Oxidative Stress and Natural Antioxidants in Osteoporosis: Novel Preventive and Therapeutic Approaches.

This review reports in detail the cellular and molecular mechanisms which regulate the bone remodeling process in relation to oxidative stress (OS), inflammatory factors, and estrogen deficiency. OS is considered an important pathogenic factor of osteoporosis, inducing osteocyte apoptosis and varying levels of specific factors, such as receptor activator κB ligand (RANKL), sclerostin, and, according to recent evidence, fibroblast growth factor 23, with consequent impairment of bone remodeling and high bone resorption. Bone loss increases the risk of fragility fractures, and the most commonly used treatments are antiresorptive drugs, followed by anabolic drugs or those with a double effect. In addition, recent data show that natural antioxidants contained in the diet are efficient in preventing and reducing the negative effects of OS on bone remodeling and osteocytes through the involvement of sirtuin type 1 enzyme. Indeed, osteocytes and some of their molecular factors are considered potential biological targets on which antioxidants can act to prevent and reduce bone loss, as well as to promote bone anabolic and regenerative processes by restoring physiological bone remodeling. Several data suggest including antioxidants in novel therapeutic approaches to develop better management strategies for the prevention and treatment of osteoporosis and OS-related bone diseases. In particular, anthocyanins, as well as resveratrol, lycopene, oleuropein, some vitamins, and thiol antioxidants, could have protective and therapeutic anti-osteoporotic effects.

Extracellular Acidosis Differentially Regulates Estrogen Receptor ß-Dependent EMT Reprogramming in Female and Male Melanoma Cells.

Clinical outcomes of melanoma patients pointed out a gender disparity that supports a correlation between sex hormone activity on estrogen receptors (ER) and melanoma development and progression. Here, we found that the epithelial-to-mesenchymal transition (EMT) of melanoma cells induced by extracellular acidosis, which is a crucial hallmark of solid cancers, correlates with the expression of ERß, the most representative ER on melanoma cells. Extracellular acidosis induces an enhanced expression of ERß in female cells and EMT markers remain unchanged, while extracellular acidosis did not induce the expression of ERß in male cells and EMT was strongly promoted. An inverse relationship between ERß expression and EMT markers in melanoma cells of different sex exposed to extracellular acidosis was revealed by two different technical approaches: florescence-activated cell sorting of high ERß expressing cell subpopulations and ERß receptor silencing. Finally, we found that ERß regulates EMT through NF-κB activation. These results demonstrate that extracellular acidosis drives a differential ERß regulation in male and female melanoma cells and that this gender disparity might open new perspectives for personalized therapeutic approaches.

miR-214-Enriched Extracellular Vesicles Released by Acid-Adapted Melanoma Cells Promote Inflammatory Macrophage-Dependent Tumor Trans-Endothelial Migration.

The understanding of the molecular mechanisms leading to melanoma dissemination is urgently needed in view of the identification of new targets and the development of innovative strategies to improve patients' outcomes. Within the complexity of tumor intercellular communications leading to metastatic dissemination, extracellular vesicles (EV) released by tumor cells are central players. Indeed, the ability to travel through the circulatory system conveying oncogenic bioactive molecules even at distant sites makes EV capable of modulating recipient cells to facilitate metastatic dissemination. The dynamic remodeling of the tumor microenvironment might influence, along with a number of other events, tumoral EV release. We observed that, in melanoma, extracellular acidosis increases the release of EV enriched in miR-214, an onco-miRNA involved in melanoma metastasis. Then, miR-214-enriched EV were found to induce a state of macrophage activation, leading to an overproduction of proinflammatory cytokines and nitric oxide. Such an inflammatory microenvironment was able to alter the endothelial cell permeability, thereby facilitating the trans-endothelial migration of melanoma cells, a crucial step in the metastatic cascade. The use of synthetic miR-214 inhibitors and miR-214 overexpression allowed us to demonstrate the key role of miR-214 in the EV-dependent induction of macrophage activation. Overall, our in vitro study reveals that the release of tumor miR-214-enriched EV, potentiated by adapting tumor cells to extracellular acidosis, drives a macrophage-dependent trans-endothelial migration of melanoma cells. This finding points to miR-214 as a potential new therapeutic target to prevent melanoma intravasation.

Extra Virgin Olive Oil and Cardiovascular Protection in Chronic Kidney Disease.

The high mortality related to chronic kidney disease (CKD) is not only due to the disease itself; in fact, CKD also represents an important risk factor for cardiovascular (CV) morbidity and mortality. Among the functional foods that seems to have cardioprotective action, extra virgin olive oil (EVOO) plays a pivotal health-promoting role. The aim of this study was to evaluate the possible cardioprotective effects of an EVOO containing a very high content (>900 ppm) of minor phenolic compounds (MPCs). The selected EVOO was analyzed by HPLC-DAD-MS to establish the MPC content. The Olea extract obtained from the selected EVOO was tested against the RAW 264.7 cell line in order to investigate its anti-inflammatory activity. We enrolled 40 CKD patients under conservative therapy for in vivo clinical testing. All CKD patients consumed 40 mL/day of raw EVOO for 9 weeks (T1). At baseline (T0) and at T1, we monitored the patients' blood and urinary parameters. The patients' body composition was assessed using bioelectrical impedance analysis and the carotid intima-media thickness (CIMT) using ultrasound imaging. At T1, we observed a decrease in inflammatory parameters, CIMT, and oxidative stress biomarkers. We also noticed improvements in lipid and purine metabolism, atherogenic indices, and body composition. Thus, this study highlighted the cardioprotective action of EVOO in nephropathic patients.

An Oleocanthal-Enriched EVO Oil Extract Induces the ROS Production in Gastric Cancer Cells and Potentiates the Effect of Chemotherapy.

Oleocanthal, a minor polar compound in extra-virgin olive (EVO) oil, contains anticancer properties, which should be encouraged in its use in oncology. Gastric Cancer (GC), a very aggressive human cancer, is often diagnosed at advanced stages, when surgery is substituted or supported by chemotherapy (CT). However, CT frequently fails due to the patient's resistance to the treatment. Thus, the aim of this study is to verify whether an OC-enriched EVO oil extract fraction (OCF) may be useful in order to overcome a resistance to GC. We evaluated the OCF effects on an AGS gastric adenocarcinoma cell line wild type (AGS wt) and on its subpopulations resistant to 5-fluorouracil (5FUr), Paclitaxel (TAXr) or cisplatin (CISr). We found that a 60 µM dose of the OCF acts on the AGS wt, 5FUr and TAXr, leading to the cell cycle inhibition and to a ROS production, but not on CISr cells. Resistance of CISr to the OCF seems to be due to higher levels of antioxidant-enzymes that can counteract the OCF-induced ROS production. Moreover, using the OCF plus 5-fluorouracil, Paclitaxel or cisplatin, we found a potentiating effect compared with a mono-treatment in all resistant GC cells, including CISr. In conclusion, the use of the OCF in the management of GC has shown very interesting advantages, opening-up the possibility to evaluate the efficacy of the OCF in vivo, as a valid adjuvant in the treatment of resistant GC.

Oxidative Stress and Inflammation as Targets for Novel Preventive and Therapeutic Approaches in Non-Communicable Diseases II.

Non-communicable diseases (NCDs) are non-infectious chronic pathologies-including obesity, metabolic syndrome, chronic kidney disease (CKD), cardiovascular (CV) diseases, cancer, and chronic respiratory diseases-which represent the main cause of death and disability for the general population [...].

Oleuropein from olive leaf extracts and extra-virgin olive oil provides distinctive phenolic profiles and modulation of microbiota in the large intestine.

The interest in the modulation of gut microbiota by polyphenols from olives and derived products is increasing. In this work, phenolic leaf extracts (PLE) were in vitro faecal fermented to evaluate the changes in phenolic profiles and the impact on microbiota, using a commercial extra-virgin olive oil (EVOO) as reference. The in vitro fermentation decreased oleuropein content in PLE, determining an increase of hydroxytyrosol and other phenolic metabolites. An increase (p < 0.05) of hydroxytyrosol (LogFC = 6.02; VIP score = 1.05) was also observed in fermented EVOO. Besides, PLE significantly (p < 0.05) changed amino acids (LogFC = 6.1) and fatty acids (LogFC = 5.9) profile of the faeces. Metagenomic sequencing revealed that Coriobacteriaceae at the family level, and Collinsella at the genus level, were the most affected by PLE fermentation. These findings support the modulation of the gut microbiota exerted by phenolics from PLE and EVOO.

uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells.

Malignant melanoma is a highly aggressive skin cancer characterized by an elevated grade of tumor cell plasticity. Such plasticity allows adaptation of melanoma cells to different hostile conditions and guarantees tumor survival and disease progression, including aggressive features such as drug resistance. Indeed, almost 50% of melanoma rapidly develop resistance to the BRAFV600E inhibitor vemurafenib, with fast tumor dissemination, a devastating consequence for patients outcomes. Vasculogenic mimicry (VM), the ability of cancer cells to organize themselves in perfused vascular-like channels, might sustain tumor spread by providing vemurafenib-resistant cancer cells with supplementary ways to enter into circulation and disseminate. Thus, this research aims to determine if vemurafenib resistance goes with the acquisition of VM ability by aggressive melanoma cells, and identify a driving molecule for both vemurafenib resistance and VM. We used two independent experimental models of drug-resistant melanoma cells, the first one represented by a chronic adaptation of melanoma cells to extracellular acidosis, known to drive a particularly aggressive and vemurafenib-resistant phenotype, the second one generated with chronic vemurafenib exposure. By performing in vitro tube formation assay and evaluating the expression levels of the VM markers EphA2 and VE-cadherin by Western blotting and flow cytometer analyses, we demonstrated that vemurafenib-resistant cells obtained by both models are characterized by an increased ability to perform VM. Moreover, by exploiting the CRISPR-Cas9 technique and using the urokinase plasminogen activator receptor (uPAR) inhibitor M25, we identified uPAR as a driver of VM expressed by vemurafenib-resistant melanoma cells. Thus, uPAR targeting may be successfully leveraged as a new complementary therapy to inhibit VM in drug-resistant melanoma patients, to counteract the rapid progression and dissemination of the disease.

Oleuropein-Rich Leaf Extract as a Broad Inhibitor of Tumour and Macrophage iNOS in an Apc Mutant Rat Model.

Oleuropein, the major compound found in olive leaves, has been reported to exert numerous pharmacological properties, including anti-inflammatory, anti-diabetic and anti-cancer effects. The purpose of this study was to evaluate, for the first time, the effect of oleuropein-rich leaf extracts (ORLE) in already-developed colon tumours arising in Apc (adenomatous polyposis coli) mutated PIRC rats (F344/NTac-Apcam1137). Here, we were able to investigate in parallel the anti-cancer effect of ORLE, both in vivo and in vitro, and its anti-inflammatory effect on macrophages, representing a critical and abundant population in most solid tumour microenvironment. We found that in vivo ORLE treatment promoted apoptosis and attenuated iNOS activity both in colon tumours as in peritoneal macrophages of PIRC rats. We this confirmed in vitro using primary RAW264.7 cells: ORLE reduced iNOS activity in parallel with COX-2 and pro-inflammatory cytokines, such as IL-1ß, IL-6 and TGF-ß. These findings suggest that ORLE possess a strong anti-inflammatory activity, which could be crucial for dampening the pro-tumourigenic activity elicited by a chronic inflammatory state generated by either tumour cells or tumour-associated macrophages.

Effects of an Olive By-Product Called Pâté on Cardiovascular Risk Factors.

BACKGROUND: The nutraceutical effects of Olea europaea L. products are mainly due to phenolic compounds. During olive milling, most of the total phenols remain in the process by-products. AIM: We aimed to evaluate the effects of a specific by-product of olive oil called "pâté" (OlP) administered as tablets, on cardiovascular and metabolic risk factors. METHODS: The study was a crossover trial with 2 intervention periods. Nineteen participants (mean age: 38 years) took 4 tablets/day of either olive pâté (corresponding to 30 mg/day of hydroxytyrosol) or placebo for 2 months followed by a 2-month washout and another 2 months of crossed over treatment. RESULTS: After the intervention with pâté, participants showed a statistically significant reduction in plasma levels of total cholesterol (-10.8 mg/dL), LDL cholesterol (-10.8 mg/dL) and urea (-4.1 mg/dL), and a significant increase in calcium levels (+0.3 mg/dL). Leukocyte response to exogenous oxidative stress was significantly reduced (-12.8%) and levels of the antioxidant transcription factor Nrf-2 increased by 88.9%. Plasma levels of the pro-inflammatory protein MCP-1 were significantly reduced (-9.0 pg/mL). CONCLUSION: In conclusion, the intake of OlP showed positive effects on several cardiovascular risk factors, demonstrating the nutraceutical potential of a widely available but, to date, underestimated olive oil by-product.

Modelling genetic diseases for drug development: Hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the commonest genetic cardiac disease, with a prevalence of 1/500. It is caused by over 1400 different mutations, mainly involving the genes coding for sarcomere proteins. The main pathological features of HCM are left ventricular hypertrophy, diastolic dysfunction and the increased ventricular arrhythmogenesis. Predicting the risk of heart failure and lethal arrhythmias is the most challenging clinical task for HCM patient management. Moreover, there are no disease-modifying therapies that can prevent disease progression or sudden arrhythmic death in HCM patients. In this review, we will illustrate the most advanced research models and methods that have been employed for HCM studies, including preclinical tests of novel or existing drugs, along with visionary future development based on gene editing approaches. Acknowledging the advantages and limitations of the different models, and a critical consideration of the different, often conflicting result obtained using different approaches is essential for a deep understanding of HCM pathophysiology and for obtaining meaningful information on novel treatments, in order to improve patient risk stratification and therapeutic management.

The Carbonic Anhydrase IX inhibitor SLC-0111 as emerging agent against the mesenchymal stem cell-derived pro-survival effects on melanoma cells.

Mesenchymal stem cells (MSC) take part to solid tumour-associated stroma and critically influence progression of malignancy. Our study represents a striking example of melanoma progression to a more malignant and resistant phenotype promoted by MSC and the possibility to contrast this diabolic liaison using CAIX inhibitors. In particular, we demonstrated that melanoma cells exposed to a MSC-conditioned medium switch to a more malignant phenotype, characterised by resistance to programmed cell death and endowed with an epithelial-to-mesenchymal transition and stem cell characteristics. These effects were reversed abrogating MSC CAIX activity using SLC-0111, a CAIX inhibitor. Moreover, the acquisition by melanoma cells of a Vemurafenib-resistant phenotype upon MSC-conditioned medium exposure was removed when MSC were treated with SLC-0111. Therefore, MSC may profoundly reprogramme melanoma cells towards a wide resistant phenotype through CAIX involvement, as the use of SLC-0111 is able to contrast the development of this highly risky adaptation for disease progression.

Oxidative Stress and Inflammation as Targets for Novel Preventive and Therapeutic Approches in Non Communicable Diseases.

As recently reported by the World Health Organization (WHO), Non-Communicable Diseases (NCDs) has been rising over the last century representing the main cause of death and disability for the general population regardless of age, region, or gender [...].

Cancer Glycolytic Dependence as a New Target of Olive Leaf Extract.

Oleuropein (Ole), the main bioactive phenolic component of Olea europaea L. has recently attracted the scientific attention for its several beneficial properties, including its anticancer effects. This study is intended to investigate whether an olive leaf extract enriched in Ole (OLEO) may counteract the aerobic glycolysis exploited by tumor cells. We found that OLEO decreased melanoma cell proliferation and motility. OLEO was also able to reduce the rate of glycolysis of human melanoma cells without affecting oxidative phosphorylation. This reduction was associated with a significant decrease of glucose transporter-1, protein kinase isoform M2 and monocarboxylate transporter-4 expression, possible drivers of such glycolysis inhibition. Extending the study to other tumor histotypes, we observed that the metabolic effects of OLEO are not confined to melanoma, but also confirmed in colon carcinoma, breast cancer and chronic myeloid leukemia. In conclusion, OLEO represents a natural product effective in reducing the glycolytic metabolism of different tumor types, revealing an extended metabolic inhibitory activity that may be well suited in a complementary anti-cancer therapy.

A potentiated cooperation of carbonic anhydrase IX and histone deacetylase inhibitors against cancer.

The emergence of tumour recurrence and resistance limits the survival rate for most tumour-bearing patients. Only, combination therapies targeting pathways involved in the induction and in the maintenance of cancer growth and progression might potentially result in an enhanced therapeutic efficacy. Herein, we provided a prospective combination treatment that includes suberoylanilide hydroxamic acid (SAHA), a well-known inhibitor of histone deacetylases (HDACs), and SLC-0111, a novel inhibitor of carbonic anhydrase (CA) IX. We proved that HDAC inhibition with SAHA in combination with SLC-0111 affects cell viability and colony forming capability to greater extent than either treatment alone of breast, colorectal and melanoma cancer cells. At the molecular level, this therapeutic regimen resulted in a synergistically increase of histone H4 and p53 acetylation in all tested cell lines. Overall, our findings showed that SAHA and SLC-0111 can be regarded as very attractive combination providing a potential therapeutic strategy against different cancer models.

Oleuropein, a Bioactive Compound from Olea europaea L., as a Potential Preventive and Therapeutic Agent in Non-Communicable Diseases.

Growing scientific literature data suggest that the intake of natural bioactive compounds plays a critical role in preventing or reducing the occurrence of human chronic non-communicable diseases (NCDs). Oleuropein, the main phenolic component of Olea europaea L., has attracted scientific attention for its several health beneficial properties such as antioxidant, anti-inflammatory, cardio- and neuro-protective, and anti-cancer. This article is a narrative review focused on the current literature concerning the effect of oleuropein in NCDs, such as neuro- and cardiovascular diseases, diabetes mellitus, chronic kidney diseases, and cancer, by its putative antioxidant and anti-inflammatory activity, but also for its other peculiar actions such as an autophagy inducer and amyloid fibril growth inhibitor and, finally, for its anti-cancer effect. Despite the increasing number of published studies, looking at the beneficial effects of oleuropein, there is limited clinical evidence focused on the benefits of this polyphenol as a nutraceutical product in humans, and many problems are still to be resolved about its bioavailability, bioaccessibility, and dosage. Thus, future clinical randomized trials are needed to establish the relation between the beneficial effects and the mechanisms of action occurring in the human body in response to the intake of oleuropein.

Health Effects of Phenolic Compounds Found in Extra-Virgin Olive Oil, By-Products, and Leaf of Olea europaea L.

Olea europaea L. fruit is a peculiar vegetal matrix containing high levels of fatty acids (98-99% of the total weight of extra-virgin olive oil, EVOO) and low quantities (1-2%) of phenolics, phytosterols, tocopherols, and squalene. Among these minor components, phenolics are relevant molecules for human health. This review is focused on their beneficial activity, in particular of hydroxytyrosol (HT), oleuropein (OLE), oleocanthal (OLC), and lignans found in EVOO, olive oil by-products and leaves. Specifically, the cardioprotective properties of the Mediterranean diet (MD) related to olive oil consumption, and the biological activities of polyphenols recovered from olive oil by-products and leaves were described. Recent European projects such as EPIC (European Prospective Investigation into Cancer and Nutrition) and EPICOR (long-term follow-up of antithrombotic management patterns in acute coronary syndrome patients) have demonstrated the functional and preventive activities of EVOO showing the relation both between cancer and nutrition and between consumption of EVOO, vegetables, and fruit and the incidence of coronary heart disease. The data reported in this review demonstrate that EVOO, one of the pillars of the MD, is the main product of Olea europaea L. fruits; leaves and by-products are secondary but precious products from which bioactive compounds can be recovered by green technologies and reused for food, agronomic, nutraceutical, and biomedical applications according to the circular economy strategy.

Anoikis Resistance as a Further Trait of Acidic-Adapted Melanoma Cells.

Melanoma is characterized by a low extracellular pH, which contributes to the development of an aggressive phenotype characterized by several properties as the switch to an epithelial-to-mesenchymal program, the increase of apoptotic resistance, and the migratory ability together with the development of drug resistance. Here, we demonstrate that melanoma cells grown in low pH medium (pH 6.7) for a short (24 hours) or long (at least 3 months) period equally express an anoikis resistance profile. Anoikis is a form of apoptosis prompted by loss of adhesion, particularly requested by aggressive cancer cells to metastasize. Anoikis resistance was ascertained in acidic melanoma cells either grown in agarose-coated plates or incubated in rocking conditions. Both analyses indicate that acidic cells were more able to survive in a nonadherent condition than cells grown in standard pH, an effect resulting in a more cloning efficiency and migratory ability. Ability to survive during rocking was inhibited using mTOR/NF-kB inhibitors. Finally, we checked whether characteristics related to the in vitro anoikis resistance acquired by acidic melanoma cells might be also suitable for in vivo challenge. We injected acidic melanoma cells into blood stream, and then we verify how many cells survived in blood after 15 min from the injection. Only acidic cells, transient and chronic, survived, whereas melanoma cells grown in standard pH medium did not. Overall, we have had the opportunity to demonstrate that low extracellular pH represents an additional mechanism able to promote an anoikis resistance in solid tumors.

Oleuropein, the Main Polyphenol of Olea europaea Leaf Extract, Has an Anti-Cancer Effect on Human BRAF Melanoma Cells and Potentiates the Cytotoxicity of Current Chemotherapies.

Oleuropein (Ole), a secoiridoid glucoside present in Olea europaea leaves, gained scientific interest thanks to its several biological properties, including the anticancer one. We verified whether Ole might potentiate the cytotoxicity of conventional drugs used to treat melanoma, disclosing a potentially new therapeutic strategy. We tested the cytotoxic action of Ole alone or in combination with chemotherapeutics on A375 human melanoma cells. We found that Ole was able, at a dose of 500 µM, to stimulate apoptosis, while at a non-toxic dose of 250 µM, it affected cell proliferation and induced the downregulation of the pAKT/pS6 pathway. A dose of 250 µM Ole did not potentiate the effect of Vemurafenib (PLX4032), but it succeeded in increasing the cytotoxic effect of Dacarbazine (DTIC). The major effect was found in the association between Ole and Everolimus (RAD001), also on PLX4032-resistant BRAF melanoma cells, which possibly cooperate in the inhibition of the pAKT/pS6 pathway. Of interest, an olive leaf extract enriched in equimolar Ole was more effective and able to further improve DTIC and RAD001 efficacy on BRAF melanoma cells with respect to Ole alone. Therefore, Ole represents a natural product able to potentiate a wide array of chemotherapeutics against BRAF melanoma cells affecting the pAKT/pS6 pathway.

Oleuropein Aglycone Protects against MAO-A-Induced Autophagy Impairment and Cardiomyocyte Death through Activation of TFEB.

Age-associated diseases such as neurodegenerative and cardiovascular disorders are characterized by increased oxidative stress associated with autophagy dysfunction. Oleuropein aglycone (OA), the main polyphenol found in olive oil, was recently characterized as an autophagy inducer and a promising agent against neurodegeneration. It is presently unknown whether OA can have beneficial effects in a model of cardiac stress characterized by autophagy dysfunction. Here, we explored the effects of OA in cardiomyocytes with overexpression of monoamine oxidase-A (MAO-A). This enzyme, by degrading catecholamine and serotonin, produces hydrogen peroxide (H2O2), which causes oxidative stress, autophagic flux blockade, and cell necrosis. We observed that OA treatment counteracted the cytotoxic effects of MAO-A through autophagy activation, as displayed by the increase of autophagic vacuoles and autophagy-specific markers (Beclin1 and LC3-II). Moreover, the decrease in autophagosomes and the increase in autolysosomes, indicative of autophagosome-lysosome fusion, suggested a restoration of the defective autophagic flux. Most interestingly, we found that the ability of OA to confer cardioprotection through autophagy induction involved nuclear translocation and activation of the transcriptional factor EB (TFEB). Our data provide strong evidence of the beneficial effects of OA, suggesting its potential use as a nutraceutical agent against age-related pathologies involving autophagy dysfunction, including cardiovascular diseases.