Cell Signaling Inhibitors Research Articles

Pharmacologic Characterization of ALD403, a Potent Neutralizing Humanized Monoclonal Antibody Against the Calcitonin Gene-Related Peptide.

ALD403 is a genetically engineered, humanized immunoglobulin G1 monoclonal antibody that inhibits the action of human calcitonin gene-related peptide (CGRP). Clinical trial data indicate that ALD403 is effective as a preventive therapy for migraine and has an acceptable safety profile. For preclinical characterization of ALD403, rabbit antibodies targeting α-CGRP were humanized and modified to eliminate fragment crystallizable (Fc) γ receptor (FcγR) and complement interactions. The ability of ALD403 to inhibit CGRP-induced cAMP production was assessed using a cAMP bioassay (Meso Scale Discovery). The IC50 for inhibition of cAMP release was 434 and 288 pM with the rabbit-human chimera antibody and the humanized ALD403, respectively. ALD403 inhibited α-CGRP binding with an IC50 of 4.7 × 10-11 and 1.2 × 10-10 M for the α-CGRP and AMY1 receptors, respectively. ALD403 did not induce antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity and did not stably interact with any of the FcγR mediating these functions, exhibiting only weak binding to FcγRI. ALD403 significantly lowered capsaicin-induced blood flow responses in rodents at all time points starting at 5 minutes postapplication in a dose-dependent manner. In conclusion, ALD403 is a potent functional ligand inhibitor of α-CGRP‒driven pharmacology. SIGNIFICANCE STATEMENT: α-Calcitonin gene-related peptide blockade by ALD403 was assessed via radiolabeled ligand displacement, in vitro inhibition of cell signaling, and in vivo inhibition of capsaicin-induced vasodilation. Lack of engagement of fragment crystallizable-mediated immune-effector functions by ALD403 was shown.

Combined inhibition of PIM and CDK4/6 suppresses both mTOR signaling and Rb phosphorylation and potentiates PI3K inhibition in cancer cells.

Aberrant activation of mitogenic signaling pathways in cancer promotes growth and proliferation of cells by activating mTOR and S6 phosphorylation, and D-cyclin kinases and Rb phosphorylation, respectively. Correspondingly, inhibition of phosphorylation of both Rb and S6 is required for robust anti-tumor efficacy of drugs that inhibit cell signaling. The best-established mechanism of mTOR activation in cancer is via PI3K/Akt signaling, but mTOR activity can also be stimulated by CDK4 and PIM kinases. In this study, we show that the CDK4/6 inhibitor abemaciclib inhibits PIM kinase and S6 phosphorylation in cancer cells and concurrent inhibition of PIM, CDK4, and CDK6 suppresses both S6 and Rb phosphorylation. TSC2 or PIK3CA mutations obviate the requirement for PIM kinase and circumvent the inhibition of S6 phosphorylation by abemaciclib. Combination with a PI3K inhibitor restored suppression of S6 phosphorylation and synergized to curtail cell growth. By combining abemaciclib with a PI3K inhibitor, three pathways (Akt, PIM, and CDK4) to mTOR activation are neutralized, suggesting a potential combination strategy for the treatment of PIK3CA-mutant ER+ breast cancer.

Inhibition of PI3K/AKT/mTOR and MAPK signaling pathways decreases progranulin expression in ovarian clear cell carcinoma (OCCC) cell line: a potential biomarker for therapy response to signaling pathway inhibitors.

Patients with advanced stage ovarian clear cell carcinoma (OCCC) have a poor prognosis due to resistance to conventional platinum chemotherapy. Recent studies have demonstrated that PI3K/AKT/mTOR and ERK1/2 signaling pathways are involved in this chemoresistance. Progranulin (PGRN) overexpression contributes to cisplatin resistance of epithelial ovarian cancer cell lines. Also, PGRN expression is regulated by AKT/mTOR and ERK1/2 signaling pathways in different cell types. Thus, the present study was designed to identify if PGRN expression is regulated by AKT, mTOR, and ERK1/2 signaling pathways in the OCCC cell line TOV-21G. Cultured TOV-21G cells were incubated with different concentrations of pharmacological cell signaling inhibitors. PGRN expression and phosphorylation of ERK1/2, AKT, and mTOR were assessed by Western blotting. Inhibition of AKT, mTOR, and ERK1/2 significantly reduced PGRN expression. Cell viability was not affected, while cell proliferation significantly decreased with all inhibitors used in this study. These observations demonstrated that inhibition of PI3K/AKT/mTOR and ERK1/2 signaling pathways reduces PGRN expression in TOV-21G cells. Thus, PGRN could be considered as a candidate for explaining the high resistance to platinum-based treatment and a potential biomarker for therapy response to cell signaling inhibitors in patients with OCCC.

From bench to bedside: evolving therapeutic targets in autoimmune blistering disease.

Autoimmune blistering diseases comprise a group of heterogenous conditions characterized by the loss of tolerance and subsequent development of autoantibodies targeting epidermal and subepidermal adhesion proteins. Blisters and erosions form on the skin and mucous membranes leading to significant morbidity and mortality. Traditional therapies rely on systemic immunosuppression. Advancements in our understanding of the pathophysiology of pemphigus and pemphigoid have led to the development of molecules which target specific pathways involved in induction and perpetuation of disease. In this review, we outline the novel therapeutic strategies including B-cell depletion, T-regulatory cell repletion, cell signalling inhibitors and small molecular inhibitors, inhibitory monoclonal antibodies, as well as complement inhibition. We additionally review their current level of clinical evidence. We lastly review therapeutics targets gleaned from the experimental epidermolysis bullosa acquisita mouse model. These emerging treatments offer an exciting progression from basic science discoveries that have the potential to transform the treatment paradigm in autoimmune blistering diseases.

Research Progress of Diphenyl Urea Derivatives as Anticancer Agents and Synthetic Methodologies

The malignant neoplasm, which is recognized as cancer, is a serious threat to human health and frequently-occurring disease. Diphenylurea, an important link structure in the design of active substance for treating cancer due to its near-perfect binding with certain acceptors, has demonstrated many activities against several human cancer cell lines. Various novel compounds with diphenyl urea as anticancer agents were constructed with the successful development of sorafenib. Diphenylurea is utilized to treat cancer by inhibiting cell signaling transduction, such as RAS-RAFMEK- ERK signaling pathway and PI3K-Akt-mTOR pathway. In addition, this structure inhibits tumor cell growth by inhibiting receptor tyrosine kinases multiply, such as Vascular Endothelial Growth Factor Receptors (VEGFRs), Platelet-Derived Growth Factor Receptors (PDGFRs), Epidermal Growth Factor Receptors (EGFRs). It regulates the pH value in cells by inhibiting CAIX/XII and to achieve cancer therapeutic effect. Besides, the diphenyl urea structure is applied to the synthesis of reagents like Aurora kinases inhibitors and HDAC inhibitors that affect cell division and differentiation to treat cancer. To reach the goal of treating tumor, this structure is also used as a DNA-directed alkylating agent by affecting the expression of genes. An application of the most representative diphenyl urea derivatives as antitumor agents is summarized in this review, focusing on their mechanisms bound to the targets. Meanwhile, the progress of researches on methods of synthesizing diphenyl urea derivatives is provided.

Preclinical Development of U3-1784, a Novel FGFR4 Antibody Against Cancer, and Avoidance of Its On-target Toxicity.

The FGFR4/FGF19 signaling axis is overactivated in 20% of liver tumors and currently represents a promising targetable signaling mechanism in this cancer type. However, blocking FGFR4 or FGF19 has proven challenging due to its physiological role in suppressing bile acid synthesis which leads to increased toxic bile acid plasma levels upon FGFR4 inhibition. An FGFR4-targeting antibody, U3-1784, was generated in order to investigate its suitability as a cancer treatment without major side effects.U3-1784 is a high-affinity fully human antibody that was obtained by phage display technology and specifically binds to FGFR4. The antibody inhibits cell signaling by competing with various FGFs for their FGFR4 binding site thereby inhibiting receptor activation and downstream signaling via FRS2 and Erk. The inhibitory effect on tumor growth was investigated in 10 different liver cancer models in vivo The antibody specifically slowed tumor growth of models overexpressing FGF19 by up to 90% whereas tumor growth of models not expressing FGF19 was unaffected. In cynomolgus monkeys, intravenous injection of U3-1784 caused elevated serum bile acid and liver enzyme levels indicating potential liver damage. These effects could be completely prevented by the concomitant oral treatment with the bile acid sequestrant colestyramine, which binds and eliminates bile acids in the gut. These results offer a new biomarker-driven treatment modality in liver cancer without toxicity and they suggest a general strategy for avoiding adverse events with FGFR4 inhibitors.

Exploring Signaling Pathways and Pancreatic Cancer Treatment Approaches Using Genetic Models.

Despite available treatment options, the overall survival rates of pancreatic cancer patients remain dismal. Multiple counter-regulatory pathways have been identified and shown to be involved in interfering with the efficacy of therapeutic agents. In addition, various known genetic alterations in the cellular signaling pathways have been implicated in affecting the growth and progression of pancreatic cancer. Nevertheless, the significance of other unknown pathways is yet to be explored, which provides the rationale for the intervention of new approaches. Several experimental genetic models have been explored to define the impact of key signaling cascades, and their mechanisms in the pathophysiology as well as treatment approaches of pancreatic cancer. The current review highlights the recent updates, and significance of such genetic models in the therapeutic efficacy of anti-tumor agents including the standard chemotherapeutic agents, natural products, cell signaling inhibitors, immunebased therapies and the combination of these approaches in pancreatic cancer.

PS1130 STEM CELL FACTOR IS IMPLICATED IN MICROENVIRONMENTAL INTERACTIONS AND CELLULAR DYNAMICS OF CHRONIC LYMPHOCYTIC LEUKEMIA

Background:The mitogenic glycoprotein Stem Cell Factor (SCF, ligand of c‐kit receptor) has been implicated as a pro‐oncogenic driver in a variety of human cancers. Increased SCF levels have recently been reported in a small series of patients with chronic lymphocytic leukemia (CLL), however little if any evidence exists regarding its precise role in CLL pathophysiology.Aims:To analyze the expression profile of SCF in a well‐characterized cohort of CLL patients and investigate whether SCF is modulated by the CLL milieu.Methods:CD19+ primary CLL cells were negatively selected. SCF transcripts were detected by qPCR, SCF protein levels were analyzed using Western Blotting (WB) and SCF+ cells were determined by Flow Cytometry (FC). Cellular proliferation (Ki‐67+ cells) and oxidative stress (mitochondrial mass) were analyzed using FC. CLL cells were cultured in vitro with anti‐IgM, CpG, CD40 ligand and H2O2, or co‐cultured with the mesenchymal cell line HS‐5. Secreted SCF was determined using ELISA in supernatants of CLL cells.Results:Initially, we determined through qPCR (n = 14) that CLL cells predominantly express the membrane‐bound isoform of SCF, which is known to form a more stable complex with c‐Kit, compared to the soluble isoform. Next, we found significantly increased SCF protein levels in CLL cells (n = 68), compared to healthy tonsillar B cells (n = 13) (FD:9.8; p < 0.001). Within our CLL cohort, increased SCF protein levels significantly correlated with CD38 positivity (FD:2.45; p < 0.05), ZAP70 positivity (FD: 2; p < 0.05), trisomy 12 (FD:3; p < 0.05), and a shorter time to first treatment (p < 0.01). Moreover, increased SCF protein levels and SCF+ cells strongly correlated with the expression of unmutated IGHV genes (FD:6.7; p < 0.001 and FD:4.8; p < 0.01 respectively).Next, we investigated whether stimulants mimicking the CLL milieu in vitro could modulate SCF protein expression in CLL cells. Stimulation of the B cell receptor (BCR) significantly increased SCF protein levels (n = 11, FD:1.4; p < 0.001). TLR‐9 stimulation significantly increased SCF+ cells (n = 8, FD:1.5; p < 0.01) and induced the secretion of soluble SCF (n = 7, FD:9.3; p < 0.05). Co‐stimulation of TLR9/CD40 receptors induced Ki‐67+ (FD:7.1; p < 0.01) and SCF+ (FD:3.8; p < 0.01) cells. Of note, 85% of the proliferating CLL cells were also SCF+ (Ki‐67+/SCF+ vs Ki‐67+/SCF− ; p < 0.01). The in vitro modulation of SCF in CLL cells by immune determinants prompted us to investigate whether B cell signaling inhibition might also modulate SCF expression in vivo. Longitudinal profiling of 6 CLL patients under Ibrutinib therapy showed a significant decrease in SCF+ cells at +1 month under treatment, compared to the pre‐treatment samples (FD:2.2; p < 0.05), further highlighting the links between immune signaling and SCF expression in CLL.Finally, co‐culturing of CLL cells (n = 12) with the HS‐5 cells significantly reduced SCF+ cells (FD:2.2; p < 0.001), in parallel with a decrease in mitochondrial mass (r = 0.73; p < 0.01). Treatment with H2O2 abrogated the anti‐oxidant protection of HS‐5 cells and up‐regulated SCF expression (FD = 1.9; p < 0.01), indicating that SCF expression in CLL cells is modulated by oxidative stress.Summary/Conclusion:Overexpression of membrane‐bound SCF in CLL is associated with adverse prognosis and a shorter time‐to‐first treatment. Microenvironmental cues modulate the expression and secretion of SCF isoforms by CLL cells, highlighting a crucial role for this inflammatory growth factor in CLL cell homeostasis.

Cancer stem cell impact on clinical oncology.

Cancer is a widespread worldwide chronic disease. In most cases, the high mortality rate from cancer correlates with a lack of clear symptoms, which results in late diagnosis for patients, and consequently, advanced tumor disease with poor probabilities for cure, since many patients will show chemo- and radio-resistance. Several mechanisms have been studied to explain chemo- and radio-resistance to anti-tumor therapies, including cell signaling pathways, anti-apoptotic mechanisms, stemness, metabolism, and cellular phenotypes. Interestingly, the presence of cancer stem cells (CSCs), which are a subset of cells within the tumors, has been related to therapy resistance. In this review, we focus on evaluating the presence of CSCs in different tumors such as breast cancer, gastric cancer, lung cancer, and hematological neoplasias, highlighting studies where CSCs were identified in patient samples. It is evident that there has been a great drive to identify the cell surface phenotypes of CSCs so that they can be used as a tool for anti-tumor therapy treatment design. We also review the potential effect of nanoparticles, drugs, natural compounds, aldehyde dehydrogenase inhibitors, cell signaling inhibitors, and antibodies to treat CSCs from specific tumors. Taken together, we present an overview of the role of CSCs in tumorigenesis and how research is advancing to target these highly tumorigenic cells to improve oncology patient outcomes.

Bisphosphonate-Generated ATP-Analogs Inhibit Cell Signaling Pathways.

Bisphosphonates are a major class of drugs used to treat osteoporosis, Paget's disease, and cancer. They have been proposed to act by inhibiting one or more targets including protein prenylation, the epidermal growth factor receptor, or the adenine nucleotide translocase. Inhibition of the latter is due to formation in cells of analogs of ATP: the isopentenyl ester of ATP (ApppI) or an AppXp-type analog of ATP, such as AMP-clodronate (AppCCl2p). We screened both ApppI as well as AppCCl2p against a panel of 369 kinases finding potent inhibition of some tyrosine kinases by AppCCl2p, attributable to formation of a strong hydrogen bond between tyrosine and the terminal phosphonate. We then synthesized bisphosphonate preprodrugs that are converted in cells to other ATP-analogs, finding low nM kinase inhibitors that inhibited cell signaling pathways. These results help clarify our understanding of the mechanisms of action of bisphosphonates, potentially opening up new routes to the development of bone resorption, anticancer, and anti-inflammatory drug leads.

PPARgamma agonists sensitize PTEN-deficient resistant lung cancer cells to EGFR tyrosine kinase inhibitors by inducing autophagy

We aimed to develop novel drug combination strategy to overcome drug resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in the treatment of non-small cell lung cancer (NSCLC). Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor, which upon activation upregulates phosphatase and tensin homolog (PTEN) to inhibit cell signaling downstream of PI3K to mediate apoptosis. To this end, PTEN loss is a known mechanism contributing to resistance to EGFR TKIs. Therefore, PPARγ agonists are hypothesized to overcome EGFR TKI resistance. Using human NSCLC cell models with PTEN deficiency, the potentiation of EGFR TKI anticancer activity by PPARγ agonists was evaluated. PPARγ agonists were found to upregulate PTEN, subsequently inhibiting the PI3K-Akt signaling pathway, and thus enhancing the anticancer activity of gefitinib (a first generation EGFR TKI). Chemical and genetic inhibition of PPARγ were shown to prevent this potentiation of anticancer activity by PPARγ agonists, thus confirming the crucial role played by PPARγ activation. Interestingly, the tested PPARγ agonists were also found to induce autophagy, as evidenced by the increased expression of an autophagy marker LC3-II and the autophagic degradation of p62/SQSTM1. PPARγ agonists-induced autophagic cell death was believed to contribute to the circumvention of resistance in PTEN-deficient cells because the genetic silencing of ATG5 (an autophagy mediator) was found to eliminate the drug potentiation effect by the PPARγ agonists. Our findings thus provide the basis for the rational and personalized use of PPARγ agonists in combination with EGFR TKIs in lung cancer patients.

BET inhibitors as novel therapeutic agents in breast cancer.

Tumoral cells not only depend on oncogenic abnormalities to maintain its malignant phenotype but on non-oncogenic vulnerabilities. Targeting epigenomics can modify specific cellular functions required for malignant transformation. The Bromodomain (BRD) family mediates their effect by recruiting proteins of the transcription machinery, recognizing acetylated-lysine residues in nucleosomal histones. Bromodomain and extra-terminal (BET) inhibitors have shown to produce growth inhibition in several tumors through the inhibition of the expression of several transcription factors. In this review we will discuss the current knowledge regarding BET inhibitors in breast cancer. Recent data demonstrates their antiproliferative effect in several cancer subtypes, including the triple negative subtype, or when combined with cell signaling inhibitors. We will also describe options for therapeutic combinations or potential mechanisms of resistance, with special emphasis on their future clinical development.

Rare genetic variants in CX3CR1 and their contribution to the increased risk of schizophrenia and autism spectrum disorders

CX3CR1, a G protein-coupled receptor solely expressed by microglia in the brain, has been repeatedly reported to be associated with neurodevelopmental disorders including schizophrenia (SCZ) and autism spectrum disorders (ASD) in transcriptomic and animal studies but not in genetic studies. To address the impacts of variants in CX3CR1 on neurodevelopmental disorders, we conducted coding exon-targeted resequencing of CX3CR1 in 370 Japanese SCZ and 192 ASD patients using next-generation sequencing technology, followed by a genetic association study in a sample comprising 7054 unrelated individuals (2653 SCZ, 574 ASD and 3827 controls). We then performed in silico three-dimensional (3D) structural modeling and in vivo disruption of Akt phosphorylation to determine the impact of the detected variant on CX3CR1-dependent signal transduction. We detected a statistically significant association between the variant Ala55Thr in CX3CR1 with SCZ and ASD phenotypes (odds ratio=8.3, P=0.020). A 3D structural model indicated that Ala55Thr could destabilize the conformation of the CX3CR1 helix 8 and affect its interaction with a heterotrimeric G protein. In vitro functional analysis showed that the CX3CR1-Ala55Thr mutation inhibited cell signaling induced by fractalkine, the ligand for CX3CR1. The combined data suggested that the variant Ala55Thr in CX3CR1 might result in the disruption of CX3CR1 signaling. Our results strengthen the association between microglia-specific genes and neurodevelopmental disorders.

Binding of \u03b1v\u03b23 Integrin-Specific Radiotracers Is Modulated by Both Integrin Expression Level and Activation Status

PurposeMolecular imaging of αvβ3 integrin has exhibited real potential to guide the appropriate use of anti-angiogenic therapies. However, an incomplete understanding of the factors that influence binding of αvβ3 integrin-specific radiotracers currently limits their use for assessing response to therapy in cancer patients. This study identifies two fundamental factors that modulate uptake of these radiotracers.ProceduresExperiments were performed in prostate cancer (PC3) and glioblastoma (U87MG) cells, which differentially express αvβ3 integrin. αvβ3 integrin-specific radiotracers were used to investigate the effect of manipulating αvβ3 integrin expression or activation in cellular binding assays. β3 integrin and αvβ3 integrin expression were measured by western blotting and flow cytometry, respectively. The effect of select pharmacological inhibitors on αvβ3 integrin activation and expression was also determined.ResultsRadiotracer binding was proportional to αvβ3 integrin expression when it was decreased (β3 knock-down cells) or increased, either using pharmacological inhibitors of cell signalling or by culturing cells for different times. Studies with both small molecule and arginine–glycine–aspartic acid (RGD)-based radiotracers revealed increased radiotracer binding after activation of αvβ3 integrin with Mn2+ or talin head domain. Moreover, inhibition of fundamental signalling pathways (mitogen-activated protein kinase kinase (MEK), Src and VEGFR2) decreased radiotracer binding, reflecting reduced αvβ3 integrin activity.ConclusionBinding of small molecule ligands and radiolabelled RGD peptides is modulated by expression and activation status of αvβ3 integrin. αvβ3 integrin-specific radiotracers can provide otherwise inaccessible information of the effect of signalling pathways on αvβ3 integrin. This has significant implications for assessing response to anti-angiogenic therapies in clinical studies.

Abstract LB-126: PPARgamma agonists sensitize PTEN-deficient lung cancer cells resistant to EGFR tyrosine kinase inhibitors

Abstract We aimed to develop novel drug combination strategy to overcome drug resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in the treatment of lung cancer. EGFR TKIs are approved for treating advanced lung cancer that harbors the sensitizing EGFR mutations. However, resistance to EGFR TKIs is almost inevitable for all patients due to multifactorial mechanisms. The PI3K/Akt/mTOR pathway is a major signaling pathway regulated by EGFR to drive cancer survival. Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor, which upon activation upregulates PTEN to inhibit cell signaling downstream of PI3K to mediate apoptosis. To this end, PTEN loss is a known mechanism contributing to intrinsic resistance to EGFR TKIs. Therefore, PPARγ agonists are hypothesized to overcome EGFR TKI resistance. Using well defined human non-small cell lung cancer (NSCLC) cell model with PTEN deficiency, the potentiation of EGFR TKI anticancer activity by PPARγ agonists was demonstrated. PPARγ agonists were found to upregulate PTEN, subsequently inhibit the PI3K-Akt signaling pathway, and thus enhancing the anticancer activity of gefitinib (a first generation EGFR TKI). Specific PPARγ inhibitor (GW9662) and genetic silencing of PPARγ were shown to prevent this potentiation of anticancer activity by PPARγ agonists, thus confirming the crucial role played by PPARγ activation. Interestingly, the tested PPARγ agonists were also found to induce autophagy, as evidenced by the increased expression of an autophagy marker LC3-II and the autophagic degradation of p62/SQSTM1. PPARγ agonists-induced autophagic cell death was believed to contribute to the circumvention of resistance in PTEN-deficient cells because the genetic silencing of ATG5 (an autophagy mediator) was found to eliminate the drug potentiation effect by the PPARγ agonists. Our findings thus provide the basis for the rational and personalized use of PPARγ agonists in combination with EGFR TKIs in lung cancer patients with specific resistance mechanism. Citation Format: Kenneth KW To, Wing Sum Tong, William KK Wu, Herbert HF Loong. PPARgamma agonists sensitize PTEN-deficient lung cancer cells resistant to EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-126. doi:10.1158/1538-7445.AM2017-LB-126

Uncaria tomentosa (Willd. ex Schult.) DC (Rubiaceae) Sensitizes THP-1 Cells to Radiation-induced Cell Death.

Background:Uncaria tomentosa (Willd. ex Schult.) DC (Rubiaceae), known as Cat's Claw or Uña de gato, is a traditionally used medicinal plant native to Peru. Some studies have shown that U. tomentosa can act as an antiapoptotic agent and enhance DNA repair in chemotherapy-treated cells although others have shown that U. tomentosa enhanced apoptosis.Objective:To determine if treatment with U. tomentosa can significantly enhance cell death in THP-1 cells exposed to ionizing radiation.Materials and Methods:THP-1 monocyte-like cells were treated with ethanolic extracts of U. tomentosa in the presence or absence of bacterial lipopolysaccharide and then exposed to ionizing radiation. Cell proliferation was assessed by MTT and clonogenic assays and the effects on cell cycle measured by flow cytometry and immunoblotting. Changes in cell signaling were determined by immunoblotting and cytokine ELISA and activation of apoptosis measured by caspase activation and DNA fragmentation analysis.Results:Treatment of THP-1 cells with U. tomentosa had a small effect on cell proliferation. However, when the U. tomentosa-pretreated cells were also subjected to 5–9 Gy ionizing radiation, they showed a significant decrease in cell proliferation and increased cellular apoptosis as measured by DNA fragmentation and caspase activation. Treatment with U. tomentosa also decreased the expression of Cyclin E and Cyclin B, key regulators of normal cell cycle progression, and decreased the phosphorylation of various stress-activated, cell survival proteins including p38, ERK, and SAP/JNK kinase.Conclusions:These results suggest that U. tomentosa could be useful in enhancing cell death following anticancer therapies including ionizing radiation.SUMMARY Treatment of THP-1 cells with Uncaria tomentosa increases their susceptibility to X-rays. The combination of Uncaria tomentosa and X-ray exposure strongly inhibits cell signaling and promotes apoptosis. Abbreviations Used: LPS: Lipopolysaccharide, TNF: Tumor necrosis factor: IL-1, Interleukin-1: SDS: Sodium dodecylsulphate, TBS: Tris-buffered saline.

Impact of phosphoproteomics in the translation of kinase‐targeted therapies

Signaling pathways driven by protein and lipid kinases are altered in most human diseases. Therefore, pharmacological inhibitors of cell signaling are one of the most intensively pursued therapeutic approaches for the treatment of diseases such as cancer, neurodegeneration, and metabolic syndromes. Phosphoproteomics is a technique that measures the products of kinase activities and, with the appropriate bioinformatics techniques, the methodology can also provide measures of kinase pathway activation and network circuitry. Hence, due to recent technological advantages, LC-MS-based quantitative phosphoproteomics provides relevant information for the design and implementation of kinase inhibitor based therapies. Here, we review how phosphoproteome profiling is being used in translational research as a means to identify drug targets and biomarkers for personalizing therapies based on kinase inhibitors.

Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture.

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation.

Single Chain Antibodies as Tools to Study transforming growth factor-β-Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-β signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-β signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-β signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

Synthetic high-density lipoprotein-like nanoparticles potently inhibit cell signaling and production of inflammatory mediators induced by lipopolysaccharide binding Toll-like receptor 4

Toll-like receptor 4 (TLR4) plays a critical role in the innate immune system. Stimulation of TLR4 occurs upon binding lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls. Due to the potency of the induced inflammatory response, there is a growing interest in agents that can most proximally modulate this LPS/TLR4 interaction to prevent downstream cell signaling events and the production of inflammatory mediators. Building on the natural ability of human high-density lipoprotein (HDL) to bind LPS, we synthesized a suite of HDL-like nanoparticles (HDL-like NP). We identified one HDL-like NP that was particularly effective at decreasing TLR4 signaling caused by addition of purified LPS or Gram-negative bacteria to model human cell lines or primary human peripheral blood cells. The HDL-like NP functioned to inhibit TLR4-dependent inflammatory response to LPS derived from multiple bacterial species. Mechanistically, data show that the NP mainly functions by scavenging and neutralizing the LPS toxin. Taken together, HDL-like NPs constitute a powerful endotoxin scavenger with the potential to significantly reduce LPS-mediated inflammation.