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Anti-Neoplastic Effects of Topoisomerase Inhibitors in Canine Mammary Carcinoma, Melanoma, and Osteosarcoma Cell Title Lines

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Anti-Neoplastic Effects of Topoisomerase Inhibitors in Canine Mammary Carcinoma, Melanoma, and Osteosarcoma Cell Title Lines Anti-neoplastic effects of topoisomerase inhibitors in canine mammary carcinoma, melanoma, and osteosarcoma cell Title lines Ong, Siew Mei; Yamamoto, Hiroki; Saeki, Kohei; Tanaka, Yuiko; Yoshitake, Ryohei; Nishimura, Ryohei; Nakagawa, Author(s) Takayuki Citation Japanese Journal of Veterinary Research, 65(1), 17-28 Issue Date 2017-02 DOI 10.14943/jjvr.65.1.17 Doc URL http://hdl.handle.net/2115/64788 Type bulletin (article) File Information 65-1_017-028.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Japanese Journal of Veterinary Research 65(1): 17-28, 2017 REGULAR PAPER Experimental Research Anti-neoplastic effects of topoisomerase inhibitors in canine mammary carcinoma, melanoma, and osteosarcoma cell lines Siew Mei Ong1,†), Hiroki Yamamoto1,†), Kohei Saeki1), Yuiko Tanaka1), Ryohei Yoshitake1), Ryohei Nishimura1) and Takayuki Nakagawa1,＊) 1) 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 Received for publication, November 29, 2016; accepted, December 27, 2016 Abstract Numerous topoisomerase inhibitors with proven efficacy have been used extensively to treat various human neoplasms. However, among these, only doxorubicin has been used and studied extensively in veterinary oncology. The current study was performed to evaluate the responsiveness of canine osteosarcoma (cOSA), mammary gland tumour (cMGT), and malignant melanoma (cMM) cell lines to several topoisomerase inhibitors. In addition, the correlation between the sensitivity to treatment and multi-drug resistant (MDR) factors was investigated. cOSA cell lines exhibited higher sensitivity than cMGT and cMM cell lines to all the topoisomerase inhibitors tested in vitro; this was associated with the levels of multi-drug resistance protein 1 (MDR1) gene expression in the cOSA cell lines. Treatment of cOSA (HMPOS) and cMGT cell line (CHMp) xenograft mouse models with etoposide markedly delayed tumour progression in HMPOS xenografts, but failed to elicit lasting anti-tumour effects on CHMp xenograft mice. The present findings suggest that MDR1 represents a molecular signature for prediction of treatment efficacy of topoisomerase inhibitors, especially that of etoposide, which may be a clinically useful anti-tumour agent for cOSA; however, further study is necessary to refine the treatment protocol. Key Words: Topoisomerase inhibitors; canine osteosarcoma; canine mammary gland tumour; canine malignant melanoma; multi-drug resistant factors Introduction species differences in pharmacokinetic parameters and sensitivity of tumour cells to cancer The use of chemotherapeutic agents in therapeutic compounds. Furthermore, there are veterinary medicine is frequently extrapolated differences between tumours in different species, from information obtained in human medicine. and the relevance of certain canine cancers as Direct extrapolation of chemotherapeutic protocols therapeutic models for human cancer, or vice from human medicine is debatable due to inter- versa, is yet to be defined.29,44) For instance, unlike †Both authors contributed equally to the paper *Corresponding author: Takayuki Nakagawa, 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 Phone: +81 3 58415414. Fax: +81 3 58415420. E-mail: [email protected] (T. Nakagawa) doi: 10.14943/jjvr.65.1.17 18 Topoisomerase inhibitors in dog cancers in human breast cancer, no chemotherapeutic i.e. pre-existing resistance factors in the tumour agents have been shown to be effective in canine that render tumour cells unresponsive to mammary gland tumours44) (cMGT) with the therapy; or acquired, where tumour cells that exception of doxorubicin, to which a subset of were primarily sensitive develop resistance during patients have exhibited responsiveness. Adjuvant the course of treatment.24) Various resistance treatment of canine osteosarcoma (cOSA) with mechanisms have been identified: one of the doxorubicin or a platinum-based drug, alone or in factors that decrease tumour sensitivity to combination, is generally employed by veterinary topoisomerase inhibitors is the development of oncologists; however, the protocols practiced by multidrug resistance (MDR), which has been human oncologists for the same disease are more reported in both human and canine oncology. The aggressive and complex.2,3,34,39) Therefore, it is mechanism of resistance is extensively studied, necessary to explore anti-neoplastic drugs for and a wide range of molecular mechanisms have canine patients with cancer via veterinary been linked to resistance to topoisomerase oncology studies. inhibitors; in particular, mutation or decreased Topoisomerases are nuclear enzymes that expression of topoisomerase I or II, and increased are essential for relaxing supercoiled DNA during drug efflux by multidrug resistance protein 1 cell replication. Two major forms of topoisomerase (MDR1) and multidrug resistance associated have been established: topoisomerase I, which protein 1 (MRP1).9,11,17,27,40,42,49) Therefore, transforms DNA topology by introducing single- elucidation of the relationship between the strand breaks in DNA, and topoisomerase II, expression of MDR factors and sensitivity to which causes double-strand breaks.14,37,45) Various topoisomerase inhibitors may help predict topoisomerase inhibitors that inhibit either responsiveness of canine tumours to these topoisomerase I (irinotecan, topotecan, and chemotherapeutic agents. camptothecin) or topoisomerase II (doxorubicin, The aims of this study were to assess the etoposide, and daunorubicin) have been anti-tumour effects of topoisomerase inhibitors on developed to treat a wide spectrum of human cOSA, cMGT, and canine malignant melanoma neoplasms.14,15,37,48) However, only doxorubicin has (cMM) in vitro and in vivo, and evaluate the been extensively studied and frequently employed association between the expression of several in veterinary chemotherapeutic protocols for MDR factors and sensitivity of canine tumours to certain malignancies based on its efficacy against topoisomerase inhibitors. human cancers. However, its efficacy in canine patients may not be equivalent to that reported in humans. Several clinical trials have Materials and methods demonstrated the efficacy of etoposide, either as single agent or in combination with other anti- Cell lines and reagents: Three cOSA cell lines, neoplastic drugs, against canine lymphoma and namely HMPOS,12) HOS,19) and OOS19), three hemangiosarcoma.18,23) However, this drug is not cMGT cell lines (CHMp, CIPp, and CTBp),43) and extensively used in veterinary oncology due to six cMM cell lines (CMec-1, CMec-2, CMM1, lack of clinical research on its efficacy and limited CMM2, KMec, and LMec)20,32) were maintained in experience. In addition, basic research on the RPMI 1640 medium (Wako Pure Chemical, Osaka, therapeutic effects of topoisomerase inhibitors, Japan) supplemented with 10% foetal bovine including doxorubicin and etoposide, against serum (FBS) (Gibco BRL, NY, USA) and 5 mg/L canine cancers is scarce. gentamicin (Sigma-Aldrich, MO, USA), and The efficacy of chemotherapy is constrained incubated in a humidified atmosphere containing by drug resistance, which may be either intrinsic, 5% CO2 at 37°C. Topoisomerase inhibitors for in Siew Mei Ong et al. 19 Table 1. Concentrations of topoisomerase inhibitors used to determine the dose response curve Topoisomerase inhibitor Concentration (μM) Topoisomerase I inhibitors Camptothecin 0.001-10 Irinotecan 0.01-100 Topotecan 0.01-100 Topoisomerase II inhibitors Daunorubicin 0.001-25 Doxorubicin 0.001-100 Etoposide 0.01-100 vitro studies were topotecan (Tocris Bioscience, Probe qPCR Mix (Toyobo) using a Step One Plus Bristol, UK), irinotecan (LKT Laboratories, MN, Real-Time PCR system (Applied Biosystems, USA), camptothecin, doxorubicin, etoposide, and Foster City, CA, USA). Primers used to detect daunorubicin (Wako Pure Chemical), were the expression of topoisomerase I, IIα, and IIβ, reconstituted in dimethyl sulphoxide (DMSO) and MDR1, and MRP1 genes were designed using the stored at －20°C. For each experiment, drugs NCBI Primer-BLAST Tool (http://www.ncbi.nlm. were diluted with medium supplemented with nih.gov/tools/primer-blast). The primer sequences 10% FBS such that the DMSO concentration were as follows: (a) topoisomerase I forward was 0.5%. Meanwhile, etoposide for in vivo (5´-ATCACAGTGGCTTGGTGCAA-3´) and experiments was acquired from Nippon Kayaku reverse (5´-TTTTCACAGAACCCTGCCCC-3´); (b) (Tokyo, Japan). topoisomerase IIα forward (5´-ACCCCAGGTGGG AAGTATGT-3´) and reverse (5´-GTCTGCCACCC Growth inhibition assay: Tumour cells were plated TTGGAGGTA-3´); (c) topoisomerase IIβ forward at 1 × 103 to 4.5 × 103 cells per well into 96-well (5´-GTGTGTGGATACTCTTCGGGG-3´) and plates in quadruplicate. Cell seeding number for reverse (5´-AAAGAGTCCACAGAGCCACAC-3´); each cell line was optimized in preliminary (d) MDR1 forward (5´-CAGTGGTTCAGGTGGC experiments (data not shown). After 24 h, cells CCT-3´) and reverse (5´-CGAACTGTAGACAAA were treated with topoisomerase inhibitors at the CGATGAGCT-3´); (e) MRP1 forward (5´-GGCTAT concentrations indicated in Table 1. Cell viability CAAGGGCTCAGTGG-3´) and reverse (5´-GCA was determined using the Cell Counting Kit-8 CAGGCTTCAATCACAGC-3´); and (f) GAPDH assay (Dojindo Laboratories, Kumamoto, Japan) forward (5´-TGACACCCACTCTTCCACCTTC-3´)
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