Regulation of Cell-Signaling Pathways by Berbamine in Different Cancers.

Natural product research is a cornerstone of the architectural framework of clinical medicine. Berbamine is a natural, potent, pharmacologically active biomolecule isolated from Berberis amurensis. Berbamine has been shown to modulate different oncogenic cell-signaling pathways in different cancers. In this review, we comprehensively analyze how berbamine modulates deregulated pathways (JAK/STAT, CAMKII/c-Myc) in various cancers. We systematically analyze how berbamine induces activation of the TGF/SMAD pathway for the effective inhibition of cancer progression. We also summarize different nanotechnological strategies currently being used for proficient delivery of berbamine to the target sites. Berbamine has also been reported to demonstrate potent anti-cancer and anti-metastatic effects in tumor-bearing mice. The regulation of non-coding RNAs by berbamine is insufficiently studied, and future studies must converge on the identification of target non-coding RNAs. A better understanding of the regulatory role of berbamine in the modulation of non-coding RNAs and cell-signaling pathways will be advantageous in the effective translation of laboratory findings to clinically effective therapeutics.

Breast Cancer Derived Extracellular Vesicles in Bone Metastasis Induction and Their Clinical Implications as Biomarkers.

Cancer incidence and mortality are rapidly growing worldwide. The main risk factors for cancer can be associated with aging as well as the growth of the population and socioeconomic condition. Breast cancer, a crucial public health problem, is the second cause of death among women. About 70% of patients with advanced breast cancer have bone metastases. In bone metastasis, cancer cells and osteoclasts form a vicious cycle: cancer cells promote osteoclast differentiation and activation that, in turn, induce cancer cell seeding and proliferation in the bone. Growing evidence shows that extracellular vesicles (EVs) play a key role in carcinogenesis, proliferation, pre-metastatic niche formation, angiogenesis, metastasis, and chemoresistance in several tumors, such as breast, lung, prostate, and liver cancer. Here, we discuss the role of EVs released by breast cancer cells, focusing on bone metastasis induction and their clinical implications as biomarkers.

Circular RNA in Exosomes.

Circular RNAs (circRNAs) are a novel family of non-coding endogenous RNAs discovered in all eukaryotic cells and generated through a particular mechanism of alternative splicing called "back-splicing". These molecules show multiple functions, by acting as modulators of gene and miRNA expression, and may have a role in several biological processes, such as cell proliferation and invasion with, tumour development and progression, and in several mechanisms underlying other diseases. Their presence has been shown to be abundant in several body fluids such as blood and saliva. Based on their biogenesis mechanism, circRNAs may be categorized into five classes: exonic circRNAs, intronic circRNAs, antisense circRNAs, sense overlapping circRNAs and intergenic circRNAs. Recently, the presence of circRNAs, in addition to that of miRNAs and long non-coding RNAs, has been detected also in small extracellular vesicles called exosomes. Investigating the presence and expression levels of serum exosomal circRNAs could allow us, in future, to discriminate cancer patients from healthy individuals, identifying new potential exosome-based cancer biomarkers.In this chapter, we briefly will describe the major features and functions of exosomal circRNAs, discussing their potential role as molecular biomarkers for diagnosis, prognosis and monitoring of complex diseases, including cancer.

Liquid biopsies in lung cancer: the new ambrosia of researchers.

In the last decades the approach to cancer patient management has been deeply revolutionized. We are moving from a "one-fits-all" strategy to the "personalized medicine" based on the molecular characterization of the tumor. In this new era it is becoming more and more clear that the monitoring of the disease is fundamental for the success of the treatment, thus there is the need of new biomarker discovery. More precisely in the last years the scientific community has started to use the term "liquid biopsy". A liquid biopsy is a liquid biomarker that can be easily isolated from many body fluids (blood, saliva, urine, ascites, pleural effusion, etc.) and, as well as a tissue biopsy, a representative of the tissue from which it is spread. In this review we will focus our attention on circulating tumor cells, circulating tumor DNA, exosomes and secretomes with the aim to underlie their usefulness and potential application in a clinical setting for lung cancer patient management.

Exosomal shuttling of miR-126 in endothelial cells modulates adhesive and migratory abilities of chronic myelogenous leukemia cells.

BACKGROUND: Recent findings indicate that exosomes released from cancer cells contain microRNAs (miRNAs) that may be delivered to cells of tumor microenvironment. RESULTS: To elucidate whether miRNAs secreted from chronic myelogenous leukemia cells (CML) are shuttled into endothelial cells thus affecting their phenotype, we first analysed miRNAs content in LAMA84 exosomes. Among the 124 miRNAs identified in LAMA84 exosomes, we focused our attention on miR-126 which was found to be over-overexpressed in exosomes compared with producing parental cells. Transfection of LAMA84 with Cy3-labelled miR-126 and co-culture of leukemia cells with endothelial cells (EC) confirmed that miR-126 is shuttled into HUVECs. The treatment of HUVECs with LAMA84 exosomes for 24 hours reduced CXCL12 and VCAM1 expression, both at the mRNA and protein level, and negatively modulated LAMA84 motility and cells adhesion. Transfection in HUVECs of miR-126 inhibitor reversed the decrease of CXCL12 and restored the motility and adhesion of LAMA84 cells while the over-expression of miR-126, showed opposite effects. CONCLUSION: Our results show that the miR-126 shuttled by exosomes is biologically active in the target cells, and support the hypothesis that exosomal miRNAs have an important role in tumor-endothelial crosstalk occurring in the bone marrow microenvironment, potentially affecting disease progression.

Extracellular Vesicles in Lung Cancer: Implementation in Diagnosis and Therapeutic Perspectives.

Lung cancer represents the leading cause of cancer-related mortality worldwide, with around 1.8 million deaths in 2020. For this reason, there is an enormous interest in finding early diagnostic tools and novel therapeutic approaches, one of which is extracellular vesicles (EVs). EVs are nanoscale membranous particles that can carry proteins, lipids, and nucleic acids (DNA and RNA), mediating various biological processes, especially in cell-cell communication. As such, they represent an interesting biomarker for diagnostic analysis that can be performed easily by liquid biopsy. Moreover, their growing dataset shows promising results as drug delivery cargo. The aim of our work is to summarize the recent advances in and possible implications of EVs for early diagnosis and innovative therapies for lung cancer.

Contribution of proteomics to understanding the role of tumor-derived exosomes in cancer progression: state of the art and new perspectives.

Exosomes are nanometer-sized vesicles (40-100 nm diameter) of endocytic origin released from different cell types under both normal and pathological conditions. They function as cell free messengers, playing a relevant role in the cell-cell communication that is strongly related to the nature of the molecules (proteins, mRNAs, miRNAs, and lipids) that they transport. Tumor cells actively shed exosomes into their surrounding microenvironment and growing evidence indicates that these vesicles have pleiotropic functions in the regulation of tumor progression, promoting immune escape, tumor invasion, neovascularization, and metastasis. During the last few years remarkable efforts have been made to obtain an accurate definition of the protein content of tumor-derived exosomes (TDEs) by applying MS-based proteomic technologies. To date, TDEs proteomic studies have been mainly utilized to catalog TDEs proteins with the purpose of identifying disease biomarkers. The future challenge for improving our understanding and characterization of TDEs will be the implementation of new systems-driven and proteomic integrative strategies. The aim of this article is to provide an overview of the most characterized exosomes-mediated mechanisms that contribute to the pathogenesis of cancer and to review recent proteomics data that support the protumorigenic role of TDEs.

PIWI-RNAs Small Noncoding RNAs with Smart Functions: Potential Theranostic Applications in Cancer.

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a new class of small noncoding RNAs (ncRNAs) that bind components of the PIWI protein family. piRNAs are specifically expressed in different human tissues and regulate important signaling pathways. Aberrant expressions of piRNAs and PIWI proteins have been associated with tumorigenesis and cancer progression. Recent studies reported that piRNAs are contained in extracellular vesicles (EVs), nanosized lipid particles, with key roles in cell-cell communication. EVs contain several bioactive molecules, such as proteins, lipids, and nucleic acids, including emerging ncRNAs. EVs are one of the components of liquid biopsy (LB) a non-invasive method for detecting specific molecular biomarkers in liquid samples. LB could become a crucial tool for cancer diagnosis with piRNAs as biomarkers in a precision oncology approach. This review summarizes the current findings on the roles of piRNAs in different cancer types, focusing on potential theranostic applications of piRNAs contained in EVs (EV-piRNAs). Their roles as non-invasive diagnostic and prognostic biomarkers and as new therapeutic options have been also discussed.

Effects of condensates from volcanic fumaroles and cigarette smoke extracts on airway epithelial cells.

The impact of volcanic airborne products on airway epithelium homeostasis is largely unknown. This study assessed the effects of volcanic Fumarole Condensates (FC) alone or combined with Cigarette Smoke Extracts (CSE) on airway epithelial cells (16HBE and A549). Chemical composition of FC was analyzed by gas chromatography and HPLC. Cells were exposed to FC and IL-33 and IL-8 were assessed. The effects of FC and CSE on cell injury were evaluated assessing cell metabolism/cell viability, mitochondrial stress, cell apoptosis/cell necrosis, and cell proliferation. FC contained: water vapor (70-97%), CO2 (3-30%), acid gases (H2S, SO2, HCl, HF) around 1%. FC increased the intracellular IL-33 but differently modulated IL-33 and IL-8 gene expression and IL-8 release in the tested cell lines. FC without/with CSE: (a) increased cell metabolism/cell viability in 16HBE, while decreased it in A549; (b) increased mitochondrial stress in both cell types. FC with CSE increased cell necrosis in A549 in comparison to CSE alone. CSE reduced cell proliferation in 16HB,E while increased it in A549 and FC counteracted these effects in both cell types. Overall, FC induce a pro-inflammatory profile associated to a metabolic reprogramming without a relevant toxicity also in presence of CSE in airway epithelial cells.

Tyndallized Bacteria Preferentially Induce Human Macrophage M1 Polarization: An Effect Useful to Balance Allergic Immune Responses and to Control Infections.

Macrophage polarization is a dynamic process through which macrophages acquire specific features whose extremes are represented by M1 and M2 polarization. Interleukin (IL)-6, IL-1ß, IL-12 and IL-8 belong to M1 macrophages while transforming growth factor-beta (TGF-ß belongs to M2 cytokines. M2 polarization prevalence is observed in allergic diseases. Tyndallization is a thermal process able to inactivate microorganisms and to allow their use for chronic respiratory disease treatment via immune response modulation. The present study explores the effects of a blend of tyndallized bacteria (TB) on macrophage polarization. THP-1-derived macrophages were exposed to different concentrations of TB (106, 5 × 106, 107, 5 × 107, 108 CFU/mL) and then cell viability and TB phagocytosis, and IL-8, IL-1ß, IL-6, IL-12 and TGF-ß1 gene expression and release were assessed. TB were tolerated, phagocyted and able to increase IL-8, IL-1ß and IL-6 gene expression and release IL-12 gene expression, as well as decrease TGF-ß1 gene expression and release. The effects on IL-8, IL-6 and TGF-ß1 release were confirmed in human monocyte-derived macrophages (hMDMs) exposed to TB. In conclusion, TB promote M1 polarization, and this mechanism might have valuable potential in controlling allergic diseases and infections, possibly preventing disease exacerbations.

Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis.

Our study is designed to assess if exosomes released from chronic myelogenous leukemia (CML) cells may modulate angiogenesis. We have isolated and characterized the exosomes generated from LAMA84 CML cells and demonstrated that addition of exosomes to human vascular endothelial cells (HUVEC) induces an increase of both ICAM-1 and VCAM-1 cell adhesion molecules and interleukin-8 expression. The stimulation of cell-cell adhesion molecules was paralleled by a dose-dependent increase of adhesion of CML cells to a HUVEC monolayer. We further showed that the treatment with exosomes from CML cells caused an increase in endothelial cell motility accompanied by a loss of VE-cadherin and ß-catenin from the endothelial cell surface. Functional characterization of exosomes isolated from CML patients confirmed the data obtained with exosomes derived from CML cell line. CML exosomes caused reorganization into tubes of HUVEC cells cultured on Matrigel. When added to Matrigel plugs in vivo, exosomes induced ingrowth of murine endothelial cells and vascularization of the Matrigel plugs. Our results suggest for the first time that exosomes released from CML cells directly affect endothelial cells modulating the process of neovascularization.

Exosomes released by K562 chronic myeloid leukemia cells promote angiogenesis in a Src-dependent fashion.

Exosomes, microvesicles of endocytic origin released by normal and tumor cells, play an important role in cell-to-cell communication. Angiogenesis has been shown to regulate progression of chronic myeloid leukemia (CML). The mechanism through which this happens has not been elucidated. We isolated and characterized exosomes from K562 CML cells and evaluated their effects on human umbilical endothelial cells (HUVECs). Fluorescent-labeled exosomes were internalized by HUVECs during tubular differentiation on Matrigel. Exosome localization was perinuclear early in differentiation, moving peripherally in cells undergoing elongation and connection. Exosomes move within and between nanotubular structures connecting the remodeling endothelial cells. They stimulated angiotube formation over a serum/growth factor-limited medium control, doubling total cumulative tube length (P = 0.003). Treatment of K562 cells with two clinically active tyrosine kinase inhibitors, imatinib and dasatinib, reduced their total exosome release (P < 0.009); equivalent concentrations of drug-treated exosomes induced a similar extent of tubular differentiation. However, dasatinib treatment of HUVECs markedly inhibited HUVEC response to drug control CML exosomes (P < 0.002). In an in vivo mouse Matrigel plug model angiogenesis was induced by K562 exosomes and abrogated by oral dasatinib treatment (P < 0.01). K562 exosomes induced dasatinib-sensitive Src phosphorylation and activation of downstream Src pathway proteins in HUVECs. Imatinib was minimally active against exosome stimulation of HUVEC cell differentiation and signaling. Thus, CML cell-derived exosomes induce angiogenic activity in HUVEC cells. The inhibitory effect of dasatinib on exosome production and vascular differentiation and signaling reveals a key role for Src in both the leukemia and its microenvironment.

Increased expression of interleukin-32 in the inflamed ileum of ankylosing spondylitis patients.

OBJECTIVE: To study the mRNA expression and protein tissue distribution of IL-32 in ileal biopsy specimens from patients with AS. METHODS: Quantitative gene expression analysis, by real-time PCR, of IL-32, IL-1ß, IL-10, TNF-α and IFN-γ was performed on ileal biopsies of 15 AS and 15 Crohn's disease (CD) patients and 10 healthy subjects (HSs). IL-32 tissue distribution was evaluated by immunohistochemistry. The effect of IL-32 on the production of IL-10 by intestinal epithelial cell lines was also evaluated. RESULTS: In the ileal specimens of patients with AS and intestinal chronic inflammation, significant up-regulation of IL-32 at both the mRNA and protein levels was found as compared with non-inflamed AS patients and controls. IL-32 over-expression in AS was accompanied by a significant increase of IL-10 but not of cytokines involved in IL-32 induction. IL-32 stimulates intestinal epithelial cell lines in vitro to produce IL-10. CONCLUSION: Our findings suggest IL-32 as an important cytokine probably involved in the innate immune response occurring in early phases of intestinal inflammation, where it seems to play a prevalent protective role.

Extracellular Vesicles-ceRNAs as Ovarian Cancer Biomarkers: Looking into circRNA-miRNA-mRNA Code.

Ovarian cancer (OC) is one of the most lethal gynecologic malignancies in females worldwide. OC is frequently diagnosed at an advanced stage due to a lack of specific symptoms and effective screening tests, resulting in a poor prognosis for patients. Age, genetic alterations, and family history are the major risk factors for OC pathogenesis. Understanding the molecular mechanisms underlying OC progression, identifying new biomarkers for early detection, and discovering potential targets for new drugs are urgent needs. Liquid biopsy (LB), used for cancer detection and management, consists of a minimally invasive approach and practical alternative source to investigate tumor alterations by testing extracellular vesicles (EVs), circulating tumor cells, tumor-educated platelets, and cell-free nucleic acids. EVs are nanosize vesicles shuttling proteins, lipids, and nucleic acids, such as DNA, RNA, and non-coding RNAs (ncRNAs), that can induce phenotypic reprogramming of target cells. EVs are natural intercellular shuttles for ncRNAs, such as microRNAs (miRNAs) and circular-RNAs (circRNAs), known to have regulatory effects in OC. Here we focus on the involvement of circRNAs and miRNAs in OC cancer progression. The circRNA-microRNA-mRNA axis has been investigated with Circbank and miRwalk analysis, unraveling the intricate and detailed regulatory network created by EVs, ncRNAs, and mRNAs in OC.

High Mobility Group Box 1: Biological Functions and Relevance in Oxidative Stress Related Chronic Diseases.

In the early 1970s, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and named high-mobility group (HMG) proteins. High-mobility group box 1 (HMGB1) is the most studied HMG protein that detects and coordinates cellular stress response. The biological function of HMGB1 depends on its subcellular localization and expression. It plays a critical role in the nucleus and cytoplasm as DNA chaperone, chromosome gatekeeper, autophagy maintainer, and protector from apoptotic cell death. HMGB1 also functions as an extracellular alarmin acting as a damage-associated molecular pattern molecule (DAMP). Recent findings describe HMGB1 as a sophisticated signal of danger, with a pleiotropic function, which is useful as a clinical biomarker for several disorders. HMGB1 has emerged as a mediator in acute and chronic inflammation. Furthermore, HMGB1 targeting can induce beneficial effects on oxidative stress related diseases. This review focus on HMGB1 redox status, localization, mechanisms of release, binding with receptors, and its activities in different oxidative stress-related chronic diseases. Since a growing number of reports show the key role of HMGB1 in socially relevant pathological conditions, to our knowledge, for the first time, here we analyze the scientific literature, evaluating the number of publications focusing on HMGB1 in humans and animal models, per year, from 2006 to 2021 and the number of records published, yearly, per disease and category (studies on humans and animal models).

Emerging noncoding RNAs contained in extracellular vesicles: rising stars as biomarkers in lung cancer liquid biopsy.

Lung cancer has a high morbidity and mortality rate, and affected patients have a poor prognosis and low survival. The therapeutic approaches for lung cancer treatment, including surgery, radiotherapy, and chemotherapy, are not completely effective, due to late diagnosis. Although the identification of genetic drivers has contributed to the improvement of lung cancer clinical management, the discovery of new diagnostic and prognostic tools remains a critical issue. Liquid biopsy (LB) represents a minimally invasive approach and practical alternative source to investigate tumor-derived alterations and to facilitate the selection of targeted therapies. LB allows for the testing of different analytes such as circulating tumor cells, extracellular vesicles (EVs), tumor-educated platelets, and cell-free nucleic acids including DNAs, RNAs, and noncoding RNAs (ncRNAs). Several regulatory factors control the key cellular oncogenic pathways involved in cancers. ncRNAs have a wide range of regulatory effects in lung cancers. This review focuses on emerging regulatory ncRNAs, freely circulating in body fluids or shuttled by EVs, such as circular-RNAs, small nucleolar-RNAs, small nuclear-RNAs, and piwi-RNAs, as new biomarkers for early detection, prognosis, and monitoring of therapeutic strategy of lung cancer treatment.

Mesoporous Silica Particles Functionalized with Newly Extracted Fish Oil (Omeg@Silica) Reducing IL-8 Counteract Cell Migration in NSCLC Cell Lines.

Lung cancer is one of the leading forms of cancer in developed countries. Interleukin-8 (IL-8), a pro-inflammatory cytokine, exerts relevant effects in cancer growth and progression, including angiogenesis and metastasis in lung cancer. Mesoporous silica particles, functionalized with newly extracted fish oil (Omeg@Silica), are more effective than the fish oil alone in anti-proliferative and pro-apoptotic effects in non-small cell lung cancer (NSCLC) cell lines. The mechanisms that explain this efficacy are not yet understood. The aim of the present study is therefore to decipher the anti-cancer effects of a formulation of Omeg@Silica in aqueous ethanol (FOS) in adenocarcinoma (A549) and muco-epidermoid (NCI-H292) lung cancer cells, evaluating cell migration, as well as IL-8, NF-κB, and miRNA-21 expression. Results show that in both cell lines, FOS was more efficient than oil alone, in decreasing cell migration and IL-8 gene expression. FOS reduced IL-8 protein release in both cell lines, but this effect was only stronger than the oil alone in A549. In A549, FOS was able to reduce miRNA-21 and transcription factor NF-κB nuclear expression. Taken together, these data support the potential use of the Omeg@Silica as an add-on therapy for NSCLC. Dedicated studies which prove clinical efficacy are needed.

miR-126-3p and miR-21-5p as Hallmarks of Bio-Positive Ageing; Correlation Analysis and Machine Learning Prediction in Young to Ultra-Centenarian Sicilian Population.

Human ageing can be characterized by a profile of circulating microRNAs (miRNAs), which are potentially predictors of biological age. They can be used as a biomarker of risk for age-related inflammatory outcomes, and senescent endothelial cells (ECs) have emerged as a possible source of circulating miRNAs. In this paper, a panel of four circulating miRNAs including miR-146a-5p, miR-126-3p, miR-21-5p, and miR-181a-5p, involved in several pathways related to inflammation, and ECs senescence that seem to be characteristic of the healthy ageing phenotype. The circulating levels of these miRNAs were determined in 78 healthy subjects aged between 22 to 111 years. Contextually, extracellular miR-146a-5p, miR-126-3p, miR-21-5p, and miR-181a-5p levels were measured in human ECs in vitro model, undergoing senescence. We found that the levels of the four miRNAs, using ex vivo and in vitro models, progressively increase with age, apart from ultra-centenarians that showed levels comparable to those measured in young individuals. Our results contribute to the development of knowledge regarding the identification of miRNAs as biomarkers of successful and unsuccessful ageing. Indeed, they might have diagnostic/prognostic relevance for age-related diseases.

Can Be miR-126-3p a Biomarker of Premature Aging? An Ex Vivo and In Vitro Study in Fabry Disease.

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by lysosomal accumulation of glycosphingolipids in a wide variety of cytotypes, including endothelial cells (ECs). FD patients experience a significantly reduced life expectancy compared to the general population; therefore, the association with a premature aging process would be plausible. To assess this hypothesis, miR-126-3p, a senescence-associated microRNA (SA-miRNAs), was considered as an aging biomarker. The levels of miR-126-3p contained in small extracellular vesicles (sEVs), with about 130 nm of diameter, were measured in FD patients and healthy subjects divided into age classes, in vitro, in human umbilical vein endothelial cells (HUVECs) "young" and undergoing replicative senescence, through a quantitative polymerase chain reaction (qPCR) approach. We confirmed that, in vivo, circulating miR-126 levels physiologically increase with age. In vitro, miR-126 augments in HUVECs underwent replicative senescence. We observed that FD patients are characterized by higher miR-126-3p levels in sEVs, compared to age-matched healthy subjects. We also explored, in vitro, the effect on ECs of glycosphingolipids that are typically accumulated in FD patients. We observed that FD storage substances induced in HUVECs premature senescence and increased of miR-126-3p levels. This study reinforces the hypothesis that FD may aggravate the normal aging process.

Membrane vesicles containing matrix metalloproteinase-9 and fibroblast growth factor-2 are released into the extracellular space from mouse mesoangioblast stem cells.

Certain proteins, including fibroblast growth factor-2 (FGF-2) and matrix metalloproteinase-9 (MMP-9), have proved very effective in increasing the efficacy of mesoangioblast stem cell therapy in repairing damaged tissue. We provide the first evidence that mouse mesoangioblast stem cells release FGF-2 and MMP-9 in their active form through the production of membrane vesicles. These vesicles are produced and turned over continuously, but are stable for some time in the extracellular milieu. Mesoangioblasts shed membrane vesicles even under oxygen tensions that are lower than those typically used for cell culture and more like those of mouse tissues. These findings suggest that mesoangioblasts may themselves secrete paracrine signals and factors that make damaged tissues more amenable to cell therapy through the release of membrane vesicles.