The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo.

Introduction: Male reproduction is under the control of the hypothalamus-pituitary-gonadal (HPG) axis. The endocannabinoid system (ECS) and the kisspeptin system (KS) are two major signaling systems in the central and peripheral control of reproduction, but their possible interaction has been poorly investigated in mammals. This manuscript analyzes their possible reciprocal modulation in the control of the HPG axis. Materials and methods: Adolescent male rats were treated with kisspeptin-10 (Kp10) and endocannabinoid anandamide (AEA), the latter alone or in combination with the type 1 cannabinoid receptor (CB1) antagonist rimonabant (SR141716A). The hypothalamic KS system and GnRH expression, circulating sex steroids and kisspeptin (Kiss1) levels, and intratesticular KS and ECS were evaluated by immunohistochemical and molecular methods. Non-coding RNAs (i.e., miR145-5p, miR-132-3p, let7a-5p, let7b-5p) were also considered. Results: Circulating hormonal values were not significantly affected by Kp10 or AEA; in the hypothalamus, Kp10 significantly increased GnRH mRNA and aromatase Cyp19, Kiss1, and Kiss1 receptor (Kiss1R) proteins. By contrast, AEA treatment affected the hypothalamic KS at the protein levels, with opposite effects on the ligand and receptor, and SR141716A was capable of attenuating the AEA effects. Among the considered non-coding RNA, only the expression of miR145-5p was positively affected by AEA but not by Kp10 treatment. Localization of Kiss1+/Kiss1R+ neurons in the arcuate nucleus revealed an increase of Kiss1R-expressing neurons in Kp10- and AEA-treated animals associated with enlargement of the lateral ventricles in Kp10-treated animals. In the brain and testis, the selected non-coding RNA was differently modulated by Kp10 or AEA. Lastly, in the testis, AEA treatment affected the KS at the protein levels, whereas Kp10 affected the intragonadal levels of CB1 and FAAH, the main modulator of the AEA tone. Changes in pubertal transition-related miRNAs and the intratesticular distribution of Kiss1, Kiss1R, CB1, and CB2 following KP and AEA treatment corroborate the KS-ECS crosstalk also showing that the CB1 receptor is involved in this interplay. Conclusion: For the first time in mammals, we report the modulation of the KS in both the hypothalamus and testis by AEA and revealed the KP-dependent modulation of CB1 and FAAH in the testis. KP involvement in the progression of spermatogenesis is also suggested.

Activity and Selectivity of Novel Chemical Metallic Complexes with Potential Anticancer Effects on Melanoma Cells.

Human malignant melanoma cells from lymph node metastatic site (MeWo) were selected for testing several synthesized and purified silver(I) and gold(I) complexes stabilized by unsymmetrically substituted N-heterocyclic carbene (NHC) ligands, called L20 (N-methyl, N'-[2-hydroxy ethylphenyl]imidazol-2-ylide) and M1 (4,5-dichloro, N-methyl, N'-[2-hydroxy ethylphenyl]imidazol-2-ylide), having halogenide (Cl- or I-) or aminoacyl (Gly=N-(tert-Butoxycarbonyl)glycinate or Phe=(S)-N-(tert-Butoxycarbonyl)phenylalaninate) counterion. For AgL20, AuL20, AgM1 and AuM1, the Half-Maximal Inhibitory Concentration (IC50) values were measured, and all complexes seemed to reduce cell viability more effectively than Cisplatin, selected as control. The complex named AuM1 was the most active just after 8 h of treatment at 5 µM, identified as effective growth inhibition concentration. AuM1 also showed a linear dose and time-dependent effect. Moreover, AuM1 and AgM1 modified the phosphorylation levels of proteins associated with DNA lesions (H2AX) and cell cycle progression (ERK). Further screening of complex aminoacyl derivatives indicated that the most powerful were those indicated with the acronyms: GlyAg, PheAg, AgL20Gly, AgM1Gly, AuM1Gly, AgL20Phe, AgM1Phe, AuM1Phe. Indeed, the presence of Boc-Glycine (Gly) and Boc-L-Phenylalanine (Phe) showed an improved efficacy of Ag main complexes, as well as that of AuM1 derivatives. Selectivity was further checked on a non-cancerous cell line, a spontaneously transformed aneuploid immortal keratinocyte from adult human skin (HaCaT). In such a case, AuM1 and PheAg complexes resulted as the most selective allowing HaCaT viability at 70 and 40%, respectively, after 48 h of treatment at 5 µM. The same complexes tested on 3D MeWo static culture induced partial spheroid disaggregation after 24 h of culture, with almost half of the cells dead.

Differential Expression of Kisspeptin System and Kisspeptin Receptor Trafficking during Spermatozoa Transit in the Epididymis.

The hypothalamus-pituitary-testis axis controls the production of spermatozoa, and the kisspeptin system, comprising Kiss1 and Kiss1 receptor (Kiss1R), is the main central gatekeeper. The activity of the kisspeptin system also occurs in testis and spermatozoa, but currently the need of peripheral kisspeptin to produce gametes is not fully understood. Hence, we characterized kisspeptin system in rat spermatozoa and epididymis caput and cauda and analyzed the possible presence of Kiss1 in the epididymal fluid. The presence of Kiss1 and Kiss1R in spermatozoa collected from epididymis caput and cauda was evaluated by Western blot; significant high Kiss1 levels in the caput (p < 0.001 vs. cauda) and constant levels of Kiss1R proteins were observed. Immunofluorescence analysis revealed that the localization of Kiss1R in sperm head shifts from the posterior region in the epididymis caput to perforatorium in the epididymis cauda. In spermatozoa-free epididymis, Western blot revealed higher expression of Kiss1 and Kiss1R in caput (p < 0.05 vs. cauda). Moreover, immunohistochemistry revealed that Kiss1 and Kiss1R proteins were mainly localized in the secretory epithelial cell types and in contractile myoid cells, respectively. Finally, both dot blot and Elisa revealed the presence of Kiss1 in the epididymal fluid collected from epididymis cauda and caput, indicating that rat epididymis and spermatozoa possess a complete kisspeptin system. In conclusion, we reported for the first time in rodents Kiss1R trafficking in spermatozoa during the epididymis transit and Kiss1 measure in the epididymal fluid, thus suggesting a possible role for the system in spermatozoa maturation and storage within the epididymis.

Pleiotropic Outcomes of Glyphosate Exposure: From Organ Damage to Effects on Inflammation, Cancer, Reproduction and Development.

Glyphosate is widely used worldwide as a potent herbicide. Due to its ubiquitous use, it is detectable in air, water and foodstuffs and can accumulate in human biological fluids and tissues representing a severe human health risk. In plants, glyphosate acts as an inhibitor of the shikimate pathway, which is absent in vertebrates. Due to this, international scientific authorities have long-considered glyphosate as a compound that has no or weak toxicity in humans. However, increasing evidence has highlighted the toxicity of glyphosate and its formulations in animals and human cells and tissues. Thus, despite the extension of the authorization of the use of glyphosate in Europe until 2022, several countries have begun to take precautionary measures to reduce its diffusion. Glyphosate has been detected in urine, blood and maternal milk and has been found to induce the generation of reactive oxygen species (ROS) and several cytotoxic and genotoxic effects in vitro and in animal models directly or indirectly through its metabolite, aminomethylphosphonic acid (AMPA). This review aims to summarize the more relevant findings on the biological effects and underlying molecular mechanisms of glyphosate, with a particular focus on glyphosate's potential to induce inflammation, DNA damage and alterations in gene expression profiles as well as adverse effects on reproduction and development.

The Complex Interplay between Endocannabinoid System and the Estrogen System in Central Nervous System and Periphery.

The endocannabinoid system (ECS) is a lipid cell signaling system involved in the physiology and homeostasis of the brain and peripheral tissues. Synaptic plasticity, neuroendocrine functions, reproduction, and immune response among others all require the activity of functional ECS, with the onset of disease in case of ECS impairment. Estrogens, classically considered as female steroid hormones, regulate growth, differentiation, and many other functions in a broad range of target tissues and both sexes through the activation of nuclear and membrane estrogen receptors (ERs), which leads to genomic and non-genomic cell responses. Since ECS function overlaps or integrates with many other cell signaling systems, this review aims at updating the knowledge about the possible crosstalk between ECS and estrogen system (ES) at both central and peripheral level, with focuses on the central nervous system, reproduction, and cancer.

Bisphenol A induces DNA damage in cells exerting immune surveillance functions at peripheral and central level.

Bisphenol A (BPA) is a synthetic xenoestrogen diffused worldwide. Humans are chronically exposed to low doses of BPA from food and drinks, thus BPA accumulates in tissues posing human health risk. In this study, we investigated the effects of BPA on peripheral blood mononuclear cells (PBMC) from human healthy donors, and in glia and microglia of rat offspring at postnatal day 17 (17PND) from pregnant females who received BPA soon after coupling and during lactation and weaning. Results indicated that BPA affected Phytoemagglutinin (PHA) stimulated PBMC proliferation causing an S-phase cell cycle accumulation at nanomolar concentrations while BPA was almost ineffective in resting PBMC. Furthermore, BPA induced chromosome aberrations and the appearance of shattered cells characterized by high number of fragmented and pulverized chromosomes, suggesting that the compound could cause a massive genomic rearrangement by inducing catastrophic events. The BPA-induced DNA damage was observed mainly in TCD4+ and TCD8+ subsets of T lymphocytes and was mediated by the increase of ERK1/2 phosphorylation, p21/Waf1 and PARP1 protein expression. Intriguingly, we observed for the first time that BPA-induced effects were associated to a sex specific modulation of ERα and ERß in human PBMC. Immunofluorescence analysis of rat hippocampus corroborated in vitro findings showing that BPA induced É£H2AX phosphorylation in microglia and astrocytosis by decreasing ERα expression within the dentate gyrus. Overall these results suggest that BPA can alter immune surveillance functions at both peripheral and central level with a potential risk for cancer, neuroinflammation and neurodegeneration.

Innate immunity and cellular senescence: The good and the bad in the developmental and aged brain.

Ongoing studies evidence cellular senescence in undifferentiated and specialized cells from tissues of all ages. Although it is believed that senescence plays a wider role in several stress responses in the mature age, its participation in certain physiological and pathological processes throughout life is coming to light. The "senescence machinery" has been observed in all brain cell populations, including components of innate immunity (e.g., microglia and astrocytes). As the beneficial versus detrimental implications of senescence is an open question, we aimed to analyze the contribution of immune responses in regulatory mechanisms governing its distinct functions in healthy (development, organogenesis, danger patrolling events) and diseased brain (glioma, neuroinflammation, neurodeneration), and the putative connection between cellular and molecular events governing the 2 states. Particularly this review offers new insights into the complex roles of senescence both as a chronological event as age advances, and as a molecular mechanism of brain homeostasis through the important contribution of innate immune responses and their crosstalk with neighboring cells in brain parenchyma. We also highlight the impact of the recently described glymphatic system and brain lymphatic vasculature in the interplay between peripheral and central immune surveillance and its potential implication during aging. This will open new ways to understand brain development, its deterioration during aging, and the occurrence of several oncological and neurodegenerative diseases.

The isoprenoid end product N6-isopentenyladenosine reduces inflammatory response through the inhibition of the NFκB and STAT3 pathways in cystic fibrosis cells.

OBJECTIVE: N6-isopentenyladenosine (iPA) is an intermediate of the mevalonate pathway that exhibits various anti-cancer effects. However, studies on its anti-inflammatory activity are scarce and underlying molecular mechanisms are unknown. Therefore, we aimed to investigate the ability of iPA to exert anti-inflammatory effects in the human cystic fibrosis (CF) cell model of exacerbated inflammation. MATERIALS AND METHODS: TNFα-stimulated CF cells CuFi-1 and its normal counterpart NuLi-1 were pre-treated with increasing concentrations of iPA and cell viability and proliferation were assessed by MTT and BrdU assays. The effect of iPA on IL-8 and RANTES secretion was determined by ELISA, and the activation and expression of signaling molecules and selenoproteins were studied by Western blot. To assess the direct effect of iPA on NFκB activity, luciferase assay was performed on TNFα-stimulated HEK293/T cells transfected with a NFκB reporter plasmid. RESULTS: We demonstrated for the first time that iPA prevents IL-8 and RANTES release in TNFα-stimulated CF cells and this effect is mediated by increasing the expression of the direct NFκB inhibitor IκBα and decreasing the levels of STAT3. Consistent with this, we showed that iPA inhibited TNFα-mediated NFκB activation in HEK/293T cells. Finally, we also found that iPA improved the levels of glutathione peroxidase 1 and thioredoxin reductase 1 only in CF cells suggesting its ability to maintain sufficient expression of these anti-oxidant selenoproteins. CONCLUSIONS: Our findings indicate that iPA can exert anti-inflammatory activity especially in the cases of excessive inflammatory response as in CF.

BAG3 is involved in neuronal differentiation and migration.

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

Anandamide inhibits the Wnt/ß-catenin signalling pathway in human breast cancer MDA MB 231 cells.

We previously showed that methyl-F-anandamide, a stable analogue of the anandamide, inhibited the growth and the progression of cultured human breast cancer cells. As accumulating evidences indicate that the constitutive activation of the canonical Wnt pathway in human breast cancer may highlight a key role for aberrant activation of the ß-catenin-TCF cascade and tumour progression, we studied the anandamide effect on the key elements of Wnt pathway in breast cancer cells. In this study we described that the treatment of human breast cancer cells, MDA MB 231 cells, with methyl-F-anandamide reduced protein levels of ß-catenin in the cytoplasmic and nuclear fractions inhibiting the transcriptional activation of T Cell Factor (TCF) responsive element (marker for ß-catenin signalling). The anandamide treatment resulted in up-regulation of epithelial markers, like E-cadherin with a concomitant decrease in protein levels of mesenchymal markers, including vimentin and Snail1. We, furthermore, observed that the induction of experimental epithelial-mesenchymal transition by exposure to adriamycin in MCF7 human breast cancer cell line was inhibited by anandamide treatment. In the present study we reported a novel anticancer effect of anandamide involving the inhibition of epithelial-mesenchymal transition, a process triggered during progression of cancer to invasive state.

Interaction of endocannabinoid system and steroid hormones in the control of colon cancer cell growth.

Increasing evidence suggest the role of the cannabinoid receptors (CBs) in the control of cell survival or death and signaling pathways involved in tumor progression. Cancer cell lines are characterized by a subtle modulation of CB levels which produces a modified responsiveness to specific ligands, but the molecular mechanisms underlying these events are poorly and partially understood. We previously provided evidence that the endocannabinoid (EC) anandamide (AEA) exerts anti-proliferative effect likely by modulation of the expression of genes involved in the cellular fate. In this study we focused on the role of the CB1 receptor, ECs, and steroids in the mechanisms involved in colorectal cancer (CRC) cell growth inhibition in vitro. We demonstrated that, in DLD1 and SW620 cells, 17ß-estradiol induced a specific and strong up-regulation of the CB1 receptor by triggering activation of the CB1 promoting region, localized at the exon 1 of the CNR1 gene. Moreover, treatment of DLD1 and SW620 cells with Met-F-AEA, a stable AEA-analogous, or URB597, a selective inhibitor of FAAH, induced up-regulation of CB1 expression by co-localization of PPARγ and RXRα at the promoting region. Finally, increased availability of AEA, of both exogenous and endogenous sources, induced the expression of estrogen receptor-beta in both cell lines. Our results partially elucidated the role of EC system in the molecular mechanisms enrolled by steroids in the inhibition of colon cancer cell growth and strongly suggested that targeting the EC system could represent a promising tool to improve the efficacy of CRC treatments.

Pharmacological actions of statins: a critical appraisal in the management of cancer.

Statins, among the most commonly prescribed drugs worldwide, are cholesterol-lowering agents used to manage and prevent cardiovascular and coronary heart diseases. Recently, a multifaceted action in different physiological and pathological conditions has been also proposed for statins, beyond anti-inflammation and neuroprotection. Statins have been shown to act through cholesterol-dependent and -independent mechanisms and are able to affect several tissue functions and modulate specific signal transduction pathways that could account for statin pleiotropic effects. Typically, statins are prescribed in middle-aged or elderly patients in a therapeutic regimen covering a long life span during which metabolic processes, aging, and concomitant novel diseases, including cancer, could occur. In this context, safety, toxicity, interaction with other drugs, and the state of health have to be taken into account in subjects treated with statins. Some evidence has shown a dichotomous effect of statins with either cancer-inhibiting or -promoting effects. To date, clinical trials failed to demonstrate a reduced cancer occurrence in statin users and no sufficient data are available to define the long-term effects of statin use over a period of 10 years. Moreover, results from clinical trials performed to evaluate the therapeutic efficacy of statins in cancer did not suggest statin use as chemotherapeutic or adjuvant agents. Here, we reviewed the pharmacology of the statins, providing a comprehensive update of the current knowledge of their effects on tissues, biological processes, and pathological conditions, and we dissected the disappointing evidence on the possible future use of statin-based drugs in cancer therapy.

Leucine enhances aerosol performance of naringin dry powder and its activity on cystic fibrosis airway epithelial cells.

The effect of different amino acids (AAs) on the aerosol performance of N spray-dried powders was studied. Morphology, size distribution, density, dissolution rate were evaluated and correlated to process parameters. The aerosol performance was analyzed by both Single Stage Glass Impinger and Andersen Cascade Impactor. Results indicated that powders containing 5% (w/w) of leucine, proline or histidine and dried from 3:7 ethanol/water feeds showed very satisfying aerodynamic properties with fine particle fraction>60%. Both neat N (raw and spray-dried) and N-leu1 dry-powder showing good aerodynamic properties were tested in cystic fibrosis (CF) and normal bronchial epithelial cells. Cell proliferation and expression levels of the key enzymes of the NF-κB and MAPK/ERK pathways, overactivated in CF cell lines, were evaluated. N-leu1 was able to significantly inhibit the expression levels of IKKα, IKKß, as well as of the direct NF-κB inhibitor, IκBα. In addition N-Leu1 inhibited phosphorylation of ERK1/2 kinase and did not reduce cell proliferation as observed for the neat raw drug. Leucine co-spray-dried with the drug improved both aerodynamic properties and in vitro pharmacological activity of Naringin. The optimized N-Leu formulation as dry powder is potentially able to reduce hyperinflammatory status associated to CF.

Effects of anandamide on polyamine levels and cell growth in human colon cancer cells.

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.

Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and of Ras farnesylation mediate antitumor effects of anandamide in human breast cancer cells.

The endocannabinoid system regulates cell proliferation in human breast cancer cells. Recently, we described that a metabolically stable anandamide analog, 2-methyl-2'-F-anandamide, by activation of CB1 receptors significantly inhibited cell proliferation of human breast cancer cell lines. In this study, we observed that the activation of the CB1 receptor, in two human mammary carcinoma cell lines, MDA-MB-231 and MCF7, caused the inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity due to a reduction of HMG-CoA reductase transcript levels. The decrease of HMG-CoA reductase activity induced the inhibition of the prenylation of proteins, in particular of the farnesylation of Ras oncogenic protein involved in cell proliferation of these cell lines. We suggest that the inhibitory effect of anandamide analog on tumor cell proliferation could be related to the inhibition of Ras farnesylation.

Synergistic inhibition of human colon cancer cell growth by the cannabinoid CB1 receptor antagonist rimonabant and oxaliplatin.

Rimonabant (SR141716), a highly selective cannabinoid receptor antagonist, exerts along with its anti-obesity action, pleiotropic functions affecting a broad range of diseases, from obesity-related co-morbidities to drug dependence and cancer. Several studies suggested an anti-tumour activity of rimonabant in several in vitro and in vivo models. In this study, we compared the anti-proliferative effect of SR141716 in the human colon cancer cell line DLD-1 with oxaliplatin, one of the cytotoxic drugs currently used in the treatment of colorectal cancer. We show that SR141716 inhibits DLD-1 cell proliferation similarly to oxaliplatin and if administered in combination SR141716 potentiated the inhibitory effect caused by oxaliplatin. Assessment of drug interaction was performed calculating combination index that showed a strong synergistic effect between the two drugs added to cells in combination. Our findings suggest that the combined synergic effect of SR141716 and oxaliplatin improves the blocking of colon cancer cell proliferation. Therefore, this combination merits further explorations in preclinical and clinical settings.

Human sperm anatomy: ultrastructural localization of the cannabinoid1 receptor and a potential role of anandamide in sperm survival and acrosome reaction.

Recently, the endocannabinoid (EC) system and the presence of CB1 receptor (CB1-R), have been identified in human sperm. However, the effects of EC receptor ligands such as anandamide (N-arachidonoylethanolamine) and the role of EC system in male fertility is still largely unexplored. In the present study, we investigated the ultrastructural compartmentalization of CB1-R and analyzed the effects of its stimulation by using a stable analog of anandamide, 2-methylarachidonyl-2'-fluoro-ethylamide (MET-F-AEA). We focused particularly on sperm survival and acrosin activity. The study of human sperm anatomy by transmission electron microscopy with immunogold analysis revealed the location of the CB1-R prevalently in the sperm membranes of the head and interestingly on the mitochondria. The effect of different concentrations of MET-F-AEA from 100 nM to 1 microM evidenced a significant decrease of sperm survival. Interestingly, we analyzed this negative effect at molecular level, testing the EC action on different known sperm survival targets. MET-F-AEA-treatment decreased both pBCL2 and pAkt, two prosurvival proteins, and increased pPTEN expression which is the main regulator of the PI3K/Akt pathway. Moreover, a biphasic effect was observed with increasing MET-F-AEA concentrations on the acrosin activity. The blockage of the CB1-R by using its selective antagonist SR141716 (rimonabant) induced an opposite action on sperm survival supporting a role for this receptor in the biology of the male gamete.

Cannabinoid receptor CB1 antagonists state of the art and challenges.

The discovery of cannabinoid receptors led to the development of several compounds targeted against these receptors. In particular, CB1 receptor antagonists have been described to possess key functions in the treatment of obesity and obesity-related pathologies. Numerous clinical trials revealed the advantage of strategies designed to block CB1 receptor but also evidenced the limitations due to side effects exerted by these substances. Recent studies have highlighted that CB1 antagonists could have other effects and find applications even in other pathologies like hepatic fibrosis, chronic inflammatory conditions, diabetes, and cancer. Since the suspending sales of the lead compound, rimonabant, and the discontinuation of all ongoing clinical trials of CB1 blockers, alternative strategies could emerge and lead to the development of further basic research studies to redirect these compounds.

Rimonabant inhibits human colon cancer cell growth and reduces the formation of precancerous lesions in the mouse colon.

The selective CB1 receptor antagonist rimonabant (SR141716) was shown to perform a number of biological effects in several pathological conditions. Emerging findings demonstrate that rimonabant exerts antitumor action in thyroid tumors and breast cancer cells. In our study, human colorectal cancer cells (DLD-1, CaCo-2 and SW620) were treated with rimonabant and analyzed for markers of cell proliferation, cell viability and cell cycle progression. Rimonabant significantly reduced cell growth and induced cell death. In addition, rimonabant was able to alter cell cycle distribution in all the cell lines tested. Particularly, rimonabant produced a G2/M cell cycle arrest in DLD-1 cells without inducing apoptosis or necrosis. The G2/M phase arrest was characterized by a parallel enhancement of the number of mitoses associated to elevated DNA double strand breaks and chromosome misjoining events, hallmarks of mitotic catastrophe. Protein expression analyses of Cyclin B1, PARP-1, Aurora B and phosphorylated p38/MAPK and Chk1 demonstrated that rimonabant-induced mitotic catastrophe is mediated by interfering with the spindle assembly checkpoint and the DNA damage checkpoint. Moreover, in the mouse model of azoxymethane-induced colon carcinogenesis, rimonabant significantly decreased aberrant crypt foci (ACF) formation, which precedes colorectal cancer. Our findings suggest that rimonabant is able to inhibit colorectal cancer cell growth at different stages of colon cancer pathogenesis inducing mitotic catastrophe in vitro.

Use of cannabinoid receptor agonists in cancer therapy as palliative and curative agents.

Cannabinoids (the active components of Cannabis sativa) and their derivatives have received renewed interest in recent years due to their diverse pharmacological activities. In particular, cannabinoids offer potential applications as anti-tumour drugs, based on the ability of some members of this class of compounds to limit cell proliferation and to induce tumour-selective cell death. Although synthetic cannabinoids may have pro-tumour effects in vivo due to their immunosuppressive properties, predominantly inhibitory effects on tumour growth and migration, angiogenesis, metastasis, and also inflammation have been described. Emerging evidence suggests that agonists of cannabinoid receptors expressed by tumour cells may offer a novel strategy to treat cancer. In this chapter we review the more recent results generating interest in the field of cannabinoids and cancer, and provide novel suggestions for the development, exploration and use of cannabinoid agonists for cancer therapy, not only as palliative but also as curative drugs.