Mukhopadhyay, Debabrata

Inhibition of the EGFR signaling pathway is one of the attractive therapeutic targets for pancreatic cancer as recent studies demonstrated that EGFR is over‐expressed in pancreatic cancer. In this article we have demonstrated the design of targeted drug delivery system containing Bovine Serum Albumin (BSA) microspheres as delivery vehicle, gemcitabine as anticancer drug and anti‐EGFR (epidermal growth factor receptor) monoclonal antibody as targeting agent. The conjugated BSA microspheres were characterized by several physico‐chemical techniques such as scanning electron microscope, optical microscopy, fluorescent microscopy etc. Administration of these BSA microspheres containing gemcitabine and anti‐EGFR (BSA‐Gem‐EGFR) shows significant inhibition of pancreatic cancer cells (AsPC1) compared to the cells treated with only BSA microspheres, BSA with gemcitabine (BSA‐Gem), and free gemcitabine. This strategy could be used as a generalized approach for the treatment of pancreatic cancer along with other cancers which overexpress EGFR on cell surface. Copyright © 2012 John Wiley & Sons, Ltd.

Abstract The tumor microenvironment, including stromal myofibroblasts and associated matrix proteins, regulates cancer cell invasion and proliferation. Here, we report that neuropilin-1 (NRP-1) orchestrates communications between myofibroblasts and soluble fibronectin that promote α5β1 integrin–dependent fibronectin fibril assembly, matrix stiffness, and tumor growth. Tumor growth and fibronectin fibril assembly were reduced by genetic depletion or antibody neutralization of NRP-1 from stromal myofibroblasts in vivo. Mechanistically, the increase in fibronectin fibril assembly required glycosylation of serine 612 of the extracellular domain of NRP-1, an intact intracellular NRP-1 SEA domain, and intracellular associations between NRP-1, the scaffold protein GIPC, and the nonreceptor tyrosine kinase c-Abl that augmented α5β1 fibronectin fibril assembly activity. Analysis of human cancer specimens established an association between tumoral NRP-1 levels and clinical outcome. Our findings indicate that NRP-1 activates the tumor microenvironment, thereby promoting tumor growth. These results not only identify new molecular mechanisms of fibronectin fibril assembly but also have important implications for therapeutic targeting of the myofibroblast in the tumor microenvironment. Cancer Res; 72(16); 4047–59. ©2012 AACR.

Abstract VEGF/vascular permeability factor (VEGF/VPF or VEGF-A) is a pivotal driver of cancer angiogenesis that is a central therapeutic target in the treatment of malignancy. However, little work has been devoted to investigating functions of VEGF that are independent of its proangiogenic activity. Here, we report that VEGF produced by tumor cells acts in an autocrine manner to promote cell growth through interaction with the VEGF receptor neuropilin-1 (NRP-1). Reducing VEGF expression by tumor cells induced a differentiated phenotype in vitro and inhibited tumor forming capacity in vivo, independent of effects on angiogenesis. Autocrine activation of tumor cell growth was dependent on signaling through NRP-1, and Ras was determined to be a critical effector signaling molecule downstream of NRP-1. Our findings define a novel function for VEGF in dedifferentiation of tumor cells expanding its role in cancer beyond its known proangiogenic function. Cancer Res; 72(16); 3912–8. ©2012 AACR.

AbstractVEGF induces vascular permeability (VP) in ischemic diseases and cancer, leading to many pathophysiological consequences. The molecular mechanisms by which VEGF acts to induce hyperpermeability are poorly understood and in vivo models that easily facilitate real-time, genetic studies of VP do not exist. In the present study, we report a heat-inducible VEGF transgenic zebrafish (Danio rerio) model through which VP can be monitored in real time. Using this approach with morpholino-mediated gene knock-down and knockout mice, we describe a novel role of phospholipase Cβ3 as a negative regulator of VEGF-mediated VP by regulating intracellular Ca2+ release. Our results suggest an important effect of PLCβ3 on VP and provide a new model with which to identify genetic regulators of VP crucial to several disease processes.

AbstractThe Na+/H+ exchanger regulatory factor-2 (NHERF-2) is an integral component of almost all endothelial cells (ECs), yet its endothelial function is not known. Here, we found that NHERF-2, is a key regulator of endothelial homeostasis because NHERF-2–silenced ECs proliferate at a much higher rate even in the absence of mitogens such as VEGF compared with control ECs. We further show that the hyperproliferation phenotype of NHERF-2–silenced EC is because of an accelerated cell cycle that is probably caused by a combination of the following factors: increased cytoplasmic calcium, increased expression of c-Myc, increased expression of cyclin D1, and reduced expression of p27. Using an experimental mouse model of human hemangioma, we found that the endothelial neoplasms derived from NHERF-2–silenced cells were much larger in volume than those derived from control cells. Thus, NHERF-2 is a negative regulator of endothelial proliferation and may have important roles in endothelial homeostasis and vascular modeling.

Abstract We have recently shown that effective cytokine gene therapy of solid tumors in HLA-A2 transgenic (HHD) mice lacking murine MHC class I molecule expression results in the generation of HLA-A2–restricted CD8+ T effector cells selectively recognizing tumor blood vessel-associated pericytes and/or vascular endothelial cells. Using an HHD model in which HLA-A2neg tumor (MC38 colon carcinoma or B16 melanoma) cells are not recognized by the CD8+ T cell repertoire, we now show that vaccines on the basis of tumor-associated blood vessel Ags (TBVA) elicit protective Tc1-dependent immunity capable of mediating tumor regression or extending overall survival. Vaccine efficacy was not observed if (HLA-A2neg) wild-type C57BL/6 mice were instead used as recipient animals. In the HHD model, effective vaccination resulted in profound infiltration of tumor lesions by CD8+ (but not CD4+) T cells, in a coordinate reduction of CD31+ blood vessels in the tumor microenvironment, and in the “spreading” of CD8+ T cell responses to alternate TBVA that were not intrinsic to the vaccine. Protective Tc1-mediated immunity was durable and directly recognized pericytes and/or vascular endothelial cells flow-sorted from tumor tissue but not from tumor-uninvolved normal kidneys harvested from these same animals. Strikingly, the depletion of CD8+, but not CD4+, T cells at late time points after effective therapy frequently resulted in the recurrence of disease at the site of the regressed primary lesion. This suggests that the vaccine-induced anti-TBVA T cell repertoire can mediate the clinically preferred outcomes of either effectively eradicating tumors or policing a state of (occult) tumor dormancy.

Objective— Tissue factor pathway inhibitor (TFPI) is the primary regulator of the tissue factor (TF) coagulation pathway. As such, TFPI may regulate the proangiogenic effects of TF. TFPI may also affect angiogenesis independently of TF, through sequences within its polybasic carboxyl terminus (TFPI C terminus [TFPIct]). We aimed to determine the effects of TFPI on angiogenesis and the role of TFPIct. Methods and Results— Transgenic overexpression of TFPI attenuated angiogenesis in the murine hindlimb ischemia model and an aortic sprout assay. In vitro, TFPI inhibited endothelial cell migration. Peptides within the human TFPIct inhibited endothelial cell cord formation and migration in response to vascular endothelial growth factor (VEGF) 165 but not VEGF121. Furthermore, exposure to human TFPIct inhibited the phosphorylation of VEGF receptor 2 at residue Lys951, a residue known to be critical for endothelial cell migration. Finally, systemic delivery of a murine TFPIct peptide inhibited angiogenesis in the hindlimb model. Conclusion— These data demonstrate an inhibitory role for TFPI in angiogenesis that is, in part, mediated through peptides within its carboxyl terminus. In addition to its known role as a TF antagonist, TFPI, via its carboxyl terminus, may regulate angiogenesis by directly blocking VEGF receptor 2 activation and attenuating the migratory capacity of endothelial cells.

The neurotransmitter dopamine and its dopamine receptor D2 (D2DR) agonists are known to inhibit vascular permeability factor/vascular endothelial growth factor (VEGF)-mediated angiogenesis and vascular permeability. Lung injury is a clinical syndrome associated with increased microvascular permeability. However, the effects of dopamine on pulmonary edema, a phenomenon critical to the pathophysiology of both acute and chronic lung injuries, have yet to be established. Therefore, we sought to determine the potential therapeutic effects of dopamine in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compared with sham-treated controls, pretreatment with dopamine (50 mg/kg body wt) ameliorated LPS-mediated edema formation and lowered myeloperoxidase activity, a measure of neutrophil infiltration. Moreover, dopamine significantly increased survival rates of LPS-treated mice, from 0–75%. Mechanistically, we found that dopamine acts through the VEGF-VEGFR2 axis to reduce pulmonary edema, as dopamine pretreatment in LPS-treated mice resulted in decreased serum VEGF, VEGFR2 phosphorylation, and endothelial nitric oxide synthase phosphorylation. We used D2DR knockout mice to confirm that dopamine acts through D2DR to block vascular permeability in our lung injury model. As expected, a D2DR agonist failed to reduce pulmonary edema in D2DR−/− mice. Taken together, our results suggest that dopamine acts through D2DR to inhibit pulmonary edema-associated vascular permeability, which is mediated through VEGF-VEGFR2 signaling and conveys protective effects in an ALI model.