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Ganoderma lucidum or Reishi is recognized as the most potent adaptogen present in nature, and its anti-inflammatory, antioxidant, immunomodulatory and anticancer activities are well known. Moreover, lately, there has been an increasing interest from pharmaceutical companies in antiaging G. lucidum-extract-based formulations. Nevertheless, the pharmacological mechanisms of such adaptogenic and regenerative actions remain unclear. The present investigation aimed to explore its molecular and cellular effects in vitro in epidermal keratinocyte cultures by applying liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) for analysis of ethanol extracts using ganoderic acid-A as a reference compound. The G. lucidum extract showed a keratinocyte proliferation induction accompanied by an increase of cyclic kinase protein expressions, such as CDK2 and CDK6. Furthermore, a noteworthy migration rate increase and activation of tissue remodelling factors, such as matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), were observed. Finally, the extract showed an antioxidant effect, protecting from H2O2-induced cytotoxicity, preventing activation of AKT (protein kinase B), ERK (extracellular signal-regulated kinase), p53 and p21, and reducing the number of apoptotic cells. Our study paves the path for elucidating pharmacological properties of G. lucidum and its potential development as cosmeceutical skin products, providing the first evidence of its capability to accelerate the healing processes enhancing re-epithelialization and to protect cells from free-radical action.

To date stress, a highly complex process that disrupts homeostasis and involves environmental and psychosocial factors, is considered as one of the most crucial factor that affects our daily life, especially urban dweller’s life. Clinical and experimental studies widely support the notion that adrenergic stimulation due to chronic stress affects inflammation and metabolism. In this work, supported by several recent scientific evidences, we show how stress plays a positive role in cancer initiation, progression and cancer metastasis, a negative role for anti-tumor immune function and therapy response. Understanding the intricacies of this interaction could provide an additional help on how act in cancer prevention and therapy.

Cardiovascular diseases, followed by strokes, represent the leading cause of mortality worldwide. Despite its success in preventing cardiovascular diseases, the therapeutic potential of 3-Hydroxytyrosol (HT) for treating ischemic diseases is yet to be investigated in detail, especially with regard to ischemic heart disease, which is a major challenge for humans. We assessed that low concentrations (1&ndash, 5 &micro, M) of HT, generally achieved after the ingestion of olive oil, stimulate endothelial cells migration and angiogenesis in an in vitro model. At early time points (1&ndash, 6 h), HT induces the expression of different proteins such as proto-oncogene tyrosine-protein kinase Src (Src), rho-associated protein kinase (ROCK) and matrix metalloproteinase-2 (MMP-2) protein influencing cell adhesion, cytoskeletal dynamics and cell migration. We observed that at the same time, HT induces prominent vascular formation in the tube formation assay, accompanied by an increase in the expression of the vascular endothelial growth factor receptor (VEGF-R2) and PI3K-Akt-eNOS protein pathways, which are recognized for their central role in angiogenesis. Therefore, in addition to the proven capability of HT to regulate reactive oxygen species (ROS) levels, through both direct scavenging properties and indirect antioxidant efficacy, our results revealed that HT promotes angiogenesis, arguing in favor of great pharma-nutritional potential in ischemic injuries.

Cancer cell stress induced by cytotoxic agents promotes antitumor immune response. Here, we observed that N6-isopentenyladenosine (iPA), an isoprenoid modified adenosine with a well established anticancer activity, was able to induce a significant upregulation of cell surface expression of natural killer (NK) cell activating receptor NK Group 2 member D (NKG2D) ligands on glioma cells in vitro and xenografted in vivo. Specifically suboptimal doses of iPA (0.1 and 1 µM) control the selective upregulation of UL16-binding protein 2 on p53wt-expressing U343MG and that of MICA/B on p53mut-expressing U251MG cells. This event made the glioblastoma cells a potent target for NK cell-mediated recognition through a NKG2D restricted mechanism. p53 siRNA-mediated knock-down and pharmacological inhibition (pifithrin-α), profoundly prevented the iPA action in restoring the immunogenicity of U343MG cells through a mechanism that is dependent upon p53 status of malignancy. Furthermore, accordingly to the preferential recognition of senescent cells by NK cells, we found that iPA treatment was critical for glioma cells entry in premature senescence through the induction of S and G2/M phase arrest. Collectively, our results indicate that behind the well established cytotoxic and antiangiogenic effects, iPA can also display an immune-mediated antitumor activity. The indirect engagement of the innate immune system and its additional activity in primary derived patient's glioma cell model (GBM17 and GBM37), fully increase its translational relevance and led to the exploitation of the isoprenoid pathway for a valid therapeutic intervention in antiglioma research.

Over the past years, several lines of evidence support a therapeutic potential of Cannabis derivatives and in particular phytocannabinoids. ?9-THC and cannabidiol (CBD) are the most abundant phytocannabinoids in Cannabis plants and therapeutic application for both compounds have been suggested. However, CBD is recently emerging as a therapeutic agent in numerous pathological conditions since devoid of the psychoactive side effects exhibited instead by ?9-THC. In this review, we highlight the pharmacological activities of CBD, its cannabinoid receptor-dependent and -independent action, its biological effects focusing on immunomodulation, angiogenetic properties, and modulation of neuronal and cardiovascular function. Furthermore, the therapeutic potential of cannabidiol is also highlighted, in particular in nuerological diseases and cancer.

Farnesyl pyrophosphate synthase (FPPS) is a crucial enzyme for the synthesis of isoprenoids and the key target of nitrogen-containing bisphosphonates (N-BPs). N-BPs are potent and selective FPPS inhibitors that are used in the treatment of bone-related diseases, but have poor pharmacokinetic properties. Given the key role played by FPPS in many cancer-related pathways and the pharmacokinetic limits of N-BPs, hundreds of molecules have been screened to identify new FPPS inhibitors characterized by improved drug-like properties that are useful for broader therapeutic applications in solid, non-skeletal tumours. We have previously shown that N6-isopentenyladenosine (i6A) and its related compound N6-benzyladenosine (2) exert anti-glioma activity by interfering with the mevalonate pathway and inhibiting FPPS. Here, we report the design and synthesis of a panel of N6-benzyladenosine derivatives (compounds 2a-m) incorporating different chemical moieties on the benzyl ring. Compounds 2a-m show in vitro antiproliferative activity in U87MG glioma cells and, analogous to the bisphosphonate FPPS inhibitors, exhibit immunogenic properties in ex vivo γδ T cells from stimulated peripheral blood mononuclear cells (PBMCs). Using saturation transfer difference (STD) and quantitative 1H nuclear magnetic resonance (NMR) experiments, we found that 2f, the N6-benzyladenosine analogue that includes a tertbutyl moiety in the para position of the benzyl ring, is endowed with increased FPPS binding and inhibition compared to the parent compounds i6A and 2. N6-benzyladenosine derivatives, characterized by structural features that are significantly different from those of N-BPs, have been confirmed to be promising chemical scaffolds for the development of non N-BP FPPS inhibitors, exerting combined cytotoxic and immunostimulatory activities.

Malignant gliomas are highly dependent on the isoprenoid pathway for the synthesis of lipid moieties critical for cell proliferation. The isoprenoid derivative N6-isopentenyladenosine (iPA) displays pleiotropic biological effects, including a direct anti-tumor activity in several tumor models. The antiglioma effects of iPA was then explored in U87MG cells both in vitro and grafted in mice and the related molecular mechanism confirmed in primary derived patients' glioma cells. iPA powerfully inhibited tumor cell growth and induced caspase-dependent apoptosis through a mechanism involving a marked accumulation of the pro-apoptotic BIM protein and inhibition of EGFR. Indeed, activating AMPK following conversion into its iPAMP active form, iPA stimulated EGFR phosphorylation and ubiquitination along a proteasome-mediated pathway which was responsible for receptor degradation and its downstream signaling pathways inhibition, including the STAT3, ERK and AKT cascade. The inhibition of AMPK by compound C prevented iPA-mediated phosphorylation of EGFR, known to precede receptor loss. As expected the block of EGFR degradation, by exposure to the proteasome inhibitor MG132, significantly reduced iPA-induced cell death. Given the importance of receptor degradation in iPA-mediated cytotoxicity, we also documented that the EGFR expression levels in a panel of primary glioma cells confers them a high sensitivity to iPA treatment. In conclusion our study provides the first evidence of iPA antiglioma effect. Indeed, as glioma is driven by aberrant signaling of growth factor receptors, particularly the EGFR, iPA, alone or in association with EGFR targeted therapies, might be a promising therapeutic tool to achieve a potent anti-tumoral effect.

3-hydroxytyrosol (HT) is the main phenolic compound found in olive oil with known antioxidant, anti-inflammatory, and antimicrobial properties in several dermatological conditions, both when taken in the form of olive oil or pure in cosmeceutical formulations. To date, its direct effect on the wound healing process and the molecular mechanisms involved have not yet been elucidated. Thus, in the present study, we aimed to explore its effects in vitro in epidermal keratinocyte cultures focusing on the molecular mechanism implied. HT was able to induce keratinocyte proliferation in the low micromolar range, increasing the expression of cyclin dependent kinases fundamental for cell cycle progression such as CDK2 and CDK6. Furthermore, it increased cell migration through the activation of tissue remodeling factors such as matrix metalloproteinase-9 (MMP-9) protein. Then, we evaluated whether HT also showed antioxidant activity at this concentration range, protecting from H2O2-induced cytotoxicity. The HT prevented the activation of ATM serine/threonine kinase (ATM), Checkpoint kinase 1 (Chk1), Checkpoint kinase 2 (Chk2), and p53, reducing the number of apoptotic cells. Our study highlighted novel pharmacological properties of HT, providing the first evidence of its capability to induce keratinocyte migration and proliferation required for healing processes and re-epithelialization.

Mevalonate kinase deficiency (MKD) is a rare autoinflammatory genetic disorder characterized by recurrent fever attacks and systemic inflammation with potentially severe complications. Although it is recognized that the lack of protein prenylation consequent to mevalonate pathway blockade drives IL1β hypersecretion, and hence autoinflammation, MKD pathogenesis and the molecular mechanisms underlaying most of its clinical manifestations are still largely unknown. In this study, we performed a comprehensive bioinformatic analysis of a microarray dataset of MKD patients, using gene ontology and Ingenuity Pathway Analysis (IPA) tools, in order to identify the most significant differentially expressed genes and infer their predicted relationships into biological processes, pathways, and networks. We found that hematopoiesis linked biological functions and pathways are predominant in the gene ontology of differentially expressed genes in MKD, in line with the observed clinical feature of anemia. We also provided novel information about the molecular mechanisms at the basis of the hematological abnormalities observed, that are linked to the chronic inflammation and to defective prenylation. Considering the broad and unspecific spectrum of MKD clinical manifestations and the difficulty in its diagnosis, a better understanding of MKD molecular bases could be translated to the clinical level to facilitate diagnosis, and improve management and therapy.

Cancer cell stress induced by cytotoxic agents promotes antitumor immune response. Here, we observed that N6-isopentenyladenosine (iPA), an isoprenoid modified adenosine with a well established anticancer activity, was able to induce a significant upregulation of cell surface expression of natural killer (NK) cell activating receptor NK Group 2 member D (NKG2D) ligands on glioma cells in vitro and xenografted in vivo. Specifically suboptimal doses of iPA (0.1 and 1 µM) control the selective upregulation of UL16-binding protein 2 on p53wt-expressing U343MG and that of MICA/B on p53mut-expressing U251MG cells. This event made the glioblastoma cells a potent target for NK cell-mediated recognition through a NKG2D restricted mechanism. p53 siRNA-mediated knock-down and pharmacological inhibition (pifithrin-α), profoundly prevented the iPA action in restoring the immunogenicity of U343MG cells through a mechanism that is dependent upon p53 status of malignancy. Furthermore, accordingly to the preferential recognition of senescent cells by NK cells, we found that iPA treatment was critical for glioma cells entry in premature senescence through the induction of S and G2/M phase arrest. Collectively, our results indicate that behind the well established cytotoxic and antiangiogenic effects, iPA can also display an immune-mediated antitumor activity. The indirect engagement of the innate immune system and its additional activity in primary derived patient's glioma cell model (GBM17 and GBM37), fully increase its translational relevance and led to the exploitation of the isoprenoid pathway for a valid therapeutic intervention in antiglioma research.

The endocannabinoid system (ECS) represents one of the major neuromodulatory systems involved in different physiological and pathological processes. Multi-target compounds exert their activities by acting via multiple mechanisms of action and represent a promising pharmacological modulation of the ECS. In this work we report 4-substituted and 4,5-disubstituted 1,2-dihydro-2-oxo-pyridine-3-carboxamide derivatives with a broad spectrum of affinity and functional activity towards both cannabinoid receptors and additional effects on the main components of the ECS. In particular compound B3 showed high affinity for CB1R (Ki = 23.1 nM, partial agonist) and CB2R (Ki = 6.9 nM, inverse agonist) and also significant inhibitory activity (IC50 = 70 nM) on FAAH with moderate inhibition of ABHD12 (IC50 = 2.5 μΜ). Compounds B4, B5 and B6 that act as full agonists at CB1R and as partial agonists (B5 and B6) or antagonist (B4) at CB2R, exhibited an additional multi-target property by inhibiting anandamide uptake with sub-micromolar IC50 values (0.28–0.62 μΜ). The best derivatives showed cytotoxic activity on U937 lymphoblastoid cells. Finally, molecular docking analysis carried out on the three-dimensional structures of CB1R and CB2R and of FAAH allowed to rationalize the structure-activity relationships of this series of compounds.

Glioblastoma (GBM), the most aggressive brain cancer, is highly dependent on the mevalonate (MVA) pathway for the synthesis of lipid moieties critical for cell proliferation but the function and regulation of key intermediate enzymes like farnesyl-diphosphate synthase (FDPS), up to now, remained unknown. A deregulated expression and activity of FDPS was the central research idea of the present study. FDPS mRNA, protein and enzyme activity were analyzed in a cohort of stage III-IV glioma patients (N = 49) and primary derived cells. FDPS silencing helped to clarify its function in the maintenance of malignant phenotype. Interestingly, compared to tumor-free peripheral (TFB) brain and normal human astrocytes (NHA), FDPS protein expression and enzyme activity were detected at high degree in tumor mass where a correlation with canonical oncogenic signaling pathways such as STAT3, ERK and AKT was also documented. Further, FDPS knockdown in U87 and GBM primary cells but not in NHA, enhanced apoptosis. With the effort to develop a more refined map of the connectivity between signal transduction pathways and metabolic networks in cancer FDPS as a new candidate metabolic oncogene in glioblastoma, might suggest to further target MVA pathway as valid therapeutic tool.

Malignant gliomas are highly dependent on the isoprenoid pathway for the synthesis of lipid moieties critical for cell proliferation. The isoprenoid derivative N6-isopentenyladenosine (iPA) displays pleiotropic biological effects, including a direct anti-tumor activity in several tumor models. The antiglioma effects of iPA was then explored in U87MG cells both in vitro and grafted in mice and the related molecular mechanism confirmed in primary derived patients' glioma cells. iPA powerfully inhibited tumor cell growth and induced caspase-dependent apoptosis through a mechanism involving a marked accumulation of the pro-apoptotic BIM protein and inhibition of EGFR. Indeed, activating AMPK following conversion into its iPAMP active form, iPA stimulated EGFR phosphorylation and ubiquitination along a proteasome-mediated pathway which was responsible for receptor degradation and its downstream signaling pathways inhibition, including the STAT3, ERK and AKT cascade. The inhibition of AMPK by compound C prevented iPA-mediated phosphorylation of EGFR, known to precede receptor loss. As expected the block of EGFR degradation, by exposure to the proteasome inhibitor MG132, significantly reduced iPA-induced cell death. Given the importance of receptor degradation in iPA-mediated cytotoxicity, we also documented that the EGFR expression levels in a panel of primary glioma cells confers them a high sensitivity to iPA treatment. In conclusion our study provides the first evidence of iPA antiglioma effect. Indeed, as glioma is driven by aberrant signaling of growth factor receptors, particularly the EGFR, iPA, alone or in association with EGFR targeted therapies, might be a promising therapeutic tool to achieve a potent anti-tumoral effect.

We assessed the immunomodulatory and anti-inflammatory effects of 9,11-dihydrogracilin A (DHG), a molecule derived from the Antarctic marine sponge Dendrilla membranosa. We used in vitro and in vivo approaches to establish DHG properties. Human peripheral blood mononuclear cells (PBMC) and human keratinocytes cell line (HaCaT cells) were used as in vitro system, whereas a model of murine cutaneous irritation was adopted for in vivo studies. We observed that DHG reduces dose dependently the proliferative response and viability of mitogen stimulated PBMC. In addition, DHG induces apoptosis as revealed by AnnexinV staining and downregulates the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), signal transducer and activator of transcription (STAT) and extracellular signal–regulated kinase (ERK) at late time points. These effects were accompanied by down-regulation of interleukin 6 (IL-6) production, slight decrease of IL-10 and no inhibition of tumor necrosis factor-alpha (TNF-α) secretion. To assess potential properties of DHG in epidermal inflammation we used HaCaT cells; this compound reduces cell growth, viability and migration. Finally, we adopted for the in vivo study the croton oil-induced ear dermatitis murine model of inflammation. Of note, topical use of DHG significantly decreased mouse ear edema. These results suggest that DHG exerts anti-inflammatory effects and its anti-edema activity in vivo strongly supports its potential therapeutic application in inflammatory cutaneous diseases.