Anti-inflammatory actions of melatonin and its metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in macrophages
Introduction
Macrophages are potent defenders of physiological integrity in vertebrates with important roles in inflammatory reactions. Inflammation is a complex phenomenon involving numerous mediators which trigger a number of biological effects that are crucial for the host’s normal defense against insults, pathogens or stress. If the inflammatory response is not tightly regulated, chronic inflammation occurs, which accounts for a variety of different pathologies, e.g., cancer and neurodegenerative diseases (Balkwill and Coussens, 2004, Consilvio et al., 2004).
Vasoactive prostaglandins (PGs) are essential components in the regulation of vascular function under normal physiological conditions. Cyclooxygenase (COX) is the key enzyme that catalyzes the two sequential steps in the biosynthesis of PGs from arachidonic acid (AA) (Vane et al., 1998). This enzyme exists in at least two isoforms: a constitutively expressed enzyme known as COX-1, which is highly localized in endothelial cells and platelets; and an inducible form, referred to as COX-2, which is induced by a variety of stimuli associated with cell activation and inflammation. COX-1 has clear physiological functions whereas COX-2 is induced by pro-inflammatory stimuli in migratory cells and inflamed tissues (Vane et al., 1998).
Lipopolysaccharide (LPS) binds to the Toll-like receptor 4 (Tlr4) and initiates several major cellular responses which are involved in the pathogenesis of endotoxic shock, including the expression of COX-2 and the inducible isoform of nitric oxide synthase (iNOS). Both, PGs or nitric oxide (NO)-formed from L-arginine by the enzymatic action of NOS play important roles in inflammation, immune functions, blood vessel dilatation and neurotransmission. Over production of PGs and NO during inflammation is associated with local and systemic symptoms of fever, pain and edema (Kiefer and Dannhardt, 2002). Therefore, mechanisms controlling PG production are of special interest to counteract the inflammatory process (Turini and DuBois, 2002). Since both COX-2 and iNOS are inducible forms up-regulated in response to inflammatory challenge, they are traditionally associated with pathological states (Vane et al., 1998, Bakhle, 2001). To date, several cell systems have been successfully used for the study of COX-2 regulation, such as RAW 264.7 murine macrophage cells previously stimulated with LPS (von Knethen et al., 1999).
A major mechanism of action of nonsteroid anti-inflammatory drugs (NSAIDs) is the inhibition of biosynthesis of PGs (Vane and Botting, 1998, Vane et al., 1998). COX-2 specific inhibitors suppress inflammation while reducing the side effects of classical NSAIDs treatment, including gastrointestinal ulceration and bleeding, renal damage and platelet dysfunction. The unwanted side effects of NSAIDs, such as damage to the gastric mucosa and kidneys, are due to their ability to inhibit COX-1, while their anti-inflammatory (therapeutic) effects are due to inhibition of COX-2. Thus, drugs that have high potency for COX-2 and a lesser effect on COX-1 would provide potent anti-inflammatory activity with fewer side effects. COX-2 is abundantly expressed in human colon cancer cells, and NSAIDs delay the progress of colon tumors possibly by causing apoptosis of the tumor cells. Furthermore, the risk of developing Alzheimer's disease (AD), which may involve an inflammatory component, is reduced by chronic ingestion of NSAIDs.
Melatonin, or N-acetyl-5-methoxytryptamine, is an indole mainly produced in the mammalian pineal gland during the dark phase. Melatonin secretion from the pineal gland exhibits a distinctive circadian rhythm and has been classically associated with circadian and circanual rhythm regulation, and with adjustments of physiology of animals to seasonal environmental changes (Reiter, 1991). Melatonin production, however, is not confined exclusively to the pineal gland and other organs and tissues including retina, Harderian glands, gut, ovary, testes, bone marrow and lens also have been reported to produce it (Menendez-Pelaez et al., 1987, Tan et al., 1999). Melatonin is also synthesized in non-mammalian vertebrates, invertebrates and in other organisms including dinoflagellates, algae and bacteria (Hardeland and Poeggeler, 2003). Melatonin has also been shown to act as a potent antioxidant and free radical scavenger, protecting against a number of radical species in both in vivo and in vitro models of oxidative stress (Tan et al., 2002). Melatonin protects against oxidative stress-related processes in experimental models of ischemia/reperfusion, aging and neurodegenerative disorders among others. In addition to its roles as an adjustor to circadian rhythms and protector against ROS, its function in oncostasis (Blask et al., 2002) and as a modulator of the immune system (Guerrero and Reiter, 2002) have also been widely reported. Due to the potential role of melatonin as an endogenous antioxidant and a regulator of immune system and since the pro-inflammatory enzyme COX-2 plays an important role in the immune response and seems to be subjected to redox control, the aim of the present work was to determine whether melatonin and its metabolites N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), might regulate the expression and activity of COX-2 and iNOS in LPS-activated macrophages and, if so, whether this is related to its antioxidant activity.
Section snippets
Drugs and treatments
LPS (from E.coli, isotype 0111:B4) was purchased from SIGMA (SIGMA-Aldrich, St. Louis, MO, USA). Ultra pure grade melatonin was a kind gift from Helsinn Chemical (Biasca, Switzerland). All culture reagents were purchased from Invitrogen (GBCO-BRL, Rockville, MD, USA). Vitamin C, N-acetylcysteine (NAC), trolox, 3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2H-tetrazolium bromide (MTT), propidium iodide, and 2′,7′-dichlorofluorescein diacetate (DFCH-DA) were obtained from Sigma Chemical (St. Louis,
Melatonin inhibits COX-2 and iNOS expression in LPS-stimulated macrophages
As shown in Fig. 1A, the indole melatonin, in a dose-dependent manner, inhibited the LPS-induced increase of COX-2 protein expression in RAW 264.7 macrophages. Melatonin did not alter the expression pattern of the constitutive isoform, COX-1 (house-keeping), therefore revealing a specific effect on the inducible form. Since both COX-2 and iNOS are usually induced following immune stimulation, we also investigated the putative role of melatonin on the inducible isoform of NOS. Melatonin also
Discussion
Given its role in the initial steps of activation of the inflammatory process, COX-2 is thought to be an important target in diseases such as colon cancer (Sinicrope and Gill, 2004) and Alzheimer's disease (McGeer and McGeer, 2001). Furthermore, chronic use of NSAIDs in clinical studies as well as laboratory findings have proved to be beneficial for these pathologies (Peek, 2004). Therefore, the search for natural or endogenous products with a high degree of specificity on COX-2 inhibition is
Acknowledgments
RMS acknowledges support from a Fullbright Grant and the financial sponsorship from the “Spanish Ministry of Education, Culture and Sports”. The “Instituto Universitario de Oncología del Principado de Asturias” is supported by Obra Social Cajastur, Asturias, Spain.
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