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Title:Investigation of the beta 2 adrenergic receptor (B2-AR) pathway in canine hemangiosarcoma
Author(s):Portela, Roberta
Advisor(s):Wypij, Jackie M.
Department / Program:Vet Clinical Medicine
Discipline:VMS-Veterinary Clincial Medcne
Degree Granting Institution:University of Illinois at Urbana-Champaign
beta adrenergic
Abstract:Canine hemangiosarcoma is a highly metastatic cancer arising from vascular endothelial cells. It is one of the most aggressive canine cancers and most dogs die from this disease within a few months of the diagnosis. Despite advancements in veterinary oncology, there has been minimal improvement in the overall survival time even with standard treatment, which includes surgery and chemotherapy. Propranolol, an oral drug originally developed for the treatment of cardiovascular diseases, has been successfully used for the treatment of infantile hemangioma which is a benign neoplasia of vascular endothelial cells. Propranolol blocks adrenergic receptors, which would otherwise bind to catecholamines responsible for the “stress signal” leading to many physiologic changes. Stress has been implicated in many models of carcinogenesis and tumor progression. Given the relationship between stress and cancer, as well as similarities between canine hemangiosarcoma and infantile hemangioma, we sought to investigate the presence of the beta 2 adrenergic receptor and the effects of propranolol in canine hemangiosarcoma. We demonstrated the presence of the beta 2 adrenergic receptor via immunohistochemistry in all 18 tissue samples of spontaneous canine splenic hemangiosarcoma and in canine hemangiosarcoma cell lines Fitz and DEN, cell line receptor expression was also confirmed with Western blot. Src, a possible intermediary downstream protein involved in adrenergic signaling was also investigated and both Fitz and DEN exhibited the presence of the Src protein in Western blot. Activation of this pathway would involve phosphorylation of Src upon catecholamine binding to the receptor, which was investigated through Western blot. Fitz and DEN exhibited basal phosphorylation of Src and after treatment with norepinephrine, and Fitz exhibited greater phosphorylation (23% increase compared to basal control) after 60-minute exposure to the agonist. Fitz cells were pretreated with a biologically achievable dose of propranolol followed by the agonist, and a modest decrease in phosphorylation was observed (11% decrease compared to basal level). Further investigation into the biological effects of propranolol in Fitz and DEN revealed a decrease in VEGF secretion, increase in proliferation and decrease in cell migration. Reduction in VEGF secretion was evaluated via ELISA and it was present at propranolol doses greater than 10 μM for DEN and Fitz, achieving a maximum reduction of 21% in DEN and 44% in Fitz compared to untreated cells. Cell proliferation was measured through MTS assay, which revealed an increase in cell proliferation only in Fitz cells treated with 0.1 μM of NE (25% increase) and cells treated with 0.1 μM of propranolol (30% increase). Cell migration was evaluated with a scratch assay and was decreased only when cells were treated with propranolol at a high dose (100 μM). Taken together, the findings of this study show that beta 2 adrenergic receptors are expressed by canine hemangiosarcoma, Src may be involved in the downstream signaling from the receptor and blockade of the receptor leads to mild to moderate effects in cell angiogenesis, proliferation and migration.
Issue Date:2015-01-21
Rights Information:Copyright 2014 Roberta Portela
Date Available in IDEALS:2015-01-21
Date Deposited:2014-12

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