Effects of Flavonoids on Cancer, Cardiovascular and Neurodegenerative Diseases: Role of NF-κB Signaling Pathway.

Flavonoids are polyphenolic phytochemical compounds found in many plants, fruits, vegetables, and leaves. They have a multitude of medicinal applications due to their anti-inflammatory, antioxidative, antiviral, and anticarcinogenic properties. Furthermore, they also have neuroprotective and cardioprotective effects. Their biological properties depend on the chemical structure of flavonoids, their mechanism of action, and their bioavailability. The beneficial effects of flavonoids have been proven for a variety of diseases. In the last few years, it is demonstrated that the effects of flavonoids are mediated by inhibiting the NF-κB (Nuclear Factor-κB) pathway. In this review, we have summarized the effects of some flavonoids on the most common diseases, such as cancer, cardiovascular, and human neurodegenerative diseases. Here, we collected all recent studies describing the protective and prevention role of flavonoids derived from plants by specifically focusing their action on the NF-κB signaling pathway.

Nobiletin and xanthohumol counteract the TNFα-mediated activation of endothelial cells through the inhibition of the NF-κB signaling pathway.

Angiogenesis, a process characterized by the formation of new blood vessels from pre-existing ones, is a crucial step in tumor growth and dissemination. Given the ability of tumors to interfere with multiple or different molecular pathways to promote angiogenesis, there is an increasing need to therapeutically block tumor progression by targeting multiple antiangiogenic pathways. Natural polyphenols present health-protective properties, which are likely attributed to their ability to activate multiple pathways involved in inflammation, carcinogenesis, and angiogenesis. Recently, increased attention has been addressed to the ability of flavonoids, the most abundant polyphenols in the diet, to prevent cancer by suppressing angiogenesis. Here we investigate the mechanisms by which xanthohumol (the major prenylated flavonoid of the hop plant Humulus lupulus L.) and nobiletin (flavonoid from red-orange Citrus sinensis) can modulate the effects of Tumor Necrosis Factor-α (TNF-α) on human umbilical vein endothelial cells (HUVEC). The results reported in this paper show that xanthohumol and nobiletin pretreatment of HUVEC inhibits the effects induced by TNF-α on cell migration, invasion capability, and colon cancer cell adhesion on the endothelial monolayer. Moreover, the pretreatment reduces metalloproteinases and adhesion molecules' expression. Finally, our results highlight that xanthohumol and nobiletin can counteract the effects of TNF-α on angiogenesis and invasiveness, mainly through Vascular Endothelial Growth Factor and NF-κB pathways. Since angiogenesis plays an important pathological role in the progression of several diseases, our findings may provide clues for developing xanthohumol and nobiletin as therapeutic agents against angiogenesis-associated diseases.

Mir-675-5p supports hypoxia-induced drug resistance in colorectal cancer cells.

BACKGROUND: The uncontrolled proliferation of cancer cells determines hypoxic conditions within the neoplastic mass with consequent activation of specific molecular pathways that allow cells to survive despite oxygen deprivation. The same molecular pathways are often the cause of chemoresistance. This study aims to investigate the role of the hypoxia-induced miR-675-5p in 5-Fluorouracil (5-FU) resistance on colorectal cancer (CRC) cells. METHODS: CRC cell lines were treated with 5-Fu and incubated in normoxic or hypoxic conditions; cell viability has been evaluated by MTT assay. MiR-675-5p levels were analysed by RT-PCR and loss and gain expression of the miRNA has been obtained by the transfection of miRNA antagomir or miRNA mimic. Total protein expression of different apoptotic markers was analysed through western blot assay. MirWalk 2.0 database search engine was used to investigate the putative targets of the miR-675-5p involved in the apoptotic process. Finally, the luciferase assay was done to confirm Caspase-3 as a direct target of the miR-675-5p. RESULTS: Our data demonstrated that hypoxia-induced miR-675-5p counteracts the apoptotic signal induced by 5-FU, thus taking part in the drug resistance response. We showed that the apoptotic markers, cleaved PARP and cleaved caspase-3, increased combining miR-675-5p inhibition with 5-FU treatment. Moreover, we identified pro-caspase-3 among the targets of the miR-675-5p. CONCLUSION: Our data demonstrate that the inhibition of hypoxia-induced miR-675-5p combined with 5-FU treatment can enhances drug efficacy in both prolonged hypoxia and normoxia, indicating a possible strategy to partially overcome chemoresistance.

Molecular Mediators of RNA Loading into Extracellular Vesicles.

In the last decade, an increasing number of studies have demonstrated that non-coding RNA (ncRNAs) cooperate in the gene regulatory networks with other biomolecules, including coding RNAs, DNAs and proteins. Among them, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are involved in transcriptional and translation regulation at different levels. Intriguingly, ncRNAs can be packed in vesicles, released in the extracellular space, and finally internalized by receiving cells, thus affecting gene expression also at distance. This review focuses on the mechanisms through which the ncRNAs can be selectively packaged into extracellular vesicles (EVs).

Rapamycin-Loaded Polymeric Nanoparticles as an Advanced Formulation for Macrophage Targeting in Atherosclerosis.

Recently, rapamycin (Rapa) represents a potential drug treatment to induce regression of atherosclerotic plaques; however, its use requires site-specific accumulation in the vessels involved in the formation of the plaques to avoid the systemic effects resulting from its indiscriminate biodistribution. In this work, a stable pharmaceutical formulation for Rapa was realized as a dried powder to be dispersed extemporaneously before administration. The latter was constituted by mannitol (Man) as an excipient and a Rapa-loaded polymeric nanoparticle carrier. These nanoparticles were obtained by nanoprecipitation and using as a starting polymeric material a polycaprolactone (PCL)/α,ß-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) graft copolymer. To obtain nanoparticles targeted to macrophages, an oxidized phospholipid with a high affinity for the CD36 receptor of macrophages, the 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdia-PC), was added to the starting organic phase. The chemical-physical and technological characterization of the obtained nanoparticles demonstrated that: both the drug loading (DL%) and the entrapment efficiency (EE%) entrapped drug are high; the entrapped drug is in the amorphous state, protected from degradation and slowly released from the polymeric matrix; and the KOdia-PC is on the nanoparticle surface (KP-Nano). The biological characterization demonstrated that both systems are quickly internalized by macrophages while maintaining the activity of the drug. In vitro studies demonstrated that the effect of KP-Nano Rapa-loaded, in reducing the amount of the Phospo-Ser757-ULK1 protein through the inhibition of the mammalian target of rapamycin (mTOR), is comparable to that of the free drug.

Hypoxia-Induced Non-Coding RNAs Controlling Cell Viability in Cancer.

Hypoxia, a characteristic of the tumour microenvironment, plays a crucial role in cancer progression and therapeutic response. The hypoxia-inducible factors (HIF-1α, HIF-2α, and HIF-3α), are the master regulators in response to low oxygen partial pressure, modulating hypoxic gene expression and signalling transduction pathways. HIFs' activation is sufficient to change the cell phenotype at multiple levels, by modulating several biological activities from metabolism to the cell cycle and providing the cell with new characteristics that make it more aggressive. In the past few decades, growing numbers of studies have revealed the importance of non-coding RNAs (ncRNAs) as molecular mediators in the establishment of hypoxic response, playing important roles in regulating hypoxic gene expression at the transcriptional, post-transcriptional, translational, and posttranslational levels. Here, we review recent findings on the different roles of hypoxia-induced ncRNAs in cancer focusing on the data that revealed their involvement in tumour growth.

Hypoxia-Induced miR-675-5p Supports ß-Catenin Nuclear Localization by Regulating GSK3-ß Activity in Colorectal Cancer Cell Lines.

The reduction of oxygen partial pressure in growing tumors triggers numerous survival strategies driven by the transcription factor complex HIF1 (Hypoxia Inducible Factor-1). Recent evidence revealed that HIF1 promotes rapid and effective phenotypic changes through the induction of non-coding RNAs, whose contribution has not yet been fully described. Here we investigated the role of the hypoxia-induced, long non-coding RNA H19 (lncH19) and its intragenic miRNA (miR-675-5p) into HIF1-Wnt crosstalk. During hypoxic stimulation, colorectal cancer cell lines up-regulated the levels of both the lncH19 and its intragenic miR-675-5p. Loss of expression experiments revealed that miR-675-5p inhibition, in hypoxic cells, hampered ß-catenin nuclear localization and its transcriptional activity, while lncH19 silencing did not induce the same effects. Interestingly, our data revealed that miRNA inhibition in hypoxic cells restored the activity of Glycogen Synthase Kinase 3ß (GSK-3ß) reducing the amount of P-Ser9 kinase, thus unveiling a role of the miR-675-5p in controlling GSK-3ß activity. Bioinformatics analyses highlighted the serine/threonine-protein phosphatases PPP2CA, responsible for GSK-3ß activation, among the miR-675-5p targets, thus indicating the molecular mediator through which miR-675-5p may control ß-catenin nuclear localization. In conclusion, here we demonstrated that the inhibition of the hypoxia-induced non-coding RNA miR-675-5p hampered the nuclear localization of ß-catenin by regulating GSK-3ß activity, thus proposing the miR-675-5p as a new therapeutic target for the treatment of colorectal cancer.

Mesenchymal and Induced Pluripotent Stem Cells-Derived Extracellular Vesicles: The New Frontier for Regenerative Medicine?

Regenerative medicine aims to repair damaged, tissues or organs for the treatment of various diseases, which have been poorly managed with conventional drugs and medical procedures. To date, multimodal regenerative methods include transplant of healthy organs, tissues, or cells, body stimulation to activate a self-healing response in damaged tissues, as well as the combined use of cells and bio-degradable scaffold to obtain functional tissues. Certainly, stem cells are promising tools in regenerative medicine due to their ability to induce de novo tissue formation and/or promote organ repair and regeneration. Currently, several studies have shown that the beneficial stem cell effects, especially for mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) in damaged tissue restore are not dependent on their engraftment and differentiation on the injury site, but rather to their paracrine activity. It is now well known that paracrine action of stem cells is due to their ability to release extracellular vesicles (EVs). EVs play a fundamental role in cell-to-cell communication and are directly involved in tissue regeneration. In the present review, we tried to summarize the molecular mechanisms through which MSCs and iPSCs-derived EVs carry out their therapeutic action and their possible application for the treatment of several diseases.

Differences in Intercellular Communication During Clinical Relapse and Gadolinium-Enhanced MRI in Patients With Relapsing Remitting Multiple Sclerosis: A Study of the Composition of Extracellular Vesicles in Cerebrospinal Fluid.

This study was designed based on the hypothesis that changes in both the levels and surface marker expression of extracellular vesicles (EVs) isolated from the cerebrospinal fluid (CSF) may be associated with the clinical form, disease activity, and severity of multiple sclerosis (MS). The analyzes were performed on subjects affected by MS or other neurological disorders. EVs, which were isolated by ultracentrifugation of CSF samples, were characterized by flow cytometry. A panel of fluorescent antibodies was used to identify the EV origin: CD4, CCR3, CCR5, CD19, and CD200, as well as isolectin IB4. The Mann-Whitney U-test and Kruskal-Wallis test were used for statistical analyzes. EVs isolated from the CSF were more abundant in patients with progressive MS and in those with a clinically isolated syndrome than in all the other groups examined. Furthermore, an important change in the number of EVs and in their surface marker expression occurred during active phases of MS [i.e., clinical relapses and the presence of enhancing lesions on magnetic resonance imaging (MRI)]. In particular, the number of CSF-EVs increased in patients affected by MS during clinical relapse; this finding was associated with a decrease in the number of CD19+/CD200+ (naïve B cells) EVs. These markers are expressed by immature and naïve B lymphocytes, and to the best of our knowledge, this double staining has never been associated with MS, but their reduction has been observed in patients with another type of Th1 cell-mediated autoimmune disease. In contrast, the presence of lesions in the brain and spine on gadolinium-enhanced MRI was associated with an increase in the numbers of CCR3+/CCR5+ (subset of CD8 memory T cells), CD4+/CCR3+ (Th2 cells), and CD4+/CCR5+ (Th1 cells) CSF-EVs. Two points are worth emphasizing: (i) the data obtained in this study confirm that CSF-EVs represent a potentially promising tool to identify biomarkers specific for different phases of MS; and (ii) Considering the role of EVs in intercellular communication, our results provide some insights that improve our understanding of the relationships among some of the cell types that are mainly involved in MS pathogenesis (e.g., lymphocytes, glia, and neurons).

Extracellular Vesicles in Multiple Sclerosis as Possible Biomarkers: Dream or Reality?

Extracellular vesicles are recently described as specialized structures for intercellular communication. Their role in the central nervous system was diffusely studied in both physiological and pathological condition. In particular, an increased extracellular vesicle number was detected in several autoimmune diseases, including multiple sclerosis, a chronic autoimmune, inflammatory, demyelinating and neurodegenerative disease. This chapter summarizes the available information on the involvement of the extracellular vesicles in multiple sclerosis pathogenesis and their possible use as biomarker of therapy efficacy.