ASK1 Protein Research Articles

Discovery of dual rho-associated protein kinase 1 (ROCK1)/apoptosis signal–regulating kinase 1 (ASK1) inhibitors as a novel approach for non-alcoholic steatohepatitis (NASH) treatment

In the current study we suggest a novel approach to curb non-alcoholic steatohepatitis (NASH) progression, and we suggest privileged scaffolds for the design of novel compounds for this aim. NASH is an advanced form of non-alcoholic fatty liver disease that can further progress into fibrosis, cirrhosis, and hepatocellular carcinoma. It is a widely emerging disease affecting 25% of the global population and has no current approved treatments. Protein kinases are key regulators of cellular pathways, of which, Rho-associated protein kinase 1 (ROCK1) and apoptosis signal–regulating kinase 1 (ASK1) play an important role in the progression of NASH and they stand out as promising targets for NASH therapy. Interestingly, their kinase domains are found to be similar in sequence and topology; therefore, dual inhibition of ROCK1 and ASK1 is expected to be amenable and could achieve a more favourable outcome. To reach this goal, a training set of ROCK1 and ASK1 protein structures co-crystalized with type 1 (ATP-competitive) inhibitors was constructed to manually generate receptor-based pharmacophore models representing ROCK1 and ASK1 inhibitors’ common pharmacophoric features. The models produced were assessed using a test set of both ROCK1 and ASK1 actives and decoys, and their performance was evaluated using different assessment metrics. The best pharmacophore model obtained, showing a Mathew’s correlation coefficient (MCC) of 0.71, was then used to screen the ZINC purchasable database retrieving 6178 hits that were filtered accordingly using several medicinal chemistry and pharmacokinetics filters returning 407 promising compounds. To confirm that these compounds are capable of binding to the target kinases, they were subjected to molecular docking simulations at both protein structures. The results were then assessed individually and filtered, setting the spotlight on various privileged scaffolds that could be exploited as the nucleus for designing novel ROCK1/ASK1 dual inhibitors.

Uncovering the pharmacological mechanism of Shou Tai Wan on recurrent spontaneous abortion: A integrated pharmacology strategy-based research

Ethnopharmacological relevanceShou Tai Wan (STW), a traditional Chinese medicine formula, has been historically used for the treatment of recurrent spontaneous abortion (RSA). Despite its long-standing usage, the exact mechanism underlying the therapeutic effects of STW remains unclear in the existing literature. Aims of this studyTo explore the Pharmacological Mechanism of STW on RSA. MethodsA network pharmacological methodology was utilized to predict the active compounds and potential targets of STW, collect the RSA targets and other human proteins of STW, and analyze the STW related networks. The animal experiments were also performed to validate the effect of STW on RSA. ResultsThe results of network analysis showed that STW may regulate PI3K/AKT, MAPK, FoxO signaling pathways and so on. Animal experiment established the RSA model with CBA/J × DBA/2 mice. It was found that STW can reduce the embryo absorption rate of RSA group (p < 0.05) and balance the expression of Th 1/Th2 type cytokines compared with the model group. After 14 days of administration, the decidual and placental tissues were taken and the CD4+ T cells were isolated, and the phosphorylation level of signaling pathway was detected by Springbio720 antibody microarray. This experiment found that STW can significantly up-regulate the phosphorylation levels of STAT3 and STAT6 proteins in the STAT signaling pathway, and down-regulating the phosphorylation level of STAT1 protein. STW also significantly up-regulated the phosphorylation levels of Raf1, A-Raf, Ask1, Mek1, Mek2, JKK1, ERK1, ERK2, c-fos, c-Jun and CREB proteins in the MAPK signaling pathway, and down-regulate the phosphorylation levels of MEK6 and IKKb proteins. Compared with the RSA group, the STW group increased the expression levels of ERK1/2 mRNA and proteins and p-ERK1/2 proteins, and there was a statistical difference (p < 0.05). This is consistent with the chip results. ConclusionSTW may achieve therapeutic effects by interfering with the signaling pathways, biological processes and targets discovered in this study. It provides a new perspective for revealing the immunological mechanism of STW in the treatment of RSA, and also provides a theoretical basis for the clinical use of STW in the treatment of RSA.

Synthesis, characterization, molecular docking, and biological evaluation of novel ASK1 inhibitors

Apoptosis signal regulated kinase 1 (ASK1, also known as MAP3K5) is a member of the mitogen activated protein kinase (MAPK) signaling pathway that participates in cell survival, differentiation, stress responses, and apoptosis. Inhibiting ASK1 is an attractive strategy for treating diseases such as nonalcoholic steatohepatitis and multiple sclerosis. Here, we explored and synthesized a series of new skeleton compounds containing hydrogenated indole, indole, benzimidazole, tetrahydroquinoxaline, benzoxazine, tetrahydroquinoline, phenol, nitrobenzene, pyridine as ASK1 inhibitors. Among them, 44a and 44b containing 2-triazolylpyridine fragment showed the potent inhibitory activity, with IC50 values of 0.15 and 0.31 μM, respectively. In addition, the cell survival rates of 44a and 44b at different concentrations were greater than 90%, especially at 0.05, 0.1, and 0.8 μM, their cell survival rate is higher than GS-4997, indicating that they exhibit good safety in human normal liver LO2 cells. The results of flow cytometry showed that 44a and 44b significantly blocked the progression of the G1 phase cycle. Oil red O staining experiments showed that at a concentration of 8 μM for 44a, only slightly swollen cytoplasmic fat droplets were seen in LO2 cells, and there was no significant fusion between the fat droplets. In addition, 44a, as a representative molecule, was used to study the interaction modes with ASK1 protein through molecular docking. These studies and findings provide a good choice for the future development of ASK1 inhibitors.

The mechanism of action of safflower total flavonoids in the treatment of endometritis caused by incomplete abortion based on network pharmacology and 16S rDNA sequencing

Ethnopharmacological relevanceSafflower is a traditional Chinese medicine used for treating gynaecological diseases. However, its material basis and mechanism of action in the treatment of endometritis induced by incomplete abortion are still unclear. Aim of the studyThis study aimed to reveal the material basis and mechanism of action of safflower in the treatment of endometritis induced by incomplete abortion through comprehensive methods, including network pharmacology and 16S rDNA sequencing. Materials and methodsNetwork pharmacology and molecular docking methods were used to screen the main active components and potential mechanisms of action of safflower in the treatment of endometritis induced by incomplete abortion in rats. A rat model of endometrial inflammation by incomplete abortion was established. The rats were treated with safflower total flavonoids (STF) based on forecasting results, serum levels of inflammatory cytokines were analysed, and immunohistochemistry, Western blots, and 16S rDNA sequencing were performed to investigate the effects of the active ingredient and the treatment mechanism. ResultsThe network pharmacology prediction results showed 20 active components with 260 targets in safflower, 1007 targets related to endometritis caused by incomplete abortion, and 114 drug-disease intersecting targets, including TNF, IL6, TP53, AKT1, JUN, VEGFA, CASP3 and other core targets, PI3K/AKT, MAPK and other signalling pathways may be closely related to incomplete abortion leading to endometritis. The animal experiment results showed that STF could significantly repair uterine damage and reduce the amount of bleeding. Compared with the model group, STF significantly down-regulated the levels of pro-inflammatory factors (IL-6, IL-1β, NO, TNF-α) and the expression of JNK, ASK1, Bax, caspase3, and caspase11 proteins. At the same time, the levels of anti-inflammatory factors (TGF-β and PGE2) and the protein expression of ERα, PI3K, AKT, and Bcl2 were up-regulated. Significant differences in the intestinal flora were seen between the normal group and the model group, and the intestinal flora of the rats was closer to the normal group after the administration of STF. ConclusionsThe characteristics of STF used in the treatment of endometritis induced by incomplete abortion were multi-targeted and involved multiple pathways. The mechanism may be related to the activation of the ERα/PI3K/AKT signalling pathway by regulating the composition and ratio of the gut microbiota.

The apoptotic effects of soybean agglutinin were induced through three different signal pathways by down-regulating cytoskeleton proteins in IPEC-J2 cells

Soybean agglutinin (SBA) is a main anti-nutritional factor in soybean. SBA exhibits its anti-nutritional functions by binding to intestinal epithelial cells. Keratin8 (KRT8), Keratin18 (KRT18) and Actin (ACTA) are the representative SBA-specific binding proteins. Such cytoskeletal proteins act a crucial role in different cell activities. However, limited reports reveal what the signal transduction pathway of apoptosis caused by SBA when binding to KRT8, KRT18 and ACTA. We aimed to evaluate the effects of SBA on cell apoptosis and the expression of the cytoskeletal protein (KRT8, KRT18 and ACTA), reveal the roles of these cytoskeletal proteins or their combinations on SBA-induced cell apoptosis in IPEC-J2 cell line, evaluate the influences of SBA on the mitochondria, endoplasmic reticulum stress and death receptor-mediated apoptosis signal pathway and to show the roles of KRT8, KRT18 and ACTA in different apoptosis signal pathways induced by SBA. The results showed that SBA induced the IPEC-J2 cell apoptosis and decreased the mRNA expression of KRT8, KRT18 and ACTA (p < 0.05). The degree of effect of three cytoskeleton proteins on cell apoptosis was ACTA > KRT8 > KRT18. The roles of these three cytoskeletal proteins on IPEC-J2 apoptotic rates had a certain accumulation effect. SBA up-regulated mitochondrial fission variant protein (FIS1) and fusion protein (Mfn2) promoted CytC and AIF in mitochondria to enter the cytoplasm, activated caspase-9 and caspase-3, damaged or declined mitochondrial function and reduced ATP synthesis (p < 0.05). Also, SBA up-regulated the expression of GRP78, XBP-1, eIF2α, p-eIF2α and CHOP (p < 0.05), down-regulated the expression level of ASK1 protein (p < 0.05). SBA led to the recruitment of FADD to the cytoplasmic membrane and increased the expression of FasL, resulting in caspase-8 processing. SBA up-regulated the expression level of Bax protein and decreased cytosolic Bcl-2 and Bid (p < 0.05). In addition, there was a significant negative correlation between the gene expression of cytoskeleton proteins and apoptosis, as well as the expression of key proteins of apoptosis-related signal transduction pathways. In conclusion, SBA induced the activation of the mitochondria, endoplasmic reticulum stress and the death receptor-mediated apoptosis signal pathway and the crosstalk between them. The effect of SBA on these three pathways was mainly exhibited via down-regulation of the mRNA expression of the three cytoskeletal expressions. This study elucidates the molecular mechanism and signaling pathway of SBA that lead to apoptosis from the perspective of cell biology and molecular biology and provides a new perspective on the toxicity mechanism of other food-derived anti-nutrients, medical gastrointestinal health and related cancer treatment.

A new triazolothiadiazine derivative inhibits stemness and induces cell death in HCC by oxidative stress dependent JNK pathway activation

Hepatocellular carcinoma (HCC) is a highly heterogeneous cancer, and resistant to both conventional and targeted chemotherapy. Recently, nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to decrease the incidence and mortality of different types of cancers. Here, we investigated the cellular bioactivities of a series of triazolothiadiazine derivatives on HCC, which have been previously reported as potent analgesic/anti-inflammatory compounds. From the initially tested 32 triazolothiadiazine NSAID derivatives, 3 compounds were selected based on their IC50 values for further molecular assays on 9 different HCC cell lines. 7b, which was the most potent compound, induced G2/M phase cell cycle arrest and apoptosis in HCC cells. Cell death was due to oxidative stress-induced JNK protein activation, which involved the dynamic involvement of ASK1, MKK7, and c-Jun proteins. Moreover, 7b treated nude mice had a significantly decreased tumor volume and prolonged disease-free survival. 7b also inhibited the migration of HCC cells and enrichment of liver cancer stem cells (LCSCs) alone or in combination with sorafenib. With its ability to act on proliferation, stemness and the migration of HCC cells, 7b can be considered for the therapeutics of HCC, which has an increased incidence rate of ~ 3% annually.

Nutrition and PI3K/Akt signaling are required for p38-dependent regeneration.

Regeneration after damage requires early signals to trigger the tissue repair machinery. Reactive oxygen species (ROS) act as early signals that are sensed by the MAP3 kinase Ask1, which in turn activates by phosphorylation the MAP kinases p38 and JNK. The sustained or high activation of these kinases can result in apoptosis, whereas short or low activation can promote regeneration. Using the Ask1-dependent regeneration program, we demonstrate in Drosophila wing that PI3K/Akt signaling is necessary for Ask1 to activate p38, but not JNK. In addition, nutrient restriction or mutations that target Ser83 of the Drosophila Ask1 protein, a PI3K/Akt-sensitive residue, block regeneration. However, these effects can be reversed by the ectopic activation of p38, but not of JNK. Our results demonstrate that Ask1 controls the activation of p38 through Ser83, and that the phosphorylation of p38 during regeneration is nutrient sensitive. This mechanism is important for discriminating between p38 and JNK in the cells involved in tissue repair and regenerative growth.

Bisphenol A induces apoptosis through GPER-dependent activation of the ROS/Ca2+-ASK1-JNK pathway in human granulosa cell line KGN

Bisphenol A (BPA) is widely distributed in the environment and human surroundings and is closely related to the occurrence of many chronic diseases including female infertility. Although BPA-induced granulosa cell apoptosis has been widely reported, the underlying mechanisms remain unknown. In this study, we evaluated the induction effect of BPA exposure on apoptosis and mechanisms of regulation in KGN cells (a human granulosa-like tumor cell line). Our results indicated that BPA induced apoptosis of KGN cells in a dose- and time-dependent manner. BPA exposure significantly promoted the expression of pro-apoptotic proteins and decreased mitochondrial membrane potential. We also observed that high concentrations of BPA significantly promoted the generation of reactive oxygen species (ROS) and calcium ion (Ca2+) accumulation. The involvement of ROS and Ca2+ in BPA-induced KGN cell apoptosis was confirmed by pretreatment with NAC (an antioxidant) and BAPTA-AM (a calcium chelator). After inhibitors pretreatment to block the corresponding signaling pathways, it was found that BPA-induced phosphorylation of JNK and ASK1 proteins and apoptosis of KGN cells were significantly inhibited. We pretreated with G15 (a GPER inhibitor) and found that BPA-induced ROS generation and Ca2+ accumulation and apoptosis were significantly inhibited. These results suggest that BPA exposure induces KGN cell apoptosis through GPER-dependent activation of the ROS/Ca2+-ASK1-JNK signaling pathway. Our study provides mechanisms by which BPA induced apoptosis of granulosa cells and ovarian dysfunction.

Analysis of the protective mechanism of liraglutide on retinopathy based on diabetic mouse model.

In order to study the protection mechanism of liraglutide on the infectious lesion of the retina of type I diabetes, in this experiment, a mouse model of type I diabetes was established by induction with streptozotocin (STZ) and feeding with high-fat and high-sugar diet. After observing the living conditions of the modeled mice and detecting their fasting blood glucose (FBG), it was found that the modeled mice exhibited clinically similar symptoms in patients with type I diabetes, and their FBG was larger than 16.7 mmol/L, indicating that the experimental mouse model was obtained. The mice were divided into groups. The control group was divided into negative control group (A), light positive control group (B), diabetic control group (C), and diabetes care group (D) according to different treatment methods, and the experimental group was divided into treatment group 1 (LR1), treatment group 2 (LR2) and treatment group 3 (LR3) according to different injection doses. The eyes of mice in each group were extracted and retinal tissue sections were made, and the sections were stained with HE. The retinal morphology was observed and it was found that compared with group A, the outer nucleus layer was significantly thinner in group B and C, and the group D was the thinnest. After treatment with liraglutide, the outer nuclear layer of LR1 group and LR2 group LR3 group recovered significantly, indicating that liraglutide had protective effect on type I diabetes and light-induced damage of mouse retinal photoreceptor cells. Immunohistochemistry was used to detect p-Erk1/2 and ASK1 protein contents in retina. It was found that compared with the negative control group and the light control group, p-Erk1/2 protein contents in LR1, LR2 and LR3 groups were significantly increased, showing statistical significance. Compared with the negative control group and the light control group, ASK1 protein content in LR1, LR2 and LR3 groups significantly decreased. This suggested that the protective mechanism of liraglutide on retinopathy was related to up-regulation of antioxidant protein p-Erk1/2 and down-regulation of apoptosis-related protein ASK1, that is to say, the action site of liraglutide may be related to this. Through real-time quantitative detection of the Trx gene expression level in diabetic and photodamaged mice, it was found that compared with the diabetic light group, the Trx expression level in mice treated with liraglutide showed a significant up-regulated trend, suggesting that the protective mechanism of liraglutide on retinopathy was related to the up-regulated expression of antioxidant protein Trx. Therefore, liraglutide has a certain protective effect on diabetic retinal injury, and its mechanism is related to the up-regulation of p-Erk1/2 and Trx antioxidant protein, and the down-regulation of apoptosis-related protein ASK1.

635. In HIV-Infected Patients Killing of Latently HIV-Infected CD4 T Cells by Autologous CD8 T Cells Is Modulated by Nef

BackgroundThe PBMC of HIV-infected patients contain HIV-specific CD8 T cells and their potential targets, CD4 T cells latently infected by HIV. The role of HIV-specific CD8 T cells in the course of HIV infection and the way they affect the virus that resides in the latent reservoir, the resting memory CD4 T cells, is unknown. The association between HIV Nef protein and the cellular ASK1 protein protects the HIV-infected CD4 T cells from killing by CD8 T cells.MethodsCD8 and autologous CD4 T cells procured from PBMC of acute, chronic untreated, treated and AIDS patients were isolated by magnetic beads and co-incubated. Resting memory CD4 T cells (CD25−, CD69− and HLA-DR−) were isolated from activated CD4 T cells using a two-step bead depletion purification procedure. Formation of CD8-CD4 T-cell conjugates was observed by fluorescence microscopy and in situ PCR of HIV LTR DNA. Both conjugation and apoptosis were observed and quantified by imaging flow cytometry (ImageStream) using anti-human activated caspase 3 antibody and TUNEL assay. Formation of immunological synapse was observed by using anti-Perforin, anti γ-tubulin, and anti-LCK antibodies.ResultsFollowing co-incubation we observed that CD8 T cells conjugate with and induce apoptosis of autologous CD4 T cells. In patients with acute infection or AIDS the conjugation activity and apoptosis were much higher compared with chronic HIV-infected patients. In patients on anti-retroviral therapy (ART) low grade conjugation of CD4 T cells was observed by fluorescence microscopy (2.3 ± 0.3%), by in situ PCR of HIV DNA (3 ± 0.6%) and by ImageStream analysis (2.5 ± 0.5%). After co-incubation with autologous CD8 T cells 2.1 ± 0.4% of the CD4 T cells procured from patients on ART were undergoing apoptosis. Resting memory CD4 T cells were conjugated (1.9 ± 0.3%) and killed (2.2 ± 0.3%) by autologous CD8 T cells. Delivering a peptide that interferes with the Nef-ASK1 interaction, into the CD4 T cells, resulted in twofold enhancement of their apoptosis by the autologous CD8 T cells (from 2.1 ± 0.5% to 4.0 ± 0.4%), with no effect on conjugation.ConclusionCD8 T cells conjugate with and kill HIV-infected CD4 T cells throughout the course of HIV infection. We suggest that Nef inhibition may result in the elimination of the latent reservoir CD4 T cells by CD8 T cells. Disclosures All authors: No reported disclosures.

Molecular Mechanism of Apoptosis Signal‐Regulating Kinase I Oligomerization and Auto‐activation

Apoptosis Signal-Regulating Kinase I (ASK1) is a member of the Mitogen Activated Protein Kinase (MAPK) pathway. Responding to various stimulations, ASK1 activates four MAPK kinase (MKK) substrates including MKK4/7 and MKK3/7, which phosphorylate the c – Jun N terminal Kinase (JNK) and p38, respectively. The dysfunction and misregulation of ASK1 lead to various diseases such as cancers, cardiovascular diseases, neurodegenerative disorders, inflammatory diseases and diabetes. The activation of ASK1 is highly regulated by many regulatory factors. It is widely believed that the oligomerization of ASK1 is essential for its activation. However, the molecular mechanism of ASK1 activation largely remains unclear. We recently found out that the purified ASK1 is capable of auto-activating itself and it exists as a huge oligomer instead of a homodimer as previous studies suggested. To explore the mechanism of ASK1 oligomerization and auto-activation, we have generated a series of ASK1 truncates and hoping to identify the responsible domains/motifs for its oligomerization and auto-activation. We have purified two ASK1 truncates - ASK1-NK (ASK1 protein expressing N terminal and Kinase domains) and ASK1-K (ASK1 protein expressing Kinase domain only). After performing gel filtration, it was found out that ASK1-NK forms monomer and ASK1-K forms dimer in native state. We are currently developing protocols of kinase assay for different ASK1 truncates to analyze its auto-activation mechanism. In conclusion, this study will help us to unveil the detailed mechanism of ASK1 oligomerization which will surely enrich our knowledge in understanding the activation process of ASK1. Support or Funding Information This project is supported by Faculty Research Grant to Xuanzhi Zhan and Student Research Grant to Adua Rahman awarded by Tennessee Technological University. ASK1 mediated pathway Purification of ASK1 truncates. (A) ASK1-K domain was purified using Heparin and Q-sepharose column chromatography. The purified ASK1-K domain (~2μg) is shown in SDS-PAGE; (B) ASK1-NK domain was purified using Q – Sepharose and Phenyl – Sepharose column chromatography. The purified ASK1-NK domain (~2μg) is shown in SDS-PAGE. Sephacryl gel filtration chromatography of ASK1 truncates. (A) ASK1-NK is observed as monomer (116KDa). (B) Predominant form of ASK1-K is dimer (77 KDa). (C) The chromatogram of gel filtration standard and log of molecular weight vs. elution volume plot is generated to determine the molecular weights of the ASK1 truncates. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P 209 - C-terminal domain of tetanus toxin changes the apoptosis- and autophagy-related protein levels following spinal cord injury in rat brain

Traumatic spinal cord injury (SCI) can lead to post-traumatic inflammation, oxidative stress, motor neuron apoptosis, necrosis and autophagy of tissue. To promote and enhance recovery after SCI, recent development of devices and therapeutic interventions are needed. The aim of the present study was to investigate the possible role of C-terminal domain of tetanus toxin (Hc-TeTx) on cell death mechanisms including apoptosis and autophagy following SCI. Thirty five adult rats were divided into five groups (n=7 each) as follows: control, sham, trauma (SCI), SCI+Hc-TeTx and SCI+methylprednisolone groups. The functional neurological deficits due to the SCI were assessed by behavioral analysis using the BBB open-field locomotor test. The alterations in pro-/anti-apoptotic and autophagy related-protein levels were measured by western blotting technique. In our study, Hc-TeTx promotes locomotor recovery and motor neuron survival of SCI rats. Hc-TeTx decreased expression of bax, bad, bak, cleaved caspase-3, Ask1 and autophagy-related proteins including Atg5 and LC3II in brain. Our study provides an evidence that cell death mechanisms play critical roles in SCI and non-toxic peptides may exert protective effect and decrease cell death following SCI.

Purification, auto-activation and kinetic characterization of apoptosis signal-regulating kinase I

Apoptosis signal-regulating kinase I (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the downstream MAP kinase kinases (MKKs) from two MAP kinase cascades: c-Jun N-terminal kinase (JNK) and p38. The essential physiological functions of ASK1 have attracted extensive attention. However, our understanding of the molecular mechanisms of ASK1, including the activation mechanism of ASK1 and the catalytic mechanism of ASK1-mediated MKK phosphorylation, remain unclear. The lack of purified ASK1 protein has hindered the elucidation of ASK1-initiated signal transduction mechanisms. Here, we report a one-step chromatography method for the expression and purification of functional full-length ASK1 from Escherichia coli. The purified ASK1 demonstrates auto-phosphorylation activity. The kinase activity of auto-phosphorylated ASK1 (pASK1) was also evaluated on two MKK substrates, MKK4 and 7, from the JNK cascades. Our results show that MKK7 can be phosphorylated by pASK1 more effectively than MKK4. The steady-state kinetic analysis demonstrates that MKK7 is a better ASK1 substrate than MKK4. These observations are further confirmed by direct pull-down assays which shows ASK1 binds MKK7 significantly stronger than MKK4. Furthermore, robust phospho-tyrosine signal is observed in MKK4 phosphorylation by pASK1 in addition to the phospho-serine and phospho-threonine. This study provides novel mechanistic and kinetic insights into the ASK1-initiated MAPK signal transduction via highly controlled reconstructed protein systems.

Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway

Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin–Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.

Novel triazolothiadiazines act as potent anticancer agents in liver cancer cells through Akt and ASK-1 proteins

Newly designed triazolothiadiazines incorporating with structural motifs of nonsteroidal analgesic anti-inflammatory drugs were synthesized and screened for their bioactivity against epithelial cancer cells. Compounds with bioactivities less then ∼5μM (IC50) were further analyzed and showed to induce apoptotic cell death and SubG1 cell cycle arrest in liver cancer cells. Among this group, two compounds (1g and 1h) were then studied to identify the mechanism of action. These molecules triggered oxidative stress induced apoptosis through ASK-1 protein activation and Akt protein inhibition as demonstrated by downstream targets such as GSK3β, β-catenin and cyclin D1. QSAR and molecular docking models provide insight into the mechanism of inhibition and indicate the optimal direction of future synthetic efforts. Furthermore, molecular docking results were confirmed with in vitro COX bioactivity studies. This study demonstrates that the novel triazolothiadiazine derivatives are promising drug candidates for epithelial cancers, especially liver cancer.

Upregulated ROS production induced by the proteasome inhibitor MG-132 on XBP1 gene expression and cell apoptosis in Tca-8113 cells

Exposure of Tca-8113 cells to proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG-132) causing apoptosis is associated with endoplasmic reticulum (ER) stress. X-box-binding protein-1 (XBP1) is an important regulator of a subset of genes active during ER stress, which is related to cell survival and is required for tumor growth. The present study is to evaluate the effect of MG-132 on ROS production, XBP1 gene expression, tumor necrosis factor receptor-associated factor 2 (TRAF2), ASK1 and c-jun protein expression in tongue squamous cell carcinoma cell line Tca-8113 cells. ROS production was measured by reactive oxygen species assay. X-box binding protein-1 (XBP1) mRNA was analyzed by real-time–PCR, TRAF2, ASK1 and c-jun protein were investigated by western blot and immunocytochemistry respectively. The result indicated that ROS production, TRAF2, ASK1 and c-jun were elevated in MG-132 treated cells. Giving ROS scavenger N-acetyl-L-cysteine (NAC) largely prevented the effects of MG-132. Furthermore, treating with MG-132 lead to decreased XBP1 mRNA expression but could not completely block the expression of XBP1. Taken together, these findings provide the evidence that MG-132 induced ER stress lead to Tca-8113 cells apoptosis through ROS generation and TRAF2-ASK1-JNK signal pathway activation.

Brain-targeting delivery for RNAi neuroprotection against cerebral ischemia reperfusion injury

Nanoparticles (NPs) with modification of brain-targeting molecules have been extensively exploited for therapeutic gene delivery across the blood–brain barrier (BBB). As one of the effective RNA interference (RNAi) approaches, short hairpin RNA (shRNA) has been proved to be promising in the field of gene therapy. Apoptosis signal-regulating kinase 1 (Ask1) has been reported to be an important target for gene therapy against cerebral ischemia reperfusion injury. In this study, dendrigraft poly-l-lysine (DGL) was decorated by dermorphin (a μ-opiate receptor agonist) through PEG for efficient brain-targeting, then complexed with anti-Ask1 shRNA plasmid DNA, yielding the DGL-PEG-dermorphin/shRNA NPs. The DGL-PEG-dermorphin/shRNA NPs were characterized and estimated the brain-targeting ability. In vitro, increased cellular uptake and transfection efficiency were explored; in vivo, preferable accumulation and gene transfection in brain were showed in images. The DGL-PEG-dermorphin/shRNA NPs also revealed high efficiency of neuroprotection. As a result of RNAi, corresponding mRNA was distinctly degraded, expression of Ask1 protein was obviously suppressed, apoptotic cell death was apparently decreased and cerebral infarct area was significantly reduced. Above all, DGL-PEG-dermorphin/shRNA NPs were proved to be efficient and safe for brain-targeting RNAi neuroprotection against cerebral ischemia reperfusion injury.

Probucol inhibits hydrogen peroxide to induce apoptosis of vascular smooth muscle cells

The aim of this study was to investigate the effect of the antioxidant probucol on hydrogen peroxide (H2O2)-induced vascular smooth muscle cell (VSMC) apoptosis. H2O2 (1 mmol/l) was used as the VSMC apoptosis-inducing agent, and 100, 10 and 1 µmol/l probucol concentrations were incubated with cells for 6 h. The cell apoptosis was observed using Annexin V-FITC, TUNEL and Hoechst 33258 staining methods. H2O2 promoted VSMC apoptosis, ASK-1 protein expression increased and Trx-l protein expression decreased. Probucol reduced H2O2-induced apoptosis in a dose-dependent manner; while ASK-1 protein expression decreased, Trx-1 protein expression increased. Probuco1 is capable of antagonizing the H2O2-induced apoptosis in VSMCs, which may be related to the decrease in ASK-1 protein expression and the increase in Trx-1 protein expression.

MiR-20a regulates ASK1 expression and TLR4-dependent cytokine release in rheumatoid fibroblast-like synoviocytes

ObjectiveTo evaluate whether miR-20a belonging to the cluster miR-17–92 is a negative regulator of inflammation in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) by modulating expression of apoptosis signal-regulating kinase (ASK)...

The P25 Pathogenicity Factor of Beet necrotic yellow vein virus Targets the Sugar Beet 26S Proteasome Involved in the Induction of a Hypersensitive Resistance Response via Interaction with an F-box Protein

P25, a Beet necrotic yellow vein virus (BNYVV) pathogenicity factor, interacts with a sugar beet protein with high homology to Arabidopsis thaliana kelch repeat containing F-box family proteins (FBK) of unknown function in yeast. FBK are members of the Skp1-Cullin-F-box (SCF) complex that mediate protein degradation. Here, we confirm this sugar beet FBK-P25 interaction in vivo and in vitro and provide evidence for in planta interaction and similar subcellular distribution in Nicotiana tabacum leaf cells. P25 even interacts with an FBK from A. thaliana, a BNYVV nonhost. FBK functional classification was possible by demonstrating the interaction with A. thaliana orthologs of Skp1-like (ASK) genes, a member of the SCF E3 ligase. By means of a yeast two-hybrid bridging assay, a direct effect of P25 on SCF-complex formation involving ASK1 protein was demonstrated. FBK transient Agrobacterium tumefaciens-mediated expression in N. benthamiana leaves induced a hypersensitive response. The full-length F-box protein consists of one F-box domain followed by two kelch repeats, which alone were unable to interact with P25 in yeast and did not lead to cell-death induction. The results support the idea that P25 is involved in virus pathogenicity in sugar beet and suggest suppression of resistance response.