Nicotinic receptors modulate antitumor therapy response in triple negative breast cancer cells.

BACKGROUND: Triple negative breast cancer is more aggressive than other breast cancer subtypes and constitutes a public health problem worldwide since it has high morbidity and mortality due to the lack of defined therapeutic targets. Resistance to chemotherapy complicates the course of patients' treatment. Several authors have highlighted the participation of nicotinic acetylcholine receptors (nAChR) in the modulation of conventional chemotherapy treatment in cancers of the airways. However, in breast cancer, less is known about the effect of nAChR activation by nicotine on chemotherapy treatment in smoking patients. AIM: To investigate the effect of nicotine on paclitaxel treatment and the signaling pathways involved in human breast MDA-MB-231 tumor cells. METHODS: Cells were treated with paclitaxel alone or in combination with nicotine, administered for one or three 48-h cycles. The effect of the addition of nicotine (at a concentration similar to that found in passive smokers' blood) on the treatment with paclitaxel (at a therapeutic concentration) was determined using the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The signaling mediators involved in this effect were determined using selective inhibitors. We also investigated nAChR expression, and ATP "binding cassette" G2 drug transporter (ABCG2) expression and its modulation by the different treatments with Western blot. The effect of the treatments on apoptosis induction was determined by flow cytometry using annexin-V and 7AAD markers. RESULTS: Our results confirmed that treatment with paclitaxel reduced MDA-MB-231 cell viability in a concentration-dependent manner and that the presence of nicotine reversed the cytotoxic effect induced by paclitaxel by involving the expression of functional α7 and α9 nAChRs in these cells. The action of nicotine on paclitaxel treatment was linked to modulation of the protein kinase C, mitogen-activated protein kinase, extracellular signal-regulated kinase, and NF-κB signaling pathways, and to an up-regulation of ABCG2 protein expression. We also detected that nicotine significantly reduced the increase in cell apoptosis induced by paclitaxel treatment. Moreover, the presence of nicotine reduced the efficacy of paclitaxel treatment administered in three cycles to MDA-MB-231 tumor cells. CONCLUSION: Our findings point to nAChRs as responsible for the decrease in the chemotherapeutic effect of paclitaxel in triple negative tumors. Thus, nAChRs should be considered as targets in smoking patients.

Breast cancer: Muscarinic receptors as new targets for tumor therapy.

The development of breast cancer is a complex process that involves the participation of different factors. Several authors have demonstrated the overexpression of muscarinic acetylcholine receptors (mAChRs) in different tumor tissues and their role in the modulation of tumor biology, positioning them as therapeutic targets in cancer. The conventional treatment for breast cancer involves surgery, radiotherapy, and/or chemotherapy. The latter presents disadvantages such as limited specificity, the appearance of resistance to treatment and other side effects. To prevent these side effects, several schedules of drug administration, like metronomic therapy, have been developed. Metronomic therapy is a type of chemotherapy in which one or more drugs are administered at low concentrations repetitively. Recently, two chemotherapeutic agents usually used to treat breast cancer have been considered able to activate mAChRs. The combination of low concentrations of these chemotherapeutic agents with muscarinic agonists could be a useful option to be applied in breast cancer treatment, since this combination not only reduces tumor cell survival without affecting normal cells, but also decreases pathological neo-angiogenesis, the expression of drug extrusion proteins and the cancer stem cell fraction. In this review, we focus on the previous evidences that have positioned mAChRs as relevant therapeutic targets in breast cancer and analyze the effects of administering muscarinic agonists in combination with conventional chemotherapeutic agents in a metronomic schedule.

Pyridinecarboxylic Acid Derivative Stimulates Pro-Angiogenic Mediators by PI3K/AKT/mTOR and Inhibits Reactive Nitrogen and Oxygen Species and NF-κB Activation Through a PPARγ-Dependent Pathway in T. cruzi-Infected Macrophages.

Chagas disease is caused by Trypanosoma cruzi infection and represents an important public health concern in Latin America. Macrophages are one of the main infiltrating leukocytes in response to infection. Parasite persistence could trigger a sustained activation of these cells, contributing to the damage observed in this pathology, particularly in the heart. HP24, a pyridinecarboxylic acid derivative, is a new PPARγ ligand that exerts anti-inflammatory and pro-angiogenic effects. The aim of this work was to deepen the study of the mechanisms involved in the pro-angiogenic and anti-inflammatory effects of HP24 in T. cruzi-infected macrophages, which have not yet been elucidated. We show for the first time that HP24 increases expression of VEGF-A and eNOS through PI3K/AKT/mTOR and PPARγ pathways and that HP24 inhibits iNOS expression and NO release, a pro-inflammatory mediator, through PPARγ-dependent mechanisms. Furthermore, this study shows that HP24 modulates H2O2 production in a PPARγ-dependent manner. It is also demonstrated that this new PPARγ ligand inhibits the NF-κB pathway. HP24 inhibits IKK phosphorylation and IκB-α degradation, as well as p65 translocation to the nucleus in a PPARγ-dependent manner. In Chagas disease, both the sustained increment in pro-inflammatory mediators and microvascular abnormalities are crucial aspects for the generation of cardiac damage. Elucidating the mechanism of action of new PPARγ ligands is highly attractive, given the fact that it can be used as an adjuvant therapy, particularly in the case of Chagas disease in which inflammation and tissue remodeling play an important role in the pathophysiology of this disease.

Endogenous lysophosphatidic acid participates in vascularisation and decidualisation at the maternal-fetal interface in the rat.

Lysophosphatidic acid (LPA) affects several female reproductive functions through G-protein-coupled receptors. LPA contributes to embryo implantation via the lysophospholipid LPA3 receptor. In the present study we investigated the participation of endogenous LPA signalling through the LPA3 receptor in vascularisation and decidualisation, two crucial events at the maternal-fetal interface. Pregnant rats were treated with diacylglycerol pyrophosphate (DGPP), a highly selective antagonist of LPA3 receptors, on Day 5 of gestation. Pregnant rats received intrauterine (i.u.) injections of single doses of DGPP (0.1mgkg-1) in a total volume of 2µL in the left horn (treated horn) in the morning of GD5. DGPP treatment produced aberrant embryo spacing and increased embryo resorption. The LPA3 receptor antagonist decreased the cross-sectional length of the uterine and arcuate arteries and induced histological anomalies in the decidua and placentas. Marked haemorrhagic processes, infiltration of immune cells and tissue disorganisation were observed in decidual and placental tissues from sites of resorption. The mRNA expression of three vascularisation markers, namely interleukin 10 (Il10), vascular endothelial growth factor (Vegfa) and vascular endothelial growth factor receptor 1 (Vegfr1), was reduced at sites of resorption from Day 8. The results show that the disruption of endogenous LPA signalling by blocking the LPA3 receptor modified the development of uterine vessels with consequences in the formation of the decidua and placenta and in the growth of embryos.

Treatment with a New Peroxisome Proliferator-Activated Receptor Gamma Agonist, Pyridinecarboxylic Acid Derivative, Increases Angiogenesis and Reduces Inflammatory Mediators in the Heart of Trypanosoma cruzi-Infected Mice.

Trypanosoma cruzi infection induces an intense inflammatory response in diverse host tissues. The immune response and the microvascular abnormalities associated with infection are crucial aspects in the generation of heart damage in Chagas disease. Upon parasite uptake, macrophages, which are involved in the clearance of infection, increase inflammatory mediators, leading to parasite killing. The exacerbation of the inflammatory response may lead to tissue damage. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent nuclear transcription factor that exerts important anti-inflammatory effects and is involved in improving endothelial functions and proangiogenic capacities. In this study, we evaluated the intermolecular interaction between PPARγ and a new synthetic PPARγ ligand, HP24, using virtual docking. Also, we showed that early treatment with HP24, decreases the expression of NOS2, a pro-inflammatory mediator, and stimulates proangiogenic mediators (vascular endothelial growth factor A, CD31, and Arginase I) both in macrophages and in the heart of T. cruzi-infected mice. Moreover, HP24 reduces the inflammatory response, cardiac fibrosis and the levels of inflammatory cytokines (TNF-α, interleukin 6) released by macrophages of T. cruzi-infected mice. We consider that PPARγ agonists might be useful as coadjuvants of the antiparasitic treatment of Chagas disease, to delay, reverse, or preclude the onset of heart damage.

Synthetic stigmasterol derivatives inhibit capillary tube formation, herpetic corneal neovascularization and tumor induced angiogenesis: Antiangiogenic stigmasterol derivatives.

Angiogenesis plays a critical role in initiating and promoting several diseases, such as cancer and herpetic stromal keratitis (HSK). Herein, we studied the inhibitory effect of two synthetic stigmasterol derivatives on capillary tube-like structures and on cell migration in human umbilical vein endothelial cells (HUVEC): (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3ß-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2). We also studied their effect on VEGF expression in IL-6 stimulated macrophages and in LMM3 breast cancer cells. Furthermore, we investigated the antiangiogenic activity of the compounds on corneal neovascularization in the murine model of HSK and in an experimental model of tumor-induced angiogenesis in mice. Both compounds inhibited capillary tube-like formation, but only compound 1 restrained cell migration. Compound 1, unlike compound 2, was able to reduce VEGF expression. Only compound 1 not only reduced the incidence and severity of corneal neovascularization, when administered at the onset of HSK, but it also restrained the development of neovascular response induced by tumor cells in mice skin. Our results show that compound 1 inhibits angiogenesis in vitro and in vivo. Therefore, compound 1 would be a promising drug in the treatment of those diseases where angiogenesis represents one of the main pathogenic events.

Muscarinic Receptors as Targets for Metronomic Therapy in Breast Cancer.

BACKGROUND: It is actually known that acetylcholine works as a signaling molecule in non-neuronal cells and tissues, in addition to its neuronal function as neurotransmitter. It can act on two types of receptors nicotinic and muscarinic receptors (mAChRs). The latter belong to the G protein coupled receptor family and there are five subtypes genetically cloned. Their activation triggers classical and non-classical intracellular signals that could be linked to the proliferation of normal and/or transformed cells. The M3 subtype was identified in different types of tumors and its stimulation with agonists triggers cell proliferation, migration, invasion and metastasis. RESULTS: Our laboratory has extensively investigated the expression and function of mAChRs in breast tumors from animal and human origins. We found a profuse expression of mAChRs in breast tumors, but opposite to this, an absence of these receptors in normal breast cells and tissues. The stimulation of mAChRs with the cholinergic agonist carbachol for 20 h increased tumor cell death. Moreover, the combination of subthreshold concentrations of the agonist with paclitaxel potentiates cell death. The usage of low dose chemotherapy with short drug free intervals was named metronomic therapy and it has emerged as a novel regimen for cancer treatment with very low incidence of side effects. CONCLUSION: Our work and that of others indicate that mAChRs that are over-expressed in different types of tumor cells could be a useful target for metronomic therapy in cancer treatment.

Hexachlorobenzene promotes angiogenesis in vivo, in a breast cancer model and neovasculogenesis in vitro, in the human microvascular endothelial cell line HMEC-1.

Exposure to environmental pollutants may alter proangiogenic ability and promotes tumor growth. Hexachlorobenzene (HCB) is an organochlorine pesticide found in maternal milk and in lipid foods, and a weak ligand of the aryl hydrocarbon receptor (AhR). HCB induces migration and invasion in human breast cancer cells, as well as tumor growth and metastasis in vivo. In this study, we examined HCB action on angiogenesis in mammary carcinogenesis. HCB stimulates angiogenesis and increases vascular endothelial growth factor (VEGF) expression in a xenograft model with the human breast cancer cell line MDA-MB-231. Human microvascular endothelial cells HMEC-1 exposed to HCB (0.005, 0.05, 0.5 and 5µM) showed an increase in cyclooxygenase-2 (COX-2) and VEGF protein expression involving AhR. In addition, we found that HCB enhances VEGF-Receptor 2 (VEGFR2) expression, and activates its downstream pathways p38 and ERK1/2. HCB induces cell migration and neovasculogenesis in a dose-dependent manner. Cells pretreatment with AhR, COX-2 and VEGFR2 selective inhibitors, suppressed these effects. In conclusion, our results show that HCB promotes angiogenesis in vivo and in vitro. HCB-induced cell migration and tubulogenesis are mediated by AhR, COX-2 and VEGFR2 in HMEC-1. These findings may help to understand the association among HCB exposure, angiogenesis and mammary carcinogenesis.

Participation of non-neuronal muscarinic receptors in the effect of carbachol with paclitaxel on human breast adenocarcinoma cells. Roles of nitric oxide synthase and arginase.

Breast cancer is the most common type of cancer in women and represents a major issue in public health. The most frequent methods to treat these tumors are surgery and/or chemotherapy. The latter can exert not only beneficial effects by reducing tumor growth and metastasis, but also toxic actions on normal tissues. Metronomic therapy involves the use of low doses of cytotoxic drugs alone or in combination to improve efficacy and to reduce adverse effects. We have previously reported that breast tumors highly express functional muscarinic acetylcholine receptors (mAChRs) that regulate tumor progression. For this reason, mAChRs could be considered as therapeutic targets in breast cancer. In this paper, we investigated the ability of a combination of the cytotoxic drug paclitaxel plus carbachol, a cholinergic agonist, at low doses, to induce death in breast tumor MCF-7 cells, via mAChR activation, and the role of nitric oxide synthase (NOS) and arginase in this effect. We observed that the combination of carbachol plus paclitaxel at subthreshold doses significantly increased cytotoxicity in tumor cells without affecting MCF-10A cells, derived from human normal mammary gland. This effect was reduced in the presence of the muscarinic antagonist atropine. The combination also increased nitric oxide production by NOS1 and NOS3 via mAChR activation, concomitantly with an up-regulation of NOS3 expression. The latter effects were accompanied by a reduction in arginase II activity. In conclusion, our work demonstrates that mAChRs expressed in breast tumor cells could be considered as candidates to become targets for metronomic therapy in cancer treatment.

Treatment in vitro with PPARα and PPARγ ligands drives M1-to-M2 polarization of macrophages from T. cruzi-infected mice.

Trypanosoma cruzi, the etiological agent of Chagas' disease, induces a persistent inflammatory response. Macrophages are a first line cell phenotype involved in the clearance of infection. Upon parasite uptake, these cells increase inflammatory mediators like NO, TNF-α, IL-1ß and IL-6, leading to parasite killing. Although desired, inflammatory response perpetuation and exacerbation may lead to tissue damage. Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent nuclear transcription factors that, besides regulating lipid and carbohydrate metabolism, have a significant anti-inflammatory effect. This is mediated through the interaction of the receptors with their ligands. PPARγ, one of the PPAR isoforms, has been implicated in macrophage polarization from M1, the classically activated phenotype, to M2, the alternatively activated phenotype, in different models of metabolic disorders and infection. In this study, we show for the first time that, besides PPARγ, PPARα is also involved in the in vitro polarization of macrophages isolated from T. cruzi-infected mice. Polarization was evidenced by a decrease in the expression of NOS2 and proinflammatory cytokines and the increase in M2 markers like Arginase I, Ym1, mannose receptor and TGF-ß. Besides, macrophage phagocytic activity was significantly enhanced, leading to increased parasite load. We suggest that modulation of the inflammatory response by both PPARs might be due, at least in part, to a change in the profile of inflammatory macrophages. The potential use of PPAR agonists as modulators of overt inflammatory response during the course of Chagas' disease deserves further investigation.

Autoantibodies against muscarinic receptors in breast cancer: their role in tumor angiogenesis.

The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10(-8) M) and carbachol (10(-9) M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.

Muscarinic activation enhances the anti-proliferative effect of paclitaxel in murine breast tumor cells.

Muscarinic acetylcholine receptors (mAChR) are expressed in cells without nervous origin. mAChR are up-regulated in tumor cells and their stimulation can modulate tumor growth. In this work we investigated the ability of mAChR activation to induce tumor cell death. We studied the action of a combination of low doses of the muscarinic agonist carbachol plus paclitaxel, a chemotherapeutic agent frequently used in breast cancer treatment, in terms of effectiveness. Long term treatment with carbachol exerted anti-proliferative actions on LM2 and LM3 murine mammary adenocarcinoma cells, similarly to paclitaxel. The combination of carbachol with paclitaxel at submaximal concentrations, added during 20 h decreased tumor cell proliferation in a more potent manner than each drug added separately. This effect was reverted by the muscarinic antagonist atropine, and was due to a potentiation of tumor cell apoptosis tested by TUNEL assay. This treatment did not affect the proliferation of the non tumorigenic mammary cell line NMuMG. In conclusion, the combination of a muscarinic agonist plus paclitaxel should be tested as a useful therapeutic tool in breast cancer treatment.

Cholinergic drugs as therapeutic tools in inflammatory diseases: participation of neuronal and non-neuronal cholinergic systems.

Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) from acetylcoenzime A and choline. This reaction occurs not only in pre-ganglionic fibers of the autonomic nervous system and post-ganglionic parasympathetic nervous fibers but also in non neuronal cells. This knowledge led to expand the role of ACh as a neurotransmitter and to consider it as a "cytotransmitter" and also to evaluate the existence of a non-neuronal cholinergic system comprising ACh, ChAT, acetylcholinesterase, and the nicotinic and muscarinic ACh receptors, outside the nervous system. This review analyzes the participation of cholinergic system in inflammation and discusses the role of different muscarinic and nicotinic drugs that are being used to treat skin inflammatory disorders, asthma, and chronic obstructive pulmonary disease as well as, intestinal inflammation and systemic inflammatory diseases, among others, to assess the potential application of these compounds as therapeutic tools.

PPARγ ligand treatment inhibits cardiac inflammatory mediators induced by infection with different lethality strains of Trypanosoma cruzi.

Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas' disease, causes cardiac alterations in the host. Although the main clinical manifestations arise during the chronic stage, the mechanisms leading to heart damage develop early during infection. In fact, an intense inflammatory response is observed from acute stage of infection. Recently, peroxisome proliferator-activated receptors (PPARs) have attracted research interest due to their participation in the modulation of inflammation. In this work we addressed the role of 15-Deoxy-∆(12,14) ProstaglandinJ2 (15dPGJ2), a PPARγ natural ligand in the regulation of inflammatory mediators, in acute and chronic experimental mouse models of Chagas' disease with the RA and K98 T. cruzi strains, respectively. This work demonstrates that 15dPGJ2 treatment inhibits the expression and activity of inducible nitric oxide synthase (NOS2) as well as TNF-α and IL-6 mRNA levels. Also, expression and activity of metalloproteinases 2 (MMP-2) and 9 (MMP9) were inhibited by 15dPGJ2. Moreover GW9662, a specific PPARγ antagonist, revealed the participation of other signaling pathways since, in GW9662 presence, 15dPJG2 had a partial effect on the inhibition of inflammatory parameters in the acute model of infection. Accordingly, NF-κB activation was demonstrated, assessing p65 nuclear translocation in the hearts of infected mice with both T. cruzi strains. Such effect was inhibited after 15dPGJ2 treatment. Our findings support the concept that in vivo PPARγ and NF-κB pathways are implicated in the inhibitory effects of 15dPGJ2 on inflammatory mediators at different times depending on whether the infection is caused by the lethal or non-lethal T. cruzi strain.

Muscarinic regulation of SCA-9 cell proliferation via nitric oxide synthases, arginases and cyclooxygenases. Role of the nuclear translocation factor-κB.

The submandibular gland-derived tumor cell line SCA-9 is considered a useful tool to study the signaling pathways involved in proliferation, and their regulation, triggered by different stimuli. It is proposed that the non neuronal cholinergic system: acethylcholine, the enzymes that synthesize and degrade it, and the nicotinic and muscarinic receptors, play a key role in tumorigenesis. Here, we investigate the role of muscarinic receptors in SCA-9 cell proliferation, and the modulation of cholinergic signaling pathways exerted by the nuclear transcription factor κB (NF-κB). The activation of cholinergic receptors by carbachol (10⁻9M) increased cell proliferation (P<0.001). This was prevented by preincubating cells with the muscarinic antagonist atropine but not by mecamylamine, a nicotinic receptor blocker. Phospholipase C (PLC)/nitric oxide synthase (NOS)/arginase pathway is involved in this effect, since carbachol stimulated nitric oxide production, increased NOS2 and NOS3 expressions, urea production, and arginase II expression (P<0.001). Also, phospholipase A2 (PLA2)/cyclooxygenase (COX) pathway is up-regulated in carbachol-induced SCA-9 cell proliferation, because prostaglandin E2 liberation (P<0.001) is increased and COX-1 expression is turned up (P<0.001). Interactions between PLC/NOS/arginases and PLA2/COX pathways via its metabolites were detected. SCA-9 cells exhibit a constitutive activation of NF-κB, which regulates carbachol-induced NOS2 and 3, arginase II and COX-1 expressions. In addition, protein kinase C is involved in the up-regulation of NOS2 and arginase II enzymes induced by carbachol via NF-κB. In conclusion, the activation of cholinergic receptors in SCA-9 tumor cells promotes proliferation via muscarinic effector enzymes, and reveals the participation of NF-κB at this step of tumorigenesis.

Cholinergic modulation of dendritic cell function.

Dendritic cells (DCs) are highly specialized antigen-presenting cells with a unique ability to activate resting T lymphocytes. Acetylcholine (ACh) is the primary parasympathetic neurotransmitter and also a non-neural paracrine factor produced by different cells. Here, we analyzed the expression of the cholinergic system in DCs. We found that DCs express the muscarinic receptors M(3), M(4) and M(5), as well as the enzymes responsible for the synthesis and degradation of ACh, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), respectively. Differentiation of DCs in the presence of the cholinergic agonist carbachol, the synthetic analog of ACh, resulted in an increased expression of HLA-DR and CD86 and the stimulation of TNF-α and IL-8 production. All these effects were prevented by atropine, a muscarinic ACh receptor (mAChR) antagonist. Carbachol, was also able to modulate the function of DCs when added after the differentiation is accomplished; it increased the expression of HLA-DR, improved the T cell priming ability of DCs, and stimulated the production of TNF-α but not IL-12 or IL-10. By contrast, carbachol significantly inhibited the stimulation of HLA-DR expression and the enhancement in the T cell priming ability of DCs triggered by LPS. Interestingly, the TNF-α antagonist etanercept completely prevented the increased expression of HLA-DR induced by carbachol, suggesting that it promotes the phenotypic maturation of DCs by stimulating the production of TNF-α. ACh induced similar effects than carbachol; it stimulated the expression of HLA-DR and the production of TNF-α, while inhibiting the stimulation of HLA-DR expression and IL-12 production triggered by LPS. Similarly, neostigmine, an inhibitor of AChE, also stimulated the expression of HLA-DR and the production of TNF-α by DCs while inhibiting the production of TNF-α and IL-12 triggered by LPS. These results support the existence of an autocrine/paracrine loop through which ACh modulates the function of DCs.

Muscarinic receptors as targets for anti-inflammatory therapy.

ACh, the main neurotransmitter in the neuronal cholinergic system, is synthesized by pre-ganglionic fibers of the sympathetic and parasympathetic autonomic nervous system and by post-ganglionic parasympathetic fibers. There is increasing experimental evidence that ACh is widely expressed in prokaryotic and eukaryotic non-neuronal cells. The neuronal and non-neuronal cholinergic systems comprise ACh, choline acetyltransferase and cholinesterase, enzymes that synthesize and catabolize ACh, and the nicotinic and muscarinic ACh receptors (nAChRs and mAChRs, respectively), which are the targets for ACh action. This review analyzes the participation of the cholinergic system, particularly through mAChRs, in inflammation, and discusses the role of the different mAChR antagonists that have been used to treat skin inflammatory disorders, asthma and COPD, as well as intestinal inflammation and systemic inflammatory diseases, to assess the potential application of these compounds as therapeutic tools.

Immunoglobulin G from breast cancer patients in stage I stimulates muscarinic acetylcholine receptors in MCF7 cells and induces proliferation. Participation of nitric oxide synthase-derived nitric oxide.

INTRODUCTION: Muscarinic acetylcholine receptors (mAChR) belong to the G-protein-coupled receptor family and are extensively expressed in most cells in mammals. We had reported the expression of mAChR in murine and human breast tumors. METHODS: The presence of antibodies in the sera of patients with different tumors directed against self-proteins has been recently described. In this work, we investigated the presence of autoantibodies against mAChR in the sera of breast cancer patients in stage I (T1N0Mx-IgG). IgG purification was performed by affinity chromatography in protein G-agarose. We also studied the ability of these antibodies to modulate the proliferation of MCF-7 breast tumor cells by the MTS colorimetric assay. The ability of T1N0Mx-IgG to stimulate muscarinic signaling pathway via nitric oxide synthase was tested by Griess reaction. RESULTS: We demonstrated M(3) and M(4) receptors expression in MCF-7 cells. T1N0Mx-IgG promotes cell proliferation, mimicking the action of the muscarinic agonist carbachol. This effect was preferentially due to M(3) receptor activation in tumor cells via phospholipase C-induced nitric oxide liberation by calcium-dependent nitric oxide synthases. IgG from control patients was unable to produce this effect. DISCUSSION: IgG from patients with breast cancer in early stages could be promoting tumor progression by muscarinic activation, and its presence could be determining the prognosis of this illness.

Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells.

OBJECTIVE: Fibroblasts are sentinel cells that could serve as intermediaries in the immune reaction in the inflammatory process. In this work, we investigate the action of the muscarinic agonist carbachol (CARB) on the expression and function of nitric oxide synthase (NOS) and cyclooxygenase (COX) in fibroblasts under normal or inflammatory conditions. METHODS: The normal fibroblast cell line, 3T3, from NIH swiss mouse embryo, was used. The inflammatory milieu was mimicked with lipopolysaccharide (LPS) (10 ng/ml) plus interferon gamma (IFNgamma) (0.5 ng/ml). Nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production were measured by Griess reagent and radioimmunoassay, respectively. NOS, COX, and nuclear transcription factor kappa B (NF-kappaB) were studied by Western blot. RESULTS: CARB increased NO synthesis by 57 +/- 5%, while a 150 +/- 10% increase in NO liberation was triggered by LPS plus IFNgamma treatment. CARB added to LPS plus IFNgamma potentiated NO synthesis by 227 +/- 19%. CARB also upregulated NOS1 protein expression via NF-kappaB activation. In addition CARB and LPS plus IFNgamma stimulated PGE(2) synthesis by 72 +/- 9 and 42 +/- 4%, respectively, while CARB added to LPS plus IFNgamma treated cells produced a synergism in PGE(2) liberation (130 +/- 12%) via COX-2. CONCLUSION: Activation of muscarinic acetylcholine receptors can mimic mild inflammatory conditions or can deepen pre-existing inflammation, establishing a fine-tuned set-up on fibroblasts that in turn could be alerting the immune system.

Muscarinic receptors autoantibodies purified from mammary adenocarcinoma-bearing mice sera stimulate tumor progression.

The ability of tumor cells to stimulate adaptive immunity, particularly by inducing anti-tumor antibodies (Abs), has been extensively reviewed. LM3 is a tumorigenic cell line derived from a murine mammary metastatic adenocarcinoma that spontaneously overexpressed mAchR. Here we investigate the ability of Abs purified from the sera of LM3 tumor-bearing mice, directed against muscarinic acetylcholine receptors (mAchR) to modulate tumor cells' proliferation and angiogenesis. We observed that IgG from early tumor bearers (ETB), 14-day LM3 tumor, and from late tumor bearers (LTB), 28-day LM3 tumor, displaced tritiated quinuclidinyl benzilate binding to LM3 tumor cells, confirming Abs interaction with cholinoceptors, while IgG from normal mice did not modify the antagonist binding to mAchR at any concentration tested. In addition, Abs from ETB and LTB immunoblotted a protein of 70 kDa on murine tumor cells and on heart homogenates that was also recognized by a specific anti-M(2) receptor monoclonal antibody. We also observed that IgG purified from ETB-stimulated LM3 cells' proliferation in a more effective manner than the muscarinic agonist carbachol (CARB) did. IgG from LTB-potentiated LM3 cells induced angiogenesis by increasing the number of blood vessels and VEGF-A production in peritumoral skin "via" mAchR, in an agonist similar manner. All effects were blocked by preincubating cells with the non-selective antagonist atropine. In conclusion, autoAbs purified from LM3 tumor-bearing mice sera exert different pro-tumor actions depending on the stage of tumor development: in ETB, they stimulate tumor cells' proliferation, while in LTB they potentiate tumor neovascularization.