The benzoylphenylurea derivative BPU17 acts as an inhibitor of prohibitin and exhibits antifibrotic activity.

Inflammation-induced choroidal neovascularization followed by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPEs) is a cause of neovascular age-related macular degeneration (nAMD). RPE-derived myofibroblasts overproduce extracellular matrix, leading to subretinal fibrosis. We already have demonstrated that benzylphenylurea (BPU) derivatives inhibit the function of cancer-associated fibroblasts. Here, we investigated the anti-myofibroblast effects of BPU derivatives and examined such BPU activity on subretinal fibrosis. A BPU derivative, BPU17, exhibits the most potent anti-myofibroblast activity among dozens of BPU derivatives and inhibits subretinal fibrosis in a mouse model of retinal degeneration. Investigations with primary cultured RPEs reveal that BPU17 suppresses cell motility and collagen synthesis in RPE-derived myofibroblasts. These effects depend on repressing the serum response factor (SRF)/CArG-box-dependent transcription. BPU17 inhibits the expression of SRF cofactor, cysteine and glycine-rich protein 2 (CRP2), which activates the SRF function. Proteomics analysis reveals that BPU17 binds to prohibitin 1 (PHB1) and inhibits the PHB1-PHB2 interaction, resulting in mild defects in mitochondrial function. This impairment causes a decrease in the expression of CRP2 and suppresses collagen synthesis. Our findings suggest that BPU17 is a promising agent against nAMD and the close relationship between PHB function and EMT.

Curcumin Modulates the Membrane Raft Integrity via Phase Separation and Induces CD44 Shedding in Tumor Cells.

CD44 is a transmembrane cell adhesion molecule that is cleaved by the membrane proteinase, a disintegrin and metalloproteinase 10 (ADAM10), on the cell surface via ectodomain shedding after cholesterol depletion. Lipid raft-mediated CD44 shedding is essential for cancer cell invasion. As cell-cell and cell-matrix adhesions are critical for cancer progression, lipid raft-targeting agents may be effective for cancer therapy. Here, we found that curcumin and its derivatives induced the ADAM10-mediated shedding of CD44 in tumor cells. We also found that curcumin and the derivatives are membrane-active compounds whose effect depends on its planar backbone and the spatial arrangement of methoxy groups substituted on the two aromatic rings using giant unilamellar and plasma membrane vesicles. Curcumin and its derivatives with rigid backbones and hydroxy groups exerted membrane-domain-modulating activity, which may account for their pleiotropic effects via multiple signaling pathways involving membrane receptors. This study provides a basis for the use of membrane-active compounds, such as curcuminoids, to elucidate the roles of lipid rafts in cellular signaling, regulation of membrane-bound ADAM metalloproteinases, and the development of novel membrane lipid-based therapies.

CaMK phosphatase (CaMKP/POPX2/PPM1F) inhibitors suppress the migration of human breast cancer MDA-MB-231 cells with loss of polarized morphology.

CaMK phosphatase (CaMKP/POPX2/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. Accumulating evidence suggests that CaMKP is involved in the pathogenesis of various diseases, including cancer. To clarify the relationship between CaMKP activity and human breast cancer cell motility, we examined the phosphatase activity of CaMKP in cell extracts. CaMKP activity assays of the immunoprecipitates prepared from the cell extract revealed that cells exhibiting higher motility had higher CaMKP activity, with no significant differences in the specific activity being observed. Two CaMKP-specific inhibitors, 1-amino-8-naphthol-4-sulfonic acid (ANS) and 1-amino-8-naphthol-2,4-disulfonic acid (ANDS), inhibited the migration of highly invasive MDA-MB-231 breast cancer cells without significant cytotoxicity, while an inactive analog, naphthionic acid, did not. Furthermore, the cells lost their elongated morphology and assumed a rounded shape following treatment with ANS, whereas they retained their elongated morphology following treatment with naphthionic acid. Consistent with these findings, ANS and ANDS significantly enhanced the phosphorylation level of CaMKI, a cellular substrate of CaMKP, while naphthionic acid did not. The present data suggest that CaMKP could be a novel therapeutic target for cancer metastasis.

The Chemopreventive Effects of Chlorogenic Acids, Phenolic Compounds in Coffee, against Inflammation, Cancer, and Neurological Diseases.

Coffee is one of the most widely consumed beverages, which has several effects on the human body. In particular, current evidence suggests that coffee consumption is associated with a reduced risk of inflammation, various types of cancers, and certain neurodegenerative diseases. Among the various constituents of coffee, phenolic phytochemicals, more specifically chlorogenic acids, are the most abundant, and there have been many attempts to utilize coffee chlorogenic acid for cancer prevention and therapy. Due to its beneficial biological effect on the human body, coffee is regarded as a functional food. In this review article, we summarize the recent advances and knowledge on the association of phytochemicals contained in coffee as nutraceuticals, with a particular focus on phenolic compounds, their intake, and nutritional biomarkers, with the reduction of disease risk, including inflammation, cancer, and neurological diseases.

Membrane cholesterol modulates the hyaluronan-binding ability of CD44 in T lymphocytes and controls rolling under shear flow.

The adhesion of circulating lymphocytes to the surface of vascular endothelial cells is important for their recruitment from blood to secondary lymphoid organs and to inflammatory sites. CD44 is a key adhesion molecule for this interaction and its ligand-binding ability is tightly regulated. Here we show that the hyaluronan-binding ability of CD44 in T cells is upregulated by the depletion of membrane cholesterol with methyl-ß-cyclodextrin (MßCD), which disintegrates lipid rafts, i.e. cholesterol- and sphingolipid-enriched membrane microdomains. Increasing concentrations of MßCD led to a dose-dependent decrease in cellular cholesterol content and to upregulation of hyaluronan binding. Additionally, a cholesterol-binding agent filipin also increased hyaluronan binding. Cholesterol depletion caused CD44 to be dispersed from cholesterol-enriched membrane microdomains. Cholesterol depletion also increased the number of cells undergoing rolling adhesion under physiological flow conditions. Our results suggest that the ligand-binding ability of CD44 is governed by its cholesterol-dependent allocation to membrane microdomains at the cell surface. These findings provide novel insight into the regulation of T cell adhesion under blood flow.

Cholesterol lowering: role in cancer prevention and treatment.

The accumulation of cholesterol is a general feature of cancer tissue, and recent evidence suggests that cholesterol plays critical roles in the progression of cancers, including breast, prostate, and colorectal cancers. The dysregulation of metabolic pathways, including those involved in cholesterol biosynthesis, is implicated in tumor development and cancer progression. Lipid rafts are highly dynamic cholesterol-enriched domains of the cell membrane, involved in various cellular functions, including the regulation of transmembrane signaling at the cell surface. It was recently demonstrated that lipid rafts also play critical roles in cancer cell adhesion and migration. This review focuses on our current understanding of how cholesterol regulation, lipid rafts, and dysregulated cholesterol biosynthesis contribute to cancer development and progression, and the therapeutic potential of cholesterol lowering for cancer prevention and treatment.

Transmembrane signaling through single-spanning receptors modulated by phase separation at the cell surface.

A wide variety of transmembrane signals are transduced by cell-surface receptors that activate intracellular signaling molecules. In particular, receptor clustering in the plasma membrane plays a critical role in these processes. Single-spanning or single-pass transmembrane proteins are among the most significant types of membrane receptors, which include adhesion receptors, such as integrins, CD44, cadherins, and receptor tyrosine kinases. Elucidating the molecular mechanisms underlying the regulation of the activity of these receptors is of great significance. Liquid-liquid phase separation (LLPS) is a recently emerging paradigm in cellular physiology for the ubiquitous regulation of the spatiotemporal dynamics of various signaling pathways. This study describes the emerging features of transmembrane signaling through single-spanning receptors from the perspective of phase separation. Possible physicochemical modulations of LLPS-based transmembrane signaling are also discussed.

Ultrastructural analysis of nanogold-labeled cell surface microvilli in liquid by atmospheric scanning electron microscopy and their relevance in cell adhesion.

The adhesion of leukocytes circulating in the blood to vascular endothelium is critical for their trafficking in the vasculature, and CD44 is an important cell surface receptor for rolling adhesion. In this study, we demonstrate the correlative observation of CD44 distribution at the lymphocyte cell surface in liquid by fluorescence optical microscopy and immuno-electron microscopy using an atmospheric scanning electron microscope (ASEM). The ultrastructure of the cell surface was clearly imaged by ASEM using positively charged Nanogold particles. ASEM analysis demonstrated microvilli projections around the cell surface and the localization of CD44 on the microvilli. Treatment of cells with cytochalasin D resulted in a loss of the microvilli projections and concomitantly abrogated CD44-mediated adhesion to its ligand hyaluronan. These results suggest the functional relevance of microvilli in CD44-mediated rolling adhesion under shear flow.

Integrated Multimodal Omics and Dietary Approaches for the Management of Neurodegeneration.

Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are caused by a combination of multiple events that damage neuronal function. A well-characterized biomarker of neurodegeneration is the accumulation of proteinaceous aggregates in the brain. However, the gradually worsening symptoms of neurodegenerative diseases are unlikely to be solely due to the result of a mutation in a single gene, but rather a multi-step process involving epigenetic changes. Recently, it has been suggested that a fraction of epigenetic alternations may be correlated to neurodegeneration in the brain. Unlike DNA mutations, epigenetic alterations are reversible, and therefore raise the possibilities for therapeutic intervention, including dietary modifications. Additionally, reactive oxygen species may contribute to the pathogenesis of Alzheimer's disease and Parkinson's disease through epigenetic alternation. Given that the antioxidant properties of plant-derived phytochemicals are likely to exhibit pleiotropic effects against ROS-mediated epigenetic alternation, dietary intervention may be promising for the management of neurodegeneration in these diseases. In this review, the state-of-the-art applications using single-cell multimodal omics approaches, including epigenetics, and dietary approaches for the identification of novel biomarkers and therapeutic approaches for the treatment of neurodegenerative diseases are discussed.

Metabolic Reprogramming toward Aerobic Glycolysis and the Gut Microbiota Involved in the Brain Amyloid Pathology.

Alzheimer's disease (AD) is characterized by the formation of senile plaques consisting of fibrillated amyloid-ß (Aß), dystrophic neurites, and the neurofibrillary tangles of tau. The oligomers/fibrillar Aß damages the neurons or initiates an intracellular signaling cascade for neuronal cell death leading to Aß toxicity. The Aß is a 4 kDa molecular weight peptide originating from the C-terminal region of the amyloid precursor protein via proteolytic cleavage. Apart from the typical AD hallmarks, certain deficits in metabolic alterations have been identified. This study describes the emerging features of AD from the aspect of metabolic reprogramming in the main pathway of carbohydrate metabolism in the human brain. Particularly, the neurons in patients with AD favor glycolysis despite a normal mitochondrial function indicating a Warburg-like effect. In addition, certain dietary patterns are well known for their properties in preventing AD. Among those, a ketogenic diet may substantially improve the symptoms of AD. An effective therapeutic method for the treatment, mitigation, and prevention of AD has not yet been established. Therefore, the researchers pursue the development and establishment of novel therapies effective in suppressing AD symptoms and the elucidation of their underlying protective mechanisms against neurodegeneration aiming for AD therapy in the near future.

Therapeutic Implications of Probiotics in the Gut Microbe-Modulated Neuroinflammation and Progression of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the accumulation of specific proteins in the brain. A recent study revealed that manipulating gut microbiota (GM) significantly reduced tau pathology and neurodegeneration in an apolipoprotein E isoform-dependent manner. The resilience of a healthy microbiota protects it from a variety of dysbiosis-related pathologies. Convincing evidence has demonstrated the roles of GM in the pathogenesis of AD, which are partly mediated by modified microglial activity in the brain. Therefore, modulation of GM may be a promising therapeutic option for AD prevention. In addition to providing the cells with energy and affecting microglial maturation, these microbial metabolites appear to influence neuronal function. One of the potential therapeutic approaches targeting GM may involve using probiotics. Additionally, human GM and its metabolites have also become potential therapeutic targets for developing interventions for the prevention of disorders. Synbiotics and postbiotics can also be used to treat AD by modulating GM. In addition, physical activity, exercise, and physical fitness are being considered as potential nonpharmacological therapies to reduce signaling pathways related to neuroinflammation. Therefore, interventions targeting GM might be promising strategies for health promotion.

Pleiotropic Signaling by Reactive Oxygen Species Concerted with Dietary Phytochemicals and Microbial-Derived Metabolites as Potent Therapeutic Regulators of the Tumor Microenvironment.

The excessive generation of reactive oxygen species (ROS) plays a pivotal role in the pathogenesis of diseases. ROS are central to cellular redox regulation and act as second messengers to activate redox-sensitive signals. Recent studies have revealed that certain sources of ROS can be beneficial or harmful to human health. Considering the essential and pleiotropic roles of ROS in basic physiological functions, future therapeutics should be designed to modulate the redox state. Dietary phytochemicals, microbiota, and metabolites derived from them can be expected to be developed as drugs to prevent or treat disorders in the tumor microenvironment.

Fatty Acid Metabolites and the Tumor Microenvironment as Potent Regulators of Cancer Stem Cell Signaling.

Individual cancer cells are not equal but are organized into a cellular hierarchy in which only a rare few leukemia cells can self-renew in a manner reminiscent of the characteristic stem cell properties. The PI3K/AKT pathway functions in a variety of cancers and plays a critical role in the survival and proliferation of healthy cells under physiologic conditions. In addition, cancer stem cells might exhibit a variety of metabolic reprogramming phenotypes that cannot be completely attributed to the intrinsic heterogeneity of cancer. Given the heterogeneity of cancer stem cells, new strategies with single-cell resolution will become a powerful tool to eradicate the aggressive cell population harboring cancer stem cell phenotypes. Here, this article will provide an overview of the most important signaling pathways of cancer stem cells regarding their relevance to the tumor microenvironment and fatty acid metabolism, suggesting valuable strategies among cancer immunotherapies to inhibit the recurrence of tumors.

Targeting the PI3K-Akt-mTOR signaling pathway involved in vasculogenic mimicry promoted by cancer stem cells.

An accumulating body of evidence has led to the development of the cancer stem-cell (CSC) model which proposed that a subset of cells distinct from those that form the tumor mass regulated the tumor growth rate over a long period. Various types of therapy have been developed for cancer treatment. The major conventional therapies are chemotherapy, radiation therapy, and surgical excision. The other emerging therapies include targeted therapy using molecule-based agents. However, the resistance to chemotherapy and radiation therapy frequently occurs. This was most likely due to the dysregulated functioning of the multidrug efflux pumps and nucleotide repair systems resulting from the multiple interactions between the CSCs and the tumor microenvironment. Even though chimeric antigen receptor T-cell and immune checkpoint blockade therapies have succeeded remarkably for treating cancers, evidence suggested that CSCs promoted the development of resistance to these therapies and led to metastasis. The cells with stem cell-like features actively participate in vasculogenic mimicry in different types of cancer. In addition to melanoma, vasculogenic mimicry has been observed in various cancers. One of the major signaling pathways in CSCs is the phosphoinositide 3-kinase (PI3K)/Akt/PTEN pathway. PI3Ks are a family of enzymes that play a critical role in cellular growth, migration, differentiation, and vasculogenic mimicry. The PI3K-Akt pathway also plays a crucial role in epithelial-mesenchymal transition and the establishment of CSC-specific phenotypes through the PTEN/Akt mechanistic target of the rapamycin axis. Thus, targeting the PI3K pathway could be beneficial for cancer treatment through the elimination of CSCs, and such therapy might break niches which maintain the CSC, inhibit the metastasis, and suppress the recurrence of cancer.

Low cholesterol triggers membrane microdomain-dependent CD44 shedding and suppresses tumor cell migration.

CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-ß-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.

Binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH.

Ligands for L-selectin, a leukocyte adhesion molecule, are expressed in high endothelial venules (HEVs) in lymph nodes and extravascular tissues, such as renal tubules. Here, we report that the binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH. The optimal L-selectin-dependent binding of leukocytes to HEVs was observed at pH 7.4, a physiological pH in the blood. In contrast, the optimal binding of leukocytes to the renal tubules was observed at pH 5.6. Consistently, optimal binding of soluble recombinant L-selectin to a major vascular ligand, 6-sulfo sialyl Lewis X, was observed at pH 7.4. Binding to extravascular ligands, such as chondroitin sulfate (CS) B, CS E and heparan sulfate, occurred at pH 5.6. Under physiological shear stress ranging from 1 to 2 dynes/cm(2), maximal leukocyte rolling on vascular ligands was observed at pH 6.8 to 7.4, and no rolling was detected at pH conditions below 5.6. These findings suggest that the pH environment is one important factor that determines leukocyte trafficking under physiological and pathological conditions.

Elevation of rat plasma P-selectin in acute lung injury.

Acute lung injury in the rat caused by intravenous (i.v.) infusion of cobra venom factor (CVF) or lipopolysaccharide (LPS) is mediated by P-selectin-dependent neutrophil infiltration into the lung. In these lung injury models, P-selectin expression is induced on lung vascular endothelial cells after the CVF or LPS infusion, suggesting soluble P-selectin derived from inflamed sites might also be elevated. Here we established a sensitive enzyme-linked immunosorbent assay (ELISA) to measure soluble P-selectin in plasma, a potential marker of lung injury. Nine anti-rat P-selectin monoclonal antibodies that we established previously were first classified into 5 groups based on real-time biospecific interaction analyses, and used to develop a sandwich ELISA for accurately measuring the amount of soluble P-selectin in plasma. We then used this ELISA to measure the plasma P-selectin levels in Long Evans, Wistar, and Sprague-Dawley rats after the i.v. infusion of CVF or LPS. The elevation in P-selectin levels was significantly different among the strains, but it consistently correlated with the extent of lung inflammation, measured by myeloperoxidase levels in the lung tissues. Thus, our results indicate that the soluble P-selectin in plasma could serve as a sensitive biomarker reflecting lung inflammation, which is of clinical importance for detecting and preventing severe lung injury.

A simple assay for hyaluronidase activity using fluorescence polarization.

Hyaluronan is a large glycosaminoglycan and is a major constituent of the extracellular matrix, interacting with cell surface receptors such as CD44. We previously reported that fragmented hyaluronan, with the size frequently detected in cancer patients, induces CD44 cleavage and concomitantly enhances tumor cell migration. Although hyaluronan degradation to smaller fragments has been revealed to be a key reaction in regulating cancer progression, simple methods for continuously detecting hyaluronidase activity have not been established. Here, we show that fluorescently-labeled hyaluronan serves as a substrate for continuous assay of hyaluronidase activity. A very simple assay was established to measure degradation of hyaluronan based on fluorescence polarization. The developed assay method would provide a way for continuous measurement of cellular hyaluronidase activity and also for measurement of binding of hyaluronan to its receptors, and thus should be useful for investigation of the function of hyaluronan in cancer progression.

Implications of PI3K/AKT/PTEN Signaling on Superoxide Dismutases Expression and in the Pathogenesis of Alzheimer's Disease.

Alzheimer’s disease is a neurodegenerative sickness, where the speed of personal disease progression differs prominently due to genetic and environmental factors such as life style. Alzheimer’s disease is described by the construction of neuronal plaques and neurofibrillary tangles composed of phosphorylated tau protein. Mitochondrial dysfunction may be a noticeable feature of Alzheimer’s disease and increased production of reactive oxygen species has long been described. Superoxide dismutases (SODs) protect from excess reactive oxygen species to form less reactive hydrogen peroxide. It is suggested that SODs can play a protective role in neurodegeneration. In addition, PI3K/AKT pathway has been shown to play a critical role on the neuroprotection and inhibiting apoptosis via the enhancing expression of the SODs. This pathway appears to be crucial in Alzheimer’s disease because it is related to the tau protein hyper-phosphorylation. Dietary supplementation of several ordinary compounds may provide a novel therapeutic approach to brain disorders by modulating the function of the PI3K/AKT pathway. Understanding these systems may offer a better efficacy of new therapeutic approaches. In this review, we summarize recent progresses on the involvement of the SODs and PI3K/AKT pathway in neuroprotective signaling against Alzheimer’s disease.

Reactive Oxygen Species, Superoxide Dimutases, and PTEN-p53-AKT-MDM2 Signaling Loop Network in Mesenchymal Stem/Stromal Cells Regulation.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells that can differentiate to various specialized cells, which have the potential capacity to differentiate properly and accelerate recovery in damaged sites of the body. This stem cell technology has become the fundamental element in regenerative medicine. As reactive oxygen species (ROS) have been reported to adversely influence stem cell properties, it is imperative to attenuate the extent of ROS to the promising protective approach with MSCs’ regenerative therapy. Oxidative stress also affects the culture expansion and longevity of MSCs. Therefore, there is great need to identify a method to prevent oxidative stress and replicative senescence in MSCs. Phosphatase and tensin homologue deleted on chromosome 10/Protein kinase B, PKB (PTEN/AKT) and the tumor suppressor p53 pathway have been proven to play a pivotal role in regulating cell apoptosis by regulating the oxidative stress and/or ROS quenching. In this review, we summarize the current research and our view of how PTEN/AKT and p53 with their partners transduce signals downstream, and what the implications are for MSCs’ biology.