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Title:In vitro anti-cancer effects of benzimidazoles on the canine osteosarcoma D17 cell line
Author(s):Schmit, Joanna
Advisor(s):Wypij, Jackie M.
Department / Program:Vet Clinical Medicine
Discipline:VMS-Veterinary Clinical Medcne
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:M.S.
Genre:Thesis
Subject(s):canine osteosarcoma
benzimidazole
microtubule
Abstract:The high morbidity and mortality of canine osteosarcoma (OS) despite standard therapy warrants the need to investigate new treatment options. One avenue in exploring novel therapies is drug repurposing – using drugs with known dosing, toxicity profiles, and pharmacokinetics, and re-purposing them “off-label” for their pharmacologic effects for other diseases. In the search for novel therapies for canine osteosarcoma (OS), the benzimidazole (BZ) drugs, a class of safe and inexpensive anti-parasitics, were identified as potential novel therapeutics. Benzimidazole (BZ) drugs are used routinely as effective anti-parasitics in both human and veterinary medicine. Their safety is well-established and side effects are minimal in most veterinary species including dogs. Safety has been described both with short-term high doses as well as long-term chronic dosing in dogs and other species with minimal adverse effects. BZs have demonstrated in vitro and in vivo anti-cancer effects in both people and animal tumor models. The mechanism of BZs is thought to be similar to the microtubule inhibitory actions of traditional chemotherapeutic drug classes such as taxanes and vinca alkaloids, leading to metaphase arrest (G2/M phase) and tumor cell apoptosis. BZs also demonstrate indirect anti-cancer activity by vascular disruption of endothelial cells and reduction in cancer cell secretion of the angiogenic cytokine vascular endothelial growth factor (VEGF). In human OS, mitotic spindle inhibitors are routinely used as an adjuvant chemotherapy agent, and similarly mitotic spindle inhibitors demonstrate effect for canine OS. Given the proposed activity at the mitotic apparatus, BZs may have similar activity in canine osteosarcoma. In addition to direct cytotoxic effects, BZs may possess indirect anti-angiogenic effects in canine OS, including modulation of VEGF. In human OS, increased VEGF expression is a negative prognostic factor and a strong predictor of metastasis and poor survival. Similarly serum VEGF is elevated in many canine cancers including OS and correlates with poor disease free interval. This supports a role for VEGF-induced angiogenesis in the development and progression of metastatic disease in dogs with OS and provides another potential anti-neoplastic mechanism for BZs. We hypothesize that BZs have direct and indirect anti-neoplastic effects in vitro for canine OS. The aims of this study were to assess the in vitro effects of the clinically-used veterinary benzimidazoles [mebendazole (MBZ), fenbendazole (FBZ), and albendazole (ABZ)] on a canine OS cell line. Cell lines were evaluated for dose-dependent anti-neoplastic effects on the functions of cell proliferation, cell-cycle phase distribution, and cell death. Soluble VEGF secretion and the effect on tubulin polymerization were also evaluated in vitro. Specifically, the in vitro effects of ABZ, FBZ and MBZ on D17 canine OS cells were investigated by characterizing 1) cell proliferation with an MTS assay, 2) apoptosis via flow cytometry, 3) VEGF secretion via ELISA and 4) tubulin polymerization and 5) cell cycle distribution via flow cytometry. The results of this study demonstrate that treatment with BZs inhibits cell proliferation in a dose and time dependent fashion. Flow cytometry demonstrates that BZ treatment induces cells arrest in G2/M and subsequently apoptosis. Mechanistically, the BZs affect microtubules by inhibition of polymerization. Additionally, exposure to the BZs results in decreased secretion of VEGF from D17 OS cells. Our findings demonstrate that the clinically used veterinary BZs (ABZ, FBZ, and MBZ) possess anti-neoplastic activity in an OS cell line. In addition to direct effects on tubulin polymerization, cell cycle, proliferation, and cytotoxicity, BZs demonstrate indirect activity through modulation of a key pro-angiogenic cytokine. These findings are similar to what we would expect with a traditional mitotic spindle inhibitor such as a vinca alkaloid. In vitro effects are apparent at drug doses achievable in vivo with minimal expected adverse effects. This data supports the continued investigation into the use of BZs as an adjunctive therapy for canine osteosarcoma.
Issue Date:2013-08-22
URI:http://hdl.handle.net/2142/45401
Rights Information:Copyright 2013 Joanna Schmit
Date Available in IDEALS:2013-08-22
Date Deposited:2013-08


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