C-jun mRNA Research Articles

Cyclovirobuxinum D alleviates cardiac hypertrophy in hyperthyroid rats by preventing apoptosis of cardiac cells and inhibiting the p38 mitogen-activated protein kinase signaling pathway.

To investigate the underlying mechanisms of cyclovirobuxinum D (Cvb-D) on alleviating cardiac hypertrophy in rats. Sprague-Dawley rats were randomly divided into 5 groups: control group; levothyroxine-induced cardiac hypertrophy group (model); levothyroxine-induced cardiac hypertrophy + Cvb-D group (Cvb-D); levothyroxine-induced cardiac hypertrophy + captopril group (captopril); levothyroxine-induced cardiac hypertrophy + SB203580 group (SB203580), n=10 for each group. Rats were daily administered the respective drugs continuously for14 days by gastric gavage. A rat model of cardiac hypertrophy was established by intraperitoneal injection of levothyroxine to investigate whether Cvb-D protects against cardiac hypertrophy by inhibiting the p38 mitogen-activated protein kinase (MAPK) signaling pathway and preventing apoptosis of cardiac cells. Treatment with Cvb-D significantly deceased left ventricle hypertrophy, improved the histopathology, hemodynamic conditions, and cardiac function in rats with cardiac hypertrophy. Compared with the normal control group, in rats with cardiac hypertrophy, expression of bax in the heart and phospho-p38 MAPK protein levels were significantly up-regulated (P<0.01 or 0.05), whereas the bcl-2 protein level was down-regulated (P<0.01). In contrast, Cvb-D treatment reversed the changes in bax and phospho-p38 MAPK protein levels but increased the bcl-2 protein level (P<0.01 or 0.05), and these effects were similar to those of captopril and SB203580 (a specific p38MAPK inhibitor) treatment. Furthermore, both Cvb-D, captopril and SB203580 reduced mRNA expression of p38α, p38β, c-fos, and c-jun mRNA, and Cvb-D had a stronger effect (P<0.01). These results demonstrate that Cvb-D protects against cardiac hypertrophy, which is possibly mediated by prevention of cardiac cell apoptosis and inhibition of the p38MAPK signaling pathway.

Activation of c-Jun predicts a poor response to sorafenib in hepatocellular carcinoma: Preliminary Clinical Evidence.

We determined the mitogen-activated protein kinase (MAPK) gene expression profile of acquired resistance in sorafenib-sensitive hepatocellular carcinoma (HCC) cells and aimed to identify c-Jun as an important molecule mediating the efficacy of sorafenib. Differences in gene expression of the MAPK signaling between untreated and sorafenib-treated HCC cell lines were investigated using real-time polymerase chain reaction array. Western blot and real-time PCR further evaluated the expression of c-Jun. Pathological specimens from 50 patients with advanced HCC were collected to measure p-c-Jun expression. Sorafenib-resistant HCC cells demonstrated greater levels of basal c-Jun mRNA and protein compared with sorafenib-sensitive HCC cells. Sorafenib activated p-c-Jun in a dose- and time-dependent manner in PLC/PRF/5 and MHCC97H cell lines. Decreased expression levels of 6 genes after sorafenib treatment suggested a robust inhibitory impact of sorafenib on MAPK signaling in HCC cells. c-Jun and p-c-Jun expression levels were inversely correlated with the efficacy of sorafenib; a high expression level of p-c-Jun was associated with resistance to sorafenib and poor overall survival in patients with clinical HCC. p-c-Jun may act as a biomarker for predicting responses of sorafenib treatment, thus advocating targeting of JNK/c-Jun signaling as an optimal therapeutic strategy in a subset of HCC.

Abstract P5-08-17: A study of c-Jun N-terminal kinase (JNK) and c-Jun as biomarkers in early breast cancer

Abstract Background The AP-1 transcription factor c-Jun is a key downstream target of c-Jun N-terminal kinase (JNK) which mediates intracellular signalling associated with a variety of cellular functions. The JNK pathway in breast cancer (BC) can be attenuated via loss of function mutations in MAPK kinases as well as via PIK3CA mutations; however, there is contradictory information about the role of JNK pathway and its clinical implications in BC. Methods In the current study, the clinicopathological implications of JNK and JUN mRNA expressions were evaluated in multiple independent BC datasets: a) Training-set (Uppsala cohort; n=249), b) Test-set (human genome atlas database; n=540), c) External validation-set (METABRIC cohort; n=1952) and d) Multicentre pooled databases (n=5530). The clinicopathological associations of their phosphorylated proteins (p-Jnk and p-c-Jun) were assessed in the Nottingham Tenovus Primary BC Series (n= 1650) and in an ER negative cohort (n=450). Results Both JNK and c-JUN mRNA high expressions were significantly associated with PAM50-Luminal A and ER+/HER2-/low proliferation molecular BC subtypes, tubular/lobular types, and integrative molecular clusters 4 (IntClust.4), ps&amp;lt;0.001. Whereas BC that had both low JNK and c-JUN mRNA, were significantly associated with large tumour size, high grade, absence of hormonal receptors (HR), HER2 overexpression, PAM50 HER2 and PAM50 Basal molecular subtypes, and IntClust.1, 9 and 10 BCs; ps&amp;lt;0.001. There was a significant positive correlations between p-Jnk and p-c-Jun protein levels (p&amp;lt;0.0001), however; our data suggested that differential p-Jnk/p-c-Jun expression may influence BC phenotypes. BC with p-Jnk-ve/p-c-Jun-ve were associated with the most aggressive phenotypes including largest tumour size, highest grade, lympho-vascular invasion, absence of HR, basal-like-phenotype, HER2 overexpression, and loss of double strand , single stand and base excision DNA repair proteins (ps&amp;lt;0.0001). In addition p-Jnk-ve/ pc-Jun-ve phenotype was associated with the lowest levels of p-38, ATF2, and p-ATF2; ps&amp;lt;0.001. Interestingly, low levels of either c-JUN-mRNA or pc-Jun protein, was associated with, PAM50-luminal B, epithelial mesenchymal transition and TP53 mutation and loss of its downstream proteins such as MDM2, MDM4, Bcle2 and p21; ps&amp;lt;0.05. JNK+ (mRNA and p-Jnk) and c-JUN+ (mRNA and p-c-Jun) individually were associated with prolonged BC specific survival (ps&amp;lt;0.001). Multivariate cox regression models that included other validated prognostic factors and therapies revealed that c-JUN-mRNA (Uppsala: p=0.005 and METABIRIC: p=0.036) and p-c-Jun (HR: 0.69; 95% CI = 0.55-0.88; p=0.002) were independently associated with clinical outcome. Furthermore, in ER+ high risk BC, exposure to tamoxifen was associated with decreased risk of death from BC in those patients with p-c-Jun-ve BC (HR: 0.65; 95% CI: 0.45-0.95; p=0.025). Conclusion JNK and c-JUN mRNA as well as p-Jnk and p-c-Jun protein levels are associated with luminal BC, with p-c-Jun being found to be an independent prognostic factor. The interaction between p-Jnk, p-c-Jun and TP53 mutation could predict response to endocrine therapy in ER+ BC. The role of the transcriptionally active form of c-JUN warrants further investigation with regard to its role in BC. Citation Format: Palmieri C, Rudraraju B, Giannoudis A, Moore D, Shaw J, Chan S, Ellis IO, Caldas C, Coombes RC, Carroll JS, Ali S, Abdel-Fatah TMA. A study of c-Jun N-terminal kinase (JNK) and c-Jun as biomarkers in early breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-17.

Abstract 2114: Targeting c-Jun and c-Fos using microRNA's has a potential in contesting melanoma

Abstract Background and Aim: MicroRNAs (miRNAs or miRs) are an abundant class of short (17 to 25 nt) non-coding single-stranded RNAs that are significant controllers of cellular gene expression. Mature miRNA is created from long primary genomic transcripts (pri-miRNA), which are processed in the nucleus. The resulting 60-80-nucleotide precursor miRNAs (pre-miRNAs) are transferred to the cytoplasm and are treated to produce a short RNA duplex. One strand of the duplex is degraded, the remaining single-stranded miRNA molecule associates with members of the Argonaute protein family, to form the RNA-induced silencing complex (RISC). RISC plus the guide miRNA bind to the 3′untranslated region (3′-UTR) of the targeted mRNA and induce gene down-regulation by mRNA cleavage or translational repression. Serum stimulation of cancer cells has been shown to prompt positive regulatory transcription factors, such as c-Jun, c-Fos and Egr-1, as well as increasing transcriptional repressors such as YY1, NAB2 and GCF2. These transcription factors play critical parts in the early stimulation of cancer disease phenotypes and are prime targets for clarifying disease pathways and as therapeutic candidates. The identification of microRNAs regulating the mRNAs of transcription factors will improve our understanding of the mechanism of disease gene stimulation and subsequent pathway initiation leading to cancer disease phenotypes. MicroRNAs that ablate disease phenotypes are possible pharmaceutical therapies. Our aim was to identify and profile microRNAs that interact with the mRNA of transcription factors such as c-Jun and c-Fos. Results: We measured the expresion of c-Jun and c-Fos at the mRNA and protein levels (time course 0 to 6 hrs) in MM200 (human melanoma). We measured the expression of two microRNAs, miR155 and miR125b, which have been reported as playing a role in regulating c-Jun and c-Fos in Dermal Fibroblasts and melanoma cells (Song, Liu et al. Cellular Physiology and Biochemistry 2012 and Kappelmann, Kuphal et al. Oncogene 2013). We show that miR-155 and miR-125b expression is repressed following serum activation in these two cell lines (HEK293 and MM200). Loss of function and gain-of-function studies in these cancer cell lines, by supressing and overexpressing miR155/125b were performed. Subsquently, a target 3′UTR vs microRNA study was conducted that demonstrated that the miR-155 and miR-125b have the ability to recognize and repress c-Jun and c-Fos. Moreover, phenotypic studies such as migration, proliferation, cell cycle were performed. Conclusions: miR-155 and miR-125b are able to regulate c-Jun and c-Fos mRNAs and to inhibit their expression at the protein level. Citation Format: Ahmad M.N Alhendi, Leonel Prado-Lourenço, Noel Whitaker. Targeting c-Jun and c-Fos using microRNA's has a potential in contesting melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2114. doi:10.1158/1538-7445.AM2015-2114

Abstract 1505: Suppression of triple-negative breast cancer tumorigenesis by targeting cancer stem cells through JNK/Notch1 signaling inhibition

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis and lacks targeted therapies. Basal-like TNBC exhibits hyperactivated JNK (c-Jun N-terminal kinase). JNK plays a vital role in malignant transformation of different cancers. However, it is unknown whether JNK signaling is a clinically relevant target in TNBC. Here, we tested the hypotheses that JNK signaling plays a fundamental role in TNBC pathogenesis by promoting self-renewal of cancer stem cells (CSCs). Methods: The role of JNK signaling in TNBC pathogenesis was determined by examining the effects of JNK signaling inhibition using siRNA or JNK-IN-8 (an ATP-competitive JNK inhibitor) and the effects of JNK overexpression on growth, migration, and invasion of TNBC cells using cell proliferation, migration, and invasion assays. CSC regulation by JNK signaling was examined using mammosphere-forming assay. The effect of JNK signaling inhibition on tumor growth was assessed using a xenograft mouse model. The downstream molecules involved in JNK signaling-mediated CSC regulation were identified using RT-PCR stem array analysis. Association between JNK and c-Jun was analyzed using a reverse phase protein array dataset of TNBC samples (n = 81). Disease-free survival (DFS) probability in TNBC patients was analyzed by the Kaplan-Meier method using a cDNA microarray dataset (n = 79). Results: Knockdown of JNK1 or JNK2 led to significant reductions in growth, migration, and invasion of TNBC cells, mammosphere formation, and ALDH1+ CSC subpopulation. Similar results were obtained when JNK signaling was inhibited using JNK-IN-8. In contrast, overexpression of JNK1 or JNK2 enhanced these cellular activities. Importantly, JNK signaling inhibition by JNK-IN-8 (25 mg/kg) reduced tumor growth by 60.88% (P&amp;lt;0.001) in a xenograft mouse model. RT-PCR stem array analysis indicated the involvement of Notch1 in JNK signaling-mediated CSC regulation; luciferase reporter assay showed that JNK regulated Notch1 expression at the transcriptional level via c-Jun, a transcription factor and a substrate of JNK. Knockdown of c-Jun or Notch1 reduced cell growth, mammosphere formation, and ALDH1+ CSC subpopulation; overexpression of c-Jun or Notch1 blocked the inhibitory effect of JNK-IN-8 on mammosphere formation. These results suggest that JNK signaling promotes CSC self-renewal through up-regulation of Notch1 via c-Jun. Furthermore, in TNBC patients, a high level of phosphorylated JNK was associated with a high level of phosphorylated c-Jun (P = 3×10-8); low c-Jun mRNA level was associated with better DFS (P = 0.03); and there was a tendency (P = 0.17) toward better DFS with low JNK1 mRNA level but not with low JNK2 mRNA level. Conclusion: JNK signaling regulates TNBC tumorigenesis by promoting CSC self-renewal via Notch1 signaling. JNK-IN-8 is a promising therapeutic agent for TNBC by targeting JNK/Notch1 signaling. Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupunganti, Tinghu Zhang, Takahiro Kogawa, Gaurav B. Chauhan, Dionysios N. Giannoukos, Yuan Qi, Debu Tripathy, Nathanael S. Gray, Kevin N. Dalby, Chandra Bartholomeusz, Naoto T. Ueno. Suppression of triple-negative breast cancer tumorigenesis by targeting cancer stem cells through JNK/Notch1 signaling inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2015-1505

AP-1 Transcription Factors c-FOS and c-JUN Mediate GnRH-Induced Cadherin-11 Expression and Trophoblast Cell Invasion.

GnRH is expressed in first-trimester human placenta and increases cell invasion in extravillous cytotrophoblasts (EVTs). Invasive phenotypes have been reported to be regulated by transcription factor activator protein 1 (AP-1) and mesenchymal cadherin-11. The aim of our study was to investigate the roles of AP-1 components (c-FOS/c-JUN) and cadherin-11 in GnRH-induced cell invasion in human EVT cells. Phosphorylated c-FOS and phosphorylated c-JUN were detected in the cell column regions of human first-trimester placental villi by immunohistochemistry. GnRH treatment increased c-FOS, c-JUN, and cadherin-11 mRNA and protein levels in immortalized EVT (HTR-8/SVneo) cells. Moreover, GnRH treatment induced c-FOS and c-JUN protein phosphorylation and nuclear accumulation. Pretreatment with antide, a GnRH antagonist, attenuated GnRH-induced cadherin-11 expression. Importantly, basal and GnRH-induced cadherin-11 expression and cell invasion were reduced by small interfering RNA-mediated knockdown of c-FOS, c-JUN, and cadherin-11 in HTR-8/SVneo cells. Our results suggest that GnRH induces the expression and phosphorylation of the AP-1 transcription factors c-FOS and c-JUN in trophoblast cells, which contributes to GnRH-induced elevation of cadherin-11 expression and cell invasion.

Immediate expression of c-fos and c-jun mRNA in a model of intestinal autotransplantation and ischemia-reperfusion in situ

OBJECTIVE:Intestinal ischemia-reperfusion injury occurs in several clinical conditions and after intestinal transplantation. The aim of the present study was to investigate the phenomena of apoptosis and cell proliferation in a previously described intestinal ischemia-reperfusion injury autograft model using immunohistochemical markers. The molecular mechanisms involved in ischemia-reperfusion injury repair were also investigated by measuring the expression of the early activation genes c-fos and c-jun, which induce apoptosis and cell proliferation.MATERIALS AND METHODS:Thirty adult male Wistar rats were subjected to surgery for a previously described ischemia-reperfusion model that preserved the small intestine, the cecum and the ascending colon. Following reperfusion, the cecum was harvested at different time points as a representative segment of the intestine. The rats were allocated to the following four subgroups according to the reperfusion time: subgroup 1: 5 min; subgroup 2: 15 min; subgroup 3: 30 min; and subgroup 4: 60 min. A control group of cecum samples was also collected. The expression of c-fos, c-jun and immunohistochemical markers of cell proliferation and apoptosis (Ki67 and TUNEL, respectively) was studied.RESULTS:The expression of both c-fos and c-jun in the cecum was increased beginning at 5 min after ischemia-reperfusion compared with the control. The expression of c-fos began to increase at 5 min, peaked at 30 min, and exhibited a declining tendency at 60 min after reperfusion. A progressive increase in c-jun expression was observed. Immunohistochemical analyses confirmed these observations.CONCLUSION:The early activation of the c-fos and c-jun genes occurred after intestinal ischemia-reperfusion injury, and these genes can act together to trigger cell proliferation and apoptosis.

Progesterone Receptor Isoforms A and B in Pancreatic Neuroendocrine Tumor

Background: Pancreatic neuroendocrine tumors (PNETs) have been reported to express progesterone receptor (PR), and its expression has been demonstrated to be a favorable prognostic factor in these patients. We examined the status of the PR isoforms PRA and PRB in the human PNET cell line and their association with cell proliferation of the tumor cells, which is closely related to the clinical outcome of PNET patients. Methods: Quantitative RT-PCR and cell proliferation assays were performed following treatment with progesterone and RU-486 as a PR antagonist in nontransfected and PRA-transfected cells of the NET cell line QGP-1, which expresses PRB in its native state. PRA, PRB and cyclin D1 (CCND1) were immunolocalized in 87 PNET cases, and the results were compared with clinicopathological parameters. Results: CCND1, c-Fos and c-Jun mRNA levels were all significantly increased by treatment with progesterone in QGP-1 cells with PRB expression compared with PRA-transfected cells (p = 0.02, p = 0.007 and p = 0.001, respectively). The proliferative activity of QGP-1 cells with PRB expression was also significantly stimulated by the administration of progesterone (p = 0.008). PRA immunoreactivity was significantly decreased in higher-grade PNETs (p = 0.04), whereas CCND1 was significantly elevated in higher-grade PNETs (p = 0.035). Conclusion: The results of the present study demonstrate that PRA could play an inhibitory role in the cell proliferation of PNETs, possibly by inhibiting PRB-mediated signals in the presence of progesterone, which could result in decreased CCND1 expression. In addition, the status of PRA in tumor cells could be a prognostic factor in PNETs.

Voxel-based analysis of the immediate early gene, c-jun, in the honey bee brain after a sucrose stimulus.

Immediate early genes (IEGs) have served as useful markers of brain neuronal activity in mammals, and more recently in insects. The mammalian canonical IEG, c-jun, is part of regulatory pathways conserved in insects and has been shown to be responsive to alarm pheromone in honey bees. We tested whether c-jun was responsive in honey bees to another behaviourally relevant stimulus, sucrose, in order to further identify the brain regions involved in sucrose processing. To identify responsive regions, we developed a new method of voxel-based analysis of c-jun mRNA expression. We found that c-jun is expressed in somata throughout the brain. It was rapidly induced in response to sucrose stimuli, and it responded in somata near the antennal and mechanosensory motor centre, mushroom body calices and lateral protocerebrum, which are known to be involved in sucrose processing. c-jun also responded to sucrose in somata near the lateral suboesophageal ganglion, dorsal optic lobe, ventral optic lobe and dorsal posterior protocerebrum, which had not been previously identified by other methods. These results demonstrate the utility of voxel-based analysis of mRNA expression in the insect brain.

Low Frequency Repetitive Transcranial Magnetic Stimulation Modulates Brain Functional Connectivity and Functional Network Connectivity in Cerebral Palsy Patients

s / Brain Stimulation 8 (2015) 412e427 415 K groups showed a similar IEG expression profile except that K group showed greater upregulation of c-jun mRNA expression in the dentate gyrus and hippocampal CA1-3 region. Because c-jun is involved in neuronal cell death in adult brain, the increased c-jun expression suggests enhanced neuronal apoptosis in the K+Q group. Further study should determine if quenching blocks kindlinginduced seizure development by selective degeneration of kindling-activated neurons and/or pathological pathways.

CJun and CREB2 in the postsynaptic neuron contribute to persistent long-term facilitation at a behaviorally relevant synapse.

Basic region leucine zipper (bZIP) transcription factors regulate gene expression critical for long-term synaptic plasticity or neuronal excitability contributing to learning and memory. At sensorimotor synapses of Aplysia, changes in activation or expression of CREB1 and CREB2 in sensory neurons are required for long-term synaptic plasticity. However, it is unknown whether concomitant stimulus-induced changes in expression and activation of bZIP transcription factors in the postsynaptic motor neuron also contribute to persistent long-term facilitation (P-LTF). We overexpressed various forms of CREB1, CREB2, or cJun in the postsynaptic motor neuron L7 in cell culture to examine whether these factors contribute to P-LTF. P-LTF is evoked by 2 consecutive days of 5-HT applications (2 5-HT), while a transient form of LTF is produced by 1 day of 5-HT applications (1 5-HT). Significant increases in the expression of both cJun and CREB2 mRNA in L7 accompany P-LTF. Overexpressing each bZIP factor in L7 did not alter basal synapse strength, while coexpressing cJun and CREB2 in L7 evoked persistent increases in basal synapse strength. In contrast, overexpressing cJun and CREB2 in sensory neurons evoked persistent decreases in basal synapse strength. Overexpressing wild-type cJun or CREB2, but not CREB1, in L7 can replace the second day of 5-HT applications in producing P-LTF. Reducing cJun activity in L7 blocked P-LTF evoked by 2 5-HT. These results suggest that expression and activation of different bZIP factors in both presynaptic and postsynaptic neurons contribute to persistent change in synapse strength including stimulus-dependent long-term synaptic plasticity.

IL-6, IL-10, c-Jun and STAT3 expression in B-CLL.

Chronic lymphocytic leukemia is characterized by the accumulation of functionally abnormal, monoclonal B lymphocytes in the peripheral blood, bone marrow, lymph nodes and spleen, resulting in a reduction count of normal immunocompetent cells and their impaired immune function. The defect in transmission of signals from various types of extracellular receptors, leading to aberrant cytokines and transcription factors gene expression, may underlie the basis of immune failure in B-CLL. The aim of the study was to assess of IL-6, IL-10, c-Jun, and STAT3 expression. In response to antigenic stimulation IL-6, IL-10, c-Jun, and STAT3 proteins induce mutual activity. The subject of the study was subpopulations of leukemic lymphocytes (CD5+ CD19+) and CD19+ B cells from healthy donors (control group). Our results provide evidence that the regulation of IL-6, IL-10, c-Jun, and STAT3 gene expression in CLL B cells is clearly different from normal B lymphocytes. In B-CLL STAT3 expression in unstimulated lymphocytes is significantly higher (p<0.0001) compared with normal subpopulation of B cell. In contrast, IL-6, IL-10, and c-Jun mRNA expressions are statistically lower in B-CLL in comparison with the control group, in all cases (p<0.0001). When analyzing the relationship between c-Jun expression and B-CLL stage according Rai we revealed decreasing c-Jun expression, both at the mRNA and protein levels, along with advancing stage of disease.

Sphingosine-1-phosphate mediates COX-2 expression and PGE2 /IL-6 secretion via c-Src-dependent AP-1 activation.

Sphingosine-1-phosphate (S1P) has been shown to regulate cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2 ) expression and IL-6 secretion in various respiratory diseases. However, the mechanisms underlying S1P-induced COX-2 expression and PGE2 production in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here we demonstrated that S1P markedly induced COX-2 expression. S1P also induced PGE2 and IL-6 secretion which were reduced by the inhibitors of COX-2 (NS-398 and celecoxib). Pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), PYK2 (PF431396), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, PYK2, p38, p42, JNK2, c-Jun, or c-Fos reduced S1P-induced COX-2 expression and PGE2 /IL-6 secretion. Moreover, S1P induced c-Src, PYK2, p42/p44 MAPK, JNK1/2, p38 MAPK, and c-Jun phosphorylation. We observed that S1P-induced p42/p44 MAPK and JNK1/2, but not p38 MAPK activation was mediated via a c-Src/PYK2-dependent pathway. S1P also enhanced c-Fos, but not c-Jun mRNA and protein expression and the AP-1 promoter activity. S1P-induced c-Fos mRNA and protein expression, c-Jun phosphorylation, and AP-1 promoter activity was reduced by W123, CAY10444, PP1, PF431396, U0126, SP600125, or SB202190. These results demonstrated that S1P-induced COX-2 expression and PGE2 /IL-6 generation was mediated through S1PR1/3/c-Src/PYK2/p42/p44 MAPK- or JNK1/2- and S1PR1/3/c-Src/p38 MAPK-dependent AP-1 activation.

Effects of metallothionein-3 expression on biological behaviors of A549 lung cancer cells

Objective To study the efects of metallothionein-3 (MT-3) expression on biological behaviors of A549 lung cancer cells.Methods The anti-proliferation and the cytotoxicity of MT-3 on A549 cells were detected by methyl thiazol tetrazolium (MTT) assay.The MT-3 expression was observed in A549 cells,and the mRNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR).The morphological changes of the apoptotic cells were observed by invert microscope,hematoxylin and eosin (HE) stain,transmission electron microscope and acridine orange/ethidium bromide (AO/EB) double fluorescent staining.The data were analyzed by using SPSS 14.0 statistical software.Results The MT-3 expression in A549 cells transfected with MT-3 gene was confirmed by RT-PCR and immunocytochemical method,respectively.Compared with the control group,-fos and c-jun mRNA levels were decreased significantly in PEFMT-3 transfected A549 cells (x2 =2.536,P ＜ 0.05).MTT assay revealed that the inhibitory rate was enhanced obviously with the increase of effect/target rate and extension of time (x2 =2.795,P ＜0.05).The apoptosis or necrosis of A549 cells was morphologically observed after lung cancer cells were induced by MT-3 for 24 h.Conclusion MT-3 could inhibitit proliferation of A549 cells and promote apoptosis of A549 cells probably by negatively regulating the signaling pathway to ruduce the expression of c-fos and c-jun. Key words: Lung cancer; Metallothionein-3; Apoptosis

Abstract 750: JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative breast cancer

Abstract Background: Patients with triple-negative breast cancer (TNBC) have a very poor prognosis due to metastasis and limited targeted therapies. Basal-like TNBC has hyperactivated JNK (c-Jun N-terminal kinase). JNK plays a vital role in malignant transformation in different cancers and self-renewal and maintenance of glioma cancer stem cells (CSCs). However, it is not known whether JNK signaling is a clinically relevant target in TNBC. We tested the hypotheses that JNK signaling plays a fundamental role in TNBC tumorigenesis and metastasis by promoting CSCs and that suppressing JNK signaling using JNK-IN-8 inhibits tumorigenicity and metastasis of TNBC cells. Methods: We examined the effects of JNK knockdown or overexpression on cellular functions of TNBC cells and CSC subpopulations in vitro. We also evaluated the anti-TNBC efficacy of JNK-IN-8, an ATP-directed covalent small molecule inhibitor of JNK that specifically binds the Cys116 on JNK. Results: In patients with TNBC (n=79), low c-Jun mRNA level was associated with better disease-free survival (DFS) (P=0.025), and there was a tendency (P=0.167) toward better DFS with low JNK1 mRNA level. No similar trend was observed for JNK2. These results suggest that JNK1 and c-Jun may have clinical significance in TNBC. Knockdown of JNK1 or JNK2 led to reductions in mammosphere formation, migration, and invasion in HCC70 and SUM149 cells. In contrast, overexpression of constitutively active MKK7-JNK1 or -JNK2 enhanced these cellular activities and increased the ALDH1+ subpopulation. These data indicate a potential role of JNK signaling in regulating TNBC tumorigenesis and metastasis, possibly by promoting CSCs. JNK-IN-8 suppressed proliferation, anchorage-independent growth, migration, and invasion in HCC70 and SUM149 cells. In addition, JNK-IN-8 also reduced mammosphere formation and the CD44+/CD24- and ALDH1+ subpopulations, suggesting that JNK-IN-8 can inhibit JNK signaling-mediated CSC self-renewal and maintenance. Furthermore, overexpression of mutant JNK1- or JNK2-C116S, in which Cys116 was replaced with Ser116, partially abolished the inhibitory effects of JNK-IN-8 on cellular functions, suggesting that JNK-IN-8 executes antitumor activity by specifically targeting JNK. Lastly, JNK inhibition by JNK-IN-8 led to down-regulation of Notch1, suggesting involvement of Notch1 in JNK signaling-mediated CSC regulation. Conclusion: JNK signaling regulates TNBC tumorigenesis and metastasis by promoting CSCs partly via Notch signaling. Our data demonstrate that JNK is an important therapeutic target in TNBC, providing a rationale for further preclinical and clinical development of a JNK-targeted therapy for TNBC. JNK-IN-8 is an effective inhibitor targeting JNK signaling in TNBC. Our studies justify searching for JNK isoform selective inhibitors since there is evidence that JNK1 and JNK2 can have opposing roles in certain settings. Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupunganti, Tinghu Zhang, Takahiro Kogawa, Gaurav B. Chauhan, Nathanael S. Gray, Chandra Bartholomeusz, Kevin N. Dalby, Naoto T. Ueno. JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 750. doi:10.1158/1538-7445.AM2014-750

Inhibitory effects of 2,3,4',5-tetrahydroxystilbene-2-O-β-D-glucoside on angiotensin II-induced proliferation of vascular smooth muscle cells.

To investigate the effect of 2,3,4',5-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component extracted from the root of Polygonum multiflorum, on angiotensin II (Ang II)-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and to identify the potential mechanism. Cell proliferation and cell cycle were determined by cell counting, 5-bromo-2'-deoxyuridine incorporation assay, proliferating cell nuclear antigen protein expression and flow cytometry. Levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), mitogenic extracellular kinase 1/2 (MEK1/2) and Src in VSMCs were measured by Western blot. The expression of c-fos, c-jun and c-myc mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR). Intracellular reactive oxygen species (ROS) was measured by fluorescence assay. TSG significantly inhibited Ang II-induced VSMCs proliferation and arrested cells in the G /S checkpoint (P<0.05 or P<0.01). TSG decreased the levels of phosphorylated ERK1/2, MEK1/2 and Src in VSMCs (P<0.05 or P<0.01). TSG also suppressed c-fos, c-jun and c-myc mRNA expression <0.05 or P<0.01). In addition, the intracellular ROS was reduced by TSG (P<0.01). TSG inhibited Ang II-induced VSMCs proliferation. Its antiproliferative effect might be associated with down-regulation of intracellular ROS, followed by the suppression of the Src-MEK1/2-ERK1/2 signal pathway, and hence, blocking cell cycle progression.

Differential expression of AP-1 proteins in human myometrium after spontaneous term labour onset

ObjectivesThe aims of this study were (i) to determine the localisation of activator protein (AP)-1 family members (cFos, FosB, cJun, JunB and JunD) in human myometrium; and (ii) to determine the effect of human term labour on the expression of AP-1 family of transcription factors in myometrium. Study designThis localised the AP-1 family members cFos, FosB, cJun, JunB and JunD in human myometrium was performed by immunohistochemistry. The effect of term labour on the expression of these family members at the mRNA and protein level was assessed by qRT-PCR and Western blotting, respectively. The effect of pro-inflammatory stimuli on AP-1 transcriptional activity was assessed using a luciferase assay in primary human myometrial cells. ResultsImmunohistochemical expression of cFos, FosB, cJun, JunB and JunD were all present in human myometrial tissue and displayed cytoplasmic staining. FosB and JunD also displayed nuclear staining. Term labour was associated with an increase in cFos and JunB mRNA and protein expression. On the other hand, JunD mRNA and protein expression was decreased with labour. FosB mRNA was increased with labour, but there was no change at the protein level. There was no change in cJun mRNA or protein expression. AP-1 transcriptional activity was increased in human myometrial cells by the pro-inflammatory cytokine TNF-α. There was, however, no effect of the bacterial products lipopolysaccharide (LPS; TLR4 ligand), iE-DAP (NOD1 ligand), MDP (NOD2 ligand), FSL-1 (TLR2 ligand) or flagellin (TLR5 ligand) on AP-1 transcriptional activity. ConclusionThis study shows that human labour is associated with changes in AP-1 family members. Further studies are required to determine the exact role of the AP-1 family members in myometrium.

Pioglitazone reduces angiotensin II-induced COX-2 expression through inhibition of ROS production and ET-1 transcription in vascular cells from spontaneously hypertensive rats.

Glitazones have anti-inflammatory properties by interfering with the transcription of proinflammatory genes, such as cyclooxygenase (COX)-2, and with ROS production, which are increased in hypertension. This study analyzed whether pioglitazone modulates COX-2 expression in hypertension by interfering with ROS and endothelin (ET)-1. In vivo, pioglitazone (2.5 mg·kg(-1)·day(-1), 28 days) reduced the greater levels of COX-2, pre-pro-ET-1, and NADPH oxidase (NOX) expression and activity as well as O2 (·-) production found in aortas from spontaneously hypertensive rats (SHRs). ANG II increased COX-2 and pre-pro-ET-1 levels more in cultured vascular smooth muscle cells from hypertensive rats compared with normotensive rats. The ETA receptor antagonist BQ-123 reduced ANG II-induced COX-2 expression in SHR cells. ANG II also increased NOX-1 expression, NOX activity, and superoxide production in SHR cells; the selective NOX-1 inhibitor ML-171 and catalase reduced ANG II-induced COX-2 and ET-1 transcription. ANG II also increased c-Jun transcription and phospho-JNK1/2, phospho-c-Jun, and p65 NF-κB subunit nuclear protein expression. SP-600125 and lactacystin, JNK and NF-κB inhibitors, respectively, reduced ANG II-induced ET-1, COX-2, and NOX-1 levels and NOX activity. Pioglitazone reduced the effects of ANG II on NOX activity, NOX-1, pre-pro-ET-1, COX-2, and c-Jun mRNA levels, JNK activation, and nuclear phospho-c-Jun and p65 expression. In conclusion, ROS production and ET-1 are involved in ANG II-induced COX-2 expression in SHRs, explaining the greater COX-2 expression observed in this strain. Furthermore, pioglitazone inhibits ANG II-induced COX-2 expression likely by interfering with NF-κB and activator protein-1 proinflammatory pathways and downregulating ROS production and ET-1 transcription, thus contributing to the anti-inflammatory properties of glitazones.

Role of activator protein-1 in electro-acupuncture-induced up-regulation of heme oxygenase-1 expression in lung tissues in a rabbit model of endotoxic shock

Objective To evaluate the role of activator protein-1 （AP-1 ） in electro-acupuncture （EA ）-induced up-regulation of heme oxygenase-1 （HO-1 ） expression in lung tissues in a rabbit model of endotoxic shock .Methods Seventy healthy male New Zealand white rabbits ,aged 2 months ,weighing 1.5-2.0 kg ,were randomly divided into 7 groups （ n=10 each ） using a random number table ：normal control group （group C ） , endotoxic shock-induced acute lung injury （ALI ） group （group ALI ） , EA ＋ ALI group （group EL ） , non-acupoint＋EA＋ ALI group （group NEL ） , curcumin （HO-1 inhibitor ） group （group Cur ） , dimethyl sulfoxide group （group D ） ,and EA＋ALI＋curcumin group （group ELC ） .Bilateral 15 min EA stimulation of Zusanli and＆nbsp;Feishu （according to atlas of animals acupoints ） was performed （frequency 15Hz ） once a day for 5 consecutive days before lipopolysaccharide （LPS ） administration in EL and ELC groups ,while in group NEL ,EA stimulation was performed at non-acupoints located 0.5 cm lateral to Zusanli and Feishu acupoints with the same parameters . The animals were anesthetized with urethane and tracheostomized .The animals kept spontaneous breathing .Right internal carotid artery was cannulated for blood pressure monitoring . Ear vein was cannulated for drug administration .At 5 days after EA stimulation ,curcumin 25 mg/kg （in 0.5 ml of 0.1% dimethyl sulfoxide ） was injected in Cur and ELC groups ,0.1% dimethyl sulfoxide 0.5 ml was injected in D group ,and the equal volume of normal saline was given in the other groups .LPS 5 mg/kg （in 2 ml of 0.9% normal saline ） was injected intravenously at 30 min after administration in ALI ,EL ,NEL and ELC groups ,while the equal volume of normal saline was given in the other three groups .Endotoxic shock was confirmed by decrease in mean arterial pressure to 75% of the baseline value within 2 h after LPS injection .Blood samples were collected from the common carotid artery at 6 h after LPS or normal saline administration and the rabbits were then sacrificed .The lungs were removed for microscopic examination and the pathological changes were scored .The wet/dry lung weight ratio （W/D ratio ） was calculated .The malondialdehyde （MDA ） content and superoxide dismutase （SOD ） activity in lung tissues were detected ,and the expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun in lung tissues was determined by Western blot .Results Compared with group C ,the pathological score ,W/D ratio and MDA content were significantly increased ,and the SOD activity was decreased in ALI ,EL ,NEL and ELC groups ,the expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun was up-regulated in groups ALI ,EL and NEL （ P0.05） .There was no significant difference in the parameters mentioned above between Cur and D groups （ P〉0.05 ） .Compared with group ALI ,the pathological score ,W/D ratio and MDA content were significantly decreased ,and SOD activity was increased ,and the expression of HO-1 mRNA and HO-1 was up-regulated in EL and ELC groups （ P0.05） .The pathological score ,W/D ratio and MDA content were significantly higher ,and the SOD activity and expression of HO-1 mRNA ,HO-1 ,c-Jun mRNA and c-Jun were lower in group ELC than in group EL （ P〈0.05） .Conclusion EA up-regulates HO-1 expression through activating AP-1 during endotoxic shock-induced ALI in rabbits ,thus protecting the lung . Key words: Transcription factor AP-1; Electric stimulation therapy; Shock,septic; Lung; Heme oxygenase-1

Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression

Exposure to ultraviolet (UV) light can cause skin photoaging, which is associated with upregulation of matrix metalloproteinases (MMPs) and downregulation of collagen synthesis. It has been reported that MMPs, especially MMP-1, MMP-3 and MMP-9, decrease the elasticity of the dermis by degrading collagen. In this study, we assessed the effects of Pinus densiflora extract (PDE) on photoaging and investigated its mechanism of action in human skin fibroblast (Hs68) cells after UVB exposure using real-time polymerase chain reaction, Western blot analysis, and enzymatic activity assays. PDE exhibited an antioxidant activity and inhibited elastase activities in vitro. We also found that PDE inhibited UVB-induced cytotoxicity, MMP-1 production and expression of MMP-1, -3 and -9 mRNA in Hs68 cells. In addition, PDE decreased UVB-induced MMP-2 activity and MMP-2 mRNA expression. Moreover, PDE prevented the decrease of type I procollagen mediated by exposure to UVB irradiation, an effect that is linked to the upregulation and downregulation of Smad3 and Smad7, respectively. Another effect of UV irradiation is to stimulate activator protein 1 (AP-1) activity via overexpression of c-Jun/c-Fos, which, in turn, upregulates MMP-1, -3, and -9. In this study, we found that PDE suppressed UV-induced c-Jun and c-Fos mRNA expression. Taken together, these results demonstrate that PDE regulates UVB-induced expression of MMPs and type I procollagen synthesis by inhibiting AP-1 activity and restoring impaired Smad signaling, suggesting that PDE may be useful as an effective anti-photoaging agent.