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Direct regulation of IL-2 by curcumin
Biochemical and Biophysical Research Communications xxx (2017) 1e6
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Direct regulation of IL-2 by curcumin Jin-Gyo Oh a, Da-Jeong Hwang a, Tae-Hwe Heo a, b, * a
Lab of Pharmaco-Immunology, Integrated Research Institute of Pharmaceutical Sciences, BK21 PLUS Team for Creative Leader Program for Pharmacomicsbased Future Pharmacy, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea b ILAb Inc., NP513, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2017 Accepted 6 November 2017 Available online xxx
Interleukin-2 (IL-2) is a crucial growth factor for both regulatory and effector T cells. Thus, IL-2 plays a critical role in the stimulation and suppression of immune responses. Recently, anti-IL-2 antibodies (Abs) have been shown to possess strong IL-2 modulatory activities by affecting the interaction between IL-2 and IL-2 receptors. In this study, we screened an herbal library to identify a compound that regulates IL-2, which resulted in the identification of curcumin as a direct binder and inhibitor of IL-2. Curcumin is a phytochemical with well-known anti-cancer properties. In this study, curcumin mimicked or altered the binding pattern of anti-IL-2 Abs against IL-2 and remarkably inhibited the interaction of recombinant IL2 with the IL-2 receptor a, CD25. Interestingly, curcumin neutralized the biological activities of IL-2 both in vitro and in vivo. In this report, we elucidated the unsolved mechanism of the anti-cancer effect of curcumin by identifying IL-2 as a direct molecular target. Curcumin, as a small molecule IL-2 modulator, has the potential to be used to treat IL-2 related pathologic conditions. © 2017 Elsevier Inc. All rights reserved.
Keywords: Interleukin-2 (IL-2) Interleukin-2 receptor alpha (IL-2Ra) Vascular leak syndrome (VLS) Surface plasmon resonance (SPR) Curcumin
1. Introduction Interleukin-2 (IL-2) cytokine is a growth factor for T lymphocytes and natural killer (NK) cells [1]. Since IL-2 is required for the expansion and homeostasis of regulatory T (Treg) cells as well as effector T (Teff) cells, considerable effort has been made to investigate the Janus effect of IL-2 in immune suppression and activation. IL-2 binds to a heterotrimeric IL-2 receptor consisting of IL-2Ra (CD25), IL-2Rb (CD122), and a common gamma chain (IL-2Rg, CD132) with high affinity, and it also binds to the heterodimeric receptors IL-2Rb and IL-2Rg with low affinity [1]. The high-affinity IL-2R complex (IL-2Rabg) is highly expressed on Treg cells [1,2], and thus, IL-2 is mostly consumed by Treg cells at steady-state due to the low levels of IL-2 in vivo [1]. Endogenous and exogenous IL-2 could increase Treg cells in cancer-bearing hosts [3], and an increase in Treg cells induces an immunosuppressive, tumorfavorable environment [2,4]. Based on these findings, a wide range of efforts have been made to deplete or modulate Treg cells by targeting IL-2 [5]. To date, evidence has accumulated proving that high-dose IL-2
* Corresponding author. NP512, Hall of Cardinal Jin-Suk Cheong, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do, 420-743, Republic of Korea. E-mail address: [email protected] (T.-H. Heo).
shows anti-tumor activities by shifting the balance between Treg cells and Teff cells towards the dominance of Teff cells [6,7]. However, administration of IL-2 can lead to a severe side effect called vascular leak syndrome [8]. IL-2 is thought to have distinct epitopes against IL-2 mAbs, including mouse IL-2 mAb S4B6 [9]. S4B4 mAb inhibits the interaction between IL-2 and IL-2Ra, which results in skewing the inhibition of IL-2Rabg expressing immune cells [9]. These findings imply that IL-2binding substances have the potential to modulate IL-2. Curcumin is a natural compound originating from Curcuma longa and is abundant in curry spice [10]. Numerous studies have been conducted to determine the effect and mechanism of action of curcumin on the pathogenesis of cancer [11]. Although numerous pathways and molecules linked with tumorigenesis are modulated by curcumin treatment, information about key and primary binding targets of curcumin is still very limited. Very recently, it was reported that curcumin inhibits the augmentation of Treg cells, and in vitro study showed that the inhibitory effect of curcumin on the Treg activity appears to be dependent on the downregulation of IL-2 production and/or consumption by Treg cells [12]. In addition to IL-2 synthesis and IL-2Ra expression, curcumin also interferes with IL-2R downstream signaling, such as the JAK-STAT pathway, NFkB activation, and Foxp3 expression [13,14]. Despite the possible involvement of Treg cells and IL-2 in the biological activity of curcumin, the direct
https://doi.org/10.1016/j.bbrc.2017.11.039 0006-291X/© 2017 Elsevier Inc. All rights reserved.
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molecular target and the details of the process are unclear. In this study, we initially tried to discover a single compound from natural sources that would directly bind to IL-2 and modulate its function. Unexpectedly, we found that the famous phytochemical, curcumin, directly bound to IL-2 and inhibited the biological activity of IL-2 both in vitro and in vivo. These findings explain the unsolved mechanism of the effects of curcumin on IL-2 and Treg cells. 2. Materials and methods 2.1. Screening of herbal extracts library Recombinant mouse IL-2 (eBioscience, San Diego) was coated (0.1 mg/ml) on 96-well ELISA plates (BD Biosciences, San Jose, CA) at 4 C overnight. After coating, the plates were washed with PBST (PBS containing 0.05% Tween-20) and blocked with PBS containing 1% BSA (PBSA, Sigma) for 1 h at RT. After another washing with PBST, 20 mg/ml ethanol extracts from the natural products library (Plant extract bank, KRIBB, Korea) were added to the rmIL-2-coated wells for 1 h. Before washing, the rat anti-mouse IL-2 monoclonal Ab (clone S4B6, 0.1 mg/ml, Thermo Fisher Scientific, Germany) was added to each well and incubated for 2 h at RT. After washing, each well was treated with anti-rat HRP (Santa Cruz Biotechnology) for 1 h at RT. After washing, ABTS solution (Roche Diagnotics, Germany) was added to measure the absorbance at 405 nm. The results were expressed or plotted as the mean of duplicates. 2.2. ELISA for competition between curcumin and anti-IL-2 mAb against IL-2 The ELISA processes were performed as described above. After coating procedues, plates were washed, and then various concentrations of curcumin (Sigma-Aldrich, St. Louis, MO) or extract of Zedoariae Rhizoma were added to the rmIL-2-coated wells. Without washing, S4B6 mAb (rat anti-mouse IL-2 mAb, eBioscience) was added to each well and incubated for 2 h at RT. After washing, each well was treated with anti-rat HRP for 1 h at RT. After washing, ABTS solution was added to measure the absorbance at 405 nm. The results are expressed as the mean of triplicates ± standard deviation (SD). 2.3. CNBr-activated sepharose 4B bead binding assay Curcumin-conjugated CNBr-activated Sepharose 4B was prepared as described previously [15]. RmIL-2 was mixed with control CNBr beads or curcumin-conjugated beads and then rotated overnight at 4 C. After washing, each sample was eluted by boiling in sample buffer, was resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked by superblock buffer (Thermo Fisher Scientific) and then incubated overnight at 4 C with the S4B6 mAb diluted in 1:1000 by TBST containing 5% skim milk. Membranes were washed with TBST and incubated for 1 h at RT with the appropriate HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in superblock buffer. Membranes were washed with TBST, reacted with ECL reagents (Thermo Fisher Scientific) for 1 min and visualized using a Chemidoc XRS imager system (Bio-Rad Laboratories Inc.). 2.4. Surface plasmon resonance (SPR) assay To monitor the direct binding of curcumin to IL-2 and the inhibition of IL-2/IL-2Ra binding by curcumin, SPR analysis was
performed using Biacore T200 model (GE Healthcare). RmIL-2 and CD25 (PeproTech, Rocky Hill, NJ, USA) were immobilized on a CM5 sensor chip to 3983.4 and 1551 resonance units (RU) with amine coupling at pH 5.0, respectively. Curcumin (0.78e25 mM) was injected into the IL-2-immobilized flow cell with a flow rate of 30 ml/m for 100 s and allowed to dissociate for 600 s. Next, IL-2 (1.87e120 mg/ml) with or without curcumin (10 mM) was injected into the CD25-immobilized flow cell for 100 s and allowed to dissociate for 600 s solvent correction was applied prior to the evaluation, and all responses were reference-subtracted, and blankdeducted. The equilibrium dissociation constants (KD) were calculated by Langmuir 1:1 binding using BIAevaluation software (GE Healthcare). 2.5. IL-2 bioassay CTLL-2 cells (a murine cytotoxic T-cell line provided by Hanmi Pharmaceutical, Korea) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (HyClone, Thermo Fisher Scientific), penicillin-streptomycin (Gibco, Invitrogen, Grand Island, NY), and 10% T-STIM (BD Biosciences). CTLL-2 cells were added in triplicate (2 104 cells/well) to flat-bottom 96-well plates and cultured in the absence of mIL-2 for 4 h before the addition of mIL-2 (10 ng) with or without curcumin (5, 10, or 20 mM). Following 48 h of culture in a CO2 incubator, 15 ml of Ez-cytox (Daeil Lab Service, Korea) was added to each well. After 4 h, absorbance was read at 450 nm and analyzed using SOFTmax® PRO software (Molecular Devices, Sunnyval, CA). The results are expressed as the mean of triplicates ±SD, and the data were compared for statistical significance with Student's t-test. **0.001 < p < 0.01, and ***p < 0.001 were considered significant. 2.6. CTLL signal CTLL-2 cells were starved in the absence of IL-2 for 4 h and treated with IL-2 or IL-2 plus curcumin for 15 min, and then they were collected and lysed. Total proteins were extracted using RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Thermo Fisher Scientific) and applied to the WB assay, as described above. The lysates were mixed with LDS sample buffer containing reducing agent (Invitrogen), and then the boiled for 10 min 70 C. Equal amounts of protein were loaded onto 4e12% bis-Tris Plus Gels (Invitrogen) and transferred onto PVDF membranes. Membranes were blocked with superblock buffer for 1 h and then incubated overnight at 4 C with the primary antibody diluted to 1:1000 in superblock buffer. Beta-actin and phospho-JAK3 (Cell Signaling Technology, Beverly, MA) and total-JAK3 (Novus Bio, Littelon, CO, USA) were used for primary antibodies. 2.7. Induced Treg induction (ex vivo) Splenocytes isolated from C57BL/6 mice were enriched for a CD4 positive population using a magnetic activated cell sorting kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions. CD4-enriched cells were labeled by fluorescence conjugated antibody (CD4, CD25, CD44 and CD62L, eBioscience) and sorted into naive T cells by FACSAria™ III (BD Bioscience). Naive T cells were seeded to pre-coated plates with anti-CD3 and CD28 (Biolegend, CA, USA) mAb, which were then treated with curcumin at various concentrations (1e8 mM) in the presence of IL-2 (10 ng/ mL) and TGF-beta (20 ng/mL). After 72 h of culture, the cells were analyzed for Treg cell populations by flow cytometer (FACS Canto II, BD Bioscience) and flowjo software (Treestar, San Carlos, CA, USA).
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2.8. Vascular leak in vivo model The animal studies were approved by the Department of Laboratory Animals, Institutional Animal Care and Use committee at the Sungsim campus of The Catholic University of Korea (Bucheon, Korea). Eight-week-old C57BL/6 mice (Orient-Bio, Korea) were maintained under pathogen-free conditions in the animal facility and kept on a standard laboratory diet with free access to water. Five mice from each group were injected intraperitoneally with 150,000 units of rhIL-2 (Aldesleukin, Novartis), rhIL-2 with curcumin (0.6 mg) or vehicle control 3 times a day for 3 days and 1 time on the 4th day. For the TNF-a group, TNF-a (5 mg) or TNF-a with curcumin (0.6 mg) was injected 2 times a day for 2 days and 1 time on the 3rd day. Two h after the last injection, the mice were injected intravenously with 200 ml of 1% Evans blue in PBS (Sigma). After another 2 h, the hearts of the mice were perfused with 10 ml heparin-PBS (25 UI/ml, JW Pharmaceutical, Korea) under anesthesia. The lungs were harvested, washed, weighed, and placed in 5 ml formamide (Sigma-Aldrich) at 37 C for 24 h. The absorbance of the supernatants was measured at 650 nm and normalized by lung weight.
3. Results 3.1. Identification of an herbal extract and curcumin as a competitor of IL-2 mAb, S4B6 To discover a natural compound sharing the IL-2 modulatory
A
3.2. Direct binding of IL-2 with curcumin To examine the direct binding of curcumin to IL-2, we performed several experiments. First, a pull-down assay was conducted by using CNBr-activated Sepharose 4B beads. Curcumin was cross-linked with CNBr beads and then incubated with recombinant mouse IL-2. Bound IL-2 was eluted, separated, and detected by WB. IL-2 did not bind to CNBr only control, but it did bind to the curcumin on bead (Fig. 2A). Second, using SPR technique, we confirmed that curcumin directly binds to IL-2 (Fig. 2B) and suppresses the binding between IL-2 and IL-2Ra (Fig. 2C). In the curcumin binding to IL-2, ka (1/ms) was 2.35 104, kd (1/s) was
Binding activity (%)
Binding activity (%)
characteristics with S4B6 IL-2 mAb, we designed and performed the screening assay. At first, Zedoariae Rhizoma (ZR, botanical name; Curcuma zedoaria) was identified for possessing activity that interferes with the interaction between IL-2 and S4B6 mAb, and approximately 60% of S4B4 mAb binding was inhibited by the 20 mg/ml ethanol extract (Fig. 1A). The dose-dependent S4B6competing activity of ZR was confirmed by serially diluted extracts (Fig. 1B). Instead of fractionation and isolation of single active compounds from ZR, since one of the major constituents of ZR is curcumin (Fig. 1C), we tested if curcumin also exerts a similar effect (Fig. 1D). Curcumin indeed inhibited binding of S4B6 to IL-2 in a dose dependent manner (IC50 ¼ 1.37 mM). Next, we investigated whether this inhibition was the result of targeting of IL-2 by curcumin.
B 120 100 80 60 40 20
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Fig. 1. Screening of herbal extracts library and identification of a hit and its major compound, curcumin. (A) An herbal library was screened for the identification of competing extracts with anti-mouse IL-2 monoclonal antibody (S4B6 mAb) for binding to recombinant mouse IL-2 (rmIL-2). RmIL-2 was coated on ELISA plates and bound by herbal extracts. S4B6 mAb and HRP-conjugated secondary Ab were used for detection. The ethanol extract of Zedoariae Rhizoma (ZR) was found to show competitive effects on S4B6 mAb binding against rmIL-2. Representative data are expressed as the mean of duplicates compared with the S4B6 mAb alone control as 100% binding activity. (B) The dose dependent inhibitory action of ZR against S4B6 mAb binding to rmIL-2 was confirmed. Serially diluted ZR extracts were applied to the same assay as (A). (C) The molecular structure of curcumin. Among the major compounds derived from ZR, curcumin is the most well-known bioactive compound. (D) Curcumin was tested to determine if it had S4B6-inhibiting activity similar to ZR. RmIL-2 was coated on ELISA plates and added by various concentrations of curcumin. S4B6 mAb was added and HRP-conjugated secondary Ab was used for detection. Curcumin inhibited S4B6 mAb binding to rmIL-2 to a different extent. The results are expressed as the mean ± standard deviation (SD).
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A
B Fitted line
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Concentration (M) Fig. 2. Direct binding of curcumin to rmIL-2. (A) Direct binding between curcumin-conjugated CNBr beads and rmIL-2 was examined via a pull-down assay. CNBr-curcumin or CNBr-control beads were incubated with rmIL-2 overnight at 4 C. After washing, each sample was eluted, resolved, and detected by Western blot. (B) Interaction between curcumin and rmIL-2 was analyzed by a SPR device, Biacore T200. RmIL-2 was immobilized on a CM5 sensor chip, and curcumin (0.78e25 mM) was injected to the flow cell, and BIAevaluation software was used to subtract the references. Curcumin bound to rmIL-2 in a dose dependent manner and affinity was 2.18 106 (KD). (C) spr assay for identifying binding inhibition of IL-2/IL-2Ra by curcumin. CD25 was immobilized on a CM5 sensor chip and IL-2 (1.87e120 mg/ml) was injected into the flow cells in the absence or presence of curcumin (10 mM).
0.05112, and the Kd value was 2.18 nM, respectively. Steady state affinity of IL-2 against CD25 was calculated 0.82 nM, approximately, which was decreased to 1.47 nM by co-incubation with curcumin. 3.3. Curcumin inhibits in vitro and in vivo activity of IL-2 We investigated the effect of curcumin on the proliferation of the IL-2dependent cell line, CTLL-2. We treated the CTLL-2 cells with IL-2 in the absence or presence of curcumin, and cell viability was measured using a WST detection kit after 48 h. Curcumin inhibited the CTLL-2 proliferative activity of IL-2 (10 ng) up to the 50% level (Fig. 3A). Additionally, it was demonstrated that curcumin can block the phosphorylation of IL-2-induced JAK-3 in the CTLL2 cell line (Fig. 3B). Next, we observed naive T cell differentiation through curcumin co-incubation to determine the effect of curcumin on Treg survival. The population of CD4þ CD25þFOXP3þ Treg was reduced by the addition of curcumin to the IL-2 samples in a dose-dependent manner (Fig. 3C). IL-2 immunotherapy is a therapeutic option for metastatic melanoma and renal cell carcinoma, but it has a side effect of toxicity of vascular leak syndrome (VLS) [16]. To test the in vivo neutralization of IL-2 activity by curcumin, we adopted an IL2induced VLS model. Briefly, mice were injected with IL-2 with or
without curcumin, and vascular permeability was measured by Evans blue extravasation into the lung. The lungs of the mice treated with IL-2 or TNF-a showed significant increases in Evans blue content compared with those of the vehicle control group (Fig. 3D). Our laboratory demonstrated that TNF-a also induced VLS [17]. Curcumin selectively neutralized the vascular toxicity of IL-2, but it was not able to inhibit the TNF-ainduced vascular leak. 4. Discussion Although many studies, clinical trials, and traditional oriental healing knowledge have indicated that curcumin or curcumincontaining herbal products have the potential to be used as anticancer agents, the immune-related direct target of curcumin has remained unknown. Our findings demonstrate that curcumin directly interacts and interferes with IL-2 because (1) curcumin impacted on the binding of IL-2 mAbs to recombinant IL-2 protein as well as soluble IL-2Ra to IL-2, (2) curcumin directly bound to IL-2 in pull-down and SPR assays, (3) curcumin inhibited the IL2mediated the proliferation and signaling pathway of the IL-2 dependent cell line, (4) the protective effect of IL-2 on Treg cell differentiation was inhibited by curcumin co-incubation, and (5) curcumin neutralized the side effect of VLS of IL-2 in vivo. Hence,
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B Proliferation (%)
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Vehicle
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TNF-α TNF-α IL-2 + + curcumin curcumin
Fig. 3. Inhibition of IL-2-mediated activity by curcumin. (A) The outcome of curcumin binding to IL-2 was investigated by using an IL-2 bioassay. CTLL-2 cells were seeded in flatbottom 96-well plates. The cells were cultured in the absence of IL-2 for 4 h and added by media of T-STIM, IL-2 (10 ng), vehicle (DMSO), or mixture of IL-2 plus curcumin (5, 10, or 20 mM). Following 48 h of culture in a CO2 incubator, the cell viability assay was performed. The results were expressed as the mean ± SD and compared and analyzed with the IL-2 alone treated sample by Student's t-test. *0.01 < p < 0.05, **0.001 < p < 0.01, and ***p < 0.001 were considered significant. (B) Blockade of IL-2 signaling by curcumin in CTLL cells. CTLL cells were starved in the absence of IL-2 for 4 h and treated with IL-2 or IL-2 plus curcumin for 15 min, and then they were collected and lysed. Immunoblot analyses were performed on the lysates using anti-P-JAK3 antibody. (C) Inhibition of differentiation of naive T cells into regulatory T cells (Treg cells) by curcumin. Splenocytes isolated from C57BL/ 6 mice were enriched for CD4 positive population using magnetic beads. CD4 enriched cells were labeled by fluorescence conjugated antibody and sorted using FACS Aria into naive T cells. Naive T cells were seeded onto pre-coated plates with CD3 and CD28 mAb and then treated with curcumin at various concentrations (1e8 mM) in the presence of IL-2 (10 ng/ mL) and TGF-beta (20 ng/mL). After 72 h of culture, the cells were harvested and analyzed for Treg cell populations using a flow cytometer. (D) In vivo inhibition of IL-2-induced vascular leakage by curcumin in mice. C57BL/6 mice (n ¼ 5) were injected intraperitoneally with vehicle control, IL-2 (150,000 units), or IL-2 with curcumin (0.6 mg) for 4 days and a total of 10 times. TNF-a (5 mg) or TNF-a with curcumin (0.6 mg) was injected for 3 days a total of 5 times. After 2 h, Evans blue dye was injected intravenously, and the lungs were isolated and analyzed for vascular leaks with Evans blue extravasation (mean ± SEM). OD, optical density; NS, not significant.
the present study revealed a direct modulation of IL-2 by curcumin, which might explain the link between the beneficial effects of curcumin and immune-related disorders, including cancer. The initial purpose of this study was to discover a natural chemical competitor with S4B6 IL-2 mAb towards the binding site on IL-2. Since S4B6 mAb could bind and block the IL-2Rabinding epitope on IL-2, we hypothesized a natural compound mimicking S4B6 could also regulate Treg cells via blockade of the IL-2/IL-2Ra interaction. Indeed, we identified the ethanol extract of Zedoariae Rhizoma (Curcuma zedoaria), which showed significant inhibitory effects against S4B6 mAb binding to IL-2 (Fig. 1A and B). Since Zedoariae Rhizoma is a rich source of curcumin [18], and curcumin itself exhibits immunomodulatory effects [19], we selected curcumin and conducted the following experiments. In a similar fashion to Zedoariae Rhizoma, curcumin almost completely prohibited S4B6 mAb from binding to rmIL-2 (Fig. 1D). This inhibition was likely to be mediated by the direct binding of curcumin to rmIL-2 (Fig. 2A and B) and subsequent competition with an anti-rmIL-2 mAb. Curcumin can bind to recombinant mIL-2
with an affinity of approximately 2.18 nM. Curcumin also reduced the equilibrium constant (KD) between IL-2 and IL2Ra from 1.47 nM to 0.82 nM (Fig. 2C). Previously, small-molecule IL-2 inhibitors that bind close to the IL-2Ra contact region on IL-2 were designed and chemically synthesized based on the X-ray crystallographic studies of IL-2 [20]. However, several important medicinal chemistry studies did not provide biological activities or even clinical applications of these chemical IL-2 binders. Next, we examined the impact of binding and modulation of rmIL-2 by curcumin with the IL-2dependent CTLL cell line. Our data show dose-dependent inhibition of rmIL-2 activity of cell proliferation (Fig. 3A) and proximal events in rmIL-2 signaling (Fig. 3B) following curcumin co-incubation. These data reconfirm and account for the previous reports about the blockade of IL-2 signaling [14], inhibition of the JAK-STAT pathway in T cell leukemia [21], and inhibition of the IL-2 induced activation of human lymphocytes [13] by curcumin with our concept of direct binding of IL-2 and inhibition of its biological actions. The development, maintenance, survival and expansion of Treg
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cells are associated with signals mediated by IL-2/IL-2Ra binding [2]. Furthermore, IL-2 is essential for the survival and homeostasis of peripheral Treg cells [2]. We tested whether the binding and modulation of IL-2 by curcumin inhibited IL-2/IL-2R signaldependent Treg cells differentiation. Our data showed an inhibition of Treg survival by curcumin co-incubation in a dose-dependent manner (Fig. 3C). IL-2 immunotherapy has toxicities of vascular leak syndrome (VLS) [16], and curcumin selectively neutralized the vascular toxicity of IL-2 (Fig. 3D). A recent study suggested new evidence of the ill-defined mechanism of IL-2induced VLS that IL-2induced pulmonary edema is caused by direct interaction of IL-2 with IL2Rabg on lung endothelial cells in vivo [7]. Thus, the amelioration of IL-2induced VLS by curcumin (Fig. 3D) is possibly mediated by the direct binding of curcumin to IL-2, resulting in the blockade of IL-2IL-2R interaction. Taken together, these data indicate that curcumin and IL-2 are mutually antagonistic by direct interactions between them both in vitro and in vivo. Thus, curcumin could possibly have the potential to be developed as an attractive treatment option for IL-2driven pathological conditions, including cancer, autoimmune diseases, and inflammation. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1B03934803), the Bio & Medical Technology Development Program of the NRF funded by the Ministry of Science, ICT & Future Planning (NRF2016M3A9D9945476), a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI16C1761), and the Research Fund 2017 of the Catholic University of Korea. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2017.11.039. References [1] E.M. Shevach, Application of IL-2 therapy to target T regulatory cell function, Trends Immunol. 33 (2012) 626e632.
[2] S. Sakaguchi, M. Miyara, C.M. Costantino, D.A. Hafler, FOXP3þ regulatory T cells in the human immune system, Nat. Rev. Immunol. 10 (2010) 490e500. [3] M. Ahmadzadeh, S.A. Rosenberg, IL-2 administration increases CD4þ CD25(hi) Foxp3þ regulatory T cells in cancer patients, Blood 107 (2006) 2409e2414. [4] M.P. Colombo, S. Piconese, Regulatory-T-cell inhibition versus depletion: the right choice in cancer immunotherapy, Nat. Rev. Cancer 7 (2007) 880e887. [5] M. Murakami, A. Sakamoto, J. Bender, J. Kappler, P. Marrack, CD25þCD4þ T cells contribute to the control of memory CD8þ T cells, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 8832e8837. [6] J.A. Klapper, S.G. Downey, F.O. Smith, J.C. Yang, M.S. Hughes, U.S. Kammula, R.M. Sherry, R.E. Royal, S.M. Steinberg, S. Rosenberg, High-dose interleukin-2 for the treatment of metastatic renal cell carcinoma : a retrospective analysis of response and survival in patients treated in the surgery branch at the National Cancer Institute between 1986 and 2006, Cancer 113 (2008) 293e301. [7] C. Krieg, S. Letourneau, G. Pantaleo, O. Boyman, Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 11906e11911. [8] R. Baluna, E.S. Vitetta, Vascular leak syndrome: a side effect of immunotherapy, Immunopharmacology 37 (1997) 117e132. [9] O. Boyman, M. Kovar, M.P. Rubinstein, C.D. Surh, J. Sprent, Selective stimulation of T cell subsets with antibody-cytokine immune complexes, Science 311 (2006) 1924e1927. [10] B.B. Aggarwal, C. Sundaram, N. Malani, H. Ichikawa, Curcumin: the Indian solid gold, Adv. Exp. Med. Biol. 595 (2007) 1e75. [11] G. Sa, T. Das, Anti cancer effects of curcumin: cycle of life and death, Cell Div. 3 (2008) 14. [12] G.J. Zhao, Z.Q. Lu, L.M. Tang, Z.S. Wu, D.W. Wang, J.Y. Zheng, Q.M. Qiu, Curcumin inhibits suppressive capacity of naturally occurring CD4þCD25þ regulatory T cells in mice in vitro, Int. Immunopharmacol. 14 (2012) 99e106. [13] D. Ranjan, C. Chen, T.D. Johnston, H. Jeon, M. Nagabhushan, Curcumin inhibits mitogen stimulated lymphocyte proliferation, NFkappaB activation, and IL-2 signaling, J. Surg. Res. 121 (2004) 171e177. [14] N.A. Forward, D.M. Conrad, M.R. Power Coombs, C.D. Doucette, S.J. Furlong, T.J. Lin, D.W. Hoskin, Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling, Biochem. Biophys. Res. Commun. 407 (2011) 801e806. [15] M. Ye, W. Huang, W.W. Wu, Y. Liu, S.N. Ye, J.H. Xu, FM807, a curcumin analogue, shows potent antitumor effects in nasopharyngeal carcinoma cells by heat shock protein 90 inhibition, Oncotarget 8 (2017) 15364e15376. [16] P.V. Sivakumar, R. Garcia, K.S. Waggie, M. Anderson-Haley, A. Nelson, S.D. Hughes, Comparison of vascular leak syndrome in mice treated with IL21 or IL2, Comp. Med. 63 (2013) 13e21. [17] K.Y. Park, S.J. Kim, E. Oh, T.H. Heo, Induction of vascular leak syndrome by tumor necrosis factor-alpha alone, Biomed. Pharmacother. 70 (2015) 213e216. [18] R. Lobo, K.S. Prabhu, A. Shirwaikar, A. Shirwaikar, Curcuma zedoaria Rosc. (white turmeric): a review of its chemical, pharmacological and ethnomedicinal properties, J. Pharm. Pharmacol. 61 (2009) 13e21. [19] R.M. Srivastava, S. Singh, S.K. Dubey, K. Misra, A. Khar, Immunomodulatory and therapeutic activity of curcumin, Int. Immunopharmacol. 11 (2011) 331e341. [20] J.A. Wells, C.L. McClendon, Reaching for high-hanging fruit in drug discovery at protein-protein interfaces, Nature 450 (2007) 1001e1009. [21] J. Rajasingh, H.P. Raikwar, G. Muthian, C. Johnson, J.J. Bright, Curcumin induces growth-arrest and apoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cell leukemia, Biochem. Biophys. Res. Commun. 340 (2006) 359e368.
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Direct regulation of IL-2 by curcumin Jin-Gyo Oh a, Da-Jeong Hwang a, Tae-Hwe Heo a, b, * a
Lab of Pharmaco-Immunology, Integrated Research Institute of Pharmaceutical Sciences, BK21 PLUS Team for Creative Leader Program for Pharmacomicsbased Future Pharmacy, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea b ILAb Inc., NP513, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2017 Accepted 6 November 2017 Available online xxx
Interleukin-2 (IL-2) is a crucial growth factor for both regulatory and effector T cells. Thus, IL-2 plays a critical role in the stimulation and suppression of immune responses. Recently, anti-IL-2 antibodies (Abs) have been shown to possess strong IL-2 modulatory activities by affecting the interaction between IL-2 and IL-2 receptors. In this study, we screened an herbal library to identify a compound that regulates IL-2, which resulted in the identification of curcumin as a direct binder and inhibitor of IL-2. Curcumin is a phytochemical with well-known anti-cancer properties. In this study, curcumin mimicked or altered the binding pattern of anti-IL-2 Abs against IL-2 and remarkably inhibited the interaction of recombinant IL2 with the IL-2 receptor a, CD25. Interestingly, curcumin neutralized the biological activities of IL-2 both in vitro and in vivo. In this report, we elucidated the unsolved mechanism of the anti-cancer effect of curcumin by identifying IL-2 as a direct molecular target. Curcumin, as a small molecule IL-2 modulator, has the potential to be used to treat IL-2 related pathologic conditions. © 2017 Elsevier Inc. All rights reserved.
Keywords: Interleukin-2 (IL-2) Interleukin-2 receptor alpha (IL-2Ra) Vascular leak syndrome (VLS) Surface plasmon resonance (SPR) Curcumin
1. Introduction Interleukin-2 (IL-2) cytokine is a growth factor for T lymphocytes and natural killer (NK) cells [1]. Since IL-2 is required for the expansion and homeostasis of regulatory T (Treg) cells as well as effector T (Teff) cells, considerable effort has been made to investigate the Janus effect of IL-2 in immune suppression and activation. IL-2 binds to a heterotrimeric IL-2 receptor consisting of IL-2Ra (CD25), IL-2Rb (CD122), and a common gamma chain (IL-2Rg, CD132) with high affinity, and it also binds to the heterodimeric receptors IL-2Rb and IL-2Rg with low affinity [1]. The high-affinity IL-2R complex (IL-2Rabg) is highly expressed on Treg cells [1,2], and thus, IL-2 is mostly consumed by Treg cells at steady-state due to the low levels of IL-2 in vivo [1]. Endogenous and exogenous IL-2 could increase Treg cells in cancer-bearing hosts [3], and an increase in Treg cells induces an immunosuppressive, tumorfavorable environment [2,4]. Based on these findings, a wide range of efforts have been made to deplete or modulate Treg cells by targeting IL-2 [5]. To date, evidence has accumulated proving that high-dose IL-2
* Corresponding author. NP512, Hall of Cardinal Jin-Suk Cheong, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do, 420-743, Republic of Korea. E-mail address: [email protected] (T.-H. Heo).
shows anti-tumor activities by shifting the balance between Treg cells and Teff cells towards the dominance of Teff cells [6,7]. However, administration of IL-2 can lead to a severe side effect called vascular leak syndrome [8]. IL-2 is thought to have distinct epitopes against IL-2 mAbs, including mouse IL-2 mAb S4B6 [9]. S4B4 mAb inhibits the interaction between IL-2 and IL-2Ra, which results in skewing the inhibition of IL-2Rabg expressing immune cells [9]. These findings imply that IL-2binding substances have the potential to modulate IL-2. Curcumin is a natural compound originating from Curcuma longa and is abundant in curry spice [10]. Numerous studies have been conducted to determine the effect and mechanism of action of curcumin on the pathogenesis of cancer [11]. Although numerous pathways and molecules linked with tumorigenesis are modulated by curcumin treatment, information about key and primary binding targets of curcumin is still very limited. Very recently, it was reported that curcumin inhibits the augmentation of Treg cells, and in vitro study showed that the inhibitory effect of curcumin on the Treg activity appears to be dependent on the downregulation of IL-2 production and/or consumption by Treg cells [12]. In addition to IL-2 synthesis and IL-2Ra expression, curcumin also interferes with IL-2R downstream signaling, such as the JAK-STAT pathway, NFkB activation, and Foxp3 expression [13,14]. Despite the possible involvement of Treg cells and IL-2 in the biological activity of curcumin, the direct
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molecular target and the details of the process are unclear. In this study, we initially tried to discover a single compound from natural sources that would directly bind to IL-2 and modulate its function. Unexpectedly, we found that the famous phytochemical, curcumin, directly bound to IL-2 and inhibited the biological activity of IL-2 both in vitro and in vivo. These findings explain the unsolved mechanism of the effects of curcumin on IL-2 and Treg cells. 2. Materials and methods 2.1. Screening of herbal extracts library Recombinant mouse IL-2 (eBioscience, San Diego) was coated (0.1 mg/ml) on 96-well ELISA plates (BD Biosciences, San Jose, CA) at 4 C overnight. After coating, the plates were washed with PBST (PBS containing 0.05% Tween-20) and blocked with PBS containing 1% BSA (PBSA, Sigma) for 1 h at RT. After another washing with PBST, 20 mg/ml ethanol extracts from the natural products library (Plant extract bank, KRIBB, Korea) were added to the rmIL-2-coated wells for 1 h. Before washing, the rat anti-mouse IL-2 monoclonal Ab (clone S4B6, 0.1 mg/ml, Thermo Fisher Scientific, Germany) was added to each well and incubated for 2 h at RT. After washing, each well was treated with anti-rat HRP (Santa Cruz Biotechnology) for 1 h at RT. After washing, ABTS solution (Roche Diagnotics, Germany) was added to measure the absorbance at 405 nm. The results were expressed or plotted as the mean of duplicates. 2.2. ELISA for competition between curcumin and anti-IL-2 mAb against IL-2 The ELISA processes were performed as described above. After coating procedues, plates were washed, and then various concentrations of curcumin (Sigma-Aldrich, St. Louis, MO) or extract of Zedoariae Rhizoma were added to the rmIL-2-coated wells. Without washing, S4B6 mAb (rat anti-mouse IL-2 mAb, eBioscience) was added to each well and incubated for 2 h at RT. After washing, each well was treated with anti-rat HRP for 1 h at RT. After washing, ABTS solution was added to measure the absorbance at 405 nm. The results are expressed as the mean of triplicates ± standard deviation (SD). 2.3. CNBr-activated sepharose 4B bead binding assay Curcumin-conjugated CNBr-activated Sepharose 4B was prepared as described previously [15]. RmIL-2 was mixed with control CNBr beads or curcumin-conjugated beads and then rotated overnight at 4 C. After washing, each sample was eluted by boiling in sample buffer, was resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked by superblock buffer (Thermo Fisher Scientific) and then incubated overnight at 4 C with the S4B6 mAb diluted in 1:1000 by TBST containing 5% skim milk. Membranes were washed with TBST and incubated for 1 h at RT with the appropriate HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in superblock buffer. Membranes were washed with TBST, reacted with ECL reagents (Thermo Fisher Scientific) for 1 min and visualized using a Chemidoc XRS imager system (Bio-Rad Laboratories Inc.). 2.4. Surface plasmon resonance (SPR) assay To monitor the direct binding of curcumin to IL-2 and the inhibition of IL-2/IL-2Ra binding by curcumin, SPR analysis was
performed using Biacore T200 model (GE Healthcare). RmIL-2 and CD25 (PeproTech, Rocky Hill, NJ, USA) were immobilized on a CM5 sensor chip to 3983.4 and 1551 resonance units (RU) with amine coupling at pH 5.0, respectively. Curcumin (0.78e25 mM) was injected into the IL-2-immobilized flow cell with a flow rate of 30 ml/m for 100 s and allowed to dissociate for 600 s. Next, IL-2 (1.87e120 mg/ml) with or without curcumin (10 mM) was injected into the CD25-immobilized flow cell for 100 s and allowed to dissociate for 600 s solvent correction was applied prior to the evaluation, and all responses were reference-subtracted, and blankdeducted. The equilibrium dissociation constants (KD) were calculated by Langmuir 1:1 binding using BIAevaluation software (GE Healthcare). 2.5. IL-2 bioassay CTLL-2 cells (a murine cytotoxic T-cell line provided by Hanmi Pharmaceutical, Korea) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (HyClone, Thermo Fisher Scientific), penicillin-streptomycin (Gibco, Invitrogen, Grand Island, NY), and 10% T-STIM (BD Biosciences). CTLL-2 cells were added in triplicate (2 104 cells/well) to flat-bottom 96-well plates and cultured in the absence of mIL-2 for 4 h before the addition of mIL-2 (10 ng) with or without curcumin (5, 10, or 20 mM). Following 48 h of culture in a CO2 incubator, 15 ml of Ez-cytox (Daeil Lab Service, Korea) was added to each well. After 4 h, absorbance was read at 450 nm and analyzed using SOFTmax® PRO software (Molecular Devices, Sunnyval, CA). The results are expressed as the mean of triplicates ±SD, and the data were compared for statistical significance with Student's t-test. **0.001 < p < 0.01, and ***p < 0.001 were considered significant. 2.6. CTLL signal CTLL-2 cells were starved in the absence of IL-2 for 4 h and treated with IL-2 or IL-2 plus curcumin for 15 min, and then they were collected and lysed. Total proteins were extracted using RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Thermo Fisher Scientific) and applied to the WB assay, as described above. The lysates were mixed with LDS sample buffer containing reducing agent (Invitrogen), and then the boiled for 10 min 70 C. Equal amounts of protein were loaded onto 4e12% bis-Tris Plus Gels (Invitrogen) and transferred onto PVDF membranes. Membranes were blocked with superblock buffer for 1 h and then incubated overnight at 4 C with the primary antibody diluted to 1:1000 in superblock buffer. Beta-actin and phospho-JAK3 (Cell Signaling Technology, Beverly, MA) and total-JAK3 (Novus Bio, Littelon, CO, USA) were used for primary antibodies. 2.7. Induced Treg induction (ex vivo) Splenocytes isolated from C57BL/6 mice were enriched for a CD4 positive population using a magnetic activated cell sorting kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions. CD4-enriched cells were labeled by fluorescence conjugated antibody (CD4, CD25, CD44 and CD62L, eBioscience) and sorted into naive T cells by FACSAria™ III (BD Bioscience). Naive T cells were seeded to pre-coated plates with anti-CD3 and CD28 (Biolegend, CA, USA) mAb, which were then treated with curcumin at various concentrations (1e8 mM) in the presence of IL-2 (10 ng/ mL) and TGF-beta (20 ng/mL). After 72 h of culture, the cells were analyzed for Treg cell populations by flow cytometer (FACS Canto II, BD Bioscience) and flowjo software (Treestar, San Carlos, CA, USA).
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2.8. Vascular leak in vivo model The animal studies were approved by the Department of Laboratory Animals, Institutional Animal Care and Use committee at the Sungsim campus of The Catholic University of Korea (Bucheon, Korea). Eight-week-old C57BL/6 mice (Orient-Bio, Korea) were maintained under pathogen-free conditions in the animal facility and kept on a standard laboratory diet with free access to water. Five mice from each group were injected intraperitoneally with 150,000 units of rhIL-2 (Aldesleukin, Novartis), rhIL-2 with curcumin (0.6 mg) or vehicle control 3 times a day for 3 days and 1 time on the 4th day. For the TNF-a group, TNF-a (5 mg) or TNF-a with curcumin (0.6 mg) was injected 2 times a day for 2 days and 1 time on the 3rd day. Two h after the last injection, the mice were injected intravenously with 200 ml of 1% Evans blue in PBS (Sigma). After another 2 h, the hearts of the mice were perfused with 10 ml heparin-PBS (25 UI/ml, JW Pharmaceutical, Korea) under anesthesia. The lungs were harvested, washed, weighed, and placed in 5 ml formamide (Sigma-Aldrich) at 37 C for 24 h. The absorbance of the supernatants was measured at 650 nm and normalized by lung weight.
3. Results 3.1. Identification of an herbal extract and curcumin as a competitor of IL-2 mAb, S4B6 To discover a natural compound sharing the IL-2 modulatory
A
3.2. Direct binding of IL-2 with curcumin To examine the direct binding of curcumin to IL-2, we performed several experiments. First, a pull-down assay was conducted by using CNBr-activated Sepharose 4B beads. Curcumin was cross-linked with CNBr beads and then incubated with recombinant mouse IL-2. Bound IL-2 was eluted, separated, and detected by WB. IL-2 did not bind to CNBr only control, but it did bind to the curcumin on bead (Fig. 2A). Second, using SPR technique, we confirmed that curcumin directly binds to IL-2 (Fig. 2B) and suppresses the binding between IL-2 and IL-2Ra (Fig. 2C). In the curcumin binding to IL-2, ka (1/ms) was 2.35 104, kd (1/s) was
Binding activity (%)
Binding activity (%)
characteristics with S4B6 IL-2 mAb, we designed and performed the screening assay. At first, Zedoariae Rhizoma (ZR, botanical name; Curcuma zedoaria) was identified for possessing activity that interferes with the interaction between IL-2 and S4B6 mAb, and approximately 60% of S4B4 mAb binding was inhibited by the 20 mg/ml ethanol extract (Fig. 1A). The dose-dependent S4B6competing activity of ZR was confirmed by serially diluted extracts (Fig. 1B). Instead of fractionation and isolation of single active compounds from ZR, since one of the major constituents of ZR is curcumin (Fig. 1C), we tested if curcumin also exerts a similar effect (Fig. 1D). Curcumin indeed inhibited binding of S4B6 to IL-2 in a dose dependent manner (IC50 ¼ 1.37 mM). Next, we investigated whether this inhibition was the result of targeting of IL-2 by curcumin.
B 120 100 80 60 40 20
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Fig. 1. Screening of herbal extracts library and identification of a hit and its major compound, curcumin. (A) An herbal library was screened for the identification of competing extracts with anti-mouse IL-2 monoclonal antibody (S4B6 mAb) for binding to recombinant mouse IL-2 (rmIL-2). RmIL-2 was coated on ELISA plates and bound by herbal extracts. S4B6 mAb and HRP-conjugated secondary Ab were used for detection. The ethanol extract of Zedoariae Rhizoma (ZR) was found to show competitive effects on S4B6 mAb binding against rmIL-2. Representative data are expressed as the mean of duplicates compared with the S4B6 mAb alone control as 100% binding activity. (B) The dose dependent inhibitory action of ZR against S4B6 mAb binding to rmIL-2 was confirmed. Serially diluted ZR extracts were applied to the same assay as (A). (C) The molecular structure of curcumin. Among the major compounds derived from ZR, curcumin is the most well-known bioactive compound. (D) Curcumin was tested to determine if it had S4B6-inhibiting activity similar to ZR. RmIL-2 was coated on ELISA plates and added by various concentrations of curcumin. S4B6 mAb was added and HRP-conjugated secondary Ab was used for detection. Curcumin inhibited S4B6 mAb binding to rmIL-2 to a different extent. The results are expressed as the mean ± standard deviation (SD).
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A
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Concentration (M) Fig. 2. Direct binding of curcumin to rmIL-2. (A) Direct binding between curcumin-conjugated CNBr beads and rmIL-2 was examined via a pull-down assay. CNBr-curcumin or CNBr-control beads were incubated with rmIL-2 overnight at 4 C. After washing, each sample was eluted, resolved, and detected by Western blot. (B) Interaction between curcumin and rmIL-2 was analyzed by a SPR device, Biacore T200. RmIL-2 was immobilized on a CM5 sensor chip, and curcumin (0.78e25 mM) was injected to the flow cell, and BIAevaluation software was used to subtract the references. Curcumin bound to rmIL-2 in a dose dependent manner and affinity was 2.18 106 (KD). (C) spr assay for identifying binding inhibition of IL-2/IL-2Ra by curcumin. CD25 was immobilized on a CM5 sensor chip and IL-2 (1.87e120 mg/ml) was injected into the flow cells in the absence or presence of curcumin (10 mM).
0.05112, and the Kd value was 2.18 nM, respectively. Steady state affinity of IL-2 against CD25 was calculated 0.82 nM, approximately, which was decreased to 1.47 nM by co-incubation with curcumin. 3.3. Curcumin inhibits in vitro and in vivo activity of IL-2 We investigated the effect of curcumin on the proliferation of the IL-2dependent cell line, CTLL-2. We treated the CTLL-2 cells with IL-2 in the absence or presence of curcumin, and cell viability was measured using a WST detection kit after 48 h. Curcumin inhibited the CTLL-2 proliferative activity of IL-2 (10 ng) up to the 50% level (Fig. 3A). Additionally, it was demonstrated that curcumin can block the phosphorylation of IL-2-induced JAK-3 in the CTLL2 cell line (Fig. 3B). Next, we observed naive T cell differentiation through curcumin co-incubation to determine the effect of curcumin on Treg survival. The population of CD4þ CD25þFOXP3þ Treg was reduced by the addition of curcumin to the IL-2 samples in a dose-dependent manner (Fig. 3C). IL-2 immunotherapy is a therapeutic option for metastatic melanoma and renal cell carcinoma, but it has a side effect of toxicity of vascular leak syndrome (VLS) [16]. To test the in vivo neutralization of IL-2 activity by curcumin, we adopted an IL2induced VLS model. Briefly, mice were injected with IL-2 with or
without curcumin, and vascular permeability was measured by Evans blue extravasation into the lung. The lungs of the mice treated with IL-2 or TNF-a showed significant increases in Evans blue content compared with those of the vehicle control group (Fig. 3D). Our laboratory demonstrated that TNF-a also induced VLS [17]. Curcumin selectively neutralized the vascular toxicity of IL-2, but it was not able to inhibit the TNF-ainduced vascular leak. 4. Discussion Although many studies, clinical trials, and traditional oriental healing knowledge have indicated that curcumin or curcumincontaining herbal products have the potential to be used as anticancer agents, the immune-related direct target of curcumin has remained unknown. Our findings demonstrate that curcumin directly interacts and interferes with IL-2 because (1) curcumin impacted on the binding of IL-2 mAbs to recombinant IL-2 protein as well as soluble IL-2Ra to IL-2, (2) curcumin directly bound to IL-2 in pull-down and SPR assays, (3) curcumin inhibited the IL2mediated the proliferation and signaling pathway of the IL-2 dependent cell line, (4) the protective effect of IL-2 on Treg cell differentiation was inhibited by curcumin co-incubation, and (5) curcumin neutralized the side effect of VLS of IL-2 in vivo. Hence,
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J.-G. Oh et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
B Proliferation (%)
A
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IL-2
TNF-α TNF-α IL-2 + + curcumin curcumin
Fig. 3. Inhibition of IL-2-mediated activity by curcumin. (A) The outcome of curcumin binding to IL-2 was investigated by using an IL-2 bioassay. CTLL-2 cells were seeded in flatbottom 96-well plates. The cells were cultured in the absence of IL-2 for 4 h and added by media of T-STIM, IL-2 (10 ng), vehicle (DMSO), or mixture of IL-2 plus curcumin (5, 10, or 20 mM). Following 48 h of culture in a CO2 incubator, the cell viability assay was performed. The results were expressed as the mean ± SD and compared and analyzed with the IL-2 alone treated sample by Student's t-test. *0.01 < p < 0.05, **0.001 < p < 0.01, and ***p < 0.001 were considered significant. (B) Blockade of IL-2 signaling by curcumin in CTLL cells. CTLL cells were starved in the absence of IL-2 for 4 h and treated with IL-2 or IL-2 plus curcumin for 15 min, and then they were collected and lysed. Immunoblot analyses were performed on the lysates using anti-P-JAK3 antibody. (C) Inhibition of differentiation of naive T cells into regulatory T cells (Treg cells) by curcumin. Splenocytes isolated from C57BL/ 6 mice were enriched for CD4 positive population using magnetic beads. CD4 enriched cells were labeled by fluorescence conjugated antibody and sorted using FACS Aria into naive T cells. Naive T cells were seeded onto pre-coated plates with CD3 and CD28 mAb and then treated with curcumin at various concentrations (1e8 mM) in the presence of IL-2 (10 ng/ mL) and TGF-beta (20 ng/mL). After 72 h of culture, the cells were harvested and analyzed for Treg cell populations using a flow cytometer. (D) In vivo inhibition of IL-2-induced vascular leakage by curcumin in mice. C57BL/6 mice (n ¼ 5) were injected intraperitoneally with vehicle control, IL-2 (150,000 units), or IL-2 with curcumin (0.6 mg) for 4 days and a total of 10 times. TNF-a (5 mg) or TNF-a with curcumin (0.6 mg) was injected for 3 days a total of 5 times. After 2 h, Evans blue dye was injected intravenously, and the lungs were isolated and analyzed for vascular leaks with Evans blue extravasation (mean ± SEM). OD, optical density; NS, not significant.
the present study revealed a direct modulation of IL-2 by curcumin, which might explain the link between the beneficial effects of curcumin and immune-related disorders, including cancer. The initial purpose of this study was to discover a natural chemical competitor with S4B6 IL-2 mAb towards the binding site on IL-2. Since S4B6 mAb could bind and block the IL-2Rabinding epitope on IL-2, we hypothesized a natural compound mimicking S4B6 could also regulate Treg cells via blockade of the IL-2/IL-2Ra interaction. Indeed, we identified the ethanol extract of Zedoariae Rhizoma (Curcuma zedoaria), which showed significant inhibitory effects against S4B6 mAb binding to IL-2 (Fig. 1A and B). Since Zedoariae Rhizoma is a rich source of curcumin [18], and curcumin itself exhibits immunomodulatory effects [19], we selected curcumin and conducted the following experiments. In a similar fashion to Zedoariae Rhizoma, curcumin almost completely prohibited S4B6 mAb from binding to rmIL-2 (Fig. 1D). This inhibition was likely to be mediated by the direct binding of curcumin to rmIL-2 (Fig. 2A and B) and subsequent competition with an anti-rmIL-2 mAb. Curcumin can bind to recombinant mIL-2
with an affinity of approximately 2.18 nM. Curcumin also reduced the equilibrium constant (KD) between IL-2 and IL2Ra from 1.47 nM to 0.82 nM (Fig. 2C). Previously, small-molecule IL-2 inhibitors that bind close to the IL-2Ra contact region on IL-2 were designed and chemically synthesized based on the X-ray crystallographic studies of IL-2 [20]. However, several important medicinal chemistry studies did not provide biological activities or even clinical applications of these chemical IL-2 binders. Next, we examined the impact of binding and modulation of rmIL-2 by curcumin with the IL-2dependent CTLL cell line. Our data show dose-dependent inhibition of rmIL-2 activity of cell proliferation (Fig. 3A) and proximal events in rmIL-2 signaling (Fig. 3B) following curcumin co-incubation. These data reconfirm and account for the previous reports about the blockade of IL-2 signaling [14], inhibition of the JAK-STAT pathway in T cell leukemia [21], and inhibition of the IL-2 induced activation of human lymphocytes [13] by curcumin with our concept of direct binding of IL-2 and inhibition of its biological actions. The development, maintenance, survival and expansion of Treg
Please cite this article in press as: J.-G. Oh, et al., Direct regulation of IL-2 by curcumin, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.039
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cells are associated with signals mediated by IL-2/IL-2Ra binding [2]. Furthermore, IL-2 is essential for the survival and homeostasis of peripheral Treg cells [2]. We tested whether the binding and modulation of IL-2 by curcumin inhibited IL-2/IL-2R signaldependent Treg cells differentiation. Our data showed an inhibition of Treg survival by curcumin co-incubation in a dose-dependent manner (Fig. 3C). IL-2 immunotherapy has toxicities of vascular leak syndrome (VLS) [16], and curcumin selectively neutralized the vascular toxicity of IL-2 (Fig. 3D). A recent study suggested new evidence of the ill-defined mechanism of IL-2induced VLS that IL-2induced pulmonary edema is caused by direct interaction of IL-2 with IL2Rabg on lung endothelial cells in vivo [7]. Thus, the amelioration of IL-2induced VLS by curcumin (Fig. 3D) is possibly mediated by the direct binding of curcumin to IL-2, resulting in the blockade of IL-2IL-2R interaction. Taken together, these data indicate that curcumin and IL-2 are mutually antagonistic by direct interactions between them both in vitro and in vivo. Thus, curcumin could possibly have the potential to be developed as an attractive treatment option for IL-2driven pathological conditions, including cancer, autoimmune diseases, and inflammation. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1B03934803), the Bio & Medical Technology Development Program of the NRF funded by the Ministry of Science, ICT & Future Planning (NRF2016M3A9D9945476), a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI16C1761), and the Research Fund 2017 of the Catholic University of Korea. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2017.11.039. References [1] E.M. Shevach, Application of IL-2 therapy to target T regulatory cell function, Trends Immunol. 33 (2012) 626e632.
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Please cite this article in press as: J.-G. Oh, et al., Direct regulation of IL-2 by curcumin, Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/j.bbrc.2017.11.039