Masitinib as a chemosensitizer of canine tumor cell lines: a proof of concept study

Masitinib, a selective tyrosine kinase inhibitor, has previously been shown to enhance the antiproliferative effects of gemcitabine in human pancreatic cancer, demonstrating potential as a chemosensitizer. This exploratory study investigated the ability of masitinib to sensitize various canine cancer cell lines to doxorubicin, vinblastine, and gemcitabine. Masitinib strongly sensitized histiocytic sarcoma cells to vinblastine (>70-fold reduction in IC(50) at 5 μM masitinib), as well as osteosarcoma and mammary carcinoma cells to gemcitabine (>70-fold reduction at 5-10 μM). In addition, several cell lines were sensitized to doxorubicin (2-10-fold reduction at 10 μM). These data establish proof-of-concept that masitinib in combination with chemotherapeutic agents can generate synergistic growth inhibition in various canine cancers, possibly through chemosensitization. The findings justify further investigation into those combinations that may potentially yield therapeutic benefit.     

Rapid visualization of mammary gland tumor lesions of dogs using the enzyme-activated fluorogenic probe; γ-glutamyl hydroxymethyl rhodamine green

Since gamma-glutamyl transpeptidase (GGT) is highly and locally expressed in human breast cancer, a GGT-enzymatically activatable fluorescent probe, gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), has been developed to detect the human breast cancer area with high performance. In this study, GGT expression and the efficacy of gGlu-HMRG on visualization were investigated in canine mammary gland tumors (MGT). Seventeen non-fixed fresh-frozen MGT specimens and each peritumoral control tissue were utilized. The GGT mRNA levels were highly observed in the tumor specimens compared with the control. GGT immunostaining was mostly observed on the cell membrane and cytosol of the alveolar and duct mammary epithelium of MGT tissues. These signals were strongly positive in several cases while they were mild to not observed in other cases. When gGlu-HMRG solution was dropped to the non-fixed tissue pieces of MGT or control tissues, the fluorescence intensities (FIs) were measured using Maestro in-vivo imaging device. FIs in MGT tissues were significantly higher than each control tissue 20 min after treatment. Based on Youden index method said that the maximum sensitivity and specificity of FI was 82.4% and 82.4%. These findings suggest that GGT is highly expressed in several MGTs in dogs and gGlu-HMRG could visualize at least a part of MGT tissues in dogs. Nevertheless, it should be needed to assess the false-negative areas more carefully in canine than human cases.     

Effects of gemcitabine and gemcitabine in combination with carboplatin on five canine transitional cell carcinoma cell lines

Objective-To evaluate in vitro effects of gemcitabine alone and in combination with carboplatin on canine transitional cell carcinoma (TCC) cell lines. Sample-In vitro cultures of 5 canine TCC cell lines. Procedures-Cells were treated with gemcitabine, carboplatin, or a combination of both at various concentrations. Cell proliferation was assessed via a fluorescence-based microplate cell proliferation assay. Cell cycle was evaluated via propidium iodide staining, and apoptosis was assessed by measurement of caspase 3 and 7 enzymatic activity. Synergy between gemcitabine and carboplatin was quantified via combination index analyses. Results-Treatment of 5 canine TCC cell lines with gemcitabine or carboplatin decreased cell proliferation, increased apoptosis, and induced cell cycle arrest. Cell cycle arrest and apoptosis were markedly increased when cell lines were treated with both gemcitabine and carboplatin simultaneously or sequentially. Order of administration during sequential treatment did not consistently affect cell proliferation results in TCC cell lines. When TCC cell lines were treated with gemcitabine and carboplatin in combination at therapeutically relevant concentrations (gemcitabine concentration, < 10μM; carboplatin concentration, < 250μM), a significant decrease in cell proliferation was observed, compared with cell proliferation following treatment with gemcitabine or carboplatin alone. In combination, the effects of gemcitabine and carboplatin were synergistic in 3 of 5 cell lines and additive in the other 2. Conclusions and Clinical Relevance-Gemcitabine had antitumor effects on canine TCC cells in vitro, and the combination of gemcitabine and carboplatin had synergistic activity at biologically achievable concentrations.     

The expression of sialyl Lewis X in canine and feline mammary gland tumors

The expression of sialyl Lewis X (sLe(x)) in 93 canine and 15 feline mammary gland tumors (MGT) obtained by surgical resection at Veterinary Medical Center, the University of Tokyo was examined by immunohistochemistry. Their clinicopathological features and prognosis were also reviewed. Approximately 60% of MGT tissues showed sLe(x) positive expressions, while all normal mammary gland tissues were negative. However, its expression was not correlated with clinicopathological features and prognosis significantly. This study suggests that sLe(x) may be a tumor-associated antigen in canine and feline MGTs.     

Comparison of chemotherapeutic drug resistance in cells transfected with canine ABCG2 or feline ABCG2

ABCG2 (ATP binding cassette subfamily G, member 2) mediates resistance to a variety of cytotoxic agents. Although human ABCG2 is well characterized, the function of canine ABCG2 has not been studied previously. Feline ABCG2 has an amino acid substitution in the adenosine triphosphate‐binding domain that decreases its transport capacity relative to human ABCG2. Our goal was to compare canine ABCG2‐mediated chemotherapeutic drug resistance to feline ABCG2‐mediated chemotherapeutic drug resistance. HEK‐293 cells stably transfected with plasmid containing canine ABCG2, feline ABCG2 or no ABCG2 were exposed to carboplatin, doxorubicin, mitoxantrone, toceranib or vincristine, and cell survival was subsequently determined. Canine ABCG2 conferred a greater degree of chemotherapy resistance than feline ABCG2 for mitoxantrone. Neither canine nor feline ABCG2 conferred resistance to doxorubicin, vincristine or toceranib. Canine, but not feline, ABCG2 conferred resistance to carboplatin, a drug that is not reported to be a substrate for ABCG2 in other species.     

Pharmacokinetic properties of toceranib phosphate (Palladia™, SU11654), a novel tyrosine kinase inhibitor,in laboratory dogs and dogs with mast cell tumors

Yancey, M. F., Merritt, D. A., Lesman, S. P., Boucher, J. F., Michels, G. M. Pharmacokinetic properties of toceranib phosphate (Palladia?, SU11654), a novel tyrosine kinase inhibitor, in laboratory dogs and dogs with mast cell tumors. J. vet. Pharmacol. Therap. 33 , 162–171. Toceranib phosphate (Palladia?, SU11654), an oral tyrosine‐kinase inhibitor, is under investigation for the treatment of mast cell tumors in dogs. The pharmacokinetics of toceranib phosphate has been characterized in dogs. Means of the following pharmacokinetic parameters were estimated following a 1.0 mg/kg i.v. dose to laboratory beagles: plasma clearance of 1.45 L/kg/h, volume of distribution of 29.7 L/kg, and terminal half‐life of 17.7 h. Following single oral doses of 3.25 mg/kg administered to laboratory beagles, mean C_max estimates ranged from 68.6 ng/mL to 112 ng/mL with t_max ranging from 5.3 h and 9.3 h postdose. Terminal half‐life was estimated at 31 h. Oral bioavailability was 76.9%. There were no statistically significant (P > 0.05) differences with any pharmacokinetic parameter due to fed/fasted state or with time during 13 weeks of every‐other‐day dosing at 3.25 mg/kg. Toceranib concentrations were proportional with dose over the range of 2.0 to 6.0 mg/kg. The pharmacokinetics of toceranib in client‐owned dogs of a variety of pure and mixed breeds with mast cell tumors was similar to that in healthy laboratory dogs. In summary, toceranib phosphate exhibited moderate clearance, a high volume of distribution, and a moderate elimination half‐life. After a single oral dose at 3.25 mg/kg, the concentration vs. time curve showed broad, sustained exposure with measurable concentrations for more than 48 h. These pharmacokinetic parameters support every‐other‐day administration of toceranib phosphate at an initial dose of 3.25 mg/kg for the treatment of mast cell tumors in dogs.     

Calreticulin expression in neoplastic versus normal dog mammary glands: a cDNA subtraction-based study

This is the first report describing the expression of canine calreticulin (cCRT) in canine mammary gland tumour (MGT). Using cDNA subtraction method, it is found that mRNAs of CRT, cathepsin A, ovostatin, and lactotransferrin were differentially expressed in mammary adenocarcinoma as compared to hyperplasia, both of which were obtained from the dog. Furthermore, the mRNA expression levels of CRT and cathepsin A were significantly higher in canine MGT samples than in nontumour samples. In contrast, immunohistochemical studies have indicated that the expression of cCRT protein found to be detected in most of mammary gland tissues and was not correlated to the types of canine MGTs. Furthermore, cCRT was molecularly cloned, and the amino acid sequence of cCRT was found to be very similar to those of other species. Further studies are required to elucidate additional roles of cCRT in canine MGT.     

The oncolytic effects of reovirus in canine solid tumor cell lines

Oncolytic virotherapy is a new strategy for cancer treatment for humans and dogs. Reovirus has been proven to be a potent oncolytic virus in human medicine. Our laboratory has previously reported that canine mast cell tumor and canine lymphoma were susceptible to reovirus. In this study, canine solid tumor cell lines (mammary gland tumor, osteosarcoma and malignant melanoma) were tested to determine their susceptibility towards reovirus. We demonstrated that reovirus induces more than 50% cell death in three canine mammary gland tumors and one canine malignant melanoma cell line. The reovirus-induced cell death occurred via the activation of caspase 3. Ras activation has been shown to be one of the important mechanisms of reovirus-susceptibility in human cancers. However, Ras activation was not related to the reovirus-susceptibility in canine solid tumor cell lines, which was similar to reports in canine mast cell tumor and canine lymphoma. The results of this study highly suggest that canine mammary gland tumor and canine malignant melanoma are also potential candidates for reovirus therapy in veterinary oncology.     

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.     

Photodynamic detection of canine mammary gland tumours after oral administration of 5‐aminolevulinic acid

5‐Aminolevulinic acid (5‐ALA) is widely used in photodynamic detection (PDD) and therapy. We evaluated the pharmacokinetics of 5‐ALA‐induced porphyrins and its effectiveness in PDD in dogs with mammary gland tumours (MGTs) following oral administration. Healthy dogs and those with MGTs (nine each) were orally administered 4 mg kg^?1 5‐ALA. Protoporphyrin IX (PpIX) was not detected in the plasma of healthy dogs but it peaked in dogs with MGT at 2 h after 5‐ALA administration. In the PDD study, 16 dogs with MGT were orally administered 40 mg kg^?1 5‐ALA, and MGT but not normal tissue showed red fluorescence after 2–4 h. Photon counts were 6635–63 890 and 59–4011 (median, 19 943 and 919) for MGT and non‐tumour tissues, respectively. Cell density strongly correlated with PpIX photon counts of MGT tissue of the dogs (R = 0.743, P = 0.0009). We suggest that 5‐ALA‐PDD might be an effective diagnostic tool for MGTs.     

Genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib in a canine mast cell tumor cell line

One of the potential mechanisms underlying acquired resistance to toceranib in canine mast cell tumor (MCT) is the emergence of a secondary mutation in the KIT gene. Here, genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib were investigated in the toceranib‐susceptible canine MCT cell line VI‐MC, which carries a KIT‐activating mutation resulting in a predicted p.(Asn508Ile) amino acid change in the receptor tyrosine kinase protein KIT. Two sublines were cloned from VI‐MC and toceranib‐resistant sublines then were established by continuous exposure to toceranib. The mutation status of KIT in parental VI‐MC and its sublines was investigated using next‐generation sequencing (NGS). Additionally, effects of secondary mutations on toceranib sensitivity in p.(Asn508Ile)‐mutant KIT were examined. KIT secondary mutations, including those encoding p.(Asn679Lys)‐, p.(Asp819Val)‐, and p.(Asp819Gly)‐mutant KIT, that confer toceranib insensitivity to p.(Asn508Ile)‐mutant KIT emerged only in toceranib‐resistant VI‐MCs. These mutations were not detected by NGS in the parental VI‐MC line or in the toceranib‐naive cloned VI‐MCs, although the parental line and sublines exhibited genetic heterogeneity in KIT that may have been caused by genetic evolution during clonal expansion. VI‐MC clones with these secondary mutations in KIT appear to have arisen from subclones during treatment with toceranib rather than being pre‐existing. However, further study using a higher resolution technique will be needed to confirm the developmental mechanism of KIT secondary mutation in canine MCT cells with acquired resistance to toceranib.     

Association of prognostic features and treatment on survival time of dogs with systemic mastocytosis: A retrospective analysis of 40 dogs

Systemic mastocytosis is a rare phenomenon, with limited information regarding prognostic features and effective treatment of canine patients with this disease. The objective of this study is to determine the impact of certain features and treatments on dogs with systemic mastocytosis. The medical records of 40 dogs from 4 northeastern US veterinary hospitals, with evidence of systemic mast cell disease, were evaluated retrospectively. Variables analysed with relation to overall survival and prognostic significance included treatment protocol used, substage, presence of a cutaneous or visceral tumour, presence of multiple cutaneous Mast cell tumours, grade of the primary tumour and metastatic site(s). Dogs with metastatic disease confined to distant lymph nodes lived longer than those with circulating mast cells in the blood (P = .001), and those with metastatic disease evident in more than 2 sites had a worse prognosis than those with disease in a single location (P = .005). Additionally, administration of chemotherapeutic agents led to increased survival over prednisone therapy alone (P = .008), with the combination of lomustine, vinblastine and prednisone prolonging survival over the tyrosine kinase inhibitor, toceranib phosphate (P = .002). Presence of mast cells in the blood and/or evidence of disease in more than 2 sites indicate widespread dissemination suggesting their use as negative prognostic features. Furthermore, a chemotherapy protocol including combination lomustine and vinblastine therapy may be more effective than toceranib phosphate for the treatment of dogs with disseminated mast cell disease. Overall, patients with systemic mastocytosis have a grave prognosis and more effective treatment options are needed.     

Pharmacokinetics of gemcitabine and its primary metabolite in dogs after intravenous bolus dosing and its in vitro pharmacodynamics

Gemcitabine is a chemotherapeutic agent used to treat a variety of cancers in humans and dogs. In this study, the plasma pharmacokinetics of gemcitabine and its inactive metabolite, 2',2'-difluorodeoxyuridine (dFdU), were investigated in dogs after intravenous bolus gemcitabine doses of 3, 10, and 30 mg/kg. Furthermore, the intracellular accumulation of the active metabolite gemcitabine triphosphate, as a surrogate pharmacodynamic endpoint, was also determined in vitro in canine melanoma cells. Gemcitabine was characterized by linear kinetics, while dFdU dose proportionality remains unknown. The average gemcitabine clearance was 0.560 L/h.kg and volume of distribution at steady-state of 1.27 L/kg. The average terminal elimination half-life, depending on dose, ranged from 1.75 to 3.23 h. Plasma concentrations of dFdU peaked at approximately 2 h post-dosing. In vitro intracellular gemcitabine triphosphate accumulation was saturated with increasing extracellular gemcitabine concentrations. These data can be used to rationally design gemcitabine dosage regimes for canine oncology patients and as a basis for future investigations on the in vivo intracellular accumulation of gemcitabine triphosphate in dogs.     

Combination vinblastine and palladia for high-grade and metastatic mast cell tumors in dogs

High-grade and metastatic canine mast cell tumors carry a guarded prognosis because of their unpredictable biologic behavior. An ideal chemotherapy regime is yet to be established. The aim of this study was to review the efficacy and toxicity of combination vinblastine and toceranib for high-grade and metastatic mast cell tumors. Twenty-eight dogs were categorized with either high-grade, lymph node metastasis or Stage IV disease. Demographics, disease, and treatment variables were compared between categories (Kruskal-Wallis test for continuous data and Fisher’s Exact test for categorical data). Survival times and progression-free intervals (PFI) were calculated and compared between groups (log rank test). The PFI was 310 d [95% confidence interval (CI): 155 to 1425] and overall survival was 373 d (95% CI: 226 to 1219). There was no difference between disease categories for PFI (P = 0.9) or survival (P = 0.5). The protocol was well-tolerated with increased liver enzyme activity and gastrointestinal toxicity most frequently observed. Progression-free intervals and survival times were similar in dogs with high-grade, metastatic and Stage IV disease.     

Microsatellite instability in canine mammary gland tumors

BACKGROUND: Genomic instability is a hallmark of cancer and may be required for the accumulation of cancer-causing mutations within cells. One form of genomic instability occurs in tandem nucleotide repeats and is known as microsatellite instability (MSI). HYPOTHESIS: We hypothesized that MSI can be observed in canine mammary gland tumors (MGT) and represents a potential carcinogenic mechanism in dogs. ANIMALS: Thirty-five dogs with MGTs and 9 dogs with other tumors were recruited from the University of Minnesota Veterinary Medical Center and referring veterinary clinics. METHODS: A panel of 21 canine microsatellite (MS) markers was amplified by polymerase chain reaction (PCR) from deoxyribonucleic acid obtained from blood and from fresh or formalin-fixed, paraffin-embedded tumor tissues. PCR products were evaluated by using capillary electrophoresis, and the chromatograms were analyzed by using genotyping software. MS genotypes obtained from fresh and formalin-fixed tumor tissues were compared, as were MS genotypes from normal tissue and tumor tissue. RESULTS: Genotypes obtained from formalin-fixed and fresh tissues were identical for all MS in 9 tumors evaluated, suggesting excellent concordance between the 2 sample types. For the 35 canine mammary tumors evaluated, 13 (37%) had stable genotypes; 22 (63%) exhibited aberrations in 1 or 2 MS; and 4 tumors (11%) demonstrated high-level instability, with aberrations in 29 to 61% of MS. CONCLUSIONS AND CLINICAL IMPORTANCE: Although some low-level MSI often is observed, high-level MSI is an infrequent finding in canine mammary tumors. Further evaluations are required to better characterize this phenomenon and to determine its relevance to canine carcinogenesis.     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

Distribution,metabolism, and excretion of toceranib phosphate (Palladia™, SU11654), a novel tyrosine kinase inhibitor,in dogs

Yancey, M. F., Merritt, D. A., White, J. A., Marsh, S. A., Locuson, C. W. Distribution, metabolism, and excretion of toceranib phosphate (Palladia?, SU11654), a novel tyrosine kinase inhibitor, in dogs. J. vet. Pharmacol. Therap. 33 , 154–161. Toceranib phosphate (Palladia?, SU11654), a multireceptor tyrosine kinase inhibitor with anti‐tumor and anti‐angiogenic activity, has been developed for the treatment of mast cell tumors in dogs. An overview of the distribution, metabolism, and excretion of toceranib phosphate in dogs is presented. When [¹⁴C]‐toceranib was orally administered to dogs, the majority of the radioactivity (92%) was excreted in feces and only a small portion (7%) was excreted in urine. Seven days after a single 3.25 mg/kg oral dose, radioactivity was the highest in bile and liver, with measurable concentrations in lymph nodes, colon, adrenals, bone marrow, kidneys, lungs, spleen, pancreas, and skin. Plasma protein binding of toceranib in fresh plasma ranged from 90.8% to 92.8% at concentrations between 20 ng/mL and 500 ng/mL and was independent of concentration. Microsomal and hepatocyte incubations resulted in the formation of a single metabolite. Spectrometric analysis of the metabolite was consistent with the formation of an alicyclic N‐oxide of toceranib. The combination of the high rate of fecal excretion and the long elimination half‐life of toceranib indicate enterohepatic recirculation of the parent compound and/or the N‐oxide metabolite.     

Antitumour effects of Liporaxel (oral paclitaxel) for canine melanoma in a mouse xenograft model

Paclitaxel, a member of the taxane family, exhibits antitumour effects by targeting the microtubules in cancer cells. Recently, oral paclitaxel has been developed to overcome the side effects of intravenous paclitaxel administration in human patients. The objective of this study was to investigate the antitumour effects of oral paclitaxel in vitro and in vivo. Three weeks after inoculation, oral paclitaxel (25 and 50 mg/kg) or saline was administered every week for three consecutive weeks. To explore the underlying mechanism, tumour angiogenesis was examined by immunohistochemistry with an anti‐CD31 antibody. Tumour cell apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP Nick‐End Labeling assay, and cell cycle arrest was confirmed by western blot analysis. Oral paclitaxel treatment of canine melanoma cells exerted mediated antiproliferative effects and mediated cell cycle arrest in vitro. In animal experiments, after oral paclitaxel administration, the average tumour size decreased to approximately 30% of that in the control. Histologically, oral paclitaxel showed anti‐angiogenic effects and induced the apoptosis in tumour tissues. Oral paclitaxel also downregulated the intratumoural expression of cyclin D1 and inhibited cell proliferation. The study findings support potential application of oral paclitaxel as a novel chemotherapeutic strategy to treat canine melanoma. This is the first study to investigate the potential of oral paclitaxel as a therapeutic drug against canine tumours.     

Pharmacokinetics of gemcitabine and its primary metabolite in dogs after intravenous infusion

Gemcitabine is a relatively new chemotherapeutic compound used to treat a variety of cancers in dogs. Previous work presented in a companion paper explored the plasma kinetics of gemcitabine and its inactive metabolite, 2',2'-difluorodeoxyuridine (dFdU), in dogs after an intravenous bolus gemcitabine dose and demonstrated the saturation of intracellular dFdCTP (2',2'-difluorodeoxycytidine 5'-triphosphate) occurs in vitro with increasing extracellular gemcitabine exposure in canine melanoma cells. In this study, the plasma pharmacokinetics (PKs) of gemcitabine and dFdU are further explored after gemcitabine doses of 10, 30, and 60 mg/kg administered by intravenous infusion with a loading dose. Gemcitabine displayed linear PKs, while the kinetics of dFdU were not dose proportional. The overall clearance, volume of distribution at steady-state, and terminal elimination half-life (t(1/2)) for gemcitabine were 0.421 L/h.kg, 0.822 L/kg, and 1.49 h, respectively. Plasma concentrations of dFdU peaked at approximately 2 h postdosing and had a t(1/2) of 14.9 h.