Resveratrol, piceatannol, resminostat, entinostat, mocetinostat, vorinostat, curcumin, garcinol, anacardic acid and Tip60i were purchased from Selleckchem (Houston, TX, USA). MB-3 and BMS-345541 were purchased from MilliporeSigma (Burlington, MA, USA). Pterostilbene and myricetin were from LKT Laboratories (St. Paul, MN, USA) and trimethoxy-resveratrol (trans-3,5,4′-trimethoxystilbene) was from Cayman Chemical Co. (Ann Arbor, MI, USA). Stock solutions of the chemicals were prepared based on the information provided by the manufacturer and maintained at −20°C. The antibodies for human PD-L1 (E1L3N, 13684), p38 MAPK (D13E1, 8690), NF-κB p65 (D14E12, 8242), γH2AX (20E3, 9718), cleaved caspase 3 (D3E9, 9579), IRF-1 (D5E4, 8478) and rabbit IgG isotype monoclonal antibody (DA1E, 5742) conjugated to PE were obtained from Cell Signaling Technology (Beverly, MA, USA). c-Myc antibody (9E10, sc-40) was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Fetal bovine serum, RPMI-1640, DMEM, streptomycin and penicillin were from Gibco/Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals and solvents were of analytical grade.

Human BT549 (breast cancer), BT474 (invasive ductal carcinoma), SKBR3 (breast cancer), HCT116 (colon cancer), SW480 (colon cancer), HT29 (recto-sigmoid adenocarcinoma) and SW620 (colon cancer) cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Human Cal51 (breast cancer) cells were from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The cells were maintained in RPMI-1640 or DMEM culture media supplemented with penicillin, streptomycin and 10% heat-inactivated fetal bovine serum according to the manufacturer's instructions. The cells were split once a week and the media were changed every 3-4 days.

In all experiments described in this study, parent/parental or DMSO treated cells all refer to untreated, control cells. For treated cells, the conditions (dose and treatment duration) and whether any reagents were used together at specific doses were as indicated in the figure legends.

Paraffin-embedded SW620 colon cancer cells were immunohistochemically stained to evaluate the protein expression of PD-L1, c-Myc, p38 MAPK, γH2AX and cleaved caspase 3. Following deparaffinization and rehydration, sections of SW620 cells were prepared. The slides were heated in the Retriever 2000 pressure cooker (Electron Microscopy Sciences, Hatfield, PA, USA) in Borg buffer pH 9.5 (Biocare Medical, Concord, CA, USA) and cooled to room temperature. Endogenous peroxidase activity was inactivated with Peroxidazed 1 (Biocare Medical) for 10 min. Non-specific protein interactions were blocked for 10 min with Background Punisher (Biocare Medical). The sections were incubated with the primary antibodies, indicated above, at a dilution of 1/200 for 1 h, washed in TBS and incubated with SignalStain Boost IHC Detection Reagent (Cell Signaling Technology) for 30 min. Following washes in TBS, immunoreactivity was visualized by development with 3,3′-diaminobenzidine (DAB⁺, Dako, Carpinteria, CA, USA) for 5 min. Immunostained sections were briefly counterstained with CAT hematoxylin, washed in tap water, dehydrated in a graded alcohol series, cleared in xylene and coverslipped with Permount mounting medium (Fisher Scientific Co. L.L.C., Pittsburgh, PA, USA).

The cells were harvested and fixed in 4% parafor-maldehyde for 10 min. The cells were then rinsed twice with phosphate-buffered saline (PBS) before a 30-min staining was performed to prepare the samples for flow cytometry using a rabbit anti-human PD-L1 monoclonal antibody (E1L3N) conjugated to phycoerythrin (PE). As controls, cell samples were also stained for 30 min using rabbit IgG isotype monoclonal antibody (DA1E) conjugated to PE. Following labeling with antibody, the cells were rinsed twice with PBS and re-suspended with PBS. The data shown as the geometric means from n=3-4 independent experiments were acquired on a DB LSR Fortessa X-20, and analyzed with FlowJo version 10 software (BD Biosciences, San Jose, CA, USA).

The basal PD-L1 mRNA expression levels shown in Fig. 2B, presented as transcript per million (TPM) were obtained from a public Cancer Cell Line Encyclopedia (CCLE, https://portals.broadinstitute.org/ccle/home) database for cancer cell lines tested.

The A549-Dual cells purchased from InvivoGen (San Diego, CA, USA) were used. These are derivatives of the A549 human lung carcinoma cells containing the stable integration of two inducible reporter constructs. The constructs allow for the co-expression of a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the IFN-β minimal promoter fused 5 five NF-κB binding sites, and, a Lucia luciferase gene encoding a secreted luciferase whose transcription is driven by an ISG54 promoter fused to 5 IFN-stimulated response elements. The cells were treated for the specified amount of time (8, 24 and 48 h) with resveratrol or piceatannol, each at 100 μM or combined, each at 50 μM (referred to as combo-100) to yield a concentration of 100 μM and the secreted alkaline phosphatase and Lucia luciferase in the supernatant of the control and treated cells were detected using the Quanti-Blue reagent from InvivoGen. The results were scored by the fluorescence intensity on a Perkin Elmer EnSpire set at a wavelength of 650 nm (PerkinElmer Inc., Shelton, CT, USA). To determine the role of NF-κB in mediating the induction of PD-L1, the IKK inhibitor, BMS-345541, was administered for 24 h in vitro prior to exposure to the combination of piceatannol and resveratrol, each at 50 μM to yield a concentration of 100 μM, for a further 48 h. BMS-345541 was added at increasing concentrations (1, 4 and 8 μM), while the dose of the combination was kept constant.

The cells were harvested and washed with PBS then re-suspended in cold 1% formaldehyde in PBS solution for 15 min at 4°C. The cells were washed twice in PBS and re-suspended in ice-cold 70% ethanol and stored at −20°C for 2 h prior to analysis. Prior to fluorescence measurement the cells were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). The intensity of blue fluorescence emission of DAPI stained DNA, excited with the UV laser (355 nm) was measured, recorded and analyzed on a MoFlo flow cytometer (Beckman Coulter Life Sciences, Indianapolis IN, USA) using Kaluza fluorescence intensity analysis software (48). Experiments were repeated and representative data are presented.

All data are presented as the mean ± the standard error of the mean. A Student's t-test or two-way ANOVA with Bonferroni correction were performed to determine statistical significance between frequencies or mean fluorescence intensities of assessed cell populations using GraphPad Prism 6 (GraphPad Software). Statistical significance of results was as indicated in each figure.

We first assessed any alterations in PD-L1 levels using the Cal51 breast cancer and HCT116 colon cancer cells treated with stilbenoids, specifically, resveratrol, piceatannol, pterostilbene and 3,5,4′-trimethoxystilbene, for comparison with the flavonoid, myricetin (the chemical structures of the compounds are shown in Fig. 1A). The surface expression of PD-L1 in the control (DMSO-treated, also referred to as parent/parental cells) and treated Cal51 and HCT116 cells was assayed by flow cytometry. Resveratrol significantly increased the expression of PD-L1 in the Cal51 cells, while treatment with piceatannol resulted in a marked increase in the PD-L1 level in HCT116 cells (Fig. 1B).

To determine whether the upregulation of PD-L1 by resveratrol and piceatannol was broadly or uniquely observed in specific breast or colon cancer cell lines, we assayed any alterations in PD-L1 expression using a panel of breast (Cal51, BT549, BT474 and SKBR3) and colorectal (HCT116, SW480, HT29 and SW620) cancer cell lines. In addition, we also determined whether the synergistic upregulation of PD-L1 may result from treatment with the two stilbenoids. The differential increase in PD-L1 expression induced by resveratrol or piceatannol was observed in 2/4 breast and 3/4 colorectal cancer cell lines treated with either of the stilbenoids as a single agent (Fig. 2A). The combination of resveratrol and piceatannol acted synergistically; 50 μM each of resveratrol and piceatannol combined and referred to as 'combo-100' resulted in significantly greater induction of PD-L1 expression; specifically, ≥4.5-fold in the Cal51 and ≥3.5-fold in the SW620 cells than 50 μM of either stilbenoid added alone (Fig. 2A). Gene expression analyses frequently reveal that the relative abundance of mRNA is only weakly or even inversely associated with the level of protein expression (49–51). Thus, in this study, to determine whether the differential expression level of endogenous PD-L1 mRNA might contribute to the observed induction of PD-L1 by resveratrol and piceatannol in these two cell lines, relative to the panel of the other studied cell lines with the same cancer type grouping, the basal PD-L1 mRNA expression levels, shown as TPM were obtained from a public Cancer Cell Line Encyclopedia (CCLE, https://portals.broadinstitute.org/ccle/home) database for cancer cell lines tested. In the breast cancer cell lines, the endogenous level of PD-L1 mRNA ranked as follows: Cal51 ≤ BT474 < SKBR3 ≤ BT549 (Fig. 2B); however, the induction of PD-L1 by co-treatment with resveratrol and piceatannol yielded the opposite result: Cal51 (~7-fold) ≥ BT474 (~ 4-fold) > SKBR3 (~2-fold) ≥ BT549 (~2-fold) (Fig. 2A). A similar trend was also observed in the colorectal cancer cells; namely, the endogenous PD-L1 mRNA ranking was as follows: SW620 ≤ HCT116 < HT29 ≤ SW480 (Fig. 2B), whereas the relative induction of PD-L1 decreased from high to low as follows: SW620 (~4-fold) > HCT116 (~3-fold) > SW480 (~3-fold) > HT29 (~1.5-fold) (Fig. 2A). These results suggest that tumors with a low endogenous mRNA level of PD-L1 are more likely to be affected by resveratrol and/or piceatannol, alone or in combination.

IFN-γ is known to induce PD-L1 expression by upregulating its transcription through the activation of interferon regulatory transcription factor (IRF-1) (52,53). Thus, in this study, we investigated whether the same mechanism may contribute to the resveratrol-and/or piceatannol-mediated induction of PD-L1 expression. The results of immunohistochemistry revealed no change in IRF-1 expression in the SW620 cells treated for 48 h with 'combo-100' compared to the control (data not shown). Since the NF-κB consensus sequence is also present in the PD-L1 gene promoter (52,53), and NF-κB plays a major role in the transcription of PD-L1 by IFN-γ (54-56), in this study, we examined whether the induction of PD-L1 by resveratrol and/or piceatannol was due to the activation of NF-κB. The A549 cells co-expressing the SEAP reporter gene and Lucia luciferase gene were used to investigate the association between NF-κB activity and the PD-L1 expression levels following treatment with the two stilbenoids, alone or combination. In this dual reporter assay, the secreted SEAP and Lucia luciferase in the culture supernatant were separately measured to provide a quantitative readout of the transcriptional impact of NF-κB and the IFN signaling pathways. The time-dependent (≥24 h) and synergistic induction of NF-κB expression induced by piceatannol alone and by combined treatment with resveratrol was observed (Fig. 3A). In response to stimuli, the inhibitory protein IκB is degraded, which leads to the release/translocation of heterodimer p65/p50 from the cytoplasm to the nucleus (57,58). Therefore, the activation of NF-κB by the combined stilbenoids was examined in the SW620 cells by immunohistochemistry to analyze changes in the localization of p65; 48 h of exposure to 'combo-100' resulted in an increase in the translocation and nuclear accumulation of p65, as shown in Fig. 3B (inset).

The small molecule, BMS-345541, is an IKK kinase inhibitor (59) that prevents IκB phosphorylation, to effectively suppress the translocation of NF-κB into the nucleus for participation in transcriptional activation of NF-κB-responsive genes (57,58). Thus, in this study, we then examined whether the stilbenoid-induced PD-L1 expression can be blocked by BMS-345541. In SW620 cells, the induction of PD-L1 by 'combo-100' was inhibited by 48% with 1 μM BMS-345541 (Fig. 3C). Furthermore, a dose-dependent inhibition was observed with the 1, 4 and 8 μM of IKK inhibitor concentration range (Fig. 3C). These results, showing that the inhibition of IKK significantly decreased PD-L1 expression in SW620 cells suggest that NF-κB activation is involved in the induction of PD-L1 expression by resveratrol or piceatannol either alone, or in combination.

The expression of PD-L1 can be regulated via histone acetylation/deacetylation (60,61) and resveratrol is an activator of HDAC (62). To investigate whether the induction of PD-L1 by the combination of resveratrol and piceatannol is blocked by inhibitors of HDAC or HAT, HDACis (vorinostat, mocetinostat, resminostat and entinostat) and HATis (curcumin, garcinol, anacardic acid, MB-3 and Tip60i) were used to assess their effects on the modulation of PD-L1 by the combined use of the stilbenoids. The cells were pre-treated for 48 h with individual HDACis/HATis alone or in combination with 60 μM of either of the stilbenoids, followed by the flow cytometric analysis of PD-L1 expression. The addition of HDACis or HATis alone did not affect PD-L1 expression compared to the untreated controls (Fig. 4). When the SW620 cells were pre-treated with histone modification inhibitors, the ability of the stilbenoids to induce PD-L1 was markedly reduced by two HDACis (5 μM of resminostat and entinostat) and also by the HATi, anacardic acid (1 μM) (Fig. 5). These results demonstrated that histone modification inhibitors can suppress the induction of PD-L1 expression by stilbenoids. The data are consistent with the interpretation that the upregulation of PD-L1 by stilbenoids involves transcriptional control.

The upregulation of PD-L1 may allow cancers to evade the host immune system and acquire resistance to anticancer drugs. Having demonstrated that the upregulation of PD-L1 expression by stilbenoids in the SW620 colon cancer cells, we then investigated whether stilbenoids affect the survival status of cells by analyzing two biomarkers related to apoptosis, namely, the expression of the DNA damage indicator γH2AX, and that of cleaved caspase 3. In addition, markers associated with cell survival, p38-MAPK and c-Myc, were also assessed using immunohistochemistry. Treatment of the SW620 cells for 48 h with 'combo-100' resveratrol and piceatannol increased the expression of γH2AX and that of cleaved caspase 3, and downregulated the p38-MAPK and c-Myc levels (Fig. 6A). The induction of γH2AX is characteristic of DNA fragmentation and damage during apoptosis, and thus supports the interpretation that exposure to resveratrol and/or piceatannol causes DNA damage and apoptosis via the activation of caspase 3. We then assessed whether treatment with the stilbenoids altered cell cycle distribution by flow cytometric analysis. An increase in the percentage of cells in the S phase of the cell cycle, from 19 to ~30%, a distinct reduction in the proportion of G₁ phase cells (from 66 to ~30%), and a marked decrease in the percentage of G₂M phase cells, from 12 to ~3% were observed. There was also an increase in the percentage of cells with fractional DNA content ('sub-G₁ cells'), an indication of apoptosis from 2.85% in the control cells to 31.84, 35.08 and 36.55% in the cells treated with 50 μM resveratrol, 50 μM piceatannol or 50 μM of these two compounds for 48 h, respectively (Fig. 6B). We also examined and determined that low-dose (10 μM) resveratrol and/or piceatannol did not induce PD-L1 expression, whereas the analysis of cell cycle phase distribution changes using Cal51 and SW620 cell cultures treated for 48 h revealed an apparent increase in the proportion of S-phase cells concomitant with the reduction of G₂M-phase cells in cultures treated with resveratrol alone, and to an even greater extent following treatment with both stilbenoids (Fig. 7). In addition, the combination of resveratrol and piceatannol induced apoptosis in both cell types to a much greater degree than each of them alone, as demonstrated by an increase in the percentage of cells with fractional DNA content ('sub-G₁ cells'), an indication of apoptosis (compare Fig. 7A with Fig. 7B–D) (48,63,64). Taken together, our findings indicate that stilbenoids not only increase PD-L1 expression, but may also induce DNA damage, leading to increased cell death in tumor cells, such as SW620 colon cancer cells.