Anti-tumour efficacy of etoposide alone and in combination with piroxicam against canine osteosarcoma in a xenograft model
Introduction
Osteosarcoma (OSA) accounts for 80–90% of canine primary bone neoplasms, with large and giant breed dogs at greater risk of developing the disease (Ru et al., 1998; Morris and Dobson, 2001; McNeill et al., 2007; Rosenberger et al., 2007). The locally invasive nature of canine OSA requires extensive surgical excision, such as limb amputation, which is the most effective treatment for the primary tumour and pain relief (Mauldin et al., 1988; Szewczyk et al., 2015). The 1-year survival rate of canine OSA patients after treatment with surgery alone is approximately 11–21%. A combination of surgery and adjuvant chemotherapy using doxorubicin or platinum-based drugs extends overall survival of dogs with OSA to 35–50% at 1 year, but fails to hinder the development of fatal metastasis (Straw et al., 1991; Berg et al., 1992, Berg et al., 1995; Bergman et al., 1996; Boston et al., 2006; Moore et al., 2007; Bacon et al., 2008; Phillips et al., 2009; Oblak et al., 2012; Szewczyk et al., 2015). This highlights the need to explore other therapeutic options for canine OSA.
Etoposide is a topoisomerase II inhibitor with proven efficacy for a wide range of neoplasms including small cell lung cancer, testicular cancers, Hodgkin's and non-Hodgkin's lymphomas, and acute leukaemia in humans (Hande, 1998; Gerritsen-van Schieveen and Royer, 2011). It has also been employed in some human OSA treatment protocols (Le Deley et al., 2007; O'Kane et al., 2015; Whelan et al., 2015; Schwartz et al., 2016). A clinical trial published earlier has demonstrated the efficacy of etoposide against recurrent canine lymphoma, indicating that etoposide may be a potential anti-neoplastic agent for clinical application in veterinary oncology (Hohenhaus and Matus, 1990).
While chemotherapy using cytotoxic anti-cancer agents is one of the major treatment modalities for cancer, monotherapy with a single drug is insufficient for cure (Hanahan and Weinberg, 2000; Zimmermann et al., 2007). It has been suggested that anti-cancer therapy using combination of drugs with different cytotoxic mechanisms and less overlapping toxicity can achieve better response rate than monotherapy (Yamanaka et al., 2011). Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammation and pain, including cancer pain in veterinary patients (Gaynor, 2008). In recent years, the role of NSAIDs in cancer treatment has been explored extensively and has shown promising results in experimental and clinical studies. Numerous studies have demonstrated that NSAIDs inhibit tumour cell proliferation and angiogenesis, promote apoptosis, and chemosensitise tumour cells to chemotherapeutic agents (Hanif et al., 1996, Jones et al., 1999, Hida et al., 2000, Knapp et al., 2000, Souza et al., 2000, Caicedo-Granados et al., 2011). Piroxicam, one of the NSAIDs, exhibits anti-neoplastic activity against canine transitional cell carcinoma, mammary adenocarcinoma, and oral squamous cell carcinoma (Schmidt et al., 2001; Mohammed et al., 2002; Souza et al., 2009). It also enhances the cytotoxicity of anti-neoplastic agents against transitional cell carcinoma in dogs and human mesothelioma xenografts (Knapp et al., 2000; Henry et al., 2003; Spugnini et al., 2006).
We have recently shown that etoposide exerts a cytotoxic effect on canine OSA cell lines, and combination treatment with piroxicam enhances the apoptotic activity through suppression of an inhibitor of apoptosis protein, survivin; however, this synergistic effect is only evident at a concentration of piroxicam that exceeds the physiological attainable concentration (Ong et al., 2016). Therefore, the current study was undertaken to evaluate in vivo the therapeutic potential of etoposide on canine OSA and to determine whether combination treatment with piroxicam could enhance the anti-neoplastic effect.
Section snippets
Animal study
This study was performed upon approval of the University of Tokyo Animal Care and Use Committee (reference: P16–265). Twenty-four five-week-old female BALB/c nu/nu mice (SLC Japan) were maintained under specific pathogen-free condition at 24 ± 1 °C with 40–70% humidity and a 12 h: 12 h light:dark cycle throughout all experiments. Sterilized food (CL-2; Clea Japan) and distilled water were provided ad libitum. Sub-confluent HMPOS cells (Barroga et al., 1999) were trypsinised, washed once with
Etoposide alone and in combination with piroxicam inhibit growth of canine OSA xenografts
All mice developed firm, dome-shaped, noncalcified subcutaneous tumours at the site of injection. The mean tumour volume (total tumour volume per group/number of treated animal per group, n) at the endpoint was 1036 ± 367 mm3 in the control group, 1131 ± 585 mm3 in the piroxicam treatment group, 317 ± 236 mm3 in the etoposide treatment group, and 273 ± 77 mm3 in the etoposide and piroxicam combination treatment group (mean ± SD; n = 6) (Fig. 1). Etoposide single agent treatment and combination treatment with
Discussion
Etoposide has been used and studied extensively to treat various human cancers; however, investigation into its potential use for the treatment of canine neoplasms is currently limited. In the present study, we examined the anti-tumour efficacy of etoposide against canine osteosarcoma in nude mouse models. We showed that etoposide single agent treatment and its combination with piroxicam effectively reduced tumour cell proliferation activity that predominantly contributed to slower tumour
Conclusions
The findings of this in vivo study support the efficacy of the anti-tumour effect of etoposide on canine OSA. The mechanism of inhibition is predominantly through suppression of neoplastic cell proliferation activity. Combination treatment with piroxicam fails to enhance the anti-tumour effect exhibited by etoposide. We postulate that the intra-tumoural concentration of piroxicam was inadequate to chemosensitise canine OSA tumour cells to etoposide. The outcome of this study provides a platform
Conflict of interest statement
None of the authors of this paper have any financial or personal relationships with other people or organisations that could inappropriately influence or bias the content of the paper.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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