Abstract

BackgroundCancer is the leading cause of death in older dogs and its prevalence is increasing. There is clearly a need to develop more effective anti-cancer drugs in dogs. SG2000 (SJG-136) is a sequence selective DNA minor groove cross-linking agent. Based on its in vitro potency, the spectrum of in vivo and clinical activity against human tumours, and its tolerability in human patients, SG2000 has potential as a novel therapeutic against spontaneously occurring canine malignancies.ResultsIn vitro cytotoxicity was assessed using SRB and MTT assays, and in vivo activity was assessed using canine tumour xenografts. DNA interstrand cross-linking (ICL) was determined using a modification of the single cell gel electrophoresis (comet) assay. Effects on cell cycle distribution were assessed by flow cytometry and measurement of γ-H2AX by immunofluorescence and immunohistochemistry.SG2000 had a multi-log differential cytotoxic profile against a panel of 12 canine tumour cell lines representing a range of common tumour types in dogs. In the CMeC-1 melanoma cell line, DNA ICLs increased linearly with dose following a 1 h treatment. Peak ICL was achieved within 1 h and no removal was observed over 48 h. A relationship between DNA ICL formation and cytotoxicity was observed across cell lines. The formation of γ-H2AX foci was slow, becoming evident after 4 h and reaching a peak at 24 h.SG2000 exhibited significant anti-tumour activity against two canine melanoma tumour models in vivo. Anti-tumour activity was observed at 0.15 and 0.3 mg/kg given i.v. either once, or weekly x 3. Dose-dependent DNA ICL was observed in tumours (and to a lower level in peripheral blood mononuclear cells) at 2 h and persisted at 24 h. ICL increased following the second and third doses in a repeated dose schedule. At 24 h, dose dependent γ-H2AX foci were more numerous than at 2 h, and greater in tumours than in peripheral blood mononuclear cells. SG2000-induced H2AX phosphorylation measured by immunohistochemistry showed good correspondence, but less sensitivity, than measurement of foci.ConclusionsSG2000 displayed potent activity in vitro against canine cancer cell lines as a result of the formation and persistence of DNA ICLs. SG2000 also had significant in vivo antitumour activity against canine melanoma xenografts, and the comet and γ-H2AX foci methods were relevant pharmacodynamic assays. The clinical testing of SG2000 against spontaneous canine cancer is warranted.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0534-2) contains supplementary material, which is available to authorized users.

Highlights

  • Cancer is the leading cause of death in older dogs and its prevalence is increasing

  • A clear increase in the number of cells in G2/M was observed in the LMeC, KMeC and CMeC-1 cell lines as has been observed γ-H2AX foci formation following SG2000 treatment of canine cancer cell lines We have previously shown that exposure of cells to conventional cross-linking agents and SG2000 can induce γH2AX foci, clusters of modified histones formed at sites of complex DNA damage [23,24,25]

  • We have previously shown that the peak of γ-H2AX foci formation is approximately 1 h following the peak of interstrand cross-linking (ICL) for nitrogen mustard and platinum drugs [24]

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Summary

Introduction

There is clearly a need to develop more effective anti-cancer drugs in dogs. Based on its in vitro potency, the spectrum of in vivo and clinical activity against human tumours, and its tolerability in human patients, SG2000 has potential as a novel therapeutic against spontaneously occurring canine malignancies. Cancer is the leading cause of death in older dogs and its prevalence is increasing [1, 2]. Current therapies in canine oncology, including chemotherapy, are often extrapolated from those known to be effective in the corresponding human disease. There are currently few veterinary licenced drugs for the treatment of cancer in dogs. Some types of tumours are intrinsically drug resistant (e.g. malignant melanoma and many carcinomas). There is clearly a need to develop novel effective agents with an acceptable toxicity profile targeted towards the most common canine malignancies

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