Tsuyoshi Uchide

A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): a pilot study in a canine model.

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P<0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [(3)H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P<0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.

Diagnostic utility of NT-proBNP and ANP in a canine model of chronic embolic pulmonary hypertension

The information needed to diagnose pulmonary arterial hypertension (PAH) in dogs based on N-terminal pro B-type natriuretic peptide (NT-proBNP) and atrial natriuretic peptide (ANP) levels is unclear. In this study, serial changes in plasma NT-proBNP and ANP concentrations were evaluated in association with the development of chronic embolic pulmonary hypertension (CEPH). Six Beagle dogs underwent percutaneous pulmonary artery catheterization. CEPH was induced by the repeated injection of 300 μm microspheres into the pulmonary artery via the catheter. Measured peak systolic pulmonary arterial pressure (PAPs) was elevated up to 80 mm Hg at 90 days by repeated injection of microspheres. Echocardiographic examination showed significant increase in the main pulmonary artery enlargement, right ventricular dilation, transtricuspid late diastolic flow, and ventricular late diastolic myocardial velocity. Plasma concentrations of NT-proBNP and ANP were significantly increased by microsphere-induced severe CEPH, but not by mild CEPH. Measured PAPs correlated weakly with plasma NT-proBNP and ANP concentrations (r=0.63 and 0.69, respectively) and with several echocardiographic variables. Our results indicated that plasma ANP and NT-proBNP responded to severe PAH, but that they were not sensitive for mild PAH.

Epidermal Growth Factor Receptor Expression in Canine Transitional Cell Carcinoma

Transitional cell carcinoma (TCC), a urinary bladder tumor with high mortality, is encountered commonly in dogs. Whereas overexpression of epidermal growth factor receptor (EGFR) is associated with development of human urinary bladder cancer, information on EGFR expression in canine TCC is lacking. In this study, EGFR protein and mRNA expression in canine normal bladder (n=5), polypoid cystitis (n=5) and TCC (n=25) were examined by immunohistochemistry and real-time polymerase chain reaction. EGFR protein expression was significantly higher in TCC than that in normal healthy bladder (P<0.001) and polypoid cystitis (P<0.005). High EGFR protein expression was significantly (P<0.01) associated with TCC with a sensitivity of 72% and specificity of 100%. Comparative analysis of protein and mRNA expression levels in TCC showed significant positive correlation (r=0.88, P<0.05) between mRNA and protein expression. These findings suggest that intense expression of EGFR protein could be used as a marker to help canine TCC diagnosis.

Ultrasonographic findings related to prognosis in canine transitional cell carcinoma.

In human bladder cancer patients, ultrasonography is extensively used not only to identify tumor masses but also to evaluate tumor size, shape, echogenicity, location, and degree of tumor invasion into the bladder wall. The information revealed by ultrasonography delineates the tumors biological features and facilitates prediction of prognosis. However, in veterinary medicine the feasibility of using ultrasonography for these purposes has not been fully investigated. In this retrospective study, we reviewed cases of dogs with histologically confirmed bladder mass lesions, including transitional cell carcinoma (n = 22) and polypoid cystitis (n = 5), to determine whether ultrasonography could reliably predict bladder wall involvement. By following patients with transitional cell carcinoma until death, we also determined whether ultrasonographic tumor size, shape, echogenicity, and mass location were related to prognosis. Wall involvement as revealed by ultrasound was significantly (P = 0.00005) associated with histological muscular layer involvement with a sensitivity of 93% (95% Confidence interval, 79-98%) and specificity of 92% (95% Confidence interval, 76-98%). Ultrasonographic wall involvement (P = 0.03, vs. noninvolvement), heterogeneous mass (P = 0.02, vs. homogeneous mass), and trigone location (P = 0.01, vs. other locations) characteristics were significantly associated with shorter survival times in transitional cell carcinoma cases. Findings indicated that ultrasonographic characteristics such as wall involvement, heterogeneous mass, and trigone location could be reliable prognostic indicators in canine transitional cell carcinoma.

Extranodal Lymphoma with Peripheral Nervous System Involvement in a Dog

ABSTRACT An 8-year-old neutered female Cavalier King Charles spaniel was evaluated for progressing right forelimb lameness. Magnetic resonance imaging revealed that the right-side radial nerves and the caudal brachial plexus were swollen. The histological and molecular biological diagnosis by partial biopsy of the C8 spinal nerve was T-cell lymphoma. Coadministration of lomustine and irradiation was started. However, this therapy was ineffective. At necropsy, neoplastic tissues were seen extending into the subarachnoid space of the spinal cord, liver, pancreas and kidneys as gross findings. A large mass was also identified occupying the caudal thorax. Histologic findings included infiltration in these organs and the mass by neoplastic lymphocytes. To date, involvement of peripheral nerves (neurolymphomatosis) is rarely reported in veterinary species.

Serum big endothelin-1 as a clinical marker for cardiopulmonary and neoplastic diseases in dogs

AIMS Many studies of human subjects have demonstrated the utility of assessing serum levels of endothelin-1 (ET-1) and big ET-1 as clinical biomarkers in cardiopulmonary and neoplastic diseases. In this study we explored the feasibility of using serum big ET-1 as a reliable veterinary marker in dogs with various cardiopulmonary and neoplastic diseases. MAIN METHODS Serum big ET-1 levels were measured by ELISA in dogs with cardiopulmonary (n=21) and neoplastic diseases (n=57). Dogs exhibiting cardiopulmonary disease were divided into two groups based on the velocity of tricuspid valve regurgitation (3.0>m/s) measured by ultrasound: without and with pulmonary hypertension. Big ET-1 levels for the dogs with the diseases were compared with levels in normal healthy dogs (n=17). KEY FINDINGS Dogs with cardiopulmonary disease (4.6±4.6 pmol/l) showed a significantly (P<0.01) higher level of big ET-1 than healthy control dogs (1.1±0.53 pmol/l). Serum levels in the dogs with pulmonary hypertension (6.2±5.3 pmol/l) were significantly (P<0.01) higher than those without pulmonary hypertension (2.0±0.6 pmol/l). Dogs with hemangiosarcoma (5.6±2.2 pmol/l), adenocarcinoma (2.0±1.8 pmol/l), histiocytic sarcoma (3.3±1.9 pmol/l), chondrosarcoma or osteosarcoma (3.0±1.6 pmol/l) and hepatocellular carcinoma (2.7±1.8 pmol/l) showed significantly (P<0.05) higher levels than healthy control dogs. SIGNIFICANCE These findings point to the potential of serum big ET-1 as a clinical marker for cardiopulmonary and neoplastic diseases in dogs.

L-type amino acid transporter 1 (LAT1): A new therapeutic target for canine mammary gland tumour

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities, such as cellular growth, proliferation and maintenance. LAT1 has recently received attention because of its preferential and upregulated expression in a variety of human tumours which is in contrast to its limited distribution and low-level expression in normal tissues. In this study, the feasibility of using an LAT1 inhibitor as a new therapeutic agent was explored for mammary gland tumours (MGT). [(3)H]l-leucine uptake by CHM, a cell line established from MGT, and effects on cell growth were analysed in the presence or absence of two LAT1 inhibitors, namely, BCH (2-amino-2-norbornane-carboxylic acids) or melphalan (LPM). [(3)H]l-leucine uptake and cellular growth activities in CHM were inhibited in a dose-dependent manner by both LAT1 inhibitors. The inhibitory growth activities of various conventional anti-cancer drugs used for MGT treatment, including carboplatin, cyclophosphamide, doxorubicin, mitoxantrone, vinblastine and vincristine, were significantly enhanced by combining use with BCH or LPM. The findings suggest that LAT1 could be a new therapeutic target for canine MGT.

Characterization of feline cytochrome P450 2B6

Abstract 1. Little is known about drug metabolism in carnivores. Although the domestic cat (Felis catus) is an obligate carnivore and is the most common companion animal, usage and dosage of many drugs are determined according to information obtained from humans and dogs. We determined the complete cDNA sequence of CYP2B6 from the feline lung. 2. Feline CYP2B6 consists of 494 deduced amino acids, showing highest identity with the dog CYP2B ortholog, followed by those of horse, pig, primate and human. 3. Feline CYP2B6 transcripts were expressed predominantly in the lung and slightly in the small intestine but not in the liver without significant sex-dependent differences. Western blot analysis with an anti-human CYP2B6 antibody confirmed the presence of CYP2B protein in the lung but not in the liver. 4. Feline CYP2B6 proteins heterologously expressed in Escherichia coli metabolized several substrates specific to human CYP2B6, including 7-ethoxy-4-(trifluoromethyl) coumarin (EFC). The metabolic activity was strongly inhibited by medetomidine and atipamezole, potent inhibitors of canine CYP2B11 (now officially CYP2B6) as well as by ticlopidine and sertraline, inhibitors selective to human CYP2B6. 5. The results suggest that feline CYP2B6 is a functional CYP2B ortholog that plays a role in the local defense mechanism in the cat respiratory system and intestine.

Identification and functional characterization of novel feline cytochrome P450 2A

Abstract 1. Cytochrome P450s are the major metabolizing enzymes for xenobiotics in humans and other mammals. Although the domestic cat Felis catus, an obligate carnivore, is the most common companion animal, the properties of cytochrome P450 subfamilies are largely unknown. 2. We newly identified the feline CYP2A13, which consists of 494 deduced amino acids, showing the highest identity to CYP2As of dogs, followed by those of pigs, cattle and humans. 3. The feline CYP2A13 transcript and protein were expressed almost exclusively in the liver without particular sex-dependent differences. 4. The feline CYP2A13 protein heterogeneously expressed in Escherichia coli showed metabolic activity similar to those of human and canine CYP2As for coumarin, 7-ethoxycoumarin and nicotine. 5. The results indicate the importance of CYP2A13 in systemic metabolism of xenobiotics in cats.

Big endothelin-1 as a tumour marker for canine haemangiosarcoma

Haemangiosarcoma (HSA) is an important malignant neoplasm of dogs that originates from vascular endothelial cells. This study explored the suitability of using serum big endothelin-1 (ET-1) as a tumour marker for canine spontaneous HSA. Serum big ET-1 was measured in dogs with splenic HSA (n = 14), splenic malignant tumours other than HSA (n = 10), benign splenic lesions (n = 11) and normal healthy dogs (n = 17) by ELISA. Serum big ET-1 levels in dogs with HSA were significantly (P < 0.01) higher than in other dogs. High sensitivity (100%, 95% confidence interval 86-100%) and specificity (95%, 95% confidence interval 86-95%) for HSA diagnosis were obtained using a cut-off of 17 pg/mL according to receiver operating characteristic (ROC) curves (area under ROC curve 0.93). PPET1, ETA, VEGF and Hif1-α mRNA expression, measured by real-time PCR, were elevated in HSA compared with normal tissues. These findings suggest that elevated serum big ET-1 could be used as a diagnostic marker for canine HSA.