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Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor
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Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Inflammation and immunopharmacology
Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor Hadar Einia,* , Valeria Frishmana , Robert Yulzarib , Leonid Kachkoc, Eli C. Lewisa , Cidio Chaimovitzb , Amos Douvdevania,b a b c
Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel Department of Nephrology, Soroka Medical Center Beer-Sheva, Israel Department of Pathology, Soroka Medical Center Beer-Sheva, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 May 2015 Accepted 13 August 2015 Available online xxx
Epidemiologic studies depict a negative correlation between caffeine consumption and incidence of tumors in humans. The main pharmacological effects of caffeine are mediated by antagonism of the adenosine receptor, A2AR. Here, we examine whether the targeting of A2AR by caffeine plays a role in antitumor immunity. In particular, the effects of caffeine are studied in wild-type and A2AR knockout (A2AR/ ) mice. Tumor induction was achieved using the carcinogen 3-methylcholanthrene (3-MCA). Alternatively, tumor cells, comprised of 3-MCA–induced transformed cells or B16 melanoma cells, were inoculated into animal footpads. Cytokine release was determined in a mixed lymphocyte tumor reaction (MLTR). According to our findings, caffeine-consuming mice (0.1% in water) developed tumors at a lower rate compared to water-consuming mice (14% vs. 53%, respectively, p = 0.0286, n = 15/group). Within the caffeine-consuming mice, tumor-free mice displayed signs of autoimmune alopecia and pronounced leukocyte recruitment intocarcinogen injection sites. Similarly, A2AR/ mice exhibited reduced rates of 3-MCA-induced tumors. In tumor inoculation studies, caffeine treatment resulted in inhibition of tumor growth and elevation in proinflammatory cytokine release over water-consuming mice, as depicted by MLTR. Addition of the adenosine receptor agonist, NECA, to MLTR resulted in a sharp decrease in IFNg levels; this was reversed by the highly selective A2AR antagonist, ZM241385. Thus, immune response modulation through either caffeine or genetic deletion of A2AR leads to a Th1 immune profile and suppression of carcinogen-induced tumorigenesis. Taken together, our data suggest that the use of pharmacologic A2AR antagonists may hold therapeutic potential in diminishing the rate of cancer development. ã 2015 Elsevier Inc. All rights reserved.
Keywords: Adenosine A2AR Caffeine 3-MCA Anti-tumor immunity
1. Introduction Prospective epidemiologic studies regarding the effects of caffeinated coffee on the incidence of cancer showed a negative correlation between coffee consumption and incidence of tumors in humans. In particular, meta-analysis studies comprised of >800,000 subjects revealed that caffeine consumption is associated with significantly decreased risk for cutaneous malignant melanoma [1], liver cancer [2,3] and pancreatic cancer [4]. Coffee is
* Corresponding author at: Department of Clinical Biochemistry and Pharmacology, Soroka University Medical Center and Ben-Gurion University of the Negev, P.O. Box 151, Beer-Sheva 84101, Israel. Fax: +972 8 640321. E-mail addresses: [email protected] (H. Eini), fi[email protected] (V. Frishman), [email protected] (R. Yulzari), [email protected] (L. Kachko), [email protected] (E.C. Lewis), [email protected] (C. Chaimovitz), [email protected] (A. Douvdevani).
a leading source of methylxanthines, such as caffeine (1,3,7trimethylxanthine), and while the immunomodulatory properties of many of the methylxanthines have been widely investigated, very few studies have directly examined the effects of caffeine on anti-tumor immune cell functions [5]. Out of the four subtypes of adenosine receptors, A2A receptor (A2AR) is the main pharmacological target of caffeine; the A2AR is blocked by caffeine with a KD of close to 2.4 mM and 8.1 mM in humans and rats, respectively [6]. Indeed, caffeine antagonizes adenosine receptors at moderate to heavy levels of coffee consumption [7]. Adenosine, the ligand of A2AR, is an endogenous purine nucleoside which is released from almost all cell types. Over the past few years a vast number of investigations have reported of its involvement in inflammatory processes [8]. Adenosine is constitutively present in extracellular spaces at low concentrations, but its extracellular levels dramatically increase in metabolically stressful conditions [9]. For example, adenosine accumulates
http://dx.doi.org/10.1016/j.bcp.2015.08.092 0006-2952/ ã 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: H. Eini, et al., Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.08.092
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in the hypoxic microenvironment of solid tumors [10], where its extracellular concentration is 10- to 20-fold higher than that found in adjacent normal tissue [11]. Following both its release from cells or after being derived extracellularly from related molecules, interstitial adenosine diffuses to the cell membranes of surrounding cells where it can bind to four specific G-coupled receptors, including A2AR [12]. Anti-tumor immune responses are coordinated by both innate immunity and adaptive immunity, and are mainly mediated by cytotoxic CD8+ T cells (CTLs), natural killer (NK) cells and natural killer T (NKT) cells. In addition, dendritic cells (DCs) are important for the generation and maintenance of anti-tumor immune responses as they are able to regulate adaptive immunity and induce a CTLs response [13]. It was shown that the anti-tumor activity of these cells is strongly affected by adenosine; A2AR activation during T-cell stimulation significantly inhibits cytotoxicity and cytokine production [14] and has been reported to inhibit T-cell proliferation [15,16]. Similarly, using an A2AR agonist, CGS21680, Csoka et al. showed that A2AR activation suppresses the development of Th1 immune responses in vitro and in vivo [17]. In addition, T-cells that are activated in the presence of CGS21680 fail to proliferate, and then fail to produce IFNg upon reactivation, essentially reaching T-cell anergy. Importantly, these cells maintain a suppressed phenotype after removal of the A2AR agonist [14,18]. In contrast, in vivo activation of A2AR was shown to drive CD4+ T-cell differentiation towards Tregs, probably due to an increase in TGFb and a decrease in IL-6 levels following A2AR activation [18]. In addition, A2AR has been shown to suppress NK cell functions, including IFNg production [19,20]. NKT cells that are deficient in A2AR were found to produce significantly lower levels of IL-4, IL-10 and TGFb, but appear to maintain IFNg production upon stimulation with a-galactosylceramide [21]. The positive effect of caffeine and the role of A2AR in cancer immunity were previously demonstrated in mice inoculated with tumor cells. Ohata et al. have shown that 60% of A2AR/ mice completely rejected inoculated tumor cells and had survived, whereas WT mice displayed no tumor rejection and did not survive; importantly, Ohata et al. also showed that caffeine, as well as a specific A2AR antagonist, ZM241385, enhanced inoculated tumor cell destruction by tumor antigen-specific CD8+ T cells [10]. Moreover, blockade of A2AR with its antagonists, SCH58261, and by caffeine, augmented the efficacy of adoptive T-cell therapy [22]. In a recent study, Beavis et al. found that CD73, which catalyzes the conversion of extracellular nucleosides to adenosine, promoted metastasis through the activation of both A2A and A2B adenosine receptors. A2AR/ mice were strongly protected against tumor metastasis whilst the blockade of A2AR with SCH58261 resulted in enhanced NKcell activity in vitro and in vivo and reduced metastasis in a perforindependent manner [23]. The combination of A2A-adenosinergic pathway inhibitors and blockade of negative immunologic regulators represents a new approach to cancer treatment. It was recently reported that combined therapy with the A2AR antagonist SCH58261 and PD-1 monoclonal antibody (mAb) significantly inhibited the development of B16 melanoma lung metastases and prolonged the life of 4T1.2 tumor-bearing mice compared with either monotherapy alone. Interestingly, the combination was only effective when the tumor expressed high levels of CD73 [24]. Consistent with these findings, Allard et al. showed that anti-CD73 mAb significantly enhanced the activity of both anti-CTLA-4 and anti-PD-1 mAbs against MC38-OVA and RM-1 subcutaneous tumors, and established metastatic 4T1.2 breast cancer. Furthermore, activation of A2AR was found to enhance PD-1 expression on tumor-specific CD8 + T cells and CD4+ Tregs [25]. Since evidence show that the A2AR pathway suppresses antitumor immunity, we hypothesize that part of the effects of caffeine
on reducing tumor incidence may be mediated by its blockade of A2AR. In contrast to previous studies that focused on models of tumor cell line injection, we aimed to investigate the effect of caffeine during the early stages of tumor initiation and progression in a carcinogen-induced tumor model. The role of A2AR in subversion of the immune response is directly examined as a possible mechanism for tumor evasion from immune-surveillance. 2. Materials and methods 2.1. Mice ICR and C57BL/6J wild type mice were purchased from Harlan, Jerusalem, Israel; A2AR/ mice (ICR background) were kindly donated by Catherine Ledent (UniversitéLibre de Bruxelles) [26]. Mice were housed in the animal facility at the Soroka Medical Center (Beer-Sheva, Israel). Heterozygote A2AR+/ mice were generated by crossbreeding with wild type ICR mice. Six- to eight-week—old ICR female mice were used in the carcinogeninduced tumor model. In the tumor cell line inoculation models, Six- to eight week-old C57BL/6J and ICR female mice were used. Animal use conformed to the guidelines established by Institutional Animal Care and Use Committee. 2.2. Adenosine and adenosine analogues Adenosine was purchased from Sanofi Winthrop (Adenocor, Auckland, NZ). Adenosine analog 50 -(N-Ethylcarboxamido) adenosine (NECA) was purchased from Sigma (Rehovot, Israel). A2AR/ A2BR antagonist 4-(2-([7-Amino-2-(2-furyl)[1,2,4]triazin-5-ylamino) ethyl]) phenol) (ZM241385) was purchased from Tocris Cookson (Ellisville, MS, USA), and Caffeine was purchased from Sigma (cat# C0750, purity 99%). 2.3. Carcinogen-induced tumor model Mice were injected intramuscularly into the left hip with 200 mg of the carcinogen 3-methylcholanthrene (3-MCA; cat# 213942, Sigma) or vehicle (olive oil) (total volume 200 ml) [27]. Local fibrosarcomas developed within 3–5 months. Mice were inspected twice a week for tumor development by caliper measurement. When tumors reached 1.8 cm diameter, mice were sacrificed and cell lines were generated from the removed tumor cells. For histological examination, samples were obtained 95 days post-carcinogen injection. 2.4. Caffeine treatment protocol Mice were injected with the carcinogen 3-MCA or with tumor cell lines, and were then randomly divided into two groups; one group was allowed to drink ad-libitum water containing caffeine (0.1% wt/vol) and the other group water with no added caffeine. 2.5. Generation of MCA-induced fibrosarcoma cell lines Tumor tissue was aseptically removed and processed for the establishment of cell lines, as previously described [27], with minor modifications. Disaggregation was performed by continuous stirring of the minced tissue for 1 h at 37 C in the presence of 0.2% collagenase B (Boehringer Mannheim, F.R.G) in DMEM medium. The disaggregated tissue was separated from tissue debris by 40 mm BD Falcon cell strainers and was then subjected to enzymatic digestion by trypsin (10 min at 37 C). DMEM medium and trypsin were purchased from Biological Industries (Beit Haemek, Israel). One of the established cell lines (designated MCA-12.12) was used in the current study.
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2.6. Histology Samples from the site of 3-MCA injections were fixed in 10% formalin and embedded in paraffin. Four-micron sections were stained with H&E. Histological preparations were analyzed for tumor cells and leukocyte infiltration by two histopathologists in a blinded and random manner. 2.7. Tumor cell line maintenance B16-F10 melanoma cells (purchased from the ATCC) and the established MCA-12.12 cell line were grown in complete DMED medium containing 10% heat-inactivated fetal calf serum, 2 mMLglutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. All supplements were purchased from Biological Industries. Experiments were performed using cells from the second to fourth passages. The cultures were incubated in 5% CO2 at 37 C, and maintained in exponential state by sub-culturing periodically. Both cell lines were routinely tested for the presence of mycoplasma and for their ability to generate characteristic tumors in recipient mice. The appearance of a black melanin B16-F10 cell pellet was validated for each passage. 2.8. Tumor cell line injection model Using 1 ml insulin syringes fitted with 25-gauge needles (BD, Franklin, NJ, USA), 5 104 B16 melanoma cells in a volume of 0.05 ml were injected into footpads of C57BL/6 mice. ICR mice were injected using the same method with MCA-12.12 cell line (0.25 106 cells/mouse). Tumor volume was measured three times a week with a caliper and calculated using the following formula: width2 length 0.52. The volume of footpad without tumor cells was subtracted from all measurements. 2.9. Quantitative real-time PCR Total RNA was extracted from tumor tissue using the Versagene RNA cell kit (Gentra systems, Minneapolis, MN). cDNA was prepared from 1 mg of total RNA as template using 5-prime Masterscript kit (Hilden, Germany). Quantitative real-time PCR (qPCR) assays were carried out with Thermo-Start master mix (ABgene, Surrey, UK) in Rotor-Gene Real-Time PCR apparatus (Corbett-Research, Northlake, Australia). Quantification of transcripts were performed using the following mouse-specific primers pairs: IL-15 sense:50 -TGC AAT GAA CTG CTT TCT CCT GGA-30 ,IL-15 anti-sense:50 -CCT CAC ATT CCT TGC AGC CAG ATT-30 ; IL-15Ra sense:50 -GCC AGC GCC ACC CTC CAC AGT AA-30 ,IL-15Ra anti-sense:50 -GCC AGC GGG GGA GTT TGC CTT GAC-30 ; IFNg sense:50 -CAT CAG CAA CAA CAT AAG CGT CA-30 ,IFNg anti-sense:50 CTC CTT TTC CGC TTC CTG A-30 ; CD3 sense:50 -GCC TCA GAA GCA TGA TAA GC-30 ,CD3 anti-sense:50 -CCC AGA GTG ATA CAG ATG TC-30 ; foxp3 sense: 50 -CCC ACC TAC AGG CCC TTC TC-30 , foxp3 anti-sense: 50 -GGC ATG GGC ATC CAC AGT-30 ; IL-17 sense: 50 -TTC ATC TGT GTC TCT GAT GCT-30 ,IL-17 anti-sense:5'-CAC AGA GGG ATA TCT ATC AAG GG-30 ; GAPDH sense: 50 -CAA TGC ATC CTG CAC CAC CAA-30 ,GAPDH anti-sense:50 -GTC ATT GAG AGC AAT GCC AGC-30 .
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cells. Cultures were incubated at 37 C in 5% CO2 in complete RPMI medium containing 10% heat-inactivated fetal calf serum, 2 mMLglutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (Biological Industries), and 50 mM b-mercaptoethanol (Sigma). After a48-h incubation, IFNg protein levels in culture supernatants were determined by ELISA (R&D Systems, Minneapolis, MN, USA). Protein levels of IL-6, MCP-1, TNF-a, IL-10, IL-17 and IL-15 were measured by Quansys multiplex assays according to the Quansys Bioscience protocol (Logan, UT, USA). MLTR was repeated in the presence of adenosine analog NECA with the exception that isolated splenocytes were incubated with the antagonist ZM241385 (1 mM) 1-h before B16 target cells and NECA (3 mM) were added to the culture. 2.11. Statistical analysis Results are expressed as mean S.E. Statistical analysis was performed by t-test and Kaplan–Meier Test. P values of <0.05 were considered significant. 3. Results 3.1. The effect of caffeine or A2AR deletion on the progression of carcinogen-induced tumors To study the effect of caffeine on tumor development, mice (ICR) were injected with 3-MCA carcinogen or vehicle, and were then allowed to drink ad-libitum water with or without 0.1% caffeine (n = 15 in each group). As shown in Fig. 1A, 250 days following inoculation of the carcinogen, the caffeine-treated group displayed a significantly lower incidence rate of carcinogeninduced tumors compared to the water-drinking group (P = 0.0286, 14% vs. 53% tumor growth rate, respectively). After 250 days, caffeine treatment was replaced with water in all groups. As shown, no additional tumors were observed in the caffeine-treated group. Within the group of caffeine-treated 3-MCA-injected mice, a subset of mice that did not develop tumors was found to display signs of autoimmune lesions. Specifically, alopecia was detected in the head and neck regions and in the site of injection on day
2.10. MLTR assays Single-cell suspensions of splenocytes from C57BL/6 mice that underwent the course of B16 cells inoculation were prepared as previously described [28], and mixed with B16 target cells in vitro. MLTR assay was conducted using splenocytes isolated from caffeine- and water-administered mice 34 days after B16 cell injection. Splenocytes (2 105) were co-cultured in round-bottom 96-microwell plates (Corning, NY, USA) with 0.6105 B16 target
Fig. 1. Caffeine treatment significantly decreases the rate of tumor-bearing mice in a carcinogen model. Wild-type ICR mice were administered either water or 0.1% caffeine in drinking water, and were injected 3-MCA or vehicle into the hip tissue (i. m. 200 mg/mouse) (n = 15 per group). (A) Tumor follow-up. Replacement of caffeine with water is indicated. P = 0.0286, Kaplan–Meier Test. (B) Signs of spontaneously resolved autoimmunity. Representative pictures of (i) alopecia on day 95. Arrow, alopecia. (ii) skin lesion on day 250. Arrows indicate lesion.
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95 post-3-MCA administration. Subsequently, complete body hairloss and vast skin lesions were observed on day 250 and lasted throughout the experiment (Fig. 1B). Importantly, none of these symptoms were observed in mice that consumed caffeine but were not injected with the carcinogen (data not shown). As shown in Fig. 2, a blinded histological examination of tissue biopsies that were obtained from the site of 3-MCA injection, revealed extensive tumor-mediated muscle injury in most of the samples of waterconsuming mice. In contrast, most of the samples obtained from caffeine-treated mice showed no signs of muscle injury and only a few of them showed perivascular tumor cell clusters. Tumors in both groups were characterized by a leukocytic infiltrate that was comprised predominantly of lymphocytes, as well as scattered neutrophils and macrophages. Importantly, lymphocytes were more abundant in samples of caffeine-treated mice than those of water-consuming mice. To specifically depict the role of A2AR in cancer initiation and progression, we compared tumor development in A2AR/ mice, heterozygote mice (A2AR+/) and WT mice (n = 20), all on ICR background. As shown in Fig. 3, tumors were first detected in the WT group on day 83, and then later in the A2AR+/ and A2AR/ groups (day 110 and 127, respectively). The delay in time between tumor detection in A2AR/ mice and WT mice is statistically significant (P = 0.0351). 3.2. The effect of caffeine on tumor progression and immune profile in cell-injection models Since carcinogenesis studies involve extremely prolonged time lines, we generated thirteen cell lines from solid tumors induced in WT ICR mice by 3-MCA. Following in vivo screening, we selected the MCA12.12 line by its high growth rate following inoculation (not shown). To examine the effect of caffeine on tumor cell growth and immune responses, we inoculated MCA12.12 cells into footpads of WT mice. As shown in Fig. 4A, by day-17 caffeine-treated mice displayed lower median tumor volume compared to control mice. Since several mice from both groups rejected the tumor at various
Fig. 3. Reduced tumor incidence in A2AR/ mice following 3-MCA administration. WT (n = 20), heterozygote (A2AR+/,n = 20), and knockout (A2AR/,n = 18) mice were injected 3-MCA into the hip tissue (i.m. 200 mg/mouse). Tumor detection was followed weekly. P = 0.0351 between WT and KO, Kaplan–Meier Test.
time points, comparison of tumor volumes between the two groups did not reach statistical significance. At this time point, tumors were harvested and gene expression profiles were examined by qPCR. As shown in Fig. 4B and C, the mean mRNA levels of CD3, IL-15, IL-15Ra and IL-17 in tumors that were explanted from caffeine-treated mice were higher in compared to those of the water-drinking group. However, differences between the two groups of mice did not reach statistical significance. Consistent with the caffeine-treated group being skewed towards an enhanced immune response, IFNg mRNA levels were markedly elevated in caffeine-treated mice (P < 0.05) (Fig. 4C). Since in vivo screenings with MCA12.12 cells led to highvariance outcomes, most probably due to excessive immunogenicity of the cells, we turned to examine the highly metastatic B16 melanoma cell line. As shown in Fig. 5, caffeine-treated mice were remarkably resistant to B16 tumor growth; while 7 out of 8 mice in the control group presented tumors, only 1 out of 8 mice in the caffeine group developed a tumor that was detectable on day 34.
Fig. 2. Caffeine treatment is associated with leukocytic infiltration at the site of 3-MCA injection. Wild-type mice were administered either water or 0.1% caffeine in drinking water, and were injected 3-MCA or vehicle into the hip tissue (i.m. 200 mg/mouse) (n = 5 per group). On day-95, sections of biopsies from the site of injection were stained with haematoxylin and eosin (H&E) and analyzed for tumor cells and leukocyte infiltration. Representative histology images are shown. Typical areas are displayed at 40 and 10 magnification. M, muscle; T, tumor; L, lymphocytes. Arrow, blood vessel.
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Fig. 4. Effect of caffeine after 3-MCA—induced transformed cells line injection. Wild-type mice were administered either water (n = 9) or 0.1% caffeine (n = 8) in drinking water, and were injected into the footpads with MCA12.12 cells (1 106 cells/mouse). Tumor volume was measured three times a week. (A) follow-up, expressed as median tumor volume. On day-17, tumors were harvested and qPCR was performed with primers specific for (B) T-cell markers and (C) cytokine-related genes. Results are normalized to GAPDH transcript levels. Mean S.E. *P < 0.05 from WT.
In order to examine whether the observed beneficial effect of caffeine had involved a heightened anti-tumor immune response, we established a mixed lymphocyte tumor reaction (MLTR) assay. Isolated splenocytes from caffeine- and water-administered B16challenged mice were re-challenged with fresh B16 target cells in vitro (n = 5 for each group). Induction of IFNg production was then determined. As shown in Fig. 6A, MLTR resulted in a sharp elevation of IFNg. However, MLTR that incorporated splenocytes from B16-challenged caffeine-treated mice produced 2-fold greater levels of IFNg than the MLTR that was comprised of splenocytes from B16-challenged water-consuming mice. IFNg was below detection in supernatants of B16 cells and splenocytes that were incubated separately (data not shown). In order to examine the sensitivity of the reacting cells in MLTR to adenosine, the assay was repeated in the presence of a stable adenosine agonist, NECA. As shown in Fig. 6B, the addition of NECA to the culture medium almost completely abolished the production of
IFNg by splenocytes from both water- and caffeine-consuming mice. Of note, splenocytes obtained from caffeine-treated mice were not exposed to caffeine in culture; it is therefore presumed that they expressed intact functional A2A receptors. Pretreatment of splenocytes from both groups of mice with the A2AR/A2BR antagonist, ZM241385, reversed NECA-induced IFNg down-regulation. In contrast to water-treated samples, IFNg down-regulation was only partly reversed in caffeine-treated samples following ZM241385 addition. In addition to IFNg, the production of several other cytokines was tested following incubation of splenocytes from B16-challenged mice with target cells in vitro. As shown in Fig. 6C, mixed cultures of splenocytes from caffeine-treated mice produced elevated levels of IL-6, MCP-1, TNFa and IL-10 compared to mixed cultures of splenocytes from water-treated mice (n = 5 for each group). Splenocytes isolated from control mice exhibited lower cytokine levels compared to splenocytes from both caffeine- and water-
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Fig. 5. Effect of caffeine on B16 tumor volume. Wild-type mice were administered either water or 0.1% caffeine in drinking water (n = 8 per group), and were injected into the footpads with B16 cells (5 104 cells/mouse). Tumor volume was measured three times a week. Left, follow-up. Right, representative images of tumors on day 34. Arrows, macroscopically identifiable tumors. Figure shows mean S.E (n = 8 in each group) of a representative experiment out of three independent experiments with similar results. **P < 0.01 and ***P < 0.01 between water and caffeine groups.
consuming mice. No significant difference in IL-17 levels was observed. IL-15 was undetectable in supernatants. 3. Discussion Several prospective epidemiological studies report a negative correlation between caffeine consumption and risk of various cancers in humans [2,3]. In order to experimentally represent the etiology of tumor initiation, 3-MCA, a complete carcinogen that mediates both initiation and promotion of tumor development, was used in the present study [29]. Our results demonstrate that tumor incidence in 3-MCA-injected mice is dramatically reduced during caffeine treatment. Based on three separate experimental observations, it is suggested that the primary effect of caffeine responsible for the marked reduction of cancer rates in this model, is the enhancment of anti-tumor immunity. For example, leukocyte infiltrates, composed primarily of lymphocytes, were more abundant in samples of caffeine-treated mice than those found in water-consuming mice. In addition, signs of spontaneously resolved autoimmunity, such as skin lesions and alopecia, were observed in the entire subset of caffeine-treated mice that were injected with the carcinogen and did not develop tumors, indicating that treatment with caffeine most probably leads to an enhanced immune response against epitopes shared by the tumor and normal tissues. Spontaneous appearance of alopecia with skin lesions was previously described in one of the major autoimmune lupus-prone mouse models, the MRL/lpr mouse [30,31]. In addition, these observations are similar to reports of autoimmunity in melanoma patients who underwent immunotherapy with melanoma antigen-specific T cells [32], as well as in CL-81 melanoma-rejecting A2AR/ mice [10]. Lastly, further evidence in support of immune-related benefits for caffeine in the present study can be found at the point where the system was challenged by removal of caffeine; no additional tumors were observed in the caffeine group, further supporting the impression that effective immunity prevented tumor development. We suggest that the mechanism behind the positive shift in immunity relates to the antagonism of adenosine receptors by caffeine. The well-described protocol of low levels 0.1% caffeine in drinking water was chosen in the present study in order to promote preferential antagonism of A2AR [10], the adenosine receptor that was shown to be involved in suppressing key stages of the inflammatory process, including leukocyte recruitment, phagocytosis, cytokine production and immune cell proliferation [33]. Similar to caffeine-treated mice, 3-MCA-challenged A2AR/ mice displayed significantly fewer tumors as well as delayed tumor
development compared to their WT littermates. Comparison between the genetically deficient animal and caffeine treatment, however, did not fully overlap: 60% of A2AR/ mice presented with tumors, whereas 14% of caffeine-treated mice presented with tumors (day 150 and 250, respectively). Since differences in tumor onset were observed between the control groups of these experiments (both composed of water-treated WT mice), this discrepancy might be attributed to a stronger effect of the carcinogen in the genetically-targeted mice, or to A2AR-independent anti-tumor effects of caffeine. The latter could also explain the lack of long-term differences in tumor development between A2AR/ and WT mice. Indeed, it was recently shown that A2BR blockade reverses the immunosuppressive effects of adenosine and inhibits tumor progression [34]. Caffeine blocks A2BR, albeit with a lower affinity than A2AR. Thus, it is possible that in the present study, caffeine partially blocked A2BR in treated animals. Of note, we cannot exclude that in addition to the immunomodulatory effects of caffeine presented here, inhibition of replication checkpoint function by caffeine, which was shown in recent studies [35,36], may have also contributed to the suppression of tumor in the carcinogenesis model. Caffeine is a non-selective phosphodiesterase (PDE) inhibitor. It acts by competing with adenine for access to the catalytic site of PDEs and prevents PDEs from inactivating cAMP or cGMP [37]. High intracellular level of cAMP and cGMP could arrest growth, induce apoptosis and attenuate cancer cell migration [38,39]. Caffeine is also known as a stimulator of the ryanodine receptor (RyRs) Ca2+ release-channels that have been previously implicated in the proliferation of human T lymphocytes and melanocytes. Interestingly, RyR was recently suggested to serve as prognostic indicator and as a target for breast cancer treatment [40]. Another possible mechanism for the reduction of cancer incidence in an experimental model of carcinogenesis is a direct effect of the therapy agent on mutagenesis. Literature exhibits an inconclusive array of evidence regarding the effect of caffeine on mutagenesis; it was previously demonstrated that caffeine can act as a comutagenic, nonmutagenic, and/or antimutagenic, depending upon test system and protocol [41]. We have no conclusive data that support the direct reduction of mutagenesis by caffeine treatment. Taking into consideration the long latency period associated with the development of cancer after 3-MCA exposure, we chose to further assess the effect of caffeine on anti-tumor response in a cancer cell inoculation models. Similar to its effects on tumor progression in the 3-MCA model, we observed that caffeine treatment is able to improve tumor inhibition and modulate
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Fig. 6. Adenosine receptors agonist, NECA, blocks cytokines production by splenocytes from B16-challenged mice in an A2AR-dependent manner. Wild-type mice were administered either water or 0.1% caffeine in drinking water (n = 5 per group), and were introduced with B16 cells (5 104 cells/mouse) into the footpads. On day-34, splenocytes were isolated from mice and were then (A, C) co-incubated in triplicate with B16 target cells (control, splenocytes from unchallenged mice) or (B) preincubated with ZM241385 (1 mM) and mixed with B16 target cells in the presence of NECA (3 mM). Control, mixed cultures in the absence of NECA or ZM241385. (A, B) IFNg in supernatants after 48 h of incubation were measured by ELISA. Figure shows mean S.E (n = 5 in each group) of a representative experiment out of three independent experiments with similar results. ***P < 0.001 from water. **P < 0.01 and ***P < 0.001 from control. ^^P < 0.01 and ^^^P < 0.001 from NECA. (C) Cytokine levels in supernatants after 48 h of incubation were measured by Quansys multiplex assay. Mean S.E (n = 5 in each group). *P < 0.05, **P < 0.01 between water and caffeine groups.
immune responses during the progression of cancer cell growth. Here, caffeine-treated mice injected with MCA12.12 cells exhibited lower tumor volumes in comparison to water-consuming mice. Differences in MCA12.12 rejection by caffeine- and water-treated mice were also reflected in the extent of intratumoral IFNg mRNA expression levels. When the well established C57BL/6-derived B16 melanoma model was used, treatment with caffeine induced nearly complete rejection of tumors. This remarkable augmentation of tumor rejection was unexpected, since the B16 melanoma model is known to produce poorly immunogenic tumors that are notoriously aggressive and difficult to treat. With remarkable relevance, our findings support two large epidemiological studies that report an inverse correlation between coffee consumption and decreased risk of cutaneous malignant melanoma in women [1,42]. In
addition, it was previously reported that both intraperitoneal and oral administration of caffeine (100 and 50 mg/kg body weight, respectively) for 10 consecutive days reduces tumor volume in B16 melanoma-bearing mice. Accordingly, mice injected with B16 melanoma cells through lateral tail vein and exposed to the above indicated caffeine treatment, showed a reduction in the number of lung tumor nodules [43]. Here, we observed that the enhanced inhibition of B16 tumor growth following caffeine treatment is associated with increased production of IFNg by splenocytes from caffeine-treated mice that were re-challenged with B16 target cells in the MLTR assay. In order to test the sensitivity of anti-tumor immune cells to adenosine, the MLTR assay was repeated in the presence of the stable adenosine agonist, NECA. Our results show that the addition of NECA to the culture medium almost completely abolished the production of IFNg by
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splenocytes from both water- and caffeine-consuming mice. Furthermore, pretreatment of splenocytes with the A2AR/A2BR antagonist, ZM241385, reversed NECA-induced IFNg down regulation in both groups, suggesting an involvement of A2AR/A2BR in mediating IFNg down-regulation. Similar to the effect of caffeine on IFNg expression, mixed cultures of splenocytes from caffeine-treated mice produced elevated levels of IL-6, MCP-1, TNFa and IL-10 compared to mixed cultures of splenocytes from water-treated mice. As expected, IL15, which performs most of its biological functions in a membraneassociated form, was undetectable in supernatants. Importantly, MLTR resulted in a sharp elevation of IFNg from the pg/ml range to the ng/ml range whereas levels of the other tested cytokines were increased <2-fold. This observation implies that a role for immune cell secretion of IFNg in the B16 melanoma model is of significance, holding great importance since IFNg is known to possess antiproliferative, as well as anti-angiogenic and immune-inducing activities and its neutralization in many tumor models is followed by enhanced aggressiveness of tumors [44–46]. Taken together, we show here for the first time that caffeine treatment promotes an effective anti-tumor immune response during tumor initiation, partly through the antagonism of A2AR. We demonstrate that caffeine treatment leads to increased IFNg production and to a heightened tumor-specific memory T cell response. In addition, the results suggest a role for A2AR activation in preventing autoimmunity. Thus, immune modulation through targeting of the A2AR pathway holds a promising potential for cancer therapy, and fine-tuning of this pathway may allow the adjusting of local inflammatory responses. These outcomes support important recent epidemiological studies that identify a correlation between caffeinated-coffee consumption and low incidence of tumors in humans. Conflict of interest The authors have no conflict of interest associated with this study to declare. Acknowledgments We would like to thank Dr. Catherine Ledent from Université Libre de Bruxelles for sharing with us the A2AR/ mice. This work was supported by the Dr. Montague Robin Fleisher Kidney Transplant Unit Fund. References [1] M.B. Veierod, D.S. Thelle, P. Laake, Diet and risk of cutaneous malignant melanoma: a prospective study of 50,757 Norwegian men and women, Int. J. Cancer 71 (1997) 600–604. [2] I.S. Cadden, N. Partovi, E.M. Yoshida, Review article: possible beneficial effects of coffee on liver disease and function, Aliment. Pharmacol. Ther. 26 (1) (2007) 1–8. [3] T. Imatoh, S. Tanihara, M. Miyazaki, Y. Momose, Y. Uryu, H. Une, Coffee consumption but not green tea consumption is associated with adiponectin levels in Japanese males, Eur. J. Nutr. 50 (2011) 279–284. [4] J. Dong, J. Zou, X.F. Yu, Coffee drinking and pancreatic risk: a meta-analysis of cohort studies, World J. Gastroenterol. 17 (2011) 1204–1210. [5] L.A. Horrigan, J.P. Kelly, T.J. Connor, Caffeine suppresses TNF-alpha production via activation of the cyclic AMP/protein kinase A pathway, Int. Immunopharmacol. 4 (2004) 1409–1417. [6] B.B. Fredholm, K. Battig, J. Holmen, A. Nehlig, E.E. Zvartau, Actions of caffeine in the brain with special reference to factors that contribute to its widespread use, Pharmacol. Rev. 51 (1999) 83–133. [7] M.V. Sitkovsky, J. Kjaergaard, D. Lukashev, A. Ohta, Hypoxia-adenosinergic immunosuppression: tumor protection by T regulatory cells and cancerous tissue hypoxia, Clin. Cancer Res. 14 (2008) 5947–5952. [8] G. Hasko, P. Pacher, E.A. Deitch, E.S. Vizi, Shaping of monocyte and macrophage function by adenosine receptors, Pharmacol. Ther. 113 (2007) 264–275.
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Biochemical Pharmacology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Inflammation and immunopharmacology
Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor Hadar Einia,* , Valeria Frishmana , Robert Yulzarib , Leonid Kachkoc, Eli C. Lewisa , Cidio Chaimovitzb , Amos Douvdevania,b a b c
Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel Department of Nephrology, Soroka Medical Center Beer-Sheva, Israel Department of Pathology, Soroka Medical Center Beer-Sheva, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 May 2015 Accepted 13 August 2015 Available online xxx
Epidemiologic studies depict a negative correlation between caffeine consumption and incidence of tumors in humans. The main pharmacological effects of caffeine are mediated by antagonism of the adenosine receptor, A2AR. Here, we examine whether the targeting of A2AR by caffeine plays a role in antitumor immunity. In particular, the effects of caffeine are studied in wild-type and A2AR knockout (A2AR/ ) mice. Tumor induction was achieved using the carcinogen 3-methylcholanthrene (3-MCA). Alternatively, tumor cells, comprised of 3-MCA–induced transformed cells or B16 melanoma cells, were inoculated into animal footpads. Cytokine release was determined in a mixed lymphocyte tumor reaction (MLTR). According to our findings, caffeine-consuming mice (0.1% in water) developed tumors at a lower rate compared to water-consuming mice (14% vs. 53%, respectively, p = 0.0286, n = 15/group). Within the caffeine-consuming mice, tumor-free mice displayed signs of autoimmune alopecia and pronounced leukocyte recruitment intocarcinogen injection sites. Similarly, A2AR/ mice exhibited reduced rates of 3-MCA-induced tumors. In tumor inoculation studies, caffeine treatment resulted in inhibition of tumor growth and elevation in proinflammatory cytokine release over water-consuming mice, as depicted by MLTR. Addition of the adenosine receptor agonist, NECA, to MLTR resulted in a sharp decrease in IFNg levels; this was reversed by the highly selective A2AR antagonist, ZM241385. Thus, immune response modulation through either caffeine or genetic deletion of A2AR leads to a Th1 immune profile and suppression of carcinogen-induced tumorigenesis. Taken together, our data suggest that the use of pharmacologic A2AR antagonists may hold therapeutic potential in diminishing the rate of cancer development. ã 2015 Elsevier Inc. All rights reserved.
Keywords: Adenosine A2AR Caffeine 3-MCA Anti-tumor immunity
1. Introduction Prospective epidemiologic studies regarding the effects of caffeinated coffee on the incidence of cancer showed a negative correlation between coffee consumption and incidence of tumors in humans. In particular, meta-analysis studies comprised of >800,000 subjects revealed that caffeine consumption is associated with significantly decreased risk for cutaneous malignant melanoma [1], liver cancer [2,3] and pancreatic cancer [4]. Coffee is
* Corresponding author at: Department of Clinical Biochemistry and Pharmacology, Soroka University Medical Center and Ben-Gurion University of the Negev, P.O. Box 151, Beer-Sheva 84101, Israel. Fax: +972 8 640321. E-mail addresses: [email protected] (H. Eini), fi[email protected] (V. Frishman), [email protected] (R. Yulzari), [email protected] (L. Kachko), [email protected] (E.C. Lewis), [email protected] (C. Chaimovitz), [email protected] (A. Douvdevani).
a leading source of methylxanthines, such as caffeine (1,3,7trimethylxanthine), and while the immunomodulatory properties of many of the methylxanthines have been widely investigated, very few studies have directly examined the effects of caffeine on anti-tumor immune cell functions [5]. Out of the four subtypes of adenosine receptors, A2A receptor (A2AR) is the main pharmacological target of caffeine; the A2AR is blocked by caffeine with a KD of close to 2.4 mM and 8.1 mM in humans and rats, respectively [6]. Indeed, caffeine antagonizes adenosine receptors at moderate to heavy levels of coffee consumption [7]. Adenosine, the ligand of A2AR, is an endogenous purine nucleoside which is released from almost all cell types. Over the past few years a vast number of investigations have reported of its involvement in inflammatory processes [8]. Adenosine is constitutively present in extracellular spaces at low concentrations, but its extracellular levels dramatically increase in metabolically stressful conditions [9]. For example, adenosine accumulates
http://dx.doi.org/10.1016/j.bcp.2015.08.092 0006-2952/ ã 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: H. Eini, et al., Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.08.092
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in the hypoxic microenvironment of solid tumors [10], where its extracellular concentration is 10- to 20-fold higher than that found in adjacent normal tissue [11]. Following both its release from cells or after being derived extracellularly from related molecules, interstitial adenosine diffuses to the cell membranes of surrounding cells where it can bind to four specific G-coupled receptors, including A2AR [12]. Anti-tumor immune responses are coordinated by both innate immunity and adaptive immunity, and are mainly mediated by cytotoxic CD8+ T cells (CTLs), natural killer (NK) cells and natural killer T (NKT) cells. In addition, dendritic cells (DCs) are important for the generation and maintenance of anti-tumor immune responses as they are able to regulate adaptive immunity and induce a CTLs response [13]. It was shown that the anti-tumor activity of these cells is strongly affected by adenosine; A2AR activation during T-cell stimulation significantly inhibits cytotoxicity and cytokine production [14] and has been reported to inhibit T-cell proliferation [15,16]. Similarly, using an A2AR agonist, CGS21680, Csoka et al. showed that A2AR activation suppresses the development of Th1 immune responses in vitro and in vivo [17]. In addition, T-cells that are activated in the presence of CGS21680 fail to proliferate, and then fail to produce IFNg upon reactivation, essentially reaching T-cell anergy. Importantly, these cells maintain a suppressed phenotype after removal of the A2AR agonist [14,18]. In contrast, in vivo activation of A2AR was shown to drive CD4+ T-cell differentiation towards Tregs, probably due to an increase in TGFb and a decrease in IL-6 levels following A2AR activation [18]. In addition, A2AR has been shown to suppress NK cell functions, including IFNg production [19,20]. NKT cells that are deficient in A2AR were found to produce significantly lower levels of IL-4, IL-10 and TGFb, but appear to maintain IFNg production upon stimulation with a-galactosylceramide [21]. The positive effect of caffeine and the role of A2AR in cancer immunity were previously demonstrated in mice inoculated with tumor cells. Ohata et al. have shown that 60% of A2AR/ mice completely rejected inoculated tumor cells and had survived, whereas WT mice displayed no tumor rejection and did not survive; importantly, Ohata et al. also showed that caffeine, as well as a specific A2AR antagonist, ZM241385, enhanced inoculated tumor cell destruction by tumor antigen-specific CD8+ T cells [10]. Moreover, blockade of A2AR with its antagonists, SCH58261, and by caffeine, augmented the efficacy of adoptive T-cell therapy [22]. In a recent study, Beavis et al. found that CD73, which catalyzes the conversion of extracellular nucleosides to adenosine, promoted metastasis through the activation of both A2A and A2B adenosine receptors. A2AR/ mice were strongly protected against tumor metastasis whilst the blockade of A2AR with SCH58261 resulted in enhanced NKcell activity in vitro and in vivo and reduced metastasis in a perforindependent manner [23]. The combination of A2A-adenosinergic pathway inhibitors and blockade of negative immunologic regulators represents a new approach to cancer treatment. It was recently reported that combined therapy with the A2AR antagonist SCH58261 and PD-1 monoclonal antibody (mAb) significantly inhibited the development of B16 melanoma lung metastases and prolonged the life of 4T1.2 tumor-bearing mice compared with either monotherapy alone. Interestingly, the combination was only effective when the tumor expressed high levels of CD73 [24]. Consistent with these findings, Allard et al. showed that anti-CD73 mAb significantly enhanced the activity of both anti-CTLA-4 and anti-PD-1 mAbs against MC38-OVA and RM-1 subcutaneous tumors, and established metastatic 4T1.2 breast cancer. Furthermore, activation of A2AR was found to enhance PD-1 expression on tumor-specific CD8 + T cells and CD4+ Tregs [25]. Since evidence show that the A2AR pathway suppresses antitumor immunity, we hypothesize that part of the effects of caffeine
on reducing tumor incidence may be mediated by its blockade of A2AR. In contrast to previous studies that focused on models of tumor cell line injection, we aimed to investigate the effect of caffeine during the early stages of tumor initiation and progression in a carcinogen-induced tumor model. The role of A2AR in subversion of the immune response is directly examined as a possible mechanism for tumor evasion from immune-surveillance. 2. Materials and methods 2.1. Mice ICR and C57BL/6J wild type mice were purchased from Harlan, Jerusalem, Israel; A2AR/ mice (ICR background) were kindly donated by Catherine Ledent (UniversitéLibre de Bruxelles) [26]. Mice were housed in the animal facility at the Soroka Medical Center (Beer-Sheva, Israel). Heterozygote A2AR+/ mice were generated by crossbreeding with wild type ICR mice. Six- to eight-week—old ICR female mice were used in the carcinogeninduced tumor model. In the tumor cell line inoculation models, Six- to eight week-old C57BL/6J and ICR female mice were used. Animal use conformed to the guidelines established by Institutional Animal Care and Use Committee. 2.2. Adenosine and adenosine analogues Adenosine was purchased from Sanofi Winthrop (Adenocor, Auckland, NZ). Adenosine analog 50 -(N-Ethylcarboxamido) adenosine (NECA) was purchased from Sigma (Rehovot, Israel). A2AR/ A2BR antagonist 4-(2-([7-Amino-2-(2-furyl)[1,2,4]triazin-5-ylamino) ethyl]) phenol) (ZM241385) was purchased from Tocris Cookson (Ellisville, MS, USA), and Caffeine was purchased from Sigma (cat# C0750, purity 99%). 2.3. Carcinogen-induced tumor model Mice were injected intramuscularly into the left hip with 200 mg of the carcinogen 3-methylcholanthrene (3-MCA; cat# 213942, Sigma) or vehicle (olive oil) (total volume 200 ml) [27]. Local fibrosarcomas developed within 3–5 months. Mice were inspected twice a week for tumor development by caliper measurement. When tumors reached 1.8 cm diameter, mice were sacrificed and cell lines were generated from the removed tumor cells. For histological examination, samples were obtained 95 days post-carcinogen injection. 2.4. Caffeine treatment protocol Mice were injected with the carcinogen 3-MCA or with tumor cell lines, and were then randomly divided into two groups; one group was allowed to drink ad-libitum water containing caffeine (0.1% wt/vol) and the other group water with no added caffeine. 2.5. Generation of MCA-induced fibrosarcoma cell lines Tumor tissue was aseptically removed and processed for the establishment of cell lines, as previously described [27], with minor modifications. Disaggregation was performed by continuous stirring of the minced tissue for 1 h at 37 C in the presence of 0.2% collagenase B (Boehringer Mannheim, F.R.G) in DMEM medium. The disaggregated tissue was separated from tissue debris by 40 mm BD Falcon cell strainers and was then subjected to enzymatic digestion by trypsin (10 min at 37 C). DMEM medium and trypsin were purchased from Biological Industries (Beit Haemek, Israel). One of the established cell lines (designated MCA-12.12) was used in the current study.
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2.6. Histology Samples from the site of 3-MCA injections were fixed in 10% formalin and embedded in paraffin. Four-micron sections were stained with H&E. Histological preparations were analyzed for tumor cells and leukocyte infiltration by two histopathologists in a blinded and random manner. 2.7. Tumor cell line maintenance B16-F10 melanoma cells (purchased from the ATCC) and the established MCA-12.12 cell line were grown in complete DMED medium containing 10% heat-inactivated fetal calf serum, 2 mMLglutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. All supplements were purchased from Biological Industries. Experiments were performed using cells from the second to fourth passages. The cultures were incubated in 5% CO2 at 37 C, and maintained in exponential state by sub-culturing periodically. Both cell lines were routinely tested for the presence of mycoplasma and for their ability to generate characteristic tumors in recipient mice. The appearance of a black melanin B16-F10 cell pellet was validated for each passage. 2.8. Tumor cell line injection model Using 1 ml insulin syringes fitted with 25-gauge needles (BD, Franklin, NJ, USA), 5 104 B16 melanoma cells in a volume of 0.05 ml were injected into footpads of C57BL/6 mice. ICR mice were injected using the same method with MCA-12.12 cell line (0.25 106 cells/mouse). Tumor volume was measured three times a week with a caliper and calculated using the following formula: width2 length 0.52. The volume of footpad without tumor cells was subtracted from all measurements. 2.9. Quantitative real-time PCR Total RNA was extracted from tumor tissue using the Versagene RNA cell kit (Gentra systems, Minneapolis, MN). cDNA was prepared from 1 mg of total RNA as template using 5-prime Masterscript kit (Hilden, Germany). Quantitative real-time PCR (qPCR) assays were carried out with Thermo-Start master mix (ABgene, Surrey, UK) in Rotor-Gene Real-Time PCR apparatus (Corbett-Research, Northlake, Australia). Quantification of transcripts were performed using the following mouse-specific primers pairs: IL-15 sense:50 -TGC AAT GAA CTG CTT TCT CCT GGA-30 ,IL-15 anti-sense:50 -CCT CAC ATT CCT TGC AGC CAG ATT-30 ; IL-15Ra sense:50 -GCC AGC GCC ACC CTC CAC AGT AA-30 ,IL-15Ra anti-sense:50 -GCC AGC GGG GGA GTT TGC CTT GAC-30 ; IFNg sense:50 -CAT CAG CAA CAA CAT AAG CGT CA-30 ,IFNg anti-sense:50 CTC CTT TTC CGC TTC CTG A-30 ; CD3 sense:50 -GCC TCA GAA GCA TGA TAA GC-30 ,CD3 anti-sense:50 -CCC AGA GTG ATA CAG ATG TC-30 ; foxp3 sense: 50 -CCC ACC TAC AGG CCC TTC TC-30 , foxp3 anti-sense: 50 -GGC ATG GGC ATC CAC AGT-30 ; IL-17 sense: 50 -TTC ATC TGT GTC TCT GAT GCT-30 ,IL-17 anti-sense:5'-CAC AGA GGG ATA TCT ATC AAG GG-30 ; GAPDH sense: 50 -CAA TGC ATC CTG CAC CAC CAA-30 ,GAPDH anti-sense:50 -GTC ATT GAG AGC AAT GCC AGC-30 .
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cells. Cultures were incubated at 37 C in 5% CO2 in complete RPMI medium containing 10% heat-inactivated fetal calf serum, 2 mMLglutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (Biological Industries), and 50 mM b-mercaptoethanol (Sigma). After a48-h incubation, IFNg protein levels in culture supernatants were determined by ELISA (R&D Systems, Minneapolis, MN, USA). Protein levels of IL-6, MCP-1, TNF-a, IL-10, IL-17 and IL-15 were measured by Quansys multiplex assays according to the Quansys Bioscience protocol (Logan, UT, USA). MLTR was repeated in the presence of adenosine analog NECA with the exception that isolated splenocytes were incubated with the antagonist ZM241385 (1 mM) 1-h before B16 target cells and NECA (3 mM) were added to the culture. 2.11. Statistical analysis Results are expressed as mean S.E. Statistical analysis was performed by t-test and Kaplan–Meier Test. P values of <0.05 were considered significant. 3. Results 3.1. The effect of caffeine or A2AR deletion on the progression of carcinogen-induced tumors To study the effect of caffeine on tumor development, mice (ICR) were injected with 3-MCA carcinogen or vehicle, and were then allowed to drink ad-libitum water with or without 0.1% caffeine (n = 15 in each group). As shown in Fig. 1A, 250 days following inoculation of the carcinogen, the caffeine-treated group displayed a significantly lower incidence rate of carcinogeninduced tumors compared to the water-drinking group (P = 0.0286, 14% vs. 53% tumor growth rate, respectively). After 250 days, caffeine treatment was replaced with water in all groups. As shown, no additional tumors were observed in the caffeine-treated group. Within the group of caffeine-treated 3-MCA-injected mice, a subset of mice that did not develop tumors was found to display signs of autoimmune lesions. Specifically, alopecia was detected in the head and neck regions and in the site of injection on day
2.10. MLTR assays Single-cell suspensions of splenocytes from C57BL/6 mice that underwent the course of B16 cells inoculation were prepared as previously described [28], and mixed with B16 target cells in vitro. MLTR assay was conducted using splenocytes isolated from caffeine- and water-administered mice 34 days after B16 cell injection. Splenocytes (2 105) were co-cultured in round-bottom 96-microwell plates (Corning, NY, USA) with 0.6105 B16 target
Fig. 1. Caffeine treatment significantly decreases the rate of tumor-bearing mice in a carcinogen model. Wild-type ICR mice were administered either water or 0.1% caffeine in drinking water, and were injected 3-MCA or vehicle into the hip tissue (i. m. 200 mg/mouse) (n = 15 per group). (A) Tumor follow-up. Replacement of caffeine with water is indicated. P = 0.0286, Kaplan–Meier Test. (B) Signs of spontaneously resolved autoimmunity. Representative pictures of (i) alopecia on day 95. Arrow, alopecia. (ii) skin lesion on day 250. Arrows indicate lesion.
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95 post-3-MCA administration. Subsequently, complete body hairloss and vast skin lesions were observed on day 250 and lasted throughout the experiment (Fig. 1B). Importantly, none of these symptoms were observed in mice that consumed caffeine but were not injected with the carcinogen (data not shown). As shown in Fig. 2, a blinded histological examination of tissue biopsies that were obtained from the site of 3-MCA injection, revealed extensive tumor-mediated muscle injury in most of the samples of waterconsuming mice. In contrast, most of the samples obtained from caffeine-treated mice showed no signs of muscle injury and only a few of them showed perivascular tumor cell clusters. Tumors in both groups were characterized by a leukocytic infiltrate that was comprised predominantly of lymphocytes, as well as scattered neutrophils and macrophages. Importantly, lymphocytes were more abundant in samples of caffeine-treated mice than those of water-consuming mice. To specifically depict the role of A2AR in cancer initiation and progression, we compared tumor development in A2AR/ mice, heterozygote mice (A2AR+/) and WT mice (n = 20), all on ICR background. As shown in Fig. 3, tumors were first detected in the WT group on day 83, and then later in the A2AR+/ and A2AR/ groups (day 110 and 127, respectively). The delay in time between tumor detection in A2AR/ mice and WT mice is statistically significant (P = 0.0351). 3.2. The effect of caffeine on tumor progression and immune profile in cell-injection models Since carcinogenesis studies involve extremely prolonged time lines, we generated thirteen cell lines from solid tumors induced in WT ICR mice by 3-MCA. Following in vivo screening, we selected the MCA12.12 line by its high growth rate following inoculation (not shown). To examine the effect of caffeine on tumor cell growth and immune responses, we inoculated MCA12.12 cells into footpads of WT mice. As shown in Fig. 4A, by day-17 caffeine-treated mice displayed lower median tumor volume compared to control mice. Since several mice from both groups rejected the tumor at various
Fig. 3. Reduced tumor incidence in A2AR/ mice following 3-MCA administration. WT (n = 20), heterozygote (A2AR+/,n = 20), and knockout (A2AR/,n = 18) mice were injected 3-MCA into the hip tissue (i.m. 200 mg/mouse). Tumor detection was followed weekly. P = 0.0351 between WT and KO, Kaplan–Meier Test.
time points, comparison of tumor volumes between the two groups did not reach statistical significance. At this time point, tumors were harvested and gene expression profiles were examined by qPCR. As shown in Fig. 4B and C, the mean mRNA levels of CD3, IL-15, IL-15Ra and IL-17 in tumors that were explanted from caffeine-treated mice were higher in compared to those of the water-drinking group. However, differences between the two groups of mice did not reach statistical significance. Consistent with the caffeine-treated group being skewed towards an enhanced immune response, IFNg mRNA levels were markedly elevated in caffeine-treated mice (P < 0.05) (Fig. 4C). Since in vivo screenings with MCA12.12 cells led to highvariance outcomes, most probably due to excessive immunogenicity of the cells, we turned to examine the highly metastatic B16 melanoma cell line. As shown in Fig. 5, caffeine-treated mice were remarkably resistant to B16 tumor growth; while 7 out of 8 mice in the control group presented tumors, only 1 out of 8 mice in the caffeine group developed a tumor that was detectable on day 34.
Fig. 2. Caffeine treatment is associated with leukocytic infiltration at the site of 3-MCA injection. Wild-type mice were administered either water or 0.1% caffeine in drinking water, and were injected 3-MCA or vehicle into the hip tissue (i.m. 200 mg/mouse) (n = 5 per group). On day-95, sections of biopsies from the site of injection were stained with haematoxylin and eosin (H&E) and analyzed for tumor cells and leukocyte infiltration. Representative histology images are shown. Typical areas are displayed at 40 and 10 magnification. M, muscle; T, tumor; L, lymphocytes. Arrow, blood vessel.
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Fig. 4. Effect of caffeine after 3-MCA—induced transformed cells line injection. Wild-type mice were administered either water (n = 9) or 0.1% caffeine (n = 8) in drinking water, and were injected into the footpads with MCA12.12 cells (1 106 cells/mouse). Tumor volume was measured three times a week. (A) follow-up, expressed as median tumor volume. On day-17, tumors were harvested and qPCR was performed with primers specific for (B) T-cell markers and (C) cytokine-related genes. Results are normalized to GAPDH transcript levels. Mean S.E. *P < 0.05 from WT.
In order to examine whether the observed beneficial effect of caffeine had involved a heightened anti-tumor immune response, we established a mixed lymphocyte tumor reaction (MLTR) assay. Isolated splenocytes from caffeine- and water-administered B16challenged mice were re-challenged with fresh B16 target cells in vitro (n = 5 for each group). Induction of IFNg production was then determined. As shown in Fig. 6A, MLTR resulted in a sharp elevation of IFNg. However, MLTR that incorporated splenocytes from B16-challenged caffeine-treated mice produced 2-fold greater levels of IFNg than the MLTR that was comprised of splenocytes from B16-challenged water-consuming mice. IFNg was below detection in supernatants of B16 cells and splenocytes that were incubated separately (data not shown). In order to examine the sensitivity of the reacting cells in MLTR to adenosine, the assay was repeated in the presence of a stable adenosine agonist, NECA. As shown in Fig. 6B, the addition of NECA to the culture medium almost completely abolished the production of
IFNg by splenocytes from both water- and caffeine-consuming mice. Of note, splenocytes obtained from caffeine-treated mice were not exposed to caffeine in culture; it is therefore presumed that they expressed intact functional A2A receptors. Pretreatment of splenocytes from both groups of mice with the A2AR/A2BR antagonist, ZM241385, reversed NECA-induced IFNg down-regulation. In contrast to water-treated samples, IFNg down-regulation was only partly reversed in caffeine-treated samples following ZM241385 addition. In addition to IFNg, the production of several other cytokines was tested following incubation of splenocytes from B16-challenged mice with target cells in vitro. As shown in Fig. 6C, mixed cultures of splenocytes from caffeine-treated mice produced elevated levels of IL-6, MCP-1, TNFa and IL-10 compared to mixed cultures of splenocytes from water-treated mice (n = 5 for each group). Splenocytes isolated from control mice exhibited lower cytokine levels compared to splenocytes from both caffeine- and water-
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Fig. 5. Effect of caffeine on B16 tumor volume. Wild-type mice were administered either water or 0.1% caffeine in drinking water (n = 8 per group), and were injected into the footpads with B16 cells (5 104 cells/mouse). Tumor volume was measured three times a week. Left, follow-up. Right, representative images of tumors on day 34. Arrows, macroscopically identifiable tumors. Figure shows mean S.E (n = 8 in each group) of a representative experiment out of three independent experiments with similar results. **P < 0.01 and ***P < 0.01 between water and caffeine groups.
consuming mice. No significant difference in IL-17 levels was observed. IL-15 was undetectable in supernatants. 3. Discussion Several prospective epidemiological studies report a negative correlation between caffeine consumption and risk of various cancers in humans [2,3]. In order to experimentally represent the etiology of tumor initiation, 3-MCA, a complete carcinogen that mediates both initiation and promotion of tumor development, was used in the present study [29]. Our results demonstrate that tumor incidence in 3-MCA-injected mice is dramatically reduced during caffeine treatment. Based on three separate experimental observations, it is suggested that the primary effect of caffeine responsible for the marked reduction of cancer rates in this model, is the enhancment of anti-tumor immunity. For example, leukocyte infiltrates, composed primarily of lymphocytes, were more abundant in samples of caffeine-treated mice than those found in water-consuming mice. In addition, signs of spontaneously resolved autoimmunity, such as skin lesions and alopecia, were observed in the entire subset of caffeine-treated mice that were injected with the carcinogen and did not develop tumors, indicating that treatment with caffeine most probably leads to an enhanced immune response against epitopes shared by the tumor and normal tissues. Spontaneous appearance of alopecia with skin lesions was previously described in one of the major autoimmune lupus-prone mouse models, the MRL/lpr mouse [30,31]. In addition, these observations are similar to reports of autoimmunity in melanoma patients who underwent immunotherapy with melanoma antigen-specific T cells [32], as well as in CL-81 melanoma-rejecting A2AR/ mice [10]. Lastly, further evidence in support of immune-related benefits for caffeine in the present study can be found at the point where the system was challenged by removal of caffeine; no additional tumors were observed in the caffeine group, further supporting the impression that effective immunity prevented tumor development. We suggest that the mechanism behind the positive shift in immunity relates to the antagonism of adenosine receptors by caffeine. The well-described protocol of low levels 0.1% caffeine in drinking water was chosen in the present study in order to promote preferential antagonism of A2AR [10], the adenosine receptor that was shown to be involved in suppressing key stages of the inflammatory process, including leukocyte recruitment, phagocytosis, cytokine production and immune cell proliferation [33]. Similar to caffeine-treated mice, 3-MCA-challenged A2AR/ mice displayed significantly fewer tumors as well as delayed tumor
development compared to their WT littermates. Comparison between the genetically deficient animal and caffeine treatment, however, did not fully overlap: 60% of A2AR/ mice presented with tumors, whereas 14% of caffeine-treated mice presented with tumors (day 150 and 250, respectively). Since differences in tumor onset were observed between the control groups of these experiments (both composed of water-treated WT mice), this discrepancy might be attributed to a stronger effect of the carcinogen in the genetically-targeted mice, or to A2AR-independent anti-tumor effects of caffeine. The latter could also explain the lack of long-term differences in tumor development between A2AR/ and WT mice. Indeed, it was recently shown that A2BR blockade reverses the immunosuppressive effects of adenosine and inhibits tumor progression [34]. Caffeine blocks A2BR, albeit with a lower affinity than A2AR. Thus, it is possible that in the present study, caffeine partially blocked A2BR in treated animals. Of note, we cannot exclude that in addition to the immunomodulatory effects of caffeine presented here, inhibition of replication checkpoint function by caffeine, which was shown in recent studies [35,36], may have also contributed to the suppression of tumor in the carcinogenesis model. Caffeine is a non-selective phosphodiesterase (PDE) inhibitor. It acts by competing with adenine for access to the catalytic site of PDEs and prevents PDEs from inactivating cAMP or cGMP [37]. High intracellular level of cAMP and cGMP could arrest growth, induce apoptosis and attenuate cancer cell migration [38,39]. Caffeine is also known as a stimulator of the ryanodine receptor (RyRs) Ca2+ release-channels that have been previously implicated in the proliferation of human T lymphocytes and melanocytes. Interestingly, RyR was recently suggested to serve as prognostic indicator and as a target for breast cancer treatment [40]. Another possible mechanism for the reduction of cancer incidence in an experimental model of carcinogenesis is a direct effect of the therapy agent on mutagenesis. Literature exhibits an inconclusive array of evidence regarding the effect of caffeine on mutagenesis; it was previously demonstrated that caffeine can act as a comutagenic, nonmutagenic, and/or antimutagenic, depending upon test system and protocol [41]. We have no conclusive data that support the direct reduction of mutagenesis by caffeine treatment. Taking into consideration the long latency period associated with the development of cancer after 3-MCA exposure, we chose to further assess the effect of caffeine on anti-tumor response in a cancer cell inoculation models. Similar to its effects on tumor progression in the 3-MCA model, we observed that caffeine treatment is able to improve tumor inhibition and modulate
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Fig. 6. Adenosine receptors agonist, NECA, blocks cytokines production by splenocytes from B16-challenged mice in an A2AR-dependent manner. Wild-type mice were administered either water or 0.1% caffeine in drinking water (n = 5 per group), and were introduced with B16 cells (5 104 cells/mouse) into the footpads. On day-34, splenocytes were isolated from mice and were then (A, C) co-incubated in triplicate with B16 target cells (control, splenocytes from unchallenged mice) or (B) preincubated with ZM241385 (1 mM) and mixed with B16 target cells in the presence of NECA (3 mM). Control, mixed cultures in the absence of NECA or ZM241385. (A, B) IFNg in supernatants after 48 h of incubation were measured by ELISA. Figure shows mean S.E (n = 5 in each group) of a representative experiment out of three independent experiments with similar results. ***P < 0.001 from water. **P < 0.01 and ***P < 0.001 from control. ^^P < 0.01 and ^^^P < 0.001 from NECA. (C) Cytokine levels in supernatants after 48 h of incubation were measured by Quansys multiplex assay. Mean S.E (n = 5 in each group). *P < 0.05, **P < 0.01 between water and caffeine groups.
immune responses during the progression of cancer cell growth. Here, caffeine-treated mice injected with MCA12.12 cells exhibited lower tumor volumes in comparison to water-consuming mice. Differences in MCA12.12 rejection by caffeine- and water-treated mice were also reflected in the extent of intratumoral IFNg mRNA expression levels. When the well established C57BL/6-derived B16 melanoma model was used, treatment with caffeine induced nearly complete rejection of tumors. This remarkable augmentation of tumor rejection was unexpected, since the B16 melanoma model is known to produce poorly immunogenic tumors that are notoriously aggressive and difficult to treat. With remarkable relevance, our findings support two large epidemiological studies that report an inverse correlation between coffee consumption and decreased risk of cutaneous malignant melanoma in women [1,42]. In
addition, it was previously reported that both intraperitoneal and oral administration of caffeine (100 and 50 mg/kg body weight, respectively) for 10 consecutive days reduces tumor volume in B16 melanoma-bearing mice. Accordingly, mice injected with B16 melanoma cells through lateral tail vein and exposed to the above indicated caffeine treatment, showed a reduction in the number of lung tumor nodules [43]. Here, we observed that the enhanced inhibition of B16 tumor growth following caffeine treatment is associated with increased production of IFNg by splenocytes from caffeine-treated mice that were re-challenged with B16 target cells in the MLTR assay. In order to test the sensitivity of anti-tumor immune cells to adenosine, the MLTR assay was repeated in the presence of the stable adenosine agonist, NECA. Our results show that the addition of NECA to the culture medium almost completely abolished the production of IFNg by
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splenocytes from both water- and caffeine-consuming mice. Furthermore, pretreatment of splenocytes with the A2AR/A2BR antagonist, ZM241385, reversed NECA-induced IFNg down regulation in both groups, suggesting an involvement of A2AR/A2BR in mediating IFNg down-regulation. Similar to the effect of caffeine on IFNg expression, mixed cultures of splenocytes from caffeine-treated mice produced elevated levels of IL-6, MCP-1, TNFa and IL-10 compared to mixed cultures of splenocytes from water-treated mice. As expected, IL15, which performs most of its biological functions in a membraneassociated form, was undetectable in supernatants. Importantly, MLTR resulted in a sharp elevation of IFNg from the pg/ml range to the ng/ml range whereas levels of the other tested cytokines were increased <2-fold. This observation implies that a role for immune cell secretion of IFNg in the B16 melanoma model is of significance, holding great importance since IFNg is known to possess antiproliferative, as well as anti-angiogenic and immune-inducing activities and its neutralization in many tumor models is followed by enhanced aggressiveness of tumors [44–46]. Taken together, we show here for the first time that caffeine treatment promotes an effective anti-tumor immune response during tumor initiation, partly through the antagonism of A2AR. We demonstrate that caffeine treatment leads to increased IFNg production and to a heightened tumor-specific memory T cell response. In addition, the results suggest a role for A2AR activation in preventing autoimmunity. Thus, immune modulation through targeting of the A2AR pathway holds a promising potential for cancer therapy, and fine-tuning of this pathway may allow the adjusting of local inflammatory responses. These outcomes support important recent epidemiological studies that identify a correlation between caffeinated-coffee consumption and low incidence of tumors in humans. Conflict of interest The authors have no conflict of interest associated with this study to declare. Acknowledgments We would like to thank Dr. Catherine Ledent from Université Libre de Bruxelles for sharing with us the A2AR/ mice. This work was supported by the Dr. Montague Robin Fleisher Kidney Transplant Unit Fund. References [1] M.B. Veierod, D.S. Thelle, P. Laake, Diet and risk of cutaneous malignant melanoma: a prospective study of 50,757 Norwegian men and women, Int. J. Cancer 71 (1997) 600–604. [2] I.S. Cadden, N. Partovi, E.M. Yoshida, Review article: possible beneficial effects of coffee on liver disease and function, Aliment. Pharmacol. Ther. 26 (1) (2007) 1–8. [3] T. Imatoh, S. Tanihara, M. Miyazaki, Y. Momose, Y. Uryu, H. Une, Coffee consumption but not green tea consumption is associated with adiponectin levels in Japanese males, Eur. J. Nutr. 50 (2011) 279–284. [4] J. Dong, J. Zou, X.F. Yu, Coffee drinking and pancreatic risk: a meta-analysis of cohort studies, World J. Gastroenterol. 17 (2011) 1204–1210. [5] L.A. Horrigan, J.P. Kelly, T.J. Connor, Caffeine suppresses TNF-alpha production via activation of the cyclic AMP/protein kinase A pathway, Int. Immunopharmacol. 4 (2004) 1409–1417. [6] B.B. Fredholm, K. Battig, J. Holmen, A. Nehlig, E.E. Zvartau, Actions of caffeine in the brain with special reference to factors that contribute to its widespread use, Pharmacol. Rev. 51 (1999) 83–133. [7] M.V. Sitkovsky, J. Kjaergaard, D. Lukashev, A. Ohta, Hypoxia-adenosinergic immunosuppression: tumor protection by T regulatory cells and cancerous tissue hypoxia, Clin. Cancer Res. 14 (2008) 5947–5952. [8] G. Hasko, P. Pacher, E.A. Deitch, E.S. Vizi, Shaping of monocyte and macrophage function by adenosine receptors, Pharmacol. Ther. 113 (2007) 264–275.
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