Expression Of Apoptosis Signal-regulating Kinase Research Articles

Hyperoside alleviates doxorubicin-induced myocardial cells apoptosis by inhibiting the apoptosis signal-regulating kinase 1/p38 pathway.

Cardiotoxicity is a side effect of the anthracycline broad-spectrum anti-tumor agent, doxorubicin (DOX). Hyperoside, a flavonoid glycoside extracted from many herbs, has anti-apoptotic and anticancer properties. However, its impact on the alleviation of DOX-induced apoptosis in cardiomyocytes remains elusive. The HL-1 cell line was treated with 100 µ M hyperoside for 1 h prior to treatment with 100 µ M hyperoside and 1 µ M DOX for 24 h. The cell counting kit-8 (CCK-8) assay was used to detect cell viability; DCFH-DA fluorescent probe was used to detect (reactive oxygen species) ROS; biochemical methods were used to detect the activity of glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA); the degree of apoptosis following DOX insult was assessed using immunofluorescence staining and terminal deoxynucleotidyl transferase mediated deoxy uridine triphosphate nick end labeling (TUNEL) assay; the change in protein expression of apoptosis signal-regulating kinase 1 (ASK1), p38, and apoptosis markers was determined using western blot. Hyperoside ameliorated DOX-induced oxidative stress in HL-1 cells, up-regulated GSH, SOD and CAT activity, reduced ROS production and inhibited MDA overproduction. Moreover, in addition to promoting HL-1 cell apoptosis, DOX administration also increased B-cell lymphoma (Bcl)-2-associated X-protein and cleaved caspase-3 protein levels and decreased Bcl-2 protein level. Hyperoside therapy, however, significantly reversed the impact of DOX on the cardiomyocytes. Mechanically, DOX treatment increased the phosphorylation of the ASK1/p38 axis whereas hyperoside treatment attenuated those changes. In a further step, hyperoside synergizes with DOX to kill MDA-MB-231 cells. Hyperoside protects HL-1 cells from DOX-induced cardiotoxicity by inhibiting the ASK1/p38 signaling pathway. Meanwhile, hyperoside maintained the cytotoxicity of DOX in MDA-MB-231 cells.

Liraglutide combined with human umbilical cord mesenchymal stem cell transplantation inhibits beta-cell apoptosis via mediating the ASK1/JNK/BAX pathway in rats with type 2 diabetes.

Accumulating evidence suggests an association between beta-cell apoptosis and the ASK1/JNK/BAX pathway. The aim of this study was to investigate the effects of a combined therapy of liraglutide and human umbilical cord mesenchymal stem cells (hUC-MSCs) on the glucose metabolism and islet beta-cell apoptosis, and further explore its relationship to the ASK1/JNK/BAX pathway. Type 2 diabetes mellitus (T2DM) rat model was induced by a high-sugar and high-fat diet and intraperitoneal injection of low-dose streptozotocin (STZ) (30 mg/kg). Three days after STZ injection, diabetic rats were randomly treated with subcutaneous injection of liraglutide (200 μg/kg/12 h) for 8 weeks and or hUC-MSCs (1 × 106 /rat) at the first and fifth weeks. Diabetes-related physical and biochemical parameters, pancreatic histopathological changes, immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blot were used to measure the expression of apoptosis signal-regulating kinase 1 (ASK1), Jun N-terminal kinase (JNK), Bcl-2 associated X protein (BAX), and B-cell lymphoma-2 (Bcl-2). Eight weeks after liraglutide or human umbilical cord mesenchymal stem cell administration, FPG, HbA1c , glucagon, body weight, and pancreatic ASK1, JNK, and BAX mRNA and proteins were significantly decreased, and the levels of serum C-p, INS and GLP-1, ratio of insulin positive area, and Bcl-2 expression were significantly increased in three treatment groups compared with T2DM group (P<.05). Liraglutide combined with hUC-MSCs improve glucose metabolism and inhibit islet beta-cell apoptosis in a ASK1/JNK/BAX pathway-dependent manner.

Effect of CXCR4 silencing with shRNA on MAPK signaling in ovarian cancer.

Our previous study demonstrated that short hairpin RNA (shRNA) targeting of C-X-C chemokine receptor type 4 (CXCR4) significantly inhibited cell proliferation, metastasis and invasion. On the basis of these results, the aim of the present study was to determine the effects of shRNA-CXCR4 silencing on mitogen-activated protein kinase (MAPK) signaling in human SW626 ovarian cancer cells. Following silencing the CXCR4 gene with shRNA, the mRNA expression of apoptosis signal-regulating kinase 1 (ASK1) was determined using the reverse transcription-quantitative polymerase chain reaction, whereas the protein expression of extracellular-signal-regulated kinase (ERK)1/2 and phosphorylated (p)-c-Jun were determined using immunocytochemistry and western blotting. SW626 cells transfected with shRNA-CXCR4 exhibited significantly increased ASK1 mRNA expression (P<0.05), significantly increased p-c-Jun protein expression (P<0.05), and significantly decreased ERK1/2 protein expression (P<0.05). Silencing the CXCR4 gene with shRNA significantly inhibited cell proliferation, promoted cell apoptosis and may be mediated by the MAPK signaling pathway.

The effect and mechanism of chaetocin on the intrahepatic cholangiocarcinoma cells

Objective To investigate the effect of chaetocin on the intrahepatic cholangiocarcinoma CCLP-1 and explore the potential mechanism. Methods The concentration of chaetocin were divided into 4 groups: Group 1: 0 nmol/L, Group 2: 50 nmol/L, Group 3: 100 nmol/L and Group 4: 200 nmol/L, and all were co-cultured with CCLP-1 cell, respectively. Cell viability was detected by Cell Counting Kit-8 assay; Giemsa staining was used to observe the cell morphological and flow cytometry technique were used to detect cell apoptosis; The intracellular reactive oxygen species (ROS) level was detected using fluorescent probe under fluorescence microscope; Western blotting were used to measure the expression of Apoptosis signal-regulating kinase 1 (ASK-1) and c-Jun N-terminal kinase (JNKs). Results The proliferation of CCLP-1 cell was inhibited by chaetocin in dose dependent (F=102.369, P=0.000) or in time dependent (F=141.377, P=0.000). Furthermore, both have interaction (F=15.418, P=0.000). Giemsa staining showed chaetocin brought about CCLP-1 cell to present morphological characteristics of apoptosis, and flow cytometry indicated title rate of apoptosis of CCLP-1 cell were (6.42±3.10)%, (10.82±2.84)%, (13.67±1.71)% and (17.91±6.16)%, and apoptosis was significantly increased (F=4.788, P=0.034). The intracellular ROS level of treatment group (24.126±9.587) was much higher than that of control group (5.114±1.937) and had significantly difference (P=0.006). Western blotting showed the expressions of p-ASK-1 were 0.255±0.150, 0.680±0.039, 1.356±0.125 and 1.916±0.367. The expressions of JNKs were 0.493±0.233, 1.542±0.573, 1.639±0.342 and 2.480±1.001.Both proteins expressed increasingly as dose of chaetocin increased. (ASK-1: F=36.946, P=0.000, JNKs: F=5.291, P=0.027). Conclusion Chaetocin could inhibit CCLP-1 cell and induce it apoptosis via promote ROS mediate ASK-1/JNK signal. Key words: Chaetocin; Human intrahepatic cholangiocarcinoma cell; Apoptosis; Oxygen species

Hypothermic machine perfusion increases A20 expression which protects renal cells against ischemia/reperfusion injury by suppressing inflammation, apoptosis and necroptosis.

There is an urgent need to improve the quality of donor organs obtained after cardiac death. In the present study, we examined the potential mechanisms through which A20 protects renal cells against ischemia/reperfusion injury (IRI) following either hypothermic machine perfusion (HMP) or static cold storage (CS) of the kidneys in a rabbit model. The expression of markers of apoptosis, necroptosis and inflammation in frozen kidney tissues were detected by western blot analysis, RT-qPCR and ELISA. Compared with the CS group, A20 expression was significantly higher in the tissue from the HMP group (P<0.01). By contrast, the expression of nuclear factor-κB (NF-κB) and tumor necrosis factor-α (TNF-α) was significantly lower in HMP group (P<0.01), whereas IκBα expression was significantly higher (P<0.01). The expression of apoptosis signal-regulating kinase 1 (ASK1), phosphorylated (p-)c-Jun N-terminal kinase (JNK) and activated caspase-3 in the HMP group was significantly downregulated compared with that in the CS group (all P<0.01). In addition, A20 inhibited receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis in the kidney. RIPK3 expression in the HMP group was significantly lower than that in the CS group (P<0.01), although the levels in both groups were higher than those in the sham group (P<0.01). Based on these findings, we propose a novel mechanism underlying the anti-apoptotic effect of A20 in renal cells in which A20 binds to ASK1 and promotes the degradation of ASK1 leading to the suppression of JNK activation and eventually, to the blockade of apoptosis. Thus, HMP reduces inflammation, apoptosis and necroptosis by upregulating the expression of A20; this mechanism may be responsible for protecting the kidney against IRI.

Involvement of miR-Let7A in inflammatory response and cell survival/apoptosis regulated by resveratrol in THP-1 macrophage.

BACKGROUND/OBJECTIVESResveratrol, a natural polyphenol, has multiple functions in cellular responses including apoptosis, survival, and differentiation. It also participates in the regulation of inflammatory response and oxidative stress. MicroRNA-Let-7A (miR-Let7A), known as a tumor suppressor miRNA, was recently reported to play a crucial role in both inflammation and apoptosis. Therefore, we examined involvement of miR-Let7A in the modulation of inflammation and cell survival/apoptosis regulated by resveratrol.MATERIALS/METHODSmRNA expression of pro-/anti-inflammatory cytokines and sirtuin 1 (SIRT1), and protein expression of apoptosis signal-regulating kinase 1 (ASK1), p-ASK1, and caspase-3 and cleaved caspase-3 were measured, and cell viability and Hoechst/PI staining for apoptosis were observed in Lipopolysaccharide (LPS)-stimulated human THP-1 macrophages with the treatment of resveratrol and/or miR-Let7A overexpression.RESULTSPre-treatment with resveratrol (25-200 µM) resulted in significant recovery of the reduced cell viabilities under LPS-induced inflammatory condition and in markedly increased expression of miR-Let7A in non-stimulated or LPS-stimulated cells. Increased mRNA levels of tumor necrosis factor-α and interleukin (IL)-6 induced by LPS were significantly attenuated, and decreased levels of IL-10 and brain-derived neurotrophic factor were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. Decreased expression of IL-4 mRNA by LPS stimulation was also significantly increased by miR-Let7A overexpression co-treated with resveratrol. In addition, decreased SIRT1 mRNA levels, and increased p-ASK1 levels and PI-positive cells by LPS stimulation were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination.CONCLUSIONSmiR-Let7A may be involved in the inflammatory response and cell survival/apoptosis modulated by resveratrol in human THP-1 macrophages.

Expression of apoptosis signal-regulating kinase 1 in dibated cardiomyoprthy and its correlation with left ventricular ejection fraction

Objective To observe the changes of apoptosis signal-regulating kinase 1(ASK1) expression in left ventricular myocardium of the dilated cardiomyopathy(DCM) rats induced by adriamycin and its correlation with left ventricular ejection fraction(LVEF). Methods One hundred and twenty SPF Wistar rats were divided into 2 groups as follows: control group(n=15), model group(n=105). Adriamycin was administered in the model group by intraperitoneal injection for 3 times in a week and repeated with a two-week interval for 6 times, while 9 g/L saline were administered in the control group in the same pattern, thus DCM rats model were constructed by the end of the 8th week.Both the model group and control group rats were drawn out and their LVEF were tested by the end of the 8th week and 12th week.Apoptosis index (AI) and the ASK1 in myocardium were tested respectively by apoptotic cells situ labeling, semi-quantitative analysis and Western blot. Results The AI of the control group, the 8th weekend model group and the 12th weekend model group were 0.53±0.27, 16.13±1.72, 19.54±2.24.The AI of the 8th and the 12th weekend mo-del groups were obviously higher than that in the control group.Comparing the differences among the 3 groups were statistically significant (F=18.98, P<0.05). The relative ASK1 expressions in ventricular myocardium of the control group, the 8th weekend model group and the 12th weekend model group were 0.169±0.010, 0.649±0.071, 0.781±0.077.Comparing the differences among the 3 groups were statistically significant (F=27.72, P<0.01). The LVEF(%)results of the control group, the 8th weekend model group and the 12th weekend model group were 75.41±2.02, 53.25±3.74, 39.21±6.13.Comparing the differences among the 3 groups were statistically significant(F=154.87, P<0.01). There was a negative correlation between the ASK1 expression and LVFE in model group at the end of 12 weeks(r=-0.86, P<0.01). Conclusions The ASK1 expression in myocardium of the DCM group increases obviously, thus apoptosis signal-regulating probably participates in the pathological process of DCM induced by ASK1. Key words: Cardiomyopathy; Apoptosis signal-regulating kinase 1; Apoptosis index; Adriamycin; Apoptosis

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)...

ASK1 is Involved in EBV LMP1-induced NF-κB Activation

Epstein-Barr virus (EBV) latent infection transforms B lymphocytes into proliferating lymphoblastoid cell lines (LCLs). EBV latent infection membrane protein 1 (LMP1) is required for EBV-mediated B lymphocyte transformation, and LMP1-induced NF-κB activation is essential for LCL survival. Previously, it was reported that the level of reactive oxygen species (ROS) and the expression of apoptosis signal-regulating kinase 1 (ASK1) are elevated in EBV-positive Burkitt’s lymphoma (BL) cells, the potential role of ASK1 in LMP1-induced NF-κB activation was thus investigated in this study. In EBV-positive BL cells, ASK1 was highly expressed and activated. In addition, TRAF6-ASK1 interaction was significantly increased in EBV-positive BL cells. Interestingly, the expression of LMP1 alone facilitated ASK1 activation. The expression of a dominant negative ASK1 mutant (ASK1KM) strongly blocked LMP1-induced NF-κB activation. Furthermore, LMP1-induced NF-κB activation was significantly reduced in ASK1 knock out (ASK1-/-) mouse embryonic fibroblasts (MEFs). Taken together, these results demonstrate that ASK1 is activated by LMP1 and is critical for LMP1-induced NF-κB activation.

Expression of apoptosis signal-regulating kinase 1 in pulmonary tissue of premature newborn rat exposed to hyperoxia

Objective To investigate the changes and potential roles of the expression of apoptosis signal-regulating kinase 1(ASK1),thioredoxin(Trx)and thioredoxin reductase(TrxR)in the pathogenesis of lung injury of premature newborn rats exposed to hyperoxia. Methods In the first day after delivery,preterm SD rats were randomly divided into air group and hyperoxia group with 64 rats in each.The rats were respectively sacrificed at 1,4,7,10 and 14 days after air or hyperoxia exposure.Sections of 1ungs were stained with HE to observe the histologic changes.Trx and TrxR mRNA were detected by reverse transcription-polymerase chain reaction(RT-PCR).Immunohistochemistry was used to detect the expression and distribution of ASK1.Western blot was used to detect the expression of ASK1 protein. Results Rats in hyperoxia group showed typical lung injury,which was characterized by alveolitis and delayed of lung development.Immunohistochemistry detected that ASK1 expressed generally in the cytoplasm of both alveolar epithelial cells and vascular endothelial cells:ASK1 protein expression in hyperoxia group at 1th and 4th day were 0.4382±0.0227 and 0.5270±0.0432,higher than in the air group(0.3458±0.0263 and 0.3760±0.0058)(P<0.01),and it was until 7th day that the expression became weaker(0.4343±0.0254),but still higher compared with the air group(0.3473±0.0220)(P<0.01).Compared with the air group,Trx and TrxR mRNA of the hyperoxia group increased significantly and peaked at lOth day(0.6860±0.0811)and 7th day(2.0351±0.1600),respectively(P<0.05).ASK1 protein expressions resulting Key words: Hyperoxia; Infant,premature; Thioredoxins; Thioredoxin-disulfide reductase; Lung diseases

Expression of Apoptosis Signal-Regulating Kinase 1 in Mouse Spinal Cord Under Chronic Mechanical Compression

To examine apoptosis signal cascade in neurons and oligodendrocytes under the chronic spinal cord compression of tiptoe-walking Yoshimura (TWY) mouse, which is model of progressive cervical cord compression. To clarify the biologic mechanisms of apoptosis, which may produce destructive changes in the spinal cord under chronic mechanical compression, with a resulting irreversible neurologic deficit. The stress-activated mitogen-activated protein kinase pathways including ASK1 transmitted apoptosis signals after acute spinal cord injury. Apoptosis in acute spinal cord injury induced both secondary degeneration around the site of injury and chronic demyelination. Chronic spinal cord compression showed myelin destruction, loss of axons, and oligodendrocytes in white matter, and loss of neurons in gray matter. Apoptosis associated with chronic spinal cord compression contributes to these changes. However, the biologic mechanisms of apoptosis in the spinal cord under chronic mechanical compression remain unclear. We examined the expression of phosphorylated-apoptosis signal-regulating kinase 1 (ASK1), phosphorylated-c-Jun N-terminal kinase (JNK), phosphorylated-p38 mitogen-activated protein kinase (p38), and activated caspase-3 immunohistologically in TWY mice, an animal model of progressive cervical spinal cord compression, since the ASK1-JNK and -p38 signaling cascades participate in the signaling pathway leading to apoptosis in neural tissue and neuronal culture. Double immunohistochemistry for phosphorylated-ASK1, phosphorylated-JNK, phosphorylated-p38, activated-caspase3, and cell-specific markers confirmed the presence of apoptosis signals in both neurons and oligodendrocytes in compressed spinal cord cells. We found that mitogen-activated protein kinase pathways including ASK1, JNK, and p38 were activated in destructive spinal cord under chronic compression.