Anti-tumour efficacy of etoposide alone and in combination with piroxicam against canine osteosarcoma in a xenograft model

Accepted Manuscript Anti-tumour efficacy of etoposide alone and in combination with piroxicam against canine osteosarcoma in a xenograft model

S.M. Ong, K. Saeki, M.K. Kok, Y. Tanaka, N. Choisunirachon, R. Yoshitake, R. Nishimura, T. Nakagawa PII: DOI: Reference:

S0034-5288(17)30225-4 doi: 10.1016/j.rvsc.2017.09.019 YRVSC 3430

To appear in:

Research in Veterinary Science

Received date: Revised date: Accepted date:

27 February 2017 14 September 2017 16 September 2017

Please cite this article as: S.M. Ong, K. Saeki, M.K. Kok, Y. Tanaka, N. Choisunirachon, R. Yoshitake, R. Nishimura, T. Nakagawa , Anti-tumour efficacy of etoposide alone and in combination with piroxicam against canine osteosarcoma in a xenograft model. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yrvsc(2017), doi: 10.1016/j.rvsc.2017.09.019

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ACCEPTED MANUSCRIPT Anti-tumour efficacy of etoposide alone and in combination with piroxicam against canine osteosarcoma in a xenograft model S.M. Ong a, K. Saeki a, M.K. Kok b, Y. Tanaka a, N. Choisunirachon a, R. Yoshitake a, R. Nishimura a, T. Nakagawa a* a

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(Siew Mei Ong) (Kohei Saeki) (Mun Keong Kok) (Yuiko Tanaka) (Nan Choisunirachon) (Ryohei Yoshitake) (Ryohei Nishimura) (Takayuki Nakagawa)

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Email addresses: [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected]

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Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

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* Corresponding author. Tel.: +81 3 58415414. E-mail address: [email protected] (T. Nakagawa)

ACCEPTED MANUSCRIPT Abstract Osteosarcoma (OSA) in dogs is locally invasive and highly malignant. Distant metastasis is the most common cause of death. To date, the survival rate in dogs with OSA remains poor. The cytotoxic effects of etoposide against canine OSA cell lines, either alone or in combination with piroxicam, have been previously demonstrated in

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vitro. The aim of this study was to evaluate the anti-tumour effect of etoposide alone and in combination with piroxicam on canine OSA using murine models. Etoposide single agent treatment significantly delayed tumour progression with a marked reduction in Ki-67 immunoreactivity in tumour tissue. Concomitant treatment with

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piroxicam did not enhance the anti-tumour efficacy of etoposide. Etoposide single agent treatment and combination treatment with piroxicam down-regulated survivin

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expression, but was not followed by increased apoptotic activity. These findings indicate that etoposide might be a promising novel therapeutic for canine OSA.

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Further investigations into its potential for clinical application in veterinary oncology

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are warranted.

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Keywords: canine osteosarcoma, etoposide, piroxicam, survivin

ACCEPTED MANUSCRIPT 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

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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-

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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,

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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

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the need to explore other therapeutic options for canine OSA. Etoposide is a topoisomerase II inhibitor with proven efficacy for a wide range of

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neoplasms including small cell lung cancer, testicular cancers, Hodgkin’s and non-

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Hodgkin’s lymphomas, and acute leukaemia in humans (Hande, 1998; Gerritsen-van Schieveen et al., 2011). It has also been employed in some human OSA treatment

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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 antineoplastic 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

ACCEPTED MANUSCRIPT 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

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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

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chemosensitise tumour cells to chemotherapeutic agents (Hida et al., 2000; Knapp et al., 2000; Caicedo-Granados et al., 2011). Piroxicam, one of the NSAIDs, exhibits

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anti-neoplastic activity against canine transitional cell carcinoma, mammary adenocarcinoma, and oral squamous cell carcinoma (Schmidt et al., 2001;

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Mohammed et al., 2002; Souza et al., 2009). It also enhances the cytotoxicity of antineoplastic agents against transitional cell carcinoma in dogs and human mesothelioma

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xenografts (Knapp et al., 2000; Henry et al., 2003; Spugnini et al., 2006).

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We have recently shown that etoposide exerts a cytotoxic effect on canine OSA cell lines, and combination treatment with piroxicam enhances the apoptotic activity

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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.

ACCEPTED MANUSCRIPT Materials and methods Cell culture and reagents The canine OSA cell line HMPOS (Highly Metastasizing POS cells) (Barroga et al., 1999) was maintained in RPMI 1640 medium (Wako Pure Chemical), supplemented with 10% foetal bovine serum (FBS) (Gibco BRL) and 5 mg/L

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gentamicin (Sigma-Aldrich), and grown in a humidified atmosphere containing 5% CO2 at 37 °C. Etoposide (Nippon Kayaku) was diluted in saline immediately prior to use, while piroxicam (Sigma-Aldrich) was reconstituted with dimethyl sulfoxide into

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150 mM, stored at -20 °C, and mixed with sesame oil before administration.

Animal study

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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

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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

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experiments. Sterilized food (CL-2; Clea Japan) and distilled water were provided ad

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libitum. Sub-confluent HMPOS cells were trypsinised, washed once with phosphate buffer saline (PBS), and resuspended in PBS at the density of 1 × 107 cells/mL.

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HMPOS cells were inoculated subcutaneously into the right flank of all mice (1 × 106 cells per mouse). Three days later, mice were randomly assigned to one of the following 4 groups of 6 mice each: (1) negative control (saline), (2) piroxicam (0.3 mg/kg/day, orally, daily) (Choisunirachon et al., 2015), (3) etoposide (20 mg/kg, intraperitoneally, every 5 days), and (4) combination. Tumour volume and body weight were measured every three days until the endpoint. Tumour volume was assessed using a calliper and calculated according to the following formula: (length ×

ACCEPTED MANUSCRIPT width2 )/2. All mice were humanely euthanized after 35 days of treatment. Tumour samples were harvested and fixed in 10% neutral buffered formalin, routinely processed and embedded in paraffin. Decalcification was not performed on any of the tumour masses.

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Immunohistochemistry

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Paraffin-embedded tumour sections (4 μm thick) were dewaxed and rehydrated in xylene and graded ethanol followed by antigen retrieval using citrate buffer pH 6 at 121 °C for 10 min. After washing with tris-buffered saline (TBS), endogenous

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peroxidase was blocked with 3% hydrogen peroxide in methanol for 10 min. Slides were then washed with TBS and incubated in 8% skimmed milk for 1 h at 37 °C to

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reduce non-specific binding before overnight incubation with either mouse antihuman Ki-67 (clone MIB-1; ready-to-use; IS626; Dako) or rabbit anti-survivin

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(1:800; NB500-201; Novus Biologicals) (Scase et al., 2006; Bongiovanni et al., 2009) primary antibody at 4 °C in a humidified chamber. A negative control was incubated

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with TBS under identical conditions. For survivin immunostaining, a section of

2006).

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canine cutaneous squamous cell carcinoma was used as positive control (Scase et al.,

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After washing with TBS, sections were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse (Envision+ System labelled polymer; K4001; Dako) or anti-rabbit antibody (Envision+ System labelled polymer; K4003; Dako) for 40 min at 37 °C. Thereafter, the sections were washed with TBS, incubated with 3,3’-diaminobenzidine (DAB; Dojindo Laboratories) solution, and counterstained with Mayer’s haematoxylin. The proliferation index was determined as number of Ki-67 positive nuclei (per 400× microscopic field) × 100 per total number of nuclei,

ACCEPTED MANUSCRIPT counting at least 1000 cells. The expression of survivin was quantified as number of survivin-positive nuclei (per 400× microscopic field) × 100 per total number of nuclei, counting at least 1000 cells.

Terminal deoxyribonucleotide transferase-mediated dUTP nick-end labelling assay

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Apoptotic cells in the xenograft tumour samples were identified by terminal deoxyribonucleotide transferase-mediated dUTP nick-end labelling (TUNEL) reaction using DeadEndT M Colorimetric TUNEL System (Promega). Briefly, formalin- fixed and paraffin-embedded 4-μM thick sections were dewaxed and

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rehydrated in xylene and graded ethanol. Sections were incubated with 20 μg/ml proteinase K at room temperature for 10 min, washed with PBS, followed by the

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TUNEL reaction mixture at 37 °C for 1 h in humidified chamber. For the negative control, the section was incubated in the reaction mixture without TdT enzyme. The

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reaction was stopped by immersing the sections in 2× saline sodium citrate buffer for 15 min. After washing with PBS, endogenous peroxidase was blocked with 3%

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hydrogen peroxide for 10 min. Subsequently, sections were washed with PBS, treated

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with HRP-labelled streptavidin at room temperature for 30 min, and followed by washing with PBS. Thereafter, sections were developed with DAB substrate (Liquid

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DAB+ substrate chromogen system; Dako) and counterstained with Mayer’s haematoxylin. The apoptotic index was determined as number of TUNEL-positive area (per 400× microscopic field) × 100 per total number of nuclei, counting at least 1000 cells.

Statistical analysis

ACCEPTED MANUSCRIPT Statistical analyses were performed using SPSS statistical software (version 23, SPSS Inc.). Statistical significance was calculated using Dunnett’s test or Student’s t test for unpaired data. Data are presented as mean ± standard deviation (SD). Values of P < 0.05 were considered statistically significant.

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Results

Etoposide alone and in combination with piroxicam inhibit growth of canine OSA xenografts

All mice developed firm, dome-shaped, noncalcified subcutaneous tumours at

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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

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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

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combination treatment group (mean ± SD; n = 6) (Fig. 1). Etoposide single agent treatment and combination treatment with piroxicam significantly delayed HMPOS

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xenograft growth, with final tumour volumes that were 30% and 26% of saline-treated

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control mice, respectively. The average tumour volume of the combination treatment group was the smallest but not substantially different from the etoposide treatment

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group. Meanwhile, treatment with piroxicam alone failed to inhibit growth of the HMPOS xenograft tumours. None of the mice displayed weight loss or signs of toxicity (Fig. 2). In addition, no distant metastasis was detected macroscopically at necropsy.

Effect of treatments on cell proliferation and apoptosis

ACCEPTED MANUSCRIPT Treatment with etoposide alone and in combination with piroxicam markedly suppressed the cell proliferation activity of the HMPOS xenograft tumours (Fig. 3 and Fig. 6). In addition, the expression of survivin protein was significantly decreased in these two treatment groups (Fig. 4 and Fig. 6). Both the proliferation index and survivin expression in the xenografts of the combination treatment group were lower

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than the etoposide single agent treatment group, but these differences were not statistically significant. Meanwhile, piroxicam single agent treatment did not alter the protein expression of both Ki-67 and survivin. Figures 5 shows the apoptotic indices of the xenograft tumours. Although there was a slight elevation in the apoptotic index

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of the combination treatment group (P = 0.054), none of the treatments significantly

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enhanced the apoptotic activity of the xenografts.

Discussion

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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

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currently limited. In the present study, we examined the anti-tumour efficacy of

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etoposide against canine osteosarcoma in nude mouse models. We showed that etoposide single agent treatment and its combination with piroxicam effectively

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reduced tumour cell proliferation activity that predominantly contributed to slower tumour growth. Unexpectedly, the in vivo anti-tumour effect demonstrated was not a consequent of increased apoptosis, which has been demonstrated otherwise in our previous work in vitro (Ong et al., 2016). Survivin is an inhibitor of apoptosis protein, which is implicated in the regulation of the mitotic spindle checkpoint and apoptosis (Altieri, 2003; Mita et al., 2008). It is abundantly expressed in a wide range of human and canine tumours,

ACCEPTED MANUSCRIPT including lung, pancreas, colon, bladder, prostate, skin, bone and breast cancers, and its expression is associated with unfavourable prognosis (Ambrosini et al., 1997; Kawasaki et al., 1998; Adida et al., 2000; Sarela et al., 2000; Rankin et al., 2008; Rebhun et al., 2008; Bongiovanni et al., 2009; Shoeneman et al., 2012; Bongiovanni et al., 2015). Suppression of survivin results in apoptosis and sensitises tumour cells

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to chemotherapeutic agents in vitro and in vivo (Olie et al., 2000; Hayashi et al., 2005; Sharma et al., 2005; Nakahara et al., 2011; Arora et al., 2012; Dresang et al., 2013; Yamazaki et al., 2015; Zhang et al., 2015). Our findings correspond with previous investigations in which chemotherapy down-regulated the expression of survivin

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(Wang et al., 2016; Yang et al., 2017); however, it did not significantly contribute to apoptosis.

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The lack of association between the frequency of apoptosis in vivo and chemotherapy-induced tumour growth inhibition has been reported previously

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(Szepeshazi et al., 1991; Guerriero et al., 2008). Furthermore Matsubara et al. (1994) demonstrated that exposure of leukaemia cells to chemotherapeutic agents induced

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apoptosis in vitro, but apoptosis was not detected in vivo. The reason for this is

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unclear, but there are several possible explanations. Apoptosis requires active synthetic processes that can be abrogated by drugs that inhibit protein or RNA

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synthesis (Wyllie, 1986). Besides, tumour microenvironment is more resistant to apoptosis than cell culture (Orth et al., 2011). This could be due to the presence of biological machinery that attenuates apoptotic death in the in vivo environment. Apoptotic response to chemotherapy is time-dependent. It has been shown that apoptosis declines to normal level 3–4 days after administration of chemotherapy in human mammary carcinoma xenografts (Milas et al., 1995); while in human breast cancer patients, apoptotic response completes or ends by 96 h (Symmans et al., 2000).

ACCEPTED MANUSCRIPT In our study, tumour samples were collected 4 days after the final etoposide treatment, where the apoptotic response could have diminished. Furthermore, the apoptotic bodies could have been cleared by neighbouring macrophages. We have previously revealed that piroxicam enhances the cytotoxicity of etoposide only at concentration that exceeds achievable serum concentration. In

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concordance with our observation in vitro, concomitant treatment with piroxicam did not improve the anti-tumour effect of etoposide in vivo. The findings of this in vivo study suggest that intra-tumoural concentration of piroxicam may not exceed the serum concentration; therefore, co-administration with etoposide fails to reiterate the

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synergistic anti-tumour effect shown in the in vitro study.

Likewise, piroxicam single agent treatment had little effect on xenograft tumour

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growth. This finding stands in contrast to previous results conducted on canine transitional cell carcinoma, mammary adenocarcinoma, and squamous cell carcinoma

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(Schmidt et al., 2001; Mohammed et al., 2002; Souza et al., 2009). Despite the fact that piroxicam treatment did not display a substantial impact on canine OSA tumour

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growth, co-administration with etoposide was well tolerated by mice without signs of

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toxicity. This indicates that prescription of piroxicam to control mild to moderate pain in dogs with OSA receiving etoposide therapy is feasible. However, one limitation of

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this study is that it was conducted on one cell line only.

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

ACCEPTED MANUSCRIPT 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 for further investigations on etoposide as a novel therapeutic for canine OSA, either alone or in combination with existing chemotherapeutic regimens,

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and its potential for clinical application in veterinary oncology.

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

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content of the paper.

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Funding

This research did not receive any specific grant from funding agencies in the

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public, commercial, or not-for-profit sectors.

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Mean tumour volume of etoposide single treatment and combination treatment groups

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is significantly smaller than the control. Dunnett’s test, * P < 0.05 versus control.

Figure 2 Average body weight of xenograft mouse models exposed to different

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treatment regimen. Treatments have little influence on the body weight of xenograft

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mice. Data are expressed as mean ± standard deviation (SD).

Figure 3 Effect of treatments on Ki-67 protein expression in xenograft tumours. Data

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control, ** P < 0.01 versus control.

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Figure 4 Etoposide single agent treatment and combination treatment suppresses expression of survivin in xenograft tumours. Data are expressed as mean ± standard

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deviation (SD). Student’s t test, *** P < 0.001 versus control.

Figure 5 Effect of treatments on apoptotic activity of HMPOS xenograft tumours. There is no significant difference in the apoptotic index between treatment and control groups. Data are expressed as mean ± standard deviation (SD).

ACCEPTED MANUSCRIPT Figure 6 Representative photomicrographs of immunohistochemistry analysis for Ki67, survivin and apoptotic cells (TUNEL). Nuclei were counterstained with Mayer’s

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haematoxylin (400×). Bar = 100 μm.

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ACCEPTED MANUSCRIPT Highlights 

Canine osteosarcoma (OSA) is highly malignant and has a poor overall survival rate.

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The anti-tumour effect of etoposide alone and in combination with piroxicam was evaluated in vivo. Etoposide single agent treatment delayed tumour progression.

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Concomitant treatment with piroxicam did not improve the anti-tumour

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Treatment with etoposide, either alone or in combination with piroxicam,

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down-regulated survivin expression.

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efficacy of etoposide.