Biphasic α2β1 Integrin Expression in Breast Cancer Metastasis to Bone

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.     

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8β1 and α11β1, Induced Specifically on Activated Stellate Cells

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvβ1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFβ activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, “disease specificity” has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbβ3 on platelets, α4β1, α4β7 and αLβ2 on leukocytes. Herein, “disease specific” integrins would serve as attractive targets. α8β1 and α11β1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather “pathology specific” nature of these new integrins. The monoclonal antibodies against α8β1 and α11β1 in preclinical examinations may illuminate the road to the first medical agents.     

The Influence of Interaction between Cadmium with 17β-Estradiol, 2-Methoxyestradiol and 16α-Hydroxyestrone on Viability and p-Glycoprotein in Ovarian Cancer Cell Line

Occupational and environmental exposure to xenoestrogens, a subgroup of endocrine disruptors (EDCs), can affect the endocrine system and increase the risk of cancer, primarily the hormone-dependent kind. This type of cancer includes ovarian cancer, which is the leading cause of death from gynecological tumors. The aim of this study was to assess the role of 17β-estradiol and its metabolites: 2-MeOE2, 16α-OHE1 in exposure to the metalloestrogen cadmium. The effect of interactions of cadmium with estrogens on the viability of cells in malignant ovarian cancer cells SKOV-3 was investigated, both in simultaneous action and in the pre-incubation model. There are no known interactions between estrogens and cadmium in ovarian cancer cells. Due to the frequent occurrence of multidrug resistance (MDR) in ovarian cancer, the effects of estrogens and cadmium on MDR in SKOV-3, measured as P-glycoprotein (P-gp), were assessed. An interaction study showed that E2 had an antagonistic effect on cadmium-induced cell damage, while 2-MeOE2 showed less of a protective effect in combination with CdCl₂ than E2. There were two types of interaction: toxic synergism and beneficial antagonism. E2 and cadmium increased P-gp expression in SKOV-3 cells, while 2-MeOE2 decreased P-gp expression to a potentially beneficial effect on MDR prevention. The obtained results constitute an interesting starting point for further research in the field of interactions between estrogens and xenoestrogens in ovarian cancer.     

Eugenol β-Amino/β-Alkoxy Alcohols with Selective Anticancer Activity

Eugenol, 4-allyl-2-methoxyphenol, is the main constituent of clove essential oil and has demonstrated relevant biological activity, namely anticancer activity. Aiming to increase this activity, we synthesized a series of eugenol β-amino alcohol and β-alkoxy alcohol derivatives, which were then tested against two human cancer cell lines, namely gastric adenocarcinoma cells (AGS) and lung adenocarcinoma cells (A549). An initial screening was performed to identify the most cytotoxic compounds. The results demonstrated that three β-amino alcohol derivatives had anticancer activity that justified subsequent studies, having been shown to trigger apoptosis. Importantly, the most potent molecules displayed no appreciable toxicity towards human noncancer cells. Structure-activity relationships show that changes in eugenol structure led to enhanced cytotoxic activity and can contribute to the future design of more potent and selective drugs.     

Synthesis of Oxidized 3β,3′β-Disteryl Ethers and Search after High-Temperature Treatment of Sterol-Rich Samples

It was proven that sterols subjected to high-temperature treatment can be concatenated, which results in polymeric structures, e.g., 3β,3′β-disteryl ethers. However, it was also proven that due to increased temperature in oxygen-containing conditions, sterols can undergo various oxidation reactions. This study aimed to prove the existence and perform quantitative analysis of oxidized 3β,3′β-disteryl ethers, which could form during high-temperature treatment of sterol-rich samples. Samples were heated at 180, 200 and 220 °C for 0.5 to 4 h. Quantitative analyses of the oxidized 3β,3′β-disteryl ethers were performed with liquid extraction, solid-phase extraction and liquid chromatography coupled with mass spectrometry. Additionally, to perform this analysis, the appropriate standards of all oxidized 3β,3′β-disteryl ethers were prepared. Eighteen various oxidized 3β,3′β-disteryl ethers (derivatives of 3β,3′β-dicholesteryl ether, 3β,3′β-disitosteryl ether and 3β,3′β-distigmasteryl ether) were prepared. Additionally, the influence of metal compounds on the mechanism of ether formation at high temperatures was investigated.     

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee β-cell Function and Protection

Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic β-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to β-cells improves whole-body glucose homeostasis, enhances β-cell function, and surprisingly, protection of β-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of β-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.     

Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.     

Neuronal ApoE Regulates the Cell-to-Cell Transmission of α-Synuclein

The presence of protein inclusions, called Lewy bodies (LBs) and Lewy neurites (LNs), in the brain is the main feature of Parkinson’s disease (PD). Recent evidence that the prion-like propagation of α-synuclein (α-syn), as a major component of LBs and LNs, plays an important role in the progression of PD has gained much attention, although the molecular mechanism remains unclear. In this study, we evaluated whether neuronal ApoE regulates the cell-to-cell transmission of α-syn and explored its molecular mechanism using in vitro and in vivo model systems. We demonstrate that neuronal ApoE deficiency attenuates both α-syn uptake and release by downregulating LRP-1 and LDLR expression and enhancing chaperone-mediated autophagy activity, respectively, thereby contributing to α-syn propagation. In addition, we observed that α-syn propagation was attenuated in ApoE knockout mice injected with pre-formed mouse α-syn fibrils. This study will help our understanding of the molecular mechanisms underlying α-syn propagation.     

Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

The tumor microenvironment plays a critical role in defining the growth and malignancy of solid tumors. Extracellular matrix (ECM) proteins such as collagen, vitronectin, and fibronectin are major components of the tumor microenvironment. Tumor growth-promoting reciprocal interaction between ECM and cytoplasmic proteins is regulated by the cell surface receptors called integrins. This study investigated the mechanism by which integrin β1 promotes pancreatic tumor growth. In MIA PaCa-2 pancreatic cancer cell line, the loss of integrin β1 protein reduced the ability of cells to proliferate in a 3D matrix and compromised the ability to form a focal adhesion complex. Decreased expression of integrin α5 was observed in KO cells, which resulted in impaired cell spreading and adhesion on vitronectin and fibronectin. Reduced expression of the integrin-associated protein, kindlin-2 was also recorded. The downregulation of kindlin-2 decreased the phosphorylation of Smad2/3 by reducing the expression of TGF-β receptor 2. These results unravel a new mechanism of integrin β1 in tumor growth by modifying the expression of kindlin-2 and TGF-β receptor 2 signaling.     

Cellular Integrin α5β1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size−exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5β1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.     

Quantitative Analysis of the Cardiac Phosphoproteome in Response to Acute β-Adrenergic Receptor Stimulation In Vivo

β-adrenergic receptor (β-AR) stimulation represents a major mechanism of modulating cardiac output. In spite of its fundamental importance, its molecular basis on the level of cell signalling has not been characterised in detail yet. We employed mass spectrometry-based proteome and phosphoproteome analysis using SuperSILAC (spike-in stable isotope labelling by amino acids in cell culture) standardization to generate a comprehensive map of acute phosphoproteome changes in mice upon administration of isoprenaline (ISO), a synthetic β-AR agonist that targets both β1-AR and β2-AR subtypes. Our data describe 8597 quantitated phosphopeptides corresponding to 10,164 known and novel phospho-events from 2975 proteins. In total, 197 of these phospho-events showed significantly altered phosphorylation, indicating an intricate signalling network activated in response to β-AR stimulation. In addition, we unexpectedly detected significant cardiac expression and ISO-induced fragmentation of junctophilin-1, a junctophilin isoform hitherto only thought to be expressed in skeletal muscle. Data are available via ProteomeXchange with identifier PXD025569.     

The Landscape of microRNAs in βCell: Between Phenotype Maintenance and Protection

Diabetes mellitus is a group of heterogeneous metabolic disorders characterized by chronic hyperglycaemia mainly due to pancreatic β cell death and/or dysfunction, caused by several types of stress such as glucotoxicity, lipotoxicity and inflammation. Different patho-physiological mechanisms driving β cell response to these stresses are tightly regulated by microRNAs (miRNAs), a class of negative regulators of gene expression, involved in pathogenic mechanisms occurring in diabetes and in its complications. In this review, we aim to shed light on the most important miRNAs regulating the maintenance and the robustness of β cell identity, as well as on those miRNAs involved in the pathogenesis of the two main forms of diabetes mellitus, i.e., type 1 and type 2 diabetes. Additionally, we acknowledge that the understanding of miRNAs-regulated molecular mechanisms is fundamental in order to develop specific and effective strategies based on miRNAs as therapeutic targets, employing innovative molecules.     

Cell-Free DNA Fragments as Biomarkers of Islet β-Cell Death in Obesity and Type 2 Diabetes

Type 2 diabetes (T2D) typically occurs in the setting of obesity and insulin resistance, where hyperglycemia is associated with decreased pancreatic β-cell mass and function. Loss of β-cell mass has variably been attributed to β-cell dedifferentiation and/or death. In recent years, it has been proposed that circulating epigenetically modified DNA fragments arising from β cells might be able to report on the potential occurrence of β-cell death in diabetes. Here, we review published literature of DNA-based β-cell death biomarkers that have been evaluated in human cohorts of islet transplantation, type 1 diabetes, and obesity and type 2 diabetes. In addition, we provide new data on the applicability of one of these biomarkers (cell free unmethylated INS DNA) in adult cohorts across a spectrum from obesity to T2D, in which no significant differences were observed, and compare these findings to those previously published in youth cohorts where differences were observed. Our analysis of the literature and our own data suggest that β-cell death may occur in subsets of individuals with obesity and T2D, however a more sensitive method or refined study designs are needed to provide better alignment of sampling with disease progression events.     

The PPARβ/δ-AMPK Connection in the Treatment of Insulin Resistance

The current treatment options for type 2 diabetes mellitus do not adequately control the disease in many patients. Consequently, there is a need for new drugs to prevent and treat type 2 diabetes mellitus. Among the new potential pharmacological strategies, activators of peroxisome proliferator-activated receptor (PPAR)β/δ show promise. Remarkably, most of the antidiabetic effects of PPARβ/δ agonists involve AMP-activated protein kinase (AMPK) activation. This review summarizes the recent mechanistic insights into the antidiabetic effects of the PPARβ/δ-AMPK pathway, including the upregulation of glucose uptake, muscle remodeling, enhanced fatty acid oxidation, and autophagy, as well as the inhibition of endoplasmic reticulum stress and inflammation. A better understanding of the mechanisms underlying the effects resulting from the PPARβ/δ-AMPK pathway may provide the basis for the development of new therapies in the prevention and treatment of insulin resistance and type 2 diabetes mellitus.     

Recombinant Reg3α Prevents Islet β-Cell Apoptosis and Promotes β-Cell Regeneration

Progressive loss and dysfunction of islet β-cells has not yet been solved in the treatment of diabetes. Regenerating protein (Reg) has been identified as a trophic factor which is demonstrated to be associated with pancreatic tissue regeneration. We previously produced recombinant Reg3α protein (rReg3α) and proved that it protects against acute pancreatitis in mice. Whether rReg3α protects islet β-cells in diabetes has been elusive. In the present study, rReg3α stimulated MIN6 cell proliferation and resisted STZ-caused cell death. The protective effect of rReg3α was also found in mouse primary islets. In BALB/c mice, rReg3α administration largely alleviated STZ-induced diabetes by the preservation of β-cell mass. The protective mechanism could be attributed to Akt/Bcl-2/-xL activation and GRP78 upregulation. Scattered insulin-expressing cells and clusters with small size, low insulin density, and exocrine distribution were observed and considered to be neogenic. In isolated acinar cells with wheat germ agglutinin (WGA) labeling, rReg3α treatment generated insulin-producing cells through Stat3/Ngn3 signaling, but these cells were not fully functional in response to glucose stimulation. Our results demonstrated that rReg3α resists STZ-induced β-cell death and promotes β-cell regeneration. rReg3α could serve as a potential drug for β-cell maintenance in anti-diabetic treatment.     

Pathomechanisms of Immunological Disturbances in β-Thalassemia

Thalassemia, a chronic disease with chronic anemia, is caused by mutations in the β-globin gene, leading to reduced levels or complete deficiency of β-globin chain synthesis. Patients with β-thalassemia display variable clinical severity which ranges from asymptomatic features to severe transfusion-dependent anemia and complications in multiple organs. They not only are at increased risk of blood-borne infections resulting from multiple transfusions, but they also show enhanced susceptibility to infections as a consequence of coexistent immune deficiency. Enhanced susceptibility to infections in β-thalassemia patients is associated with the interplay of several complex biological processes. β-thalassemia-related abnormalities of the innate immune system include decreased levels of complement, properdin, and lysozyme, reduced absorption and phagocytic ability of polymorphonuclear neutrophils, disturbed chemotaxis, and altered intracellular metabolism processes. According to available literature data, immunological abnormalities observed in patients with thalassemia can be caused by both the disease itself as well as therapies. The most important factors promoting such alterations involve iron overload, phenotypical and functional abnormalities of immune system cells resulting from chronic inflammation oxidative stress, multiple blood transfusion, iron chelation therapy, and splenectomy. Unravelling the mechanisms underlying immune deficiency in β-thalassemia patients may enable the designing of appropriate therapies for this group of patients.     

Skin Resident γδ T Cell Function and Regulation in Wound Repair

The skin is a critical barrier that protects against damage and infection. Within the epidermis and dermis reside γδ T cells that play a variety of key roles in wound healing and tissue homeostasis. Skin-resident γδ T cells require T cell receptor (TCR) ligation, costimulation, and cytokine reception to mediate keratinocyte activity and inflammatory responses at the wound site for proper wound repair. While both epidermal and dermal γδ T cells regulate inflammatory responses in wound healing, the timing and factors produced are distinct. In the absence of growth factors, cytokines, and chemokines produced by γδ T cells, wound repair is negatively impacted. This disruption in γδ T cell function is apparent in metabolic diseases such as obesity and type 2 diabetes. This review provides the current state of knowledge on skin γδ T cell activation, regulation, and function in skin homeostasis and repair in mice and humans. As we uncover more about the complex roles played by γδ T cells in wound healing, novel targets can be discovered for future clinical therapies.     

β-Catenin-Specific Inhibitor,iCRT14, Promotes BoHV-1 Infection-Induced DNA Damage in Human A549 Lung Adenocarcinoma Cells by Enhancing Viral Protein Expression

Oncolytic bovine herpesvirus type 1 (BoHV-1) infection induces DNA damage in human lung adenocarcinoma cell line A549. However, the underlying mechanisms are not fully understood. We found that BoHV-1 infection decreased the steady-state protein levels of p53-binding protein 1 (53BP1), which plays a central role in dictating DNA damage repair and maintaining genomic stability. Furthermore, BoHV-1 impaired the formation of 53BP1 foci, suggesting that BoHV-1 inhibits 53BP1-mediated DNA damage repair. Interestingly, BoHV-1 infection redistributed intracellular β-catenin, and iCRT14 (5-[[2,5-Dimethyl-1-(3-pyridinyl)-1H-pyrrol-3-yl]methylene]-3-phenyl-2,4-thiazolidinedione), a β-catenin-specific inhibitor, enhanced certain viral protein expression, such as the envelope glycoproteins gC and gD, and enhanced virus infection-induced DNA damage. Therefore, for the first time, we provide evidence showing that BoHV-1 infection disrupts 53BP1-mediated DNA damage repair and suggest β-catenin as a potential host factor restricting both virus replication and DNA damage in A549 cells.