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5-Azacytidine is well known for its clinical usage in cancer treatments. The present study investigates the role of 5-azacytidine as a cardioprotective agent to ameliorate ischemia/reperfusion (I/R) injury. The cardioprotective effect of 5-azacytidine was evaluated in three experimental models: in vitro, ex vivo, and in vivo. The cardioprotective effect was evaluated via cell viability, hemodynamic indices, infarct size measurement, and assessment of histopathology, oxidative stress, and mitochondrial function. The experiments were repeated in the presence of PI3K/GSK3β and mitochondrial KATP (mtKATP ) cardioprotective signaling pathway inhibitors to understand the underlying mechanism. 5-Azacytidine improved the cell viability by 29% in I/R-challenged H9C2 cells. Both isolated rat heart and LAD ligation model confirmed the infarct sparing effect of 5-azacytidine against I/R. It also provided a beneficial effect by normalizing the altered hemodynamics, reducing the infarct size and cardiac injury markers, reversing the perturbation of mitochondria, reduced oxidative stress, and improved the pPI3K and pAKT protein expression from I/R. In addition, it also augmented the activation of PI3K/AKT and mtKATP signaling pathway, confirmed by using wortmannin (PI3K inhibitor), SB216763 (GSK3β inhibitor), and glibenclamide (mtKATP channel closer). The effectiveness of 5-azacytidine as a cardioprotective agent is attributed to its activation of the PI3K/GSK3β and mtKATP channel signaling axis, thereby preserving mitochondrial function and reducing oxidative stress.

Autophagy has been recognized as a stress tolerance mechanism that maintains cell viability, which contributes to tumor progression, dormancy, and treatment resistance. The inhibition of autophagy in cancer has the potential to improve the therapeutic efficacy. It is therefore of great significance to search for new autophagy inhibitors. In the present study, after screening a series of curcumin derivatives synthesized in our laboratory, (E)-3-((E)-4-chlorobenzylidene)-5-((5-methoxy-1H-indol-3-yl)methylene)-1-methylpiperidin-4-one (CB-2) was selected as a candidate for further study. We found that CB-2 increased the LC3B-II and SQSTM1 levels associated with the accumulation of autophagosomes in non-small cell lung cancer (NSCLC) A549 cells. The increased level of LC3B-II induced by CB-2 was neither eliminated when autophagy initiation was suppressed by wortmannin nor further increased when autophagosome degradation was inhibited by chloroquine (CQ). CB-2 enhanced the accumulation of LC3B-II under starvation conditions. Further studies revealed that CB-2 did not affect the levels of the key proteins involved in autophagy induction but significantly blocked the fusion of autophagosomes with lysosomes. High-dose CB-2 induced the apoptosis and necrosis of A549 cells, while a lower dose of CB-2 mainly impaired the migrative capacity of A549 cells, which only slightly induced cell apoptosis. CB-2 increased the levels of mitochondrial-derived reactive oxygen species (ROS) while decreasing the mitochondrial membrane potential (MMP). Scavenging ROS via N-acetylcysteine (NAC) reversed CB-2-induced autophagy inhibition and its inhibitory effect against A549 cells. In conclusion, CB-2 serves as a new late-stage autophagy inhibitor, which has a strong inhibitory potency against A549 cells.

The main danger of cold stress to animals in cold regions is systemic metabolic changes and protein synthesis inhibition. Cold-induced RNA-binding protein is a cold shock protein that is rapidly up-regulated under cold stimulation in contrast to the inhibition of most proteins and participates in multiple cellular physiological activities by regulating targets. Therefore, this study was carried out to investigate the possible mechanism of CIRP-mediated glucose metabolism regulation and survival promotion in skeletal muscle after acute cold exposure. Skeletal muscle and serum from mice were obtained after 0, 2, 4 and 8 h of acute hypothermia exposure. Subsequently, the changes of CIRP, metabolism and apoptosis were examined. Acute cold exposure increased energy consumption, enhanced glycolysis, increased apoptosis, and up-regulated CIRP and phosphorylation of AKT. In addition, CIRP overexpression in C2C12 mouse myoblasts at each time point under 37°C and 32°C mild hypothermia increased AKT phosphorylation, enhanced glucose metabolism, and reduced apoptosis. CIRP knockdown by siRNA interference significantly reduced the AKT phosphorylation of C2C12 cells. Wortmannin inhibited the AKT phosphorylation of skeletal muscle after acute cold exposure, thereby inhibiting glucose metabolism and aggravating apoptosis. Taken together, acute cold exposure up-regulates CIRP in mouse skeletal muscle, which regulates glucose metabolism and maintains energy balance in skeletal muscle cells through the AKT signaling pathway, thus slowing down the apoptosis of skeletal muscle cells.

Ischemic stroke is a leading cause of mortality and disability. Diabetes mellitus, characterized by hyperglycemia, is a common concomitant disease of ischemic stroke, which is associated with autophagy dysfunction and blood-brain barrier (BBB) damage following cerebral ischemia/reperfusion (I/R) injury. At present, there is no effective treatment strategy for the disease. The purpose of the present study was to explore the molecular mechanisms underlying the protective effects of selenium on the BBB following I/R injury in hyperglycemic rats. Middle cerebral artery occlusion was performed in diabetic Sprague-Dawley rats. Treatment with selenium and the autophagy inhibitor 3-methyladenine significantly reduced cerebral infarct volume, brain water content and Evans blue leakage, while increasing the expression of tight junction (TJ) proteins and decreasing that of autophagy-related proteins (P

One of the most important mechanisms of preconditioning-mediated neuroprotection is the attenuation of cell apoptosis, inducing brain tolerance after a subsequent injurious ischemia. In this context, the antiapoptotic PI3K/AKT signaling pathway plays a key role by regulating cell differentiation and survival. Active AKT is known to increase the expression of murine double minute-2 (MDM2), an E3-ubiquitin ligase that destabilizes p53 to promote the survival of cancer cells. In neurons, we recently showed that the MDM2–p53 interaction is potentiated by pharmacological preconditioning, based on subtoxic stimulation of NMDA glutamate receptor, which prevents ischemia-induced neuronal apoptosis. However, whether this mechanism contributes to the neuronal tolerance during ischemic preconditioning (IPC) is unknown. Here, we show that IPC induced PI3K-mediated phosphorylation of AKT at Ser473, which in turn phosphorylated MDM2 at Ser166. This phosphorylation triggered the nuclear stabilization of MDM2, leading to p53 destabilization, thus preventing neuronal apoptosis upon an ischemic insult. Inhibition of the PI3K/AKT pathway with wortmannin or by AKT silencing induced the accumulation of cytosolic MDM2, abrogating IPC-induced neuroprotection. Thus, IPC enhances the activation of PI3K/AKT signaling pathway and promotes neuronal tolerance by controlling the MDM2–p53 interaction. Our findings provide a new mechanistic pathway involved in IPC-induced neuroprotection via modulation of AKT signaling, suggesting that AKT is a potential therapeutic target against ischemic injury., This research was funded by the Instituto de Salud Carlos III (PI18/00103 to M.D.-E.; PI18/00285 and RD16/0019/0018 to A.A.; CB16/10/00282 to J.P.B.), MICINN (PID2019-105699RB-I00/AEI/10.13039/501100011033 and RED2018-102576-T to J.P.B.; SAF2017-90794-REDT to A.A.), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03; CSI151P20), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA (to J.P.B.), Fundación Ramón Areces (to A.A.P. and J.P.B.), European Regional Development Fund, and European Union’s Horizon 2020 Research and Innovation Program (Grant Agreement 686009 to A.A.).

Context The grey mullet, Mugil cephalus, is an edible fish of high economic importance. Breeding biology with reference to hormonal/growth factor regulation of oocyte maturation needs to be known for its commercial production. Objective The present study was conducted to examine the potency of maturation inducing hormones, chorionic gonadotropin (hCG), bovine-insulin, and insulin like growth factor1 (h-IGF-1) I on ovarian steroidogenesis and oocyte maturation. Design The role of hormones and growth factors on steroidogenesis and oocyte maturation was investigated using specific inhibitors, Wortmannin for phosphatidylinositol-3 (PI3) kinase, trilostane for 3β-hydroxysteroid dehydrogenase, 1-octanol and 1-heptanol for gap junctions, actinomycin D for transcription and cycloheximide for translation of signal molecules. Methods Actions of hormonal and growth factors were examined for steroidogenesis, by radioimmunoassay and oocyte maturation by germinal vesicle breakdown (GVBD). Specific inhibitors were used to determine the cell signaling pathways, PI3 kinase. Results All the inhibitors attenuated the hCG-induced oocyte maturation (GVBD%), steroidogenesis including transcription, translation, gap junctions and PI3 kinase signaling. These inhibitors failed to inhibit h-IGF-I and b-insulin-induced oocyte maturation, steroidogenesis, translation and PI3 kinase signaling. Conclusion hCG induces oocyte maturation via steroid dependent pathway involving gap junctions, transcription, translation and PI3 kinase signaling, unlike h-IGF-I and b-insulin in the mullet.

Delayed healing of diabetic foot ulcers (DFUs) is one of the major consequences of angiopathy caused by hyperglycemia stemming from insulin resistance. Interventions that improve blood supply and hyperglycemia are essential for treating DFUs. Low-frequency vibration (LFV) promotes peripheral blood flow and wound healing in DFUs, regardless of hyperglycemia. We hypothesized that LFV promotes non-insulin-mediated glucose uptake, which is also referred to as AMPK-mediated glucose uptake, in adipocytes at wound sites, thereby alleviating hyperglycemia, which, in turn, accelerates wound healing. The objective of this in vitro study was to identify LFVs that optimally promote glucose uptake in adipocytes and investigate the mechanism underlying enhanced glucose uptake caused by LFV. 3T3-L1 adipocytes were used in this study. LFV was applied at 50 Hz for 40 min/d to investigate the most effective vibration intensity (0–2000 mVpp) and duration (0–7 d) of glucose uptake. We comparatively assessed 2-deoxyglucose (2-DG) uptake in control and vibration groups. To elucidated the mechanism underlying 2-DG uptake induced by LFV, wortmannin and compound C were used to inhibit insulin-mediated GLUT4 translocation and AMPK activation, respectively. Additionally, GLUT4 translocation to the plasma membrane was assessed using immunofluorescence image analysis. Our results indicated that 2-DG uptake in the 1000 and 1500 mVpp groups was higher than that in the control group (p = 0.0372 and 0.0018, respectively). At 1000 mVpp, 2-DG uptake in the 5- and 7-d groups was higher than that in the non-vibration group (p = 0.0169 and 0.0452, respectively). Although wortmannin did not inhibit 2-DG uptake, compound C did. GLUT4 translocation to the plasma membrane was not observed in the vibration group adipocytes treated with compound C. Thus, our results indicated that an LFV of 50 Hz, 1000 mVpp, 40 min/d, over 5 d was optimal for accelerating AMPK-mediated GLUT4 translocation and glucose uptake in adipocytes.

Spinocerebellar ataxia type 3 (SCA3), a hereditary and lethal neurodegenerative disease, is attributed to the abnormal accumulation of undegradable polyglutamine (polyQ), which is encoded by mutated ataxin-3 gene (ATXN3). The toxic fragments processed from mutant ATXN3 can induce neuronal death, leading to the muscular incoordination of the human body. Some treatment strategies of SCA3 are preferentially focused on depleting the abnormal aggregates, which led to the discovery of small molecule n-butylidenephthalide (n-BP). n-BP-promoted autophagy protected the loss of Purkinje cell in the cerebellum that regulates the network associated with motor functions. We report that the n-BP treatment may be effective in treating SCA3 disease. n-BP treatment led to the depletion of mutant ATXN3 with the expanded polyQ chain and the toxic fragments resulting in increased metabolic activity and alleviated atrophy of SCA3 murine cerebellum. Furthermore, n-BP treated animal and HEK-293GFP-ATXN3-84Q cell models could consistently show the depletion of aggregates through mTOR inhibition. With its unique mechanism, the two autophagic inhibitors Bafilomycin A1 and wortmannin could halt the n-BP-induced elimination of aggregates. Collectively, n-BP shows promising results for the treatment of SCA3.

The current study aimed to examine the anticancer activity of HTF-1, a cardiac glycoside (CG) isolated from Helleborus thibetanus Franch, using a cell-based model and to discover the underlying mechanisms with specific focus on autophagy. We found that HTF-1 was able to potently decrease the viability of several cancer cell lines especially for HeLa cervical carcinoma cells. It was discovered that HTF-1 dose dependently induced overproduction of ROS in HeLa cells, and the cell viability can be rescued when adding ROS scavenger N-acetyl-l-cysteine (NAC). More, we found that HTF-1 induced ROS-independent autophagy in concentration- and time-dependent manners in HeLa cells. This can be collectively verified by LC3-II and p62 abundance and also eGFP-LC3 puncta assay, bafilomycin clamp experiment, and acidotropic dye fluorescent labeling experiment. Additionally, TEM examination showed more autophagic vacuoles for HTF-1-treated HeLa cells. In HeLa cells, pretreatment with wortmannin (an inhibitor of the initial stages of autophagy to block autophagosome formation, thus, it should weaken the autophagy induction effect of HTF-1) decreased the autophagic flux and partially antagonized cell death induced by HTF-1, indicating that autophagy induced by HTF-1 played a cancer-suppressing role. Furthermore, coadministration of BAF (as a distal inhibitor of autophagy) with HTF-1 demonstrated a synergistic anticancer effect against HeLa cells. We believe that our work will enrich the understanding of CGs and especially anticarcinoma activity, also, pave the way for natural-product-based anticancer drug development.

Summary CD4+ T cells play critical roles in directing immunity, both as T helper and as regulatory T (Treg) cells. Here, we demonstrate that hepatocytes can modulate T cell populations through engulfment of live CD4+ lymphocytes. We term this phenomenon enclysis to reflect the specific enclosure of CD4+ T cells in hepatocytes. Enclysis is selective for CD4+ but not CD8+ cells, independent of antigen-specific activation, and occurs in human hepatocytes in vitro, ex vivo, and in vivo. Intercellular adhesion molecule 1 (ICAM-1) facilitates T cell early adhesion and internalization, whereas hepatocytes form membrane lamellipodia or blebs to mediate engulfment. T cell internalization is unaffected by wortmannin and Rho kinase inhibition. Hepatocytes engulf Treg cells more efficiently than non-Treg cells, but Treg cell-containing vesicles preferentially acidify overnight. Thus, enclysis is a biological process with potential effects on immunomodulation and opens a new field for research to fully understand CD4+ T cell dynamics in liver inflammation., Graphical Abstract, Highlights • Hepatocytes engulf live CD4+ T cells, with a preference for regulatory cells • We called this process enclysis and compared it with known cell-in-cell processes • ICAM-1 and β-catenin accumulated at the point of T cell engulfment • Enclysis was seen in health, in liver cancer, and especially in autoimmune disorders, Enclysis describes the enclosure of live CD4+ T cells in intracellular vesicles. Davies et al. show that hepatocytes engulf regulatory T (Treg) cells and preferentially delete them. Enclysis is distinct from entosis, efferocytosis, and suicidal emperipolesis and may be targeted to control T cell populations in the liver during inflammation.

Objectives: To explore the role of mTOR signaling pathway in modulating epileptogenesis in an N-methyl-D-aspartic acid (NMDA)-induced infant spasm (IS) rat model. Methods: After inducing IS successfully, the phosphorylation status of PI3K, Akt, mTOR and S6K of brain and hippocampus tissues was assessed using western blot and immunochemistry analysis, respectively. The possible mechanism of mTOR signaling pathway was evaluated by the, inhibitors for mTOR and PI3K, rapamycin and wortmannin, respectively. The inhibitors were injected into the intraperitoneal space of the rats to examine the effects of PI3K and mTOR in IS rat model. Results: The phosphorylated levels of mTOR and PI3K in hippocampus increased significantly (P < 0.05) 7 days after IS induction in rats. After administration of wortmannin, the phosphorylated levels of PI3K and mTOR decreased. However, only the phosphorylated level of mTOR decreased obviously after rapamycin administration. No obvious neurogenesis was found after IS induction. Discussion: Results of the present study suggest that hippocampal PI3K may be another potential target for IS treatment.

This study aimed to establish a high-fat diet (HFD)-fed obese mouse model and a cell culture model of insulin resistance (IR) in mature 3T3-L1 adipocytes. A dual-luciferase reporter assay (DLRA) was confirmed interaction between miR-27a and the 3'-untranslated region (UTR) of Peroxisome proliferator-activated receptor (PPAR)-γ. The inhibition of PPAR-γ expression by microRNA (miR)-27a in IR cells at both the protein and mRNA levels was confirmed by a mechanistic investigation. Moreover, the 3'-UTR of PPAR-γ was found to be a direct target of miR-27a, based on the DLRA. Furthermore, antagomiR-27a upregulated the activation of PI3K/Akt signaling and glucose transporter type 4 (GLUT4) expression at the protein and mRNA levels. Additionally, the PPAR inhibitor T0070907 repressed the insulin sensitivity upregulated by antagomiR-27a, which was accompanied by the inhibition of PPAR-γ expression and increased levels of AKT phosphorylation and GLUT4. The PI3K inhibitor wortmannin reduced miR-27a-induced increases in AKT phosphorylation, glucose uptake, and GLUT4. miR-27a is considered to be involved in the PPAR-γ-PI3K/AKT-GLUT4 signaling axis, thus leading to increased glucose uptake and decreased IR in HFD-fed mice and 3T3-L1 adipocytes. Therefore, miR-27a is a novel target for the treatment of IR in obesity and diabetes.

Pompe disease (OMIM # 232300) is a glycogen storage disease caused by autosomal recessive mutations of the gene encoding alpha-1,4-glucosidase (GAA; EC 3.2.1.20). Despite the relatively effective employment of enzyme replacement therapy, some critical medical issues still exist in patients with this disease, including the persistence of abnormalities in the central nervous system (CNS), probably because of the inability of the recombinant GAA to pass through the blood–brain barrier. To address this issue, identification of more therapeutic agents that target the CNS of patients with Pompe disease may be required. In this study, we derived neuronal cells from Pompe disease-induced pluripotent stem cells (Pom-iPSCs) and proved that they are able to recapitulate the hallmark cellular and biochemical phenotypes of Pompe disease. Using the Pom-iPSC-derived neurons as an in vitro drug-testing model, we then identified three compounds, ebselen, wortmannin and PX-866, with therapeutic potential to alleviate Pompe disease-associated pathological phenotypes in the neurons derived from Pom-iPSCs. We confirmed that all three compounds were able to enhance the GAA activity in the Pom-iPSC-derived neurons. Moreover, they were able to enhance the GAA activity in several important internal organs of GAA-deficient mice when co-injected with recombinant human GAA, and we found that intraperitoneal injection of ebselen was able to promote the GAA activity of the GAA-heterozygous mouse brain. Our results prove the usefulness of Pom-iPSC-derived neuronal populations for identifying new compounds with therapeutic potential.

BACKGROUND It is well-known that severe acute pancreatitis (SAP) due to infection is mainly caused by intestinal bacterial translocation. The intestinal barrier is tasked with preventing intestinal pathogenic bacteria and toxins from reaching the parenteral tissues through the intestinal wall. Therefore, maintaining intestinal barrier function may be the key to preventing damage from acute pancreatitis (AP). The phosphatidylinositol 3-kinase/protein kinase B pathway (PI3K/PKB) plays a role in AP. However, the exact effect of PI3K/PKB on injury associated with SAP has not yet been found. OBJECTIVES The present study was aimed at investigating the impact of wortmannin (WT), a PI3K/PKB inhibitor, on intestinal barrier function in SAP rats. MATERIAL AND METHODS The rats were divided into 3 groups: 1) the Sham Surgery group (SS), whose duodenum and pancreas were flipped 3 times (n = 18); 2) the pancreatitis group (SAP), who were injected through retrograde pancreatic duct injection with 5% sodium taurocholate (n = 18); and 3) the WSAP intervention group (SAP+WT). Serum alpha-amylase levels, plasma endogenous endotoxin, hematoxylin-eosin (H&E) staining, intestinal mucosa electron microscopy, intestinal permeability, and expression of p-PKB (phosphorylated protein kinase B) were measured. RESULTS In our findings under an electron microscope, the SAP group presented cell edema and mitochondrial vacuolated degeneration, sparsely arranged microvilli, tight junction damage, and widening, while the WSAP group exhibited less change and lower pancreas scores (7.4 ±1.14, 10.2 ±1.48 and 12.0 ±1.58 for 3 h, 6 h and 12 h, respectively) (p < 0.05). Furthermore, the plasma endogenous endotoxin levels and Evans blue content of the WSAP group was significantly lower at all timepoints than in the SAP group (p < 0.05). Western blotting showed that p-PKB expression was lower in the WSAP group than in the SAP group (p < 0.05). Our study suggests that WT relieves intestinal permeability changes in SAP rats and may be dose-dependent. CONCLUSIONS The PI3K/PKB signal pathway may involve SAP-related intestinal injuries and WT may relieve SAP intestinal injuries through the PI3K/PKB pathway.

Chondrocyte death due to apoptosis is central for osteoarthritis (OA) pathogenesis. The family with sequence similarity 3A (FAM3A) is a mitochondrial protein that plays an important role for cellular adaptation to stress and cell survival. Yet, whether FAM3A is associated with chondrocyte apoptosis and OA pathogenesis remains uncharacterized. In this study, we found that FAM3A expression was downregulated in cartilage tissue from an experimental OA mouse model. Besides, FAM3A expression was also reduced in chondrocytes treated with interleukin-1β (IL-1β), an inflammatory cytokine that promotes cartilage degradation. Moreover, we discovered that FAM3A attenuated chondrocyte apoptosis induced by IL-1β treatment in vitro, suggesting a protective effect of FAM3A against chondrocyte apoptosis. Moreover, mechanistically, FAM3A activated PI3K/Akt/mTOR pathway in IL-1β-treated chondrocytes, and blockade of PI3K/Akt/mTOR pathway with specific inhibitors, wortmannin and LY294002, diminished FAM3A effect on IL-1β-induced chondrocyte apoptosis, hence demonstrating that FAM3A attenuates IL-1β-induced chondrocyte apoptosis through activating the pro-survival PI3K/Akt/mTOR pathway. In conclusion, our study may identify FAM3A as a potential regulator of chondrocyte apoptosis involved in OA pathogenesis.

Selenocyanates and diselenides are potential antitumor agents. Here we report two series of selenium derivatives related to selenocyanates and diselenides containing carboxylic, amide and imide moieties. These compounds were screened for their potency and selectivity against seven tumor cell lines and two non-malignant cell lines. Results showed that MCF-7 cells were especially sensitive to the treatment, with seven compounds presenting GI50 values below 10 μM. Notably, the carboxylic selenocyanate 8b and the cyclic imide 10a also displayed high selectivity for tumor cells. Treatment of MCF-7 cells with these compounds resulted in cell cycle arrest at S phase, increased levels of pJNK and pAMPK and caspase independent cell death. Autophagy inhibitors wortmannin and chloroquine partially prevented 8b and 10a induced cell death. Consistent with autophagy, increased Beclin1 and LC3-IIB and reduced SQSTM1/p62 levels were detected. Our results point to 8b and 10a as autophagic cell death inducers. The research leading to these results has received funding from 'La Caixa' Banking Foundation. P. Garnica wishes to express his gratitude to the Asociación de Amigos de la Universidad de Navarra for the pre-doctoral fellowship. Furthermore, the authors wish to express their gratitude to the Plan de Investigación de la Universidad de Navarra, PIUNA (Ref 2014–26 ) as well as Calgary Foundation for financial support for the project.

Bone marrow mesenchymal stem cells (BM MSCs) can differentiate into multi-lineage tissues. However, obtaining BM MSCs by aspiration is difficult and can be painful; therefore peripheral blood (PB) MSCs might provide an easier alternative for clinical applications. Here, we show that circulating PB MSCs proliferate as efficiently as BM MSCs in the presence of extracellular matrix (ECM) and that differentiation potential into osteoblast in vitro and in vivo. Both BM MSCs and PB MSCs developed into new bone when subcutaneously transplanted into immune-compromised mice using hydroxyapatite/tricalcium phosphate as a carrier. Furthermore, LY294002 and Wortmannin blocked mesenchymal stem cell attachment in a dose-dependent manner, suggesting a role of phosphatidylinositol 3-kinase in MSC attachment. Our data showed that the growth of PB MSCs could be regulated by interaction with the ECM and that these cells could differentiate into osteoblasts, suggesting their potential for clinical applications.

Double FYVE-containing protein 1 (DFCP1) is ubiquitously expressed, participates in intracellular membrane trafficking and labels omegasomes through specific interactions with phosphatidylinositol-3-phosphate (PI3P). Previous studies showed that subcellular DFCP1 proteins display multi-organelle localization, including in the endoplasmic reticulum (ER), Golgi apparatus and mitochondria. However, its localization and function on lipid droplets (LDs) remain unclear. Here, we demonstrate that DFCP1 localizes to the LD upon oleic acid incubation. The ER-targeted domain of DFCP1 is indispensable for its LD localization, which is further enhanced by double FYVE domains. Inhibition of PI3P binding at the FYVE domain through wortmannin treatment or double mutation at C654S and C770S have no effect on DFCP1's LD localization. These show that the mechanisms for DFCP1 targeting the omegasome and LDs are different. DFCP1 deficiency in MEF cells causes an increase in LD number and reduces LD size. Interestingly, DFCP1 interacts with GTP-bound Rab18, an LD-associated protein. Taken together, our work demonstrates the dynamic localization of DFCP1 is regulated by nutritional status in response to cellular metabolism.

Targeting of extrinsic apoptosis pathway by TNF-related apoptosis-inducing ligand (TRAIL) is an attractive approach for cancer therapy. However, two TRAIL drug candidates failed in clinical trials due to lack of efficacy. We identified 17-hydroxy wortmannin (17-HW) in a drug repurposing screen that resensitized TRAIL's response in the resistant colon cancer cells. The deficiency of caspase-8 in drug-resistant cells along with defects in apoptotic cell death was corrected by 17-HW, an inhibitor of PIK3C3-beclin 1 (BECN1) complex and autophagy activity. Further study found that BECN1 significantly increased in the TRAIL-resistant cells, resulting in increased autophagosome formation and enhanced autophagy flux. The extracellular domain (ECD) of BECN1 directly bound to the caspase-8 catalytic subunit (p10), leading to sequestration of caspase-8 in the autophagosome and its subsequent degradation. Inhibition of BECN1 restored the caspase-8 level and TRAIL's apoptotic response in the resistant colon cancer cells. An analysis of 120 colon cancer patient tissues revealed a correlation of a subgroup of patients (30.8%, 37/120) who have high BECN1 level and low caspase-8 level with a poor survival rate. Our study demonstrates that the increased BECN1 accompanied by enhanced autophagy activity is responsible for the TRAIL resistance, and a combination of TRAIL with a PIK3C3-BECN1 inhibitor is a promising therapeutic approach for the treatment of colon cancer.

Maintenance of glucose homeostasis is reciprocally regulated by insulin and glucagon-like peptide-1 (GLP-1). We previously reported that GLP-1 secretion in response to an oral glucose load was impaired following an administration of 3-deoxyglucosone (3DG), an independent factor associated with the development of pre-diabetes. Here we investigated the effects of 3DG on insulin signaling and insulin-induced GLP-1 secretion under high-glucose conditions in the enteroendocrine L cell line STC-1. STC-1 cells were exposed to 3DG (80, 300, and 1000 ng/ml) in the presence of 10−7 M insulin and 25 mM glucose. GLP-1 secretion was determined by ELISA, glucose uptake was monitored with 2-NBDG (2-(N(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose), glucose consumption was detected by glucoseoxidase, and protein expression of insulin signaling molecules was examined by western blot. Results showed a decrease in insulin-induced GLP-1 secretion and insulin receptor phosphorylation after 3DG treatment. Concomitantly, 3DG treatment inhibited insulin-induced phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway activation. In the presence, but not absence, of insulin, 3DG treatment decreased insulin-stimulated glucose consumption. Inhibition of PI3K with Wortmannin attenuated insulin-induced increment in glucose transporter 2 (GLUT2) expression and 2-NBDG uptake. Accordingly, insulin-induced increase in GLUT2 expression and 2-NBGD uptake was significantly inhibited by 3DG treatment. 3DG-mediated reduction in GLUT2 expression contributes to the attenuation of insulin-induced GLP-1 secretion under high-glucose conditions in part through the insulin-PI3K/Akt/GLUT2 pathway in STC-1 cells. We conclude that 3DG interferes with insulin signaling and attenuates insulin action on glucose-induced GLP-1 secretion in STC-1 cells.

Painful peripheral neuropathy is the most dose-limiting side effect of paclitaxel (PTX), a widely used anti-cancer drug to treat solid tumours. The understanding of the mechanisms involved in this side effect is crucial to the development of new therapeutic approaches. CXCL1 chemokine and its receptor CXCR2 have been pointed as promising targets to treat chronic pain. Herein, we sought to evaluate the possible involvement of CXCL1 and CXCR2 in the pathogenesis of PTX-induced neuropathic pain in mice. PTX treatment led to increased levels of CXCL1 in both dorsal root ganglion and spinal cord samples. Systemic treatment with the anti-CXCL1 antibody (10 μg/kg, i.v.) or the selective CXCR2 antagonist (SB225002, 3 mg/kg, i.p.) had minor effect on PTX-induced mechanical hypersensitivity. On the other hand, the intrathecal (i.t.) treatment with anti-CXCL1 (1 ng/site) or SB225002 (10 μg/site) consistently inhibited the nociceptive responses of PTX-treated mice. Similar results were obtained by inhibiting the PI3Kγ enzyme a downstream pathway of CXCL1/CXCR2 signalling with either the selective AS605240 (5 μg/site, i.t.) or the non-selective wortmannin PI3K inhibitor (0.4 μg/site, i.t.). Overall, the data indicates that the up-regulation of CXCL1 is important for the development and maintenance of PTX-induced neuropathic pain in mice. Therefore, the spinal blockage of CXCL1/CXCR2 signalling might be a new innovative therapeutic approach to treat this clinical side effect of PTX.

Previously, we reported that adenosine N1-oxide (ANO), which is found in royal jelly, inhibited the secretion of inflammatory mediators by activated macrophages and reduced lethality in lipopolysaccharide (LPS)-induced endotoxin shock. Here, we examined the regulatory mechanisms of ANO on the release of pro-inflammatory cytokines, with a focus on the signaling pathways activated by toll-like receptor (TLR)4 in response to LPS. ANO inhibited both tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion from LPS-stimulated RAW264.7 cells without affecting cell proliferation. In this response, phosphorylation of mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulated kinase (ERK)1/2, p38 and SAPK/c-Jun N-terminal kinase (JNK)) and nuclear factor-κB (NF-κB) p65 was not affected by treatment with ANO. In contrast, phosphorylation of Akt (Ser473) and its downstream molecule glycogen synthase kinase-3β (GSK-3β) (Ser9) was up-regulated by ANO, suggesting that ANO stimulated GSK-3β phosphorylation via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The phosphorylation of GSK-3β on Ser9 has been shown to negatively regulate the LPS-induced inflammatory response. Activation of PI3K/Akt signaling pathway has also been implicated in differentiation of mesenchymal stem cells into osteoblasts and adipocytes. As expected, ANO induced alkaline phosphatase activity and promoted calcium deposition in a mouse pre-osteoblastic MC3T3-E1 cell line. The ANO-induced differentiation into osteoblasts was abrogated by coincubation with Wortmannin. Furthermore, ANO promoted insulin/dexamethasone-induced differentiation of mouse 3T3-L1 preadipocytes into adipocytes at much lower concentrations than adenosine. The protective roles of PI3K/Akt/GSK-3β signaling pathway in inflammatory disorders have been well documented. Our data suggest that ANO may serve as a potential candidate for the treatment of inflammatory disorders. Promotion of osteogenic and adipocyte differentiation further suggests its application for regenerative medicine.

Despite multiple antitumor activities, interferon-alpha (IFNα) therapy alone is less effective in solid tumors. Autophagy has been reported to play a key role in tumor chemoresistance. Therefore, it is meaningful to explore whether autophagy can be activated by IFNα in head and neck squamous cell carcinoma (HNSCC) and serve as a potential target to improve efficacy of IFNα therapy. In this study, we report that IFNα not only exhibits anti-proliferation activity and induces apoptosis, but also activates autophagy in HNSCC cells. Moreover, silencing autophagy-related protein 5 (ATG5) and signal transducer and activator of transcription 1 (STAT1) suppresses autophagy flux. Furthermore, IFNα and autophagy inhibitors (hydroxychloroquine and wortmannin) show clear synergistic effects on inhibiting growth and promoting apoptosis in HNSCC cells and xenograft models. Our findings indicate that IFNα-induced autophagy plays a cytoprotective role and blocking autophagy flux promotes IFNα-mediated apoptosis in HNSCC. These results suggest that the combination of IFNα and autophagy inhibitors represents a novel strategy for HNSCC treatment.

Apoptosis is involved in 2,2',4,4'- tetrabromodiphenyl ether (PBDE-47)-induced developmental neurotoxicity. However, little is known about the role of autophagy, especially its relationship with apoptosis underlying such neurotoxic process. Methods: Using female Sprague-Dawley rats exposed to low-dose PBDE-47 (0.1, 1.0 and 10 mg/kg/day) from pre-pregnancy until weaning of offspring to mimic human exposure, we investigated the effects of PBDE-47 on autophagy and apoptosis in relation to cognitive impairment of adult offspring rats. We also evaluated relationship between autophagy and apoptosis using neuroendocrine pheochromocytoma (PC12) cells, a widely used neuron-like cell line for neuronal development. Results: In vivo, perinatal exposure to PBDE-47 induced memory deficits in adult rats. This is accompanied by hippocampal neuronal loss partly as a result of apoptosis, as evidenced by caspase-3 activation and PARP cleavage. Further study identified that PBDE-47 triggered autophagic vesicles accumulation, increased levels of microtubule-associated protein 1 light chain 3 (LC3)-II, an essential protein for autophagosomes formation, and autophagy substrate sequestosome 1 (SQSTM1/p62), but reduced levels of autophagy-related protein (ATG) 7, a key protein for autophagosomes elongation, suggestive of autophagy impairment. These findings were further demonstrated by an in vitro model of PBDE-47-treated PC12 cells. Mechanistically, autophagy alteration is more sensitive to PBDE-47 treatment than apoptosis induction. Importantly, while stimulation of autophagy by the chemical inducer rapamycin and adenovirus-mediated Atg7 overexpression aggravated PBDE-47-induced apoptosis and cell death, inhibition of autophagy by the chemical inhibitor wortmannin and siRNA knockdown of Atg7 reversed PBDE-47-produced detrimental outcomes. Interestingly, blockage of apoptosis by caspase-3 inhibitor Ac-DEVD-CHO ameliorated PBDE-47-exerted autophagy impairment and cell death, though in combination with autophagy inhibitor did not further promote cell survival. Conclusion: Our data suggest that autophagy impairment facilitates apoptosis, which, in turn, disrupts autophagy, ultimately resulting in cell death, and that autophagy may act as a promising therapeutic target for PBDE-47-induced developmental neurotoxicity.

Fibrosis is the major pathological feature of chronic kidney disease (CKD). Aloe-emodin (AE), one of the main active compounds in Rhubarb, is widely used for renal protection. However, mechanisms implied in the modulation of kidney fibrosis after AE treatment for CKD remain elusive. Here, we explored the protective effects of AE for renal fibrosis and the involved mechanisms in vivo and in vitro. The renal fibrosis mice model was established by unilateral ureteral obstruction (UUO). We found that AE administration significantly ameliorated UUO-induced impairment of kidney, evidenced by improved histopathological abnormalities, body weight, and abnormal renal function in mice model. Immunohistochemical staining showed that TGF-β1 and Fibronectin expressions were significantly decreased in UUO mice compared with sham group. Meanwhile, we found that AE suppressed the activation of the PI3K/Akt/mTOR pathway induced by TGF-β1 in vivo. AE improved cell survival and decreased the level of fibrosis-related proteins under TGF-β1-induced fibrosis in HK-2 cells as well as in vitro. Furthermore, both wortmannin, an inhibitor of PI3K, and short-hairpin RNAs of PI3K knockdown abrogated TGF-β1-induced phosphorylation of Akt and mTOR, and decreased the suppression of fibrosis. These findings indicated that AE alleviated fibrosis by inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro, which may provide a potential therapeutic option for CKD.

Tunneling nanotubes (TNTs) are actin-based membranous structures bridging distant cells for intercellular communication. We define roles for TNTs in stress adaptation and treatment resistance in prostate cancer (PCa). Androgen receptor (AR) blockade and metabolic stress induce TNTs, but not in normal prostatic epithelial or osteoblast cells. Co-culture assays reveal enhanced TNT formation between stressed and unstressed PCa cells as well as from stressed PCa to osteoblasts. Stress-induced chaperones clusterin and YB-1 localize within TNTs, are transported bi-directionally via TNTs and facilitate TNT formation in PI3K/AKT and Eps8-dependent manner. AR variants, induced by AR antagonism to mediate resistance to AR pathway inhibition, also enhance TNT production and rescue loss of clusterin- or YB-1-repressed TNT formation. TNT disruption sensitizes PCa to treatment-induced cell death. These data define a mechanistic network involving stress induction of chaperone and AR variants, PI3K/AKT signaling, actin remodeling and TNT-mediated intercellular communication that confer stress adaptative cell survival.

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein–lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro. We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

Autophagy plays a key role in the stress response of tumor cells, which contributes to cancer cell survival and resistance to chemotherapy by degrading cytoplasmic proteins to provide energy and clear the hazardous substances. Therefore, combined treatment of chemotherapeutics and autophagy inhibitors is thought to obtain a desirable antitumor effect. Nanoparticles (NPs) show potential in tumor-targeting drug delivery because of the enhanced permeability and retention (EPR) effect. However, NPs with fixed particle size cannot achieve optimal delivery effect. Herein, a strategy based on Cu (I)-catalyzed click chemistry-triggered aggregation of azide/alkyne-modified micelles was developed for the co-delivery of the chemotherapeutic drug doxorubicin (Dox) and the autophagy inhibitor wortmannin (Wtmn). In vitro experiments showed that the size of micelles increased in a time-dependent manner, which enhanced micelle accumulation in both B16F10 and 4 T1 cells. The fluorescence resonance energy transfer (FRET) experiment and biodistribution study further demonstrated that the aggregation of micelles through click cycloaddition significantly improved the accumulation of drug-loading micelles at the tumor region. Furthermore, the decreased amount of autophagosomes observed by transmission electron microscopy (TEM), the declined expression of LC3-II, and the increased level of p62 by western blotting and immunohistochemistry (IHC) confirmed the obvious inhibition of autophagy induced by Dox/Wtmn co-loaded size-adjustable micelles, which had a synergistic effect in cancer suppression. In addition, the co-loaded size-adjustable micelles showed outstanding cytotoxicity and antitumor effect. Therefore, this strategy effectively suppressed melanoma and breast cancer in mice. Statement of Significance The therapeutic effects of chemotherapy can be limited by autophagy; hence, combined use of autophagy inhibitors with chemotherapeutics achieves desirable anticancer efficacy. In the present study, we designed size-adjustable micelles by modifying the click reaction substrate azide group and the alkyne group on the surface of micelles, and subsequently, the autophagy inhibitor wortmannin and the chemotherapeutic drug doxorubicin were co-loaded. The micelles could aggregate by click reaction at the tumor site when the catalysts were intratumorally injected. The results showed that the size-adjustable micelles achieved efficient drug delivery, penetration, and retention in tumors; through the combined effect of wortmannin-mediated autophagy inhibition and doxorubicin-mediated cytotoxicity, this strategy exerted significant anticancer effect in melanoma and breast cancer treatment.

alpha-Melanocyte-stimulating hormone (-MSH) has been shown to be involved in nociception, but the underlying molecular mechanisms remain largely unknown. In this study, we report that -MSH suppresses the transient outward A-type K+ current (I-A) in trigeminal ganglion (TG) neurons and thereby modulates neuronal excitability and peripheral pain sensitivity in rats. Exposing small-diameter TG neurons to -MSH concentration-dependently decreased I-A. This -MSH-induced I-A decrease was dependent on the melanocortin type 4 receptor (MC4R) and associated with a hyperpolarizing shift in the voltage dependence of A-type K+ channel inactivation. Chemical inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin or of class I PI3Ks with the selective inhibitor CH5132799 prevented the MC4R-mediated I-A response. Blocking G(i/o)-protein signaling with pertussis toxin or by dialysis of TG neurons with the G-blocking synthetic peptide QEHA abolished the -MSH-mediated decrease in I-A. Further, -MSH increased the expression levels of phospho-p38 mitogen-activated protein kinase, and pharmacological or genetic inhibition of p38 abrogated the -MSH-induced I-A response. Additionally, -MSH significantly increased the action potential firing rate of TG neurons and increased the sensitivity of rats to mechanical stimuli applied to the buccal pad area, and both effects were abrogated by I-A blockade. Taken together, our findings suggest that -MSH suppresses I-A by activating MC4R, which is coupled sequentially to the G complex of the G(i/o)-protein and downstream class I PI3K-dependent p38 signaling, thereby increasing TG neuronal excitability and mechanical pain sensitivity in rats.

Purpose: To investigate the protective effects of dexmedetomidine (Dex) on post cardiopulmonary bypass (CPB) lung injury in rats and to explore the possibility of underlying mechanisms involving ph...

To comprehensively understand the endocytosis of Sapelovirus A (PSV) entry into PK-15 cells, we studied PSV infection in the context of cell perturbations through drug inhibition, siRNA silencing and overexpression of dominant negative (DN) mutants. We showed here that PSV infection of PK-15 cells was unaffected by pretreated with chlorpromazine, EIPA, knockdown of the clathrin heavy chain or overexpression of Eps15 DN mutant. Conversely, PSV infection was sensitive to NH4Cl, chloroquine, dynasore, nystatin, MβCD and wortmannin with reduced PSV VP1 expression levels and virus titer. Additionally, PSV invasion leaded to rapid actin rearrangement and disruption of the cellular actin network enhanced PSV infection. After internalization the virus was transported to late endosomes and/or cycling endosomes that requires the participation of Rab7 and Rab11. Our findings demonstrate that PSV uses caveolae-dependent endocytosis as the predominant entry portal into PK-15 cells which requires low pH, dynamin, Rab7 and Rab11.

Chronic exposure to pesticides is implicated in the etiopathogenesis of Parkinson’s disease (PD). Previously, we showed that dieldrin induces dopaminergic neurotoxicity by activating a cascade of apoptotic signaling pathways in experimental models of PD. Here, we systematically investigated endosulfan’s effect on the interplay between apoptosis and autophagy in dopaminergic neuronal cell models of PD. Exposing N27 dopaminergic neuronal cells to endosulfan rapidly induced autophagy, indicated by an increased number of autophagosomes and LC3-II accumulation. Prolonged endosulfan exposure (>9 h) triggered apoptotic signaling, including caspase-2 and -3 activation and protein kinase C delta (PKCδ) proteolytic activation, ultimately leading to cell death, thus demonstrating that autophagy precedes apoptosis during endosulfan neurotoxicity. Furthermore, inhibiting autophagy with wortmannin, a phosphoinositide 3-kinase inhibitor, potentiated endosulfan-induced apoptosis, suggesting that autophagy is an early protective response against endosulfan. Additionally, Beclin-1, a major regulator of autophagy, was cleaved during the initiation of apoptotic cell death, and the cleavage was predominantly mediated by caspase-2. Also, caspase-2 and caspase-3 inhibitors effectively blocked endosulfan-induced apoptotic cell death. CRISPR/Cas9-based stable knockdown of PKCδ significantly attenuated endosulfan-induced caspase-3 activation, indicating that the kinase serves as a regulatory switch for apoptosis. Additional studies in primary mesencephalic neuronal cultures confirmed endosulfan’s effect on autophagy and neuronal degeneration. Collectively, our results demonstrate that a functional interplay between autophagy and apoptosis dictate pesticide-induced neurodegenerative processes in dopaminergic neuronal cells. Our study provides insight into cell death mechanisms in environmentally linked neurodegenerative diseases.

Insulin-like growth factor 1 (IGF-1) mediates the generation of reactive oxygen species (ROS) and the activation of growth promoting signaling pathways. Histone deacetylases (HDACs) regulate gene transcription by deacetylating lysine residues in histone and nonhistone proteins and a heightened HDAC activation, notably of HDAC5, is associated with vascular disorders, such as atherosclerosis. Although the contribution of IGF-1 in these pathologies is well documented, its role in HDAC phosphorylation and activation remains unexplored. Here, we examined the effect of IGF-1 on HDAC5 phosphorylation in vascular smooth muscle cells (VSMCs) and identified the signaling pathways involved in controlling HDAC5 phosphorylation and nuclear export. Treatment of A10 VSMCs with IGF-1 enhanced HDAC5 phosphorylation. Blockade of the IGF-1 receptor tyrosine kinase (TK) activity with the specific pharmacological inhibitor, AG1024, significantly inhibited IGF-1-induced HDAC5 phosphorylation, whereas the epidermal growth factor receptor (EGFR) TK antagonist, AG1478, had no effect. Inhibition of the mitogen-activated protein kinase pathway with U0126, SP600125, or SB203580, did not affect HDAC5 phosphorylation, whereas two inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathways, wortmannin and SC66, almost completely attenuated IGF-1-induced responses as confirmed by immunoblotting of phospho-HDAC5 and by small interfering RNA (siRNA)-induced AKT silencing. Moreover, the NAD(P)H oxidase (Nox) inhibitor, diphenyleneiodonium (DPI), and Nox4 siRNA, attenuated IGF-1-induced phosphorylation of HDAC5 and AKT. The HDAC5 phosphorylation resulted in its nuclear export, which was reversed by SC66 and DPI. Our results indicate that IGF-1-induced phosphorylation and nuclear export of HDAC5 involve Nox4-dependent ROS generation and PI3K/AKT signaling pathways.

Aims The impaired angiogenesis is the major cause of diabetic delayed wound healing. The molecular insight remains unknown. Previous study has shown that high glucose (HG) activates Na+/H+ exchanger 1 (NHE1) and induces intracellular alkalinization, resulting in endothelial dysfunction. The aim of this study is to investigate whether activation of NHE1 in endothelial cells by HG damages the angiogenesis in vitro and in vivo. Methods and results We used western blot to detect the phosphorylations of both Akt and Girdin, and pH-sensitive BCECF fluorescence to assay NHE1 activity and pHi value, respectively. The angiogenesis was evaluated by measuring the number of tube formation in vitro, and blood perfusion by laser doppler and neovascularization by staining CD31 in vivo. Our results indicated that induction of intracellular acidosis (IA) increased p-Akt and p-Girdin in human umbilical vein endothelial cells (HUVEC). HG activated NHE1 and increased pHi value in a time-dependent manner, associated with the decreased phosphorylations of both Akt and Gridin, while inhibition of NHE1 by amiloride abolished the HG-induced reductions of p-Akt and p-Girdin. However, silence of Akt by siRNA transfection or pharmacological inhibitors (wortmannin and LY294002) bypassed IA-induced Girdin phosphorylation. Overexpression of constitutively active Akt abolished HG-reduced Girdin phosphorylation. In addition, upregulation of Akt or inhibition of NHE1 remarkably attenuated HG-impaired tube formation in HUVEC. In vivo study revealed that amiloride dramatically rescued hyperglycemia-delayed blood perfusion and neovascularization by augmenting ischemia-induced angiogenesis. Conclusion IA promotes ischemia-induced angiogenesis via Akt-dependent Girdin phosphorylation in diabetic mice.

Sustained ligand-activated preconditioning (SLP), induced with chronic opioid receptor (OR) agonism, enhances tolerance to ischemia/reperfusion injury in young and aged hearts. Underlying mechanisms remain ill-defined, although early data implicate phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) during the induction phase, and β2-adrenoceptor (β2-AR), Gs alpha subunit (Gαs), and protein kinase A (PKA) involvement in subsequent cardioprotection. Here, we tested for induction of a protective β2-AR/Gαs/PKA signaling axis with SLP to ascertain whether signaling changes were PI3K-dependent (by sustained cotreatment with wortmannin), and whether the downstream PKA target Rho kinase (ROCK) participates in subsequent cardioprotection (by acute treatment with fasudil). A protected phenotype was evident after 5 days of OR agonism (using morphine) in association with increased membrane versus reduced cytosolic levels of total and phosphorylated β2-ARs; increased membrane and cytosolic expression of 52 and 46 kDa Gαs isoforms, respectively; and increased phosphorylation of PKA and Akt. Nonetheless, functional sensitivities of β2-ARs and adenylyl cyclase were unchanged based on concentration-response analyses for formoterol, fenoterol, and 6-[3-(dimethylamino)propionyl]-forskolin. Protection with SLP was not modified by ROCK inhibition, and changes in β2-AR, Gαs, and PKA expression appeared insensitive to PI3K inhibition, although 5 days of wortmannin alone exerted unexpected effects on signaling (also increasing membrane β2-AR and PKA expression/phosphorylation and Gαs levels). In summary, sustained OR agonism upregulates cardiac membrane β2-AR expression and phosphorylation in association with increased Gαs subtype levels and PKA phosphorylation. While Akt phosphorylation was evident, PI3K activity appears nonessential to OR upregulation of the β2-AR signal axis. This opioidergic remodeling of β2-AR signaling may explain β2-AR, Gαs, and PKA dependence of SLP protection.

Mechanisms of programmed cell death differ between animals, plants and fungi. In animals, apoptotic cell death depends on caspases and Bcl-2 family proteins. These protein families are only found in multicellular animals, including cnidarians, insects and mammals. In contrast, members of the TMBIM-family of transmembrane proteins are conserved across all eukaryotes. Sequence comparisons of cell death related proteins between phyla indicate strong conservation of the genes involved. However, often it is not known whether this is paralleled by conservation of function. Here we present the first study to support an anti-apoptotic function of Bcl-2 like proteins in the cnidarian Hydra within a physiological context. We used transgenic Hydra expressing GFP-tagged HyBcl-2-like 4 protein in epithelial cells. The protein was localised to mitochondria and able to protect Hydra epithelial cells from apoptosis induced by either the PI(3) kinase inhibitor wortmannin or by starvation. Moreover, we identified members of the TMBIM-family in Hydra including HyBax-Inhibitor-1, HyLifeguard-1a and -1b and HyLifeguard 4. Expressing these TMBIM-family members in Hydra and human HEK cells, we found HyBax-inhibitor-1 protein localised to ER-membranes and HyLifeguard-family members localised to the plasma membrane and Golgi-vesicles. Moreover, HyBax-inhibitor-1 protected human cells from camptothecin induced apoptosis. This work illustrates that the investigated Bcl-2- and TMBIM-family members represent evolutionarily conserved mitochondrial, ER, Golgi and plasma membrane proteins with anti-apoptotic functions. The participation of ER and Golgi proteins in the regulation of programmed cell death might be a very ancient feature.

Melatonin is present in mammalian follicular fluid and plays an important role in regulating steroidogenesis in follicular cells. In this study, we report the effect of melatonin on steroidogenesis in the theca interna (TI) in small bovine follicles and theca cells (TCs) cultured in vitro. Treatment with melatonin significantly increased the expression of steroidogenic acute regulatory protein (STAR) and the production of progesterone in both TI and in TCs. Melatonin stimulated the phosphorylation of AKT but not ERK1/2, and the addition of luzindole (a nonspecific MT1 and MT2 inhibitor) or 4P-PDOT (specific MT2 inhibitor) reduced melatonin-induced STAR expression, progesterone secretion, and PI3K/AKT pathway activation. The effect of melatonin on the TI in follicles was more obvious than on the TCs in vitro. Results indicate that melatonin stimulates the steroidogenesis of TCs mainly via the activation of the PI3K/AKT pathway by MT1 and MT2.

Background and Aim Caloric restriction (CR) improves insulin sensitivity and is one of the dietetic strategies most commonly used to enlarge life and to prevent aging-induced cardiovascular alterations. The aim of this study was to analyze the possible beneficial effects of caloric restriction (CR) preventing the aging-induced insulin resistance in the heart of male Wistar rats. Methods and results Three experimental groups were used: 3 months old rats (3m), 24 months old rats (24m) and 24 months old rats subjected to 20% CR during their three last months of life (24m-CR). After sacrifice hearts were mounted in a perfusion system (Langendorff) and heart function in basal conditions and in response to accumulative doses of insulin (10−9-10−7 M), in the presence or absence of Wortmannin (10−6 M), was recorded. CR did not attenuate the aging-induced decrease in coronary artery vasodilation in response to insulin administration, but it prevented the aging-induced downregulation of cardiac contractility (dp/dt) through activation of the PI3K/Akt intracellular pathway. Insulin stimulated in a greater extent the PI3K/Akt pathway vs the activation of the MAPK pathway and increased the protein expression of IR, GLUT-4 and eNOS in the hearts of 3m and 24m-CR rats, but not in the hearts of 24m rats. Furthermore, CR prevented the aging induced increase in endothelin-1 protein expression in myocardial tissue. Conclusion In conclusion CR partially improves cardiac insulin sensitivity and prevents the aging induced decrease in myocardial contractility in response to insulin administration through activation of PI3K/Akt pathway.

Background: The aim of the study was to examine the dependency of p53 status and the usefulness of mild hyperthermia (MHT) as an inhibitor of recovery from radiation-induced damage, referring to the response of quiescent (Q) tumor cell population. Methods: Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/ mp53 ) or with neo vector (SAS/ neo ) were injected subcutaneously into left hind legs of nude mice. Tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells. They received high dose-rate I³-ray irradiation (HDR) immediately followed by localized MHT (40 °C for 2 h), or caffeine or wortmannin administration, or low dose-rate gamma-ray irradiation simultaneously with localized MHT or caffeine or wortmannin administration. Nine hours after the start of irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. Results: SAS/ neo tumor cells, especially intratumor Q cell populations, showed a marked reduction in sensitivity due to the recovery from radiation-induced damage, compared with the total or Q tumor cells within SAS/ mp53 tumors that showed little recovery capacity. The recovery from radiation-induced damage was thought to be a p53- dependent event. In both total and Q tumor cells within SAS/ neo tumors, especially the latter, MHT efficiently suppressed the reduction in sensitivity caused by leaving an interval between HDR irradiation and the assay and decreasing the irradiation dose-rate, as well as the combination with wortmannin administration. Conclusions: From the viewpoint of solid tumor control as a whole, including intratumor Q-cell control, non-toxic MHT is useful for suppressing the recovery from radiation-induced damage, as well as wortmannin treatment combined with I³-ray irradiation. World J Oncol. 2019;10(3):132-141 doi: https://doi.org/10.14740/wjon1203

Autophagy may represent a common cellular response to nanomaterials. In the present study, it was demonstrated that zinc oxide nanoparticle (ZON)-elicited autophagy contributes to tumor cell killing by accelerating the intracellular dissolution of ZONs and reactive oxygen species (ROS) generation. In particular, ZONs could promote Atg5-regulated autophagy flux without the impairment of autophagosome-lysosome fusion, which is responsible for ZON-elicited cell death in cancer cells. On the other hand, a further study revealed that a significant free zinc ion release in lysosomal acid compartments and sequential ROS generation in cells treated with ZONs were also associated with tumor cytotoxicity. Intriguingly, the colocalization between FITC-labeled ZONs and autophagic vacuoles indicates that the intracellular fate of ZONs is associated with autophagy. Moreover, the chemical or genetic inhibition of autophagy significantly reduced the level of intracellular zinc ion release and ROS generation separately, demonstrating that ZON-induced autophagy contributed toward cancer cell death by accelerating zinc ion release and sequentially increasing intracellular ROS generation. The modulation of autophagy holds great promise for improving the efficacy of tumor chemotherapy. Herein, ZONs were verified to enhance chemotherapy in both normal and drug-resistant cancer cells via synergistic autophagy elicitation. Further, this elicitation resulted in tremendous zinc ion release and ROS generation, which accounted for enhancing the tumor chemotherapy and overcoming drug resistance. No obvious changes in the expression level of P-gp proteins or the amount of doxorubicin uptake induced by ZONs in MCF-7/ADR cells also indicated that the increased zinc ion release and ROS generation via synergistic autophagy induction were responsible for overcoming the drug resistance. Finally, in vivo experiments involving animal models of 4T1 tumor cells revealed that the antitumor therapeutic effect of a combinatory administration obviously outperformed those of ZONs or free doxorubicin treatment alone at the same dose, which could be attenuated by the autophagy inhibitor wortmannin or ion-chelating agent EDTA. Taken together, our results reveal the mechanism wherein the autophagy induction by ZONs potentiates cancer cell death and a novel biological application for ZONs in adjunct chemotherapy in which autophagy reinforces zinc ion release and ROS generation.

Wortmannin, a known inhibitor of phosphoinositide 3-kinases (PI3Ks), their low selectivity and high toxicity is still problematic and less is known about their effects on PI3Ks in cellular systems. Hence, we have synthesized a series of multifunctional wortmannin probes with the ability to self-activate, by installing a clickable handle at C11 site, and secondary amine and cancer-targeting moiety at C20 site, respectively. MTT assay first confirmed that self-activating probes have better inhibition potency and biotin enhanced their cancer cell uptake. Further experiments showed most of probes can target PI3K/Akt/mTOR pathway with prolonged turn-over time. Protein profiling and pull-down results were observed that the derivatives not only labelled four PI3Ks with selectivity, but also engaged in covalent interactions with numerous cellular proteins which could be the major reason of their high toxicity.

The endoplasmic reticulum stress (ERS)-induced autophagy and apoptosis are favorable for the suppression of many cancer types. Salidroside (Salid) has been proven to be capable of inducing the apoptosis of many cancer cells. However, the underlying mechanisms and whether Salid can activate the autophagic system have still not been explained thoroughly. Herein, the inhibition effect of Salid on the growth and progress of gastric cancer and the underlying mechanisms were investigated. With the SGC-7901 cells acting as the cancer model cells, we ascertained that Salid exerted a superior antagonism effect on the growth and migration of gastric cancer cells in a dose-dependent manner. Additionally, Salid exhibited strong capacity to induce cell apoptosis by the down-regulation of proliferation-related genes (Ki67 and PCNA), increase in the pro-apoptotic protein C-caspase-3, and changing the levels of other related genes. A mechanism study revealed that the levels of the ERS-related genes, such as CHOP, C-caspase-12, GADD34, and BiP, in the SGC-7901 cells dramatically changed post-treatment by Salid, indicating the involvement of ERS in Salid-inducing cell apoptosis. In addition, the increased LC3+ autophagic vacuoles, enhanced conversion of LC3-I to LC3-II, and inhibition of the PI3K/Akt/mTOR pathway further confirmed the activation of autophagy induced by Salid. Importantly, the effect of Salid in regulating the levels of autophagy-related proteins or the signaling pathway could be markedly depressed by co-incubating with Wortmannin (Wort), an autophagy inhibitor. The final evaluation of the tumor therapy efficacy exhibited satisfactory cancer growth inhibition by Salid with negligible toxicity to normal tissues. In summary, the present work provides a comprehensive effective evaluation of Salid for treating gastric cancer. The detailed investigation of the underlying mechanisms may offer a rational reference for the future applications of Salid in clinic.

Volatile anesthetics, including isoflurane, have been reported to have negative effects on cognitive dysfunction characterized by cognitive deficits following anesthesia. The aim of the current study was to investigate the effects involved with disintegrin and metallopeptidase domain 2 (ADAM2) silencing on isoflurane-induced cognitive dysfunction via the P13 K/Akt signaling pathway in immature rats. One week old healthy Sprague-Dawley (SD) rats were recruited and administered isoflurane anesthesia. The rats were then subjected to shADAM2 or wortmannin (PI3K/Akt signaling pathway inhibitor) to identify the effects of ADAM2 and the PI3K/Akt signaling pathway on the cognitive function of rats. Morris water maze and passive-avoidance tests were performed to examine the cognitive function of the rats. TUNEL staining was conducted to detect neuronal apoptosis in the hippocampal CA1 region. The obtained experimental results demonstrated that isoflurane anesthesia led to increased escape latency, reaction time, number of errors and TUNEL-positive neurons, along with a decreased latency time. In response to treatment with shADAM2, escape latency, reaction time, number of errors and TUNEL-positive cells were all noted to have decreased, in addition to elevated latency time, while contrasting trends were observed in regard to treatment with wortmannin. Taken together, the key findings of the present study revealed that shADAM2 activated the PI3K/Akt signaling pathway, resulting in elevated expressions of PI3K and Akt. Our study ultimately identified that ADAM2 silencing alleviates isoflurane-induced cognitive dysfunction by activating the P13 K/Akt signaling pathway in immature rats.

Therapeutic failure and drug resistance are common sequelae to insulin resistance associated with type 2 diabetes mellitus (T2DM). Consequently, there is an unmet need of alternative strategies to overcome insulin resistance associated complications.To demonstrate whether Kutkin (KT), iridoid glycoside enriched fraction of Picrorhiza kurroa extract (PKE) has potential to increase the insulin sensitivity vis à vis glucose uptake in differentiated adipocytes.Molecular interaction of KT phytoconstituents, picroside-I (P-I)picroside- II (P-II) with peroxisome proliferator-activated receptor gamma (PPARγ), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) were analyzed in silico. Cellular viability and adipogenesis were determined by following 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium bromide (MTT) assay and Oil Red-O staining. Further, ELISA kit based triglycerides and diacylglycerol-O-Acyltransferase-1 (DGAT1) were assessed in differentiated adipocytes. ELISA based determination were performed to check the levels of adiponectin and tumor necrosis factor alpha (TNF-α). However, Flow cytometry and immunofluorescence based assays were employed to measure the glucose uptake and glucose transporter 4 (glut4) expression in differentiated adipocytes, respectively. Further to explore the targeted signaling axis, mRNA expression levels of PPARγ, CCAAT/enhancer binding protein α (CEBPα), and glut4 were determined using qRT-PCR and insulin receptor substrate-1 (IRS-1), Insulin receptor substrate-2 (IRS-2), PI3K/Akt, AS160, glut4 followed by protein validation using immunoblotting in differentiated adipocytes.In silico analysis revealed the binding affinities of major constituents of KT (P-IP-II) with PPARγ/PI3K/Akt. The enhanced intracellular accumulation of triglycerides with concomitant activation of PPARγ and C/EBPα in KT treated differentiated adipocytes indicates augmentation of adipogenesis in a concentration-dependent manner. Additionally, at cellular level, KT upregulated the expression of DAGT1, and decreases fatty acid synthase (FAS), and lipoprotein lipase (LPL), further affirmed improvement in lipid milieu. It was also observed that KT upregulated the levels of adiponectin and reduced TNFα expression, thus improving the secretory functions of adipocytes along with enhanced insulin sensitivity. Furthermore, KT significantly promoted insulin mediated glucose uptake by increasing glut4 translocation to the membrane via PI3/Akt signaling cascade. The results were further validated using PI3K specific inhibitor, wortmannin and findings revealed that KT treatment significantly enhanced the expression and activation of p-PI3K/PI3K and p-Akt/Akt even in case of treatment with PI3K inhibitor wortmannin alone and co-treatment with KT in differentiated adipocytes and affirmed that KT as activator of PI3K/Akt axis in the presence of inhibitor as well.Collectively, KT fraction of PKE showed anti-diabetic effects by enhancing glucose uptake in differentiated adipocytes via activation of PI3K/Akt signaling cascade. Therefore, KT may be used as a promising novel natural therapeutic agent for managing T2DMand to the best of our knowledge, this is the first report, showing the efficacy and potential molecular mechanism of KT in enhancing insulin sensitivity and glucose uptake in differentiated adipocytes.

Chaetoglobosin K (ChK) is a natural product that has been shown to promote F-actin capping, inhibit growth, arrest cell cycle G2 phase, and induce apoptosis. ChK also has been shown to downregulate two important kinases involved in oncogenic pathways, Akt and JNK. This report investigates how ChK is involved in the receptor tyrosine kinase pathway (RTK/PI3K/mTORC2/Akt) to the centrally located protein kinase, Akt. Studies have reported that ChK does not inhibit PI3K comparable to wortmannin and does not affect PDK1 activation. PDK1 is responsible for phosphorylation on Akt T308, while mTORC2 phosphorylates Akt S473. Yet, Akt's two activation sites, T308 and S473, are known to be affected by ChK treatment. It was our hypothesis that ChK acts on the mTORC2 complex to inhibit the phosphorylation seen at Akt S473. This inhibition at mTORC2 should decrease phosphorylation at both these proteins, Akt and mTORC2 complex, compared to a known mTOR specific inhibitor, Torin1. Human lung adenocarcinoma H1299 and H2009 cells were treated with IGF-1 or calyculin A to increase phosphorylation at complex mTORC2 and Akt. Pretreatment with ChK was able to significantly decrease phosphorylation at Akt S473 similarly to Torin1 with either IGF-1 or calyculin A treatment. Moreover, the autophosphorylation site on complex mTORC2, S2481, was also significantly reduced with ChK pretreatment, similar to Torin1. This is the first report to illustrate that ChK has a significant effect at mTORC2 S2481 and Akt S473 comparable to Torin1, indicating that it may be a mTOR inhibitor.

Peste des petits ruminants (PPR) is an acute and highly contagious disease in small ruminants that causes significant economic losses in developing countries. An increasing number of studies have demonstrated that both autophagy and apoptosis are important cellular mechanisms for maintaining homeostasis, and they participate in the host response to pathogens. However, the crosstalk between apoptosis and autophagy in host cells during PPRV infection has not been clarified. In this study, autophagy was induced upon virus infection in caprine endometrial epithelial cells (EECs), as determined by the appearance of double- and single-membrane autophagy-like vesicles, LC3-I/LC3-II conversion, and p62 degradation. We also found that PPRV infection triggered a complete autophagic response, most likely mediated by the non-structural protein C and nucleoprotein N. Moreover, our results suggest that autophagy not only promotes the replication of PPRV in EECs but also provides a potential mechanism for inhibiting PPRV-induced apoptosis. Inhibiting autophagosome formation by wortmannin and knocking down the essential autophagic proteins Beclin-1 and ATG7 induces caspase-dependent apoptosis in EECs in PPRV infection. However, inhibiting autophagosome and lysosome fusion by NH4Cl and chloroquine did not increase the number of apoptotic cells. Collectively, these data are the first to indicate that PPRV-induced autophagy inhibits caspase-dependent apoptosis and thus contributes to the enhancement of viral replication and maturity in host cells.

The resolution of inflammation is closely linked with tissue repair. Recent studies have revealed that macrophages suppress inflammatory reactions by producing lipid mediators, called specialized proresolving mediators (SPMs); however, the biological significance of SPMs in tissue repair remains to be fully elucidated in the heart. In this study, we focused on maresin-1 (MaR1) and examined the reparative effects of MaR1 in cardiomyocytes. The treatment with MaR1 increased cell size in cultured neonatal rat cardiomyocytes. Since the expression of fetal cardiac genes was unchanged by MaR1, physiological hypertrophy was induced by MaR1. SR3335, an inhibitor of retinoic acid-related orphan receptor α (RORα), mitigated MaR1-induced cardiomyocyte hypertrophy, consistent with the recent report that RORα is one of MaR1 receptors. Importantly, in response to MaR1, cardiomyocytes produced IGF-1 via RORα. Moreover, MaR1 activated phosphoinositide 3-kinase (PI3K) / Akt signaling pathway and wortmannin, a PI3K inhibitor, or triciribine, an Akt inhibitor, abrogated MaR1-induced cardiomyocyte hypertrophy. Finally, the blockade of IGF-1 receptor by NVP-AEW541 inhibited MaR-1-induced cardiomyocyte hypertrophy as well as the activation of PI3K/Akt pathway. These data indicate that MaR1 induces cardiomyocyte hypertrophy through RORα/IGF-1/PI3K/Akt pathway. Considering that MaR1 is a potent resolving factor, MaR1 could be a key mediator that orchestrates the resolution of inflammation with myocardial repair.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer with a poor prognosis and a high recurrence rate. PIK3CA gene is frequently mutated in breast cancer, with PIK3CA H1047R as the hotspot mutation reported in TNBC. We used the ZINC database to screen natural compounds that could be structurally modified to develop drugs targeting the PIK3CA H1047R mutant protein in the PI3K pathway. The LibDock module showed that 2,749 compounds could strongly bind to the PIK3CA H1047R protein. Ultimately, the top 20 natural ligands with high LibDock scores were used for further analyses including assessment of ADME (absorption, distribution, metabolism, and excretion), toxicity, stability, and binding affinity. ZINC000004098448 and ZINC000014715656 were selected as the safest drug candidates with strong binding affinity to PIK3CA H1047R, no hepatotoxicity, less carcinogenicity, better plasma protein binding (PPB) properties, and enhanced intestinal permeability and absorption than the two reference drugs, PKI-402 and wortmannin. Moreover, their lower potential energies than those of PIK3CA H1047R confirmed the stability of the ligand-receptor complex under physiological conditions. ZINC000004098448 and ZINC000014715656 are thus safe and stable leads for designing drugs against PIK3CA H1047R as part of a targeted therapeutic approach for patients with TNBC.

We have shown that cholesterol regulates the activity of ion channels in mouse cortical collecting duct (CCD) mpkCCDc14 cells and that the transient receptor potential melastatin 4 (TRPM4) channel is expressed in these cells. However, whether TRPM4 channel is regulated by cholesterol remains unclear. Here, we performed inside-out patch-clamp experiments and found that inhibition of cholesterol biosynthesis by lovastatin significantly decreased, whereas enrichment of cholesterol with exogenous cholesterol significantly increased, TRPM4 channel open probability (Po) by regulating its sensitivity to Ca2+ in mpkCCDc14 cells. In addition, inside-out patch-clamp data show that acute depletion of cholesterol in the membrane inner leaflet by methyl-β-cyclodextrin (MβCD) significantly reduced TRPM4 Po, which was reversed by exogenous cholesterol. Moreover, immunofluorescence microscopy, Western blot, cell-surface biotinylation, and patch clamp analysis show that neither inhibition of intracellular cholesterol biosynthesis with lovastatin nor application of exogenous cholesterol had effect on TRPM4 channel protein abundance in the plasma membrane of mpkCCDc14 cells. Sucrose density gradient centrifugation studies demonstrate that TRPM4 was mainly located in cholesterol-rich lipid rafts. Lipid-protein overlay experiments show that TRPM4 directly interacted with several anionic phospholipids, including PI(4,5)P2. Depletion of PI(4,5)P2 with either wortmannin or PGE2 abrogated the stimulatory effects of exogenous cholesterol on TRPM4 activity, whereas exogenous PI(4,5)P2 (diC8-PI(4,5)P2, a water-soluble analog) increased the effects. These results suggest that cholesterol stimulates TRPM4 via a PI(4,5)P2-dependent mechanism.