Calcitriol (1,25‐dihydroxycholecalciferol) enhances mast cell tumour chemotherapy and receptor tyrosine kinase inhibitor activity in vitro and has single‐agent activity against spontaneously occurring canine mast cell tumours*

Calcitriol potentiates the effect of multiple chemotherapy agents in a variety of tumour models. In this study, we examine whether calcitriol increases chemotherapy or tyrosine kinase inhibitor in vitro cytotoxicity in canine mastocytoma C2 cells. We also evaluate the in vivo effect of DN101, a highly concentrated oral formulation of calcitriol designed specifically for cancer therapy, as a single‐agent therapy in dogs with mast cell tumours (MCTs). Calcitriol exhibits synergistic, antiproliferative activity when used in combination with CCNU, vinblastine, imatinib or toceranib in vitro. The concentrations required for 50% growth inhibition were generally two‐ to six‐fold lower when the drugs were used in combination than when used individually. High‐dose oral calcitriol induced remission in 4 of 10 dogs (one complete remission, three partial remissions), although the majority experienced toxicity, necessitating discontinuation of the trial. Further evaluation of calcitriol in combination therapy for dogs with MCTs is warranted.     

Masitinib reverses doxorubicin resistance in canine lymphoid cells by inhibiting the function of P‐glycoprotein

Overexpression of ABC‐transporters including Pgp, MRP1, and BCRP has been associated with multidrug resistance (MDR) in both human and canine oncology. Therapeutic interventions to reverse MDR are limited, but include multidrug protocols and the temporary concomitant use of inhibitors of ABC‐transporters. Recently, the use of tyrosine kinase inhibitors has been proposed to overcome MDR in human oncology. One of the tyrosine kinase inhibitors, masitinib, is licensed for veterinary use in the treatment of canine mast cell tumors. Therefore, this study aimed to assess the potential of masitinib to revert MDR in canine malignant lymphoma using an in vitro model with canine lymphoid cell lines. Masitinib had a mild antiproliferative effect on lymphoid cells, inhibited Pgp function at concentrations equal to or exceeding 1 μm and was able to reverse doxorubicin resistance. The current findings provide the rationale for a combined use of masitinib with doxorubicin in the treatment of dogs with doxorubicin‐resistant malignant lymphoma but await confirmation in clinical trials.     

Non‐immunosuppressive FTY720‐derivative OSU‐2S mediates reactive oxygen species‐mediated cytotoxicity in canine B‐cell lymphoma

OSU‐2S is a FTY720 (Fingolimod) derivative that lacks immunosuppressive properties but exhibits strong anti‐tumour activity in several haematological and solid tumour models. We have recently shown OSU‐2S to mediate potent cytotoxicity in human mantle cell lymphoma cell lines and primary cells. We report here the pre‐clinical activity of OSU‐2S in spontaneous B‐cell lymphoma of dogs which shares many characteristics of human lymphoma. OSU‐2S mediated apoptosis in canine B‐cell lines and primary B‐cell lymphoma cells obtained from spontaneous lymphoma bearing dogs. OSU‐2S induced reactive oxygen species (ROS) in canine lymphoma cells and inhibition of ROS partially rescued OSU‐2S‐mediated cell death. These studies provide a rational basis for the use of spontaneous lymphoma in pet dogs as a preclinical large animal model for the development of OSU‐2S as small molecule for treating people and dogs with lymphoma.     

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild‐type p53

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.     

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.     

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.     

Prognostic significance of hypermethylation of death‐associated protein kinase (DAPK) gene CpG island in dogs with high‐grade B‐cell lymphoma

Death‐associated protein kinase (DAPK) is a serine/threonine kinase and a tumour suppressor gene. Diffuse large B‐cell lymphomas with inactivated DAPK through hypermethylation of a CpG island is known to result in a biologically aggressive phenotype in humans. This retrospective study was carried out to analyse the prognostic significance of DAPK CpG island hypermethylation in canine lymphoma. We hypothesized that DAPK CpG island hypermethylation can be a negative prognostic indicator in dogs with nodal high‐grade B‐cell lymphoma. Forty‐seven dogs with high‐grade B‐cell lymphoma, according to the updated Kiel classification, were evaluated after being treated with a CHOP (vincristine, cyclophosphamide, doxorubicin and prednisolone)‐based chemotherapy protocol. The methylation status of the DAPK CpG island was examined by methylation‐specific PCR. Progression‐free survival (PFS) and overall survival (OS) were compared using the Kaplan‐Meier analysis and log‐rank test. The cox proportional hazard regression model was used to evaluate the effect of multiple variables. Hypermethylation of the DAPK CpG island was detected in 21 of the 47 dogs. The PFS and OS in dogs with the hypermethylation (median: 220 and 266 days, respectively) were significantly shorter than those of dogs without hypermethylation (median: 301 and 412 days, respectively) (PFS, P = .036; OS, P = .007). In the multivariate analysis, hypermethylation of the DAPK CpG island remained an independent prognostic factor in predicting shortened PFS (P = .047) and OS (P = .021) as well as clinical substage b. Overall, hypermethylation of the DAPK CpG island was a negative prognostic factor in canine high‐grade B‐cell lymphoma.     

Tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine‐derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine‐derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8). CDV replication‐induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV‐induced cancer therapy.     

Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation

Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (D_m) values ranging from 4.6–15.8 μM and 2.1–5.1 μM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with D_m values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.     

The selective cyclooxygenase‐2 inhibitor mavacoxib (Trocoxil) exerts anti‐tumour effects in vitro independent of cyclooxygenase‐2 expression levels

The inducible inflammatory enzyme cyclooxygenase‐2 (COX‐2) and its product prostaglandin E2 (PGE₂) are prominent tumour promoters, and expression of COX‐2 is elevated in a number of tumours of both humans and canines. Targeting COX‐2 in cancer is an attractive option because of readily available non‐steroidal anti‐inflammatory drugs (NSAIDs), and there is a clear epidemiological link between NSAID use and cancer risk. In this study, we aim to establish the anti‐tumourigenic effects of the selective, long‐acting COX‐2 inhibitor mavacoxib. We show here that mavacoxib is cytotoxic to a panel of human and canine osteosarcoma, mammary and bladder carcinoma cancer cell lines; that it can induce apoptosis and inhibit the migration of these cells. Interestingly, we establish that mavacoxib can exert these effects independently of elevated COX‐2 expression. This study highlights the potential novel use of mavacoxib as a cancer therapeutic, suggesting that mavacoxib may be an effective anti‐cancer agent independent of tumour COX‐2 expression.     

In vitro effects of 2-methoxyestradiol on canine tumour cells

The in vitro antiproliferative, apoptotic and cell‐cycle effects of 2‐methoxyestradiol (2ME₂), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME₂. Inhibition of tumour cell proliferation was evaluated using a tetrazolium‐based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V‐fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell‐cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME₂ ranging from 0.88 to 7.67 µM, depending on the cell line tested. Profound G₂/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose‐dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.     

Flow cytometric techniques for detection of candidate cancer stem cell subpopulations in canine tumour models

The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of ‘CSC’. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell‐associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker‐defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context‐dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria.     

Tumour necrosis factor‐related apoptosis‐inducing ligand induces apoptosis in canine hemangiosarcoma cells in vitro

Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is an apoptosis‐inducing cytokine that shows potential therapeutic value for human neoplasms, and is effective in some canine tumours; however, its potential for killing canine hemangiosarcoma (HSA) cells is unknown. Thus, we evaluated the proapoptotic effect of TRAIL in nine canine HSA cell lines. Cells (JuA1, JuB2, JuB2‐1, JuB4, Re11, Re12, Re21, Ud2 and Ud6) were cultured with three recombinant human TRAILs (rhTRAILs): TRAIL‐TEC derived from Escherichia coli, TRAIL‐TL derived from mammalian cells and isoleucine zipper recombinant human TRAIL (izTRAIL) containing an isoleucine‐zippered structure that facilitates trimerization. TRAIL‐TEC did not decrease the cell viability in any of the cell lines tested, whereas the other two rhTRAILs effectively decreased the viability of all cell lines as assessed by the WST‐1 assay. In canine HSA cells, izTRAIL induced apoptosis more effectively than TRAIL‐TL. In JuB4, Re12, and Ud6 cells, izTRAIL increased the activation of caspase‐3 and caspase‐8 and caused poly (ADP‐ribose) polymerase degradation. Moreover, izTRAIL treatment increased the proportion of Annexin V+/ Propidium iodide (PI)? apoptotic cells and nuclear fragmentation in izTRAIL‐sensitive cells. These results show that rhTRAIL can induce apoptosis in canine HSA cells, but the sensitivity of TRAIL was different depending on the cell lines. Therefore, TRAIL could be an effective therapeutic agent against canine HSA, but the specific mechanism of resistance should be determined to clarify under what conditions this treatment would be most effective.     

The in vitro effects of piroxicam and meloxicam on canine cell lines

OBJECTIVES: To analyse the direct antiproliferative effects of both piroxicam and meloxicam at a variety of concentrations on a series of canine cancer cell lines and the mechanism of cell death. METHODS: The in vitro effects of piroxicam and meloxicam at various concentrations on canine cell cultures (Madin-Darby canine kidney cells, osteosarcoma, mammary carcinoma, and lymphoma) were assessed with respect to proliferation inhibition and apoptosis induction. Western blot analysis of cyclooxygenase-1 and cyclooxygenase-2 expression was performed on all cell lines. RESULTS: All cell lines used in this study were cyclooxygenase-1 and cyclooxygenase-2 positive apart from Madin-Darby canine kidney cells which were negative for both cyclooxygenase-1 and cyclooxygenase-2. Both meloxicam and piroxicam were able to inhibit proliferation in cell lines in a dose-dependent manner. However, the drug concentration required for a given effect was cell line dependent. CLINICAL SIGNIFICANCE: The results suggest that significant inhibition of proliferation and induction of apoptosis would only occur when drug concentrations were in excess of those that can be achieved in vivo following maximum recommended dose rates. It is possible, however, that local or topical treatment or altered dosing regimens may offer alternative approaches to the use of these drugs as antineoplastic agents.     

Positive association between a glutathione‐S‐transferase polymorphism and lymphoma in dogs

Glutathione‐S‐transferase enzymes (GSTs) play an important role in the detoxification of environmental carcinogens. Defective GST genotypes are over‐represented in human cancers; in particular, low activity GSTT1 genotypes are risk factors for non‐Hodgkin lymphoma. We hypothesized that defective GSTT1 genotypes would be associated with lymphoma risk in dogs. To address this, we resequenced the exons, splice junctions, and 3′‐UTR of canine GSTT1 in dogs with lymphoma (n = 93) and age‐matched unaffected dogs (n = 86). Of 27 canine GSTT1 variants identified, the I2+28 G>A was significantly associated with lymphoma [odds ratio (OR) 6.26, 95% confidence interval (CI), 1.77–22.2], with the AA genotype found in 18.3% of affected dogs but only 3.5% of controls (P = 0.002). This intronic variant was predicted to perturb GSTT1 mRNA splicing, and may increase lymphoma risk by impairing detoxification of environmental chemicals. Confirmation of this finding in a larger population of dogs may support the inclusion of GSTT1 genotyping in epidemiologic studies of canine lymphoma risk.     

Drug dose and drug choice: Optimizing medical therapy for veterinary cancer

Although novel agents hold great promise for the treatment of animal neoplasia, there may be room for significant improvement in the use of currently available agents. These improvements include altered dosing schemes, novel combinations, and patient‐specific dosing or selection of agents. Previous studies have identified surrogates for “individualized dose intensity,”, for example, patient size, development of adverse effects, and pharmacokinetic parameters, as potential indicators of treatment efficacy in canine lymphoma, and strategies for patient‐specific dose escalation are discussed. Strategies for treatment selection in individual patients include conventional histopathology, protein‐based target assessment (eg, flow cytometry, immunohistochemistry, and mass spectrometry), and gene‐based target assessment (gene expression profiling and targeted or global sequencing strategies). Currently available data in animal cancer evaluating these strategies are reviewed, as well as ongoing studies and suggestions for future directions.