Effect of low dose metronomic therapy on MCF-7 tumor cells growth and angiogenesis. Role of muscarinic acetylcholine receptors.

The non-neuronal cholinergic system refers to the presence of acetylcholine, choline acetyltransferase, acetylcholinesterase and cholinergic receptors, nicotinic and muscarinic (mAChRs) expressed in non-neuronal cells. The presence of mAChRs has been detected in different type of tumor cells and they are linked with tumorigenesis. We had previously documented the expression of mAChRs in murine and human mammary adenocarcinomas and the absence of these receptors in normal mammary cells of the same origins. We also demonstrated that mAChRs are involved in breast cancer progression, pointing to a main role for mAChRs as oncogenic proteins. Since the long term treatment of breast cancer cells with the muscarinic agonist carbachol promoted cell death, here we investigated the ability of low doses of this agonist combined with paclitaxel (PX), a taxane usually administered to treat breast cancer, to inhibit the progression of human MCF-7 tumor cells. We demonstrated that PX plus carbachol reduced cell viability and tumor growth in vitro probably due to a down-regulation in cancer stem cells population and in the expression of ATP "binding cassette" G2 drug extrusion pump; also a reduction in malignant-induced angiogenesis was produced by the in vivo administration of the mentioned combination in a metronomic schedule to MCF-7 tumor-bearing NUDE mice. Our results confirm that mAChRs could be considered as therapeutic targets for metronomic therapy in breast cancer as well as the usefulness of a muscarinic agonist as repositioning drug in the treatment of this type of tumors.

Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX.

BACKGROUND AND PURPOSE: LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. EXPERIMENTAL APPROACH: NIH3T3 cells were treated with LPS (10 ng·mL(-1)) plus IFN-γ (0.5 ng·mL(-1)) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. KEY RESULTS: The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. CONCLUSIONS AND IMPLICATIONS: Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation.

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.

Parasympathetic modulation of amylase secretion by IFN gamma in murine submandibular glands.

IFN gamma is a pleiotropic cytokine that exerts immunologic and non-immunologic functions. We show here that at low doses (10 U/ml), it stimulates amylase secretion in murine submandibular glands (SMG) "via" muscarinic receptor activation, comparable to that produced by the muscarinic agonist carbachol. Both effects are blocked by atropine. NG-monomethyl-L-arginine (L-NMMA) and EGTA inhibited the cytokine effect on amylase secretion, involving the participation of a calcium-dependent isoform of nitric oxide synthase (NOS). We confirm NOS activation because IFN gamma stimulates nitrite production and enzyme activity in SMG. Carbachol (10(-7) M) did not modify basal nitric oxide production. In addition, both IFN gamma and carbachol increase prostaglandin E2 production in SMG, but while indomethacin potentiates IFN gamma effect on amylase secretion, it blunted amylase secretion exerted by carbachol. Thus, IFN gamma and carbachol stimulate IFN gamma secretion on SMG in a dose-dependent manner. Our results are pointing to neuroregulatory functions of IFN gamma in murine SMG, because it regulates its own levels in oral cavity, perhaps to exert a local immuno-surveillance.

Participation of nitric oxide synthase and cyclo-oxygenase in the signal transduction pathway of ileal muscarinic acetylcholine receptors.

Parasympathetic activation of ileal motility is essential for intestinal physiology. We have previously demonstrated that carbachol activates muscarinic acetylcholine receptors (mAChR) of rat intestine and stimulates ileal motility via phospholipase C. This activation induces phosphoinositide turnover and intracellular calcium mobilization. We show here that carbachol stimulation of rat ileal motility is potentiated by the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl arginine. Thus, we confirm that carbachol increases, in a dose-dependent manner, the activity of a NOS isoform that depends on calcium-calmodulin binding. Its product, nitric oxide (NO), activates not only guanylyl cyclase, inducing cGMP synthesis, but also cyclo-oxygenase, producing prostaglandin E(2). The prostanoid probably cooperates with NO to induce ileal smooth muscle relaxation.

Protein kinase C regulates NO-cGMP pathway in muscarinic receptor activation by HIV+-IgA.

In this work we demostrate that IgA purified from HIV infected patients recognizes a band with a molecular weight corresponding to radiolabelled ileal muscarinic acethylcholine receptors (mAChR) by immunoblotting. HIV+-IgA triggers the signals that are the consequence of mAChR stimulation in the intestine, regulating protein kinase C (PKC) and nitric oxide synthase (NOS) activity as well as 3',5'-cyclic guanosine monophosphate (cGMP) formation. On the one hand PKC activation by HIV+-IgA induces NOS inhibition and as a consequence, low amounts of NO that could improve local immunosuppression in the intestine; on the other hand HIV+-IgA stimulates cGMP production which could potentiate ileal motility and loss of water/electrolytes involved in intestinal damage in AIDS.