Cellular physiological approach for treatment of gastric cancer

Recent studies show that ion channels/transporters play important roles in fundamental cellular functions that would be involved in the cancer process. We review the evidence for their expression and functioning in human gastric cancer (GC), and evaluate the potential of cellular physiological approach in clinical management. Various types of ion channels, such as voltage-gated K⁺ channels, intracellular Cl^- channels and transient receptor potential channels have been found to express in GC cells and tissues, and to control cell cycles. With regard to water channels, aquaporin 3 and 5 play an important role in the progression of GC. Regulators of intracellular pH, such as anion exchanger, sodium-hydrogen exchanger, vacuolar H⁺-ATPases and carbonic anhydrases are also involved in tumorigenesis of GC. Their pharmacological manipulation and gene silencing affect cellular behaviours, suggesting their potential as therapeutic targets for GC. Our studies indicate the intracellular Cl^- concentration could act as a mediator of cellular signaling and control cell cycle progression in GC cells. Further, we demonstrate the cytocidal effects of hypotonic shock on GC cells, and indicate that the blockade of Cl^- channels/transporters enhances these effects by inhibiting regulatory volume decrease. A deeper understanding of molecular mechanisms may lead to the discovery of these cellular physiological approaches as a novel therapeutic strategy for GC.     

To explore the Radix Paeoniae Rubra-Flos Carthami herb pair's potential mechanism in the treatment of ischemic stroke by network pharmacology and molecular docking technology

To explore the Radix Paeoniae Rubra-Flos Carthami herb pair''s (RPR-FC) potential mechanism in treating ischemic stroke (IS) by network pharmacology and molecular docking technology.The Traditional Chinese Medicine Systems Pharmacology Database was used to screen the active components of the RPR-FC, and Cytoscape 3.8 software was used to construct a network map of its active components and targets of action. The GeneCards and OMIM databases were used to identify disease targets of IS, and the common targets were chosen as research targets and imported into the STRING database to construct a protein–protein interaction network map of these targets. R language software was used to analyze the enrichment of GO terms and KEGG pathways, and explore the mechanisms of these targets. Molecular docking technology was used to verify that the RPR-FC components had a good bonding activity with their potential targets.A total of 44 active components, which corresponded to 197 targets, were identified in the RPR-FC. There were 139 common targets between the herb pair and IS. GO functional enrichment analysis revealed 2253 biological process entries, 72 cellular components entries, and 183 molecular functions entries. KEGG pathway enrichment analysis was mainly related to the NF-kappa B signaling pathway, the TNF signaling pathway, apoptosis, the MAPK signaling pathway, the PI3K-Akt signaling pathway, the VEGF signaling pathway, etc. The molecular docking results showed the components that docked well with key targets were quercetin, luteolin, kaempferol, and baicalein.The active components (quercetin, luteolin, kaempferol, and baicalein) of the RPR-FC and their targets act on proteins such as MAPK1, AKT1, VEGFA, and CASP3, which are closely related to IS.¹ These targets are closely related to the NF-kappa B signaling pathway, the MAPK signaling pathway, the PI3K-Akt signaling pathway, the VEGF signaling pathway, and other signaling pathways. These pathways are involved in the recovery of nerve function, angiogenesis, and neuronal apoptosis and the regulation of inflammatory factors, which may have a therapeutic effect on IS.     

Update on antiarrhythmic drug pharmacology

Cardiac arrhythmias constitute a major public health problem. Pharmacological intervention remains mainstay to their clinical management. This, in turn, depends upon systematic drug classification schemes relating their molecular, cellular, and systems effects to clinical indications and therapeutic actions. This approach was first pioneered in the 1960s Vaughan‐Williams classification. Subsequent progress in cardiac electrophysiological understanding led to a lag between the fundamental science and its clinical translation, partly addressed by The working group of the European Society of Cardiology (1991), which, however, did not emerge with formal classifications. We here utilize the recent Revised Oxford Classification Scheme to review antiarrhythmic drug pharmacology. We survey drugs and therapeutic targets offered by the more recently characterized ion channels, transporters, receptors, intracellular Ca²⁺ handling, and cell signaling molecules. These are organized into their strategic roles in cardiac electrophysiological function. Following analysis of the arrhythmic process itself, we consider (a) pharmacological agents directly targeting membrane function, particularly the Na⁺ and K⁺ ion channels underlying depolarizing and repolarizing events in the cardiac action potential. (b) We also consider agents that modify autonomic activity that, in turn, affects both the membrane and (c) the Ca²⁺ homeostatic and excitation‐contraction coupling processes linking membrane excitation to contractile activation. Finally, we consider (d) drugs acting on more upstream energetic and structural remodeling processes currently the subject of clinical trials. Such systematic correlations of drug actions and arrhythmic mechanisms at different molecular to systems levels of cardiac function will facilitate current and future antiarrhythmic therapy.     

Domestication of the cardiac mitochondrion for energy conversion

The control of mitochondria energy conversion by cytosolic processes is reviewed. The nature of the cytosolic and mitochondrial potential energy homeostasis over wide ranges of energy utilization is reviewed and the consequences of this homeostasis in the control network are discussed. An analysis of the major candidate cytosolic signaling molecules ADP, Pi and Ca²⁺ are reviewed based on the magnitude and source of the cytosolic concentration changes as well as the potential targets of action within the mitochondrial energy conversion system. Based on this analysis, Ca²⁺ is the best candidate as a cytosolic signaling molecule for this process based on its ability to act as both a feedforward and feedback indicator of ATP hydrolysis and numerous targets within the matrix to provide a balanced activation of ATP production. These targets include numerous dehydrogenases and the F1-F0-ATPase. Pi is also a good candidate since it is an early signal of a mismatch between cytosolic ATP production and ATP synthesis in the presence of creatine kinase and has multiple targets within oxidative phosphorylation including NADH generation, electron flux in the cytochrome chain and a substrate for the F1-F0-ATPase. The mechanism of the coordinated activation of oxidative phosphorylation by these signaling molecules is discussed in light of the recent discoveries of extensive protein phosphorylation sites and other post-translational modifications. From this review it is clear that the control network associated with the maintenance of the cytosolic potential energy homeostasis is extremely complex with multiple pathways orchestrated to balance the sinks and sources in this system. New tools are needed to image and monitor metabolites within sub-cellular compartments to resolve many of these issues as well as the functional characterization of the numerous matrix post-translational events being discovered along with the enzymatic processes generating and removing these protein modifications.     

Kinase inhibitors for cardiovascular disease

Over the last decade, there has been substantial progress toward understanding the pathophysiology and treatment of cardiovascular diseases (CVDs). Elucidating cellular responses to the extracellular environment and signal transduction mechanisms have provided the opportunity to explore novel molecular therapeutic approaches for the treatment of CVDs. Neurohormonal stimulation has been implicated in these diseases; blockade of the renin-angiotensin and beta-adrenergic systems are examples of therapeutic effectiveness. There are multiple cell signaling cascades, some of which are beneficial or compensatory and others deleterious. The balance between these pathways, which in large part is dictated by the cellular environment, determines the outcome as a diseased or non-diseased state. Selective targeting of signaling pathways using protein kinase inhibitors, would have a potential advantage over receptor blockers. We review potential protein kinase targets and recent evidence supporting therapeutic interventional value in CVDs.     

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I–V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD⁺- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1–NAD⁺ and Ndi1–UQ2 complexes show overlapping binding sites for the NAD⁺ and quinone substrates.     

Immunity,Inflammation, and Oxidative Stress in Heart Failure: Emerging Molecular Targets

Purpose

Heart failure (HF) remains a major cause of morbidity and mortality worldwide. Although various therapies developed over the last two decades have shown improved long term outcomes in patients with established HF, there has been little progress in preventing the adverse cardiac remodeling that initiates HF. To fill the gap in treatment, current research efforts are focused on understanding novel mechanisms and signaling pathways. Immune activation, inflammation, oxidative stress, alterations in mitochondrial bioenergetics, and autophagy have been postulated as important pathophysiological events in this process. An improved understanding of these complex processes could facilitate a therapeutic shift toward molecular targets that can potentially alter the course of HF.

Methods

In this review, we address the role of immunity, inflammation, and oxidative stress as well as other novel emerging concepts in the pathophysiology of HF that may have therapeutic implications.

Conclusion

Based on the experimental and clinical studies presented here, we anticipate that a better understanding of the pathophysiology of HF will open the door for new therapeutic targets. A one-size-fits-all approach may not be appropriate for all patients with HF, and further clinical trials utilizing molecular targeting in HF may result in improved outcomes.

     

Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase

Calcium oscillations suppress mitochondrial movements along the microtubules to support on-demand distribution of mitochondria. To activate this mechanism, Ca²⁺ targets a yet unidentified cytoplasmic factor that does not seem to be a microtubular motor or a kinase/phosphatase. Here, we have studied the dependence of mitochondrial dynamics on the Miro GTPases that reside in the mitochondria and contain two EF-hand Ca²⁺-binding domains, in H9c2 cells and primary neurons. At resting cytoplasmic [Ca²⁺] ([Ca²⁺]_c), movements of the mitochondria were enhanced by Miro overexpression irrespective of the presence of the EF-hands. The Ca²⁺-induced arrest of mitochondrial motility was also promoted by Miro overexpression and was suppressed when either the Miro were depleted or their EF-hand was mutated. Miro also enhanced the fusion state of the mitochondria at resting [Ca²⁺]_c but promoted mitochondrial fragmentation at high [Ca²⁺]_c. These effects of Miro on mitochondrial morphology seem to involve Drp1 suppression and activation, respectively. In primary neurons, Miro also caused an increase in dendritic mitochondrial mass and enhanced mitochondrial calcium signaling. Thus, Miro proteins serve as a [Ca²⁺]_c-sensitive switch and bifunctional regulator for both the motility and fusion-fission dynamics of the mitochondria.     

Role of Hepatitis C virus core protein in viral‐induced mitochondrial dysfunction

Summary. Hepatitis C virus (HCV) infection results in several changes in mitochondrial function including increased reactive oxygen species (ROS) production and greater sensitivity to oxidant, Ca²⁺ and cytokine‐induced cell death. Prior studies in protein over‐expression systems have shown that this effect can be induced by the core protein, but other viral proteins and replication events may contribute as well. To evaluate the specific role of core protein in the context of viral replication and infection, we compared mitochondrial sensitivity in Huh7‐derived HCV replicon bearing cells with or without core protein expression with that of cells infected with the JFH1 virus strain. JFH1 infection increased hydrogen peroxide production and sensitized cells to oxidant‐induced loss of mitochondrial membrane potential and cell death. An identical phenomenon occurred in genome‐length replicons‐bearing cells but not in cells bearing the subgenomic replicons lacking core protein. Both cell death and mitochondrial depolarization were Ca²⁺ dependent and could be prevented by Ca²⁺ chelation. The difference in the mitochondrial response of the two replicon systems could be demonstrated even in isolated mitochondria derived from the two cell lines with the ‘genome‐length’ mitochondria displaying greater sensitivity to Ca²⁺‐induced cytochrome c release. In vitro incubation of ‘subgenomic’ mitochondria with core protein increased oxidant sensitivity to a level similar to that of mitochondria derived from cells bearing genome‐length replicons. These results indicate that increased mitochondrial ROS production and a reduced threshold for Ca²⁺ and ROS‐induced permeability transition is a characteristic of HCV infection. This phenomenon is a direct consequence of core protein interactions with mitochondria and is present whenever core is expressed, either in infection, full‐length replicon‐bearing cells, or in over‐expression systems.     

Mitochondria-Rich Extracellular Vesicles From Autologous Stem Cell–Derived Cardiomyocytes Restore Energetics of Ischemic Myocardium

BackgroundMitochondrial dysfunction results in an imbalance between energy supply and demand in a failing heart. An innovative therapy that targets the intracellular bioenergetics directly through mitochondria transfer may be necessary.ObjectivesThe purpose of this study was to establish a preclinical proof-of-concept that extracellular vesicle (EV)-mediated transfer of autologous mitochondria and their related energy source enhance cardiac function through restoration of myocardial bioenergetics.MethodsHuman-induced pluripotent stem cell–derived cardiomyocytes (iCMs) were employed. iCM-conditioned medium was ultracentrifuged to collect mitochondria-rich EVs (M-EVs). Therapeutic effects of M-EVs were investigated using in vivo murine myocardial infarction (MI) model.ResultsElectron microscopy revealed healthy-shaped mitochondria inside M-EVs. Confocal microscopy showed that M-EV–derived mitochondria were transferred into the recipient iCMs and fused with their endogenous mitochondrial networks. Treatment with 1.0 × 10⁸/ml M-EVs significantly restored the intracellular adenosine triphosphate production and improved contractile profiles of hypoxia-injured iCMs as early as 3 h after treatment. In contrast, isolated mitochondria that contained 300× more mitochondrial proteins than 1.0 × 10⁸/ml M-EVs showed no effect after 24 h. M-EVs contained mitochondrial biogenesis-related messenger ribonucleic acids, including proliferator-activated receptor γ coactivator-1α, which on transfer activated mitochondrial biogenesis in the recipient iCMs at 24 h after treatment. Finally, intramyocardial injection of 1.0 × 10⁸ M-EVs demonstrated significantly improved post-MI cardiac function through restoration of bioenergetics and mitochondrial biogenesis.ConclusionsM-EVs facilitated immediate transfer of their mitochondrial and nonmitochondrial cargos, contributing to improved intracellular energetics in vitro. Intramyocardial injection of M-EVs enhanced post-MI cardiac function in vivo. This therapy can be developed as a novel, precision therapeutic for mitochondria-related diseases including heart failure.     

Reperfusion injury as a therapeutic challenge in patients with acute myocardial infarction

Cardiomyocyte death secondary to transient ischemia occurs mainly during the first minutes of reperfusion, in the form of contraction band necrosis involving sarcolemmal rupture. Cardiomyocyte hypercontracture caused by re-energisation and pH recovery in the presence of impaired cytosolic Ca²⁺ control as well as calpain-mediated cytoskeletal fragility play prominent roles in this type of cell death. Hypercontracture can propagate to adjacent cells through gap junctions. More recently, opening of the mitochondrial permeability transition pore has been shown to participate in reperfusion-induced necrosis, although its precise relation with hypercontracture has not been established. Experimental studies have convincingly demonstrated that infarct size can be markedly reduced by therapeutic interventions applied at the time of reperfusion, including contractile blockers, inhibitors of Na⁺/Ca²⁺ exchange, gap junction blockers, or particulate guanylyl cyclase agonists. However, in most cases drugs for use in humans have not been developed and tested for these targets, while the effect of existing drugs with potential cardioprotective effect is not well established or understood. Research effort should be addressed to elucidate the unsolved issues of the molecular mechanisms of reperfusion-induced cell death, to identify and validate new targets and to develop appropriate drugs. The potential benefits of limiting infarct size in patients with acute myocardial infarction receiving reperfusion therapy are enormous.     

TGF‐β & BMP Receptors Endoglin and ALK1: Overview of their Functional Role and Status as Antiangiogenic Targets

The formation of new blood vessels from existing vasculature, angiogenesis, is facilitated through a host of different signaling processes. Members of the TGF‐β superfamily, TGF‐β1, TGF‐β3, and BMP9, are key propagators of both inhibition and initiation of angiogenesis. HHT, characterized by AVM and capillary bed defects, is caused by germline mutations in the ENG and ACVRL1/ALK1 genes, respectively. Clinical symptoms include epistaxis and GI hemorrhage. The membranous receptors endoglin and ALK1 activate proliferation and migration of endothelial cells during the angiogenic process via the downstream intracellular SMAD signaling pathway. Endothelial cell senescence or activation is dependent on the type of cytokine, ligand concentration, cell–cell interaction, and a multitude of other signaling molecules. Endoglin and ALK1 receptor levels in tumor vasculature correlate inversely with prognosis in humans, whereas in mice, endoglin deficiency decelerates tumor progression. Therefore, endoglin and ALK1 have been identified as potential therapeutic targets for antibody treatment in various cancers. Early phase clinical trials in humans are currently underway to evaluate the efficacy and safety of biological therapy targeting endoglin/ALK1‐mediated cells signaling.     

Diabetes reduces β-cell mitochondria and induces distinct morphological abnormalities,which are reproducible by high glucose in vitro with attendant dysfunction

We investigated the impact of a diabetic state with hyperglycemia on morphometry of β cell mitochondria and modifying influence of a K⁺-ATP channel opener and we related in vivo findings with glucose effects in vitro. For in vivo experiments islets from syngeneic rats were transplanted under the kidney capsule to neonatally streptozotocin-diabetic or non-diabetic recipients. Diabetic recipients received vehicle, or tifenazoxide (NN414), intragastrically for 9 weeks. Non-diabetic rats received vehicle. Transplants were excised 7 d after cessation of treatment (wash-out) and prepared for electron microscopy. Morphological parameters were measured from approx. 25,000 mitochondria. Rat islets were cultured in vitro for 2–3 weeks at 27 or 11 (control) mmol/l glucose. Transplants to diabetic rats displayed decreased numbers of mitochondria (-31%, p < 0.05), increased mitochondrial volume and increased mitochondrial outer surface area, p < 0.001. Diabetes increased variability in mitochondrial size with frequent appearance of mega-mitochondria. Tifenazoxide partly normalized diabetes-induced effects, and mega-mitochondria disappeared. Long-term culture of islets at 27 mmol/l glucose reproduced the in vivo morphological abnormalities. High-glucose culture was also associated with reduced ATP and ADP contents, reduced oxygen consumption, reduced signaling by MitoTracker Red and reduction of mitochondrial proteins (complexes I–IV), OPA 1 and glucose-induced insulin release.

We conclude that (1) a long-term diabetic state leads to a reduced number of mitochondria and to distinct morphological abnormalities which are replicated by high glucose in vitro; (2) the morphological abnormalities are coupled to dysfunction; (3) K⁺-ATP channel openers may have potential to partly reverse glucose-induced effects.     

Identification of tumor-associated,MHC class II-restricted phosphopeptides as targets for immunotherapy

The activation and recruitment of CD4⁺ T cells are critical for the development of efficient antitumor immunity and may allow for the optimization of current cancer immunotherapy strategies. Searching for more optimal and selective targets for CD4⁺ T cells, we have investigated phosphopeptides, a new category of tumor-derived epitopes linked to proteins with vital cellular functions. Although MHC I-restricted phosphopeptides have been identified, it was previously unknown whether human MHC II molecules present phosphopeptides for specific CD4⁺ T cell recognition. We first demonstrated the fine specificity of human CD4⁺ T cells to discriminate a phosphoresidue by using cells raised against the candidate melanoma antigen mutant B-Raf or its phosphorylated counterpart. Then, we assessed the presence and complexity of human MHC II-associated phosphopeptides by analyzing 2 autologous pairs of melanoma and EBV-transformed B lymphoblastoid lines. By using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing, and comparative analysis, a total of 175 HLA-DR-associated phosphopeptides were characterized. Many were derived from source proteins that may have roles in cancer development, growth, and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome “fingerprints.” We then generated HLA-DRβ1*0101-restricted CD4⁺ T cells specific for a phospho-MART-1 peptide identified in both melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen-presenting cells as well as for intact melanoma cells. This previously undescribed demonstration of MHC II-restricted phosphopeptides recognizable by human CD4⁺ T cells provides potential new targets for cancer immunotherapy.     

Diabetes induces IL-17A-Act1-FADD-dependent retinal endothelial cell death and capillary degeneration

PurposeDiabetes leads to progressive complications such as diabetic retinopathy, which is the leading cause of blindness within the working-age population worldwide. Interleukin (IL)-17A is a cytokine that promotes and progresses diabetes. The objective of this study was to determine the role of IL-17A in retinal capillary degeneration, and to identify the mechanism that induces retinal endothelial cell death. These are clinically meaningful abnormalities that characterize early-stage non-proliferative diabetic retinopathy.MethodsRetinal capillary degeneration was examined in vivo using the streptozotocin (STZ) diabetes murine model. Diabetic-hyperglycemia was sustained for an 8-month period in wild type (C57BL/6) and IL-17A^?/? mice to elucidate the role of IL-17A in retinal capillary degeneration. Further, ex vivo studies were performed in retinal endothelial cells to identify the IL-17A-dependent mechanism that induces cell death.ResultsIt was determined that diabetes-induced retinal capillary degeneration was significantly lower in IL-17A^?/? mice. Further, retinal endothelial cell death occurred through an IL-17A/IL-17R ? Act1/FADD signaling cascade, which caused caspase-mediated apoptosis.ConclusionThese are the first findings that establish a pathologic role for IL-17A in retinal capillary degeneration. Further, a novel IL-17A-dependent apoptotic mechanism was discovered, which identifies potential therapeutic targets for the early onset of diabetic retinopathy.     

Molecular signaling network complexity is correlated with cancer patient survivability

The 5-y survival for cancer patients after diagnosis and treatment is strongly dependent on tumor type. Prostate cancer patients have a >99% chance of survival past 5 y after diagnosis, and pancreatic patients have <6% chance of survival past 5 y. Because each cancer type has its own molecular signaling network, we asked if there are “signatures” embedded in these networks that inform us as to the 5-y survival. In other words, are there statistical metrics of the network that correlate with survival? Furthermore, if there are, can such signatures provide clues to selecting new therapeutic targets? From the Kyoto Encyclopedia of Genes and Genomes Cancer Pathway database we computed several conventional and some less conventional network statistics. In particular we found a correlation (R² = 0.7) between degree-entropy and 5-y survival based on the Surveillance Epidemiology and End Results database. This correlation suggests that cancers that have a more complex molecular pathway are more refractory than those with less complex molecular pathway. We also found potential new molecular targets for drugs by computing the betweenness—a statistical metric of the centrality of a node—for the molecular networks.     

Regulation of GPCR signal networks via membrane trafficking

G-protein-coupled receptors (GPCRs) are a superfamily of cell surface signaling proteins that act as central molecular activators and integrators in all endocrine systems. Membrane trafficking of GPCRs is a fundamental process in shaping extensive signaling networks activated by these receptors. Mounting evidence has identified an increasingly complex network of pathways and protein interactions that a GPCR can traverse and associate with, indicating a multi-level system of regulation. This review will discuss the recent developments in how GPCRs are trafficked to the cell surface as newly synthesized receptors, their recruitment to the clathrin-mediated pathway for endocytosis, and their sorting to subsequent divergent post-endocytic fates, focusing primarily on hormone-activated GPCRs. Current models depicting the classic roles membrane trafficking plays in GPCR signaling have evolved to a highly regulated and complex system than previously appreciated. These developments impart key mechanistic information on how spatial and temporal aspects of GPCR signaling may be integrated and could provide pathway-specific targets to be exploited for therapeutic intervention.