) J-Lat A2 cells were exposed to resveratrol derivatives for 48 h. The percentage of GFP-positive cells was examined by flow cytometry analysis. (C) J-Lat A2 cells were mock treated or treated with triacetyl resveratrol (20 μM), prostratin (0.5 μM), JQ1 (0.5 μM), SAHA (1 μM), triacetyl resveratrol/prostratin, triacetyl resveratrol/JQ1, or triacetyl resveratrol/SAHA for 48 h. The percentage of GFP-positive cells was measured by flow cytometry. Data represent the mean ± SD of three independent experiments. (∗) p < 0.05 versus triacetyl resveratrol treatment alone; (∗∗) p < 0.01 versus triacetyl resveratrol treatment alone. (D) Calculation of synergy for triacetyl resveratrol and prostratin, JQ1, or SAHA using the Bliss independence model. Data are presented as the difference between the experimentally observed and predicted fractional effects. Synergy is defined as Δfaxy > 0. Statistical significance was calculated using a t test comparing the experimentally observed and predicted fractional effects. (∗) p < 0.05; (∗∗) p < 0.01.

tails within the HIV promoter area,49 which prompted us to propose that increasing histone acetylation induced by resveratrol might be in part associated with HSF1 activation. However, we found that HSF1 activation and increasing histone acetylation induced by resveratrol might be independent because inhibition of HSF1 by KRIBB11 showed no inhibitory activity against resveratrol-mediated histone acetylation (data not shown). Further investigation of other mechanisms contributed to resveratrol-mediated reactivation

In addition, several cellular stimuli trigger the phosphorylation of HSF1, thus resulting in the translocation of HSF1 into the nucleus and inducing the transcription of many genes by binding to specific consensus sequences in their promoter regions, including the HIV gene.19,36 We revealed that activation of the HSF1 pathway might be one of the mechanisms during resveratrol-mediated activation of latent HIV expression. It was previously found that transcription factors would recruit the cellular HATs to acetylate the histone 4391

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Journal of Agricultural and Food Chemistry Notes

of latent HIV should be pursued owing to the fact that resveratrol has multiple targets, including the mitogen-activated protein kinases (MAPK) pathway, which is involved in regulating HIV transcription.50,51 Although resveratrol is a potent LRA, data on its pharmacokinetics show that resveratrol has a low bioavailbility as it would be rapidly and extensively metabolized into metabolites (glucuronides and sulfates) in the body.52,53 Therefore, it is important to research whether resveratrol derivatives could reactivate latent HIV. Our data showed that triacetyl resveratrol presented a similar reactivation effect on latent HIV with resveratrol. The result is in accordance with previous observations reporting that triacetyl resveratrol was as efficient as resveratrol in the cytostatic and cytotoxic activities,54,55 Furthermore, triacetyl resveratrol also showed synergistic effects with conventional LRAs. These results are of great interest because esterification of the phenol groups could lead to higher hydrophobicity than the parent molecules, thus resulting in better absorption into the body.56 However, the other derivatives lost reactivation effects on latent HIV. By comparison with the structures of resveratrol trimethyl ether, pterostilbene, polydatin, and resveratrol, we supposed that the hydroxyl group in the meta-position of the benzene ring might be critical for maintaining the pharmacological activity. Additionally, the different reactivation effects between dihydroresveratrol and resveratrol on latent HIV indicated that the double bond between two benzene rings might be of great importance in maintaining the spatial conformation of two benzene rings because the saturation of double bonds would break the planar construction of these two benzene rings. Piceatannol could not reactivate latent HIV, which was likely related to the impediment of interaction with its target proteins. In summary, resveratrol is a potent antagonist of HIV latency through increasing histone acetylation and activating HSF1 without causing global T cell activation. We also demonstrate that resveratrol synergizes with prostratin, JQ1, and SAHA in the induction of HIV transcription. However, it is necessary to extend these observations to latently HIV infected cells from HIV-infected patients on suppressive cART.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Shibo Jiang at Fudan University, China, for kindly gifting the essential cells. We also thank Dr. Heqing Huang at Sun Yat-Sen Univesity, China, for kindly providing us with polydatin.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00418. Cell viability of J-Lat A2 cells treated with EX527, SRT1720, or KRIBB11 alone or in combination with resveratrol or carfilzomib (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(S.L.) Phone: +86-020-61648538. Fax: +86-020-61648655. Email: [email protected]. ORCID

Yuanxin Tian: 0000-0003-3847-559X Shuwen Liu: 0000-0001-6346-5006 Funding

This study was financially supported by grants from the Natural Science Foundation of China (31370781), the National S&T Key Special Foundation (2014ZX09509001-004), and the Natural Science Foundation of Guangdong Province (S2011020005207) to S.L. 4392

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