Protein Expression Levels Of p-STAT3 Research Articles

The LINC00501-HSP90B1-STAT3 positive feedback loop promotes malignant behavior in gastric cancer cells

Long non-coding RNAs (lncRNAs) have been implicated in gastric cancer (GC) carcinogenesis and progression. However, the role of LINC00501 in GC growth and metastasis remains unclear. In this study, we found that LINC00501 was frequently upregulated in GC cells and tissues and was closely related to adverse GC clinicopathological features. Aberrant overexpression of LINC00501 promoted GC cell proliferation, invasion, and metastasis both in vitro and in vivo. Mechanistically, LINC00501 stabilized client protein STAT3 from deubiquitylation by directly interacting with cancer chaperone protein HSP90B1. Furthermore, the LINC00501-STAT3 axis modulated GC cell proliferation and metastasis. In turn, STAT3 bound directly to the LINC00501 promoter and positively activated LINC00501 expression, thus forming a positive feedback loop, thereby accelerating tumor growth, invasiveness, and metastasis. In addition, LINC00501 expression was positively correlated with STAT3 and p-STAT3 protein expression levels in gastric clinical samples. Our results reveal that LINC00501 acts as an oncogenic lncRNA and that the LINC00501-HSP90B1-STAT3 positive feedback loop contributes to GC development and progression, suggesting that LINC00501 may be a novel potential biomarker and treatment target for GC.

Abietic acid attenuates LPS-induced acute lung injury by restoring Th17/Treg balance

Purpose: To investigate the mechanism of action and effects of abietic acid (AA) in a mouse acute lung injury (ALI) model.
 Methods: A mouse ALI model was established by lipopolysaccharide (LPS) induction. Lung tissues were examined for histological alterations and scored based on the degree of injury. Myeloperoxidase (MPO), IL-6, IL-1β, and TNF-α levels were measured by enzyme-linked immunosorbent assay (ELISA) while the numbers of Th17 and Treg cells were assessed by immunofluorescence. Protein expression levels of p-STAT3, p-STAT5, RORrt, and FOXP3 were analyzed by immunoblot assay. Expression of peroxisome proliferator-activated receptor γ (PPARγ) was assessed by immunoblot.
 Results: AA ameliorated LPS-induced lung injury in mice. Furthermore, AA ameliorated LPS-induced pneumonia in mice (p < 0.05) and restored Th17/Treg balance and Th17/Treg transcription factor expression that was altered by LPS induction. AA also activated PPARγ expression to restore Th17/Treg balance (p < 0.05).
 Conclusion: The results indicate that AA attenuates LPS-induced ALI in mice by restoring Th17/Treg balance. Thus, AA is a potential drug for the management of ALI; however, AA must first be evaluated in clinical studies.

Gut microbiota changes and biological mechanism in hepatocellular carcinoma after transarterial chemoembolization treatment.

Background and aimsIntestinal flora is closely associated with the occurrence and development of hepatocellular carcinoma (HCC). However, gut microbial changes and biological mechanisms in HCC after transarterial chemoembolization (TACE) treatment are rarely reported.MethodsWe evaluated changes in intestinal flora after TACE in rabbit HCC models and assessed the impact of these changes on the disease. Twenty-four rabbit VX2 HCC models were established and intestinal flora structures, intestinal barrier function, changes in blood lipopolysaccharide (LPS) levels, Toll-like receptor 4 (TLR4), Cyclooxygenase-2 (COX-2), and p-signal transducer and activator of transcription 3(p-STAT3) protein expression levels were studied after TACE treatment.ResultsCompared with healthy rabbits, the intestinal flora in HCC models exhibited structural changes; intestinal barrier function was decreased, and increased LPS levels entered the circulation. A short-term follow-up after TACE showed the procedure partially reversed the intestinal microflora disorder caused by the tumor: intestinal barrier and liver functions were improved, intestinal LPS levels in the blood were reduced, and liver metabolism toward LPS was enhanced. Correlation analyses of the first 75 significantly changed bacteria with clinical factors showed that harmful bacteria had decreased and beneficial bacteria increased. Blood LPS levels and downstream signaling molecule TLR4, COX-2, and p-STAT3 protein expression levels were reduced, which correlated with tumor drug resistance and invasion capabilities.ConclusionsWe first characterized gut microbiota changes and biological mechanisms in HCC after TACE treatment. Our data provide a theoretical research basis for TACE combined with an intestinal flora intervention and systemic chemotherapy.

The IL-6/STAT3 signaling pathway mediated by radiotherapy regulates the expression of PD-L1 in esophageal cancer cells

Objective: To explore the possible mechanism of radiotherapy regulating the expression of PD-L1 in esophageal carcinoma. Methods: Three esophageal cancer cell lines (Eca109, Kyse150, TE1) were irradiated with different doses of X-rays, and 6 Gy+ AG490 group was set. The mRNA expression of PD-L1 was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The protein expressions of PD-L1, STAT3, p-STAT3 were detected by western blotting and the protein level of IL-6 was detected by ELISA. Results: The mRNA expressions of PD-L1 in Eca109, Kyse150 and TE1 were 2.86±0.30, 960.01±21.27 and 106.78±6.67, higher than 1.07±0.15 in normal esophageal cell line HET-1A (P<0.01). The protein expressions of PD-L1 in Eca109, Kyse150 and TE1 were 0.091±0.036, 1.533±0.079 and 0.914±0.035, higher than 0.063±0.01 in normal esophageal cell line HET-1A (P<0.01). After 48 hours of 6 Gy irradiation, the protein expression levels of PD-L1 in Eca109, Kyse150 and TE1 were 0.135±0.007, 1.66±0.06 and 1.32±0.06, higher than 0.09±0.01, 1.21±0.05 and 0.93±0.03 of the 0 Gy group (P<0.01), while the protein expression levels of p-STAT3 in Eca109, Kyse150 and TE1 were 1.44±0.26, 0.75±0.04 and 1.92±0.17, higher than 0.18±0.05, 0.48±0.02 and 0.36±0.06 of the 0 Gy group (P<0.01). IL-6 protein expression increased significantly after different doses of irradiation (P<0.01). After the IL-6/STAT3 signaling pathway was blocked by the specific inhibitor AG490, the expressions of PD-L1 of Eca109, Kyse150 and TE1 in the 6 Gy+ AG490 groups were 0.11±0.03, 1.07±0.08 and 0.96±0.11, without significant differences of 0.09±0.01, 0.96±0.05 and 0.85±0.09 of the 0 Gy group (P>0.05), while the protein expressions of p-STAT3 were 0.76±0.11, 0.59±0.06 and 0.96±0.12, without significant differences of 0.67±0.08, 0.54±0.06 and 0.84±0.11 of the 0 Gy group (P>0.05). Conclusion: Radiotherapy may regulate the expression of PD-L1 in esophageal cancer cells through IL-6 / STAT3 signaling pathway.

Sesquiterpene lactones isolated from Carpesium abrotanoides L. by LC–MS combined with HSCCC inhibit liver cancer through suppression of the JAK2/STAT3 signaling pathway

Carpesium abrotanoides L. is rich in sesquiterpene lactones, the experiments of anti-tumor activity in vitro showed that total sesquiterpene lactone extracts exhibited the most cytotoxicity activities against HepG-2 cells (IC50 = 4.2 μg/mL). In order to isolated the active compounds quickly, the strategy of HPLC-ESI-Q-TOF-MS/MS combined with high-speed counter-current chromatography (HSCCC) was implemented. Three sesquiterpene lactone compounds, 2α,5α-dihydroxy-11αH-eudesma-4(15)-en-12,8β-olide (1), telekin (2), oxoeudesm-11(13)-eno-12,8α-lactone (3) were obtained. Their structures were elucidated by detailed analysis of 1D, 2D NMR and HRMS data. In addition, compounds 1–3 were evaluated for their cytotoxic activities against HepG-2 cell line with IC50 value of 9.83, 2.95, and 4.15 μM, respectively. Moreover, all compounds not only can reduce the expression of JAK2 and STAT3 mRNA but also inhibit the p-JAK2 and p-STAT3 protein expression levels in a dose‐dependent manner in the HepG-2 cells. In conclusion, three sesquiterpene lactone compounds inhibited the proliferation of HepG-2 cells via suppressing the JAK2/STAT3 signaling pathway.

Minimally modified low-density lipoprotein upregulates mouse mesenteric arterial 5-HT1B receptor in vivo via activation of the JAK2/STAT3 pathway.

To explore whether minimally modified low-density lipoprotein (mmLDL) upregulates mesenteric arterial 5-hydroxytryptamine 1B (5-HT1B) receptor expression by activating the JAK2/STAT3 signaling pathway. Mice were randomly divided into the following groups: the normal saline (NS), LDL, mmLDL, mmLDL+galiellactone (GL, a JAK2/STAT3 pathway inhibitor), and mmLDL+DMSO groups. The dose-response curve of mesenteric arterial ring constriction after administration of 5-carboxamidotryptamine (5-CT), an agonist of 5-HT1B, was recorded with a microvascular tensiometer. JAK2, p-JAK2, STAT3, p-STAT3, and 5-HT1B receptor protein expression levels were determined by Western blotting. 5-HT1B receptor mRNA levels were measured by RT-PCR. 5-HT1B receptor protein expression was determined by immunofluorescence. Injection of mmLDL into the tail vein significantly increased the contractile dose-response curve after 5-CT stimulation, as the Emax was 82.15 ± 6.15% in the NS group and 171.88 ± 5.78% in the mmLDL group (P < 0.01); significantly elevated 5-HT1B receptor mRNA and protein expression levels; and significantly increased p-JAK2 and p-STAT3 protein expression levels. After intraperitoneal injection of GL, the vasoconstrictive response was significantly reduced compared with that in the mmLDL group, as the Emax was decreased to 97.14 ± 1.20% (P < 0.01); 5-HT1B receptor mRNA and protein expression levels were significantly reduced; STAT3 phosphorylation and p-JAK2 and p-STAT3 protein expression were not significantly changed; and 5-HT1B receptor expression was altered via inhibition of p-STAT3 binding to DNA, which suppressed transcription. mmLDL can upregulate 5-HT1B receptor expression in mouse mesenteric arteries by activating the JAK2/STAT3 signaling pathway.

Promotion of Adropin Expression by Hyperglycemia Is Associated with STAT3 Activation in Diabetic Rats.

BackgroundAdropin is a secreted polypeptide that has been demonstrated to play an important role in energy homeostasis and lipid metabolism. Signal transducer and activator of transcription 3 (STAT3) may promote the transcription of target genes including adropin. In the current study, we investigated the effect of adropin on glucose metabolism in diabetic rats and the mechanism that governs this effect was subsequently assessed.Materials and MethodsRats received a single injection of streptozotocin to induce type 1 diabetes. The diabetic rats were treated with insulin or phloridzin, another antidiabetic agent through inhibition of glucose reabsorption, for 7 days. Plasma glucose levels and adropin levels were measured. The interaction between STAT3 and adropin was evaluated using the human hepatoma HepG2 cell line. HepG2 cells were pretreated with the specific antagonist Stattic or with STAT3-specific siRNAs to knockout STAT3. Changes in energy homeostasis-associated gene expression were measured using real-time PCR. The protein expression levels of pSTAT3 and STAT3 were measured using Western blotting.ResultsIn diabetic rats, the serum concentrations of adropin were increased in the vehicle-treated group and decreased in the insulin- or phloridzin-treated group. In liver tissues, the Enho expression level and the activity of STAT3 also showed similar tendencies. After HepG2 cells were treated with medium containing high glucose, the ratio of p-STAT3 to STAT3, Enho mRNA levels and reactive oxygen species expression levels in HepG2 cells were significantly increased in conjunction with increased glucose levels. The effect was inhibited after pretreatment with Stattic or knockdown with STAT3-specific siRNAs.ConclusionSTAT3 is involved in the genetic regulation of adropin, increasing the levels of circulating adropin and promoting Enho expression in the livers of diabetic rats.

STAT3 Inhibition by Centipede Scolopendra Extract in Liver Cancer HepG2 Cells and Orthotopic Mouse Models of Hepatocellular Carcinoma

ObjectiveTo observe the effects of Centipede Scolopendra extraction (CSE) on human liver cancer HepG2 cells and the nude mouse tumor model of liver orthotopic transplantation, and to explore the anti-liver cancer mechanism of the extract. MethodsHepG2 cells were respectively treated with CSE250 (250 μg/mL), CSE500 (500 μg/mL) and 5-FU, and control group was established. An enzymatic hydrolysis and acetone precipitation method was used to separate and purify CSE, which was then used to treat HepG2 cells. The CCK8 assay was used to detect the inhibition of cell proliferation and the half maximal inhibitory concentration (IC50) was calculated. Flow cytometry was used to analyze the cell cycle, and western blot was used to detect the expression of signal transduction and activator of transcription 3 (STAT3) pathway-related proteins in HepG2 cells treated with CSE. A nude mouse model with an orthotopic liver tumor was prepared. The mice were randomly divided into four groups, each containing 12 animals: the model group, the 5-FU group, the CSE10 group [10 mg/(kg·d)] and the CSE50 group [50 mg/(kg·d)]. The volume and mass changes in the nude mice with orthotopic transplanted tumors were observed. Western blot method was used to test the protein expression levels of p-STAT3 and p38 mitogen-activated protein kinase (p38MAPK). Tissues from the liver of mice in the model group and the CSE50 group were analyzed by using a protein tyrosine kinase (PTK) chip, and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) function enrichment analysis of the differentially expressed proteins was performed. ResultsThis study showed that CSE significantly inhibited the proliferation of HepG2 cells (P < 0.05). After 48 h of CSE treatment, the cell cycle of HepG2 cells manifested as S phase and G2/M phase; p-STAT3 protein levels in the CSE groups were significantly lower than that in the control group (P < 0.05). Analysis of the tumor inhibition in the mice showed that the tumor masses and volume in CSE groups were lower (P < 0.05). The protein levels of p-STAT3 and p38MAPK in CSE50 group and 5-FU group decreased significantly (P < 0.05). PTK antibody chip screening results showed that CSE groups had a bidirectional regulation trend, and there were 23 up-regulated PTKs and six down-regulated PTKs. The GO and KEGG analyses showed that CSE exerted its anticancer effects through regulation of biological processes, including mitogen-activated protein kinase (MAPK) cascade, chemotaxis, cell invasion, cell adhesion, angiogenesis and other biological processes, and through signaling pathways, including the MAPK, phosphatidylinositol-3-kinase/serine threonine protein kinase (PI3K/AKT), and RAS signaling pathways. ConclusionsCSE can effectively inhibite the proliferation of HepG2 cells and effectively inhibite the growth of liver cancer orthotopic transplantation tumor. Its mechanism may be closely related to the regulation of STAT3, MAPK and PI3K/AKT signaling pathways.

Erchen decoction plus huiyanzhuyu decoction inhibits the cell cycle, migration and invasion and induces the apoptosis of laryngeal squamous cell carcinoma cells

Ethnopharmacological relevanceLaryngeal carcinoma (LC) is one of the most common malignant head and neck cancers with high incidence and mortality rates. Erchen decoction plus Huiyanzhuyu decoction (EHD) is commonly used for treating LC patients and produces beneficial results. However, the mechanisms underlying the effects of EHD remain unclear. Aim of the studyThe present study aimed to analyse the anticancer effects of EHD on the LC cell cycle, apoptosis, migration and invasion in vitro and to explore the underlying biological mechanisms. Materials and methodsTU212 and Hep-2 cells were used. The antitumour effects of EHD were detected by CCK8, microscopy, flow cytometry, EdU incorporation, Hoechst 33342 staining, wound-healing, and transwell assays to assess viability, morphology, apoptosis, cell cycle, migration and invasion, respectively. Furthermore, STAT3 and related proteins were evaluated in laryngeal squamous cell carcinoma (LSCC) cells by Western blot (WB) analysis. ResultsEHD treatment significantly decreased STAT3 and p-STAT3 protein expression levels in LSCC cells. EHD blocked the cell cycle at the G0/G1 phase and induced LSCC apoptosis. Moreover, the viability, migration, and invasion of LSCC cells were markedly inhibited by EHD. In addition, the expression of the cell cycle-related proteins cyclin D1 and cyclin B1 was downregulated in LSCC cells, but P27 expression was increased after EHD treatment. Regarding apoptosis-related proteins, EHD also reduced Bcl-2 expression but upregulated Bax and caspase-3 expression in LSCC cells. In the migration- and invasion-related protein analyses, EHD downregulated MMP-9 expression and upregulated E-cadherin expression. ConclusionsThese results suggest that EHD has an anticancer effect in LSCC. EHD treatment induces apoptosis and inhibits the cell cycle, migration and invasion of LSCC cells, but further work is warranted to address the mechanisms.

The mechanism and tumor inhibitory study of Lagopsis supine ethanol extract on colorectal cancer in nude mice

BackgroundThis study was aimed to determination the tumor inhibitory effect and explore the potential mechanisms of Lagopsis supine ethanol extract (Ls) on colorectal cancer.MethodsThe cell growth inhibition experiment of Ls in colorectal cancer cell lines was determined by MTT method in the time course of 24, 48 and 72 h in four gradient drug concentrations. The protein expression levels of pSTAT3, pJAK2, STAT3, JAK2, Bcl-2 and caspase 3 were measured by Western blot method. The mRNA levels of the downstream genes of STAT3 were detected through semi-quantitative RT PCR. Sixty Balb/c-nude mice were xenograft with HCT116 colorectal cancer cells through subcutaneously. The xenografts were divided into five groups: model group, positive group (capecitabine 300 mg/kg) and three dosages of Ls treated groups (75, 150 and 300 mg/kg). Tumor size and tumor weight were calculated for evaluation the anti-tumor effects. H & E staining and immunohistochemical analysis were used to determine the histopathological changes and the levels of pSTAT3 and pJAK2 in the tumor tissues.ResultsLs exhibited a significant anti-proliferation effect in HCT116 and SW480 cells in vitro. The protein levels of pSTAT3, pJAK2 and Bcl-2, and the mRNA levels of Bcl-2 and Bak notably reduced with a dose-dependent manner. While the protein levels of caspase 3, and mRNA levels of Bax and caspase-3 remarkably increased in the gradient dosage of Ls in HCT116 cells. HCT116 in vivo xenografts experiment showed that the growth of the tumors significantly inhibited by Ls administration, which with no any significant body weight changes in each experiment group. The histopathology analysis displayed that Ls significantly reduced the inflammatory cells in tumor tissue. Furthermore, Ls also significantly down-regulate the protein levels of pSTAT3 and pJAK2 in the tumor tissues, compared with the model group.ConclusionsThis work shows that Ls inhibited the cell proliferation of colorectal cancer in vitro and significantly reduced the tumor growth in HCT116 xenografts in vivo, which is probably related with the JAK/STAT signal pathway.

Gastrodin Inhibits Inflammasome Through the STAT3 Signal Pathways in TNA2 Astrocytes and Reactive Astrocytes in Experimentally Induced Cerebral Ischemia in Rats.

This study was aimed to determine Gastrodin (GAS) and its underlying signaling pathway involved in suppression of inflammasome specifically in reactive astrocytes that are featured prominently in different neurological conditions or diseases including cerebral ischemia. For this purpose, TNA2 astrocytes in cultures were exposed to oxygen-glucose-deprivation (OGD) mimicking hypoxic cerebral ischemia. Separately, TNA2 cells were pretreated with GAS prior to OGD exposure. Additionally, Stattic, an inhibitor of STAT3 signaling pathway, was used to ascertain its involvement in regulating inflammasome in astrocytes exposed to OGD. In parallel to the above, adult rats subjected to middle cerebral artery occlusion (MCAO) with or without GAS pretreatment were sacrificed at different time points to determine the effects of GAS on astrocyte inflammasome. TNA2 astrocytes in different treatments as well as reactive astrocytes in MCAO were processed for immunofluorescence labeling and Western blot analysis for various protein markers. In the latter, protein expression levels of p-STAT3, NLRP3, and NLRC4 were markedly increased in TNA2 astrocytes exposed to OGD. Remarkably, the expression levels of these biomarkers were significantly suppressed by GAS. Of note, GAS especially at dose 20μM inhibited NLRP3 and NLRC4 expression levels most substantially. Moreover, GAS inhibited the downstream proteins caspase-1 and IL-18. Concomitantly, GAS significantly suppressed the expression of STAT3 and NF-κB signaling pathway. It is noteworthy that Stattic at dose 100μM inhibited STAT3 pathway and NF-κB activation in TNA2 astrocytes, an effect that was shared by GAS. In MCAO, GAS was found to effectively attenuate p-STAT3 immunofluorescence intensity in reactive astrocytes. Arising from the above, it is concluded that GAS is anti-inflammatory as it effectively suppresses inflammasome in OGD-stimulated astrocytes as well as in reactive astrocytes in MCAO via STAT3 and NF-κB signaling expression coupled with decreased expression of caspase-1 and IL-18.

Isoalantolactone induces apoptosis through ROS-mediated ER stress and inhibition of STAT3 in prostate cancer cells

BackgroundProstate cancer is one of the most commonly diagnosed cancers in men worldwide. Currently available therapies for metastatic prostate cancer are only marginally effective. Therefore, new therapeutic agents are urgently needed to improve patient outcome. Isoalantolactone (IATL), an active sesquiterpene naturally present in many vegetables and medicinal plants, is known to induce cell death and apoptosis in various cancer cell lines. Nevertheless, antitumor mechanisms initiated by IATL in cancer cells have not been fully defined.MethodsCell apoptosis and cellular ROS levels were analyzed by flow cytometry. Western blot and qRT-PCR were used to analyze the protein and mRNA levels of indicated molecules, respectively. Nude mice xenograft model was used to test the effects of IATL on prostate cancer cell growth in vivo.ResultsIn this study, we found that IATL dose-dependently inhibited cancer cell growth and induced apoptosis in PC-3 and DU145 cells. Mechanistically, our data found that IATL induced reactive oxygen species (ROS) production, resulting in the activation of endoplasmic reticulum stress pathway and eventually cell apoptosis in prostate cancer cells. IATL also decreased the protein expression levels of p-STAT3 and STAT3, and the effects of IATL were reversed by pretreatment with N-acetyl-L-cysteine (NAC). In vivo, we found that IATL inhibited the growth of prostate cancer xenografts without exhibiting toxicity. Treatment of mice bearing human prostate cancer xenografts with IATL was also associated with induction of ER stress and inhibtion of STAT3.ConclusionIn summary, our results unveil a previously unrecognized mechanism underlying the biological activity of IATL, and provide a novel anti-cancer candidate for the treatment of prostate cancer.

Honokiol protects against renal ischemia/reperfusion injury via the suppression of oxidative stress, iNOS, inflammation and STAT3 in rats.

Honokiol is the predominant active ingredient in the commonly used traditional Chinese medicine, Magnolia, which has been confirmed in previous studies to exhibit anti-oxidation, antimicrobial, antitumor and other pharmacological effects. However, its effects on renal ischemia/reperfusion injury (IRI) remain to be elucidated. The present study aimed to examine the effects of honokiol on renal IRI, and to investigate its potential protective mechanisms in the heart. Male adult Wistar albino rats were induced into a renal IRI model. Subsequently, the levels of serum creatinine, blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), and the levels of serum nitrite and the kidney nitrite were examined in the IRI group. The levels of oxidative stress, inducible nitric oxide synthase (iNOS), inflammatory factors and caspase-3 were evaluated using a series of commercially available kits. The levels of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and the protein expression levels of STAT3 were determined using western blotting. Pretreatment with honokiol significantly reduced the levels of serum creatinine, BUN, ALT, AST and ALP, and the level of nitrite in the kidney of the IRI group, compared with the control group. The levels of malondialdehyde, the activity of myeloperoxidase, and the gene expression and activity of iNOS were reduced in the IRI rats, compared with the sham-operated rats, whereas the levels of superoxide dismutase and catalase were increased following treatment with honokiol in the IRI rats. In addition, the expression levels of tumor necrosis factor-α and interleukin-6 in the IRI rats were increased by honokiol. Treatment with honokiol suppressed the protein expression levels of p-STAT3 and caspase-3 in the IRI rats. These findings indicated that honokiol protects against renal IRI via the suppression of oxidative stress, iNOS, inflammation and STAT3 in the rat.

Increased expression of activated pSTAT3 and PIM-1 in the pulmonary vasculature of experimental congenital diaphragmatic hernia

PurposeSignal transducer and activator of transcription (STAT) protein family (STAT1-6) regulates diverse cellular processes. Recently, the isoform STAT3 has been implicated to play a central role in the pathogenesis of pulmonary hypertension (PH). In human PH activated STAT3 (pSTAT3) was shown to directly trigger expression of the provirus integration site for Moloney murine leukemia virus (Pim-1), which promotes proliferation and resistance to apoptosis in SMCs. We designed this study to investigate the hypothesis that pSTAT3 and Pim-1 pulmonary vascular expression is increased in nitrofen-induced CDH. MethodsPregnant rats were exposed to nitrofen or vehicle on D9.5. Fetuses were sacrificed on D21 and divided into nitrofen (n=16) and control group (n=16). QRT-PCR, western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene and protein expression levels of pSTAT3 and Pim-1. ResultsPulmonary Pim-1 gene expression was significantly increased in the CDH group compared to controls. Western blotting and confocal-microscopy confirmed increased pulmonary protein expression of Pim-1 and increased activation of pSTAT3 in CDH lungs compared to controls. ConclusionMarkedly increased gene and protein expression of Pim-1 and activated pSTAT3 in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that pSTAT3 and Pim-1 are important mediators of PH in nitrofen-induced CDH.

Protective effect of Salvia miltiorrhiza aqueous extract on myocardium oxidative injury in ischemic–reperfusion rats

Salvia miltiorrhiza has strong antioxidative activity. They may have a strong potential as cardioprotective agents in ischemic-reperfusion injury. Experiments were carried out in Sprague-Dawley rats with myocardium ischemia reperfusion (IR). Myocardial injuries during IR were determined by changes in electrocardiogram analysis of arrhythmias, antioxidant enzyme activities, AST, CK-MB, lactate dehydrogenase (LDH) levels, and myocyte apoptosis. Results showed that S. miltiorrhiza aqueous extract (SAME) pre-treatment significantly decreased the ST-segment (ΣST120) and myocardium MDA, AST, CK-MB, lactate dehydrogenase (LDH) levels, increased myocardium antioxidant enzyme activities, and inhibit myocardium cell apoptosis. Furthermore, the SAME pre-treatment significantly upregulated p-JAK2 and p-STAT3 protein expression, decreased myocardium TNF-α and IL-6 concentrations in IR rats. The levels of TNF-α and IL-6 were positively correlated with the changes in myocardium p-JAK2 and p-STAT3 protein expression levels in IR rats. It can be concluded that the SAME pre-treatment has anti-ischemic and anti-apoptosis activity in heart IR rats. SAME pre-treatment protects heart against IR injury, at least in part, through its stimulating effects on injury-induced deactivation of JAK2/STAT3 signaling pathway.

STAT3 activation in cardiac hypertrophy induced by ryanodine receptor 2 mutation

Ryanodine receptor 2 (RyR2) is a 2,200 kDa ion channel that is activated during an action potential to release Ca2+ required for cardiac muscle to contract. RyR2 mutant mice (RyR2‐ W3587A/L3591D/F3603A or Ryr2ADA) impaired in regulation by calmodulin show cardiac hypertrophy and survive about two weeks. We hypothesized that signal transducer and activator of transcription‐3 (STAT3) may play a role in cardiac hypertrophy of Ryr2ADA/ADA mice. We found that the protein expression levels of pSTAT3 and IL‐6, an upstream effector of STAT3, were increased in 10 day old Ryr2ADA/ADA mouse hearts but STAT3 expression was maintained. Corresponding with an increased nuclear translocation of pSTAT3 in Ryr2ADA/ADA mice, mRNA expression levels of ANP and c‐fos in left ventricular hearts were greatly increased in Ryr2ADA/ADA mice. The mutant mice also showed higher protein expression level of c‐fos and Chip assay indicated enrichment of STAT3 target genes ANP and c‐fos in the nuclei. The results suggest that activated STAT3 signaling pathway may play an important role in the development of cardiac hypertrophy in mice with dysfunctional ryanodine receptor. Supported by NIH.

Antioxidant N-Acetylcysteine Attenuates the Reduction of Brg1 Protein Expression in the Myocardium of Type 1 Diabetic Rats

Brahma-related gene 1 (Brg1) is a key gene in inducing the expression of important endogenous antioxidant enzymes, including heme oxygenase-1 (HO-1) which is central to cardioprotection, while cardiac HO-1 expression is reduced in diabetes. It is unknown whether or not cardiac Brg1 expression is reduced in diabetes. We hypothesize that cardiac Brg1 expression is reduced in diabetes which can be restored by antioxidant treatment with N-acetylcysteine (NAC). Control (C) and streptozotocin-induced diabetic (D) rats were treated with NAC in drinking water or placebo for 4 weeks. Plasma and cardiac free15-F2t-isoprostane in diabetic rats were increased, accompanied with increased plasma levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6), while cardiac Brg1, p-STAT3 and HO-1 protein expression levels were significantly decreased. Left ventricle weight/body weight ratio was higher, while the peak velocities of early (E) and late (A) flow ratio was lower in diabetic than in C rats. NAC normalized tissue and plasma levels of 15-F2t-isoprostane, significantly increased cardiac Brg1, HO-1 and p-STAT3 protein expression levels and reduced TNF-alpha and IL-6, resulting in improved cardiac function. In conclusion, myocardial Brg1 is reduced in diabetes and enhancement of cardiac Brg1 expression may represent a novel mechanism whereby NAC confers cardioprotection.

Abstract 929: Repression of CD44 is a key molecular action of a novel Gemini vitamin D BXL0124 to inhibit breast cancer cell invasion induced by HGF/Met signaling

Abstract CD44, a transmembrane glycoprotein, is highly expressed in breast tumors, and the overexpression of CD44 has been associated with poor clinical outcome. CD44 has been known to play a key role in invasive growth of cancer by interacting with extracellular matrix, matrix metalloproteinases (MMPs) and other membrane receptors. In addition, CD44 has been shown to be required for the activation of hepatocyte growth factor (HGF)/Met signaling pathway which is associated with invasive phenotype of human breast cancer. Our previous study demonstrated that a novel Gemini vitamin D BXL0124 repressed the expression of CD44 in MCF10DCIS.com human breast cancer cells and inhibited MCF10DCIS xenograft tumor growth in vivo. In the present study, we investigated the effect of CD44 repression by BXL0124 on HGF/Met mediated invasion in MCF10DCIS cells. The effects of HGF and BXL0124 on the invasive phenotype of MCF10DCIS cells were investigated with invasion assays. To identify key molecules in HGF-induced cancer cell invasion, invasive markers and downstream of HGF/Met signaling pathway were evaluated. The treatment of HGF increased CD44 protein expression and invasive growth of MCF10DCIS cells, which was inhibited by BXL0124 treatment. Stat3 DNA-binding and nuclear translocalization experiments demonstrated that the activation of Stat3, one of the key downstream molecules of HGF/Met signaling pathway, was also significantly decreased by BXL0124 treatment. To further study a specific role of CD44 on breast cancer cell invasion, the effects of CD44 repression were investigated by using MCF10DCIS CD44-knockdown cells in vitro and in vivo. The induction of cancer cell invasion by HGF was blocked when CD44 was knockdown in MCF10DCIS cells. In addition, invasion markers, MMP-9 and uPA, were significantly down-regulated in MCF10DCIS CD44 knockdown cells. For the in vivo study, MCF10DCIS control or CD44-knockdown cells were injected into mammary fat pads in immunodeficient mice. Five weeks later, mammary tumors were collected for evaluating the expression level of invasion markers. The xenograft tumor volume and weight with MCF10DCIS CD44-knockdown cells were significantly smaller by 41% (p&amp;lt;0.01) and 36% (p&amp;lt;0.05), respectively, than those from MCF10DCIS control tumors (n=5). The MCF10DCIS CD44-knockdown tumors also showed markedly decreased protein expression levels of pStat3 and MMP-9. The mRNA expression levels of MMP-9 and uPA were significantly down-regulated in the MCF10DCIS CD44-knockdown tumors. The present study demonstrated the critical role of CD44 in HGF-induced breast cancer cell invasion, suggesting that the repression of CD44 might be a key molecular action of BXL0124 to inhibit breast cancer invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 929. doi:1538-7445.AM2012-929

Abstract A32: Notch pathway regulates pancreatic acinar-to-ductal metaplasia

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States and, to date, without effective treatment. PDAC develops through defined preneoplastic lesions of which pancreatic intraepithelial neoplasia (PanIN) are the most common precursors. Acinar-to-ductal metaplasia (ADM) appears to be an important prerequisite for PanIN development, especially under inflammatory conditions. The Notch signaling pathway plays a key role in cell fate and differentiation and is activated during ADM and PanIN development, suggesting a role in initiation of transformation processes. We have recently shown that Notch2 but not Notch1 regulates PanIN progression in a Kras-driven model of endogenous PDAC. Methods: To determine the role of Notch2 in acinar plasticity as well as in PanIN development, R0SA26R-LSL-N2-IC mice were crossed to Ela-CreERT mice to produce mice with an inducible pancreas-specific constitutively active Notch2 intracellular domain (ElaCreERT; N2-IC). Additionally, ElaCreERT; N2-IC mice were crossed to floxed Hes1 mice to assess the role of Hes1. Morphological and molecular analyses were performed using immunohistochemistry, immunofluorecence, Western Blot, real time quantitative PCR (qRT-PCR) and microarray based gene profiling. Results: Starting 3 days post tamoxifen induction in adult acinar cells, ADM developed in ElaCreERT;N2-IC mice. Acinar cells down-regulated typical acinar genes such as amylase, increasingly expressed the ductal marker CK19 and showed a high proliferative rate. The ADM cells showed high expression of the Notch target gene HES1 and the progenitor marker SOX9 but stained negative for MUC5AC, a marker found in PanIN lesions. In order to investigate the role of the downstream target Hes1 in ADM, ElaCreERT;N2-IC mice were crossed with Hes1lox/Iox mice to produce mice with additional deletion of Hes1. ElaCreERT;N2IC; Hes1lox/iox mice showed a significant attenuation, but no complete rescue of ADM development. Using qRT-PCR, we assayed for Notch downstream target genes and found a strong up-regulation of the Hes family member Hes5. In accordance with other studies of ADM, protein-expression levels of pSTAT3 were up-regulated in ductal cells in ElaCreERT;N2-IC mice. Interestingly, pSTAT3 was expressed in the acinar cells in ElaCreERT; N2IC; Hes1lox/lox mice, which had not undergone N2-IC mediated ADM, suggesting the expression of HES1 to be a decisive part in ADM induction and progression. Conclusion: We have thus shown that Notch2-IC is sufficient for ADM induction in adult acinar cells. The mechanism of phenotype rescue in mice with additional deletion of Hes1 needs to be further investigated but may involve regulation of Stat3. Hes5 was increasingly up-regulated in Hes1-deficient cells, pointing to a potential role in ADM development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A32.

Abstract PR11: Egfr is essential for Ras-driven pancreatic cancer development

Abstract Introduction: Oncogenic activation of Kras is a key event in the development of pancreatic ductal adenocarcinoma (PDAC), leading to initiation and progression of pancreatic intraepithelial neoplasia (PanIN), the preneoplastic precursor to PDAC. Endogenous mouse models with activating KrasG12D mutations recapitulate this process very well. Epidermal growth factor receptor (Egfr) is known to be elevated in PDAC and is to date the only approved target for targeted therapy, although the exact mechanism of action remains unknown. In this study we thus investigated the effect of conditional Egfr ablation in a genetically engineered mouse (GEM) model of PDAC. Experimental design: Using a Cre/lox approach, we deleted Egfr from Ptf1a-positive pancreatic progenitor cells in different GEMs of PDAC and in mice with caerulein-induced acute pancreatitis. Acinar epithelial explants of mutant mice were used to investigate the role of Egfr in vitro. In vivo therapy-studies in GEM were performed using different inhibitors of Egfr. Results: Unexpectedly, KrasG12D;EgfrΔ/Δ mice revealed complete absence of PanIN lesions and showed only rare fields of ductal-like lesions at 6 months of age. In a mouse model of induced acute pancreatitis KrasG12D;EgfrΔ/Δ mice did not develop PanINs as did KrasG12D control mice. In a highly aggressive KrasG12D;p53Δ/Δ GEM model for PDAC, tumor initiation and progression rate was reduced although not completely blocked in EGFR-ablated mice. In vivo, early therapy/chemoprevention of KrasG12D;p53Δ/Δ mice with either cetuximab, an antibody directed against the Egfr ligand-binding domain, or erlotinib, a small molecule tyrosine kinase domain inhibitor, displayed significantly reduced development of acino-ductal metaplasia (ADM) in cetuximab-treated mice and to a even higher extend in erlotinib-treated animals, indicating a ligand-independent role of Egfr in ADM- and PanIN-induction. To closer investigate the underlying mechanisms in vitro, we isolated acinar epithelial explants of EgfrΔ/Δ and KrasG12D; EgfrΔ/Δ mice and corresponding controls. Acinar epithelial explants of EgfrΔ/Δ and KrasG12D;EgfrΔ/Δ did not undergo ADM in opposite to KrasG12D or Egf-stimulated wildtype acinar-epithelial explants. Closer examination revealed that initiating events like the expression of the progenitor cell marker nestin in acinar explants of KrasG12D;EgfrΔ/Δ mice and the expression of pStat3 in acinar tissue of KrasG12D; EgfrΔ/Δ mice were still detectable, similar to KrasG12D controls, but progression to ductal-like structures was blocked. Protein-expression levels of pStat3 and pErk were reduced in KrasG12D;EgfrΔ/Δ mice although active GTP-bound Kras levels were not altered in KrasG12D;EgfrΔ/Δ mice. Additionally, we performed immunofluorescence and confocal analysis of Egfr and Kras in acinar-epithelial explants of KrasG12D and KrasG12D;EgfrΔ/Δ mice, which did not reveal Egfrdependent differences in localization of Kras. Conclusions: These data provide strong evidence that Egfr seems to be essential for induction of ADM and PanIN development in a Kras-independent manner that needs to be further investigated. Even additional stimuli such as caerulein-induced inflammation did not lead to the development of precursor lesions suggesting an early requirement of Egfr in the carcinogenic process. This abstract is also presented as Poster A44. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR11.