Activation Of p-ERK1 Research Articles

Annexin A1 upregulates hematoma resolution via the FPR2/p-ERK(1/2)/DUSP1/CD36 signaling pathway after germinal matrix hemorrhage

Germinal matrix hemorrhage (GMH) is one of the leading causes of morbidity and mortality in preterm infants in the United States, with little progress made in its clinical management. Blood clots disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage are key contributors towards post-hemorrhagic hydrocephalus development. n-formyl peptide receptor 2 (FPR2), a G-protein-coupled receptor, has been associated with the activation of p-ERK1/2, which in turn promotes the transcription of the DUSP1 gene, which may play a role in CD36 signaling. CD36 scavenger, a transmembrane glycoprotein, plays an essential role in microglia phagocytic blood clot clearance after GMH. FPR2's role in blood clot clearance after hemorrhagic stroke is unknown. We hypothesize that FPR2 activation by FPR2 agonist Annexin A1 (AnxA1) will enhance hematoma resolution via the upregulation of the CD36 signaling pathway, thereby improving short- and long-term neurological outcomes. Bacterial collagenase (0.3 U) was infused intraparenchymally into the right hemispheric ganglionic eminence in P7 rat pups to induce GMH. AnxA1 and FPR2 Inhibitor (Boc2) were given at 1-h post-GMH via intranasal administration. FPR2 CRISPR was given 48-h prior to GMH induction. Short-term neurological deficits were assessed using negative geotaxis test. Hematoma volume was assessed using hemoglobin assay. Protein expression was assessed using western blots. Long-term neurocognitive deficits and motor coordination were assessed using Morris water maze, rotarod, and foot fault tests. We have demonstrated that AnxA1 treatment enhances hematoma resolution and improved short and long-term outcomes. Lastly, FPR2 agonist AnxA1 treatment resulted in the upregulation of the FPR2/p-ERK(1/2)/DUSP1/CD36 signaling pathway.

In the presence of TGF-\u03b21, Asperosaponin VI promotes human mesenchymal stem cell differentiation into nucleus pulposus like- cells

BackgroundThe regeneration of nucleus pulposus (NP) cells is an effective method to prevent intervertebral disc degeneration (IVDD). In this study, we investigated the role of Asperosaponin VI (ASA VI), isolated from a traditional Chinese medicine (TCM), the root of Dipsacus asper Wall, in promoting human mesenchymal stem cell (HMSC) proliferation and differentiation into NP-like cells and explored the possible mechanism of action.MethodsThe effects of ASA VI on HMSC viability and proliferation were determined by the XTT method and EDU staining. Then, Real-time qPCR, immunocytochemistry and immunofluorescence assays were used to measure the effect of ASA VI on the expression of extracellular matrix (ECM) components, such as COL2A1, aggrecan, SOX9, KRT19, PAX1, and glycosaminoglycans (GAGs), in NP cells. In addition, Western blot assay was used to measure the expression of p-ERK1/2 and p-smad2/3.ResultsASA VI was able to promote the proliferation and differentiation of HMSCs into NP-like cells, and the optimum concentration was 1 mg/L. Western blot assay indicated that the possible mechanism might be related to the activation of p-ERK1 / 2 and p-Smad2 / 3.ConclusionsASA VI can promote the proliferation and differentiation of HMSCs into NP-like cells, which can potentially be used as a treatment for IVDD.

Abstract P230: Depletion Of Cyclic-gmp Levels And The Inhibition Of Cgks Activate P21 Cip1 /p27 Kip1 Pathways And Trigger High Blood Pressure With Renal Fibrosis And Dysfunction

Targeted-deletion of Npr1 gene (coding for guanylyl cyclase/natriuretic peptide receptor-A, GC-A/NPRA) exhibits hypertrophic and proliferative effects in target organs of Npr 1 gene-knockout mice. Fibrosis and hypertrophy are regulated by p21 Cip1 and p27 Kip1 , cell-cycle regulatory proteins that inhibit target cyclin and cyclin-dependent kinase (cyclin-CDK) complex. We examined the activation of CDK blocker (p21 Cip1 /p27 Kip1 ) in Npr1 gene-knockout (0-copy; Npr 1 -/- ) mice and guanylyl cyclase (GC) inhibitor, A71915-treated and cGMP-dependent protein kinase (cGK) inhibitor, Rp-8-Br-cGMPS (Rp)-treated wild-type 2-copy ( Npr 1 +/+ ) and gene-duplicated 4-copy ( Npr 1 ++/++ ) mice. Blood pressure (BP) was significantly higher in 0-copy mice (138.6 ± 3.1 mmHg) and lower in 4-copy mice (86.0 ± 2.8 mmHg) than 2-copy mice (102.2 ± 1.7 mmHg). Treatment with A71915 and Rp showed significant changes in BP in 2-copy mice but caused only small increase in 4-copy mice. We found a significant decrease in renal cGMP levels with diminished cGK activity in 0-copy mice (p<0.0001) as well as A71915-treated (p<0.001) and Rp-treated (p<0.05) 2-copy and 4-copy mice as compared with controls animals. While significant activation of p-Erk1/2 (3-fold), p-p38MAPK (4-fold), p21 Cip1 (6-fold), and p27 Kip1 (5-fold) occurred in 0-copy, A71915-treated 2-copy, and A71915-treated 4-copy mice but Rp treatment caused minimal changes compared to control mice. There were significant increases in the proinflammatory cytokines, including TNF-α (6-fold), and IL-6 (3-fold) and profibrotic cytokine TGF-β1 (4-fold) in plasma and kidneys of 0-copy and A791915-treated 2-copy mice, but less in A71915-treated 4-copy mice than controls. Progressive renal pathology, including fibrosis, mesangial matrix expansion, tubular hypertrophy, and perivascular infiltration were significantly scored in 0-copy and A71915-treated 2-copy mice, but did so minimally in 4-copy mice compared with controls. The present results suggest that Npr1 has a pivotal role in inhibiting the renal fibrosis and pathology and exerts renal protective effects through the cGMP/cGK axis by repressing the CDK inhibitors, p21 Cip1 and p27 Kip1 . This work was supported by NIH grant (HL062147).

MGL1 Receptor Plays a Key Role in the Control of T. cruzi Infection by Increasing Macrophage Activation through Modulation of ERK1/2, c-Jun, NF-κB and NLRP3 Pathways.

Macrophage galactose-C type lectin (MGL)1 receptor is involved in the recognition of Trypanosoma cruzi (T. cruzi) parasites and is important for the modulation of the innate and adaptive immune responses. However, the mechanism by which MGL1 promotes resistance to T. cruzi remains unclear. Here, we show that MGL1 knockout macrophages (MGL1−/− Mφ) infected in vitro with T. cruzi were heavily parasitized and showed decreased levels of reactive oxygen species (ROS), nitric oxide (NO), IL-12 and TNF-α compared to wild-type macrophages (WT Mφ). MGL1−/− Mφ stimulated in vitro with T. cruzi antigen (TcAg) showed low expression of TLR-2, TLR-4 and MHC-II, which resulted in deficient splenic cell activation compared with similar co-cultured WT Mφ. Importantly, the activation of p-ERK1/2, p-c-Jun and p-NF-κB p65 were significantly reduced in MGL1−/− Mφ exposed to TcAg. Similarly, procaspase 1, caspase 1 and NLRP3 inflammasome also displayed a reduced expression that was associated with low IL-β production. Our data reveal a previously unappreciated role for MGL1 in Mφ activation through the modulation of ERK1/2, c-Jun, NF-κB and NLRP3 signaling pathways, and to the development of protective innate immunity against experimental T. cruzi infection.

Abstract P3-09-06: CXCL1 chemokine stimulates migration and invasion in ER-negative breast cancer through activation of the ERK/MMP-2/9 signaling axis

Abstract Chemokine (C-X-C motif) ligand 1 (CXCL1), a member of the CXC chemokine family, has been reported to be a critical factor in inflammatory diseases and tumor progression. However, its functions and molecular mechanism in estrogen receptor α (ER) negative breast cancer (BC) has little been known. In this study, we discovered that CXCL1 is overexpression in ER-negative breast cancer tissues and cell lines compared with ER-positive patient tissues and cell lines. Treatment with recombinant human CXCL1 protein promotes ER-negative BC cell migration and invasion in dose-dependent manner and stimulates the activation of p-ERK1/2, but not p-STAT3 or p-AKT. Whereas, knockdown of CXCL1 in BC cells attenuates these effects. Moreover, CXCL1 induces the expression of MMP2/9 via ERK1/2 pathway. The blockage of ERK by its antagonists (U0126) can abolish the effect of CXCL1 on MMP2/9 expression. Furthermore, immunohistochemical (IHC) analysis revealsa strong positive correlation between CXCL1 and p-ERK1/2 expressions in BC tissues. In conclusion, our study illustrates that CXCL1 is highly expressed in ER-negative BC and stimulates cell migration and invasion through ERK/MMP2/9 pathway, which may serve as a potential therapeutictarget in ER-negative breast cancer. Citation Format: Liu S, Yang C, Guo S, Chen R. CXCL1 chemokine stimulates migration and invasion in ER-negative breast cancer through activation of the ERK/MMP-2/9 signaling axis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-09-06.

Myocardial apoptosis and anti-apoptotic mechanism of captopril on cardiac arrest after resuscitation of pulmonary embolism

Objective To observe the myocardial apoptosis and the molecular mechanism of captopril inhibiting myocardial apoptosis on cardiac arrest (CA) after resuscitation in a porcine acute pulmonary embolism (APE) model. Methods In this study, 29 inbred Beijing Landrace were randomly (random number)divided into four groups (n=5, each group): control, APE-CA, restoration of spontaneous circulation (ROSC)-captopril, and ROSC-saline. The model of CA and ROSC was induced by APE through injection of thrombus followed by cardiopulmonary resuscitation and thrombolytic therapy (urokinase, 15 000 U/kg, iv). Ten of 19 pigs with CA recovered to spontaneous circulation were divided randomly into the ROSC-captopril and ROSC-saline groups. Pigs in the ROSC-captopril group were treated with captopril (22.22 mg/kg) via porcine femoral vein at 30 min after ROSC. Pigs in the ROSC-saline group were treated with equal normal saline at 30 min after ROSC. The myocardial tissues were evaluated at 6 h after ROSC. Western blot was used to evaluate the protein levels of Bax, Bcl-2, Caspase-3, phosphorylated (p)-Src and phosphorylated extracellular regulated protein kinase (p-ERK1/2). Immunohistochemistry was used to evaluate the protein expression of p-Src and p-ERK1/2. Enzyme-linked immunosorbent assay was used to detect myocardial Na+-K+-ATPase levels. Statistical analysis was performed using one-way analysis of variance and pearson correlation test. Results Compared with the control group, the protein expression of Bax (0.25±0.01, 0.53±0.01, 0.37±0.05, F=14.16, P<0.05) and Caspase-3 (0.24±0.01, 0.33±0.01, 0.34±0.06, F=7.32, P<0.05) in the APE-CA and ROSC- saline group were increased significantly, and the Bcl-2 expression was significantly decreased (0.56±0.02, 0.19±0.01, 0.37±0.10, F=6.68, P<0.05). Captopril reduced the protein levels of Caspase-3 and Bax, while stimulated the Bcl-2 expression (all P<0.05). Compared with the control group, the protein expression of p-Src and p-ERK1/2 were higher and the Na+-K+-ATPase level was decreased on CA and ROSC induced by APE (all P<0.05). Compared with the APE-CA group, the p-Src expression in the ROSC-captopril group (0.46±0.01 vs. 0.35±0.06, P<0.05) was decreased significantly. Captopril inhibited the activation of p-ERK1/2 than saline group (0.41±0.10 vs. 0.26±0.07, P<0.05), but has no effect on the Na+-K+-ATPase level. The protein expression of p-Src and p-ERK1/2 were positively correlated with the Bax, and negatively correlated with the Bcl-2 respectively. The myocardial Na+-K+-ATPase level negatively correlated with Caspase-3 protein expression. Conclusions The molecular mechanism of cardiomyocyte apoptosis on CA and ROSC induced by APE might be related to decreased Na+-K+-ATPase level and activation of p-Src and p-ERK1/2. The cardiomyocyte apoptosis were inhibited by captopril through reducing the expression of p-Src and p-ERK1/2 in myocardium. Key words: Acute pulmonary embolism; Cardiac arrest; Spontaneous circulation recovery; Myocardial apoptosis; Molecular mechanism

Fibronectin Promotes the Malignancy of Glioma Stem-Like Cells Via Modulation of Cell Adhesion, Differentiation, Proliferation and Chemoresistance.

Glioma stem-like cells (GSCs) are regarded as the sources of oncogenesis, recurrence, invasion and chemoresistance in malignant gliomas. Growing evidence suggests that the microenvironment surrounding GSCs interacts with tumor cells to influence biological behavior; however, the functional mechanisms involved are still unclear. In the present study, we investigated the modulation of GSCs triggered by fibronectin (FN), a main component of the extracellular matrix (ECM), in terms of cell adhesion, differentiation, proliferation and chemoresistance. We demonstrated that pre-coated FN prompted increased adherence by GSCs, with increased matrix metallopeptidases (MMPs)-2 and -9 expression, in a concentration-dependent manner. Decreases in sox-2 and nestin levels, and increased levels of glial fibrillary acidic protein (GFAP) and β-tubulin were also found in GSCs, indicating cell differentiation driven by FN. Further investigation revealed that FN promoted cell growth, as demonstrated by the elevation of Ki-67, with the activation of p-ERK1/2 and cyclin D1 also evident. In addition, FN suppressed p53-mediated apoptosis and upregulated P-glycoprotein expression, making GSCs more chemoresistant to alkylating agents such as carmustine. In contrast, this effect was reversed by an integrin inhibitor, cilengitide. Activation of the focal adhesion kinase/paxillin/AKT signaling pathway was involved in the modulation of GSCs by FN. Focusing on the interactions between tumor cells and the ECM may be an encouraging aspect of research on novel chemotherapeutic therapies in future.

Regulation of OPN expression in MDA‐MB‐435: role of genistein

Breast cancer is the most common cause of cancer‐related mortality in women worldwide and metastatic spread is the leading cause of breast cancer deaths. Osteopontin (OPN) plays a functional role in normal mammary gland development, progression of breast cancer and in malignancy of breast cancer. High expression of OPN is observed in high‐grade metastatic malignant human gliomas and in the highly invasive and metastatic human breast cancer cell lines (e.g. MDA‐MB‐435). Soy isoflavone genistein, a tyrosine kinase inhibitor and agonist of estrogen receptor‐β (ERβ), is known to have antitumoural properties and also induce apoptotic cell death of ERα‐negative breast cancer cells via p53‐dependent pathway. The isoflavone has been reported to exert antiproliferative activity in some breast cancer cell lines; however, its role with respect to OPN expression is not well understood. Therefore, to address this, the expression level of OPN was investigated upon genistein treatment in MDA‐MB‐435. The cells were treated with different concentrations of genistein for different time intervals; the expression of OPN, the signaling pathways and the promoter regions involved in the regulation of OPN were studied. Genistein displayed an inhibitory effect on OPN protein and mRNA levels in a dose dependent manner and treatment with 50 μM genistein for 24 h showed to be promising. Analysis of the signaling pathways showed no activation of Akt/PKB pathway, no alteration in p‐MEK½ expression whilst an increase in the expression of p‐ERK½ was observed in genistein treated group. These results indicate that genistein acts as an anti‐metastatic agent by inhibiting OPN expression through the activation of p‐ERK½ in MDA‐MB‐435 cells. The transcription factors responsible for this event are being studied in details.Support or Funding InformationDBT (GoI) No. BT/253/NE/TBP/2011 dated May 07, 2012This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aldose reductase deficiency leads to oxidative stress-induced dopaminergic neuronal loss and autophagic abnormality in an animal model of Parkinson's disease

Fungicide exposure causes degeneration of dopaminergic neurons and contributes to Parkinson's disease (PD). Benomyl inhibits enzymes responsible for detoxifying the reactive dopamine metabolite 3,4-dihydroxyphenylacetaldehyde. Aldose reductase (AR) is known as tetrahydrobiopterin (BH4) reductase that generates BH4, a cofactor for tyrosine hydroxylase (TH) involved in dopamine synthesis. AR also acts as an aldehyde reductase involved in detoxifying 3,4-dihydroxyphenylacetaldehyde. In PD patients, the level of AR is significantly lower in the cerebellum. To determine if AR deficiency contributes to PD, AR wild-type (AR+/+) and knockout (AR−/−) mice were administrated with 1-methyl-4-phenyl -1,2,3,6- tetrahydropyridine (MPTP). The MPTP-treated AR−/− mice showed more severe behavioral deficits and brain damage than that of AR+/+ mice. Contrary to expectation, under normal or MPTP-treated condition, AR−/− mice showed a significant elevation of BH4 and dopamine in the midbrain, suggesting that either AR does not contribute to BH4 production, or other BH4 synthetic pathways are induced. The AR−/− brain showed upregulation of peroxynitrite, inducible nitric oxide synthase and downregulation of antioxidant enzymes, Cu/Zn superoxide dismutase (SOD) and peroxiredoxin 2 (Prx2), which indicate an increase in oxidative stress. In line with the animal data, pretreating the SH-SY5Y cells with AR inhibitors (Fidarestat or Epalrestat) before MPP+ treatment, increased severe cell death and mitochondrial fragmentation with downregulation of SOD were observed when compared to the MPP+ treatment alone. Cycloxygenase 2 (COX2), which can lead to the oxidation of dopamine, was upregulated in AR−/− brains. Autophagic proteins, beclin-1 and LC3B were also downregulated. The loss of dopaminergic neurons was associated with activation of p-ERK1/2. These findings suggest that AR plays an important role in protecting dopaminergic neuron against neurotoxic metabolites in PD.

C3G overexpression promotes the survival of rat‐derived H9C2 cardiomyocytes by p‐ERK1/2

Integrin β1 subunit and its downstream molecules, such as integrin-linked kinase and focal adhesion kinase, are imperative for promotion of cell proliferation, survival and anti-apoptosis in cardiomyocytes by activation of their downstream pro-survival signalling molecules, such as the phosphorylated extracellular signal-regulated kinase1/2 (p-ERK1/2). As a component of the integrin pathway, C3G (Crk-SH3 domain guanine nucleotide exchange factor) protein may be involved in the promotion of cell proliferation and survival and anti-apoptosis in the H9C2 cardiomyocytes. Rat-derived H9C2 cardiomyocytes were transfected with pCXN2-flag-hC3G, a human C3G overexpression eukaryotic recombinant plasmid. Apoptosis, cell proliferation and survival were analysed in the H9C2 cardiomyocytes either treated with hypoxia/reoxygenation (H/R). Human C3G mRNA overexpression significantly elevated C3G protein expression in H9C2 cardiomyocytes whether treated with H/R or not. C3G overexpression promoted proliferation and survival and anti-apoptosis, and attenuated the proliferative and survival inhibition, and apoptosis induced by H/R by activation of its downstream pro-survival signalling molecule, p-ERK1/2. The results suggest that C3G acts as a pro-survival molecule in H9C2 cardiomyocytes by activation of p-ERK1/2.

Alternate Estrogen Receptors Promote Invasion of Inflammatory Breast Cancer Cells via Non-Genomic Signaling

Although Inflammatory Breast Cancer (IBC) is a rare and an aggressive type of locally advanced breast cancer with a generally worst prognosis, little work has been done in identifying the status of non-genomic signaling in the invasiveness of IBC. The present study was performed to explore the status of non-genomic signaling as affected by various estrogenic and anti-estrogenic agents in IBC cell lines SUM149 and SUM190. We have identified the presence of estrogen receptor α (ERα) variant, ERα36 in SUM149 and SUM190 cells. This variant as well as ERβ was present in a substantial concentration in IBC cells. The treatment with estradiol (E2), anti-estrogenic agents 4-hydroxytamoxifen and ICI 182780, ERβ specific ligand DPN and GPR30 agonist G1 led to a rapid activation of p-ERK1/2, suggesting the involvement of ERα36, ERβ and GPR30 in the non-genomic signaling pathway in these cells. We also found a substantial increase in the cell migration and invasiveness of SUM149 cells upon the treatment with these ligands. Both basal and ligand-induced migration and invasiveness of SUM149 cells were drastically reduced in the presence of MEK inhibitor U0126, implicating that the phosphorylation of ERK1/2 by MEK is involved in the observed motility and invasiveness of IBC cells. We also provide evidence for the upregulation of p-ERK1/2 through immunostaining in IBC patient samples. These findings suggest a role of non-genomic signaling through the activation of p-ERK1/2 in the hormonal dependence of IBC by a combination of estrogen receptors. These findings only explain the failure of traditional anti-estrogen therapies in ER-positive IBC which induces the non-genomic signaling, but also opens newer avenues for design of modified therapies targeting these estrogen receptors.

Abstract 4264: CXCL12-CXCR4 axis in sunitinib-sensitive and -resistant hepatocarcinoma cells

Abstract Background: Even though sunitinib is a multikinase inhibitor with activity in hepatocarcinoma, acquired resistance may eventually develop within 4-6 months. Increased expression of CXCR4 has been associated with poor prognosis and resistance to several anticancer agents. In addition, high plasma levels of CXCL12 (SDF1α) characterize the lack of sunitinib efficacy in patients with hepatocarcinoma. This study aimed to identify the mechanisms associated with acquired resistance to sunitinib in hepatocarcinoma cell lines. Materials and Methods: SK-HEP1 cells were exposed to increasing sunitinib concentrations for more than 6 months to obtain a resistant cell line named as SK-suni. In these cell lines, cell proliferation, clonogenic survival, mRNA levels of selected genes and invasion of cells were evaluated. Protein levels were analyzed by Western Blot. CXCR4 expression was evaluated by qRT-PCR, Western blot and flow cytometry. Transient siRNA transfection was carried out to target CXCR4. Results: Exposure to 48-hour sunitinib led to IC50s of 4.4 and 10.5 µM in SK-HEP1 and SK-suni cells, respectively and further confirmed by clonogenic assay. Sk-suni was 4-fold more invasive in matrigel as compared to parental cells. Protracted exposure to sunitinib led to a 3-fold increase mRNA expression of CXCR4, CXCL12, MIF, PDGFRα, RET, IGF1R, VEGFR3, CDH1 in SK-suni as compared to parental SK-HEP1 cells. In addition, a constitutive activation of p-ERK1/2 and p-AKT levels were detected in SK-suni cells. Using 200 ng/ml CXCL12 as chemoatractant, invasion index increased by ∼2.5 and ∼3 times in SK-HEP1 and SK-suni cells, whereas it was markedly suppressed by AMD3100, a CXCR4 antagonist. These findings indicate that sensitive and resistant cells harbour a functional CXCR4, although mRNA and protein levels of CXCR4 are higher in SK-suni. Upon CXCL12 treatment, a prompt activation of p-ERK was observed in SK-suni. However, AMD3100 pretreatment did not inhibit the proliferation and had no effect on the baseline up-regulation of p-ERK levels in SK-suni. To further investigate the function of CXCR4, small interfering RNAs was employed to specifically inhibit CXCR4 expression in vitro, obtaining a 80% inhibition of CXCR4 mRNA expression in SK-HEP1 and SK-suni. siRNA CXCR4 did not modify the IC50 of sunitinib in SK-suni. Matrigel invasion assays showed a significant decrease of CXCL12 induce cell invasion in transfected cells (p&amp;lt;0.05). Conclusions: In hepatocarcinoma cells, CXCL12/CXCR4 axis is expressed and functional in sunitinib-sensitive and -resistant cells. Inhibiting CXCR4 might counteract increased invasion associated with sunitinib resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4264. doi:10.1158/1538-7445.AM2011-4264

Mitomycin C-induced fibroblast apoptosis and mechanism

Objective To elucidate the mitomycin C (MMC) -induced apoptosis of fibroblasts and the mechanism. Methods The cultured fibroblasts were exposed for 12 h to 0-0. 3 g/L MMC, respectively. The cell proliferation was measured by CCK-8 assay and apoptotic cell death was analyzed by Ho-echst33342 stain on the fisherbrand covergla. The expression of Caspase-3 and the activation of p-ERK1/2, p-Akt were detected by Western blotting. Results Treatment of fibroblasts with MMC induced a significant decrease in proliferation. With the increases of MMC from 0. 001-0. 2 g/L MMC, Hoechst 33342 staining displayed nuclei with abnormal morphologies: bean-shaped nuclei with highly condensed chromatin or nuclei with irregular clumps of dense chromatin. 0.2g/L MMC, 100 nmol/L PD98059 (ERK kinase inhibitor) , 100 nmol/L LY294002 (AKT kinase inhibitor) induced a significant increase of Caspase-3 as compared with control group, accompanied by an increase in cell apoptosis. 0.2 g/L MMC, PD98059, LY294002 caused decrease of p-ERKl/2 and p-Akt. Conclusion 0. 2 g/L MMC can induce fibroblast apoptosis by reducing the phosphorylation of p-ERKl/2 and p-Akt, resulting in the increase in the Caspase-3 expression. MMC can inhibit fibroblast proliferation and induce its apoptosis, which may be one of mechanisms by which MMC prevents peridural adhesion after laminectomy. Key words: Fibroblast; Mitomycin C; Apoptosis

Regulation of angiotensin converting enzyme II by angiotensin peptides in human cardiofibroblasts

Numerous studies have suggested that angiotensin peptides modulate the expression of angiotensin converting enzyme II (ACE2) in the cardiovascular system, but the molecular mechanisms remain poorly understood. In the present study, human cardiac fibroblasts (HCF) were used to test the regulatory effects of angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)] on ACE2 expression. The results show that Ang II upregulates ACE2 expression. This action is modulated through activation of Ang II type 1 receptor (AT1R). Ang II-mediated ACE2 upregulation was blocked by antagonists of AT1R and ERK-MAPK signaling pathways. Additionally, Ang-(1-7) increased ACE2 expression, and this upregulation was inhibited by Ang-(1-7) Mas receptor blockade. Our results further reveal that the activation of p-ERK1/2 proteins plays a critical role in upregulating ACE2 in Ang-(1-7)-stimulated HCF cells. This effect occurs independently of the Ang II-AT1R signaling pathway. In conclusion, we propose that Ang II-upregulated ACE2 may increase Ang-(1-7) formation from Ang II, and that ACE2 expression is further enhanced by Ang-(1-7) in a positive feedback loop.

Cyclic strain promotes migration of human periodontal ligament cell via extracellular signal-regulated kinase (ERK) signaling pathway

To study the effect of cyclic strain on migration of human periodontal ligament cell(hPDLC) and underlying mechanism. The cultured hPDLC were subjected to 10% or 20%-elongation magnitude cyclic strain at frequency of 0.1 Hz by FX-4000T system for 6 or 24 hours-duration respectively, while the static group serves as control. hPDLC migration was assayed by wound healing method. The expressions of matrix metalloproteinases-9 (MMP-9) and p-ERK1/2 in hPDLC without or with cyclic strain were analyzed by Western blotting. To investigate the effect of ERK signaling pathway and MMP-9 on migration of hPDLC, the cells were incubated with PD98059, a specific extracellular signal-regulated kinase (ERK) kinase inhibitor, or doxycycline, a MMP inhibitor. Then the expressions of p-ERK1/2 and MMP-9 and hPDLC migration were analyzed. In wound healing tests, the migration of hPDLC exposed to 10% or 20%-cyclic strain at 0.1 Hz-frequency for 6 hours was not apparent but became significantly different for 24 hours (P < 0.05) compared to control. Furthermore, the 20%-elongation magnitude of cyclic strain had more remarkable effect on migration of hPDLC than 10%-elongation magnitude at 24 hours-duration (P < 0.05). Cyclic strain obviously increased the expression of MMP-9 in hPDLC (P < 0.05). PD98059 could repress not only the activation of p-ERK1/2 but also the expression of MMP-9 induced by cyclic strain in hPDLC. The migration of hPDLC enhanced by cyclic strain was repressed by DOX or PD98059 in wound healing tests. Cyclic strain promotes the migration of hPDLC through activating ERK signaling pathway and inducing the expression of MMP-9.

Abstract B84: Withaferin-A is a novel HSF-1 inhibitor with potent antitumor effects in melanoma in vivo

Abstract Introduction: Withaferin A (WA) is a natural product withanolide compound with historical anti-inflammatory activity and recent anticancer activity in vitro. The purpose of this study was to evaluate the mechanism of action of withaferin A in melanoma cancer cells and test its efficacy in a murine orthotopic model of metastatic melanoma. Methods: Human melanoma cells lines SKMEL28, NPA, DRO and the murine B16F10 cell line were used for in vitro analysis. MTS cell viability assay and cell proliferation analysis was performed to determine the concentration-dependent effect of withaferin A on melanoma cells. Effects on cell cycle arrest and apoptosis were evaluated by PI/Annexin V staining with flow cytometry and confirmed through Western analysis. MAPkinase and heat shock protein expression after WA treatment was evaluated by Western analysis. Inhibitors of MEK1/2, JNK and p38 MAP kinase were used to examine the relationship between WA-mediated MAPkinase protein expression and modulation of heat shock proteins. In vivo analysis was performed on BalbC mice injected s.c. with 5 million B16F10 melanoma cells, allowing tumors to grow and metastasize. Treatment was with 5mg/kg/d WA i.p. for 3 weeks. Tumor growth and survival was followed prospectively. In vitro and in vivo variables were statistically evaluated using SPSS 17.0 software and significance was defined for p&amp;lt;0.05. Results: Withaferin A induces reduction of cell viability and cell proliferation in all melanoma cell lines tested [Range of IC50(B16F10 =209.7±16nM); (SKMEL-28=650.1 ±25nM)]. Mechanistic studies demonstrate that WA induced a cell cycle shift from G0/G1 arrest to G2/M arrest in all melanoma cells lines tested. Annexin V /PI staining on FC as well as caspase activation and PARP cleavage on Western studies confirm that WA induces apoptosis in melanoma cells. WA reduced p-Akt levels and total Akt levels in all cell lines tested in a concentration and time-dependent manner. Withaferin A modulates MAP kinase proteins, inducing activation of p-ERK1/2 , p38 MAPkinase and SAPK/JNK in all melanoma cell lines. HSP expression analysis yielded strong induction of HSP72 expression in all cell lines, reduction of HSP90 in SKMEL28 cells, and strong reduction of HSF1 cellular levels in a dose dependant manner with almost complete reduction at IC50 concentrations. Confocal microscopy analysis showed that withaferin A-induced expression of HSP72 was increased in the cytoplasm and that HSP72 was transported to the nucleus upon withaferin A stimulation. In vivo studies demonstrate almost complete tumor regression with CR in 80% of animals treated with 5 mg/kg/day WA compared to controls (p&amp;lt;0.01). Additionally at 5/mg/kg dosing, there was complete reversal of organ and lymphatic metastatic disease by inspection after 3 weeks treatment in the 80% of animals responding without progression once the treatment was discontinued. Conclusion: Our present results show that withaferin A is a potent therapy against melanoma cells in vitro and in vivo through induction of cell cycle arrest, apoptosis, and modulation of MAPkinase and HSP expression. This is the first report of inhibition of HSF1 expression in cancer cells induced by withaferin A treatment. These results support future proof of concept studies for translation to potential therapies for melanoma patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B84.

Activation of p-ERK1/2 by nicotine in pancreatic tumor cell line AR42J: effects on proliferation and secretion

The objectives of the present study were to determine the effect of nicotine on MAPK signaling and on the proliferation of AR42J cells as well as to assess the relationship between MAPK activation and exocrine secretion in these cells. AR42J cells were incubated with nicotine and analyzed for the activation of MAPK by Western blot analysis using their respective antibodies and confirmed by immunohistochemistry. The effect of nicotine on cell proliferation was determined by the spectrophotometric method, and cell function was assessed by cholecystokinin (CCK)-stimulated amylase release into the culture medium. Nicotine at a dose of 100 microM induced phospho-ERK1/2 activation maximally in 3 min compared with untreated cells. Furthermore, immunofluorescence study confirmed the nicotine-induced increase in translocation of phospho-ERK1/2 to the nucleus. Activation of phospho-ERK1/2 was inhibited by an ERK1/2 pathway inhibitor but not by a nicotine receptor antagonist. At the same dose, there was significantly enhanced proliferation of AR42J cells until 72 h without toxic effect, as the percentage of lactate dehydrogenase release remained unchanged. Other MAPKs, c-Jun NH2-terminal kinase 1/2 and p38 MAPK, were not affected by nicotine treatment. At a nicotine dose of 100 microM, the CCK-stimulated release of amylase was maximal at 6 min, and, although a nicotinic receptor antagonist inhibited this response, it was not inhibited by the ERK1/2 pathway inhibitor. We conclude that nicotine treatment induced activation of ERK1/2 and increased the proliferation of AR42J cells. The data further indicate that MAPK signaling by nicotine is independent of the secretory response.