Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors

We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half‐maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152‐induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi‐agent protocols is warranted.     

The tyrosine kinase inhibitor sorafenib decreases cell number and induces apoptosis in a canine osteosarcoma cell line

Canine osteosarcoma, an aggressive cancer with early distant metastasis, shows still despite good chemotherapy protocols poor long term survival. The aim of our study was to determine whether sorafenib, a novel multikinase inhibitor, has any effect on D-17 canine osteosarcoma cells.A cell proliferation kit was used for detecting surviving cells after treatment for 72 h with sorafenib or carboplatin or their combination. A significant decrease of neoplastic cells was observed after incubation with 0.5-16 μM sorafenib or with 80-640 μM carboplatin. Using immunocytochemistry for activated caspase 3 to evaluate apoptosis, we found significantly more positive cells in the sorafenib treated groups. Paradoxically, expression of the nuclear proliferation marker Ki-67 was also significantly higher in sorafenib treated cells. The drug sorafenib showed potent antitumour activity against D-17 canine osteosarcoma cells in vitro, suggesting a potential as a therapeutic tool in the treatment of bone cancer in dogs.     

Osteosarcoma of the proximal radius in a donkey

A mature donkey was presented for evaluation of a forelimb lameness and presence of a large mass on the left forelimb distal to the elbow joint. Clinical examination, radiographs and cytological examination of ultrasound‐guided fine needle aspirates of the mass led to a presumptive diagnosis of osteosarcoma. The donkey was subsequently subjected to euthanasia. Post mortem examination confirmed the diagnosis of osteosarcoma in the proximal radius with expansion of the mass through the ulnar cortex and metastases to liver and regional lymph nodes, areas of metastasis previously unreported in the equine literature.     

Antioxidant 1 copper chaperone gene expression and copper levels in dog osteosarcoma patients

Twenty-four dogs with OS underwent limb amputation. Serum, OS tumour, and normal bone were harvested at time of surgery. RNA was extracted and gene expression was performed using quantitative polymerase chain reaction (qPCR). Tissue and blood copper concentrations were also determined with spectrophotometry. Compared to bone, tumour samples had significantly higher expressions of antioxidant 1 copper chaperone (ATOX1, p = .0003). OS tumour copper levels were significantly higher than that of serum (p < .010) and bone (p = .038). Similar to our previous observations in mouse and human OS, dog OS demonstrates overexpression of genes that regulate copper metabolism (ATOX1), and subsequent copper levels. Dogs with OS may provide a robust comparative oncology platform for the further study of these factors, as well as potential pharmacologic interventions.     

Radioresistance of cancer stem‐like cell derived from canine tumours

Cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye‐based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage‐independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct‐4 and CD133 gene than those of adherent‐cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent‐cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent‐cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.     

Toxicity and efficacy of a novel doxorubicin and carboplatin chemotherapy protocol for the treatment of canine appendicular osteosarcoma following limb amputation

Objective To evaluate the safety and efficacy of a novel doxorubicin and carboplatin chemotherapy protocol for the treatment of dogs with appendicular osteosarcoma following limb amputation. Design Retrospective study. Procedure Dogs diagnosed with appendicular osteosarcoma, with no evidence of metastatic disease, treated with amputation and adjuvant chemotherapy consisting of two doses of doxorubicin given 14 days apart, followed by four doses of carboplatin at 3‐weekly intervals between September 2003 and December 2009 were identified from the medical records of Perth Veterinary Oncology. Haematological and gastrointestinal toxicities were assessed based on information in the medical records and recorded complete blood count results. The efficacy of the protocol was assessed by determining the median disease‐free interval (DFI) and overall survival time (OST) using the Kaplan‐Meier product‐limit method. Results In total, 33 dogs met the inclusion criteria. The median DFI was 231.5 days and the median OST was 247 days. With regard to haematological toxicity, 56% of dogs had a grade 1–2 neutropenia recorded as their highest marrow toxicity and 9% of dogs experienced a grade 3–4 neutropenia, all subsequent to doxorubicin administration. The highest gastrointestinal toxicity was grade 1–2 in 15 dogs (47%) and 5 dogs (16%) experienced grade 3–4 gastrointestinal toxicity. Conclusion This chemotherapy protocol did not result in a longer time to disease recurrence or OST in this population of dogs. Dual‐agent protocols have failed to improve survival times and therefore we conclude that a single‐agent protocol using carboplatin may be equally effective with less toxicity.     

Spontaneous extraskeletal osteosarcoma with various histological growth patterns in the abdominal wall of an ICR mouse

Extraskeletal osteosarcoma is extremely rare in mice. This case report demonstrates a spontaneous murine extraskeletal osteosarcoma that exhibited various histological growth patterns in an ICR mouse. At necropsy, the tumor mass was located in the abdominal wall and was 45 × 30 × 25 mm in size. Histopathologically, the tumor showed the following four growth patterns: a solid pattern of polygonal cells embedded in an osteoid eosinophilic matrix with calcification, an irregular sheet pattern of short spindle cells accompanying some eosinophilic multinucleated cells, a fascicular pattern of spindle cells and a cystic pattern lined by short spindle cells. Immunohistochemically, most of the tumor cells were positive for vimentin, proliferating cell nuclear antigen and osterix. The multinucleated cells mentioned above were desmin positive and were regarded as regenerative striated muscles but not tumor cells. Since no clear continuity with normal bone tissues was observed, the tumor was diagnosed as an “extraskeletal osteosarcoma.”     

RADIATION INDUCED VERTEBRAL OSTEOSARCOMA FOLLOWING TREATMENT OF AN INTRADURAL EXTRAMEDULLARY SPINAL CORD TUMOR IN A DOG

A 2-year-old neutered female Rottweiler diagnosed with an intradural extramedullary spinal cord tumor at T12-T13 was successfully treated with cytoreductive surgery followed by Cobalt 60 teletherapy. The dog was euthanised 5-and-a-half years later following diagnosis of an osteosarcoma involving the L1 and L2 vertebrae. Evidence of the initial tumor was not present at necropsy. The vertebral neoplasm fulfilled all of the accepted criteria for a radiation induced tumor. It was concluded that adjunctive irradiation should be considered for treatment of intradural extramedullary tumors of young dogs when total surgical resection is not possible. Although tumor induction is a rare late effect of radiation therapy, the risk of this occurrence should be considered when irradiating young animals. Radiation induced tumors in dogs have been associated with coarse fractionation schemes, or when large intraoperative doses have been administered. A lower dose per fraction, e.g., 3 Gy/fraction or less, is advisable when irradiating young dogs or any dog in which the life expectancy is 3-5 or more years after irradiation.     

In vitro effects of the activity of novel platinum (II) complex in canine and human cell lines

The anticancer activity of novel platinum derivative, a complex of platinum with tris(2‐carboxyethyl)phosphine (Pt‐TCEP), has been evaluated in canine (D‐17) and human osteosarcoma (U2‐OS) cell lines. Viability of cells after incubation for 24 or 72 hours with increasing concentrations (0.625, 1.25, 2.50, 5, 10 and 20 μM) of Pt‐TCEP was tested in an MTT assay and compared to effect of cisplatin. Longer‐term effect of Pt‐TCEP was evaluated in the colony‐forming unit assay after 24 hours exposure to the Pt‐TCEP (2 and 3 μM) and subsequent incubation for 2 weeks. The influence of the compound on the cell cycle was measured after 24 hours treatment with Pt‐TCEP (3 μM). Its pro‐apoptotic activity was examined after 24 hours treatment with Pt‐TCEP (1.25, 2.50, 5, 10 and 20 μM) using flow cytometry. Expression of main proteins involved in apoptosis was measured after exposure for 24 hours to 3 or 5 μM Pt‐TCEP in Western Blot. The compound much more effectively decreased cell viability than cisplatin in case of both cell lines. IC₅₀ of Pt‐TCEP was 5.93 ± 0.12 in D‐17 and 3.45 ± 0.14 in U2‐OS cell lines after 24 hours, and 1.77 ± 0.14 in D‐17 and 1.53 ± 0.11 in U2‐OS after 72 hours (P < .05). The compound arrested cells in the G2/M phase and inhibited the ability of cells to form colonies. Pt‐TCEP induced caspase‐dependent apoptosis. The expression of the anti‐apoptotic Bcl‐X_L protein was decreased after Pt‐TCEP treatment in both cell lines. The results confirmed anti‐cancer activity of Pt‐TCEP against canine and human osteosarcoma cell lines.