Anti-inflammatory actions of melatonin and its metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in macrophages

https://doi.org/10.1016/j.jneuroim.2005.05.002Get rights and content

Abstract

Inflammation is a complex phenomenon involving multiple cellular and molecular interactions which must be tightly regulated. Cyclooxygenase-2 (COX) is the key enzyme that catalyzes the two sequential steps in the biosynthesis of PGs from arachidonic acid. The inducible isoform of COX, namely COX-2, plays a critical role in the inflammatory response and its over-expression has been associated with several pathologies including neurodegenerative diseases and cancer. Melatonin is the main product of the pineal gland with well documented antioxidant and immuno-modulatory effects. Since the action of the indole on COX-2 has not been previously described, the goal of the present report was to test the effect of melatonin on the activities of COX-2 and inducible nitric oxide synthase (iNOS), using lipopolysaccharide (LPS)-activated RAW 264.7 macrophages as a model. Melatonin and its metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), prevented COX-2 activation induced by LPS, without affecting COX-1 protein levels. The structurally related compound 6-methoxy-melatonin only partially prevented the increase in COX-2 protein levels induced by the toxin. Likewise melatonin prevented iNOS activation and reduced the concentration of products from both enzymes, PGE2 and nitric oxide. Another endogenous antioxidant like N-acetyl-cysteine (NAC) did not reduced COX-2 significantly. The current finding corroborates a role of melatonin as an anti-inflammatory agent and, for the first time, COX-2 and iNOS as molecular targets for either melatonin or its metabolites AFMK and AMK. These anti-inflammatory actions seem not to be exclusively mediated by the free radical scavenging properties of melatonin. As a consequence, the present work suggests these substances as a new class of potential anti-inflammatory agents without the classical side effects due to COX-1 inhibition.

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.

References (53)

  • J. Saldeen et al.

    Nicotinamide-induced apoptosis in insulin producing cells is associated with cleavage of poly(ADP-ribose) polymerase

    Mol. Cell. Endocrinol.

    (1998)
  • K. Senior

    COX-2 inhibitors: cancer prevention or cardiovascular risk?

    Lancet Oncol.

    (2005)
  • S.O. Silva et al.

    Neutrophils as a specific target for melatonin and kynuramines: effects on cytokine release

    J. Neuroimmunol.

    (2004)
  • D.X. Tan et al.

    Identification of highly elevated levels of melatonin in bone marrow: its origin and significance

    Biochim. Biophys. Acta

    (1999)
  • D.X. Tan et al.

    Melatonin directly scavenges hydrogen peroxide: a potentially new metabolic pathway of melatonin biotransformation

    Free Radic. Biol. Med.

    (2000)
  • J.R. Vane et al.

    The mechanism of action of aspirin

    Thromb. Res.

    (2003)
  • A. Wolfler et al.

    Prooxidant activity of melatonin promotes fas-induced cell death in human leukemic Jurkat cells

    FEBS Lett.

    (2001)
  • M. Allegra et al.

    The chemistry of melatonin's interaction with reactive species

    J. Pineal Res.

    (2003)
  • Y.S. Bakhle

    COX-2 and cancer: a new approach to an old problem

    Br. J. Pharmacol.

    (2001)
  • F. Balkwill et al.

    Cancer: an inflammatory link

    Nature

    (2004)
  • A. Barlas et al.

    Melatonin protects against pancreaticobiliary inflammation and associated remote organ injury in rats: role of neutrophils

    J. Pineal Res.

    (2004)
  • D.E. Blask et al.

    Melatonin as a chronobiotic/anticancer agent: cellular, biochemical, and molecular mechanisms of action and their implications for circadian-based cancer therapy

    Curr. Top. Med. Chem.

    (2002)
  • S.Y. Chung et al.

    Melatonin attenuates kainic acid-induced hippocampal neurodegeneration and oxidative stress through microglial inhibition

    J. Pineal Res.

    (2003)
  • E. Crespo et al.

    Melatonin inhibits expression of the inducible NO synthase II in liver and lung and prevents endotoxemia in lipopolysaccharide-induced multiple organ dysfunction syndrome in rats

    FASEB J.

    (1999)
  • S. Cuzzocrea et al.

    Potential therapeutic effect of antioxidant therapy in shock and inflammation

    Curr. Med. Chem.

    (2004)
  • H. Ding et al.

    Celecoxib derivatives induce apoptosis via the disruption of mitochondrial membrane potential and activation of caspase 9

    Int. J. Cancer

    (2005)
  • Cited by (291)

    View all citing articles on Scopus
    View full text