Phosphoprotein Levels Research Articles

Morphine self-administration alters the expression of translational machinery genes in the amygdala of male Lewis rats.

Addiction is a chronic disorder with a high risk of relapse. The neural mechanisms mediating addictions require protein synthesis, which could be relevant for the development of more effective treatments. The mTOR signaling pathway regulates protein synthesis processes that have recently been linked to the development of drug addiction. To assess the effects of morphine self-administration and its subsequent extinction on the expression of several genes that act in this pathway, and on the levels of specific phosphoproteins (Akt, Gsk3α/β, mTOR, PDK1 and p70 S6 kinase) in the amygdala, nucleus accumbens, and the prefrontal cortex. Male Lewis rats underwent morphine self-administration (1 mg/kg) for 19 days. They subsequently were submitted to extinction training for 15 days. Rats were killed either after self-administration or extinction, their brains extracted, and gene expression or phosphoprotein levels were assessed. We found an increase in Raptor and Eif4ebp2 expression in the amygdala of rats that self-administered morphine, even after extinction. The expression of Insr in the amygdala of control animals decreased over time while the opposite effect was seen in the rats that self-administered morphine. Our results suggest that morphine self-administration affects the gene expression of some elements of the translational machinery in the amygdala.

Epigallocatechin-3-gallate inhibits the growth and increases the apoptosis of human thyroid carcinoma cells through suppression of EGFR/RAS/RAF/MEK/ERK signaling pathway

BackgroundThyroid cancer is the most common type of endocrine malignancy and the incidence rate is rapidly increasing worldwide. Epigallocatechin-3-gallate (EGCG) could suppress cancer growth and induce apoptosis in many types of cancer cells. However, the mechanism of action of EGCG on the growth of human thyroid carcinoma cells has not been fully illuminated.MethodsCell proliferation and viability were detected by EdU and MTS assays. Cell cycle distribution was measured by flow cytometry. Migration and invasion were evaluated by scratch and transwell assays. Apoptotic levels were detected by TUNEL staining and western blotting. The protein levels of EGFR/RAS/RAF/MEK/ERK signaling pathway were detected by western blotting. The in vivo results were determined by tumor xenografts in nude mice. The in vivo proliferation, tumor microvessel density, and apoptosis were detected by immunohistochemistry.ResultsEGCG inhibited the proliferation, viability, and cell cycle progression in human thyroid carcinoma cells. EGCG decreased the migration and invasion, but increased the apoptosis of human thyroid carcinoma cells. EGCG reduced the protein levels of phospho (p)-epidermal growth factor receptor (EGFR), H-RAS, p-RAF, p-MEK1/2, and p-extracellular signal-regulated protein kinase 1/2 (ERK1/2) in human thyroid carcinoma cells. EGCG inhibited the growth of human thyroid carcinoma xenografts by inducing apoptosis and down-regulating angiogenesis.ConclusionsEGCG could reduce the growth and increase the apoptosis of human thyroid carcinoma cells through suppressing the EGFR/RAS/RAF/MEK/ERK signaling pathway. EGCG can be developed as an effective therapeutic agent for the treatment of thyroid cancer.

A dynamic view of the proteomic landscape during differentiation of ReNcell VM cells, an immortalized human neural progenitor line

The immortalized human ReNcell VM cell line represents a reproducible and easy-to-propagate cell culture system for studying the differentiation of neural progenitors. To better characterize the starting line and its subsequent differentiation, we assessed protein and phospho-protein levels and cell morphology over a 15-day period during which ReNcell progenitors differentiated into neurons, astrocytes and oligodendrocytes. Five of the resulting datasets measured protein levels or states of phosphorylation based on tandem-mass-tag (TMT) mass spectrometry and four datasets characterized cellular phenotypes using high-content microscopy. Proteomic analysis revealed reproducible changes in pathways responsible for cytoskeletal rearrangement, cell phase transitions, neuronal migration, glial differentiation, neurotrophic signalling and extracellular matrix regulation. Proteomic and imaging data revealed accelerated differentiation in cells treated with the poly-selective CDK and GSK3 inhibitor kenpaullone or the HMG-CoA reductase inhibitor mevastatin, both of which have previously been reported to promote neural differentiation. These data provide in-depth information on the ReNcell progenitor state and on neural differentiation in the presence and absence of drugs, setting the stage for functional studies.

The Drug Candidate BGP-15 Delays the Onset of Diastolic Dysfunction in the Goto-Kakizaki Rat Model of Diabetic Cardiomyopathy.

Background and Aims: Diabetic cardiomyopathy (DCM) is an emerging problem worldwide due to an increase in the incidence of type 2 diabetes. Animal studies have indicated that metformin and pioglitazone can prevent DCM partly by normalizing insulin resistance, and partly by other, pleiotropic mechanisms. One clinical study has evidenced the insulin-senzitizing effect of the drug candidate BGP-15, along with additional animal studies that have confirmed its beneficial effects in models of diabetes, muscular dystrophy and heart failure, with the drug affecting chaperones, contractile proteins and mitochondria. Our aim was to investigate whether the inzulin-senzitizer BGP-15 exert any additive cardiovascular effects compared to metformin or pioglitazone, using Goto-Kakizaki (GotoK) rats. Methods: Rats were divided into five groups: (I) healthy control (Wistar), (II) diseased (GotoK), and GotoK rats treated with: (III) BGP-15, (IV) metformin, and (V) pioglitazone, respectively, for 12 weeks. Metabolic parameters and insulin levels were determined at the endpoint. Doppler echocardiography was carried out to estimate diabetes-associated cardiac dysfunction. Thoracotomy was performed after the vascular status of rats was evaluated using an isolated aortic ring method. Furthermore, western blot assays were carried out to determine expression or phosphorylation levels of selected proteins that take part in myocyte relaxation. Results: BGP-15 restored diastolic parameters (e′/a′, E/e′, LAP, E and A wave) and improved Tei-index compared to untreated GotoK rats. Vascular status was unaffected by BGP-15. Expression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) and phosphodiesterase 9A (PDE9A) were unchanged by the treatments, but the phosphorylation level of vasodilator-stimulated phosphoprotein (VASP) and phospholamban (PLB) increased in BGP-15-treated rats, in comparison to GotoK. Conclusions: Even though the BGP-15-treatment did not interfere significantly with glucose homeostasis and vascular status, it considerably enhanced diastolic function, by affecting the SERCA/phospholamban pathway in GotoK rats. Although it requires further investigation, BGP-15 may offer a new therapeutic approach in DCM.

Synthesis of new derivatives of boehmeriasin A and their biological evaluation in liver cancer

Two series of boehmeriasin A analogs have been synthesized in short and high yielding processes providing derivatives differing either in the alkaloid's pentacyclic scaffold or its peripheral substitution pattern. These series have enabled, for the first time, comparative studies into key biological properties revealing a new lead compound with exceptionally high activity against liver cancer cell lines in the picomolar range for both well (Huh7, Hep3B and HepG2) and poorly (Mahlavu, FOCUS and SNU475) differentiated cells. The cell death was characterized as apoptosis by cytochrome-C release, PARP protein cleavage and SubG1 cell cycle arrest. Subsequent testing associated apoptosis via oxidative stress with in situ formation of reactive oxygen species (ROS) and altered phospho-protein levels. Compound 19 decreased Akt protein phosphorylation which is crucially involved in liver cancer tumorigenesis. Given its simple synthetic accessibility and intriguing biological properties this new lead compound could address unmet challenges within liver cancer therapy.

Post-treatment de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma

BackgroundDasatinib (Sprycel) was developed as a tyrosine kinase inhibitor targeting Bcr-Abl and the family of Src kinases. Dasatinib is commonly used for the treatment of acute lymphoblastic and chronic myelogenous leukemia. Previous clinical studies in melanoma returned inconclusive results and suggested that patients respond highly heterogeneously to dasatinib as single agent or in combination with standard-of-care chemotherapeutic dacarbazine. Reliable biomarkers to predict dasatinib responsiveness in melanoma have not yet been developed.ResultsHere, we collected comprehensive in vitro data from experimentally well-controlled conditions to study the effect of dasatinib, alone and in combination with dacarbazine, on cell proliferation and cell survival. Sixteen treatment conditions, covering therapeutically relevant concentrations ranges of both drugs, were tested in 12 melanoma cell lines with diverse mutational backgrounds. Melanoma cell lines responded heterogeneously and, importantly, dasatinib and dacarbazine did not synergize in suppressing proliferation or inducing cell death. Since dasatinib is a promiscuous kinase inhibitor, possibly affecting multiple disease-relevant pathways, we also determined if basal phospho-protein amounts and treatment-induced changes in phospho-protein levels are indicative of dasatinib responsiveness. We found that treatment-induced de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma.ConclusionsLoss of p53 phosphorylation might be an interesting candidate for a kinetic marker of dasatinib responsiveness in melanoma, pending more comprehensive validation in future studies.

Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii.

Rapamycin is an inhibitor of the evolutionary conserved Target of Rapamycin (TOR) kinase which promotes and coordinates translation with cell growth and division. In heterotrophic organisms, TOR regulation is based on intra- and extracellular stimuli such as amino acids level and insulin perception. However, how plant TOR pathways have evolved to integrate plastid endosymbiosis is a remaining question. Despite the close association of the TOR signaling with the coordination between protein turn-over and growth, proteome and phosphoproteome acclimation to a rapamycin treatment have not yet been thoroughly investigated in Chlamydomonas reinhardtii. In this study, we have used in vivo label-free phospho-proteomic analysis to profile both protein and phosphorylation changes at 0, 24, and 48 h in Chlamydomonas cells treated with rapamycin. Using multivariate statistics we highlight the impact of TOR inhibition on both the proteome and the phosphoproteome. Two-way ANOVA distinguished differential levels of proteins and phosphoproteins in response either to culture duration and rapamycin treatment or combined effects. Finally, protein–protein interaction networks and functional enrichment analysis underlined the relation between plastid and mitochondrial metabolism. Prominent changes of proteins involved in sulfur, cysteine, and methionine as well as nucleotide metabolism on the one hand, and changes in the TCA cycle on the other highlight the interplay of chloroplast and mitochondria metabolism. Furthermore, TOR inhibition revealed changes in the endomembrane trafficking system. Phosphoproteomics data, on the other hand, highlighted specific differentially regulated phosphorylation sites for calcium-regulated protein kinases as well as ATG7, S6K, and PP2C. To conclude we provide a first combined Chlamydomonas proteomics and phosphoproteomics dataset in response to TOR inhibition, which will support further investigations.

Involvement of MAPK/ERK1/2 pathway in microcystin-induced microfilament reorganization in HL7702 hepatocytes

ABSTRACTSeveral studies previously demonstrated that microcystin (MC)-LR produced cytoskeletal damage, especially to actin filaments. However, the underlying mechanisms of MC-induced cytoskeletal reorganization remain to be determined. The aim of this study was to examine the effects of 5 or 10 µM MC-LR on microfilament depolarization and expression of microRNA-451a (miR-451a) which plays a crucial role in cellular processes including cell proliferation, apoptosis and tumorigenesis in HL7702 liver cells after 24 hr treatment. Data demonstrated that MC-LR increased microfilament depolarization, elevated phosphorylation levels of mitogen-activated protein kinase (MAPK/ERK1/2) and vasodilator-stimulated phosphoprotein (VASP) but lowered miR-451a RNA expression levels. These molecular processes were associated with no marked changes in total protein ERK1/2. Data demonstrate that transfection with miR-451a may not be effective in the presence of MC-LR as evidenced by the inability of excess microRNA to prevent toxin-induced inhibition of threonine protein phosphatases1 (PP1) and 2A (PP2A) and microfilament reorganization in HL7702 cells.

Combining fluorescent cell barcoding and flow cytometry-based phospho-ERK1/2 detection at short time scales in adherent cells.

Detection of levels of intracellular phospho-proteins is key to analyzing the dynamics of signal transduction in cellular systems. Cell-to-cell variability in the form of differences in protein level in each cell affects signaling and is implicated in prognosis of many diseases. Quantitative analysis of such variability necessitate measuring the protein levels at single-cell resolution. Single-cell intracellular protein abundance detection in statistically significant number of adherent cells for short time sampling points post stimulation using classical flow cytometry (FCM) technique has thus far been a challenge due to the detrimental effects of cell detachment methods on the cellular machinery. We systematically show that cell suspension obtained by noninvasive temperature-sensitive detachment of adherent cells is amenable to high-throughput phospho-ERK1/2 protein detection at single-cell level using FCM in these short time sampling points. We demonstrate this on three adherent cell lines, viz., HeLa, A549, and MCF7, from distinct lineages having characteristically different elasticity at 37 °C. In particular, we use a right combination of multiplexing via fluorescent cell barcoding (FCB) and intracellular antibody staining for simultaneous detection of phospho-ERK1/2 (pERK) stimulated by epidermal growth factor (EGF) in multiple samples. Based on systematic characterization using Alexa 350 dye, we arrive at two conditions that must be satisfied for correct implementation of FCB. Our study reveals that the temperature-sensitive detachment of HeLa cells correctly captures the expected pronounced bimodal pERK distribution as an early response to EGF, which the enzymatic treatment methods fail to detect. © 2018 International Society for Advancement of Cytometry.

Lycopene treatment inhibits activation of Jak1/Stat3 and Wnt/β-catenin signaling and attenuates hyperproliferation in gastric epithelial cells

Helicobacter pylori (H pylori) colonizes the human stomach and increases the risk of gastric diseases including gastric cancer. H pylori increases reactive oxygen species (ROS), which activate Janus-activator kinase 1 (Jak1)/signal transducers and activators of transcription 3 (Stat3) in gastric epithelial cells. ROS mediate hyperproliferation, a hallmark of carcinogenesis, by activating Wnt/β-catenin signaling in various cells. Lycopene is a potent antioxidant exhibiting anticancer effects. We hypothesized that lycopene may inhibit H pylori–induced hyperproliferation by suppressing ROS-mediated activation of Jak1/Stat3 and Wnt/β-catenin signaling, and β-catenin target gene expression in gastric epithelial cells. We determined cell viability, ROS levels, and the protein levels of phospho- and total Jak1/Stat3, Wnt/β-catenin signaling molecules, Wnt-1, lipoprotein-related protein 5, and β-catenin target oncogenes (c-Myc and cyclin E) in H pylori–infected gastric epithelial AGS cells. The Jak1/Stat3 inhibitor AG490 served as the control treatment. The significance of the differences among groups was calculated using the 1-way analysis of variance followed by Newman-Keuls post hoc tests. The results show that lycopene reduced ROS levels and inhibited Jak1/Stat3 activation, alteration of Wnt/β-catenin multiprotein complex molecules, expression of c-Myc and cyclin E, and cell proliferation in H pylori–infected AGS cells. AG490 similarly inhibited H pylori–induced cell proliferation, alteration of Wnt/β-catenin multiprotein complex molecules, and oncogene expression. H pylori increased the levels of Wnt-1 and its receptor lipoprotein-related protein 5; this increase was inhibited by either lycopene or AG490 in AGS cells. In conclusion, lycopene inhibits ROS-mediated activation of Jak1/Stat3 and Wnt/β-catenin signaling and, thus, oncogene expression in relation to hyperproliferation in H pylori–infected gastric epithelial cells. Lycopene might be a potential and promising nutrient for preventing H pylori–associated gastric diseases including gastric cancer.

Phosphokinome Analysis of Barth Syndrome Lymphoblasts Identify Novel Targets in the Pathophysiology of the Disease.

Barth Syndrome (BTHS) is a rare X-linked genetic disease in which the specific biochemical deficit is a reduction in the mitochondrial phospholipid cardiolipin (CL) as a result of a mutation in the CL transacylase tafazzin. We compared the phosphokinome profile in Epstein-Barr-virus-transformed lymphoblasts prepared from a BTHS patient with that of an age-matched control individual. As expected, mass spectrometry analysis revealed a significant (>90%) reduction in CL in BTHS lymphoblasts compared to controls. In addition, increased oxidized phosphatidylcholine (oxPC) and phosphatidylethanolamine (PE) levels were observed in BTHS lymphoblasts compared to control. Given the broad shifts in metabolism associated with BTHS, we hypothesized that marked differences in posttranslational modifications such as phosphorylation would be present in the lymphoblast cells of a BTHS patient. Phosphokinome analysis revealed striking differences in the phosphorylation levels of phosphoproteins in BTHS lymphoblasts compared to control cells. Some phosphorylated proteins, for example, adenosine monophosphate kinase, have been previously validated as bonafide modified phosphorylation targets observed in tafazzin deficiency or under conditions of reduced cellular CL. Thus, we report multiple novel phosphokinome targets in BTHS lymphoblasts and hypothesize that alteration in the phosphokinome profile may provide insight into the pathophysiology of BTHS and potential therapeutic targets.

Abstract 5667: High-throughput analysis of MAPK and JAK-STAT signaling in CT26 tumors using a combination of immunophenotyping and phospho-flow cytometry

Abstract The development of robust platforms that enable the analysis of cell signaling proteins is crucial for small molecule drug development. Analysis of phospho-proteins in solid tumors is one area that remains a challenge due to the complex nature of the tissue. The most common approaches used to measure the effects of in vivo therapy are immunoblot and ELISA-based assays. However, these bulk analysis methods have drawbacks. This is primarily due to their inability to distinguish between phospho-protein levels in different cell subsets that exist in the tumor. Using the CT26 syngeneic model for colorectal cancer, we describe a novel high-throughput phospho-flow platform that can simultaneously analyze the phosphorylation status of up to 6 signaling proteins in both tumor cells and immune cells. To first validate the phospho-flow assay, splenocytes from naïve mice were treated in vitro with various stimuli that preferentially target distinct signaling proteins. We then tested the platform in vivo. To this end, CT26 tumor-bearing mice were dosed with the MEK inhibitor trametinib or vehicle. At the appropriate time point post-dosing, tumors were harvested and dissociated into single cell suspensions. Live tumor cells and CD8+ T cells were first identified by flow cytometry using antibodies against CD45, CD3, and CD8. Following fixation, MAPK and JAK-STAT signaling activity was analyzed by measuring the levels of phosphorylated MEK, ERK1/2, STAT1, STAT2, STAT5, and STAT6 proteins using phospho-flow. Our results demonstrated that trametinib triggered a reduction in both pMEK and pERK1/2 levels in tumor cells that was evident 24 hours after the final trametinib dose. We also found phosphorylated STAT1, STAT3, and STAT5 was readily detectable in tumor cells, while STAT6 phosphorylation was not obvious. In contrast, we found that the CD8+ T cell levels of pMEK and pERK1/2 were not affected by trametinib. Furthermore, when JAK-STAT signaling was analyzed, only phosphorylation of STAT3 was detected in CD8+ T cells, which was also unaffected by trametinib. These results demonstrate successful combination of immunophenotyping and phospho-protein analysis in solid tumor-derived cells using flow cytometry. This platform is a tool that can be used to provide valuable insight into the effects that small molecule inhibitors may have on distinct cell subsets within a heterogenous tumor microenvironment. Citation Format: David Draper, Alden Wong, Philip Lapinski, Sheri R. Barnes, Maryland Rosenfeld Franklin, Scott C. Wise. High-throughput analysis of MAPK and JAK-STAT signaling in CT26 tumors using a combination of immunophenotyping and phospho-flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5667.

Abstract 2838: Discovery of adaptive resistance pathways and anti-resistance combination therapies in cancer from phosphoproteomic data

Abstract Drug resistance to targeted therapies, either intrinsic (existing before treatment) or acquired (resulting from adaptation to therapy), is a major challenge in patient care. Drug combinations offer a possible solution to prevent drug resistance by blocking multiple routes to tumor proliferation. Discovery of effective combinations remains a challenging task due to complexity of the underlying biological processes and inter-tumor heterogeneity. Here, we developed a statistical pathway analysis method that (i) reveals pathways involved in drug response and adaptive resistance and (ii) nominates combination targets and therapies to down-regulate the resistance pathways. The method is based on the rationale that (i) use of therapies targeting specific genomic aberrations can lead to compensatory responses (e.g. through feedback loop activation in the short term or the development of oncogenic alterations in the long term) leading to drug resistance, and (ii) collective changes in pathway activities are better predictors of resistance and mitigation strategies than individual responses. We construct a pathway model of signaling interactions for measured molecular species through automated extraction of pathway information from Pathway Commons plus manual expert curation. Next, we combine the pathway model with the cell-type-specific drug response data to calculate a TargetScore (TS) for each protein. The score quantifies the adaptive pathway responses to a perturbation by integrating the change in the level of a (phospho)protein along with its pathway neighborhood in response to the single drug. A high TS corresponds to involvement in adaptive response (e.g. upregulation of RTK expression by MEK inhibitor via a feedback loop) and a low TS corresponds to the activity of the drug (e.g. inhibition of ERK phosphorylation by MEK inhibitor). Finally, by identifying the sub-pathways with enriched, high TS values, we determine the adaptive resistance pathways. We test the resulting predictions (combinations of the original single drug with drugs targeting members of the resistance pathways) experimentally. TargetScore is amenable to calculations for hundreds of samples treated with individual (or combinations of) drugs in multiple doses and/or time points and assayed for hundreds to thousands of molecular entities (mRNA or proteomic). Analysis of longitudinal data may allow us to trace the evolution of drug resistance and, potentially, the optimum time points for intervention. The current protocol is defined with a focus on proteomic RPPA data, but can be adapted to other kinds of molecular data associated with adaptive responses. We applied our method to BET-BRD inhibition in ovarian cancer to compare resistance/response pathways in cells with varying degrees sensitivity. The analysis nominated cell-type-specific, anti-resistance combinations involving BET inhibitors. Citation Format: Augustin Luna, Özgün Babur, Gonghong Yan, Emek Demir, Chris Sander, Anil Korkut. Discovery of adaptive resistance pathways and anti-resistance combination therapies in cancer from phosphoproteomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2838.

PO-180 Dissecting the effects of EGF starvation on EGFR signalling in the mouse small intestine using 3D organoid culture systems

IntroductionThe epidermal growth factor receptor (EGFR) signalling pathway has been established as a key driver of cell proliferation, growth and migration. Activating mutations as well as amplifications of the receptor gene have been found in a number of solid tumours such as glioblastoma, non-small cell lung cancer and colorectal cancer. For this reason, this pathway has been exploited therapeutically in various malignancies through the use of anti-EGFR monoclonal antibodies and small molecule tyrosine kinase inhibitors. Understanding how the pathway components are wired in both normal and cancerous tissues is crucial to develop effective therapies and overcome drug resistance. However, one understudied aspect of EGFR signalling is how it operates in settings where the endogenous ligand is deficient. Thus, the aim of the current study is to investigate how such network is wired in the mouse intestinal tissue in settings where growth factors are supplemented versus depleted.Material and methodsIntestinal organoid cultures were prepared from the mouse small intestine according to previously published protocols. We starved the organoids for various time periods of endogenous EGF and perturbed the pathway with inhibitors and growth factors. The levels of phospho-ERK (pERK) were determined using a capillary Western blot assay, while levels of other phosphoproteins besides ERK were measured by a magnetic bead-based immunoassay.Results and discussionsEGF starvation of mouse intestinal organoids for 24 hours resulted in an unexpected upregulation of pERK levels compared to EGF supplied controls. Blocking EGFR tyrosine kinase activity in starved organoids resulted in a significant reduction of pERK levels. Similarly, MEK inhibition caused a profound drop of pERK in starved settings. In contrast, inhibition of phosphoinositide 3-kinase (PI3K) did not seem to affect pERK. These results show that increased pERK levels upon EGF starvation of organoids are mediated via an activation of EGFR tyrosine kinase domain and the downstream RAS-RAF-MEK axis. Furthermore, it was found that EGF addition to starved organoids resulted in a decrease in pERK levels in a time-dependent manner. This reduction in pERK was observed specifically following EGF addition and not addition of other ligands.ConclusionEGF depletion of mouse intestinal organoids paradoxically activates the EGFR signalling and MAPK axis resulting in an increase in pERK levels. The mechanism by which this paradoxical activation occurs is still under current investigation.

Mechanism of Yanghe Pingchuan granules treatment for airway remodeling in asthma.

PurposeYanghe Pingchuan granules (YPG), a hospital preparation developed by The First Affiliated Hospital, Anhui University of Chinese Medicine, has been used for the clinical treatment of bronchial asthma (BA) for several decades. This study aimed to explore the mechanism of action of YPG in the treatment of BA.Materials and methodsMale Sprague Dawley rats (n=60) were randomly divided into six groups (n=10 per group): control, a BA model, positive drug control (Guilong Kechuanning capsules; a proven effective treatment for BA), and model rats treated with a high, medium, or low dose of YPG. H&E staining was used to detect pathological changes in the bronchial tubes. The mRNA expression levels of PI3K, PKB, PCNA, and AR were determined by real-time PCR, and the protein levels of phospho- (p-)PI3K, p-PKB, p-PCNA, and p-AR were detected by Western blotting. ELISAs were used to detect the expression of PIP2, PIP3 IL-6, IL-8, IL-1β, and epinephrine (EPI).ResultsH&E staining demonstrated that BA can be ameliorated using YPG. Real-time PCR, Western blotting, and ELISA indicated that use of YPG decreased expression of the phosphoinositide 3-kinase (PI3K) signaling pathway and PCNA, and can also ameliorate the condition kidney Yang deficiency, which is associated with BA in Chinese traditional medicine.ConclusionYPG can attenuate BA therapeutically in a dose-dependent manner. The mechanism underlying its therapeutic effect comprises influences on three features that contribute to BA: the PI3K signaling pathway, cell proliferation, and “kidney-Yang deficiency”.

Evaluation of NCI-7 Cell Line Panel as a Reference Material for Clinical Proteomics.

Reference materials are vital to benchmarking the reproducibility of clinical tests and essential for monitoring laboratory performance for clinical proteomics. The reference material utilized for mass spectrometric analysis of the human proteome would ideally contain enough proteins to be suitably representative of the human proteome, as well as exhibit a stable protein composition in different batches of sample regeneration. Previously, The Clinical Proteomic Tumor Analysis Consortium (CPTAC) utilized a PDX-derived comparative reference (CompRef) materials for the longitudinal assessment of proteomic performance; however, inherent drawbacks of PDX-derived material, including extended time needed to grow tumors and high level of expertise needed, have resulted in efforts to identify a new source of CompRef material. In this study, we examined the utility of using a panel of seven cancer cell lines, NCI-7 Cell Line Panel, as a reference material for mass spectrometric analysis of human proteome. Our results showed that not only is the NCI-7 material suitable for benchmarking laboratory sample preparation methods, but also NCI-7 sample generation is highly reproducible at both the global and phosphoprotein levels. In addition, the predicted genomic and experimental coverage of the NCI-7 proteome suggests the NCI-7 material may also have applications as a universal standard proteomic reference.

Effect and mechanism of the aβ2‑GP I/rhβ2‑GP I complex on JEG‑3 cell proliferation, migration and invasion.

Antiphospholipid antibody (aPL)-mediated antiphospholipid syndrome (APS) is an autoimmune disease. Upon binding to aPL, the primary antigen of aPL, β2-glycoprotein I (β2-GP I), induces abnormal immune function, which further activates downstream signaling pathways in the cell and eventually leads to APS. The present study aimed to determine whether β2-GP I antigen and anti-β2-glycoprotein I antibody (aβ2-GP I), which belong to the aPL class of antibodies, may affect human chorionic epithelium cell (JEG-3) proliferation, migration and invasion. Recombinant human (rh)β2-GP I protein was expressed using a prokaryotic expression system and aβ2-GP I antibody was purified from the blood serum of 10 patients with recurrent pregnancy loss. JEG-3 cells were stimulated with rhβ2-GP I and aβ2-GP I separately or simultaneously, and serum immunoglobulin G of normal pregnant women was used as negative control. Using cell counting kit-8, cell cycle and transwell assays in addition to EdU staining, it was determined that aβ2-GP I/rhβ2-GP I complex markedly increased JEG-3 cell proliferation, migration and invasion. The results revealed that mRNA levels of inhibitor of nuclear factor (NF)-κB kinase subunit (IKKβ), myeloid differentiation primary response protein MyD88 (MyD88), NF-κB and NF-κB inhibitor α (IκBα), as well as the protein levels of MyD88, IκBα and phospho(p)-IκBα in JEG-3 cells increased following incubation with the aβ2-GP I/rhβ2-GP I complex. The observed upregulation of p-IκBα protein suggested that IκBα-mediated inhibition of NF-κB was weakened. Furthermore, JEG-3 cells were transfected with PGMLV-NF-κB-Lu vector. Luciferase activity in JEG-3-NFκB-Luc1 and JEG-3-NFκB-Luc2 cells was enhanced following treatment with aβ2-GP I/rhβ2-GP I complex. The present study demonstrated that aβ2-GP I/rhβ2-GP I complex activates NF-κB through MyD88 signal transduction pathway, which further enhances JEG-3 cell proliferation, migration and invasion.

Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease

TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised.

Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells.

To investigate the combinatorial effects of lipopolysaccharide (LPS) and extracted dentine matrix proteins (eDMP) on regenerative and inflammatory responses in human dental pulp stem cells (DPSCs). Culture media were supplemented with several concentrations of LPS, eDMP and combinations of both. Cell viability was assessed over 1week by MTT assay; cell survival was evaluated after 24h and 7days by flow cytometry. The expression of mineralization-associated marker genes was determined by real-time quantitative polymerase chain reaction (RT-qPCR). To analyse the inflammatory response, secretion of interleukin 6 (IL-6) was quantified in the initial and the late phase of cell culture by enzyme-linked immunosorbent assay (ELISA). Data were treated nonparametrically and Mann-Whitney U-tests were performed to compare all experimental groups (α=0.05). Whereas LPS had no impact on viability, eDMP led to a concentration-dependent decrease, which was significant after 7days (P≤0.024). A moderate decline of cell survival induced by LPS was detected after 48h (P≤0.026), whereas eDMP was able to reverse this effect. eDMP alone caused increased expression of tested marker genes, LPS had no regulatory effect. Combined eDMP and LPS induced an upregulation of collagen type I and osteocalcin, whereas expression levels of dentine matrix acidic phosphoprotein and dentine sialophosphoprotein were similar to the control. IL-6-secretion was increased by LPS over time. eDMP markedly elevated initial production of IL-6 (P≤0.002), but suppressed LPS-induced cytokine production in the later phase. Lipopolysaccharide did not affect cell viability but interfered with odontoblast-like cell differentiation of DPSCs. Proteins from the dentine matrix may have a protective effect, attenuate the detrimental impact of LPS and thus play an important role during pulp repair.

Abstract P2-07-03: Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line

Abstract Several publications have addressed concerns surrounding drug response screens by pointing out sources of variability and by presenting recommendations for better experimental methods and more robust analytical approaches. In the presented profiling effort, we integrated the latest advances in drug response measurement and focused on data diversity and quality rather than on breadth. We selected 32 breast cancer cell lines with a strong bias towards triple negative lines as well as 4 cell lines established from relevant patient-derived xenografts. We evaluated a panel of clinically relevant kinase inhibitors using a microscopy-based dose response assay to measure drug potency, and to quantify drug efficacy in terms of growth inhibition (GR metrics) and cell death. The use of the GR metrics to quantify drug sensitivity enabled us to identify and study differences between cytostatic and cytotoxic responses. This systematic dose response dataset is complemented by measurements of baseline transcript expression levels by mRNAseq, quantification of absolute abundance of ˜12,000 proteins, and relative phosphoprotein levels by shotgun mass spectrometry across all cell lines. Additionally, the baseline activity of transcription factors and kinases were inferred from the mRNA (using VIPER) and phosphoprotein (using kinase enrichment analysis) data, respectively. The complementarity of these multi-omics data has allowed us to address questions about the landscape of breast cancer cell lines such as: Where do the patient-derived lines lay relative to the conventional cell lines? How consistent are the landscapes defined by each dataset? How does integration across datasets provide mechanistic insight into signaling pathways that are active in each cancer subtypes? The measured and inferred baseline data were used to build predictors of the observed drug responses with the goal of identifying the biological processes responsible for the differences in sensitivity across drugs and cell lines. Overall the dataset that has been collected is a valuable resource for understanding drug response in triple negative breast cancer, and the molecular mechanisms that influence it. Citation Format: Mills CE, Subramanian K, Hafner M, Chung M, Boswell SA, Everley RA, Juric D, Sorger PK. Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-03.