P38 Extracellular Signal-regulated Kinase Research Articles

Sulforaphene-Carboplatin Combination Synergistically Enhances Apoptosis by Disruption of Mitochondrial Membrane Potential and Cell Cycle Arrest in Human Non-Small Cell Lung Carcinoma.

Worldwide non-small cell lung cancer (NSCLC) causes substantial morbidity and mortality among human populations. Due to the severe side effects and low survival rate of patients with the conventional drugs, implementation of new combination therapies is much needed. The aim of this study was to evaluate the efficacy of a combination therapy with a conventional drug and a natural medicine. We compared the combination of chemotherapy drug carboplatin and the radish-derived isothiocyanate compound sulforaphene, which synergistically induces higher apoptosis and growth inhibition in A549, to the drug alone in human NSCLC cells. We found that this combination group significantly induced higher depolarization of mitochondrial membrane potential (MMP) and intracellular reactive oxygen species generation than the single drug dose, followed by cell cycle arrest at the G0/G1 phase after 24 h of incubation. In addition to that, the Western blot assays showed that combination treatment inhibited the expression of Bcl-2 and successively upregulated the expression of Bax, cytochrome C, apoptosis-inducing factor, caspase-9 and -3, and cleaved poly ADP ribose polymerase. It also modulated the expression of PI3K, p-extracellular signal-regulated kinase (1/2), and p-c-Jun N-terminal kinase indicating the involvement of antiproliferative properties. Further pretreatment with pan-caspase inhibitor Z-VAD-fmk was carried out to confirm the effect of caspases in the combination therapy-induced apoptosis. To summarize, this is the first report that sulforaphene-carboplatin combination treatment synergistically promotes enhanced apoptosis and antiproliferative effect over single drug treatment against A549, human NSCLC cells through caspase activation, MMP disruption, and cell cycle arrest. This study demonstrates that the duel character of this combination therapy may be an effective replacement for conventional therapy alone against NSCLC.

CXCL16 induces angiogenesis in autocrine signaling pathway involving hypoxia-inducible factor 1α in human umbilical vein endothelial cells.

Chemokine (C-X-C motif) ligand 16 (CXCL16) is a new angiogenic factor inducing angiogenesis via extracellular signal-regulated kinases pathway. To further understand the molecular mechanism underlying CXCL16‑induced angiogenesis, we explored involvement of other relevant pathways in CXCL16-induced angiogenesis. In the present study, we investigated the mechanisms underlying CXCL16-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). CXCL16 promoted HUVEC proliferation, tube formation and migration. Enzyme-linked immunosorbent assay revealed that CXCL16 induced vascular endothelial growth factor secretion from HUVECs. Western blot analysis showed that CXCL16 increased the level of hypoxia‑inducible factor 1α, p-extracellular signal-regulated kinases (ERK), p-p38 and p-Akt dose- and time-dependently. ERK-, p38- and Akt-selective inhibitors significantly suppressed HUVEC proliferation, migration, tube formation and hypoxia-inducible factor 1α (HIF-1α) expression induced by CXCL16. Furthermore, CXCL16 peptides induced CXCL16 secretion via ERK, p38 and Akt pathways, which was suppressed by HIF-1α-selective inhibitor PX12. Our data suggest that CXCL16 induces angiogenesis in autocrine manner via ERK, Akt, p38 pathways and HIF-1α modulation.

Thyroid Hormone-Induced Differentiation of Astrocytes is Associated with Transcriptional Upregulation of β-arrestin-1 and β-adrenergic Receptor-Mediated Endosomal Signaling.

Thyroid hormones (TH) promote differentiation of astrocytes. We have previously reported that a downstream role β-adrenergic receptor (β-AR) system in such effects of TH. Although evidences indicate strong interaction between TH and the β-ARs, the underlying mechanism is poorly understood. In the present study, we further explored the influence of TH on β-AR signaling during the differentiation process. Unlike β1-AR, binding of (125)I-pindolol to β2-AR in cell membranes was significantly decreased at 2h of exposure to TH which came back to control values after 24h. The initial decrease in β2-AR in membranes resulted in a concomitant increase in β2-AR levels in the cytosol, suggesting that TH may induce endocytosis of the receptor. qRT-PCR as well as Western blot analysis demonstrated that unlike β-adrenergic receptor kinase (β-ARK)1 and β-ARK2, the messenger RNA (mRNA) and protein levels of β-arrestin-1 in the astrocyte cultures increased on exposure to TH. Knockdown of β-arrestin gene suggested requirement of both β-arrestin-1 and β-arrestin-2 isoforms during endocytosis of β2-AR, thereby facilitating cell differentiation. Endocytic inhibitors blocked the delayed but sustained activation of p-extracellular signal-regulated kinase (ERK) observed during cell differentiation. Observations suggest that TH upregulate β-arrestin-1 in astrocytes to facilitate endocytosis of β2-AR, required for endosomal ERK activation to drive the differentiation process.

Insulin-Like Growth Factor-1 Contributes to Mucosal Repair by β-Arrestin2–Mediated Extracellular Signal-Related Kinase Signaling in Experimental Colitis

Insulin-like growth factor-1 (IGF-1) possesses the ability to attenuate intestinal damage and promote mucosal repair of colitis. β-Arrestins, as the scaffolding proteins of G protein-coupled receptors or non-G protein-coupled receptors signaling, can be involved in IGF-1-mediated signaling pathways. However, the interaction of IGF-1 and β-arrestin2 in the mucosal repair of experimental colitis remains unexplored. Ulcerative colitis was induced in β-arrestin2 wild-type mice and β-arrestin2 knockout littermates by using 3% dextran sulfate sodium for 5 days, followed by regular water consumption for 1, 2, 3, and 4 weeks to analyze the mucosal repair from experimental colitis. Disease activity index and histologic score analyses were performed. Apoptosis and proliferation were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Ki-67 staining, respectively. The expressions of β-arrestin2, phospho (p)-IGF-1R, and p-extracellular signal-regulated kinase (ERK)1/2 were examined. Furthermore, β-arrestin2 was overexpressed or altered in HCT116 cells by transfection before IGF-1 treatment in vitro. IGF-1 and β-arrestin2 expression was up-regulated in the repairing phase of experimental colitis. Targeted deletion of β-arrestin2 delayed the repair of colitis by inhibiting cell proliferation without affecting the levels of IGF-1 and p-IGF-1R. The β-arrestin2/ERK signaling pathway was involved in IGF-1-mediated mucosal repair through promoting epithelial cell and goblet cell regeneration from experimental colitis. These results indicate that IGF-1 contributes to the mucosal repair by β-arrestin2-mediated ERK signaling in experimental colitis.

FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans.

BackgroundPeriodontitis is a bacteria-driven inflammatory bone loss disease. Previous studies showed that the oral pathogen Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) stimulated the generation of sphingosine 1 phosphate (S1P). In addition, S1P signaling regulated the migration of osteoclast precursors and affected osteoclastogenesis. Furthermore, treatment with FTY720 (also called fingolimod, a modulator of multiple S1P receptors) alleviated osteoporosis and suppressed arthritis in animals. This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation.MethodsMurine bone marrow-derived monocytes and macrophages (BMMs) were treated with vehicle ethanol or FTY720, and were either unstimulated or stimulated for 0.5 to 6 h with A. actinomycetemcomitans. The protein levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the media of BMMs were quantified by enzyme-linked immunosorbent assay (ELISA). Protein expressions, including phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, p-extracellular signal-regulated kinase (p-ERK), PI3K, Akt, and ERK were evaluated by Western blot. In addition, murine bone marrow-derived pre-osteoclasts were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) for three days. Then the cells were treated with either vehicle or FTY720 and were either unstimulated or stimulated with A. actinomycetemcomitans for 4 to 24 h. Control cells were treated with M-CSF alone with or without bacterial stimulation. Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of osteoclastogenic factors, including nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 (Nfatc1), cathepsin K (Ctsk), acid phosphatase 5 (Acp5), osteoclast-associated receptor (Oscar), and RANKL were quantified by quantitative real-time polymerase chain reaction (PCR).ResultsFTY720 dose-dependently inhibited IL-1β, IL-6, and TNF-α protein levels induced by A. actinomycetemcomitans in BMMs compared with controls. Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans. Furthermore, FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts with or without bacterial stimulation and reduced the mRNA levels of Nfatc1, Ctsk, Acp5, and Oscar, but not RANKL in bone marrow-derived pre-osteoclasts.ConclusionFTY720 inhibited proinflammatory cytokine production and suppressed osteoclastogenesis, supporting FTY720 as a potential therapy for inflammatory bone loss diseases.

Laminin α4 (LAMA4) expression promotes trophoblast cell invasion, migration, and angiogenesis, and is lowered in preeclamptic placentas

IntroductionThe laminin α4 subunit (LAMA4) has been shown to promote migration, proliferation, and survival of various cell types. This study investigated LAMA4's role in trophoblast cells during placental development. MethodsLAMA4 expression was immunohistochemically assessed in the first trimester and term human placentas. LAMA4 siRNA was applied to silence LAMA4 expression in extravillous explants and HTR8/SVneo cells. Hypoxia–reoxygenation (H/R) conditions were applied to mimic preeclampsia. LAMA4 expression and trophoblast cell invasion, migration, and tube formation (a measure of angiogenesis) were assessed in HTR8/SVneo cells. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was used to study the mechanism underlying LAMA4 activity. LAMA4 promoter methylation was assessed by bisulfite-sequencing polymerase chain reaction (PCR) or methylation-specific PCR. ResultsLAMA4 levels in preeclamptic placentas were significantly lower than those in controls. LAMA4 silencing significantly inhibited extravillous explant outgrowth as well as HTR8/SVneo cell invasion and migration. H/R conditions significantly lowered LAMA4 expression. Application of either H/R conditions or LAMA4 silencing both significantly decreased HTR8/SVneo cell invasion, migration, and tube formation, decreased MMP2 and MMP9 expression, and increased TIMP2 expression. SB203580 significantly reduced LAMA4 expression. LAMA4 silencing significantly decreased p-p38, p-c-Jun N-terminal kinase (JNK), and p-extracellular signal-regulated kinase (ERK) expressions; by contrast, H/R conditions induced significant upregulation of p-p38 and p-ERK but decreased p-JNK. LAMA4 promoter methylation was not significantly altered in preeclamptic placentas compared to controls. ConclusionsLAMA4 expression is lowered in preeclamptic placentas and promotes trophoblast cell invasion, migration, and angiogenesis. H/R conditions decrease LAMA4 expression and appear to decouple the positive relationship between LAMA4 expression and p38 and ERK activation.

Expression of Mitogen-Activated Protein Kinases in Chronic Subdural Hematoma Outer Membranes.

Growth factors and inflammatory cytokines activate the mitogen-activated protein kinase (MAPK) cascade. Previous studies have shown that chronic subdural hematoma (CSDH) fluid contains these factors and cytokines. In this study, expression of three major MAPK cascade transmitters in the outer membrane of CSDH was assessed. Eleven patients whose outer membrane and CSDH fluid were successfully obtained during trepanation surgery were included in this study. Expression of extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK, and actin was examined by Western blot and immunostaining. We examined whether CSDH fluid could activate MAPKs in cultured endothelial cells (ECs) or fibroblasts in vitro. Western blot analysis showed that p-ERK was present in all samples, whereas p-p38 and p-JNK were detected, but not in all cases. Immunostaining showed that all three p-MAPKs were expressed in vascular endothelium. However, only p-ERK was expressed in fibroblasts. Expression of p-extracellular signal-regulated kinase kinase (MEK) and p-ERK in ECs and fibroblasts was significantly induced immediately after treatment with CSDH fluid, whereas p-p38 and p-JNK expression was significantly induced in ECs 60 min after treatment, but not in fibroblasts. Activation of MEK was significantly inhibited by treatment with antibodies directed against interleukin-6 and vascular endothelial growth factor in ECs, but not in fibroblasts. Inflammatory cytokines and growth factors in CSDH fluids might activate major MAPKs in ECs, which might be associated with neovascularization in the outer membrane of CSDH. These MAPK pathways could become novel targets for treatment of CSDHs.

Particulate matter 2.5 induces autophagy via inhibition of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin kinase signaling pathway in human bronchial epithelial cells.

Particulate matter 2.5 (PM2.5) is a significant risk factor for asthma. A recent study revealed that autophagy was associated with asthma pathogenesis. However, the specific mechanisms underlying PM2.5-induced autophagy in asthma have remained elusive. In the present study, PM2.5-induced autophagy was evaluated in Beas-2B human bronchial epithelial cells and the potential molecular mechanisms were investigated. Using electron microscopy, immunofluorescence staining and immunoblot studies, it was confirmed that PM2.5 induced autophagy in Beas-2B cells as a result of PM2.5-mediated inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway in Beas-2B cells. LY294002, a PI3K inhibitor, reduced the accumulation of microtubule-associated protein 1 light chain 3 II and attenuated the effect of PM2.5. Phosphorylated (p-)p38, p-extracellular signal-regulated kinase and p-c-Jun N-terminal kinase were dephosphorylated following exposure to PM2.5. The roles of p53, reactive oxygen species scavenger tetramethylthiourea and autophagy inhibitor 3-methyladenine in PM2.5-induced autophagy in Beas-2B cells were also investigated. The results suggested that the PI3K/Akt/mTOR signaling pathway may be a key contributor to PM2.5-induced autophagy in Beas-2B cells. The results of the present study therefore provided an a insight into potential future clinical applications targeting these signaling pathways, for the prevention and/or treatment of PM2.5-induced lung diseases.

The effects and mechanisms of G protein-coupled receptor 91 on blood-retinal barrier in diabetic rats

Objective To investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats. Methods A lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed. Healthy male Sprague-Dawley (SD) rats were selected in this study. The 60 rats were randomized into 4 groups and treated as follows: (1) control group (Group A, n=15), the rats received injections of an equal volume of 0.1% citrate buffer; (2) streptozocin (STZ) group (Group B, n=15), the rats received injections of STZ; (3) LV.shScrambled group (Group C, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes; (4) LV.shGPR91 group (Group D, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml pGCSIL-GFP-shGPR91 lentiviral particles. At 12 weeks after intravitreal injection, immunohistochemistry and Western blot were used to assess the expression of GPR91, p-extracellular signal-regulated kinase(ERK)1/2, t-ERK1/2, p-Jun N-terminal kinase (JNK), t-JNK, p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK. Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel. Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF. Results Immunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer. Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31, P<0.01). HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina, while the telangiectatic vessels attenuated in Group D. It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)% compared with Group C and there was significant difference (F=30.35, P<0.05). The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15, P<0.05). The results of Western blot indicated that compared with Group A, the expressions of p-ERK1/2, p-JNK and p-p38 MAPK were significantly upregulated (q=6.38, 2.94, 3.45; P<0.05). Meanwhile, the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50, P<0.05). Conclusions The intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats. GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway. Key words: Diabetic retinopathy/physiopathology; RNA, small interfering; Receptors, G-protein-coupled; Vascular Endothelial Growth Factors; p38 Mitogen-Activated Protein Kinases; Animal experimentation

TSH Signaling Overcomes B-RafV600E–Induced Senescence in Papillary Thyroid Carcinogenesis through Regulation of DUSP6

B-RafV600E oncogene mutation occurs most commonly in papillary thyroid carcinoma (PTC) and is associated with tumor initiation. However, a genetic modification by B-RafV600E in thyrocytes results in oncogene-induced senescence (OIS). In the present study, we explored the factors involved in the senescence overcome program in PTC. First of all, we observed down-regulation of p-extracellular signal-regulated kinases 1/2 and up-regulation of dual specific phosphatase 6 (DUSP6) in the PTC with B-RafV600E mutation. DUSP6 overexpression in vitro induced extracellular signal-regulated kinases 1/2 dephosphorylation and inhibited B-RafV600E–induced senescence in thyrocytes. Although DUSP6 protein was degraded by B-RafV600E–induced reactive oxygen species (ROS), thyroid-stimulating hormone (TSH) stabilized DUSP6 protein by increasing Mn superoxide dismutase expression and inhibited B-RafV600E–induced senescence. Although serum TSH was not increased, its receptor was markedly upregulated in PTC with B-RafV600E. Furthermore, TSH together with DUSP6 reactivated Ras signaling, resulted in activation of Ras/AKT/glycogen synthase kinase 3β, and stabilized c-Myc protein by inhibiting its degradation. These observations led us to conclude that increased TSH signaling overcomes OIS and is essential for B-RafV600E–induced papillary thyroid carcinogenesis.

Quercetin enhances adriamycin cytotoxicity through induction of apoptosis and regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase/c-Jun N-terminal kinase signaling in multidrug-resistant leukemia K562 cells.

Multidrug resistance (MDR) has become a significant challenge in chemotherapeutic treatment of cancer. Quercetin, a naturally occurring flavonoid, has been found to possess anti-proliferative, anti-inflammatory and immunoregulatory bioactivities. The present study was performed to examine the effect of quercetin on human leukemic MDR K562/adriamycin (ADR) cells. Treatment of K562/ADR cells with a combination of quercetin and ADR resulted in potentiation of cytotoxicity, which was measured using a cell counting kit-8 assay. Flow cytometric analysis revealed that quercetin dose-dependently promoted cell apoptosis and treatment with a combination of quercetin and ADR caused synergistic enhancement of the apoptotic effect. In addition, treatment of K562/ADR cells with quercetin alone or in combination with ADR resulted in loss of mitochondrial membrane potential, activation of caspase-8, -9 and -3, reduced expression of the anti-apoptotic proteins B-cell lymphoma (Bcl)-2 and Bcl-extra large and enhanced expression of the pro-apoptotic proteins Bcl-2-interacting mediator of cell death, Bcl-2-associated death promoter and Bcl-2-associated X protein in the cells. Furthermore, the combined treatment of quercetin and ADR synergistically increased the expression of phosphorylated (p-)c-Jun N-terminal kinase and p-p38 mitogen-activated protein kinase and decreased the expression of p-extracellular signal-regulated kinase in the K562/ADR cells. In addition, the expression of P-glycoprotein was significantly decreased following treatment with quercetin alone or in combination with ADR. These findings demonstrated that quercetin is important in MDR and may be developed into a new reversal agent for cancer chemotherapy.

Effect of nilotinib on airway remodeling in a murine model of chronic asthma

ABSTRACTObjective: The tyrosine kinase inhibitor nilotinib has potent inhibitory activity against the stem cell growth factor receptor c-Kit and platelet-derived growth factor receptor (PDGFR). The present study aimed to determine whether nilotinib suppresses airway remodeling and whether its effect is associated with the c-Kit and PDGFR pathways. We also aimed to compare the effect of nilotinib and imatinib on remodeling. Methods: We developed a mouse model of airway remodeling, which includes smooth muscle thickening, in which ovalbumin (OVA)-sensitized mice were repeatedly exposed to intranasal OVA administration twice a week for 3 months. Mice were treated with nilotinib or imatinib during the OVA challenge. Results: Compared with control mice, the mice chronically exposed to OVA developed sustained eosinophilic airway inflammation, airway hyperresponsiveness (AHR), and exhibited features of airway remodeling, including thickening of the peribronchial smooth muscle layer. Administration of nilotinib significantly inhibited eosinophilic inflammation, AHR, and remodeling in mice chronically exposed to OVA. Nilotinib showed a trend of more potent effect than imatinib on attenuating remodeling in hydroxyproline assay and smooth muscle staining. Nilotinib treatment significantly reduced the expression of phosphorylated (p)-c-Kit, p-PDGFRβ, and p-extracellular signal-regulated kinase 1/2. The expression levels of the genes encoding c-Kit and PDGFRβ were also reduced by nilotinib treatment. Treatment with nilotinib did not affect significantly the level of OVA-specific IgE and IgG1 in serum. In vitro, nilotinib significantly inhibited cell proliferation of fibroblast. Conclusions: These results suggest that nilotinib administration can prevent airway inflammation, AHR, and airway remodeling associated with chronic allergen challenge.

Farnesol induces apoptosis of DU145 prostate cancer cells through the PI3K/Akt and MAPK pathways

The aim of this study was to investigate the effect of farnesol on the induction of apoptosis in DU145 prostate cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay showed that cell proliferation decreased significantly in a dose- and time-dependent manner. 4',6-Diamidino-2-phenylindole staining showed that chromatin condensation in cells treated with 60 µM of farnesol was markedly higher than in the control groups. Farnesol increased the expression of p53, p-c-Jun N-terminal kinase, cleaved-caspase-3, Bax, and cleaved-caspase-9, but decreased the expression of p-phosphatidylinositol-3-kinase (PI3K), p-Akt, p-p38, Bcl-2, and p-extracellular signal-regulated protein kinase, in a dose-dependent manner. The apoptotic cell ratio increased in a dose-dependent manner. The tumor growth inhibitory effect of farnesol was investigated in a mouse model. Compared to the control group, tumor volume decreased significantly in the group administered 50 mg/kg farnesol. Apoptosis was frequently detected in this same group by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The results indicated that farnesol induced apoptosis of DU145 prostate cancer cells through the PI3K/Akt and mitogen-activated protein kinase signaling pathways.

Substantial Neuroprotection Against K+ Deprivation‐Induced Apoptosis in Primary Cerebellar Granule Neurons by Novel Dimer Bis(propyl)‐Cognitin Via the Activation of VEGFR‐2 Signaling Pathway

Neuronal loss via apoptosis in CNS is the fundamental mechanism underlying various neurodegenerative diseases. Compounds with antiapoptotic property might have therapeutic effects for these diseases. In this study, bis(propyl)-cognitin (B3C), a novel dimer that possesses anti-AChE and anti-N-methyl-d-aspartate receptor activities, was investigated for its neuroprotective effect on K(+) deprivation-induced apoptosis in cerebellar granule neurons (CGNs). Cerebellar granule neurons were switched to K(+) deprived medium with or without B3C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, fluorescein diacetate (FDA)/propidium iodide (PI) staining, Hoechst staining, and DNA laddering assays were applied to detect cytotoxicity and apoptosis. Additionally, the expression of p-VEGFR-2, p-Akt, p-glycogen synthase kinase 3β (GSK3β), and p-extracellular signal-regulated kinase (ERK) was examined in CGNs. Switching CGNs to K(+) deprived medium resulted in remarkable apoptosis, which could be substantially blocked by B3C treatment (IC50 , 0.37 μM). Moreover, a rapid decrease in p-Tyr1054-VEGFR-2 was observed after the switch. B3C significantly reversed the inhibition of p-Tyr1054-VEGFR-2 as well as Akt and ERK pathways. VEGFR-2 inhibitor PTK787/ZK222584, as well as PI3-K inhibitor LY294002 and MEK inhibitor PD98059, each abolished the neuroprotective effect of B3C. Our results demonstrate that B3C blocks K(+) deprivation-induced apoptosis in CGNs through regulating VEGFR-2/Akt/GSK3β and VEGFR-2/ERK signaling pathways, providing a molecular insight into the therapeutic potential of B3C for the treatment of neurodegenerative diseases.

Differences of protein expression of renal cell carcinoma (RCC) by tumor location and treatment.

e15556 Background: Resistance to targeted therapy in RCC is primarily related to tumor and environmental changes. We assessed the difference of morphology and protein analyte expression by metastatic site and treatment types. Methods: Archival tissues from primary and metastatic RCC biopsies were studied. Immunohistochemical probes included antibodies for the protein analytes: phospholipase D2; fatty acid synthase; phosphorylated (p)-Akt (Ser 473); p-mTOR [Ser 2448]); p-p70S6K (Thr 389); p-extracellular signal-regulated kinase (ERK) 1/2 (Thr 202/Tyr 204); p-signal transducer and activator of transcription (STAT)3 (Tyr 705); vascular endothelial growth factor (VEGF)-A; stem cell markers CD44 and CD56; and intratumoral FOXP3 vs. CD8 lymphocytes. Results: A total of 66 patients with available tissue biopsies were assessed. Fourteen patients had additional biopsy at progression. Morphoproteomics was assessed on a total of 81 specimens. The nuclear distribution of phospholipase D2 was statistically significant different by biopsy site (p=0.0046) with highest expression in bone and lung. Similarly, the cytoplasmic expression fatty acid synthase was significantly different by biopsy site (p=0.0167) with highest expression in lymph node and adrenal. More importantly, nuclear mTOR activity was different by tumor biopsy site (p=0.001) with predominant expression in lung, lymph nodes and brain. Compared to mTORC1, which was more expressed in primary tumors, mTORC2 is more expressed in lung and bone metastasis. None of the other protein analytes expression was different based on tumor location. Further analysis on patients with multiple biopsies showed that the plasmalemmal CD56 expression increased with therapy but none of the other protein expression. Conclusions: This analysis emphasized the role of the microenvironment in tumor biology. Prospective treatment trial is underway based on the different molecular fingerprints.

Morphoproteomics of mTORC2 pathway as resistance signature and activated ERK and STAT3 prosurvival/antiapoptotic pathways in metastatic renal cell carcinoma (mRCC) progressing on rapalogs.

e15548 Background: Resistance to rapalog therapies (rapamycin, temsirolimus or everolimus) in mRCC may be due to adaptive switching from mammalian target of rapamycin complex (mTORC)1 to mTORC2. A preclinical study using an agent targeting both complexes demonstrated the combination is more effective than temsirolimus in vitro but not in xenograft models (Zhang 2013 PLos One). Methods: Morphoproteomic analysis of mRCC biopsies showed progression while the patients were treated with everolimus or temsirolimus at the time of biopsy. Constitutive activation evidenced by expression (≤3) of phosphorylated (p)-mTOR (Ser 2448) in the nuclear compartment coupled with concomitant expression of p-Akt (Ser 473) favors mTORC2 and constitutive activation by p-extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) and p-signal transducer and activator of transcription (STAT)3 (Tyr705) in the nuclear compartments (≤3) indicates convergent signaling through prosurvival/antiapoptotic pathways. Results: Overexpression of mTORC2 with strong expression of p-ERK1/2 and p-STAT3 indicates adaptive switch from mTORC1 to mTORC2 pathway and constitutive activation of prosurvival/antiapoptotic pathways (Table). Overexpression of mTORC1 with strong expression of p-ERK 1/2 and p-STAT3 indicates adaptive resistance to everolimus. Equal expression of mTORC 1 and 2 with concomitant strong expression of p-ERK1/2 and p-STAT3 indicates resistance to everolimus and constitutive activation of the adaptive prosurvival/antiapoptotic pathways. Conclusions: Morphoproteomics confirms mTORC2 as resistance signature to rapalog therapy and uncovers constitutive activation of the prosurvial ERK and STAT3 pathways as additional adaptive mechanisms to such treatment, indicating the need for a combinatorial therapeutic approach in mRCC progressing on rapalogs. [Table: see text]

Morphoproteomics of VEGF pathway mediators as evidence of reversible resistance to VEGF-TKIs demonstrated by expression of activated ERK and STAT3 in metastatic renal cell carcinoma (mRCC).

e15552 Background: Resistance to vascular endothelial growth factors (VEGF)-tyrosine kinase inhibitors (TKIs) (e.g., sunitinib, sorafenib) may be a result of changes in the tumor microenvironment and to the selection of resistant clones and permanent genetic changes. This has been demonstrated in mouse xenograft models where there was tumor shrinkage following reintroduction of sorafenib after a 1 week break (Zhang 2011 PLoS One). Methods: We applied morphoproteomic analysis to biopsy specimens of mRCC after a treatment break with VEGF-TKIs. Constitutive activation evidenced by expression (≤3) of p-extracellular signal-regulated kinase (ERK) 1/2 and p-signal transducer and activator of transcription (STAT) 3 in the nuclear compartments (≤3) indicates signaling through the VEGF pathway. Results: Strong overexpression of p-ERK1/2 and p-STAT3 with VEGF tyrosine kinase inhibitors indicates reactivation of the VEGF pathways (Table). Conclusions: Morphoproteomics confirms constitutive activation of the prosurvival ERK and STAT3 pathways after a treatment break indicating reversibility in resistance patterns to therapy with VEGF-TKIs indicating that the drugs can be reintroduced after having progressed on the same therapy. [Table: see text]

A comprehensive immunohistochemical approach of AKT/mTOR pathway and p-STAT3 in mycosis fungoides

Although the expression pattern of phosphorylated (p)-mTOR pathway components has attracted scientific interest in several neoplasms, to our knowledge, there is no published information regarding its significance in mycosis fungoides (MF). We sought to perform a comprehensive simultaneous assessment of key members of AKT/mTOR pathway along with p-extracellular signal-regulated kinase (ERK), NOTCH1, and p-STAT3 in patients with MF. In all, 54 skin biopsy specimens (21 tumors, 30 plaques, and 3 folliculotropic MF) from 50 patients with MF were analyzed immunohistochemically for p-mTOR, its upstream p-AKT, its downstream effectors p-p70S6K and p-4E-BP1, and for p-ERK1/2, NOTCH1, and p-STAT3. p-mTOR was coexpressed with p-p70S6K in 67.3% of lesions, but coexpression with other molecules was less common. p-p70S6K and marginally NOTCH1 displayed higher H-scores in tumors than in plaques. Significant correlations were recorded between p-ERK and p-4E-BP1, as well as between NOTCH1 and p-p70S6K or p-4E-BP1. NOTCH1, p-4E-BP1, and p-p70S6K expression were associated with advanced stage. In survival analysis simultaneous overexpression of p-AKT and p-p70S6K, along with p-4E-BP1 positivity, adversely affected cancer-specific, disease-free, and progression-free survival in advanced-stage cases. A limitation may be the small number of cases included in our investigation, precluding multivariate survival analysis. Activation of AKT/mTOR pathway in MF appears to be correlated with NOTCH1, p-ERK, and p-STAT3 and is implicated in the acquisition of a more aggressive phenotype. The combination of p-AKT, p-p70S6K, and p-4E-BP1 emerges as a significant potential prognostic marker in patients with advanced stage.

The saponin monomer of dwarf lilyturf tuber, DT-13, inhibits angiogenesis under hypoxia and normoxia via multi-targeting activity

The saponin monomer of dwarf lilyturf tuber, DT-13, exhibits anticancer activity by reducing human breast cancer cell adhesion and migration under hypoxia. To further investigate the anticancer activity of DT-13, we investigated whether DT-13 exhibits anti-angiogenic activity. DT-13 showed no effect on human umbilical vein endothelial cell proliferation but inhibited tube formation and migration under normoxia and hypoxia. Moreover, DT-13 significantly reduced density of vessels invivo observed from a chicken chorioallantoic membrane model. Western blotting results showed that DT-13 suppressed the increased level of hypoxia-inducible factor 1α, p-extracellular signal-regulated kinase 1/2 and p-Akt induced by hypoxia. Enzyme-linked immunosorbent assay revealed that vascular endothelial growth factor excretion was suppressed by DT-13. DT-13 inhibited migration and tube formation induced by vascular endothelial growth factor under normoxia and hypoxia. In addition, DT-13 reduced the level of p-vascular endothelial growth factor receptor 2 and p-Akt induced by vascular endothelial growth factor. Our data suggest that DT-13 inhibits angiogenesis under normoxia and hypoxia and also inhibits angiogenesis induced by vascular endothelial growth factor via targeting at multi elements.

A comprehensive immunohistochemical and molecular approach to the PI3K/AKT/mTOR (phosphoinositide 3‐kinase/v‐akt murine thymoma viral oncogene/mammalian target of rapamycin) pathway in bladder urothelial carcinoma

What's known on the subject? and What does the study add? A few published studies investigating single or various PI3K/AKT/mTOR signalling components have produced inconsistent results. Moreover, PI3K regulatory subunit p85a and activated p70S6K expression levels have not been previously examined in urothelial carcinoma (UC). The present study addresses simultaneously all key members of PI3K/AKT/mTOR signalling cascade supporting a differential implication of PI3K/AKT/mTOR pathway components in urothelial tumorigenesis. Furthermore, we propose p-4E-BP1 as a potential prognostic marker in UC, which might assist the selection of patients more likely to benefit from chemotherapy regimens based on PI3K/AKT/mTOR pathway inhibition. Finally, the present study indicates PIK3CA/AKT1 mutational status as a potential predictive marker for time-to-recurrence. • To perform a comprehensive simultaneous assessment of all key members of phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway along with AKT homolog 1 (AKT1) and PIK3 catalytic alpha polypeptide (PIK3CA) mutations in bladder urothelial carcinoma (UC). • Published information is limited to a few studies looking into single or various combinations of members of this pathway with inconsistent results. In particular the expression status of phosphorylated (p-)p70S6 kinase (p70S6K) and p85a subunit of PI3K has not been tested in UC. • Paraffin-embedded transurethral resection tissue from 113 patients with UC was investigated for the association of p85aPI3K, p-AKT, p-mTOR, p-p70S6K and p-4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) expression status, as well as PIK3CA and AKT1 mutations with p-extracellular signal-regulated kinase 1/2 (ERK1/2), fibroblast growth factor receptor 3 (FGFR3), pathological features, recurrence and cancer-specific survival. • With the exception of p-p70S6K, all others components of the PI3K/AKT/mTOR pathway were upregulated in UCs as compared with normal urothelium. • p-mTOR expression strongly correlated with its upstream p-AKT and marginally with its downstream p-p70S6K. p85aPI3K and p-ERK1/2 levels were also marginally correlated. • PIK3CA and AKT1 mutations were distinctly uncommon and mutually exclusive, without any association with pathological features. However, the presence of AKT1 mutations was associated with increased FGFR3 levels and was restricted to p85aPI3K immunonegative cases, whereas PIK3CA mutant cases had marginally lower p85aPI3K levels. • The presence of PIK3CA single or combined with AKT1 mutations was associated with shorter recurrence-free survival in univariate survival analysis. An inverse relationship was established between p-4E-BP1 immunopositivity and histological grade or T category, as well as between p-p70S6K levels and T category, the latter relationship being of marginal significance. • p-4E-BP1 nuclear expression was marginally associated with the presence of lymphovascular invasion and adversely affected survival in multivariate, but not in univariate analysis. • PI3K/AKT/mTOR signalling components appear to be differentially implicated in urothelial tumorigenesis and, with the exception of p85aPI3K, are unrelated to the PIK3CA or AKT1 mutational status. • Our findings propose p-4E-BP1 as a potential prognostic marker in UC independent of its association with pathological features, which might assist the selection of patients more likely to benefit from PI3K/AKT/mTOR axis inhibition. • PIK3CA/AKT1 mutational status may have a place in the prediction of time-to-recurrence.