Neurokinin‐1 receptor expression and antagonism by the NK‐1R antagonist maropitant in canine melanoma cell lines and primary tumour tissues

We interrogated the neurokinin‐1 receptor (NK‐1R)/substance P (SP) pathway in canine melanoma tumour tissues and cell lines. NK‐1R messenger RNA (mRNA) and protein expression were observed in the majority of tumour tissues. Immunohistochemical assessment of archived tissue sections revealed NK‐1R immunoreactivity in 11 of 15 tumours, which may have diagnostic, prognostic and therapeutic utility. However, we were unable to identify a preclinical in vitro cell line or in vivo xenograft model that recapitulates NK‐1R mRNA and protein expression documented in primary tumours. While maropitant inhibited proliferation and enhanced apoptosis in cell lines, in the absence of documented NK‐1R expression, this may represent off‐target effects. Furthermore, maropitant failed to suppress tumour growth in a canine mouse xenograft model derived from a cell line expressing mRNA but not protein. While NK‐1R represents a novel target, in the absence of preclinical models, in‐species clinical trials will be necessary to investigate the therapeutic potential for antagonists such as maropitant.     

Pevonedistat targeted therapy inhibits canine melanoma cell growth through induction of DNA re‐replication and senescence

MLN4924 (pevonedistat) is a potent and selective NEDD8‐activating enzyme (NAE) inhibitor. The NEDD8‐regulated neddylation system is responsible for the regulated degradation of intracellular proteins with important cellular functions in cancer cell growth, apoptosis, angiogenesis and metastasis. In human melanoma, inhibition of NAE results in induction of DNA re‐replication, S phase cell cycle arrest, DNA damage and apoptosis. The study aimed to assess the anti‐cancer effect of MLN4924 on canine malignant melanoma cell lines and patient samples and to elucidate the underlying mechanisms. Canine melanoma cell lines and primary patient samples were evaluated for cell viability after incubation with varying concentrations of MLN4924 or dimethyl sulfoxide. Apoptosis, cell proliferation and senescence assays were performed to address underlying mechanisms of MLN4924‐mediated anti‐tumour effects. Gene expression of seven previously identified deregulated genes in human melanoma was compared in sensitive vs resistant samples. MLN4924 treatment significantly reduced the viability of canine melanoma cell lines and primary samples in a dose‐ and time‐dependent manners. MLN4924 promoted cell apoptosis and inhibited cell growth through induction of DNA re‐replication and cell senescence. While the majority of canine melanoma samples demonstrated sensitivity at nanomolar ranges, some samples were resistant to the treatment. Modulation of P21 levels correlated with canine melanoma cell sensitivity. These results provided justification for further exploration of MLN4924 as a treatment of canine melanoma.     

Anti‐tumour activity of oncolytic reovirus against canine histiocytic sarcoma cells

Canine histiocytic sarcoma is an aggressive, fatal neoplastic disease with a poor prognosis. Lomustine is generally accepted as the first‐line systemic therapy, although this compound does not provide complete regression. Therefore, research into a novel approach against canine histiocytic sarcoma is needed. However, anti‐tumour effects of oncolytic therapy using reovirus against histiocytic sarcoma are unknown. Here, we showed that reovirus has oncolytic activity in canine histiocytic sarcoma cell lines in vitro and in vivo. We found that reovirus can replicate and induce caspase‐dependent apoptosis in canine histiocytic sarcoma cell lines. A single intra‐tumoural injection of reovirus completely suppressed the growth of subcutaneously grafted tumours in NOD/SCID mice. Additionally, we demonstrated that susceptibility to reovirus‐induced cell death was attributable to the extent of expression of type I interferons induced by reovirus infection in vitro. In conclusion, oncolytic reovirus appears to be an effective treatment option for histiocytic sarcoma, and therefore warrants further investigation in early clinical trials.     

Analysis of radiosensitivity of cancer stem‐like cells derived from canine cancer cell lines

Cancer stem‐like cells (CSCs) are self‐renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere‐forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs‐like properties by expression of CSCs markers (real‐time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53‐binding protein 1) after X‐ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X‐ray irradiation as compared with AC. In addition, the amount of DNA double‐strand breaks after X‐ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs‐like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double‐strand break after X‐ray exposure.     

Masitinib mesylate does not enhance sensitivity to radiation in three feline injection-site sarcoma cell lines under normal growth conditions

Masitinib, a selective tyrosine kinase inhibitor, was investigated as a radiosensitizer in three primary feline injection-site sarcoma (ISS) cell lines. Sensitivity to masitinib was previously assessed via cell growth inhibition assays with mean IC₅₀ values of 5.5–8.6 μM. Clonogenic assays were performed to determine the effect of masitinib and radiation on cell survival. Single dose radiation (0–12 Gy) experiments were carried out under normal growth conditions in control ISS cells and in cells incubated with 1 or 6 μM masitinib for 72 h prior to irradiation. Radiation administered either alone or in combination with masitinib induced a dose-dependent reduction in clonogenic survival. Survival from the combined masitinib and radiation treatment was not significantly different from that of radiation alone. Results suggest that masitinib does not directly enhance ISS cell radiosensitivity under normal in vitro conditions, although this does not preclude the utility of further investigations to assess sensitization properties under altered conditions.     

The radiosensitizing effect of the aurora kinase inhibitors,ENMD‐2076, on canine mast cell tumours in vitro

ENMD‐2076 is an aurora kinase inhibitor that also has multi‐target tyrosine kinase inhibitor properties. In this study, the mRNA and the protein expression of aurora‐A and aurora‐B were evaluated in three canine mast cell tumour cell lines. Dose‐dependent cytotoxicity was seen in the cells treated, and it affected the cell cycle with cells in the G2/M phase being selectively killed. The cells were also evaluated for radiosensitivity with/without ENMD‐2076, and radiosensitization was seen after 3 Gy and 6 Gy exposures with ENMD‐2076 for 48 h. Protein expression of caspase‐3 was gradually increased, and the expression intensity was highest at 24 h post irradiation in cells without ENMD‐2076 treatment, which indicates that radiation exposure with ENMD‐2076‐induced cell death faster than radiation treatment alone. Our study results suggest the potential usefulness of treating canine mast cell tumours with aurora kinase inhibitors alone or in conjunction with radiation therapy.     

The effects of baicalein on canine osteosarcoma cell proliferation and death

Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti‐inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage‐independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response.     

Masitinib demonstrates anti-proliferative and pro-apoptotic activity in primary and metastatic feline injection-site sarcoma cells

Dysregulation of platelet-derived growth factor receptor (PDGFR) may play a role in feline injection-site sarcoma (ISS) cell growth and viability. Masitinib, a tyrosine kinase inhibitor approved for treatment of canine mast cell tumours, is highly selective for the PDGFR signalling pathway and may offer a new therapeutic approach for this disease. The in vitro effects of masitinib on growth, apoptosis and PDGFR signalling in two novel ISS cell lines were investigated. PDGFR expression was confirmed by Western blot in cell lines derived from a primary ISS tumour (JB) and a corresponding, histologically confirmed ISS lung metastasis (JBLM). Masitinib inhibited cell growth and PDGFR phosphorylation in both cell lines. Higher drug concentrations were required to inhibit growth than to modulate ligand-induced autophosphorylation of PDGFR. These in vitro data suggest that masitinib displays activity against both primary and metastatic ISS cell line and may aid in the clinical management of ISS.     

Effects of ibrutinib on proliferation and histamine release in canine neoplastic mast cells

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE‐dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI‐1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC₅₀ values ranging between 0.1 and 1 μM in primary MCT cells and between 1 and 3 μM in C2 and NI‐1 cells. In C2 cells, the combination “ibrutinib + midostaurin” produced synergistic growth‐inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE‐dependent histamine release in primary MCT cells, with IC₅₀ values ranging from 0.05 to 0.1 μM in NI‐1 cells, and from 0.05 to 1 μM in primary MCT cells. In summary, ibrutinib exerts anti‐proliferative effects in canine neoplastic MCs and counteracts IgE‐dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials.     

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.     

Intrinsic radiosensitivity and repair of sublethal radiation-induced damage in canine osteosarcoma cell lines

OBJECTIVE: To characterize the radiosensitivity and capacity for sublethal damage repair (SLDR) of radiation-induced injury in 4 canine osteosarcoma cell lines. SAMPLE POPULATION: 4 canine osteosarcoma cell lines (HMPOS, POS, COS 31, and D17). PROCEDURES: A clonogenic colony-forming assay was used to evaluate the cell lines' intrinsic radiosensitivities and SLDR capacities. Dose-response curves for the cell lines were generated by fitting the surviving fractions after radiation doses of 0 (control cells), 1, 2, 3, 6, and 9 Gy to a linear quadratic model. To evaluate SLDR, cell lines were exposed to 2 doses of 3 Gy (split-dose experiments) at an interval of 0 (single 6-Gy dose), 2, 4, 6, or 24 hours, after which the surviving fractions were assessed. RESULTS: Mean surviving fraction did not differ significantly among the 4 cell lines at the radiation doses tested. Mean surviving fraction at 2 Gy was high (0.62), and the alpha/beta ratios (predictor of tissue sensitivity to radiation therapy) for the cell lines were low (mean ratio, 3.47). The split-dose experiments revealed a 2.8- to 3.9-fold increase in cell survival when the radiation doses were applied at an interval of 24 hours, compared with cell survival after radiation doses were applied consecutively (0-hour interval). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that these canine osteosarcoma cell lines are fairly radioresistant; alpha/beta ratios were similar to those of nonneoplastic, late-responding tissues. Future clinical investigations should involve increasing the fraction size in a manner that maximizes tumor killing without adverse effects on the nonneoplastic surrounding tissues.     

Radiosensitivity and capacity for radiation-induced sublethal damage repair of canine transitional cell carcinoma (TCC) cell lines

Understanding the inherent radiosensitivity and repair capacity of canine transitional cell carcinoma (TCC) can aid in optimizing radiation protocols to treat this disease. The objective of this study was to evaluate the parameters surviving fraction at 2 Gy (SF(2) ), α/β ratio and capacity for sublethal damage repair (SLDR) in response to radiation. Dose-response and split-dose studies were performed using the clonogenic assay. The mean SF(2) for three established TCC cell lines was high at 0.61. All the three cell lines exhibited a low to moderate α/β ratio, with the mean being 3.27. Two cell lines exhibited statistically increased survival at 4 and 24 h in the dose-response assay. Overall, our results indicate that the cell lines are moderately radioresistant, have a high repair capacity and behave similarly to a late-responding normal tissue. These findings indicate that the radiation protocols utilizing higher doses with less fractionation may be more effective for treating TCC.     

Evaluation of the gene for inducible nitric oxide synthase as a radiosensitizer under hypoxic and oxic conditions

The radiosensitizing effect of inducible nitric oxide synthase (iNOS) was evaluated, in vitro, in a feline vaccine‐associated sarcoma (VAS) cell line and a canine osteosarcoma cell line (D17). The gene encoding the human iNOS was cloned into an expression plasmid under the control of a cytomegalovirus immediate early promoter. Transient transfections were performed in feline VAS cells and D17 cells. Nitric oxide was measured in the supernatant media 48 h later as an indirect measurement of iNOS expression. Cells were irradiated using cobalt‐60 under hypoxic or oxic conditions, and clonogenic assays were used to evaluate the effects of gene transfer on the sensitivity of cells to radiation. The results demonstrated that iNOS had no significant effect on improving the radiosensitivity of cells under oxic conditions. However, under hypoxic conditions, iNOS gene transfer significantly improved radiosensitization in osteosarcoma cells. These results demonstrate the feasibility of improving the outcome of radiotherapy in dogs with large bulky tumours using iNOS gene therapy.     

Establishment of four pairs of canine mammary tumour cell lines derived from primary and metastatic origin and their E-cadherin expression

Four new pairs of canine mammary carcinoma cell lines derived from both primary and metastatic lesions were established. The cells were cultured in RPMI‐1640 with 10% fetal bovine serum and they showed stable growth for more than 120 passages. Using these cell lines, the expression of E‐cadherin was measured by flow cytometry and the function of E‐cadherin was evaluated by cell aggregation assay and results from the primary and metastatic lesions were compared statistically. E‐cadherin was strongly expressed in all of the cell lines, without a notable difference between cells of primary and metastatic origin. In the cell aggregation assay, the function of E‐cadherin was significantly weaker in the cells of primary origin (p < 0.05), as compared with cells of metastatic origin. The present results suggest that a reduction in E‐cadherin function may be implicated in the invasive and metastatic potential of canine mammary tumour cells; however, further study will be needed to clarify E‐cadherin function in the context of the metastasis of canine mammary carcinoma.     

Expression and regulation of cyclooxygenase-2 in normal and neoplastic canine keratinocytes

Squamous cell carcinoma (SCC) is one of the most common cancers in dogs, yet relatively little is known about the molecular events involved in its development. Increasing evidence implicates cyclooxygenase‐2 (COX‐2) in the pathogenesis of various cancers in humans and animals. COX‐2 overexpression has recently been demonstrated in canine SCCs. The objective of our study was to characterize the expression and regulation of COX‐2 in normal and neoplastic canine keratinocytes (CKs) to provide an in vitro system to investigate the implication of COX‐2 in SCC oncogenesis in dogs. Cell lines derived from normal CKs and neoplastic CKs (SCCs) were cultured in the absence or presence of agonists, and immunoblots, immunocytochemistry, radioimmunoassays and a cell proliferation assay were used to characterize COX‐2 expression and action. Results showed that neoplastic keratinocytes had a higher basal COX‐2 expression than normal keratinocytes. In both cell lines, stimulation with the tumour promoter phorbol 12‐myristate 13‐acetate induced a time‐dependent increase in COX‐2 protein, with COX‐2 induction being stronger in cancerous SCC than in normal CK cells. Moreover, SCC cells produced significantly more PGE₂ than CK cells, under both baseline and stimulated conditions (P < 0.05). NS‐398, a selective COX‐2 inhibitor, inhibited prostaglandin (PG)E₂ synthesis and decreased proliferation of CK and SCC cells (P < 0.05). Collectively, our results indicate that the canine neoplastic keratinocyte SCC cell line expresses more COX‐2 and produces more PGE₂ than the normal keratinocyte CK cell line, thus providing an in vitro system to study the molecular basis of elevated COX‐2 expression in SCCs in dogs.     

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.     

The second-generation curcumin analogue RL71 elicits G2/M cell cycle arrest and apoptosis in canine osteosarcoma cells

Canine osteosarcoma is an aggressive cancer, comprising 85% of canine bone neoplasms. Current treatment practices of surgery and chemotherapy increase 1-year survival by only 45%. The curcumin analogue RL71, has demonstrated potent in vitro and in vivo efficacy in several models of human breast cancer through increased apoptosis and cell cycle arrest. Thus, the present study aimed to investigate efficacy of curcumin analogues in two canine osteosarcoma cell lines. Osteosarcoma cell viability was assessed using the sulforhodamine B assay and mechanisms of action were determined by analysing the levels of cell cycle and apoptotic regulatory proteins via Western blotting. Further evidence was obtained using flow cytometry to detect cell cycle distribution and the number of apoptotic cells. RL71 was the most potent curcumin analogue with EC₅₀ values of 0.64 ± 0.04 and 0.38 ± 0.009 μM (n = 3) in D-17 (commercial) and Gracie canine osteosarcoma cells, respectively. RL71 significantly increased the ratio of cleaved-caspase 3 to pro-caspase 3 and the level of apoptotic cells at the 2× and 5× EC₅₀ concentration (p < 0.001, n = 3). Furthermore, at the same concentration, RL71 significantly increased the number of cells in the G2/M phase. In conclusion, RL71 has potent cytotoxic activity in canine osteosarcoma cells triggering G2/M arrest and apoptosis at concentrations achievable in vivo. Future research should further investigate molecular mechanisms for these changes in other canine osteosarcoma cell lines prior to in vivo investigation.     

Wnt5a expression in canine osteosarcoma

Osteosarcoma is the most common primary malignancy of bone in dogs and is associated with poor long‐term outcomes due to its highly metastatic nature. A better understanding of the signalling pathways and proteins involved with osteosarcoma pathogenesis may aid in improved outcomes through the use of targeted therapies. The Wnt5a protein, a ligand for the non‐canonical Wnt signalling pathway, is implicated in mediating the aggressiveness of cancer cell lines, including those of human osteosarcoma origin. Given the close relationship between human and canine osteosarcoma, the primary goal of this study was to characterize Wnt5a expression in canine osteosarcoma. Second, if Wnt5a expression was present in canine osteosarcoma, the study aimed to determine any potential association with clinical outcome and clinical variables in similarly treated osteosarcoma‐bearing dogs. Wnt5a expression was present in 26 of the 48 (54%) cases of canine osteosarcoma. Wnt5a expression was not associated with progression‐free survival (P = 0.4) or overall survival (P = 0.1).