Amit Choudhury

Regulation of vascular endothelial growth factor receptor 2 trafficking and angiogenesis by Golgi localized t-SNARE syntaxin 6

Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiologic and pathologic angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory transport through the Golgi apparatus. To date, the mechanism whereby the VEGFR2 traffics through the Golgi apparatus remains incompletely characterized. We show in human endothelial cells that binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2 traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of syntaxin 6, a Golgi-localized target membrane-soluble N-ethylmaleimide attachment protein receptor (t-SNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell migration, and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory form of syntaxin 6 reduced VEGF-induced neovascularization and permeability. Our data demonstrate the importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory form of syntaxin 6 has good potential as an antiangiogenic agent.

Interleukin-6 counteracts therapy-induced cellular oxidative stress in multiple myeloma by up-regulating manganese superoxide dismutase.

IL (interleukin)-6, an established growth factor for multiple myeloma cells, induces myeloma therapy resistance, but the resistance mechanisms remain unclear. The present study determines the role of IL-6 in re-establishing intracellular redox homoeostasis in the context of myeloma therapy. IL-6 treatment increased myeloma cell resistance to agents that induce oxidative stress, including IR (ionizing radiation) and Dex (dexamethasone). Relative to IR alone, myeloma cells treated with IL-6 plus IR demonstrated reduced annexin/propidium iodide staining, caspase 3 activation, PARP [poly(ADP-ribose) polymerase] cleavage and mitochondrial membrane depolarization with increased clonogenic survival. IL-6 combined with IR or Dex increased early intracellular pro-oxidant levels that were causally related to activation of NF-κB (nuclear factor κB) as determined by the ability of N-acetylcysteine to suppress both pro-oxidant levels and NF-κB activation. In myeloma cells, upon combination with hydrogen peroxide treatment, relative to TNF (tumour necrosis factor)-α, IL-6 induced an early perturbation in reduced glutathione level and increased NF-κB-dependent MnSOD (manganese superoxide dismutase) expression. Furthermore, knockdown of MnSOD suppressed the IL-6-induced myeloma cell resistance to radiation. MitoSOX Red staining showed that IL-6 treatment attenuated late mitochondrial oxidant production in irradiated myeloma cells. The present study provides evidence that increases in MnSOD expression mediate IL-6-induced resistance to Dex and radiation in myeloma cells. The results of the present study indicate that inhibition of antioxidant pathways could enhance myeloma cell responses to radiotherapy and/or chemotherapy.

Secretion of Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1/sFlt1) Requires Arf1, Arf6, and Rab11 GTPases

The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1) is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [35S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [35S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA), a drug which blocks trafficking between the endoplasmic reticulum (ER) and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.

Regulation of intracellular membrane trafficking and cell dynamics by syntaxin-6

Intracellular membrane trafficking along endocytic and secretory transport pathways plays a critical role in diverse cellular functions including both developmental and pathological processes. Briefly, proteins and lipids destined for transport to distinct locations are collectively assembled into vesicles and delivered to their target site by vesicular fusion. SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins are required for these events, during which v-SNAREs (vesicle SNAREs) interact with t-SNAREs (target SNAREs) to allow transfer of cargo from donor vesicle to target membrane. Recently, the t-SNARE family member, syntaxin-6, has been shown to play an important role in the transport of proteins that are key to diverse cellular dynamic processes. In this paper, we briefly discuss the specific role of SNAREs in various mammalian cell types and comprehensively review the various roles of the Golgi- and endosome-localized t-SNARE, syntaxin-6, in membrane trafficking during physiological as well as pathological conditions.

The myosin motor Myo1c is required for VEGFR2 delivery to the cell surface and for angiogenic signaling.

Vascular endothelial growth factor receptor-2 (VEGFR2) is a receptor tyrosine kinase that is expressed in endothelial cells and regulates angiogenic signal transduction under both physiological and pathological conditions. VEGFR2 turnover at the plasma membrane (PM) is regulated by its transport through endocytic and secretory transport pathways. Short-range cargo trafficking along actin filaments is commonly regulated by motor proteins of myosin superfamily. In the current study, performed in primary human endothelial cells, we demonstrate that unconventional myosin 1c (Myo1c; class I family member) regulates the localization of VEGFR2 at the PM. We further demonstrate that the recruitment of VEGFR2 to the PM and its colocalization with Myo1c and caveolin-1 occur in response to VEGF-A (VEGF) stimulation. In addition, VEGF-induced delivery of VEGFR2 to the cell surface requires Myo1c; surface VEGFR2 levels are reduced in the absence of Myo1c and, more importantly, are restored by the overexpression of wild-type but not mutant Myo1c. Subcellular density gradient fractionation revealed that partitioning of VEGFR2 into caveolin-1- and Myo1c-enriched membrane fractions is dependent on VEGF stimulation. Myo1c depletion resulted in increased VEGF-induced VEGFR2 transport to the lysosomes for degradation and was rescued by applying either brefeldin A, which blocks trafficking between the endoplasmic reticulum and the Golgi complex, or dynasore, an inhibitor of dynamin-mediated endocytosis. Myo1c depletion also reduced VEGF-induced VEGFR2 phosphorylation at Y1175 and phosphorylation-dependent activation of ERK1/2 and c-Src kinase, leading to reduced cell proliferation and cell migration. This is the first report demonstrating that Myo1c is an important mediator of VEGF-induced VEGFR2 delivery to the cell surface and plays a role in angiogenic signaling.

Elucidating the interplay between IgG-Fc valency and FcγR activation for the design of immune complex inhibitors

An engineered trivalent Fc drug candidate is a potent inhibitor of FcγR-driven immune cell activation and autoimmune diseases in animal models. Third Fc’s the charm The activation of Fcγ receptors by autoantibody immune complexes plays a pathogenic role in multiple autoimmune diseases. In an attempt to systematically evaluate potential therapeutic approaches, Ortiz et al. tested different shapes and sizes of Fc-containing molecules. One particular design, a trimer with a Y-shaped configuration, was particularly effective at binding to Fcγ receptors and blocking them without causing inappropriate activation. To demonstrate therapeutic potential, the authors tested this Fc trimer construct in mouse models of three different autoimmune diseases: immune thrombocytopenic purpura, collagen-induced arthritis, and epidermolysis bullosa acquisita, all with promising results. Autoantibody immune complex (IC) activation of Fcγ receptors (FcγRs) is a common pathogenic hallmark of multiple autoimmune diseases. Given that the IC structural features that elicit FcγR activation are poorly understood and the FcγR system is highly complex, few therapeutics can directly block these processes without inadvertently activating the FcγR system. To address these issues, the structure activity relationships of an engineered panel of multivalent Fc constructs were evaluated using sensitive FcγR binding and signaling cellular assays. These studies identified an Fc valency with avid binding to FcγRs but without activation of immune cell effector functions. These observations directed the design of a potent trivalent immunoglobulin G–Fc molecule that broadly inhibited IC-driven processes in a variety of immune cells expressing FcγRs. The Fc trimer, Fc3Y, was highly efficacious in three different animal models of autoimmune diseases. This recombinant molecule may represent an effective therapeutic candidate for FcγR-mediated autoimmune diseases.

Syntaxin 16 Regulates Lumen Formation during Epithelial Morphogenesis

The formation and maintenance of cell-cell junctions, both under physiological and pathological conditions, requires the targeting and trafficking of junctional proteins. Proteins of the syntaxin (Stx)-family localize to a variety of subcellular membranes and contribute to intracellular transport of cargo by regulating vesicle fusion events at these sites. Unlike plasma membrane localized Stxs, the roles of endosome- and Golgi-localized stx proteins in epithelial morphogenesis are less understood. Here we show that Stx16– an endosome- and Golgi-localized target-membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) that plays a role in membrane trafficking between these compartments – is essential for lumen development. In cultured Madin Darby Canine Kidney (MDCK) cells, Stx16 was selectively upregulated as sparsely plated cells attained confluency. Stx16-depleted confluent monolayers consistently showed lower transepithelial resistance than control monolayers, and failed to maintain endogenous and ectopically expressed E-cadherin at the adherens junctions due to decreased recycling. We further found that whereas cysts formed by MDCK cells cultured in Matrigel have a single hollow lumen, those formed by stx16-depleted counterparts had multiple lumens, due to abnormal orientiation of the mitotic spindle. Finally, a similar role for stx16 function in vivo is indicated by our analysis of pronephric-duct development in zebrafish expressing the claudinB:lynGFP transgene; lack of stx16 function in this structure (in stx16-morphant embryos) led to the development of enlarged, torturous pronephric ducts with more than one lumen. Taken together, our in vitro and in vivo studies establish a role for Stx16 in maintaining the integrity of cell-cell junctions, and thereby in morphogenesis of the kidney epithelial lumen.

Recognition of the parasite infected cell surface determinants by homologous antiserum raised against infected cell membranes.

Identification of neo-antigenic determinant(s) on parasite infected cell surface is important to control intracellular infections. Such determinant(s) on the surface of intact Plasmodium berghei infected erythrocytes have not been conclusively demonstrated. To generate polyclonal antiserum selectively recognizing the parasite infected cell surface determinant(s), in natural state, we have examined the efficacy of the homologous immunizations, in BALB/c mice, with the membrane rich preparation of: i) erythrocytes in vivo infected with Plasmodium berghei and, ii) macrophages in vitro infected with Leishmania donovani. Anti-infected erythrocyte membrane antiserum specifically recognized, albeit at low level, the infected cell surface as determined by flow cytometry and immunoelectron microscopy. Immunoprecipitation of radiolabeled antigens revealed at least three parasite proteins of >205 kDa, 160 kDa and 100 kDa specifically present on infected erythrocyte surface. Normal uninfected erythrocytes did not react with the antiserum. Anti-L. donovani-infected macrophage membrane antiserum also recognized only infected macrophage surface and not the normal macrophages. Thus, the approach may find wide application in delineating disease specific determinant(s) on the infected cell surface, particularly to those where animal models are available.

Role of SNARE proteins in epithelial apical lumen formation

G cancer of the colon is the third leading cause of cancer in males and the fourth leading cause of cancer in females. Unfortunately, only few patients experience complete pathologic responses to chemotherapy, mainly because of their resistance to treatment. Hence, it is imperative to look for novel agents high efficiency and less side effects. We have shown here that PAC, a novel curcumin analogue, has potent anti-colon cancer properties both in vitro and in vivo. Indeed, PAC triggered apoptosis in colon cancer cells through the mitochondrial pathway and down-regulated cyclin D1 as well as other important oncogenes, such as survivin and C-Myc. Interestingly, knocking down of cyclin D1 with specific siRNA enhanced the pro-apoptotic effect of PAC, suggesting that this effect is mediated through the inhibition of cyclin D1. Additionally, PAC delayed the cell cycle at G2/M phase in colon cancer cells and up-regulated the tumor suppressor proteins p16 and p21. These effects seem to be mediated through the inhibition of the STAT3/JAK2 pathway, since PAC inhibited these 2 important protein kinases. Importantly, these effects were also observed in vivo in tumor xenografts. These results indicate that PAC could constitute a powerful, yet not toxic, new chemotherapeutic agent against colorectal tumors.M attention has been paid to the prediction of function based on nucleic acid sequence or modelling 3D structure of protein from its nucleic acid sequence. The general belief is that structural amino acid characteristics which are an intermediate stage between DNA/RNA and advanced protein structure are too simple and restricted for precise modelling of protein function. Opposite to the general belief, we demonstrated the possibility of estimating accurate amino acid-based models by improving the following steps: (1) Extraction of a large number of amino acid characteristics trough computational biology, (2) Selection of the important amino acid characteristics trough attribute weighting, and (3) Testing different combinations of machine learning with attribute weighting algorithms to find the best model. Our procedure showed high performance in different biological examples such as prediction of protein thermostability and halostability, prediction of different influenza types, as well as monitoring breast and lung cancers progress. This finding has the potential to be efficiently used in protein genetic engineering, discovery of amino acid based-biomarkers, increasing the accuracy 3D protein prediction, and developing websites/softwares for prediction of the result of amino acid mutation. In addition, important discovered amino acid features can be employed as clues for discovering key DNA mutations in cancer research. Esmaeil Ebrahimie, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.026Background: Novel strategies for early detection of EOC, the most common and second most lethal cancer in Indian women, are urgently needed. Silencing tumor suppressor genes via DNA methylation has established hypermethylation as one of the most frequent molecular alterations that may initiate and drive many types of human neoplasia including EOC .To determine the alterations of tumor suppressor gene p16INK4A in EOC patients to explore the possibilities of identifying potential minimally invasive markers in blood of the patients, which could help in the clinical practice as a diagnostic and prognostic marker.T assess the efficacy and the safety of percutaneous cryoablation (PCC) and 125I seeds implantation combined with chemotherapy in the treatment of advanced pancreatic cancer, ninety six patients (male 58, average age 56.9) with advanced pancreatic cancer (55 with pancreatic head cancer, 80 in stage IV) underwent PCC and I125 seeds implantation combined with concomitant chemotherapy (gemcitabine hydrochloride and DDP) were analyzed. One hundred and seventeen percutaneous procedures of cryosurgeries combined I125 seeds implantation were performed. Clinical benefit response (CBR), therapy-related complications, postcryoablative CT imaging and survival rate were assessed. Eighty seven patients were followed up successfully. Median survival was 10.5, and 6-month and 1-year survival was 69.6% (stage III vs. IV, 66.7% vs. 71.4%) and 43.1% (stage III vs. IV, 50.0% vs. 40.1%), respectively. The maximum survival reached 47 months. CR, PR and SD were achieved in 9, 26 and 53 patients, respectively. Sixty eight and 63 in 79 patients experienced a ≥50% reduction of pain score and analgesic consumption, respectively, 26 patients experienced a ≥2kg weight gaining, and average KPS increased significantly (P<0.05). No serious therapy-related complications except pancreatic fistula accompanied abdominal hemorrhage, bile leakage, acute pancreatitis and 125I seeds leaved over in needle track occurred in 1, 1, 2 and 1 case, respectively. PCC and 125I seeds implantation combined with chemotherapy are effective and safe for the treatment of advanced pancreatic cancer.E morphogenesis is a fundamental process characterized by the emergence of ducts and acini, in which a polarized epithelial cell layer attached to a basal lamina surrounds a luminal space. Acute injury of major epithelial organ systems is one of the most important causes of death worldwide, and understanding the polarization of epithelia will be important in analyzing the response of a tissue to acute injury and in developing regeneration-based therapies. Studies of epithelial cells in 3D cultures and in vivo models have shown that lumen formation depends on a complex series of events, including: stimulation of morphogenesis, for example by cues such as cell attachment (to either other cells or the extracellular matrix); apical-basal polarization; lumen expansion. It is also clear that intracellular vesicle transport pathways are important for epithelial-gland morphogenesis, but our understanding of how they are regulated remains unclear. Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins contribute to intracellular cargo transport by regulating the fusion of donor vesicles to their target membranes. Our published studies using human endothelial cells have shown that syntaxin 6 (Stx6), a member of the target membrane-associated SNARE (t-SNARE) family protein plays an important role in VEGFR2/KDR and alpha5 beta1 integrin trafficking and angiogenesis [Blood 117, 1425-35 (2011); J Biol Chem. 286, 36749-61 (2011)]. In contrast, confluent 2D and 3D epithelial cell cyst culture show downregulation of Stx6 with concomitant up-regulation of Stx16, suggesting a role for Stx16 in morphogenesis. Furthermore, Stx16 knockdown in epithelial cells show defect in morphogenesis and forms multiple lumens in 3D cysts. Ongoing studies in the lab are investigating role for Stx16 in the protein and lipid trafficking patterns that are required to maintain the cell-cell adhesion and cell polarity, events that are essential to achieve epithelial lumen formation. These studies may provide a better understanding of the fundamental mechanisms that regulate cell polarization, information that will be needed to improve the response of tissues to acute injury as well as to identify ways to avoid fibrosis and EMT after chronic injury.O squamous cell carcinoma and is the third most common malignancy in India. The most common mode of metastasis is through the cervical lymph nodes.The complexity in predicting the presence of metastatic disease in clinically negative necks has led to wide spread use of elective neck dissection. This present study is intended to evaluate the status of clinically not detectable lymph nodes using Computerized Tomography (CT) and also to compare accuracy between clinical examination and CT. 40 patients who have been histopathologically diagnosed with Oral squamous cell carcinoma were included in the study. The findings of both clinical as well as CT examinations were correlated with pathological findings from the neck dissection. Number of True positives detected by clinical examination (CE) versus CT is 7 and 13 respectively (p=0.17).Number of True negatives detected by CE versus CT is 18 and 18 respectively (p=1). Number of False positives detected by CE versus CT is 5 and 5 respectively (p=1). Number of False negatives detected by CE versus CT is 10 and 4 respectively (p=0.1).Sensitivity for CE versus CT is 41.1% and 68.4% respectively (p=0). Specificity for CE versus CT is 78.2% and 78.2% respectively (p=1). Positive Predictive Value for CE versus CT is 58.3% and 72.2% respectively (p=0.001). Negative Predictive Value for CE versus CT is 64.2% and 75% respectively (p=0.001). Accuracy of CE versus CT is 62.5% and 77.5% respectively (p=0.001).The present indicates that CT is an important image tool for detection and statistically significant over the clinical examination.P cancer is the most prevalent cancer in men. In 2009, 40,841 men were diagnosed with prostate cancer and 10,382 men actually died from it in the UK alone. Cancer immunotherapy aims at producing a long lasting and potent tumour-specific immune response by targeting antigens that are over-expressed by tumour cells. Furthermore, prostate cancer develops slowly and this could be used to the advantage of clinicians by giving them more opportunities to vaccinate the patients.