Inhibitory Effects of a Reengineered Anthrax Toxin on Canine and Human Osteosarcoma Cells.

Jonathan Mackowiak Da Fonseca,Ivone Izabel Mackowiak Da Fonseca,Maria Lucia Zaidan Dagli,Cristina De Oliveira Massoco,Shihui Liu,Marcia Kazumi Nagamine,Jerrold Michael Ward,Stephen Howard Leppla,Adriana Tomoko Nishiya,Thomas Henrik Bugge

doi:10.3390/toxins12100614

Abstract

Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered anthrax toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered anthrax toxin on OSA cells; this reengineered toxin could be further tested as a new therapy for OSA.

Highlights

Canine spontaneous tumours are considered good models for human disease due to their similar morphology and behavior
Since osteosarcomas are so prevalent and aggressive in dogs and there are only a few effective treatments, we aimed to investigate if the reengineered anthrax toxin could exert any inhibitory effects on canine osteosarcoma cells in comparison to human osteosarcoma cells
Since it is known that urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs) are overexpressed in a variety of tumour cells and are rarely present in normal cells [20], we first evaluated their expression in a tissue microarray (TMA) containing

Summary

Introduction

Canine spontaneous tumours are considered good models for human disease due to their similar morphology and behavior. Canine tumours may represent a better alternative to rodent tumours for studying cancer biology and therapy [1,2]. Osteosarcoma (OSA) is the most common primary malignant bone tumour in dogs [3,4,5], being characterised by a high metastatic potential and poor prognosis [1,6,7]. Most dogs that develop canine OSA die because of lung metastases. The recurrence rate is high and the median survival time ranges from 3 months to 1 year [8,9,10]. New treatments are necessary for canine osteosarcomas, and targeted therapies could be adequate options

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