Antitumor activity of xanthone derivatives: effects on the immune microenvironment

VIVIANA VILAR DA SILVA ANTITUMOR ACTIVITY OF XANTHONE DERIVATIVES: EFFECTS ON THE IMMUNE MICROENVIRONMENT Dissertação de Candidatura ao grau de Mestre em Oncologia (Especialização em Oncologia Molecular) submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador: Doutor Rui Medeiros Professor Associado Convidado de Ciências Médicas Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal. Co-orientadores: Doutora Fátima Cerqueira Professora Associada de Ciências Médicas Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Porto, Portugal. Doutora Madalena M.M. Pinto Professora Catedrática em Química Orgânica e Farmacêutica Faculdade de Farmácia, Universidade do Porto, Porto, Portugal This work was developed in the Center of Investigation in Biomedicine of Fernando Pessoa – Energy, environment and health research unit (CEBIMED of FP-ENAS), in Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia da Universidade do Porto, and in the Centro de Química Medicinal da Universidade do Porto – CEQUIMED-UP and was funded through national funds from Fernando Pessoa Fundation, FCT – Fundação para a Ciência e a Tecnologia under the project CEQUIMED – PEstOE/SAU/UI4040/2014, FEDER, POCI, POPH/FSE/QREN. Agradecimentos Não posso deixar de agradecer a quem direta ou indiretamente colaborou para a realização deste trabalho e que, assim, contribuiu para o meu crescimento tanto profissional como pessoal: Ao Instituto de Ciências Biomédicas Abel Salazar, na pessoa do Digníssimo Diretor Professor Doutor António Sousa Pereira, e à coordenação do mestrado de Oncologia, na pessoa da Professora Doutora Berta Silva, pela oportunidade de aprofundar os meus conhecimentos ao longo destes dois anos de mestrado. À Universidade Fernando Pessoa, na pessoa do Digníssimo Reitor Professor Doutor Salvato Trigo, pelas condições de trabalho que me proporcionou. Ao Professor Doutor Rui Medeiros, agradeço a orientação e a oportunidade que me proporcionou, a simpatia com que sempre me recebeu e todos os conhecimentos que me transmitiu. À Professora Doutora Fátima Cerqueira, minha coorientadora, o meu sincero agradecimento pelo apoio constante, pela disponibilidade, pela amabilidade e pelos indispensáveis conselhos que forma determinantes na realização deste trabalho. À Professora Doutora Madalena Pinto, pela coorientação, porque me permitiu integrar este projeto, obrigada por toda a sabedoria que me transmitiu e pela enorme simpatia. Aos meus colegas de trabalho, nomeadamente à Jani Silva, Nair Campos, Liliana e Carla Gabriel pelos conselhos, pelo apoio e principalmente pelos bons momentos. Aos restantes membros do FP-ENAS/CEBIMED, aos professores, técnicos e auxiliares o meu muito obrigado pela simpatia e pela disponibilidade. À Mariana Santos, porque a tua amizade é indispensável, assim como os teus conselhos, obrigada por toda a ajuda e por, apesar de seres a que está mais longe, estares sempre presente. Ao Raphael Costa, pela enorme paciência, por me ajudares em tudo que podes, por estares ao meu lado em todos os momentos e porque o teu amor me ajudou a superar os piores momentos. Aos meus pais, porque as palavras não são suficientes para vos agradecer por tudo que fizeram e fazem por mim, por serem um exemplo de humildade e determinação, porque sempre me apoiaram incondicionalmente e porque me dão força para nunca desistir. Agradeço do fundo do coração todo o esforço que fazem diariamente para me proporcionarem uma vida melhor. Table of contents Nomenclature ................................................................................................................. i Resumo ........................................................................................................................ iii Abstract ......................................................................................................................... v I. General Introduction ...............................................................................................1 Chemistry of xanthones: General considerations .......................................................2 Biological activities of xanthones ...............................................................................3 Melanoma ..................................................................................................................5 Immune System .........................................................................................................6 Tumor immunology ..................................................................................................11 References ..............................................................................................................14 II. State of art ............................................................................................................23 Xanthones as potential agents in melanoma treatment ............................................24 Importance of immunomodulation in melanoma treatment .......................................25 References ..............................................................................................................27 III. Objectives and outline ..........................................................................................29 IV. Materials and Methods .........................................................................................31 Chemicals and reagents ..........................................................................................32 Xanthones ...............................................................................................................32 Cell lines ..................................................................................................................32 Cell growth assay ....................................................................................................33 Antitumor effect of conditioned macrophages culture medium .................................34 NO production assay ...............................................................................................34 NO scavenging assay ..............................................................................................35 Human mononuclear cells MTT-proliferation assay .................................................35 Cytokine quantification .............................................................................................35 MTT-viability assay ..................................................................................................36 Statistical analysis ...................................................................................................36 Ethics .......................................................................................................................36 References ..............................................................................................................37 V. Natural Xanthones: alpha-mangostin ....................................................................39 Paper 1 (submitted to Fitoterapia in 29-09-2014): Mangosteen extract: “angel or demon”? The role of xanthones of mangosteen in potential adverse effects.................40 Paper 2 (draft): “Alpha-mangostin antitumor activity: cytotoxicity and influence on the immune system microenvironment” ..............................................................................69 VI. Synthetic xanthone: 1,2-dihydroxyxanthone .........................................................89 Paper 3 (draft): “1,2-dihydroxyxanthone antitumor activity: cytotoxicity and influence on the immune system microenvironment” ...................................................................90 VII. Conclusions ........................................................................................................ 105 List of figures Figure 1 Xanthone basic skeleton .................................................................................2 Figure 2: Structure of the main xanthones from mangosteen........................................3 Figure 3: Polarization of macrophages and corresponding functions. Legend: GC indicate glucocorticoid; IC, immune complex; IL-1ra, IL-1 receptor antagonist; LPS, lipopolysaccharide; MR, mannose receptor; SR, scavenger receptor - adapted from (Chanmee et al. 2014). ......................................................................................................8 Figure 4 Cytokine network in immune system – adapted from (Zhang and An 2007) ..10 Figure 5: Schematic presentation of the role of immune system in cancer. Legend: APC indicates antigen -presenting cell; CTL, cytotoxic T lymphocyte or CD8+ T cell; NK, natural killer cell; Th, T helper cell; Treg, regulatory T cell and TAA, tumor-associated antigens - adapted from (Lakshmi Narendra et al. 2013)..................................................13 Figure 6 Xanthone basic skeleton ...............................................................................44 Figure 7 Structure of the main xanthones from mangosteen .......................................45 Figure 8 Effect of xanthones of mangosteen at different stages of cell cycle regulation. Legend: cdc indicate cell division cycle protein; CDK, cyclin-dependent kinases; Chk or CHEK2, checkpoint kinase; G1, gap phase; G2, gap phase 2; M, mitosis phase; MDM2, murine doble minute 2; MG, mangosteen xanthones; p21cip1, cyclin-dependent kinase inhibitor 1; p27kip1, cyclin-dependent kinase inhibitor; p53, tumor protein p53; S, DNA synthesis phase. ..............................................................................................................49 Figure 9: alpha-Mangostin ..........................................................................................71 Figure 10: Production of IL-1β, IL-10, TGF-β1 and TNF-α by THP-1 macrophages. Cytokine production was evaluated on unstimulated macrophages (basal), LPS-stimulated macrophages and macrophages treated with 3 and 6 µM of alpha-mangostin. Data are the mean ± SEM from tree independent experiments performed in duplicate. * p < 0.001; ┼ p > 0.05. ................................................................................................................................76 iv inibiu a interleucina 1 beta e a interleucina 10, estimulou ainda o fator transformador do crescimento beta 1 e o fator de necrose tumoral alfa em macrófagos. A alfa-mangostina também interferiu com a proliferação de linfócitos e a expressão da interleucina 10 e do fator de necrose tumoral alfa por estas células. Quanto ao impacto direto dessa modulação no crescimento de melanoma, os compostos apresentam uma melhoria de efeito citotóxico. No entanto, só no tratamento com 1,2-dihidroxixantona, esta melhoria pode ser associada com a modulação de macrófagos pela xantona, uma vez que o efeito da alfa-mangostina em doses baixas é semelhante aos macrófagos não tratados na linha celular em estudo. Em conclusão, estes resultados permitiram inferir um impacto potencial da alfamangostina e da 1,2-dihidroxixantona no tratamento do melanoma, devido à sua atividade citotóxica e ao sugestivo efeito imunoterapêutico em tumores. v Abstract Cancer is one of the most aggressive and lethal diseases, remaining a challenge for researchers and physicians, in spite of decades of research and investment in this area. Later detection and constrains of current therapies, associated with chemoresistance and toxic side effects, are the main problems. Melanoma is a melanocytic tumor highly immunogenic and with a great predisposition to metastasize. Its incidence is increasing worldwide and the mortality rates associated to the disease are very high. Therefore, it is imperative to find therapeutic alternatives that overcome the actual limitations of the treatment, namely improving the spectrum and efficacy of actuation and decrease the toxicity of currently used drugs. A good strategy to avoid the tumor resistance needs a drug that targets multiple pathways. Xanthones are naturally obtained from higher plants and microorganisms. Remarkable pharmacologic proprieties have been described for these compounds, mainly their antitumor potential, which contributed to their commercialization as dietary supplement, isolation and synthesis of numerous derivatives of xanthones. In cancer, their effect comprises several physiologic pathways, interfering with multiple hallmarks of cancer, including apoptosis, cell cycle, angiogenesis, inflammation and immune surveillance making them a prospective drug candidate. The main gold of this study was the evaluation of the effects of two xanthones, alphamangostin and 1,2-dihydroxyxanthone, on A375-C5 melanoma cell growth and their capacity to modulate the immune macrophages-dependent microenvironment. The ability for the compounds to interact with different immunologic parameters that may interfere with tumor treatment were evaluated, including production of nitric oxide and expression of cytokines interleukin-1β and tumor necrosis factor-α (characteristics of M1 phenotype) and interleukin-10 and transforming growth factor-β1 (characteristic of M2 phenotype). alpha-Mangostin was also tested in lymphocytes, namely in cell proliferation and cytokines production. This activity was previously described for 1,2-dihydroxyxanthone. In all assays, in vitro cellular systems were used, mostly of human origin. Xanthones caused a decrease in nitric oxide production and interfere with the expression of cytokines by stimulated macrophages and/or lymphocytes. Alpha-mangostin suppressed expression of interleukin-1β and transforming growth factor-β1, and stimulated tumor necrosis factor-α. 1,2-Dihydroxyxanthone inhibited interleukin-1β and interleukin-10, stimulated transforming growth factor-β1 and tumor necrosis factor-α in macrophages. Alpha-mangostin also interferes with lymphocytes proliferation and the expression of interleukin-10 and tumor necrosis factor-α by these cells. Concerning the direct impact of this modulation on melanoma growth, the compounds exhibit an vi improvement of cytotoxic effect. However, only in 1,2-dihydroxyxanthone treatment, this improvement could be associated with modulation of macrophages, since the effect of alpha-mangostin at lower doses is similar to non-treated macrophages in the cell line in study. In conclusion, these finds allowed to infer a prospective impact of alpha-mangostin and 1,2-dihydroxyxanthone in melanoma treatment, due to their cytotoxic activity and suggestive effect in cancer immunotherapy. I. General Introduction Xanthones, melanoma and immune system I. 2 Chemistry of xanthones: General considerations Xanthones are biologically active tricyclic molecules characterized by a dibenzo-γpyrone nucleus or 9H-xanthen-9-one (Figure 1) (Pinto et al. 2005; Mazimba et al. 2013). The diversity of xanthone derivatives is possible due to the variation of the nature and position of substituents on the A and B rings. According to that, natural xanthones may be categorized into: simple oxygenated, glycosylated, prenylated and their derivatives (xanthone dimers, xanthonolignoids, and miscellaneous). On the other hand, the synthetic xanthones can have simple groups such as hydroxyl, methoxyl, methyl, carboxyl, as well as more complex substituents such as epoxide, azole, methylidenebutyrolactone, aminoalcohol, sulfamoyl, methylthiocarboxylic acid, and dihydropyridine in their scaffold (Pinto et al. 2005). Natural xanthones of higher plants mainly occur in two families, Guttiferae and Gentianaceae, and can also be found in microorganisms as fungi and lichens (Vieira and Kijjoa 2005; Mazimba et al. 2013). The majority of these compounds was obtained from Garcinia mangostana Linn, being the most abundant and frequently studied, the αmangostin, β-mangostin, γ-mangostin, garcinone E and gartanin (Figure 2) (Shan et al. 2011). Figure 1 Xanthone basic skeleton I. 3 Xanthones have shown remarkable biological/pharmacological activities linked with their tricyclic scaffold, depending on the nature and/or position of the diverse constituents (Mazimba et al. 2013). As xanthones from natural origin are quite limited in type and position of the substituents due to the biosynthetic pathways, the syntheses of new xanthones can attempt to alter or improve their activity by having different nature and positions of the substituents on the nucleus of these compounds (Pedro et al. 2002). Biological activities of xanthones In the last decade, the interest for natural or derivative xanthones has been growing as readily confirmed by the increased numbers of scientific reports (Gutierrez-Orozco and Failla 2013). These have allowed to find a great variety of biological/pharmacological activities associated with xanthone derivatives including analgesic (Bianco et al. 1989; Garrido et al. 2001; Cui et al. 2010), antioxidant (Madan et al. 2002; Jung et al. 2006), anti-inflammatory (Lin et al. 1996; Madan et al. 2002; Teixeira et al. 2005; Chen et al. 2008), antitumor (Pinto et al. 2005; Pedraza-Chaverri et al. 2008; Shan et al. 2011; Gutierrez-Orozco and Failla 2013), antiallergic (Pfister et al. 1972; Nakatani et al. 2002), antimicrobial (Pinto et al. 2005; Pedraza-Chaverri et al. 2008), neuroprotective (Li and Ohizumi 2004; Weecharangsan et al. 2006) and immunomodulatory (Makare et al. 2001; Tang et al. 2009). In spite of all promising proprieties in the improvement of treatment of a number of pathologies as cancer (Pinto et al. 2005; Shan et al. 2011), diabetes (Ichiki et al. 1998; Figure 2: Structure of the main xanthones from mangosteen I. 4 Bumrungpert et al. 2009), cardiac (Jiang et al. 2004; Devi Sampath and Vijayaraghavan 2007), psychiatric (Chairungsrilerd et al. 1996), autoimmune (Madan et al. 2002; YusufMakagiansar et al. 2002; Leiro et al. 2004) and neurodegenerative pathologies (Weecharangsan et al. 2006; El-Seedi et al. 2010), it is important to note that the majority of the compounds were only evaluated in cell lines or animal models. According to that the safety and efficacy of these products cannot be completely assure. Vadimezan (5,6-dimethylxanthenone-4-acetic acid, DMXAA), a promising anticancer xanthone that presented important proprieties as vascular disrupting-agent is an example of the need of appropriated clinical trial. This compound was tested until phase II trial suggesting potential application in combination with paclitaxel and carboplatin for nonsmall-cell lung cancer (McKeage et al. 2009). Although, the phase III revealed a lack of utility to human use against this cancer due to a specie-specific role (Baguley and Ching; Lara et al. 2011). Antitumor activity Among all physiological activities mediated by xanthone compounds, the antitumor capacity seemed to be quite remarkable since they exert their inhibitory effect in a significant range of tumors. This activity was demonstrated in vitro and/or in vivo on breast (Pedro et al. 2002; Moongkarndi et al. 2004; García-Rivera et al. 2011), colorectal (Gobbi et al. 2002; Nakagawa et al. 2007; Watanapokasin et al. 2010), prostate (Johnson et al. 2012), colon (Chitchumroonchokchai et al. 2013), lung (Kostakis et al. 2002; Suksamrarn et al. 2006; Rajendran et al. 2008), glioma (Chao et al. 2011), hepatoma (Ho et al. 2002; Zou et al. 2004), leukemia (Kostakis et al. 2002; Seo et al. 2002; Matsumoto et al. 2003; Yao et al. 2010) and melanoma (Joseph et al. 1999; Pedro et al. 2002; Wang et al. 2011), among others (Pinto et al. 2005; Gutierrez-Orozco and Failla 2013). Analyze analyse The chemotherapeutic and chemopreventive potential owing their inhibitory effect on every steps in the process of tumorigenesis (initiation, promotion, and progression) and on multiple signaling targets (Sun et al. 2002; Liu et al. 2013). Indeed, these compounds are able to modulate a considerable number of hallmarks of cancer by induce cell cycle arrest, suppression of tumor cell proliferation, induction of apoptosis, differentiation, reduction of inflammation, and inhibition of adhesion, invasion, and metastasis (Pinto et al. 2005; Akao et al. 2008; Pedraza-Chaverri et al. 2008; Shan et al. 2011; Gutierrez-Orozco and Failla 2013). The induction of apoptosis may be possible, among other factors, due activation of caspase cascade and disruption of mitochondrial membrane and consequent release of cytochrome c (Matsumoto et al. 2004). Antiproliferative effects of xanthones were demonstrated as result of cell cycle arrest at G1 and S phases (Matsumoto et al. 2005). I. 5 The inhibition of metastatic process is likely associated to inhibition of matrix metalloproteinase (MMPs; particularly MMP-2 and MMP-9) and u-PA (urokinase - plasminogen activator) expression by JNK1/2 (Jun N-terminal kinase), NF-κB (nuclear factor kappa-B) and AP-1 (activator protein 1) activity suppression (Hung et al. 2009). Therefore, xanthones could be agents of an emerging antitumor therapy capable of alter multiple signaling targets, affect various traits of cancer and consequently prevent adaptive resistance (Hanahan and Weinberg 2011; Liu et al. 2013). Anti-inflammatory activity There is a range of evidences from the involvement of inflammatory pathways in tumorigenesis (Kundu and Surh 2008). Xanthones have demonstrated both, antitumor and anti-inflammatory potential. Several xanthone derivatives, mainly the natural ones, attenuated the expression of inflammatory mediators as TNF-α (tumor necrosis factor-α) and interleukins 6 (IL-6) in cell lines of macrophages and adipocytes and decreased the activation of signaling pathways including IL-1, mitogen-activated protein kinase (MEK), Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 1 (STAT-1), NF-κB, AP-1 in these cells (Kumar et al. 2003; Leiro et al. 2004; Bumrungpert and Kalpravidh 2010; Liu et al. 2012). Immunomodulatory effects were also observed in murine models (Jang et al. 2012). Reduction of inducible NO synthase (iNOS) mRNA was reported for several xanthones in murine macrophages cell lines (Garrido et al. 2004; Teixeira et al. 2005; Chen et al. 2008; Tewtrakul et al. 2009) or murine models (Leiro et al. 2003). Decreased levels of cyclooxygenase-2 (COX-2) were also associated with activity of xanthones (Leiro et al. 2004; Chen et al. 2008; Tewtrakul et al. 2009). This represents a small part of all the reported anti-inflammatory effects of xanthones, many others could be added to those as demonstrated in some published reviews (Pinto et al. 2005; Shan et al. 2011; Gutierrez-Orozco et al. 2013). Melanoma Melanoma is a malignancy of melanocytes or their precursors, the melanoblasts (Asnaghi et al. 2012). This is primarily located in the skin, but can also be found in ears, gastrointestinal tract, eyes, oral and genital mucosa and leptomeninges (McCourt et al. 2014). The main risk factor associated to this cancer is the excess of sun exposition as repeatedly referred in several prevention campaigns. In spite of this attempt to control I. 6 emerging cases, its incidence rate is still rising worldwide at highest levels than other tumors (Maio 2012; Liu et al. 2014). Melanocytic tumors presented a highly predisposition to metastasize, however, when early detected frequently means a successful treatment and increasing of survival. Contrariwise, metastatic tumors represent poor prognostic with high associated mortality (Korn et al. 2008; Gast et al. 2011). Melanoma tumorigenesis requires a multistep process, however, a great variety of evidences referred the crucial involvement of immune system in tumor progression (Hussein 2004). The character highly immunogenic of melanocytic tumors and the influence of host immune response and microenvironment inflammatory cells in cancer growth were verified (Hussein 2004; Dranoff 2009; Chen et al. 2011). Indeed, primary melanomas undergo spontaneous regression much more frequently than any other cancer that may be a sign of immunosurveillance or, by other way, it may be due melanoma are easier to visualize (Printz 2001; Kalialis et al. 2009). Nevertheless, other evidences corroborate the involvement of immune system, namely the relatively amount of tumor-infiltrating lymphocytes in melanoma microenvironment compared with other cancers and associated with favorable prognostic (Maio 2012; Kushnir and Merimsky 2013) and the appearance of autoimmune condition as vitiligo in melanoma patients or patients treated with immunotherapy which normally means a better disease outcome (Le Gal et al. 2001; Phan et al. 2001; Boasberg et al. 2006). In more advanced stages, this cancer became capable to avoid immunosurveillance (Reiman et al. 2007; Speeckaert et al. 2011) and its progression and metastatic potential may be supported by immune cells present in tumor microenvironment, as the tumorassociated macrophages (TAMs) since they are the most abundant leucocytes in melanoma and represent a poor prognostic (Brocker et al. 1988; Bernengo et al. 2000; Makitie et al. 2001; Varney et al. 2005; Porta et al. 2007; Solinas et al. 2009; Mantovani and Sica 2010; Qian and Pollard 2010). Immune System Immune system is a complex network of cells, tissues and organ that plays an important role in defense against multiple microorganisms and toxins and it is essential to organism homeostasis by respond not only to exogenous, but also to endogenous signals (Matzinger 2002). As is known, this system could be divided in two components, the innate and adaptive immunity. I. 7 Innate immunity is the immediate response to a “danger” and comprises a variety of cells, including dendritic cells (DC), macrophages, neutrophils and natural killer (NK) cells (Vesely et al. 2011; Lakshmi Narendra et al. 2013). Adaptive immunity is antigen specific due to somatic rearrangement on genes that codify each receptor of lymphocytes, i.e. the T cell receptor (TCR) for T lymphocytes and immunoglobin for B lymphocytes. Beyond T and B lymphocytes, adaptive immune system comprises humoral mediators including cytokines and antibodies (Vesely et al. 2011; Lakshmi Narendra et al. 2013). Macrophages Macrophages are essential cells of innate immune system that play an important role in host defense and tissue homeostasis maintenance (Gordon and Martinez 2010). They derived from circulating monocytes originates from monocytic precursors of bone marrow. Monocytes are attracted to target tissues where differentiate into mature macrophages (Murray and Wynn 2011; Davies et al. 2013). Macrophages are heterogeneous cells able to polarize in different subtypes among the classic phenotype (M1) and the alternatives (M2-like), according to the received stimuli which influence their immune response (Figure 3) (Mantovani et al. 2002; Martinez et al. 2009). The M1 phenotype is triggered by Th1 (T-helper 1 lymphocyte) cytokine interferonγ (IFN-γ), bacteria constituents (like LPS) or Toll-like receptors (TLRs) agonists. Once activated, M1 macrophages produce pro-inflammatory cytokines such as TNF-α, IL-1β, IL6 and IL-23, great quantities of MHC (major histocompatibility complex) class I and II (essential for antigen presentation), nitric oxide (NO) and pro-inflammatory chemokines. They promote the recruitment of Th1 cells, CD8+ CTL (cytotoxic T cells) and NK cells leading to an inflammatory response and antitumor immunity. (Mantovani et al. 2002; Mantovani et al. 2004; Fairweather and Cihakova 2009; Hao et al. 2012; Sica and Mantovani 2012). Conversely, M2 macrophages are activated by Th2 cytokines, including IL-4 and IL-13, or only by IL-10. They express high levels of IL-10 and TGF-β and immunosuppressive chemokines. M2-like phenotype favors the recruitment and development of Treg (regulatory T cells) and Th2, leading to a response that supports tumor growth through immunosuppression (Coffelt et al. 2009; Martinez et al. 2009; Siveen and Kuttan 2009; Hao et al. 2012; Sica and Mantovani 2012). However, is difficult to establish a linear effect of M1 and M2 macrophages in immune system. I. 14 References Akao Y, Nakagawa Y, Iinuma M et al (2008) Anti-cancer effects of xanthones from pericarps of mangosteen. Int J Mol Sci 9: 355-70. Akhurst RJ & Derynck R (2001) TGF-β signaling in cancer – a double-edged sword. Trends in Cell Biology 11: S44-S51. 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State of art III. 30 The main goal of this project was the evaluation of two xanthone derivatives, alphamangostin and 1,2-dihydroxyxanthone, in modulation of macrophages activity as well as the result in cytotoxic effect on melanoma cell line, since macrophages are key cells in tumor immune microenvironment. Therefore, the following were required: Evaluate the antitumor activity of referred compounds in the growth of A375-C5 human melanoma cell line by SRB assay; Measure the effects of xanthone-induced macrophage-conditioned medium on the proliferation of the human melanoma cell line A375-C5 by SRB assay; Compare the effects of the compounds and the macrophages-conditioned medium on the growth of A375-C5 cell line; Evaluate the interference of the compounds with NO production by LPS-stimulated RAW 264.7 murine macrophages, using Griess reagent; Access the impact of compounds on the cytokine expression profile by PMAdifferentiated THP-1 cells and their polarization into M1 (IL-1 and TNF-α) or M2 (IL10 and TGF-β1) phenotype, by an ELISA kit; Determine the antiproliferative effect of compounds on PHA stimulated human mononuclear cells by MTT assay and their interference with cytokines expression, using an ELISA kit. Define THP-1, RAW 264.7 and human mononuclear cells viability after treatment with the compounds by MTT-assay. IV. Materials and Methods IV. 32 Chemicals and reagents Reagents used in cell culture, including RPMI-1640, DMEM medium, and fetal bovine serum (FBS) were purchased from Gibco® Invitrogen Co. (Barcelona, Spain), 2mercaptoethanol for synthesis was obtained from Merck (Whitehouse Station, NJ, USA), Glutamine cell culture grade and Dimethyl sulfoxide (DMSO) from Applichem (Darmstadt, Germany). N.N-Dimetilformamida (DMF) was acquiring from Spectramol Science Incorporated. Reagents used in SRB assay as Trichloroacetic acid (TCA) and Acetic acid glacial 99100% were purchased from Prolabo (Oeiras, Portugal) and CHEM-LAB (Zedelgem, Belgium), respectively. IL-1β, IL-10, TNF-α and TGF-β1 ELISA Ready-Set-Go Kits were acquired from eBioscience (San Diego, CA, USA). All other chemicals of analytical grade used in the experiments and unless otherwise indicated were purchased from Sigma-Aldrich® (St. Louis, MO, USA). Xanthones Alpha-mangostin (α-MG) was obtained from Sigma-Aldrich® (ref.M3824) and 1,2dihydroxyxanthone (1,2-DHX) was synthetized in Laboratory of Organic and Pharmaceutic Chemistry (Faculty of Pharmacy, University of Porto) and in CEQUIMED (Centro de Química Medicinal; University of Porto) as previously described (Gales et al. 2001) Stock solution of compounds kept at -20ºC in DMSO. Just prior each assay, stock solutions were diluted in appropriate complete medium to the maximum concentration desired to test and 1:2 dilutions were serially prepared. Cell lines A375-C5 human malignant melanoma cell line and RAW 264.7 mouse macrophage cell line are part of CEQUIMED cell culture collection. THP-1 human monocyte cell line was a courtesy of Rui Appelberg. A375-C5 was routinely maintained in 25 cm2 flasks containing RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1 µL mL-1 Gentamycin at 37ºC in a humidified incubator with 5% CO2. For the THP-1 cell line the culture medium just described was also supplemented with 0.05 mM 2-mercaptoethanol. RAW 264.7 cell line was maintained in 75 cm2 flasks containing DMEM medium with the same percentage of FBS and gentamycin, and at the same conditions. IV. 33 Cell lines were sub-cultured every 2 or 3 days by trypsinization (A375-C5), replacement of the medium (THP-1) or scraping (RAW 264.7) and used for each experiment when the cells were in exponential growth. Human mononuclear cells were isolated from peripheral blood of healthy volunteers by Histopaque-1077 density centrifugation. All volunteers assign an informed consent. Mononuclear cells were ressuspended in RPMI medium supplemented with 10% FBS and 1 µL mL-1 gentamycin. Cell growth assay The effect of α-MG on the growth of A375-C5 cell line was determined by sulphorrodamine B (SRB) method adopted by the National Cancer Institute (NCI, USA) (Monks et al. 1991) and as already described by our group (Pedro et al. 2002; Gupta et al. 2008). A375-C5 cell line was plated into 96-well flat-bottom tissue culture plates at a concentration of 7,5 x 104 cells mL-1 in complete culture medium and incubated for 24 h in a 5% CO2 humidified incubator (HERA cell 150, Heraeus) at 37ºC. Once adherent, cells of one of the plates were immediately fixed for a no-growth control (designed Tzero plate) to determine the basal protein amount. Simultaneously, cell of test plates were treated with serial dilutions of xanthones and incubated for another 48h. Cells were then fixed with 50 µL of 50% TCA solution and incubated for 1h at 4ºC. Cells were washed five times with deionized water and allowed to dry. Fixed cells were exposed to 50 µL SRB dye (0,4% w/v in 1% acetic acid) for 30 minutes at room temperature. The unbound dye was removed by wash five times with 1% (v/v) acetic acid manually. Once completely dried, 100 µL of 10 mM Tris base solution was added and left for 30 minutes at room temperature to solubilize SRB. The absorbance was measured in an ELISA reader (Stat Fax 3200, Awareness Technology). Doxorubicin (1:10 dilutions) was used as positive control. Growth inhibition of 50% (GI50) was calculated by comparing, after 48 h, the absorbance of the xanthone-treated cells with the absorbance of Tzero plate. Lower absorbance after 48 h of treatment indicate occurrence of cell death instead of growth arrest (Monks et al. 1991). IV. 34 Antitumor effect of conditioned macrophages culture medium THP-1 human monocyte cell line is a simple, suitable and reliable model to study monocytes and macrophages functions or responses and the possible effects from external stimuli as new drugs. Differentiation of this cell line in mature THP-1 monocytederived macrophages can be achieve using phorbol-12-myristate-13-acetate (PMA), the most efficient differentiation agent in this case (Chanput et al. 2012; Chanput et al. 2014). THP-1 cell line was plated at 1 X 105 cells mL-1 in 96-wells flat-bottom plates and differentiated into THP-1 macrophages by adding 50 µL of 10 ng mL-1 PMA for 72h in a humidified incubator at 37ºC and 5% CO2 (He et al. 2012). Once differentiated, cells were washed twice with complete medium and they were incubated for another 24h in culture medium to obtain the resting stages of macrophages (Chanput et al. 2012; Chanput et al. 2014). Then, they were stimulated by 100 µL of a solution of 1 µg mL-1 LPS, simultaneously treated with the xanthone in test and incubated for 24h at 37ºC in a 5% CO2 humidified incubator (He et al. 2012). Plates were centrifuged to deposit the compounds and half of the volume of each well was added to A375-C5 adherent cells, previously plated as described in section “cell growth assay”. After a 48h incubation, SRB assay was performed, absorbance was measured and cell-growth inhibition determinate as just described (Pedro et al. 2002; Gupta et al. 2008). NO production assay The effect of the different compounds in nitric oxide (NO) production by RAW 264.7 was determined by the Griess reaction as previous described by our group (Teixeira et al. 2005). RAW 264.7 (1 X 106 cells mL-1) were platted on 96-well flat-bottom plates and allowed to adhere for 2h. Supernatants were removed and replaced by 100 µL of fresh medium containing 1.5 µg mL-1 LPS in order to induce iNOS synthesis by macrophages. The cell were also treated with 100 µL of each xanthone concentration (1:2 dilution) and incubated for 24h at 37ºC in a 5% CO2 humidified incubator. Then, 100 µL of supernatants were transferred to a new 96-well flat-bottom plates, 100 µL of Griess reagent (some quantities of a solution of 1% w/v sulphanilamide in 5% v/v phosphoric acid and a solution of 0.1% w/v naphtylethylenediamide in deionized water) was added and it was incubated for 10 min at room temperature (Green et al. 1982). Nitrite production was quantified by spectrophotometry at 550nm in an ELISA reader. N-nitro-L-arginine methyl ester (LNAME) (inhibitor of iNOS activity) and Dexamethasone (inhibitor of iNOS expression) were used as positive controls. Inhibition of 50% NO production was measured by comparing percentage of NO in treated cells and in non-treated cells. IV. 35 Compounds were also added 6h or 14h after LPS stimulation and significant differences detected in NO production between the different moments when xanthones were added, works to infer if compounds act in iNOS expression and/or activity. NO scavenging assay To discard NO scavenging effect by xanthones, nitrite was chemically generated, as previously described (Teixeira et al. 2005). 50 µL of sodium nitoprusside (5 mM) in PBS was added to xanthone dilution and incubated for 150 min at 25ºC. Griess reagent was added and nitrite accumulation was quantified. Sodium nitoprusside in PBS (100% production) and in ethanol/PBS (9:1; 0% production) were used as controls. Scavenging activity, determined in terms of percentage of nitrite formation, was present when the percentage of nitrite formed in the presence of the sample was less than 70% of the sodium nitroprusside control. Human mononuclear cells MTT-proliferation assay Human mononuclear cells (50 µl of a concentration of 2-3 X 106 cell mL-1) were plated in 96-well flat-bottom plates, treated with 50 µL of serial dilutions of α-MG and stimulated with 50 µL of PHA (Phytohaemagglutinin; used as a mitogen to trigger T-lymphocyte cell division) (10 µg mL-1) and incubated for 96h at 37ºC in a 5% CO2 humidified incubator. After incubation, 25 µL of 1 mg mL-1 MTT solution (3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide) was added to cells. After 4h, the formazan product was solubilized overnight with a sodium lauryl sulfate/N,N-dimethylformamide (SDS/DMF) solution and absorbance measured at 550 nm (Pedro et al. 2002; Cerqueira et al. 2003; Teixeira et al. 2005). Cyclosporin A was used as positive control. Lymphocyte toxicity, determined in terms of the percentage of viable cells, was present when the viability of the treated cells, compared with that of the non-treated control cells, was less than 70%. Cytokine quantification Culture supernatants of THP-1 differentiated, stimulated and treated cells, as previously described in section “Antitumor effect of conditioned macrophages culture medium”, were storage at - 20ºC until cytokine analyses. Levels of interleukin-1β (IL-1β), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and transforming growth Factor-β1 (TGF-β1) in culture supernatants were quantified by IV. 36 ELISA, using ELISA Ready-SET-Go kit (affymetrix, ebioscience) according to manufacturers’ instructions. The same cytokines were also evaluated in supernatants of human mononuclear cells treated with xanthones for 48h (Lou et al. 2013; Wang et al. 2013), incubation with PHA stimulus and three concentrations of α-MG. Interference of both compounds or α-MG in cytokines expression by THP-1 and lymphocytes, respectively, was accessed comparing treated with non-treated cells. MTT-viability assay Viability assays were performed as described elsewhere by our group (Cerqueira et al. 2003; Teixeira et al. 2005). For lymphocytes, MTT-viability assay was performed after 24h of cells treatment with xanthones. For all the other cells, viability assay was executed at the end of the experiments to determine the effect of the compound on the cell line. Briefly, to cells supernatants, 25 µL of MTT solution was added followed by 4 h incubation at 37ºC in a 5% CO2 humidified incubator. After incubation, 50 µL of SDS/DMF solution was added and absorbance measured at 550 nm (Pedro et al. 2002; Cerqueira et al. 2003; Teixeira et al. 2005). Cell toxicity was considered when the viability of the exposed cells was less than 70%. Statistical analysis Except otherwise stated, results are the mean ± SEM of at least three independent experiments, performed in duplicate. Statistical analysis was performed with SPSS for Windows (version 20.0). Statistical significance between two mean was calculated by Mann-Whitney Test and it is considered for p values less than 0.05. Ethics Ethics approved was obtained by the Ethic Comity of University Fernando Pessoa. IV. 37 References Cerqueira F, Cordeiro-Da-Silva A, Araujo N et al (2003) Inhibition of lymphocyte proliferation by prenylated flavones: artelastin as a potent inhibitor. Life Sci 73: 2321-34. Chanput W, Mes JJ & Wichers HJ (2014) THP-1 cell line: An in vitro cell model for immune modulation approach. Int Immunopharmacol. Chanput W, Reitsma M, Kleinjans L et al (2012) beta-Glucans are involved in immunemodulation of THP-1 macrophages. Mol Nutr Food Res 56: 822-33. Gales L, Sousa MED, Pinto MMM et al (2001) Naturally occurring 1,2,8trimethoxyxanthone and biphenyl ether intermediates leading to 1,2-dimethoxyxanthone. Acta Crystallographica Section C 57: 1319-1323. Green LC, Wagner DA, Glogowski J et al (1982) Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126: 131-8. Gupta S, Rodrigues LM, Esteves AP et al (2008) Synthesis of N-aryl-5-amino-4cyanopyrazole derivatives as potent xanthine oxidase inhibitors. Eur J Med Chem 43: 771-80. He X, Shu J, Xu L et al (2012) Inhibitory effect of Astragalus polysaccharides on lipopolysaccharide-induced TNF-a and IL-1beta production in THP-1 cells. Molecules 17: 3155-64. Lou X, Hou Y & Liang D (2013) Effects of hepatitis B virus X protein on human T cell cytokines. Can J Microbiol 59: 620-6. Monks A, Scudiero D, Skehan P et al (1991) Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 83: 757-66. Pedro M, Cerqueira F, Sousa ME et al (2002) Xanthones as inhibitors of growth of human cancer cell lines and their effects on the proliferation of human lymphocytes in vitro. Bioorganic & medicinal chemistry 10: 3725-30. Teixeira M, Cerqueira F, Barbosa CM et al (2005) Improvement of the inhibitory effect of xanthones on NO production by encapsulation in PLGA nanocapsules. J Drug Target 13: 129-35. Wang H, Daniel V, Sadeghi M et al (2013) Differences in the induction of induced human CD4(+) CD25(+) FoxP3(+) T-regulatory cells and CD3(+) CD8(+) CD28(-) Tsuppressor cells subset phenotypes in vitro: comparison of phorbol 12-myristate 13acetate/ionomycin and phytohemagglutinin stimulation. Transplant Proc 45: 1822-31. V. Natural Xanthones: alpha-mangostin Papers 1 and 2 V. 46 change according to several conditions as age, gender, stress, environment, among others. Consequently, the immune response is hard to predict and interpret (Cohen et al. 1991; Lesourd 2002; Beery 2003; Srinivasan et al. 2005). In cancer, for example, the innate and adaptive immunity have a dual effect, either preventing tumour progression or stimulating their growth, invasiveness and metastatic activity (Dranoff 2004; Gutkin and Shurin 2014). The immune function is essential for organism homeostasis, responding to exogenous and endogenous risk signals (Matzinger 2002). Therefore, the uncontrolled and unmeasured use of antiphlogistic agents, as mangosteen products, may cause severe adverse effects in consumers. Exploring recent reports of immunomodulation mediated by these products, we noticed that many of these possibly trigger body deregulation. The inhibition of pro-inflammatory genes triggered by lipopolysaccharides (LPS) was recurrently described and related with xanthones exposure in different cell lines. Many authors reported a decrease in gene expression of tumour necrosis factor α (TNF-α), several interleukins (IL-1β, IL-6, IL-8), monocyte chemoattractant protein-1 (MCP-1) and Toll-like receptor-2 (TLR-2) in primary cultures of human adipocytes, human U937 macrophages-like cells and murine RAW 264.7 macrophage-like cells treated with αand/or γ-mangostin (Sampath and Vijayaragavan 2008; Bumrungpert et al. 2009a; Bumrungpert et al. 2009b; Tewtrakul et al. 2009; Bumrungpert et al. 2010; Liu et al. 2012; Gutierrez-Orozco et al. 2013). But this effect is not considered to be exclusive of macrophages and adipocytes, Gutierrez-Orozco et al. detected similar inhibition, mediated by α-mangostin, in human monocyte cell line (THP-1) and different cancer cell lines (HepG2, Caco-2 HTB-37, and HT-29) (Gutierrez-Orozco et al. 2013). The described alterations on pro-inflammatory genes seems to be associated with the interference in some immune pathways, those involving conventional mitogen-activated protein kinases (MAPKs), activator protein 1 (AP-1), signal transducer and activator of transcription 1 (STAT-1) and nuclear factor kappa B (NF-κB) (Sampath and Vijayaragavan 2008; Bumrungpert et al. 2009a; Tewtrakul et al. 2009; Liu et al. 2012). These protein cascades are also involved in stress response and participate in crosstalk with other signal pathways (Ghosh et al. 1998; Ramana et al. 2000; Chang and Karin 2001; Li and Verma 2002; Bonizzi and Karin 2004; Wada and Penninger 2004) which means that the effect of xanthones from mangosteen, namely αand/or γ-mangostin, will probably not be confined to immune system modulation. V. 47 Additional restriction on immunity cells action seems to occur at basophils and mast cells level. Xanthonic compounds (α-, βand/or γ-mangostin) and extracts of mangosteen reduce degranulation of these cells preventing histamine release possibly by interfering with downstream signals (Nakatani et al. 2002a; Itoh et al. 2008). Moreover, the histaminergic receptors are blocked by α and γ-mangostin (Chairungsrilerd et al. 1996). As consequence of this blockage, these compounds deregulate the interaction histamine-receptors, a binding implicated in some pathological functions including cancer and intestinal ischemia promotion (Kusche et al. 1980; Raithel et al. 1998), along with several vital functions, as neurotransmission, immunomodulation, haematopoiesis, wound healing and day-night rhythm (Maintz and Novak 2007). According to that, a careful medical follow-up is needed to prevent possible complications. Anti-inflammatory activity of mangosteen extracts and αand/or γ-mangostin have been still linked with enzymatic alterations, namely through inhibition of the mRNA expression and/or activity of cyclooxygenases 1 and 2 (COX-1 and COX-2) and/ or inducible nitric oxide synthases (iNOS) (Nakatani et al. 2002b; Nakatani et al. 2004; Chen et al. 2008; Tewtrakul et al. 2009). COX-1 and COX-2 are involved in physiologic processes as well as pathophysiologic events. COX-1 is traditionally associated with homeostasis functions, including gastric cytoprotection and haemostasis. COX-2 is connected to inflammation and tumourigenesis. In spite of this, complementary functions between the two enzymes and variations on their actions depending of the specific target organs were reported (Masferrer et al. 1994; Dubois et al. 1998; Marnett 2009; Rouzer and Marnett 2009). Inducible nitric oxide synthases (iNOS) is responsive for nitric oxide (NO) production after stimulation by immunological stimuli, being an intermediary of inflammatory response, however the excess or insufficiency of nitric oxide production can cause equally cell damage (Gross and Wolin 1995). In cancer, NO seems to have dual effects: multiple reports has associated NO with cancer progression while others has defended the therapeutic benefits of NO donors in different cell lines and organisms (Wink et al. 1991; Lala and Orucevic 1998; Wink et al. 1998; Ziche and Morbidelli 2000; Lala and Chakraborty 2001; Choi et al. 2002; Mocellin et al. 2007; Coulter et al. 2008; Yasuda 2008). Considering the importance of all these enzymes in biochemical and physiological events the role of xanthones contained in mangosteen extracts should be carefully evaluated. V. 48 3.2. Cell Cycle arrest Cell cycle is a sequence of highly regulated events essential for all living beings. In multi-cell organisms are necessary several rounds of cell division to create a new individual and during their lifetime it is a crucial mechanism for maintaining appropriate cellular and tissue condition once cell death and damages are constant (Alberts 2008). Mechanisms of action of some xanthones from mangosteen are frequently associated with cell cycle arrest. Since the cell cycle deregulation is a hallmark of cancer and one of the earliest processes of oncogenesis (Syljuasen et al. 1999; Hanahan and Weinberg 2011), several authors purposed and proved this effect of xanthones in different tumor (Matsumoto et al. 2005; Wang et al. 2011; Johnson et al. 2012; Kurose et al. 2012; Leão et al. 2013). Matsumoto et al. related that xanthones induced cell-cycle arrest in a human colon cancer cell line. Mangostins α and β up-regulated p27 and down-regulated cyclin A, B1, D1 and E1 and cdc2, phospho-cdc2 (tyrosine 15) expression and consequently act at G1-phase, blocking the division progression. However, while some authors affirm that γ-mangostin acts at S-phase in human colon cancer (Matsumoto et al. 2005), others indicate a G1-phase arrest in a human melanoma cell line (Wang et al. 2011). Johnson et al. reported that a prostate cancer cell line treated with α-mangostin inhibited cyclins/cyclin-dependent kinases 4 (CDK4) binding, an vital protein complex to cell cycle progression from G1 to S-phase (Johnson et al. 2012). According to that, Kurose et al. described an up-regulation of p21cip1 and CHEK2 (regulator factors of G1-phase) in breast cancer model after treatment with α-mangostin (Kurose et al. 2012). The inhibition of p53-MDM2 interaction due to a potential binding between αmangostin and MDM2 was also detected by some of our group (Leão et al. 2013). Free p53 stops cell division at both G1 and G2 checkpoints (Agarwal et al. 1995). In accordance with our finds, further authors have reported enhance of p53 levels after colon and blander cancer treatment with α-mangostin and gartanin, respectively (Aisha et al. 2012; Liu et al. 2013). In summary, some xanthones from mangosteen are potent inhibitors of cell cycle progression by blocking the division at different levels in different cell models, as illustrated in Figure 8. V. 49 Figure 8 Effect of xanthones of mangosteen at different stages of cell cycle regulation. Legend: cdc indicate cell division cycle protein; CDK, cyclin-dependent kinases; Chk or CHEK2, checkpoint kinase; G1, gap phase; G2, gap phase 2; M, mitosis phase; MDM2, murine doble minute 2; MG, mangosteen xanthones; p21cip1, cyclin-dependent kinase inhibitor 1; p27kip1, cyclin-dependent kinase inhibitor; p53, tumor protein p53; S, DNA synthesis phase. Besides the importance of that effect in cancer treatment, normal cells have to be replaced to keep the tissue homeostasis, specially, tissues with constitutive division. Labile tissues as hematopoietic cells in the bone marrow and the majority of epithelial tissue (e.g., skin, oral cavity, vagina and cervix, salivary glands, pancreas, biliary and gastrointestinal tracts, uterus, fallopian tubes and urinary tract) must have a functional cell-cycle to replace the lost cells in injuries and aging process (Kumar et al. 2012). Hence that, xanthones of mangosteen referred above (mangostins α, β, γ and gartanin) as cell cycle arrest agents may interfere with normal cells replacement. 3.3. Antioxidant effect Oxidative stress is a natural process implicated in cell damage or death and it has been associated with several human pathologies, like cardiac and neurodegenerative diseases and cancer (Valko et al. 2007). The prevention of such stress through antioxidant products has been extensively study, however the potential benefits or risks associated are still ambiguous (Stanner et al. 2004; Bjelakovic et al. 2013). In oncologic patients the advantages of antioxidant use are unclear. Some studies admitted survival gains or no interference with chemo or radiotherapy, while others V. 50 suggest a reduction of the therapeutic effect of chemo or radiotherapy in patients simultaneous using antioxidant products (D'Andrea 2005; Moss 2007; Lawenda et al. 2008; Block et al. 2009). As referred previously, many xanthones from mangosteen have antioxidant activity (Moongkarndi et al. 2004; Jung et al. 2006; Weecharangsan et al. 2006; Devi Sampath and Vijayaraghavan 2007; Kosem et al. 2007; Chin et al. 2008). In fact, Devi Sampath and Vijayaraghavan 2007 described an attenuation of isoproterenol oxidative effect in rats treated with α-mangostin compared with no-treated rats. They demonstrated an augment of antioxidant enzymes glutathione-S-transferase (GST), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT), as well as a reduction of glutathione (GSH), lipid peroxides and serum enzymes, including lactate dehydrogenase (LDH), creatine phosphokinase (CPK), glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) (Devi Sampath and Vijayaraghavan 2007). In generally, it is known that some drugs, including cancer drug therapies, can be affected by xanthones from mangosteen antioxidant potential. These xanthonic derivatives have been connected with alterations on metabolizing enzymes responsible for the metabolism of several drugs (Foti et al. 2009). The changes that can occur in effect of drugs when co-administrated with these compounds will be discussed in the following section. Considering the referred controversial use of antioxidants, further studies are necessary to confirm the safety of the use of that herbal folk medicine. 3.4. Modifications in the metabolism of therapeutic agents On living organisms, exogenous compounds are subjected to biochemical modifications catalysed by enzymes responsible for detoxification. Alterations in activity of these drug-metabolizing enzymes may jeopardize the treatment response, namely in efficacy, toxicity or both (Lu 1998; Evans and Relling 2004). Xanthonic components of mangosteen were already associated with modulation of enzymes involved in phase I and phase II of the metabolism (Devi Sampath and Vijayaraghavan 2007; Balunas et al. 2008; Foti et al. 2009). The CYP family are phase I enzymes capable to metabolize the majority of drugs (Kalra et al. 2009) including natural xanthones, as already was referred. In spite of that, mangosteen has been associated with inhibition of enzymatic activity of some CYP members. Foti et al. showed that aqueous mangosteen extract extensible V. 51 reduced the activity of CYP2C8 and CYP2C9 in human liver microsomes; further enzymes were also inhibited but to a less extent. These authors proposed that such effect is associated with the presence of α-mangostin, β-mangostin, gartanin, 3isomangostin and 8-desoxygartanin in the extract (Foti et al. 2009). Balunas et al. also described a potent inhibition of CYP19 (aromatase) mediated by methanol and chloroform-soluble extracts of G. mangostana in a human placental microsomes and by some xanthones (specially γ-mangostin) in a human breast cancer cell line (Balunas et al. 2008). CYP2C8 and 2C9 metabolize important drugs (as indicated in Table 1) including the therapeutic agents ibuprofen (Davies 1998; Garcia-Martin et al. 2004), paclitaxel, phenytoin (Burns 1999; Foti et al. 2009) and warfarin (Burns 1999; Rettie and Tai 2006; Foti et al. 2009), as well as amitriptyline (Ghahramani et al. 1997; Olesen and Linnet 1997), cyclophosphamide (Chang et al. 1993; Chang et al. 1997), diazepam (Shou et al. 2000) and losartan (Zhou et al. 2009; Mannheimer and Eliasson 2010). Therefore, it is predictable that some of these drugs may become less or even ineffective when co-administered with mangosteen products and their toxicity can also be increased. Similarly, the aromatase inhibition mediated by mangosteen might increase the adverse effects of drugs like methadone (Nekhayeva et al. 2005) and hormones androstanolone and nortestosterone (Silberzahn et al. 1988; Douglas et al. 2005) or reduce the treatment response to others such as methadone derivate levacetylmethadol (Deshmukh et al. 2004) and the testosterone (Silberzahn et al. 1988; Rendic 2002; Morale et al. 2008) (Table 1). Additionally to the metabolic activity, CYP19 are also responsible for the bioconversion of androgens into estrogens (Simpson et al. 1994). Consequently, the potential of aromatase inhibitors, like mangosteen (α, γ-mangostin and garcinones D/E), in treatment of many hormoneresponsive tumors as breast cancer cannot be ignored (Azria et al. 2005; Brueggemeier 2006; Kendall and Dowsett 2006). Beyond the CYP family, the antioxidant effect of α-mangostin by stimulating several enzymes including the glutathione S-transferases (GSTs) (Devi Sampath and Vijayaraghavan 2007) must be considered. GSTs are phase II enzymes that catalyse the conjugation of many endogenous substances and xenobiotics with glutathione (GSH) (Uetrecht and Trager 2007; Oakley 2011; Bousova and Skalova 2012). GSHconjugation results, normally, in drug activity inhibition due to increase in solubility of compounds and consequently the facility of excretion (Barnouin et al. 1998; Peklak- V. 52 Scott et al. 2005). However in some cases, drug conjugation promotes drug activation (Morgan et al. 1998; Findlay et al. 2004; Tew 2005). The anticancer drugs, PABA/NO and TLK286 are examples of compounds activated after conjugation (Morgan et al. 1998; Findlay et al. 2004; Tew 2005) (Table 1). Consequently, the co-administration with α-mangostin could promote accumulation of the active metabolites and increasing the side effects. Table 1. Potential effects of xanthones derivatives on drugs metabolism. Effect on some drug-metabolizing enzymes Drugs metabolized by the enzymes # Therapeutic activity affected References # Decrease CYP2C8 and CYP2C9 activity Amprenavir Antiretroviral (HIV-1 inhibitor) (Fung et al. 2000) Bupropion Antidepressant tetracyclic (Preissner et al. 2010; Knox et al. 2011) Capsaicin Analgesic (natural) (Reilly et al. 2003) Cisapride Gastrointestinal prokinetic agent (Desta et al. 2000; Preissner et al. 2010) Clozapine * Antipsychotic (Fang et al. 1998; RostamiHodjegan et al. 2004) Cyamemazine Antipsychotic and anxiolytic (Arbus et al. 2007) Dapsone * Antibacterial (Winter et al. 2000) Dextromethorphan Cough suppressant (von Moltke et al. 1998; Preissner et al. 2010) Diclofenac * Analgesic, antipyretic, antiinflammatory (Mancy et al. 1999) Diltiazem * Cardiovascular (Sutton et al. 1997) Estradiol Sex hormones (estrogen) (Yamazaki et al. 1998; Cheng et al. 2001) Fluvastatin * Lipid regulating (Scripture and Pieper 2001) Ibuprofen * Non-Steroidal AntiInflammatory (Davies 1998; Garcia-Martin et al. 2004) Irbesartan * Cardiovascular (Hallberg et al. 2002; Preissner et al. 2010) Ketamine Anesthetic and analgesic (Hijazi and Boulieu 2002; Preissner et al. 2010) Ketobemidone Analgesic (Kristensen et al. 1996; Yasar et al. 2005) Lansoprazole * Gastro-intestinal (Pearce et al. 1996) Methadone Analgesic, antipyretic, antiinflammatory (Rendic 2002; Zhou et al. 2009) Omeprazole * Gastro-intestinal (Foti et al. 2009; Preissner et al. 2010) Paclitaxel Antineoplastic (Burns 1999; Foti et al. 2009) Perphenazine Antipsychotic (Olesen and Linnet 2000; Rendic 2002; Preissner et al. 2010) Phenytoin * Antiepileptic (Burns 1999; Foti et al. 2009) Phenprocoumon Cardiovascular (Ufer et al. 2004) Piroxicam * Analgesic, antipyretic, Non-steroidal antiinflammatory (Zhao et al. 1992; Perini et al. 2005; Preissner et al. 2010) Progesterone * Sex hormone (Progestagen) (Yamazaki and Shimada 1997; Preissner et al. 2010) Propofol * General anesthetic (Guitton et al. 1998) Quinidine * Cardiovascular (Nielsen et al. 1999; Preissner et al. 2010) Rosiglitazone * Antidiabetic (Baldwin et al. 1999; Malinowski and Bolesta 2000) V. 53 Selegiline, Deprenyl * Antidepressant (Rendic 2002; Salonen et al. 2003) Seratrodast * Cardiovascular (Kumar et al. 1997) Temazepam Anxiolytic, sedative, hypnotic (Ono et al. 1996; Yang et al. 1998; Rendic 2002) Terbinafine Antifungal (Vickers et al. 1999; Rendic 2002) Thalidomide Antineoplasic (Ando et al. 2002) Tolbutamide * Antidiabetic (Srivastava et al. 1991; Rendic 2002) Torasemide, Torsemide Cardiovascular (Diuretic) (Rendic 2002; Kerdpin et al. 2004; Vormfelde et al. 2004) Trimethadione, Troxidone Antiepileptic (Kurata et al. 1998; Rendic 2002; Tanaka et al. 2003) Verapamil * Cardiovascular (Tracy et al. 1999; Anthony and Berg 2002) Warfarin* Cardiovascular (Anticoagulant) (Burns 1999; Rettie and Tai 2006; Foti et al. 2009) Zafirlukast * Bronchodilators and Antiasthma (Dekhuijzen and Koopmans 2002; Preissner et al. 2010) Zopiclone Anxiolytic, sedative, hypnotic (Becquemont et al. 1999; Rendic 2002) Acetylsalicylic acid Anticoagulants and Antithrombotics (Preissner et al. 2010; Knox et al. 2011) Amitriptyline* Antidepressant (Ghahramani et al. 1997; Olesen and Linnet 1997) Cyclophosphamide * Antineoplastic (Chang et al. 1993; Chang et al. 1997) Diazepam * Anxiolytic, sedative, hypnotic (Shou et al. 2000) Ifosfamide * Antineoplastic (Chang et al. 1993) Lidocain(e) Local anesthetic (Narang et al. 1978; Rendic 2002) Losartan * Anti-Hypertensives (Zhou et al. 2009; Mannheimer and Eliasson 2010) Pioglitazone * Antidiabetic (Jaakkola et al. 2006; Preissner et al. 2010) Sulfadiazine * Antibacterial (Cribb et al. 1995; Rendic 2002) Testosterone Sex hormone (Androgen and anabolic) (Yamazaki and Shimada 1997; Rendic 2002) Troglitazone * Antidiabetic (Yamazaki et al. 1999b; He et al. 2004) Zidovudine, azidothymidine Antiviral (Eagling et al. 1994; Rendic 2002) Brompheniramine Antihistaminic (Preissner et al. 2010; Knox et al. 2011) Caffeine Xanthine (CNS stimulant) (Preissner et al. 2010; Knox et al. 2011) Carbinoxamine Antihistamine and anticholinergic (Preissner et al. 2010; Knox et al. 2011) Leflunomide * Antirheumatic (Rozman 2002) Mefenamic acid * Analgesic, antipyretic, antiinflammatory (Preissner et al. 2010; Knox et al. 2011) Mephenytoin Antiepileptic (Relling et al. 1990; Goldstein et al. 1994) Meloxicam * Analgesic, antipyretic, antiinflammatory (Turck et al. 1996; Chesne et al. 1998; Preissner et al. 2010) Mirtazapine Antidepressant (Stormer et al. 2000; Rendic 2002) Nicotine Supplementary drugs and other substances (Yamazaki et al. 1999a; Rendic 2002) Paracetamol, acetaminophen Analgesic, antipyretic, antiinflammatory (Raucy et al. 1989; Rendic 2002) Phenazone, Antipyrine Analgesic, antipyretic, non-steroidal anti- (Engel et al. 1996; Rendic 2002) V. 54 Information concerning Cytochrome P450s and Glutathione S-transferases metabolism was obtained from SuperCYP (Preissner et al. 2010), DrugBank database (Knox et al. 2011) and additional papers; all drugs inflammatory Rifampicin * Antibacterial (Preissner et al. 2010; Knox et al. 2011) Rofecoxib Non-steroidal antiinflammatory (Preissner et al. 2010; Knox et al. 2011) Sulfinpyrazon(e) * Antigout (Rendic 2002) Theophylline Xanthine (Bronchodilator) (Preissner et al. 2010; Knox et al. 2011) Trimethoprim * Antiepileptic (Rendic 2002) Decrease CYP19 (aromatase) activity Betamethason(e) * Corticosteroid (Paakki et al. 2000) Letrozole * Antineoplastic (Azria et al. 2005) Androstanolone Sex hormones (Androgen) (Douglas et al. 2005) Methadone Analgesic, antipyretic, antiinflammatory (Nekhayeva et al. 2005) Nandrolone, nortestosterone Sex hormone (Progestagen) (Silberzahn et al. 1988) Levacetylmethadol (methadone derivate) Analgesic, antipyretic, antiinflammatory (Deshmukh et al. 2004) Testosterone Sex hormone (Androgen and anabolic) (Silberzahn et al. 1988; Rendic 2002; Morale et al. 2008) Increase GST activity Adriamycin or Doxorubicin Antineoplastic (Hayes and Pulford 1995; Lien et al. 2002; Hamilton et al. 2003) Busulfan Antineoplastic (Vassord et al. 2008; Elhasid et al. 2010) Carboplatin Antineoplastic (Marsh et al. 2009) Carmustin or BCNU Antineoplastic (Hayes and Pulford 1995; Lien et al. 2002; Hamilton et al. 2003) Chlorambucil Antineoplastic (Hayes and Pulford 1995; Lien et al. 2002; Hamilton et al. 2003; Parker et al. 2008) Cisplatin Antineoplastic (Hayes and Pulford 1995; Peters et al. 2000; Lien et al. 2002; Hamilton et al. 2003) Cyclophosphamide Antineoplastic (Hayes and Pulford 1995; Lien et al. 2002; Hamilton et al. 2003) Ecteinascidin-743; ET-743 or Trabectedin Antineoplastic (Brandon et al. 2006) Ethacrynic acid Diuretic (Hayes and Pulford 1995; Lien et al. 2002; Hamilton et al. 2003) Etoposide Antineoplastic (Mans et al. 1992) Glutathione Supplementary drug (Hayes et al. 2005) Melphalan Antineoplastic (Hayes and Pulford 1995; Lien et al. 2002; Hamilton et al. 2003) Mitozantrone Antineoplastic (Hayes and Pulford 1995; Lien et al. 2002; Hamilton et al. 2003) Oxaliplatin Antineoplastic (Marsh et al. 2009) Thiotepa Antineoplastic (Hayes and Pulford 1995; Lien et al. 2002; Hamilton et al. 2003) Azathioprine Antineoplastic (Gianluigi Zaza et al. 2010) TLK286 Antineoplastic (Morgan et al. 1998; Tew 2005) PABA/NO Antineoplastic (Findlay et al. 2004) V. 55 indicated on the table are approved by FDA; in drugs metabolized by CYP2C8 and 2C9, only those that are metabolized by the two enzymes was listed because the effect of xanthones might be more significant. LEGEND: CYP: Cytochrome P450; GST: Glutathione S-transferases; * drugs that, besides substrates, are also inhibitors and/or inducers of enzyme activity; # references related to drugs metabolized by such enzyme. 3.5. Blocking serotonin receptors Serotonin is a ubiquitous molecule that plays multiple physiological roles including cardiovascular, gastrointestinal and endocrine functions, as well as development, sensory perception, behaviours such as aggression, appetite, sex, sleep, mood, cognition, and memory (Aghajanian and Sanders-Bush 2002). The inhibition of 5-HT2A serotonin receptors mediated by γ-mangostin (Chairungsrilerd et al. 1996) was described in animal models; this effect may disrupt several essential functions to human survival. Serotonergic receptor blocking agents are current drugs used in nervous system disorders (Chairungsrilerd et al. 1996) making mangosteen supplements good candidates for intervention at this level. Their use by healthy people however needs to be monitored once this neurotransmitter is intervener in a variety of essential biological processes. 3.6. Other effects Many others unfavourable proprieties have been linked with the use of mangosteen-based products. Wong, et al described a case of severe lactic acidosis in a woman who took mangosteen juice. The authors postulated that this effect was mediated by αmangostin causing mitochondrial injuries (Wong and Klemmer 2008). The loss of mitochondrial potential has been described in general by many reviews concerning G. mangostana (Pinto et al. 2005; Pedraza-Chaverri et al. 2008; Obolskiy et al. 2009; Shan et al. 2011; Chitchumroonchokchai et al. 2013; Gutierrez-Orozco and Failla 2013); even if only a case of lactic acidosis was reported until now, associated with an acute respiratory illness, it is necessary to be watchful to understand the possible conditions that improve that risk of its use (Wong and Klemmer 2008). 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V. 68 Zhou SF, Zhou ZW, Yang LP et al (2009) Substrates, inducers, inhibitors and structureactivity relationships of human Cytochrome P450 2C9 and implications in drug development. Curr Med Chem 16: 3480-675. Ziche M & Morbidelli L (2000) Nitric oxide and angiogenesis. J Neurooncol 50: 139-48. V. 69 Paper 2 (draft): “Alpha-mangostin antitumor activity: cytotoxicity and influence on the immune system microenvironment” Abstract Alpha-mangostin is known to interfere with multiple pathways of carcinogenesis. Therefore, it has been proposed as prospective cancer drug candidate. In melanoma, alpha-mangostin antitumor effect may be increased by the immunomodulation of tumor microenvironment, since melanocytic tumors are highly immunogenic. The main gold of this study was evaluate alpha-mangostin (i) cytotoxic effect on A375C5 melanoma cell line and (ii) it interference with the immune macrophages induced microenvironment of the tumor. This xanthone showed inhibition of A375-C5 melanoma growth (GI50 = 11.3 ± 0.9 µM). Concerning the direct impact of alpha-mangostin on macrophages function, it was observed an inhibition of nitric oxide production by RAW 264.7 murine macrophages (IC50 = 13.8 ± 0.7 µM) and decreased the concentration of IL-1β and TGF-β1 and stimulated TNF-α expression by THP-1 human macrophages. In order to complement the described effect of α-MG on the immune microenvironment of melanoma, it was also study in proliferation and cytokines production by human lymphocytes. Alpha-mangostin strongly inhibited PHA-stimulated human mononuclear cells proliferation (IC50 = 9.2 ± 0.3 µM) but not interfering for IC50 for IL-1β, TNF-α, IL-10 and TGF-β1. In conclusion this study proved that alpha-mangostin interferes with cytokine and NO production by activated macrophages leading to the evidence that the xanthone interferes with immune microenvironment of the tumor. Introduction Melanoma is a melanocytes’ malignant tumor that metastasizes very early in the disease process, causing elevated mortality rates. According to the Oncologic register of Portugal (2007), the world incidence of melanoma of the skin is about 4,5 cases for 100000 habitants (RORENO 2013) and, although it is a relative rare kind of cancer, it contributes for about 80% of the deaths caused by cutaneous cancer (Kuphal and Bosserhoff 2009; Gast et al. 2011; Raaijmakers et al. 2013). The tumorigenesis of this cancer requires a multistep process, in spite of alterations in immune system has a crucial involvement, namely the immunosuppressive and pro-tumor character of tumor microenvironment (Hussein 2004; Chen et al. 2011). Among inflammatory cells, V. 70 macrophages are of pivotal importance in malignancy development, being specifically referred to as tumor-associated macrophages (TAMs) (Chen et al. 2011). TAMs are the most abundant leukocytes in the melanoma and their presence represent a poor prognostic (Brocker et al. 1988; Bernengo et al. 2000; Makitie et al. 2001; Varney et al. 2005), due to induction of an immunosuppressive microenvironment that, among other consequences, inhibits tumor-specific CD8+ T cell-mediated cytotoxicity (Kono et al. 1996; Wang et al. 2012c). The growing understanding of immune involvement in tumorigenesis, contributed to therapies aiming immunologic targets (Dranoff 2009; Raaijmakers et al. 2013). However some of these therapies are only effective in a brief period of time while others have considerable side effects (Hodi et al. 2010; Solit and Rosen 2011; Graziani et al. 2012). Obviously, it is important to find new therapies for melanoma that target multiple biochemical pathways, in order to avoid tumor escape mechanisms (Smalley et al. 2006). Prenylated xanthones, including alpha-mangostin (α-MG) (Figure 9), the most studied and abundant xanthone present on mangosteen pericarp (Garcinia mangostana Linn), have been associated to multiple benefits in several pathological conditions. In cancer, its effect is through the alteration of a significant number of physiological pathways, comprising the influence in immune system (Obolskiy et al. 2009; Shan et al. 2011; Gutierrez-Orozco and Failla 2013). Beside the previous reports of α-MG effect in immune functions, as far as we know the interference of this xanthone in the immune system microenvironment of melanoma has never been studied. Recently, a number of evidences suggested that α-MG has a potent antitumor effect against melanoma. These reports evaluated the effect of the compound in different melanoma cell lines (B16-F10 murine cell line, SK-MEL-28 human cell line) and described induction of apoptosis, cell cycle arrest and differentiation, and inhibition of both cell proliferation and metastasis (Wang et al. 2011; Wang et al. 2012a; Wang et al. 2012b; Wang et al. 2013). During the development of this study, Beninati et al reported some experiments of α-MG effect on the A375 cell line (Beninati et al. 2014). In spite of these insights, and as far as we know, no studies on α-MG-dependent modulation of the immune microenvironment and its effect on melanoma growth were performed. Due to the importance of immune targets in melanoma therapies, the aim of the present work is to study α-MG effects on the immune system in order to potentiate its antitumor effect on A375-C5 melanoma cell line. V. 71 Figure 9: alpha-Mangostin Material and Methods Chemicals and reagents Reagents used in cell culture, including RPMI-1640, DMEM medium, and fetal bovine serum (FBS) were purchased from Gibco® Invitrogen Co. (Barcelona, Spain), 2mercaptoethanol for synthesis was obtained from Merck (Whitehouse Station, NJ, USA) and Glutamine cell culture grade and Dimethyl sulfoxide (DMSO) from Applichem (Darmstadt, Germany). N.N-Dimetilformamida (DMF) was acquiring from Spectramol Science Incorporated. Reagents used in SRB assay as Trichloroacetic acid (TCA), Acetic acid glacial 99100% and Trizma base were purchased from Prolabo (Oeiras, Portugal), CHEM-LAB (Zedelgem, Belgium) and Frilabo (Maia, Portugal), respectively. IL-1β, IL-10, TNF-α and TGF-β1 ELISA Ready-Set-Go Kits were acquired from eBioscience (San Diego, CA, USA). All other chemicals of analytical grade used in the experiments and unless otherwise indicated were purchased from Sigma-Aldrich® (St. Louis, MO, USA). Xanthones Alpha-mangostin (α-MG) was obtained from Sigma-Aldrich® (ref.M3824) and stock solution kept at -20ºC in DMSO. Just prior each assay, stock solution was diluted in appropriate complete medium to the maximum concentration to test and 1:2 dilutions were serially prepared. Cell lines A375-C5 human malignant melanoma cell line and RAW 264.7 mouse macrophage cell line are part of CEQUIMED cell culture collection. THP-1 human monocyte cell line was a courtesy of Rui Appelberg. A375-C5 was routinely maintained in 25 cm2 flasks containing RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1 µL mL-1 Gentamycin at 37ºC in a humidified incubator with 5% CO2. For the THP-1 cell line the culture medium just V. 78 Antitumor effect of conditioned macrophage culture medium As modulation of the immune system has been a promising approach in melanoma treatment. It was investigated the possible influence of conditioned macrophage supernatants on A375-C5 cells growth to teste the influence of soluble immunologic factors produced by macrophages on melanoma cell line. A significant (p < 0.001) decrease in melanoma cell growth was observed after exposure to supernatants from both α-MG-treated and untreated THP-1 macrophages (Figure 12), which is in favor of an α-MG-independent anti-tumor effect of soluble mediators produced by cultured THP-1 macrophages. Figure 12: Antitumor effect of alpha-mangostin, macrophages supernatants and alpha-mangostin conditioned macrophage culture medium on A375-C5 melanoma cell line. THP-1 PMA-differentiated macrophages were treated with xanthone and supernatants were added to melanoma cells. Results show mean values ± SEM (n = 3). * p < 0.001 Discussion Natural xanthones, including those from mangosteen extract have antitumor and immunomodulatory effects (Pinto et al. 2005; Obolskiy et al. 2009; Shan et al. 2011; Gutierrez-Orozco and Failla 2013). In particular, alpha-mangostin has been repeatedly associated with modulation of a great variety of physiologic pathways, including inflammation. These findings reinforce their antitumor potential, fighting tumor progression and treatment resistance (Pinto et al. 2005; Shan et al. 2011; Gutierrez-Orozco and Failla 2013). In melanoma, several evidences reported the potential of immune targets as therapeutic agents. This facts result in the current use of ipilimumab, an immunomodulating antibody that target CTLA4 on activated T cells and inhibit regulatory T cells, in spite of the severe toxicity associated (Hodi et al. 2010; Graziani et al. 2012). The character highly immunogenic of melanocytic tumors and the influence of host immune response and microenvironment inflammatory cells in cancer growth were Growth inhibition (% of control) Alpha-mangostin (6,25 μM) 5.4 ± 2.9 % Macrophages supernatants 25.0 ± 2.5 % * α-MG (6,25 μM) conditioned macrophages supernatants 24.4 ± 2.7 % * V. 79 verified (Hussein 2004; Dranoff 2009; Chen et al. 2011). Indeed, primary melanomas undergo spontaneous regression much more frequently than any other cancer that may be a sign of immunosurveillance or, by other way, it may be due melanoma are easier to visualize (Printz 2001; Kalialis et al. 2009). Nevertheless, other evidences corroborate the involvement of immune system, namely the relatively amount of tumor-infiltrating lymphocytes in melanoma microenvironment compared with other cancers and associated with favorable prognostic (Maio 2012; Kushnir and Merimsky 2013) and the appearance of autoimmune condition as vitiligo in melanoma patients or patients treated with immunotherapy which normally means a better disease outcome (Le Gal et al. 2001; Phan et al. 2001; Boasberg et al. 2006). In more advanced stages, this cancer became capable to avoid immunosurveillance (Reiman et al. 2007; Speeckaert et al. 2011) and its progression and metastatic potential may be supported by immune cells present in tumor microenvironment, as the tumor-associated macrophages (TAMs) since they are the most abundant leucocytes in melanoma and represent a poor prognostic (Brocker et al. 1988; Bernengo et al. 2000; Makitie et al. 2001; Varney et al. 2005; Porta et al. 2007; Solinas et al. 2009; Mantovani and Sica 2010; Qian and Pollard 2010). In spite of the verified interaction of immune system in melanoma treatment and the potential of xanthones as antitumor and immunomodulatory agent, any research was based on the direct effect of any immune cell treated with xanthonic compounds in the tumor growth. According to the established influence of inflammation on cancer (induces carcinogenisis, tumor progression and promotes angiogenisis) (Apte and Voronov 2008; Germano et al. 2008; Kundu and Surh 2008; Colotta et al. 2009; Mantovani 2010) and particularly in melanoma, it was investigated the potential effect of the compound in different immune pathways in human and murine macrophage cell lines and human mononuclear cells from peripheral blood. Indeed, α-MG inhibited NO production and TNFα secretion by murine macrophages cells (Chen et al. 2008; Tewtrakul et al. 2009). In spite of that finds, any studies were performed in the cytokines interleukin - 1β (IL-1β), IL10, the tumor grow factor - β1 (TGF-β1) and the tumor necrosis factor - α (TNFα) in THP-1 macrophages as performed in this paper. RAW 264.7 was used for investigation of NO production, since this cell line was referred as an appropriate model to study iNOS system (Dirsch et al. 1998; Shih et al. 2010). Human macrophages in vitro produce low quantity of NO in response to stimuli (Thomassen and Kavuru 2001). However, either in vitro murine cells and in vivo human macrophages express great quantities of NO in inflammatory conditions (Ochoa et al. 1991; Dirsch et al. 1998) V. 80 α-MG was a strong inhibitor of NO production by LPS-stimulated RAW 264.7 (IC50 = 13.8 ± 0.7 µM). Since α-MG effect was not due to cell death or scavenging of chemicaly generated NO, it may act at the level of NO production by iNOS. In inflammatory process, NO is produced by inducible NO synthetase (iNOS or NOS2) from L-arginine and molecular oxygen (Gross and Wolin 1995; Aramaki 2000). The inductible form is stimulated by imunologic factors and expressed in virtually all cells (Gross and Wolin 1995). In macrophages, its production is stimulated by a number of cytokines and LPS (Lowenstein et al. 1993; Gross and Wolin 1995). Production of NO could be a consequence of iNOS regulation at transcriptional and post-transcriptional level, depending of gene transcription, stability and translation of mRNA, stability of the protein, availability of substrates or co-factors, inhibitors that compete with substrates and the known NO feedback effect (Nathan and Xie 1994; Bogdan et al. 2000; Brunet 2001). Therefore, the underlying mechanism to α-MG inhibition of NO production needed further investigation. For that, α-MG was added at different hours: 0h (simultaneously), 6h and 14h after RAW 264.7 macrophages stimulation with LPS. In spite of α-MG exhibit a potent inhibition of NO production when added simultaneously with LPS-stimulation, this production remained stable either when it was added 6 or 14h after stimulation. This findings suggest an interference on iNOS expression since the enzyme is transcribed within 2-4 hours and translated within six hours after LPS-stimulation in macrophages (Xie et al. 1994). L-NAME (inhibitor of iNOS activity) (McCall et al. 1991), and Dexamethasone, inhibitor of iNOS expression (Korhonen et al. 2002) was used as positive controls and the obtained results was in accordance with the mechanism proposed to these inhibitors by our technique. In accordance to our finds, Chen et al and Tewtrakul et al reported an inhibition of iNOS mRNA expression on RAW 264.7 macrophages by α-MG. Inhibition of iNOS expression indicates an anti-inflammatory potential of α-MG. However, in cancer, this inhibition represents controversial effects because NO has been reported as cytotoxic in melanoma and many other tumors, but simultaneously it modulates many pro-tumor pathways including resistance to apoptosis, cell cycle progression, angiogenesis invasion, and metastasis (Ying and Hofseth 2007; Choudhari et al. 2013). So, the result of α-MG-dependent inhibition of NO production for melanoma progression needs further investigation. Macrophages, including TAMs, are dynamic and heterogeneous cells mainly due to their capacity to respond to stimulus. According to the microenvironment they may be polarized into a spectrum of phenotypes between the extremes M1 (classic) and M2 (alternative). M1 macrophages produced high levels of iNOS, TNF-α, IL-1β and other pro- V. 81 inflammatory molecules, while M2 macrophages are immunosuppressive, significantly expressing IL-10 and TGF-β (Allavena et al. 2008; Biswas et al. 2013). Several evidences indicate that macrophage phenotypes can change during tumor progression (Zaynagetdinov et al. 2011). M1 activation may induce chronic inflammation, a factor that could predispose to tumor initiation (Greten et al. 2004; Pikarsky et al. 2004). However, in early stages of tumor progression, TAMs adopt an M1-like phenotype that contributes to anti-tumor immunity. M2 phenotype is mainly expressed in established tumors and induce immunosuppressive, angiogenic and metastatic effects (Mantovani et al. 2004; Sica et al. 2006; Gordon and Mantovani 2011; Zaynagetdinov et al. 2011; Liao et al. 2014). α-MG had a dual influence on the immune system, either inhibiting IL-1β and NO responses, or inhibiting TGF-β1 and stimulating TNF-α expression. IL-1β, a pro-inflammatory cytokine mainly produced by monocytes and macrophages is an example of the pleiotropism of immune system. In melanoma its expression was associated to tumor progression and promotion of lung metastases from melanoma (Giavazzi et al. 1990; Meyer et al. 2011). Therefore, IL-1β has been associated with all steps of malignancy (carcinogenesis, progression, invasion and metastasis) and may even be expressed by the tumor cells (Apte and Voronov 2008). In contrast, M1 macrophages phenotype produced interleukin induce an immune response against malignant cells (Fairweather and Cihakova 2009). Considering all that was stated above, the inhibition of IL-1β mediated by α-MG could be beneficial so this cytokine induces tumor progression and metastasis. Melanoma is a highly metastatic kind of cancer contributing to the high mortality associated (Kuphal and Bosserhoff 2009; Raaijmakers et al. 2013), therefore a therapy that suppress or control angiogenesis and invasion may be an important approach. TGF-β1 regulates multiple cellular processes, including proliferation and differentiation. It acts as a potent inducer of differentiation in normal cells which leads to the concept that this cytokine protects against cancer in early stages of carcinogenesis (Yang et al. 2008). By the other hand, several tumors express increasing amounts of TGF-β1 which is associated with disease progression, invasion and metastasis both in vivo and in vitro (Akhurst and Derynck 2001). In melanoma, TGF-β isoforms (TGF-β1/2/3) are highly expressed and increase in parallel with tumor progression (Krasagakis et al. 1998; Javelaud et al. 2008). According to that, this cytokine is commonly produced by M2-like macrophages leads to a Th2 response, i.e., promotes an immunosuppressive microenvironment that allows the tumor immune escape (Coffelt et al. 2009; Martinez et al. 2009; Siveen and Kuttan 2009; Hao et al. 2012; Sica and Mantovani 2012). The V. 82 inhibition of TGF-β1 by α-MG may benefit the treatment of melanoma, mainly in later stages of tumorigenesis. TNF-α stimulation predicts a favorable outcome in melanoma treatment since several reports associated the use of TNF inhibitors as a factor that appear to increase the risk of skin cancer, including melanoma (Mariette et al. 2011; Kouklakis et al. 2013). In spite of that, the concentration expressed by THP-1 cells was minimal even in LPS-stimulated and non-treated macrophages. IL-10 is an anti-inflammatory cytokine able to induce tumor progression through the promotion of angiogenesis and suppression of immune surveillance. In melanoma, its influence facilitates metastasis formation (García-Hernández et al. 2002; Itakura et al. 2011). In the present study, it was observed no alteration on IL-10 concentration after treatment with α-MG. Taking into account the pattern of cytokines altered by the treatment with α-MG, it was inferred the potential underling mechanism of iNOS inhibition by xanthone. NOS2 gene expression and translation was stimulated by a number of pro-inflammatory cytokines, particularly TNF-α, IL-1β and IFN-γ, compounds of bacterial origin as LPS (Nathan 1992) and stress signaling as hypoxia (Ferreiro et al. 2001). These stimuli may activate NF-κB or JAK-STAT (janus tyrosine kinase - signal transducers and activators of transcription) pathways (Kleinert et al. 1998; Xuan et al. 2001). Additionally, MAPK pathway most likely contributes to iNOS expression (Janssen-Heininger et al. 1999; Chan et al. 2001). It is also known that TGF-β1 inhibits iNOS expression by mRNA destabilization (Perrella et al. 1994). NF-κB pathway contributes to iNOS synthesis and upregulate IL-1β level. α-MG treated THP-1 macrophages expressed lower IL-1β and NO levels comparatively to control cells leading us to hypothesize the interference of the xanthone with NF-κB pathway. More studies must be performed in order to corroborate this supposition. Macrophage activation and polarization are triggered by T helper cells (Th1 induce M1 phenotype and Th2 the M2-like phenotype). In turn, M1 macrophages recruit Th1 cells, CD8+ CTL (cytotoxic T cells) and NK cells leading to an inflammatory response and antitumor immunity, while M2-like macrophages favor the recruitment and development of Treg (regulatory T cells) and Th2, leading to a response that supports tumor growth through immunosuppression (Mantovani et al. 2002; Mantovani et al. 2004; Martinez et al. 2009; Siveen and Kuttan 2009; Sica and Mantovani 2012). In order to complement the described effect of α-MG on the immune microenvironment of melanoma, a study in influence of the compound in proliferation and cytokines production by human lymphocytes was performed. The compound strongly inhibit PHA-stimulated human mononuclear cells proliferation. V. 83 As weel as discussed to macrophages, lymphocytes have divergent roles in cancer due to their plasticity (Lakshmi Narendra et al. 2013). With the aim of establish the impact of proliferation inhibition in tumors, a set of four cytokines was evaluated in PHAstimulated mononuclear cells from peripheral blood. At IC50 (≃ 10 µM) concentration of αMG any cytokine concentration was altered. IL-10 and TNF-α was reduced to basal levels and TGF-β1 was stimulated at twice IC50 concentration. This finds indicate and absent effect of α-MG in T lymphocytes at concentrations required for inhibition of melanoma cells growth and for the immunomodulatory influence in macrophages which may indicate no significant impact of lymphocytes treated with α-MG on melanoma growth inhibition or in modulating of macrophages. Recently, Kasemwattanaroj et al, 2013 reported no alteration in IL-1β and TNF-α expression levels by conavalin A stimulated human mononuclear cells treated with α-MG (Kasemwattanaroj et al. 2013). Considering the results of cytokines modulation in THP-1 human macrophages mediated by α-MG and the established involvement of immune system in melanoma treatment (Brocker et al. 1988; Bernengo et al. 2000; Makitie et al. 2001; Varney et al. 2005; Porta et al. 2007; Solinas et al. 2009; Mantovani and Sica 2010; Qian and Pollard 2010), it was hyphotisised if condicioned macrophages culture medium may potentiate the cytotoxic activity of α-MG. Anticancer effect of α-MG was previously evaluated in different melanoma cell lines (Wang et al. 2011; Wang et al. 2012a; Wang et al. 2012b; Wang et al. 2013; Beninati et al. 2014). In this work, the antitumor activity of α-MG on melanoma A375-C5 cells was determined and compared with that of α-MG treated macrophages supernatants. α-MG strongly inhibited melanoma growth (GI50 = 11.3 ± 0.9 µM). The results also demonstrated that the supernatants od THP-1 macrophages either treated or not with α-MG significantly interfere with melanoma cell growth. This results prove that the immune microenvironment created by macrophages associated to tumors interfere with tumor cell viability. For α-MG dose tested (6.25 µM), below GI50 concentration, no significantly differences were observed for the cytotoxic activity of macrophages supernatants, when treated or not with xanthone. However, it was proved that α-MG interferes with cytokine and NO production by activated macrophages leading to the evidence that the xanthone interferes with immune microenvironment of the tumor. Therefore, it was expected a difference between the cytotoxic effect of treated and non-treated macrophages supernatants. Our results could be due to the lower dose used since for 6.25 µM only a ≃ 5% inhibition of melanoma cell growth was observed. Althought, no higher doses could be used in the assays since they were associated with a significant loss of viability of THP-1 macrophages, as determined by the MTT assay. 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Sica A, Schioppa T, Mantovani A et al (2006) Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anticancer therapy. Eur J Cancer 42: 717-27. VI. 94 measured at 510 nm in an ELISA reader (Stat FAX3200, Awareness Technology) (Cerqueira et al. 2003). NO production assay The determination of nitric oxide (NO) production by RAW 264.7 (1 X 106 cells mL-1) after treatment and stimulation with 1.5 µg mL-1 LPS was quantified by Griess assay as previous described by our group (Teixeira et al. 2005; Cerqueira et al. 2008). N-nitro-Larginine methyl ester (L-NAME), and inhibitor of inductive nitric oxide synthase (iNOS) activity, and Dexamethasone, an inhibitor of iNOS expression, were used as positive controls. Since inhibition of NO production could be a consequence of an inhibition of iNOS expression and/or activity, it was necessary to investigate 1,2-DHX mechanism. For that, the compound was also added 6h and 14h after RAW 264.7 macrophages stimulation with LPS (Cerqueira et al. 2008). NO Scavenging assay To discard NO scavenging effect by xanthone, nitrite was chemically generated using sodium nitroprusside as previously described. Nitrite was quantified by Griess assay (Teixeira et al. 2005; Cerqueira et al. 2008). MTT-viability assay Toxicity of 1,2-DHX on THP-1 cell line and RAW 254.7 cell line was evaluated by MTTviability assay (Pedro et al. 2002; Cerqueira et al. 2003; Teixeira et al. 2005). Statistical analysis Except otherwise stated, results are the mean ± SEM of at least three independent experiments, performed in duplicate. Statistical analysis was performed with SPSS for Windows (version 20.0). Statistical significance between groups was calculated by MannWhitney Test and it is considered for p values less than 0.05. Ethics Ethics approval was obtained by the Ethic Comity of University Fernando Pessoa. VI. 95 Results Antitumor effect of 1,2-DHX and conditioned macrophages culture medium The evaluation of 1,2-DHX effect on A375-C5 melanoma growth was performed by SRB assay and revealed a moderate inhibitory effect of the xanthone (Table 6). This effect showed to be dose-dependent (data not show). Table 6: Effect of 1,2-dihydroxyxanthone on the growth of A375-C5 human melanoma cell line. Growth inhibition (GI50) 1,2-DHX 55.0 ± 2.3 µM Doxorrubicin 1.8 X 10-3 ± 0.4 X 10-3 µM Results are the mean ± SEM of three independent experiments performed in duplicate. Doxorrubicin was used as positive control. As modulation of immune system has been a promising approach in melanoma treatment, it was investigate the possible influence of 1,2-DHX conditioned macrophages supernatants on A375-C5 cells growth. The results demonstrated a similar inhibition of cancer cells growth by the compound at 50 μM and non-treated macrophages. However, xanthone-treated macrophagesconditioned medium showed a significant (p < 0,001) decrease of melanoma cell growth comparatively with the other two conditions, as demonstrated in Figure 14. Figure 14: Cytotoxic activity of 1,2-Dihydroyixanthone, macrophages supernatants and 1,2dihydroxyxanthone conditioned macrophages supernatants on A365-C5 melanoma cell line.THP-1 PHAdifferentiated macrophages were treated with the compound and supernatants were added to melanoma cells. Results show mean values ± SEM (n = 3). * p < 0.001 Grow inhibition (% of control) 1,2-DHX (50 μM) 31.6 ± 4.9 % Macrophages supernatants 25.0 ± 2.5 % 1,2-DHX (50 μM) conditioned macrophages supernatants 55.4 ± 4.4 % * VI. 96 Effect on IL-1β, IL-10, TGF-β1 and TNF-α production by macrophages Further experiments were carried out to determine the underling mechanism of 1,2DHX on macrophages. Two concentrations of the compound (over and below GI50) were studied in order to evaluate the effect of this xanthone on the kinetics of cytokines production by LPS-stimulated THP-1 macrophages cell line (Figure 15). The expression of four cytokines was evaluated: IL-1β and TNF-α characteristic of M1 phenotype and IL10 and TGF-β1 characteristic of M2 phenotype. 1,2-DHX significantly inhibited the expression of IL-1β and stimulated the expression of TNF-α at the two concentrations. At concentration above GI50, xanthone stimulated expression of TGF-β1 and suppressed IL-10 by THP-1 macrophages. Figure 15: Effect of 1,2-DHX on IL-1β, IL-10, TGF-β1 and TNF-α production by THP-1 macrophages. Cytokines production was evaluated on Unstimulates macrophages (basal), LPS-stimulated macrophages (positive control) and macrophages treated with 6 and 3 µM 1,2-dihydroxyxanthone. Data are the mean ± SEM from one experiment, performed with duplicate cultures, and it representative of tree experiments carried out independently. * p < 0.001; ┼ p > 0.05 Effect on NO production by macrophages The production of NO by RAW 264.7 murine macrophages cell line was strongly inhibited by 1,2-DHX (IC50 = 22.0 ± 0.9 µM) (Table 7) in a dose-dependent manner (data not show). NO inhibiton was not associated to cell death since at IC50 values, viability was near 100% (Table 7). 1,2-DHX did not show any scavenging activity of NO generated in a cellfree system (data not show). α VI. 97 Table 7: Effect of 1,2-DHX on NO production by LPS-stimulated RAW 264.7 macrophages NO inhibition (IC50) Viability at IC50 1,2-Dihydroxyxanthone 22.0 ± 0.9 µM 100 % L-Name 62.4 ± 7.8 μM 96.0 ± 2.5 % Dexamethasone 4.3 ± 0.6 μM 100 % Results are the mean ± SEM (n = 3). L-Name and Dexamethasone was used as positive control. It was next investigated whether the inhibition of NO production was due to a decrease in iNOS expression or impairment of iNOS activity. iNOS is transcribed within 2-4 hours and translated within six hours after LPS-stimulation in macrophages (Xie et al. 1994). As such, 1,2-DHX was added at a concentration close to IC50 value and NO was measured at 0, 6 and 14 h after RAW 264.7 macrophages stimulation with LPS. When 1,2-DHX was added simultaneously with the stimulus, about 57% of NO production was inhibited. When the compound was added 6 h after stimulation, the effect significantly decreased to 26% (p < 0.001), while a lack of inhibitory effect was observed when the compound was added 14 h after stimulation (Table 8). Once again, toxicity was excluded by MTT viability assay, since cells showed viability higher than 90 % at dilution closest to IC50 of 1,2-DHX. Concentrations of xanthone and controls (L-NAME and Dexamethasone) used in this study were the dilution closest to IC50, previously determined. Table 8: Inhibitory effect of 1,2-DHX on NO production by RAW264.7. NO inhibition (% of control) 0 h 6 h 14 h 1,2-DHX at 25 μM 56.6 ± 1.8 % 25.0 ± 2.8 % * n.i. L-Name at 62,5 μM 52.1 ± 6.0 % 50.7 ± 3.8 % ┼ 24.7 ± 3.1 % * Dexamethasone at 6,25 μM 55.9 ± 2.3 % 15.7 ± 6.0 % * n.i. Macrophages were exposed to LPS and treated with 1,2-dihydroxyxanthone at different times after stimulation: 0 h (simultaneously with the stimulus), 6 h and 14 h after stimulation. Results are the mean ± SEM (n = 3). n.i.= no inhibition. * p < 0.001, ┼ p > 0.05. L-NAME and Dexamethasone was used as positive controls. VI. 98 Discussion The anticancer and immunomodulatory effect of natural and synthetic xanthones have been extensible reported in literature (Pinto et al. 2005; Teixeira et al. 2005; Castanheiro et al. 2007; Obolskiy et al. 2009; Gutierrez-Orozco and Failla 2013). In particular, 1,2-DHX inhibited the gowth of several cancer cell lines and the proliferation of stimulated T lymphocytes (Pedro et al. 2002; Sousa et al. 2002). In spite of these insights, and as far as we know, no studies were performed considering the immune microenvironment of 1,2DHX-conditionated macrophages mediated effect on melanoma cell. In the present study, the effect of 1,2-DHX on A375-C5 melanoma cell line was evaluated. However, a significant difference of potency was observed when comparing the GI50 fot A375-C5 (GI50 = 55.0 ± 2.3 µM) and UACC-62 melanoma cell line (GI50 = 14.0 ± 0.3 µM), this may be explained by the difference morphology of the cell, i.e. epithelial (A375-C5) and non-epethelial (UACC-62). Melanoma is a highly immunogenic tumor (Hussein 2004), therefore it was hyphotesised the possible improvement of anticancer capacity of 1,2-DHX by immune system involvement. It was showed that 1,2-DHX strongly interfere with macrophages immune microenvironment, resulting in a two-fold increase of the cytotoxic effect of macrophages on A375-C5 melanoma cells. This finds indicates a possible induction of an antitumor macrophages phenotype by 1,2-DHX since supernatants of non-treated macrophages by it self showed much lower inhibition comparatively to treated macrophages. In order to certify the underlying mechanism mediated by 1,2-DHX in macrophages, the expression of cytokines, namely interleukin - 1β (IL-1β) and tumor necrosis factor - α (TNFα) (characteristic of a M1 phenotype) and tumor grow factor - β1 (TGF-β1) and IL10 (characteristic of a M2-like phenotype) was evaluated. Macrophages are dynamic and heterogeneous cells mainly due to their capacity to respond to stimulus. According to the microenvironment they may be polarized into a spectrum of phenotypes ranging from the pro-inflammatory M1 (classic) to the immunossupressive M2 (alternative) (Allavena et al. 2008; Biswas et al. 2013). Several evidences indicate that macrophages phenotypes can change during tumor progression (Zaynagetdinov et al. 2011). M1 activation may induce chronic inflammation, a factor that could predispose to tumor initiation (Greten et al. 2004; Pikarsky et al. 2004). However, in early stages of tumor progression, TAMs adopt a M1-like phenotype that contributes to tumor immunity. M2 phenotype is mainly expressed in established tumors and induce immunosuppressive, angiogenic and metastatic effects (Mantovani et al. 2004; Sica et al. 2006; Gordon and Mantovani 2011; Zaynagetdinov et al. 2011; Liao et al. 2014). VI. 99 1,2-DHX suppress the expression of IL-1β to concentrations similar to basal and stimulated TNF-α expression. It also inhibited IL-10 production and stimulated TGF-β1 expression but only at concentration above GI50 (100 µM), while any alteration was detected at lower concentration. IL-1β, a pro-inflammatory cytokine mainly produced by monocytes and macrophages is an example of the pleiotropism of immune system. In melanoma its expression was associated to tumor progression and promotion of lung metastases from melanoma (Giavazzi et al. 1990; Meyer et al. 2011). Therefore, IL-1β has been associated with all steps of malignancy (carcinogenesis, progression, invasion and metastasis) and may even be expressed by the tumor cells (Apte and Voronov 2008). In contrast, it induces an immune response against malignant cells associated to the M1 macrophages phenotype (Fairweather and Cihakova 2009). TNF-α stimulation predicts a favorable outcome in melanoma treatment since several reports associated the use of TNF inhibitors as a factor that appear to increase the risk of skin cancer, including melanoma (Mariette et al. 2011; Kouklakis et al. 2013). TNF-α expression by THP-1 cells at basal and LPS-stimulated level was significantly lower when compared to 1,2-DHX treated cells. IL-10 and TGF-β1 are anti-inflammatory cytokine involved in carcinogenisis process. In melanoma, IL-10 was associated with metastatic formation (García-Hernández et al. 2002; Itakura et al. 2011) and TGF-β1 are highly expressed and increase in parallel with tumor progression (Krasagakis et al. 1998; Javelaud et al. 2008). In spite of the differences observed, the antitumor effect of 1,2-DHX conditioned macrophages medium was not due neither to IL-10 nor TGF-β1 expression, since for treatment with 50 µM of 1,2-DHX no significantly differences were observed for the expression of these cytokines by THP-1 macrophages. Production of nitric oxide (NO) by macrophages in tumors has controversial funtions. It was reported that below a critical concentration of NO, it causes DNA mutations (Wink et al. 1998), inhibits apoptosis (Choi et al. 2002), promotes angiogenesis (Ziche and Morbidelli 2000), limits immune response against cancer (Wink et al. 1991) and promotes metastasis (Lala and Orucevic 1998). When it exceeds the critical concentration, NO induce apoptosis and suppress the growth of the tumor (Choudhari et al. 2013). NO has been reported as cytotoxic in melanoma and many other tumors, but simultaneously it modulates many pro-tumor pathways including resistance to apoptosis, cell cycle progression, angiogenesis invasion, and metastasis (Ying and Hofseth 2007; Choudhari et al. 2013). In order to clarify this relation and better understand the mechanism mediated by 1,2-DHX in macrophages, production of NO by macrophages was evaluated. VI. 100 In NO production assay, RAW 264.7 was used instead of THP-1 human macrophages cell line since this was referred as an appropriet model to study iNOS system (Dirsch et al. 1998; Shih et al. 2010). Human macrophages in vitro produce low quantitie of NO in response to stimuli (Thomassen and Kavuru 2001). However either in vitro murine cells as in vivo human macrophages express great quantities of NO in inflammatory conditions (Ochoa et al. 1991; Dirsch et al. 1998) 1,2-DHX is a potent inhibitor of NO production by LPS-stimulated RAW 264.7 murine macrophages cell line (IC50 = 22.0 ± 0.9 µM). This effect was not due to cell death or to NO scavenging, thus it was proved that interference was at the level of NO production. In stimulated macrophages, NO is produced by the inducible form of NO synthetase (iNOS or NOS2) from L-arginine and molecular oxygen (Gross and Wolin 1995; Aramaki 2000). NOS2 gene regulation, as the majority of genes may occur at transcriptional and post-transcriptional level (Nathan and Xie 1994; Bogdan et al. 2000; Brunet 2001). With the purpose of evaluate the time point when the compound interfere with the enzyme to inhibit NO production, xanthone was added at different hours: 0h (simultaneously), 6h and 14h after RAW 264.7 macrophages stimulation with LPS. Although, 1,2-DHX added simultaneously has potent inhibition; when it was added 6h after stimulation the effect decreased significantly (p < 0.001) and when added after 14h, the effect was nule. This finds leads us to conclude that the compound may exerts their action by interfering with iNOS expression, once this enzyme is transcripted within 2-4 hours and translated within six hours after LPS-stimulation in macrophages (Xie et al. 1994). L-NAME (inhibitor of iNOS activity) (McCall et al. 1991), and Dexamethasone, inhibitor of iNOS expression (Korhonen et al. 2002) was used as positive controls and the obtained results was in accordance with the mechanism proposed to these inhibitors by our technique. Inhibition of iNOS indicated an anti-inflamatory potential of 1,2-DHX. However, inflammation and NO production has controversial effects in cancer. NO has been reported as cytotoxic in melanoma and many other tumors, but simultaneously it modulates many pro-tumor pathways including resistance to apoptosis, cell cycle progression, angiogenesis, invasion and metastasis (Ying and Hofseth 2007; Choudhari et al. 2013). The underling mechanism of inhibition of iNOS expression by 1,2-DHX may be explained by the stimulation of TGF-β1. This cytokine has been reported as a destabilizer of iNOS mRNA avoiding gene translation and inhibiting NO production. Involvement of NF-κB pathway may also be involved since it contributes to iNOS synthesis and upregulate IL-1β level and 1,2-DHX treated THP-1 macrophages expressed either lower IL-1β and NO levels comparatively to control cells. This finds leads us to hypothesize the VI. 101 interference of the xanthone with NF-κB pathway. More studies must be performed in order to corroborate this supposition. 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