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Methionine enkephalin (MENK) improves lymphocyte subpopulations in human peripheral blood of 50 cancer patients by inhibiting regulatory T cells (Tregs)

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Abstract

MENK, a penta-peptide is considered as being involved in the regulatory feedback loop between the immune and neuroendocrine systems, with marked modulation of various functions of human immune cells. The aim of the present work was to investigate change of lymphocyte subpopulations in peripheral blood of 50 cancer patients before and after treatment with MENK. Peripheral blood mononuclear cells (PBMCs) of peripheral blood from 50 cancer patients were isolated by density gradient centrifugation using Ficoll-Paque solution and cultured with MENK. We measured proliferation of total nucleated cells, subpopulations of individual CD4+T cells, CD8+T cells, CD4+CD25+ regulatory T cells (Treg), natural killer cells (NK) before and after treatment with 10(-12)M MENK in cell culture by flow cytometry (FCM). Our results indicated that MENK showed a strong inhibiting effect on Treg cells while it stimulated marked proliferation of other lymphocyte subpopulations. All data obtained were of significance statistically. It was therefore concluded that MENK could work as a strong immune booster with great potential in restoring damaged human immune system and we could consider MENK as a drug to treat cancer patients, whose immune systems are damaged by chemotherapy or radiotherapy. Furthermore we could consider MENK as a chemotherapy additive, which would sustain immune system of cancer patients during the process of chemotherapy to get maximized efficacy with minimized side effect.

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... However, the observation of its immunoregulatory and anti-cancer activity, has suggested potential utility for the treatment of immunerelated diseases and neoplasia. Since 2010, our research team has published a number of articles [19,33,[41][42][65][66][67][68] elucidating the role of MENK in cancer biotherapy as an immunomodulatory drug (Fig. 1). Herein we summarize recent research from our laboratory and others on the bioactivity of MENK, with a focus on immunoregulation and cancer therapy to provide baseline information to further the study of MENK. ...
... Our research concluded that MENK can inhibit Treg activity and retard tumor development by down-regulating Tregs in mice [41]. MENK can also regulate lymphocyte subpopulations in the peripheral blood of cancer patients by inhibiting Tregs [42] ( Table 1) and prolonged life in vast majority of patients was observed post-MENK treatment again supporting the hypothesis that MENK may provide a new strategy for cancer immunotherapy. ...
... During development, NK cells become "licensed" after they encounter cognate leucocyte antigen (HLA) class I, resulting in the acquisition of effector function(s). There are four major NK cell functions: 1) secretion of cytokines, 2) direct cytotoxicity by release of perforins and granzyme, 3) target killing through apoptotic pathways and 4) target killing through antibody-dependent cellular cytotoxicity [42]. Thus, blocking any of these pathways can decrease NK cell-mediated killing, such that the regulation of NK cells can be exploited for cancer therapy. ...
Article
Methionine enkephalin (MENK), an endogenous neuropeptide has a crucial role in both neuroendocrine and immune systems. MENK is believed to have an immunoregulatory activity to have cancer biotherapy activity by binding to the opioid receptors on immune and cancer cells. Clinical trial studies in cancer patients have shown that MENK activates immune cells directly and by inhibiting regulatory T-cells (Tregs). MENK may also change the tumor microenvironment by binding to opioid receptor on or in cancer cells. All of these mechanisms of action have biologic significance and potential for use in cancer immunotherapy. Furthermore, they reveal a relationship between the endocrine and immune systems. Due to the apparent role of MENK in cancer therapy we reviewed herein, the research undertaken with MENK in recent years; which has advanced our understanding of the role MENK has in cancer progression and its relationship to immunity, supporting MENK as a new strategy for cancer immunotherapy.
... The administration of δ agonists into the ventral tegmental area decreases alcohol consumption Barson et al. (2010) and Margolis, Fields, Hjelmstad, and Mitchell (2008) The expression of pro-enkephalin is downregulated in the caudate nucleus of alcoholics Sarkisyan et al. (2015) μ and δ receptors are involved in the expression and reinstatement of ethanol conditioned seeking behavior and enkephalin analogues inhibited alcohol withdrawal-induced anxiety behavior Gibula-Bruzda, Marszalek-Grabska, Gawel, et al. (2015) and Gibula-Bruzda, Marszalek-Grabska, Witkowska, Izdebski, and Kotlinska (2015) Cancer Low fasting plasma concentration of pro-enkephalin is associated with an increased risk of future breast cancer. Met-enkephalin restores the damage of the immune system induced by chemotherapy Melander et al. (2015) and Wang et al. (2014) Depression δ receptor agonists exert an anti-depressant effect Dripps and Jutkiewicz (2018) Eating disorders Involved in feeding and diet-induced obesity Mendez, Ostlund, Maidment, and Murphy (2015) Gastrointestinal disorders ...
... Additionally, it has been reported that pro-enkephalin is a risk predictor for poor outcome in heart failure (Siong Chan, Cao, & Ng, 2018); that low fasting plasma concentration of pro-enkephalin is associated with an increased risk of future breast cancer (Melander et al., 2015); that stress upregulated or downregulated, respectively, met-enkephalin and leu-enkephalin in the hippocampus ; that the administration of a non-opioid analogue of leu-enkephalin partially normalizes cerebral consequences after hypoxia (Simankova et al., 2017); and that met-enkephalin exerts an anti-tumor action against melanoma and promotes apoptotic mechanisms in glioma cells (Lu et al., 2018). Met-enkephalin has been proposed as a possible treatment for cancer patients whose immune systems have been damaged by chemotherapy, as the pentapeptide is able to restore such system (Wang et al., 2014). ...
Chapter
The pentapeptides methionine-enkephalin and leucine-enkephalin belong to the opioid family of peptides, and the non-opiate peptide adrenocorticotropin hormone (ACTH) to the melanocortin peptide family. Enkephalins/ACTH are derived from proenkephalin, pro-dynorphin or pro-opiomelanocortin precursors and, via opioid and melanocortin receptors, are responsible for many biological activities. Enkephalins exhibit the highest affinity for the δ receptor, followed by the μ and κ receptors, whereas ACTH binds to the five subtypes of melanocortin receptor, and is the only member of the melanocortin family of peptides that binds to the melanocortin-receptor 2 (ACTH receptor). Enkephalins/ACTH and their receptors exhibit a widespread anatomical distribution. Enkephalins are involved in analgesia, angiogenesis, blood pressure, embryonic development, emotional behavior, feeding, hypoxia, limbic system modulation, neuroprotection, peristalsis, and wound repair; as well as in hepatoprotective, motor, neuroendocrine and respiratory mechanisms. ACTH plays a role in acetylcholine release, aggressive behavior, blood pressure, bone maintenance, hyperalgesia, feeding, fever, grooming, learning, lipolysis, memory, nerve injury repair, neuroprotection, sexual behavior, sleep, social behavior, tissue growth and stimulates the synthesis and secretion of glucocorticoids. Enkephalins/ACTH are also involved in many pathologies. Enkephalins are implicated in alcoholism, cancer, colitis, depression, heart failure, Huntington’s disease, influenza A virus infection, ischemia, multiple sclerosis, and stress. ACTH plays a role in Addison’s disease, alcoholism, cancer, Cushing’s disease, dermatitis, encephalitis, epilepsy, Graves’ disease, Guillain-Barre's syndrome, multiple sclerosis, podocytopathies, and stress. In this review, we provide an updated description of the enkephalinergic and ACTH systems.
... If classical opioid receptors were effectively knocked down by siRNAs specific for the mu, delta, and kappa opioid receptor, and cultures exposed to short-term naltrexone, growth was inhibited, as was protein expression of OGF and OGFr. 21 Some investigators suggest that low doses of naltrexone (LDN) act directly as immunomodulating agents, 22,23 or by interaction with toll-like receptor 4. 24 Investigations focused on T and B cell proliferation in vitro 25 and in vivo 26 have demonstrated that the modulation of the immune system is likely a direct response to increased or decreased proliferation of lymphocytes as well as activation of peripheral lymphocytes and resultant cytokine production. Direct evidence for LDN's mechanistic effects emanates from preclinical work documenting that LDN administration to mice results in measurable increases in serum OGF levels. ...
Article
Impact statement: This mini-review presents information on the intermittent blockade of the opioid growth factor (OGF)-OGF receptor (OGFr) axis by low-dose naltrexone (LDN), and the role of enkephalin (i.e. OGF) in autoimmune disorders, specifically multiple sclerosis, Crohn's, and fibromyalgia. Clinical reports on subjects taking LDN have documented reduced fatigue, few side-effects, and improved overall health. Preclinical studies on mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, revealed that immunization for EAE reduces serum OGF. Intermittent OGFr blockade with LDN restores serum enkephalin levels that correlate with reduced behavioral and pathological signs of EAE; LDN also increases enkephalin levels in naïve mice. The interplay between LDN, and the onset and treatment of autoimmune diseases, chronic pain, and other addictive behaviors requires further investigation, but highlights a central role for enkephalins and intermittent blockade of the OGF-OGFr pathway in pathogenesis and treatment of these disorders.
... MENK, at a suitable range of concentrations and in a doseand time-dependent manner, had an anticancer effect that was associated with binding to opioid receptors. MENK can also regulate macrophage functions, 29,30 as well as the functions of dendritic cells, [31][32][33] CD8 + T cells, 13 CD4 + T cells, 18,34 and natural killer cells 12,35 as an immune regulator. Published data suggest that MENK also has an antiviral role during influenza 36 ...
Article
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Background: Gastric cancer (GC) is the second cause of cancer-related deaths. Methionine enkephalin (MENK), an endogenous opioid peptide, has immunological and antitumor activity. Purpose: The aim of this work was to investigate whether MENK could exhibit activity against human GC in vitro and in vivo. Materials and methods: Human GC cells were treated with MENK. Cell viability, colony formation, cell morphology, cell cycle, and apoptosis were assessed. The effects of MENK on gene expression of OGFr, Bax, BCL-2, caspase-3, PARP, Ki67, cyclin D1, c-myc, survivin were quantifed by qRT-PCR. Western blot was used to analyze the effects of MENK on protein expression of OGFr, Bax, BCL-2, caspase-3, PARP. The anti-tumor activity of MENK in gastic carcinoma was also investigated with animal experiments. Results: The results indicate that MENK could significantly inhibit the growth of human GC cells SGC7901 and HGC27 in a concentration-and time-dependent manner, decrease the number of cell colonies, and arrest cell cycle in the G0/G1 phase by causing a decrease in Ki67, cyclin D1, and c-myc mRNA. Furthermore, MENK could induce tumor cell apoptosis associated with the upregulation of Bax, a corresponding downregulation of BCL-2 and survivin, and activation of caspase-3 and PARP. Moreover, MENK upregulated the expression of opioid receptors (OGFr) in SGC7901 and HGC27 cells. The interaction between MENK and OGFr in SGC7901 and HGC27 cells appears to be essential for the antitumor activity of MENK. Conclusion: We conclude that MENK may be a potential drug for the treatment of GC.
... Moreover, enkephalins include methionine enkephalins (MENK) and leucine enkephalins (LENK). The regulatory effect of MENK on the immune system is reflected in many aspects, such as regulating macrophage phagocytosis and phenotypic polarization [60], activating NK cells and enhancing their activity [61][62][63][64], promoting the maturation of dendritic cells within a certain concentration range [65], regulating the growth of B lymphocytes bi-directionally [66] and affecting the release of cytokines [67]. Furthermore, studies on the mechanism of acupuncture's effect on the immune activity of NK cells have found that LENK can increase the lethality of NK cells [59]. ...
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As one of the conventional treatment methods, acupuncture is an indispensable component of Traditional Chinese Medicine. Currently, acupuncture has been partly accepted throughout the world, but the mechanism of acupuncture is still unclear. Since the theory of the neuro-endocrine-immune network was put forward, new insights have been brought into the understanding of the mechanism of acupuncture. Studies have proven that acupuncture is a mechanical stimulus that can activate local cell functions and neuroreceptors. It also regulates the release of related biomolecules (peptide hormones, lipid hormones, neuromodulators and neurotransmitters, and other small and large biomolecules) in the microenvironment, where they can affect each other and further activate the neuroendocrine-immune network to achieve holistic regulation. Recently, growing efforts have been made in the research on the mechanism of acupuncture. Some researchers have transitioned from studying the mechanism of acupuncture as a single linear pathway to using systems approaches, including metabolomics, genomics, proteomics and biological pathway analysis. This review summarizes the research progress on the neuro-endocrine-immune network related mechanism of acupuncture and discusses its current challenges and future directions.
... In this regard, the attainment of cardiogenesis in the presence of either chemical agents or physical stimulation encompasses the transcription and protein expression of endorphin peptides [33]. These molecules, besides their role in cardiogenesis [55][56][57], have long been shown to act as negative regulators for the development and spreading of different types of cancer [82][83][84][85][86][87]. ...
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Embryonic development and carcinogenesis share many molecular pathways and regulatory molecules. While the induction of a pluripotent state involves a significant oncogenic risk, as in induced pluripotent stem cells (iPSCs), the embryonic environment in vivo has been shown to suppress tumor development. In this review, we discuss the subtle equilibrium between the nanotopography (niche) of the hosting tissue resident stem cells and their biological dynamics, including the transformation in cancer stem cells. We review consistent findings indicating the potential for modulating the biology of human cancer stem cells by the aid of naturally occurring or synthetic molecules, including developmental stage zebrafish embryo extracts, hyaluronan, butyric acid (BA) and retinoic acid (RA), hyaluronan mixed esters of BA and RA, melatonin, vitamin D3, and endorphin peptides. Within this context, we dissect the multifaceted mechanisms orchestrated by endorphinergic systems, including paracrine cellto- cell communication, as well as the establishment of autocrine and intracrine (intracellular) peptide actions driving transcriptional responses and self-sustaining loops that behave as long-lived signals imparting features characteristic of differentiation, growth regulation and cell memory. Based upon the remarkable action of electromagnetic fields and mechanical vibration on (stem) cell signaling, differentiation, and senescence, we also consider the potential for using these physical energies as a tool to afford a fine tuning of cancer stem cell fate. On the whole, we forecast future deployment of the physical and/or chemical approaches described herein aiming at reprogramming, rather than destroying cancer stem cells, eventually placing cancer therapy within the context of Regenerative Medicine.
... miR-16-5p is considered as a reference miRNA that was related with fatty and metabolism in this study. Moreover, target genes of miRNAs were enriched in pathways characterized to be associated with cancer, including enkephalin release involved in immune system (54), endothelin (55), a number of signaling pathways like G-protein (56,57), apoptosis (58, 59) as well as ATP (60), purine (61), arginine biosynthetic processes (62,63) and glycolysis (64). The relationship between G-protein signaling pathway and fatty acids has been demonstrated in colon cancer (65). ...
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Aim The main goal of this analysis was prioritization of co-expressed genes and miRNAs that are thought to have important influences in the pathogenesis of colon and lung cancers. Background MicroRNAs (miRNAs) as small and endogenous noncoding RNAs which regulate gene expression by repressing mRNA translation or decreasing stability of mRNAs; they have proven pivotal roles in different types of cancers. Accumulating evidence indicates the role of miRNAs in a wide range of biological processes from oncogenesis and tumor suppressors to contribution to tumor progression. Colon and lung cancers are frequently encountered challenging types of cancers; therefore, exploring trade-off among underlying biological units such as miRNA with mRNAs will probably lead to identification of promising biomarkers involved in these malignancies. Methods Colon cancer and lung cancer expression data were downloaded from Firehose and TCGA databases and varied genes extracted by DCGL software were subjected to build two gene regulatory networks by parmigene R package. Afterwards, a network-driven integrative analysis was performed to explore prognosticates genes, miRNAs and underlying pathways. Results A total of 192 differentially expressed miRNAs and their target genes within gene regulatory networks were derived by ARACNE algorithm. BTF3, TP53, MYC, CALR, NEM2, miR-29b-3p and miR-145 were identified as bottleneck nodes and enriched via biological gene ontology (GO) terms and pathways chiefly in biosynthesis and signaling pathways by further screening. Conclusion Our study uncovered correlated alterations in gene expression that may relate with colon and lung cancers and highlighted the potent common biomarker candidates for the two diseases.
... The mechanistic pathways of LDN are still unclear. Some studies indicate that LDN works as an immunomodulating agent by directly bind on the OGFr within immune cells [33,34]. Additionally, evidences suggest that naltrexone acts on the body through at least two different receptor mechanisms. ...
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Historically, studies on endogenous and exogenous opioids and their receptors focused on the mediation of pain, with excess opiate consumption leading to addiction. Opioid antagonists such as naloxone and naltrexone blocked these interactions, and still are widely used to reverse drug and alcohol overdose. Although specific opioid antagonists have been designed for mu, delta, and kappa opioid receptors, the general antagonists remain the most effective. With the discovery of the opioid growth factor (OGF)-OGF receptor (OGFr) axis as a novel biological pathway involved in homeostasis of replicating cells and tissues, the role of opioid receptor antagonists was expanded. An intermittent OGFr blockade by low dosages of naltrexone resulted in depressed cell replication, whereas high (or sustained) dosages of naltrexone that conferred a continuous OGFr blockade resulted in enhanced growth. More than 3 decades of research have confirmed that the duration of opioid receptor blockade, not specifically the dosage, by general opioid antagonists determines the biotherapeutic outcome. Dysregulation of the OGF-OGFr pathway is apparent in a number of human disorders including diabetes, multiple sclerosis, and cancer, and thus opioid antagonist disruption of interaction prevails as a therapeutic intervention. We review evidence that the duration of opioid receptor blockade is correlated with the magnitude and direction of response, and discuss the potential therapeutic effectiveness of continuous receptor blockade for treatment of diabetic complications such as corneal defects and skin wounds, and of intermittent receptor blockade by low dosages of naltrexone for treatment of autoimmune diseases and cancer. Copyright © 2015. Published by Elsevier Inc.
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Methionine enkephalin (MENK), an opioid peptide, is known to function as a regulator in the immune system. As microglia are considered the most important immune cells in the central nervous system (CNS), we aimed to assess the function of MENK on microglia polarization and tumoricidal responses. Initially, we chose the most optimal condition of 10− 12 M for 48 h; however, MENK had no function on the viability and apoptosis of microglia under this treatment. However, MENK treatment markedly increased levels of M1-associated genes, such as CD86, CD40, IL-12, and TNF-α, but had no effect on M2 markers, including CD163, IL-10, and TGF-β. Moreover, microglia in the MENK-treated group showed high phagocytosis capacity, which coincided with characteristics of M1 microglia. MENK stimulation also induced up-regulation of reactive oxygen species (ROS) expression, which contributed to maintaining homeostasis. We also detected NO production by measuring the end product nitrite, and found that MENK treatment increased expression of nitrite and inducible NO synthase (iNOS), but did not influence arginase-1 (Arg1) expression. Furthermore, treatment of microglia with MENK led to a significant increase in cytotoxicity against glioblastoma cells, indicating that MENK possessed anti-tumor ability. Overall, MENK treatment could induce microglia to an M1 phenotype, modulating Th1 responses in the immune system. Additionally, microglia treated with MENK had tumoricidal activity, which provides new insight into anti-tumor immunity.
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The aim of this study was to investigate the effects of mechanisms of methionine enkephalin (MENK) on lymphocytes in human peripheral blood. We detected CD4+T cells, CD8+T cells, CD4+CD25+ regulatory T cells (Treg), dendritic cells (DCs), natural killer cells (NK), NKT cells and γδT cells before and after treatment with 10 (-12) M MENK, in cell culture by FCM and RT-PCR. Our findings show that MENK stimulating expansion of lymphocyte subpopulationns by inhibiting CD4+CD25+ regulatory T cells (Treg), which is unique discovery of our study. We may use MENK as a drug to treat cancer patients, whose immune systems are damaged by chemotherapy or radiotherapy.
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The opioid growth factor (OGF), chemically termed [Met(5)]-enkephalin, is an endogenous opioid peptide that interacts with the OGF receptor (OGFr) to delay the G(1)/S interface of the cell cycle by modulating cyclin-dependent inhibitory kinase (CKI) pathways. The OGF-OGFr axis is a tonically active, inhibitory pathway that is an important regulator during homeostasis and re-epithelialization, and plays a role in the onset and progression of autoimmune diseases and cancer. Modulation of the OGF-OGFr axis can be accomplished by a variety of pharmacological and molecular approaches including use of intermittent or continuous exposure to the opioid antagonist naltrexone, genetic manipulation of OGFr expression, and antibody neutralization of OGF. Clinically, OGF is a biological therapy that has potential application for treatment of cancer. Currently, naltrexone at low dosages is being evaluated for treatment of autoimmune diseases such as Crohn's and multiple sclerosis. High dosages of naltrexone are effective in reversing dry eye and accelerating the repair of corneal abrasions in normal and diabetic rats; these studies are under investigation in the clinical setting. Naltrexone also enhances full-thickness wound closure in animal models of Type 1 or Type 2 diabetes, and translation of this knowledge to the clinic is planned. In summary, understanding the OGF-OGFr axis as a homeostatic regulator of proliferation has substantial implications for maintaining human health and treatment of disease.
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The aim of this study is to investigate macrophages polarization induced by methionine enkephalin (MENK) that promotes tumoricidal responses in vivo and in vitro. Both phenotypic and functional activities of macrophages were assessed by the quantitative analysis of key surface molecules on macrophages with flow cytometry, immunofluorescent staining, and the production of cytokines with enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Our results showed that MENK could down-regulate the expression of CD206 and the production of arginase-1 (the markers of alternatively activated (M2) macrophage) in tumor-associated macrophages in vivo, meanwhile it could significantly up-regulate the expression of CD64, MHC-II, and the production of induced nitric oxide synthase (the markers of classically activated (M1) macrophages). Furthermore, the studies on bone marrow-derived macrophages treated with MENK (10(-12) M) in vitro had demonstrated that MENK could markedly increase tumoricidal activity. MENK could also enhance the release of reactive oxidant species and the production of interleukin-12p40, tumor necrosis factor-α, while decrease the production of interleukin-10. In conclusion, MENK could effectively induce M2 macrophages polarizing to M1 macrophages, sequentially to modulate the Th1 responses of the host immune system. Our results suggest that MENK might have great potential as a new therapeutic agent for cancer.
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MENK, the endogenous neuropeptide, is suggested to be involved in the regulatory loop between the immune and neuroendocrine systems, with modulation of various functions of cells related to both the innate and adaptive immune systems. Our present research findings show that MENK serves as an immune modulator to the pathway between DCs and CD4+T cells. We studied changes of DCs in key surface molecules, the activity of acid phosphatases (ACPs), the production of IL-12, and the effects on murine CD4+T cell expansion and their cytokine production by MENK alone, and in combination with interleukin-2 (IL-2) or interferon-γ (IFN-γ). In fact, we found that MENK could markedly induce the maturation of DCs through the addition of surface molecules such as MHC class II, CD86, and CD40 on murine DCs, the production of IL-12, and the down-regulation of ACP inside DCs, (which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation). We also found that MENK alone or in combination with IL-2 or IFN-γ, could markedly up-regulate both CD4+T cell expansion and the CD4 molecule expression in vivo and in vitro and that MENK alone, or MENK+IL-2, could enhance the production of interferon-γ from CD4+T cells. Moreover, MENK alone, or MENK+IFN-γ, could enhance the production of IL-2 from CD4+T cells. It is therefore concluded that MENK can exert positive modulation to the pathway between dendritic cells and CD4+T cells.
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Endogenous opioids are known to repress the incidence and progression of autoimmune diseases. One native opioid peptide, [Met⁵]-enkephalin, termed the opioid gowth factor (OGF), interacts with the OGF receptor (OGFr) to suppress the expression of experimental autoimmune encephalomyelitis. The present study examined the role of the OGF-OGFr axis in the regulation of B lymphocyte proliferation. Murine B lymphocytes were stimulated with lipopolysaccharide. Both OGF and OGFr were present in all B lymphocytes. OGF had a dose-dependent effect on growth, with cell number inhibited by up to 43% at 72 h; no other synthetic or native opioid altered cell proliferation. Exogenous OGF depressed cell number in cultures treated with siRNAs for the classical opioid receptors, MOR (μ), DOR (δ), and KOR (κ), however this peptide had no effect in preparations exposed to siRNA for OGFr. The decrease in cell number by exogenous OGF was dependent on p16 or p21 cyclin-dependent inhibitory kinase pathways. Exposure to the opioid antagonist, naltrexone, did not change cell number from control levels. These results suggest that the OGF-OGFr axis is present and functional in B lymphocytes, but this system is not an autocrine regulator of cell proliferation. Thus, at least exogenous OGF and perhaps endogenous OGF by paracrine/endocrine sources, can be an immunosuppressant. Modulation of the OGF-OGFr axis may be a novel paradigm for the treatment of autoimmune diseases.
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The opioid growth factor (OGF) regulates cell proliferation of human cancer cells through the cyclin-dependent kinase inhibitory pathway, with mediation of this action by the OGF receptor (OGFr). The ubiquity of the OGF-OGFr axis in human cancer is unknown. We used 31 human cancer cell lines, representative of more than 90% of neoplasias occurring in humans, and found that OGF and OGFr were detected in the cytoplasm and nucleus by immunohistochemistry. The addition of OGF to cultures depressed cell number up to 41%, whereas naltrexone (NTX) increased cell proliferation by up to 44%, a total of 85% in the modulating capacity for the OGF-OGFr axis. Neutralization of OGF by specific antibodies led to a marked increase in cell number. Knockdown of OGFr by OGFr-siRNA resulted in a significant increase in the number of cells, even in the face of the addition of exogenous OGF. The cultures to which NTX was added and subjected to OGFr-siRNA were similar to those with OGF-siRNA alone. The OGF-OGFr axis, a physiological determinant of cell-proliferative activity, is a ubiquitous feature of human cancer cells. The identification of this native biological system in neoplasia may be important in understanding the pathophysiology of neoplasia, and in designing treatment modalities that utilize the body's own chemistry.
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Ovarian cancer is the leading cause of death from gynecological malignancies. Understanding the biology of these tumors, as well as treatment modalities, has been challenging. The opioid growth factor (OGF; [Met(5)]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth-regulating pathway in homeostasis and neoplasia. In this investigation, we examined the relationship of the OGF-OGFr axis to ovarian cancer, and defined its presence, function, and mechanisms. Using OVCAR-3 and SKOV-3 ovarian cancer cell lines, we found that OGF and OGFr were present and functional. Exogenous OGF was observed to have a dose-dependent, serum-independent, reversible, and receptor-mediated inhibitory action on cell proliferation that was dependent on RNA and protein synthesis. The repressive effect of OGF on cell proliferation also was observed in SW626, CAOV-3, and HEY ovarian cancer cell lines. Endogenous OGF was found to be constitutively produced and tonically active on cell replicative activities, with neutralization of this peptide accelerating cell proliferation. Silencing of OGFr using siRNA technology stimulated cell replication, documenting its integral role. The mechanism of OGF-OGFr action on DNA synthesis was related to the cyclin-dependent kinase inhibitory pathway because knockdown of p16 or p21 in OVCAR-3 cells, and p21 in SKOV-3 cells, eliminated OGF's inhibitory effect on growth. These data are the first to report that the OGF-OGFr system is a native biological regulator of cell proliferation in human ovarian cancer. This information will be important in designing treatment strategies for this deadly disease.
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Enkephalin, a natural ligand for opiate receptors is composed of the pentapepides H-Tyr-Gly-Gly-Phe-Met-OH and H-Tyr-Gly-Gly-Phe-Leu-OH. The evidence is based on the determination of the amino acid sequence of natural enkephalin by the dansyl-Edman procedure and by mass spectrometry followed by synthesis and comparison of the natural and synthetic peptides.
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We have previously described the regulatory effect of beta-endorphin on three human cytotoxic cell populations. We confirmed the variable nature of these effects on human natural killer cell (NK) activity, showed mixed effects on the generation of cytotoxic T lymphocyte (CTL) activity, and demonstrated the reproducible suppression of lymphokine-activated killer cell (LAK) activity. We and others also observed mixed effects of beta-endorphin on the proliferative response to mitogens and in mixed leukocyte reactions. In the study reported here, we test the effects of beta-endorphin on the formation of phosphatidylinositol during cell activation. 32P-radiolabeled peripheral blood mononuclear cells obtained from normal adult donors and CD2-depleted subpopulations were activated with phytohemagglutinin or in a NK, LAK, or CTL protocol in the absence or presence of recombinant beta-endorphin. The total lipidic extract was analyzed by thin-layer chromatography and autoradiography. The results of these studies indicate that beta-endorphin blunts the formation of phosphatidylinositol by about 20% in the four systems studied and in all the donors tested. This effect is dose-dependent and is blocked in part by the opioid antagonist, naltrexone, suggesting involvement of the opioid receptor.
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Serum estradiol levels were measured in 360 men attending an infertility clinic and 68 proven fertile men to determine whether estradiol measurements are clinically useful. The normal range of estradiol levels found in fertile men was 10-82 pg/ml. Serum concentrations of estradiol in azoospermic or oligozoospermic patients were significantly lower than those in normozoospermic men (p < 0.021). Serum concentrations of testosterone were also significantly decreased in infertile patients (p < 0.025). This decrease in serum estradiol may be partly due to reduced testosterone levels in these men because estradiol is mainly formed by peripheral aromatization of testosterone in fatty and muscle tissues. However, the exact mechanism for the decrease in the serum estradiol levels remains undetermined. It is concluded that serum estradiol levels are significantly low in the men with various testicular disorders.
Article
The effect of chronic morphine exposure on natural killer (NK) activity in vivo and the generation of cytotoxic T lymphocytes (CTLs) in vitro and in vivo was investigated. Chronic exposure to morphine (10(-5) - 10(-11) M) in vitro had no effect on the generation of antigen-driven effector cells. However, the daily administration of morphine (50.0 mg/kg, s.c.) into alloimmunized mice (C57BL/6 into C3H/HeN) for 11 days resulted in a decrease in peritoneal and splenic CTL activity but not splenic NK activity. In addition, there was a 60% decrease in the number of thymocytes recovered from chronic morphine-treated mice compared to vehicle-treated controls. However, the overall percentage of CD4+CD8-, CD4-CD8+ and CD4+CD8+ thymocytes did not change between the two groups of treated animals. Pretreatment of the mice with the delta 1-selective antagonist, (E)-7-benzylidine-7-dihydronaltrexone (BNTX, 0.6 mg/kg, s.c.) did not block morphine-mediated suppression of splenic CTL activity but did block morphine-induced suppression of peritoneal lymphocyte CTL activity. In addition, BNTX pretreatment alone augmented splenic NK activity and such augmentation was blocked following chronic morphine exposure. In contrast, the delta-selective antagonist, naltrindole (20.0 mg/kg, s.c.), had no effect alone nor antagonized the action of morphine on CTL activity. Splenic CTL effector cells from either treated group of animals lysed their target (EL-4 lymphoma) through a Ca(2+)-dependent mechanism. Collectively, the results indicate morphine suppresses CTL activity through an indirect pathway, insensitive to naltrindole rather than through direct lymphocyte opioid receptors.
Article
Based on a plethora of data from many laboratories, we have proposed the following mechanisms by which morphine alters immune homeostasis and immunocompetence in vivo (Fig. 2). Specifically, the administration of morphine subcutaneously via routing through blood interacts directly with opioid receptors on cells of the immune system or on receptors within the central nervous system. Although there is currently no evidence to support the direct involvement of morphine on lymphocyte opioid receptors, in vitro studies show the existence of functional, naloxone-sensitive opioid receptors (25). In addition, pharmacological and biochemical characterization of lymphocyte opioid receptors has been shown to be consistent in many instances, with the profile of neural-derived opioid receptors (25-27). Finally, recent molecular studies using oligonucleotide primers specific for the delta-class opioid receptor cloned from NG-108-15 cells (28) have been used in reverse transcription-polymerase chain reactions to generate a 400 bp product in SL which has 100% sequence homology with a published opioid receptor cloned from a brain library (35). However, future studies are necessary to establish the role of lymphocyte opioid receptors following the in vivo administration of opioids (e.g. fentanyl, methadone, and morphine). Since the administration of morphine subcutaneously appears to predominately interact with brain opioid receptors (3) located in the mesencephalon (5), other neuroendocrine systems become candidates for activation and subsequent direct modulation of immune function: (i) the HPA axis and (ii) the sympathetic nervous system (SNS).(ABSTRACT TRUNCATED AT 250 WORDS)
Article
The immunosuppressive effect of morphine in an HIV-1 transactivator of transcription (TAT)-transgenic mouse model was investigated in order to elucidate possible mechanisms of human immunodeficiency virus (HIV)-1 disease progression. The TAT72 transgene (1-72 amino acids) was placed under the control of SV40 viral promoter to provide systemic expression. Mice were treated daily for 5 days with morphine (50.0 mg/kg) or vehicle following alloantigen immunization. In TAT-transgenic mice, morphine modestly reduced mitogen-induced IL-2 production, which correlated with reduced percentages of CD4+ and CD8+ splenic lymphocytes. TAT-transgenic animals displayed reduced splenic natural killer (NK) and peritoneal cytotoxic T-lymphocyte (CTL) activities irrespective of morphine treatment. In addition, the effect of morphine on splenic NK and CTL activity was shown to be stereospecific as defined using (+)-morphine (50 mg/kg). Pretreatment of mice with the mu-selective opioid receptor antagonist beta-funaltrexamine (40.0 mg/kg) blocked morphine-induced modulation of splenic CTL activity. Since elevated corticosterone levels have previously been associated with immunosuppression following prolonged morphine exposure, serum corticosterone levels were assessed. Reduced serum corticosterone levels were found to be associated with morphine treatment in non-transgenic mice as well as vehicle- or morphine-treated mice. Collectively, the data suggest that the presence of TAT72 compromises splenic NK activity as well as peritoneal CTL activity and leads to a reduction in serum corticosterone levels. Also, morphine-mediated modulation of the immune system in non-transgenic mice is stereoselective and due in part to mu-opioid receptors.
Article
Methionine-enkephalin (MET) modulates various functions of macrophages related to both immune and inflammatory reactions in a naloxone reversible manner, suggesting that opioid receptors are involved in the regulation of macrophage activity. Since an endogenous opioid ligand might interact with more than one type of opioid receptor, the receptor interaction determines its effect on a particular function. In the present study we have investigated the involvement of different opioid receptor types/subtypes in MET-induced modulation of H(2)O(2) and NO production in macrophages. Thioglycollate-elicited or resident rat peritoneal macrophages were treated in vitro with MET and/or specific antagonists of delta(1,2), delta(1), delta(2), mu and kappa opioid receptors. MET increased H(2)O(2)production in phorbol myristate acetate-stimulated rat peritoneal macrophages mainly through delta(1) opioid receptor. MET also enhanced NO production in rat peritoneal macrophages stimulated with lipopolysaccharide through delta(1) and mu opioid receptors. The blockade of mu and kappa receptor facilitated a potentiating effect of MET on H(2)O(2) release, and blockade of kappa receptor further raised the MET-induced increase of NO production in macrophages. It is concluded that both negative and positive functional interaction between delta, mu and kappa opioid receptors regulate the influence of MET on H(2)O(2) and NO production in rat peritoneal macrophages.
Article
Acute cholestasis is associated with increased activity of the endogenous opioid system. It is also known that opioid receptor agonists like morphine decrease the intestinal transit. The purpose of the present study was to investigate the effect of cholestasis on the small intestine transit and the possible involvement of opioid system in this phenomenon in mice. Cholestasis was induced by bile duct-ligation and intestinal transit was measured with charcoal meal and calculation of percent of transit through small intestine. The effect of chronic administration of naltrexone and acute pretreatment with morphine on intestinal transit was evaluated in bile duct-ligated (BDL) as well as unoperated (CTL) and sham-operated (SHAM) animals. The plasma alkaline phosphatase and alanine aminotransferase activities were also measured. A significant decrease in small intestine transit (%transit) was observed in BDL mice compared to SHAM animals, which was prominent even after 24 h of cholestasis. Chronic pretreatment with an opioid receptor antagonist, naltrexone, (10 mg/kg, i.p for 2, 4 or 6 days) completely restored the cholestasis-induced decrease in %transit to that of control animals. Although the acute administration of morphine (2 mg/kg, s.c.) 20 min before charcoal feeding caused a significant decrease in the intestinal transit of CTL and SHAM animals, it did not decrease the %transit of BDL animals on the day 5 after operation. Our findings show that acute cholestasis is associated with a prominent decrease in small intestine transit in mice and opioid receptors maybe involved in this phenomenon.
Identification of two related
  • Hughes J Tw Smith
  • Kosterlitz Hw
  • Morgan La Ba Fothergill
  • Morris
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Hughes J, Smith TW, Kosterlitz HW, Fothergill LA, Morgan BA, Morris HR. Identification of two related