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BACKGROUND AND PURPOSE: The endocannabinoid system and the cannabinoid CB(1) receptor have been identified in human sperm, and it is well known that endocannabinoids have pronounced adverse effects on male and female reproduction. In order to elucidate further the pathophysiological role of the endocannabinoid system in male fertility, we investigated the activity of the CB(1) receptor antagonist rimonabant (SR141716) on the fertilizing ability of human sperm. EXPERIMENTAL APPROACH: We evaluated in vitro the effects of rimonabant on motility, survival, capacitation, acrosin activity and metabolism of human sperm. Particularly, capacitation was studied by using three different approaches: intracellular free Ca(2+) content assay, cholesterol efflux assay and protein tyrosine phosphorylation analysis. KEY RESULTS: Rimonabant significantly increased sperm motility and viability through the induction of pAkt and pBcl2, key proteins of cell survival and metabolism, and it induced acrosome reaction and capacitation as well. Rimonabant reduced the triglyceride content of sperm, while enhancing lipase and acyl-CoA dehydrogenase activities, implying an overall lipolytic action in these cells. Rimonabant also affected sperm glucose metabolism by decreasing phosphorylation of glycogen synthase kinase 3 and increasing glucose-6-phosphate dehydrogenase activity, suggesting a role in inducing sperm energy expenditure. Intriguingly, agonism at the CB(1) receptor, with an anandamide analogue or a selective inhibitor of fatty acid amide hydrolase, produced opposing effects on human sperm functions. CONCLUSIONS AND IMPLICATIONS: Our data suggest that blockade of the CB(1) receptor by rimonabant induces the acquisition of fertilizing ability and stimulates energy expenditure in human sperm.

The interaction between 2',3'-cyclic nucleotide 3'-phosphodiesterase and guanine/adenine nucleotides was investigated. The binding of purine nucleotides to 2',3'-cyclic nucleotide 3'-phosphodiesterase was revealed by both direct and indirect methods. In fact, surface plasmon resonance experiments, triphosphatase activity measurements, and fluorescence experiments revealed that 2',3'-cyclic nucleotide 3'-phosphodiesterase binds purine nucleotide triphosphates with an affinity higher than that displayed for diphosphates; on the contrary, the affinity for both purine monophosphates and pyrimidine nucleotides was negligible. An interpretation of biological experimental data was achieved by a building of 2',3'-cyclic nucleotide 3'-phosphodiesterase N-terminal molecular model. The structural elements responsible for nucleotide binding were identified and potential complexes between the N-terminal domain of CNP-ase and nucleotide were analyzed by docking simulations. Therefore, our findings suggest new functional and structural property of the N-terminal domain of CNPase.

Emerging findings suggested the efficacy of the cannabinoid CB1 receptor antagonist rimonabant (SR141716) in several pathological conditions included tumours. In this study we investigated in vitro the effects of SR141716 on viability and the molecular pathways of methylcholanthrene-induced fibrosarcoma (Meth-A) cells and in vivo its anti-tumour properties in Meth-A-bearing mice. We evaluated in vitro the effect of SR141716 on Meth-A cell viability by trypan blue staining assay. Cell cycle progression and apoptosis were assessed by flow cytometry. Protein expression was investigated by Western blot. The anti-tumour efficacy of SR141716 was evaluated in vivo monitoring weight increase and survival of Meth-A injected mice. SR141716 affects Meth-A cell viability inducing apoptosis and controls cell cycle progression by modulation of the levels of the cell cycle inhibitor p21waf, cyclins E, D1 and NF-kB molecules. Importantly, SR141716 affects AKT/pFoxO1 pathway which promotes cell survival and regulates the cell cycle. The molecular effects observed are accompanied by reduced COX2 expression and induction of the CB1 receptor expression. Finally, SR141716 was able to reduce the tumour size and prolong animal survival, when administered in vivo during tumour growth. Our findings shed light on a novel molecular pathway associated with control of tumour growth by SR141716 and confirm the anti-cancer and anti-inflammatory properties of this drug suggesting its potential applications in the treatment of cancer.

In this study we investigated the role of CB1 receptor signaling in angiogenesis and the therapeutic exploitation of CB1 inactivation as an antiangiogenic strategy. We started from the observation that CB1 receptor expression is induced during angiogenesis and that the endocannabinoid anandamide stimulated bFGF-induced angiogenesis in the nanomolar physiologic range. To define the functional involvement of CB1 receptor signaling during angiogenesis, 2 different strategies have been carried out: siRNA-mediated knockdown and pharmacologic antagonism of CB1 receptors. CB1 receptors inactivation resulted in the inhibition of bFGF-induced endothelial proliferation, migration, and capillary-like tube formation, through prosurvival and migratory pathways involving ERK, Akt, FAK, JNK, Rho, and MMP-2. To corroborate the potential therapeutic exploitation of CB1 blockade as an antiangiogenic strategy, we performed in vivo assays founding that CB1 blockade was able to inhibit bFGF-induced neovascular growth in the rabbit cornea assay. A relevant finding was the ability to reduce ocular pathologic neo-vascularization in mouse oxygen-induced retinopathy. These results demonstrate that CB1 signaling participates to the proliferative response elicited by proangiogenic growth factors in angiogenesis and that for this reason CB1 receptor could represent a novel target for the treatment of diseases where excessive neoangiogenesis is the underlying pathology.

HMG-CoA reductase inhibitors, such as Lovastatin and Simvastatin, cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media. Cells are induced to pause in G1 and can readily resume growth upon removal of the enzymatic block. Estrogens, acting via their nuclear receptor, are mitogens for different normal and transformed cell types, where they foster cell cycle progression and cell division. In estrogen-responsive MCF-7 human breast cancer cells, but not in non responsive cells, 17 beta-estradiol (E2) induces cells arrested with Lovastatin or Simvastatin to proliferate in the presence of inhibitor, without restoring HMG-CoA reductase activity or affecting the protein prenylation pattern. Mitogenic stimulation of G1-arrested MCF-7 cells with E2 includes primary transcriptional activation of c-fos, accompanied by transient binding in vivo of the estrogen receptor and/or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene, as detected by dimethylsulphate genomic footprinting analysis. Mitogenic stimulation of growth-arrested MCF-7 cells by E2 occurs, under these conditions, without evident activation of ERK-1 and -2 kinases, and thus independently from the mitogen-responsive signal transduction pathways that converge on these enzymes.

BACKGROUND & AIMS: The endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit cancer cell proliferation by acting at cannabinoid receptors (CBRs). We studied (1). the levels of endocannabinoids, cannabinoid CB(1) and CB(2) receptors, and fatty acid amide hydrolase (FAAH, which catalyzes endocannabinoid hydrolysis) in colorectal carcinomas (CRC), adenomatous polyps, and neighboring healthy mucosa; and (2). the effects of endocannabinoids, and of inhibitors of their inactivation, on human CRC cell proliferation. METHODS: Tissues were obtained from 21 patients by biopsy during colonoscopy. Endocannabinoids were measured by liquid chromatography-mass spectrometry (LC-MS). CB(1), CB(2), and FAAH expression were analyzed by RT-PCR and Western immunoblotting. CRC cell lines (CaCo-2 and DLD-1) were used to test antiproliferative effects. RESULTS: All tissues and cells analyzed contain anandamide, 2-AG, CBRs, and FAAH. The levels of the endocannabinoids are 3- and 2-fold higher in adenomas and CRCs than normal mucosa. Anandamide, 2-AG, and the CBR agonist HU-210 potently inhibit CaCo-2 cell proliferation. This effect is blocked by the CB(1) antagonist SR141716A, but not by the CB(2) antagonist SR144528, and is mimicked by CB(1)-selective, but not CB(2)-selective, agonists. In DLD-1 cells, both CB(1) and CB(2) receptors mediate inhibition of proliferation. Inhibitors of endocannabinoid inactivation enhance CaCo-2 cell endocannabinoid levels and block cell proliferation, this effect being antagonized by SR141716A. CaCo-2 cell differentiation into noninvasive cells results in increased FAAH expression, lower endocannabinoid levels, and no responsiveness to cannabinoids. CONCLUSIONS: Endocannabinoid levels are enhanced in transformed colon mucosa cells possibly to counteract proliferation via CBRs. Inhibitors of endocannabinoid inactivation may prove useful anticancer agents

Anandamide and 2-arachidonoylglycerol (2-AG), two endogenous ligands of the CB1 and CB2 cannabinoid receptor subtypes, inhibit the proliferation of PRL-responsive human breast cancer cells (HBCCs) through down-regulation of the long form of the PRL receptor (PRLr). Here we report that 1) anandamide and 2-AG inhibit the nerve growth factor (NGF)-induced proliferation of HBCCs through suppression of the levels of NGF Trk receptors; 2) inhibition of PRLr levels results in inhibition of the proliferation of other PRL-responsive cells, the prostate cancer DU-145 cell line; and 3) CB1-like cannabinoid receptors are expressed in HBCCs and DU-145 cells and mediate the inhibition of cell proliferation and Trk/PRLr expression. Beta-NGF-induced HBCC proliferation was potently inhibited (IC50 = 50-600 nM) by the synthetic cannabinoid HU-210, 2-AG, anandamide, and its metabolically stable analogs, but not by the anandamide congener, palmitoylethanolamide, or the selective agonist of CB2 cannabinoid receptors, BML-190. The effect of anandamide was blocked by the CB1 receptor antagonist, SR141716A, but not by the CB2 receptor antagonist, SR144528. Anandamide and HU-210 exerted a strong inhibition of the levels of NGF Trk receptors as detected by Western immunoblotting; this effect was reversed by SR141716A. When induced by exogenous PRL, the proliferation of prostate DU-145 cells was potently inhibited (IC50 = 100-300 nM) by anandamide, 2-AG, and HU-210. Anandamide also down-regulated the levels of PRLr in DU-145 cells. SR141716A attenuated these two effects of anandamide. HBCCs and DU-145 cells were shown to contain 1) transcripts for CB1 and, to a lesser extent, CB2 cannabinoid receptors, 2) specific binding sites for [3H]SR141716A that could be displaced by anandamide, and 3) a CB1 receptor-immunoreactive protein. These findings suggest that endogenous cannabinoids and CB1 receptor agonists are potential negative effectors of PRL- and NGF-induced biological responses, at least in some cancer cells.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and mRNA levels were significantly reduced in FRTL-5 cells transformed with the Kirsten-Moloney sarcoma virus (KiMol); these cells have lost thyrotropin dependence and express high levels of p21ras. FRTL-5 cells, transformed with a temperature-sensitive mutant of the v-K-ras oncogene (Ats cells: 33 degrees C, permissive; 39 degrees C, nonpermissive), showed significant reduction of HMG-CoA reductase expression when exposed to 33 degrees C. In KiMol cells, as well as in Ats cells at 33 degrees C, the transcription driven by cAMP-responsive element was probed by measuring chloramphenicol acetyl transferase (CAT) levels after transfection with a chimeric plasmid containing the reporter gene linked to the rat reductase promoter. Basal CAT activity in KiMol cells transfected with wild-type promoter was lower than in FRTL-5 cells but was increased by forskolin to the levels attained in thyrotropin-stimulated FRTL-5 cells. Forskolin failed to increase CAT activity in KiMol cells transfected with the plasmid harboring a reductase promoter in which the cAMP-responsive element octamer was mutated to a nonpalindromic sequence. The effect of v-K-ras could be mimicked in FRTL-5 cells by tetradecanoyl phorbol acetate and reverted in KiMol and Ats cells, expressing active Ras protein, by increasing intracellular cAMP and/or by protein kinase C inhibition. The data are consistent with the contention that v-K-ras, through protein kinase C and depletion of intracellular cAMP, is inhibitory for the protein kinase A pathway. This is the first demonstration that active v-K-ras down-regulates HMG-CoA reductase expression.

HMG-CoA reductase inhibitors, such as Lovastatin and Simvastatin, cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media. Cells are induced to pause in G 1 and can readily resume growth upon removal of the enzymatic block. Estrogens, acting via their nuclear receptor, are mitogens for different normal and transformed cell types, where they foster cell cycle progression and cell division. In estrogen-responsive MCF-7 human breast cancer cells, but not in non responsive cells, 17β-estradiol (E 2) induces cells arrested with Lovastatin or Simvastatin to proliferate in the presence of inhibitor, without restoring HMG-CoA reductase activity or affecting the protein prenylation pattern. Mitogenic stimulation of G 1-arrested MCF-7 cells with E 2 includes primary transcriptional activation of c-fos, accompanied by transient binding in vivo of the estrogen receptor and/or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene, as detected by dimethylsulphate genomic footprinting analysis. Mitogenic stimulation of growth-arrested MCF-7 cells by E 2 occurs, under these conditions, without evident activation of ERK-1 and -2 kinases, and thus independently from the mitogen-responsive signal transduction pathways that converge on these enzymes.

BACKGROUND: Anandamide (AEA) is an endogenous agonist for cannabinoid receptor CB1-R and seems to be involved in the control of cancer growth. Polyamines are compounds that play an important role in cell proliferation and differentiation. Our aim was to investigate the effect of AEA on the polyamine levels (putrescine, spermidine and spermine) and cell growth of three human colon cancer cell lines, positive for CB1-R. MATERIALS AND METHODS: After AEA treatment of DLD-1, HT-29 and SW620 cells, polyamine analysis was performed by high-performance liquid chromatography (HPLC) and cell growth was measured by 3-(4,5 di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. CB1 gene expression was determined using reverse transcription and polymerase chain reaction (RT-PCR). RESULTS: AEA significantly reduced polyamine levels and cell proliferation dose-dependently when the tested cell lines were exposed for 24 h and 48 h. This inhibitory effect was mediated by CB1-R, since SR 1411716A, a selective CB-1 receptor antagonist, was able to entirely antagonize the effect of AEA. CB1-R mRNA levels were enhanced after AEA treatment in DLD-1 cells, whereas no induction was found in HT-29 and SW620 cells. CONCLUSION: It appears that mechanisms by which AEA may affect growth of colon cancer cells involve a decrease in cell proliferation rate by reducing the polyamine levels.

Cannabinoid receptors are involved in neurophatogenic mechanisms of inflammatory disorders of the central nervous system and their expression can be modulated during the disease. Brain inflammatory processes are characterized by infiltration of numerous types of cells, peripheral immune cells, brain resident immune cells, the microglial cells and numerous other neuronal cells. The disruption of the blood brain barrier favours cell infiltration in the central nervous system with consequent neuronal damage, common event in many neuro-inflammatory diseases. In this review we evidence the role of cannabinoid receptor, their expression at peripheral and central levels in order to better understand their implication in neuro-inflammation. Cannabinoids affect brain adaptive and immune response, have regulatory action on inflammatory mediators and can exert a role in blood brain barrier damage prevention. Furthermore, in numerous neurodegenerative diseases with inflammatory component the beneficial effects of cannabinoids have been widely reported, so current knowledge of cannabinoid involvement in these central nervous system disorders are also reviewed.

The microsomal enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and the low density lipoprotein (LDL) receptor pathway carry out a key role on cholesterol homeostasis in eucaryotic cells. The HMG-CoA reductase issensitive to oxidative inactivation and to phosphorylation by many kinases that are able to inactivate the protein and increase its susceptibility to proteolysis. We previously demonstrated that a calf thymus Cu,Zn SOD affects cholesterol metabolism. This protein binds with rat hepatocyte cell membrane by a specific surface membrane receptor. The involvement of Cu,Zn SOD in cholesterol metabolism is confirmed further by the presence of this antioxidant enzyme in circulating serum lipoproteins. We studied the effect of native human Cu,Zn SOD, metal-free SOD (apo SOD), and SOD-inactivated with hydrogen peroxide on cholesterol metabolism in human hepatocarcinoma HepG2 cells. Results showed that all forms of SODS used, at the concentration of 150 ng/ml, are able to affect cholesterol metabolism decreasing both HMG-CoA reductase activity and its protein levels; this inhibitory effect is accompanied by reduced cholesterol synthesis measured as [ 1 4 C]acetate incorporation into [ 1 4 C)cholesterol and by an increased [ 1 2 5 I]LDL binding to HepG2 cells. Furthermore, the inhibitory effect of Cu,Zn SOD on cholesterol synthesis was completely abolished when the cells were incubated with Cu,Zn SOD in the presence of bisindoilmaleimide (BDM), an inhibitor of protein kinase C (PKC); moreover, we demonstrated that Cu,Zn SOD as well as apo SOD was able to increase PKC activity. Overall, data demonstrate that Cu,Zn SOD affects cholesterol metabolism independently from its dismutase activity and its metal content and that the inhibitory action on cholesterol synthesis is mediated by an activation of protein kinase C.

BACKGROUND AND PURPOSE: There is growing evidence that the cannabinoid CB(1) receptor antagonist, rimonabant (SR141716) exerts potential anti-proliferative and anti-inflammatory actions. Here, we have assessed the effects of rimonabant in vitro in murine immortalized keratinocytes and in vivo by assaying the topical anti-inflammatory activity. EXPERIMENTAL APPROACH: Cell viability and death in a keratinocyte cell line (C5N cells) were measured by Trypan blue exclusion assay and cytotoxicity by sulphorhodamine B test. Cell cycle progression was assessed by flow cytometry and the expression of apoptotic and anti-apoptotic markers, cyclins, pathways of signal transduction and CB1 receptor levels were evaluated by Western blot. The topical anti-inflammatory properties of rimonabant were analysed by inhibition of croton oil-induced ear dermatitis in mice. KEY RESULTS: Rimonabant reduced cell viability and induced apoptosis as shown by the enhanced number of cells in the subG0 phase of the cell cycle, the expression of Bax and reduced levels of Bcl-2 and X-inhibitor of apoptosis protein. In addition, reduced levels of phosphorylated serine/threonine protein kinase Akt and nuclear factor-kappa B were detected associated with regulation of total nuclear factor-kappa B and inhibitor of kappa B-±, phosphorylated inhibitor of kappa B-±, cyclins D1, E and A. In croton oil-induced ear dermatitis, rimonabant significantly reduced oedema and leukocyte infiltrate. CONCLUSIONS AND IMPLICATIONS: Rimonabant reduced cell viability, inducing cell death in keratinocytes and decreased croton oil-induced ear dermatitis. Our findings suggest a potential application of rimonabant as a topical anti-inflammatory drug. We did not assess the involvement of CB(1) receptors in these effects of rimonabant.

The epidemic spread of obesity is nowadays recognized as a global health and economic burden, arising great interest in the scientific community. The rate of adult obesity steadily increases concomitantly with the cancer incidence. As has been comprehensively reported, obesity is included among the multiple cancer risk factors and can progressively cause and/or exacerbate certain cancer types, as colorectal and breast cancers. The term adiponcosis was forged precisely to emphasize the interconnection between obesity and cancer onset and progression. The underlying mechanisms of adiponcosis have not been fully elucidated yet, may vary on cancer type, and depend on body fat distribution. It has been proposed that insulin resistance and related chronic hyperinsulinemia, increased insulin-like growth factors production, chronic inflammation or increased bioavailability of steroid hormones could be responsible of cancer hallmarks. Additionally, it has been suggested that adipose tissue-derived hormones, cytokines and adipokines, such as leptin, adiponectin and inflammatory markers, may reflect mechanisms linked to tumorigenesis. This review summarizes the current evidence on pathways, hormones, cytokines and low-chronic inflammation subtending adiponconsis, focusing on breast and colorectal cancers. In addition, we analyzed the lifestyle interventions that could attenuate the driving forces of obesity-related cancer incidence and progression. Moreover, current targets and drugs, their pros and cons, as well as new mechanisms and targets with promising therapeutic potential in cancer are discussed. Depicting this complex interconnection will provide insights for establishing new therapeutic approaches to halt the obesity impacts and thwart cancer onset and progression.

Despite encouraging progresses achieved in the management of viral diseases, efficient strategies to counteract infections are still required. The current global challenge highlighted the need to develop a rapid and cost-effective strategy to counteract the SARS-CoV-2 pandemic. Lipid metabolism plays a crucial role in viral infections. Viruses can use the host lipid machinery to support their life cycle and to impair the host immune response. The altered expression of mevalonate pathway-related genes, induced by several viruses, assures survival and spread in host tissue. In some infections, statins, HMG-CoA-reductase inhibitors, reduce cholesterol in the plasma membrane of permissive cells resulting in lower viral titers and failure to internalize the virus. Statins can also counteract viral infections through their immunomodulatory, anti-inflammatory and anti-thrombotic effects. Beyond statins, interfering with the mevalonate pathway could have an adjuvant effect in therapies aimed at mitigating endothelial dysfunction and deregulated inflammation in viral infection. In this review we depicted the historical and current evidence highlighting how lipid homeostasis and mevalonate pathway targeting represents a valid approach to rapidly neutralize viruses, focusing our attention to their potential use as effective targets to hinder SARS-CoV-2 morbidity and mortality. Pros and cons of statins and Mevalonate-pathway inhibitors have been also dissected.

BACKGROUND: We have recently released the structure of a class of quinolin-2-(1H)-on-3-carboxamide derivatives and among them; the drug A2 has the highest CB2 receptor selectivity.OBJECTIVE: In this work we assessed the immuno-modulatory properties of A2 in lymphocytes isolated from peripheral blood of multiple sclerosis patients and healthy donors.METHODS: Cell proliferative response was measured by 3H-thymidine incorporation, cell viability and apoptosis by trypan blue, annexin V staining and western blot. Cell activation was investigated by flow cytometry and molecular pathways by western blot.CONCLUSION: We reported similar effects of A2 in both cell types; however, a different mechanism of action might be suggested in cells from patients concerning cell activation and CB2 receptor expression. Overall, these data suggest an anti-inflammatory profile of A2 with potential implication in multiple sclerosis.RESULTS: A2 exerted anti-proliferative effects with down-regulation of TNF-α, IL-10 and Rantes in both cell types. No relevant changes were observed in cell viability between the two cell types. In cells from healthy subjects, A2 did not induce apoptosis, inhibited the cell cycle and similarly down-regulated in CD4+T cells the markers CD69, CD25, CD49d and CD54. Indeed, A2 also inhibited the phosphorylation of Akt, NF-kB, IKKα/β, ERK and blocked the expression of Cox-2 and CB2 receptor. Published patents also describe CB2 receptor agonists like purine derivatives. Differently, in cells from patients, A2 did not affect CD49d, while potently blocked CD54 expression. A2 inhibitory effects of Akt and Cox-2 expression were confirmed, whereas unchanged level of the CB2 receptor was observed in these cells.

Background Several studies support the evidence that the endocannabinoid system and cannabimimetic drugs might have therapeutic potential in numerous pathologies. These pathologies range from neurological disorders, atherosclerosis, stroke, cancer to obesity/metabolic syndrome and others. Methods In this paper we review the endocannabinoid system signaling and its alteration in neurodegenerative disorders like multiple sclerosis, Alzheimer's disease, Parkinson's disease and Huntington's disease and discuss the main findings about the use of cannabinoids in the therapy of these pathologies. Results Despite different etiologies, neurodegenerative disorders exhibit similar mechanisms like neuro-inflammation, excitotoxicity, deregulation of intercellular communication, mitochondrial dysfunction and disruption of brain tissue homeostasis. Current treatments ameliorate the symptoms but are not curative. Interfering with the endocannabinoid signaling might be a valid therapeutic option in neuro-degeneration. To this aim, pharmacological intervention to modulate the endocannabinoid system and the use of natural and synthetic cannabimimetic drugs have been assessed. CB1 and CB2 receptor signaling contributes to the control of Ca2+ homeostasis, trophic support, mitochondrial activity, and inflammatory conditions. Conclusion Several studies and patents suggest that the endocannabinoid system has neuro-protective properties and might be a target in neurodegenerative diseases.

N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway with an isopentenyl chain, is already known to exert a suppressor effect against various tumors. In this work, we investigated whether iPA also directly interferes with the angiogenic process, which is fundamental to tumor growth and progression. To this end, using human umbilical vein endothelial cells (HUVECs) as a suitable in vitro model of angiogenesis, we evaluated their viability, proliferation, migration, invasion, tube formation in response to iPA, and molecular mechanisms involved. Data were corroborated in mice by using a gel plug assay. iPA dose- and time-dependently inhibited all the neoangiogenesis stages, with an IC50 of 0.98 ?M. We demonstrated for the first time, by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS), that iPA was monophosphorylated into 5'-iPA-monophosphate (iPAMP) by the adenosine kinase (ADK) inside the cells. iPAMP is the active form that inhibits angiogenesis through the direct activation of AMP-kinase (AMPK). Indeed, all effects were completely reversed by pretreatment with 5-iodotubercidin (5-Itu), an ADK inhibitor. The isoprenoid intermediate isopentenyl pyrophosphate (IPP), which shares the isopentenyl moiety with iPA, was ineffective in the inhibition of angiogenesis, thus showing that the iPA structure is specific for the observed effects. In conclusion, iPA is a novel AMPK activator and could represent a useful tool for the treatment of diseases where excessive neoangiogenesis is the underlying pathology.- Pisanti, S., Picardi, P., Ciaglia, E., Margarucci, L., Ronca, R., Giacomini, A., Malfitano, A. M., Casapullo, A., Laezza, C., Gazzerro, P., Bifulco, M. Anti-angiogenic effects of N6-isopentenyladenosine, an endogenous isoprenoid end-product, mediated by AMPK activation.

N6-isopentenyladenosine (i6A) is a modified nucleoside with a pentaatomic isopentenyl derived from mevalonate that induces inhibition of tumor cell proliferation and apoptosis in several tumor cell lines. In this study, we reported that N6-isopentenyladenosine inhibited the proliferation and promotes apoptosis in DLD1 human colon cancer cells. It suppressed the proliferation of cells through inhibition of DNA synthesis, causing a cell cycle arrest that correlated with a decrease in the levels of cyclin E, cyclin A and cyclin D1 and with a concomitant increase in the levels of cyclin-dependent kinase inhibitor p21waf and p27kip1. Moreover, it induced apoptosis through an increase in the number of annexin V-positive cells, a downregulation of antiapoptotic products and caspase-3 activation. The apoptotic effects of N6-isopentenyladenosine were accompanied by sustained phosphorylation and activation of c-jun N-terminal kinase (JNK) that induced phosphorylation of c-jun. Overall, our data show that JNK, could play an important role in i6A-mediated apoptosis in DLD1 human colon cancer cells © 2008 Wiley-Liss, Inc.

The endocannabinoid system regulates cell proliferation and migration in human breast cancer cells. In this study, we showed that a metabolically stable analog of anandamide, 2-methyl-2'-F-anandamide (Met-F-AEA), inhibited the RHOA activity and caused a RHOA delocalization from the cell membrane to cytosol determining a decrease in actin stress fibers. Overexpression of a dominant negative of RHOA activity and treatment of these cells with a RHO-associated protein kinase (ROCK) inhibitor, Y 27632, mimicked Met-F-AEA effects on actin organization and cell migration. We suggest that the inhibitory effect of Met-F-AEA on tumor cell migration could be related to RHOA-ROCK-dependent signaling pathway.

A common modification of eukaryotic and bacterial tRNAs is isopentenylation of the adenosine at position 37, with the formation of isopentenyladenosine. N6-Isopentenyladenosine plays a major role in posttranscriptional processes, including the function of mammalian selenocysteine tRNA. This molecule seems to have metabolic effects that, for its relationships with isoprenoid metabolism and its direct biological activities, affects mammalian cell cytoskeleton, proliferation and apoptosis. In addition, preliminary clinical observation seems to indicate a possible use of N6-Isopentenyladenosine as anticancer drug.

N6-isopentenyladenosine (iPA) is a modified adenosine with an isopentenyl moiety derived from the mevalonate pathway which displays pleiotropic biological effects, including anti-tumor and anti-angiogenic activity. Previous evidence revealed a biphasic effect of iPA on phytohemagglutinin-stimulated lymphocytes, being pro-proliferative at low doses and anti-proliferative at high doses. Analogously, we have recently shown that low iPA concentrations (

N 6 -Isopentenyladenosine (i 6 A), an adenosine and mevalonate derivative, inhibits, like adenosine, TSH-induced cAMP increase and its related events (I − uptake and DNA synthesis) in FRTL-5 cells. This inhibition is dose-dependent and is measurable at 10 −8 M. However, unlike adenosine, i 6 A prevents TSH-promoted microfilament disassembly. The effect of i 6 A on cytoskeletal structure is antagonized by pertussis toxin and could be assigned to its N 6 substitution since it can be mimicked by other synthetic N 6 -adenosine derivatives. It is suggested that a step beyond cAMP is involved, since i 6 A prevents also microfilament disassembly induced by 8-bromo-cAMP. This is the first demonstration that an adenosine derivative, which is also an end-product of the isoprenoid pathway, affects cAMP-dependent microfilament organization.

The relevance of CB2R-mediated therapeutic effects is well-known for the treatment of inflammatory and neuropathic pain and neurodegenerative disorders. In our search for new cannabinoid receptor modulators, we report the optimization of a series of 1,2-dihydro-2-oxopyridine-3-carboxamide derivatives as CB2R ligands. In particular, N-cycloheptyl-5-(4-methoxyphenyl)-1-(4- fluorobenzyl)-pyridin-2(1H)-on-3-carboxamide (17) showed high CB2R affinity (Ki = 1.0 nM), accompanied by interesting Ki(CB1R)/K i(CB2R) selectivity ratio (SI = 43.4). Compound 17 was also identified as a potent CB2R neutral antagonist/weak partial inverse agonist. Finally we found that the functionality activity of the series of 1,2-dihydro-2-oxopyridine is controlled by the presence of a substituent in position 5 of the heterocyclic nucleus. In fact when the hydrogen atom in position 5 of the unsubstituted compound 1 was replaced with a phenyl group (compound 18) the CB2R activity was shifted from agonism to inverse agonism whereas the introduction in the same position of p-methoxyphenyl group lead to compound 17 which showed a behavior as CB2R neutral antagonist/weak partial inverse agonist. © 2013 Elsevier Masson SAS. All rights reserved.

CB2 receptor ligands are becoming increasingly attractive drugs due to the potential role of this receptor in several physiopathological processes. Using our previously described series of 1,8-naphthyridin-2(1H)-on-3-carboxamides as a lead class, several nitrogen heterocyclic derivatives, characterized by different central cores, were synthesized and tested for their affinity toward the human CB1 and CB2 cannabinoid receptors. The obtained results suggest that the new series of quinolin-2(1H)-on-3-carboxamides, 4-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides and 1,2-dihydro-2-oxopyridine-3-carboxamides represent novel scaffolds very suitable for the development of promising CB2 ligands. Furthermore, the newly synthesized CB2 ligands inhibit proliferation of several cancer cell lines. In particular, it was demonstrated that in DU-145 cell line these ligands exert a CB2-mediated anti-proliferative action and decrease the CB2 receptor expression levels.

Emerging findings suggested the efficacy of the cannabinoid CB1 receptor antagonist rimonabant (SR141716) in several pathological conditions included tumours. In this study we investigated in vitro the effects of SR141716 on viability and the molecular pathways of methylcholanthrene-induced fibrosarcoma (Meth-A) cells and in vivo its anti-tumour properties in Meth-A-bearing mice. We evaluated in vitro the effect of SR141716 on Meth-A cell viability by trypan blue staining assay. Cell cycle progression and apoptosis were assessed by flow cytometry. Protein expression was investigated by Western blot. The anti-turnout efficacy of SR141716 was evaluated in vivo monitoring weight increase and survival of Meth-A injected mice. SR141716 affects Meth-A cell viability inducing apoptosis and controls cell cycle progression by modulation of the levels of the cell cycle inhibitor p21waf, cyclins E. D1 and NF-kB molecules. Importantly, SR141716 affects AKT/pFoxO1 pathway which promotes cell survival and regulates the cell cycle. The molecular effects observed are accompanied by reduced COX2 expression and induction of the CBI receptor expression. Finally, SR141716 was able to reduce the tumour size and prolong animal survival, when administered in vivo during tumour growth. Our findings shed light on a novel molecular pathway associated with control of tumour growth by SR141716 and confirm the anti-cancer and anti-inflammatory properties of this drug suggesting its potential applications in the treatment of cancer.