Inhibitory of EV-A71 virus-induced apoptosis by ZVAD through ROS mediated signaling pathways

Emerging evidence that Enterovirus A 71 (EV-A71) infection closely related to apoptosis. The ZVAD is a caspase inhibitor that can prevent apoptosis. The aims of this project were to evaluate the mechanism of the ZVAD inhibited EV-A71 virus and to provide experiment basis for finding new antiviral drugs. In this study, after treated with ZVAD in EV-A71 infected Vero cells, the viral replication was reduced, and the cell viability was higher than EV-A71 group. Additionally, ZVAD decreased the cell apoptosis and the level of inflammatory cytokines induced by EV-A71 in the infected Vero cells. ZVAD inhibited cell apoptosis by regulating ROS mediated signaling pathway and inflammation cytokines to achieve antiviral.     

Melatonin prevents mitochondrial dysfunctions and death in differentiated skeletal muscle cells

Oxidative stress increase induces cellular damage and apoptosis activation, a mechanism believed to represent a final common pathway correlated to sarcopenia and many skeletal muscle disorders. The goal of this study is to evaluate if melatonin, a ROS scavenger molecule, is able to counteract or modulate myotube death. Here, differentiated C2C12 skeletal muscle cells have been treated with melatonin before chemicals known to induce apoptotic death and oxidative stress, and its effect has been investigated by means of morpho‐functional analyses. Ultrastructural observations show melatonin protection against triggers by the reducing of membrane blebbing, chromatin condensation, myonuclei loss and in situ DNA cleavage. Moreover, melatonin is able to prevent mitochondrial dysfunctions which occur in myotubes exposed to the trigger alone. These findings demonstrate melatonin ability in preventing apoptotic cell death in skeletal muscle fibers in vitro, suggesting for this molecule a potential therapeutic role in the treatment of various muscle disorders.     

How is the AKT/mTOR pathway involved in cell migration and invasion?

As a pathway that plays a role in nutrient absorption, anabolic response, cell growth and survival, the important role of AKT/mTOR in tumorigenesis has also come to light. For cancer patients, most deaths are caused by the growth of metastatic tumors outside the primary focus. Therefore, migration and invasion in the late stage of tumor progression are the main unresolved issues in the study of tumor pathogenesis, and AKT/mTOR has been found to participate in the migration and invasion of cancer cells, which means that the study of this pathway may contribute to a solution for the problem. Because of its extensive and complex functions in the organism, this pathway can be regulated by a variety of different signals in the body, and then realize its function through different downstream signal molecules. This article reviews the proteins that can indirectly affect this pathway by regulating the common upstream signaling molecules of this pathway, and the proteins that can directly affect the level of phosphorylation of AKT/mTOR in cancer cells. We also review the proteins that can co-regulate this pathway and its downstream pathways. Through this study, we hope to gain a deeper understanding of the regulatory mechanism of the AKT/mTOR pathway in cancer cells, in hopes of finding effective and harmless cancer treatment targets in the future.     

STMN1 promotes the proliferation and inhibits the apoptosis of acute myeloid leukemiacells by activating the PI3K/Akt pathway

Recent studies have shown that the microtubule disrupting protein Stathmin 1 (STMN1) is differentially expressed in AML patients and healthy control. The aim of this study was to explore the effects and molecular mechanism of STMN1 in AML. Here, the expression of STMN1 in peripheral blood cells (PBMCs) and bone marrow of AML patients and healthy volunteers was detected by RT-PCR and Western blot. STMN1 expression was regulated by transfected with STMN1 overexpressed plasmid or shRNA in two human leukemia cell lines K562 and HL60. Cell proliferation was examined by CCK8 and Edu staining. Annexin V and TUNEL assays were applied to test cell apoptosis. Flow cytometry was used to test the cell cycle distribution. The activation of the PI3K signaling pathway and the expression levels of cell cycle and cell apoptosis-related protein were determined by Western blot. In this study, we found that STMN1 was overexpressed in PBMCs and bone marrow of AML patients. STMN1 expression was closely related to FAB subtypes, risk stratification, disease-free survival, and overall survival of AML. Functional assays showed that overexpression of STMN1 in HL60 and K562 cells enhanced cell proliferation, decreased cell apoptosis, and caused G₁ phase arrest. In contrast, suppression of STMN1reduced cell proliferation and enhanced cell apoptosis in both HL60 and K562 cells. Moreover, the PI3K/Akt pathway was activated by STMN1, while suppression of STMN1 dysregulated the PI3K/Akt pathway and upregulating the levels of caspases3 and Bax expression. In conclusion, STMN1 was confirmed to promote the proliferation and inhibit the apoptosis of HL60 and K562 cells by modulating the PI3K/Akt pathway. STMN1 might be a novel molecular target for treating AML.     

Identification of key long noncoding RNAs and their biological functions in hepatocellular carcinoma

Long noncoding RNAs (lncRNAs) are vital regulators in tumorigenesis and metastasis. However, the pathological role of lncRNAs in hepatocellular carcinoma (HCC) is still unclear. In this study, we filtered out three lncRNAs from The Cancer Genome Atlas (TCGA) data that were screened for basic expression and clinical research. We selected lncRNA-NEAT1 for further study to explore its function in HCC progression and its regulatory mechanism. We identified three differentially expressed lncRNAs (DElncRNAs) in tumor and adjacent normal tissues from the TCGA library using data mining methods: lncRNA-NEAT1, lncRNA-MAGI2-AS3 and lncRNA-HCG11. Their basic expression levels were detected by qPCR. Then, we selected lncRNA-NEAT1 as a potentially important lncRNA to verity its biological function and mechanism in HCC cell lines. lncRNA-NEAT1, lncRNA-MAGI2-AS3 and lncRNA-HCG11 were overexpressed in liver cancer tissues and cell lines. We found that silencing NEAT1 in vitro can inhibit the proliferation of HuH-7 and Li-7 cells, inhibit cell migration, and induce apoptosis as well as significantly increase the level of miR-16-5p. We also confirmed that miR-16-5p has a significant correlation with Bcl-2. When NEAT1 is silenced, the expression of Bcl-2 decreases. Inhibiting miR-16-5p can restore Bcl-2 to its original level. We conclude that miR-16-5p1/lncRNA NEAT1 plays a crucial role in regulating the delivery of Bcl-2 in HCC. Overall, the miR-16-5p/lncRNA-NEAT1/Bcl-2 signaling axis may be a promising target for HCC treatment.     

Proteomics analysis of tumor microenvironment: Implications of metabolic and oxidative stresses in tumorigenesis

Tumorigenesis is always concomitant with microenvironmental alterations. The tumor microenvironment is a heterogeneous and complex milieu, which exerts a variety of stresses on tumor cells for proliferation, survival, or death. Recently, accumulated evidence revealed that metabolic and oxidative stresses both play significant roles in tumor development and progression that converge on a common autophagic pathway. Tumor cells display increased metabolic autonomy, and the hallmark is the exploitation of aerobic glycolysis (termed Warburg effect), which increased glucose consumption and decreased oxidative phosphorylation to support growth and proliferation. This characteristic renders cancer cells more aggressive; they devour tremendous amounts of nutrients from microenvironment to result in an ever‐growing appetite for new tumor vessel formation and the release of more “waste,” including key determinants of cell fate like lactate and reactive oxygen species (ROS). The intracellular ROS level of cancer cells can also be modulated by a variety of stimuli in the tumor microenvironment, such as pro‐growth and pro‐inflammatory factors. The intracellular redox state serves as a double‐edged sword in tumor development and progression: ROS overproduction results in cytotoxic effects and might lead to apoptotic cell death, whereas certain level of ROS can act as a second‐messenger for regulation of such cellular processes as cell survival, proliferation, and metastasis. The molecular mechanisms for cancer cell responses to metabolic and oxidative stresses are complex and are likely to involve multiple molecules or signaling pathways. In addition, the expression and modification of these proteins after metabolic or oxidative stress challenge are diverse in different cancer cells and endow them with different functions. Therefore, MS‐based high‐throughput platforms, such as proteomics, are indispensable in the global analysis of cancer cell responses to metabolic and oxidative stress. Herein, we highlight recent advances in the understanding of the metabolic and oxidative stresses associated with tumor progression with proteomics‐based systems biology approaches. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:267–311, 2013     

AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background: Activated hepatic stellate cells (HSCs) are closely involved in the initiation, perpetuation, and resolution of liver fibrosis. Pro-inflammatory cytokine levels are positively correlated with the transition from liver injury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis. Methods: In this study, we investigated the effect of the pro-inflammatory cytokine interleukin (IL)-1β on the proliferation and signaling pathways involved in fibrogenesis in LX-2 cells, an HSC cell line, using western blotting and cell proliferation assays. Results: IL-1β increased the proliferation rate and α-smooth muscle actin (SMA) expression of LX-2 cells in a dose-dependent manner. Within 1 h after IL-1β treatment, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-κB (NF-κB) signaling was activated in LX-2 cells. Subsequently, protein kinase B (AKT) phosphorylation and an increase in α- SMA expression were observed in LX-2 cells. Each inhibitor of JNK, p38, or NF-κB decreased cell proliferation, AKT phosphorylation, and α-SMA expression in IL-1β-treated LX-2 cells. Conclusion: These results indicate that JNK, p38, and NF-κB signals converge at AKT phosphorylation, leading to LX-2 activation by IL-1β. Therefore, the AKT signaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.     

MicroRNA-145-3p suppresses the malignant behaviors of T-cell acute lymphoblastic leukemia Jurkat cells via inhibiting the NFkappaB signaling pathway

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological tumor caused by the malignant transformation of immature T-cell progenitor cells. Emerging studies have stated that microRNAs (miRNAs) may play key roles in T-ALL progression. This study aimed to investigate the roles of miR-145-3p in T-ALL cell proliferation, invasion, and apoptosis with the involvement of the nuclear factor-kappaB (NF-κB) signaling pathway. T-ALL Jurkat cells were harvested, and the expression of miR-145-3p and NF-κB-p65 was measured. Gain- and loss-of-functions of miR-145-3p and NF-κB-p65 were performed to identify their roles in the biological behaviors of Jurkat cells, including proliferation, apoptosis, and invasion. Consequently, the current study demonstrated that miR-145-3p was downregulated while NF-κB-p65 was up-regulated in Jurkat cells. miR-145-3p directly bound to the 3’ untranslated region of NF-κB-p65. Over-expression of miR-145-3p inhibited Jurkat cell proliferation, invasion, and resistance to apoptosis, while over-expression of NF-κB-p65 presented opposite trends. Co-transfection of miR-145-3p and NF-κB-p65 promoted the malignant behaviors of Jurkat cells compared to miR-145-3p transfection alone, while it reduced these behaviors of Jurkat cells compared to NF-κB-p65 transfection alone. Taken together, this study provided evidence that miR-145-3p could suppress proliferation, invasion, and resistance to the death of T-ALL cells via inactivating the NF- κB signaling pathway.     

Hypoxia induced apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway

The pathogenesis of high altitude-related gastric mucosal injury remains poorly understood, this study aimed to investigate the role of autophagy in hypoxia-induced apoptosis of rat gastric mucosal cells. Rats were randomized into four groups which were maintained at an altitude of 400 m (P) or received no treatment (H), autophagy inducer rapamycin (H+AI) or autophagy inhibitor 3-MA (H+AB) at an altitude of 4,300 m for 1, 7, 14 and 21 days, respectively, and the morphology, ultrastructure, autophagy, and apoptosis of gastric mucosal tissues were examined. Gastric mucosal epithelial cells CC-R039 were cultured under conditions of normoxia, 2% O2 (hypoxia), or 2% O2+anti-mTORC1 for 0, 24, 48, and 72 h, respectively, and the autophagy and apoptosis were analyzed. CC-R039 cells were transfected with siHIF-1α, siTERT, or siRNA and the autophagy was examined. The results showed that the exposure to hypoxia increased the autophagy and apoptosis of gastric mucosal cells in rats, and apoptosis was aggravated by rapamycin treatment but alleviated by 3-MA treatment. Increased duration of hypoxia from 0 to 72 h could increase the autophagy and apoptosis but decrease the proliferation of gastric mucosal cells. Inhibition of mTORC1 with rapamycin led to further increase in apoptosis and even substantial cell death, and inhibition of HIF- 1α and TERT increased mTORC1 expression and reduced autophagy. Moreover, the inhibition of HIF-1α reduced TERT expression. In conclusion, hypoxia could induce apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway     

Gene expression profiling of HepG2 cells after treatment with black tea polyphenols

This study aimed to determine the effects of black tea polyphenols on gene expression in hepatocellular cancer cells. The total RNA from HepG2 hepatocellular cancer cells treated with black tea polyphenols was subjected to Human 14K cDNA microarray analysis. Real-time PCR and Western blot analysis were conducted to verify microarray data. Black tea polyphenols treatment at the dose of 20 mg/L, 40 mg/L or 80 mg/L for one to three days inhibited the growth of HepG2 cells in a dose and time dependent manner. A total of 48 genes showed more than two-fold change after black tea polyphenols treatment, including 17 upregulated genes and 31 downregulated genes, and they were involved in the regulation of cell growth, cell cycle, apoptosis, signaling, angiogenesis, tumor invasion and metastasis. Real-time PCR analysis of the selected genes showed that their mRNA expression changes were consistent with the microarray data. In addition, Western blot analysis of the selected genes showed that their protein expression changes were consistent with mRNA expression. In conclusion, gene expression profiles provide comprehensive molecular mechanisms by which black tea polyphenols exerts growth inhibition effects on cancer cells. The novel molecular targets identified in this study may be further exploited as therapeutic strategies for hepatocellular cancer.     

Role of apoptosis in gastric epithelial turnover

Gastric epithelial turnover is a dynamic process. It is characterized by continuous cell proliferation, which is counterbalanced by cell loss. The biological principle that mediates the homeostasis of epithelium is programmed cell death, or apoptosis. Currently, several subtypes of apoptosis are distinguished, which are mediated by different mechanisms. Various subtypes of apoptosis also occur in the gastric epithelium under various conditions. In the normal stomach, apoptosis due to cell isolation (anoikis) mediates the physiological epithelial turnover. Albeit rarely seen in routine histology, approximately 2% of epithelial cells in the normal stomach are apoptotic. In Helicobacter pylori-induced gastritis, apoptosis and epithelial proliferation are moderately increased, with approximately 8% apoptotic epithelial cells. In gastritis, factors such as CD95 ligand or tumor necrosis factor (TNF) alpha act as death factors. They bind to specific receptors, CD95 and TNF-R, which are induced either by other cytokines, such as interferon gamma, or by Helicobacter pylori itself. In addition to CD95, H.pylorican also induce upregulation of CD95 ligand expression. Taken together, the upregulated expression of CD95, and the presence of CD95L in the close proximity to apoptotic gastric epithelial cells suggest a functional role of the CD95-CD95L system in the induction of apoptosis in H.pylori-gastritis. The role of other pathways to apoptosis is currently under study. Apart from being a biological phenomenon, apoptosis in the stomach may also have direct clinical consequences. An extreme example is given in gastric graft-vs.-host disease when epithelial denudement occurs.     

Dihydroartemisinin enhances cell apoptosis in diffuse large B cell lymphoma by inhibiting the STAT3 activity

Background: Dihydroartemisinin (DHA) is reported to be a potential anticancer agent, and the mechanisms underlying the effects of DHA on diffuse large B cell lymphoma however are still obscure. This study aimed to assess the antitumor effect of DHA on diffuse large B cell lymphoma cells and to determine the potential underlying mechanisms of DHA-induced cell apoptosis. Methods: Here, the Cell Counting Kit 8 assay was conducted to study cell proliferation. We performed Annexin V-FITC/propidium iodide staining, real-time polymerase chain reaction, and western blot analysis to analyze cell apoptosis and potential molecular mechanisms. Results: The results showed that DHA substantially suppressed cell proliferation and induced cell apoptosis in vitro in a time- and concentration-dependent fashion. Moreover, STAT3 activity could be inhibited after stimulation with DHA. Conclusion: These results imply that the underlying anti-tumoral effect of DHA may increase apoptosis in diffuse large B cell lymphoma cells via the STAT3 signaling pathway. In addition, DHA might be an effective drug for diffuse large B cell lymphoma therapy.     

The function of ubiquitin-specific protease 31 in intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is the most serious type of stroke. High level of thrombin is found in the ICH. Ubiquitin-specific protease (USP) 31, a member of deubiquitinating enzymes family, has been found to negatively regulate the NF-κB pathway. However, the function of USP31 in ICH remains largely unknown. In the present study, the mRNA and protein expression levels of USP31 were measured by real-time PCR and western blot. Flow cytometry was used to measure cell apoptosis and the level of reactive oxygen species (ROS). In the current study, we found the mRNA level of USP31 was decreased in peripheral blood mononuclear cells (PBMCs) from the patients with ICH. Thrombin stimulated cell apoptosis, and increased the ROS level, the productions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1), but it decreased the expression level of USP31 in BV-2 cells. In agreement, USP31 overexpressing could alleviate these effects caused by thrombin. USP31 partially reversed the thrombin induced increase of nuclear factor kappa B (NF-κB) localization. Further, NF-κB inhibitor could alleviate the effects induced by USP31 knockdown. In addition, USP31 decreased the ubiquitination level of IκBα, which might contribute to the depression of NF-κB activation. In conclusions, USP31 played a role in the ICH might via regulating NF-κB signaling pathway.     

Detection of ROS and translocation of ERP-57 in apoptotic induced human neuroblastoma (SH-SY5Y) cells

Several toxic compounds are known to induce apoptosis in mammalian cell lines. The human neuroblastoma cells (SH-SY5Y) were exposed to the phosphatase inhibiting toxin okadaic acid (OA) or hydrogen peroxide (H2O2) to induce apoptosis as well as generate reactive oxygen species (ROS). Mitoxantrone (MXT) was used as a positive control for apoptosis. The SH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide (HyPer). The ERp57, also called GRP58 (Glucose-regulated protein 58), is a stress protein induced in conditions like glucose starvation and viral infection. Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells. ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death. ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy. These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.     

Forkhead box protein O1 (FoxO1) regulates lipids metabolism and cell proliferation mediated by insulin and PI3K-Akt-mTOR pathway in goose primary hepatocytes

In order to explore the role of forkhead box protein O1 (FoxO1) in the lipid metabolism and cell proliferation, goose primary hepatocytes were isolated and incubated with insulin or PI3K-Akt-mTOR pathway dual inhibitor NVP-BEZ235, and then transfected with FoxO1 interference plasmid. The related parameters of lipid metabolism and cell proliferation were measured. The results firstly showed that FoxO1 interference increased the intracellular TG and lipids concentration (P < 0.05); and increased the proliferative index (PI), cell DNA synthesis, protein expression of Cyclin D1 in goose primary hepatocytes (P < 0.05). Secondly, the co-treatment of insulin and FoxO1 interference increased the mRNA level and protein content of Cyclin D1 (P < 0.05); however, there was no significant difference between the insulin treatment and the co-treatment of insulin and miR-FoxO1 interference in the intracellular TG and lipids concentration and PI (P > 0.05). Lastly, the decrease of intracellular TG and lipids concentration and PI induced by NVP-BEZ235 was up-regulated by FoxO1 interference significantly (P < 0.05). In summary, FoxO1 could regulate the lipids metabolism and cell proliferation mediated by PI3K-Akt-mTOR signaling pathway in goose primary hepatocytes. Further investigations are required to highlight the potential role of FoxO1 in the lipid metabolism and cell proliferation mediated by insulin in goose primary hepatocyte.     

A <i>Brachypodium distachyon</i> calcineurin B-like protein-interacting protein kinase,BdCIPK26, enhances plant adaption to drought and high salinity stress

As sessile organisms, plants possess a complex system to cope with environmental changes. Ca²⁺ functions as a vital second messenger in the stress signaling of plants, and the CBL-interacting protein kinases (CIPKs) serve as essential elements in the plant Ca²⁺ signaling pathway. In this study, calcineurin B-like protein-interacting protein kinase 26 (BdCIPK26) from Brachypodium distachyon was characterized. Overexpression of BdCIPK26 enhanced tolerance to drought and salt stress of transgenic plants. Further investigations revealed that BdCIPK26 participated in abscisic acid (ABA) signaling, conferred hypersensitivity to exogenous ABA in transgenic plants, and promoted endogenous ABA biosynthesis. Moreover, BdCIPK26 was found to maintain ROS homeostasis in plants under stress conditions. Therefore, this study indicates that BdCIPK26 functions as a positive regulator in drought and salt stress response.