Establishment of a cell line for assessing drugs as canine P‐glycoprotein substrates: proof of principle

P‐glycoprotein (P‐gp), encoded by the ABCB1 (MDR1) gene, dramatically impacts drug disposition. P‐gp is expressed in the intestines, biliary canaliculi, renal tubules, and brain capillaries where it functions to efflux substrate drugs. In this capacity, P‐gp restricts oral absorption, enhances biliary and renal excretion, and inhibits central nervous system entry of substrate drugs. Many drugs commonly used in veterinary medicine are known substrates for canine P‐gp (vincristine, loperamide, ivermectin, others). Because these drugs have a narrow therapeutic index, defective P‐gp function can cause serious adverse drug reactions due to enhanced brain penetration and/or decreased clearance. P‐gp dysfunction in dogs can be intrinsic (dogs harboring ABCB1‐1Δ) or acquired (drug interactions between a P‐gp inhibitor and P‐gp substrate). New human drug candidates are required to undergo assessment for P‐gp interactions according to FDA and EMA regulations to avoid adverse drug reactions and drug–drug interactions. Similar information regarding canine P‐gp could prevent adverse drug reactions in dogs. Because differences in P‐gp substrates have been documented between species, one should not presume that human or murine P‐gp substrates are necessarily canine P‐gp substrates. Thus, our goal was to develop a cell line for assessing drugs as canine P‐gp substrates.     

Oral bioavailability of P‐glycoprotein substrate drugs do not differ between ABCB1‐1Δ and ABCB1 wild type dogs

Mealey, K.L., Waiting, D., Raunig, D.L., Schmidt, K.R., Nelson, F.R. Oral bioavailability of P‐glycoprotein substrate drugs do not differ between ABCB1‐1Δ and ABCB1 wild type dogs. J. vet. Pharmacol. Therap. 33 , 453–460. Previous studies have indicated that intestinal P‐glycoprotein (P‐gp) limits the oral bioavailability of substrate drugs and alters systemic pharmacokinetics. In this study, dogs lacking functional P‐gp were used to determine the contribution of P‐gp to the oral bioavailability and systemic pharmacokinetics of several P‐gp substrate drugs. The P‐gp substrates quinidine, loperamide, nelfinavir, cyclosporin and the control (non P‐gp substrate) drug diazepam were individually administered intravenously and per os to ABCB1‐1Δ dogs, which have a P‐gp null phenotype and ABCB1 wildtype dogs. ABCB1‐1Δ dogs have been shown to have greater brain penetration of P‐gp substrates, but limited information is available regarding oral bioavailability of P‐gp substrate drugs in this animal model. Plasma drug concentration vs. time curves were generated and pharmacokinetic parameters were calculated for each drug. There were no differences in oral bioavailability between ABCB1‐1Δ dogs and ABCB1 wildtype dogs for any of the drugs studied, suggesting that intestinal P‐gp does not significantly affect intestinal absorption of these particular substrate drugs in ABCB1‐1Δ dogs. However, small sample sizes and individual variability in CYP enzyme activity may have affected the power of the study to detect the impact of P‐gp on oral bioavailability.     

Immune regulation of canine tumour and macrophage PD‐L1 expression

Expression of programmed cell death receptor ligand 1 (PD‐L1) on tumor cells has been associated with immune escape in human and murine cancers, but little is known regarding the immune regulation of PD‐L1 expression by tumor cells and tumor‐infiltrating macrophages in dogs. Therefore, 14 canine tumor cell lines, as well as primary cultures of canine monocytes and macrophages, were evaluated for constitutive PD‐L1 expression and for responsiveness to immune stimuli. We found that PD‐L1 was expressed constitutively on all canine tumor cell lines evaluated, although the levels of basal expression were very variable. Significant upregulation of PD‐L1 expression by all tumor cell lines was observed following IFN‐γ exposure and by exposure to a TLR3 ligand. Canine monocytes and monocyte‐derived macrophages did not express PD‐L1 constitutively, but did significantly upregulate expression following treatment with IFN‐γ. These findings suggest that most canine tumors express PD‐L1 constitutively and that both innate and adaptive immune stimuli can further upregulate PD‐L1 expression. Therefore the upregulation of PD‐L1 expression by tumor cells and by tumor‐infiltrating macrophages in response to cytokines such as IFN‐γ may represent an important mechanism of tumor‐mediated T‐cell suppression in dogs as well as in humans.     

Inhibition of P‐glycoprotein by psychotherapeutic drugs in a canine cell model

Drug–drug interactions related to long‐term therapies are of increasing concern. Psychotherapeutic drugs, licensed for the use in dogs for the management of separation anxiety and other behavioural disorders, are examples of drugs used in long‐term therapies. In an in vitro system with canine P‐glycoprotein (P‐gp) expressing cell lines, three psychotherapeutic drugs with a different mode of action were tested for their ability to inhibit the canine multidrug transporter P‐gp. At 10 μm , the selective serotonin reuptake inhibitor fluoxetine and the tricyclic antidepressant clomipramine inhibited P‐gp for 41% and 59%, respectively. In contrast, selegeline did not inhibit the function of the canine P‐gp.     

Tissue Distribution of P‐Glycoprotein in Cats

Permeability glycoprotein (P‐gp) is a membrane‐bound efflux pump that exports various substances out of the cell. Variations in P‐gp expression play an important role in susceptibility to toxic substances, drug efficacy and disease risk. In the present study, the distribution of the MDR1‐gene product P‐gp was determined in normal tissues of domestic shorthair cats using immunohistochemistry. Two monoclonal antibodies C494 and C219 were used, recognizing a different epitope on the human P‐gp molecule. A consistent positive immunolabelling was obtained. The tissue distribution and cellular locations with antibody C494 were similar to those in man and dogs; with liver, colon, adrenal cortex and brain capillaries being consistently and intensely labelled. However, the immunolabelling in the kidney was in contradiction to man and dogs. The C219 antibody seems to react with a specific form of P‐gp, only expressed in feline tissues with a barrier function, i.e. endothelia of the brain, testes and ovaries, and intestinal epithelial cells in contact with the lumen.     

Cloning and heterologous expression of the ovine (Ovis aries) P‐glycoprotein (Mdr1) in Madin–Darby canine kidney (MDCK) cells

Zahner, D., Alber, J., Petzinger, E. Cloning and heterologous expression of the ovine (Ovis aries) P‐glycoprotein (Mdr1) in Madin–Darby canine kidney (MDCK) cells. J. vet. Pharmacol. Therap. 33 , 304–311. P‐glycoprotein (P‐gp) plays a crucial role in the multidrug resistance of pathogenic helminths in sheep (Ovis aries) as well as in antiparasitic drug pharmacokinetics in the host. We cloned sheep P‐gp cDNA and expressed it stably in Madin–Darby canine kidney (MDCK) cells. The open reading frame consists of 3858 nucleotides coding for a 1285 amino acids containing protein. The sequence shows high homology to the orthologs of other mammalian species, especially cattle. Both ruminant DNA sequences show a 9 bp insertion that is lacking in all other investigated sequences. Expressed in MDCK cells, the protein displays a size of 170 kDa on Western analysis. Transfection of MDCK cells with sheep P‐gp resulted in 10‐ to 50‐fold resistance to the cytotoxic P‐gp substrates colchicin and daunorubicin, and in reduced digoxin accumulation.     

Expression of the high mobility group A1 (HMGA1) and A2 (HMGA2) genes in canine lymphoma: analysis of 23 cases and comparison to control cases

Overexpression of high mobility group A (HMGA) genes was described as a prognostic marker in different human malignancies, but its role in canine haematopoietic malignancies was unknown so far. The objective of this study was to analyse HMGA1 and HMGA2 gene expression in lymph nodes of canine lymphoma patients. The expression of HMGA1 and HMGA2 was analysed in lymph node samples of 23 dogs with lymphoma and three control dogs using relative quantitative real‐time RT‐PCR. Relative quantity of HMGA1 was significantly higher in dogs with lymphoma compared with reference samples. HMGA2 expression did not differ between lymphoma and control dogs. With the exception of immunophenotype, comparison of disease parameters did not display any differences in HMGA1 and HMGA2 expression. The present findings indicate a role of HMGA genes in canine lymphoma. This study represents the basis for future veterinary and comparative studies dealing with their diagnostic, prognostic and therapeutic values.     

Polymorphism of P53‐Ets/AP1 transactivation region of MDM2 oncogene and its immunohistochemical analysis in canine tumours

Mouse Double Minute‐2 (MDM2) is an ubiquitin ligase which is overexpressed or its promoter polymorphism has been reported in different tumours. The objective of this study was to examine the MDM2 protein expression and its promoter polymorphism in some canine tumours. Twenty specimens were collected from 20 dogs with 15 mammary gland carcinomas, 3 lymphomas, 1 transmissible venereal tumour and 1 trichoblastoma. Samples were analysed immunohistochemically using human antibody against MDM2 protein. PCR and DNA sequencing were carried out to identify MDM2 promoter polymorphism. MDM2 gene was expressed in 13 of 20 samples including 11 mammary carcinomas, 1 lymphoma and 1 trichoblastoma. We found 94% homology between canine and human sequences. Four mutations including G169C, A177G, G291T and A177G were identified in different types of breast carcinomas. An extra p53 response element was found in a mixed mammary carcinoma.     

Clinical,histopathological and immunohistochemical characterization of canine indolent lymphoma

Indolent lymphoma comprises up to 29% of all canine lymphoma; however, limited information exists regarding the subtypes and biological behaviour. This retrospective study describes the clinical characteristics, histopathological and immunohistochemical features, treatment, outcome and prognostic factors for 75 dogs with indolent lymphoma. WHO histopathological classification and immunohistochemistry (IHC) for CD79a, CD3, Ki67 and P‐glycoprotein (P‐gp) was performed. The most common histopathological subtype was T‐zone, 61.7%, (MST 33.5 months), followed by marginal zone, 25%, (MST 21.2 months), P = 0.542. The addition of IHC to preliminary histopathological classification resulted in a revised diagnosis in 20.4% of cases. The use of systemic treatment did not influence survival, P = 0.065. Dogs treated with chlorambucil and prednisone did not reach a MST, compared with a MST of 21.6 months with CHOP‐based chemotherapy, P = 0.057. The overall MST of 4.4 years confirms that this is indeed an indolent disease. However, the effect of systemic treatment must be determined through prospective trials.     

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.     

Expression of thymic stromal lymphopoietin in canine atopic dermatitis

Background – In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD). Hypothesis/Objectives – Our aim was to characterize canine TSLP and to assess its expression in CAD. Methods – Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT‐PCR. Real‐time quantitative RT‐PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs. Results – Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full‐length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll‐like receptor ligands lipopolysaccharide and poly I:C. Conclusions and clinical importance – Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.     

Brain penetration of emodepside is increased in P‐glycoprotein‐deficient mice and leads to neurotoxicosis

The antiparasitic drug emodepside (EMO) is a substrate of the P‐glycoprotein multidrug efflux carrier (P‐gp; syn. MDR1, ABCB1), which has an important function in protecting the brain from potentially toxic compounds by functional drug efflux at the blood–brain barrier (BBB). Many dogs of the Collie breed and even dogs of many other breeds have a loss‐of‐function 4‐bp deletion mutation in the MDR1 gene. In these dogs, brain penetration of many P‐gp‐transported drugs is increased and so their therapeutic usage is restricted. To elucidate the role of P‐gp at the BBB for the brain penetration of EMO, we applied EMO at 1 mg/kg to mdr1‐deficient (PGP^mut) and mdr1‐intact (PGP^WT) CF1 mice. Whereas in the brain of the PGP^WT mice, EMO was below the detection level of 10 ng/g, its concentration was at 43.7 ng/g in the PGP^mut mice. Furthermore, appearance of neurological toxicity was analyzed in these mice after application of 1 mg/kg EMO using a rotarod setup. In all PGP^mut mice, but not in the PGP^WT mice, the walking performance on the rotarod was impaired by EMO with clear differences in the degree and duration of neurological toxicity. Some of the mice were completely unable to walk on the rotarod already at 2 h after drug application and showed long‐lasting ataxia over >24 h. Others even showed significantly reduced walking performance, but completely recovered within 1 day. In conclusion, P‐gp restricts brain penetration of EMO and prevents neurological toxicity of this drug in mice.     

Molecular markers of prognosis in canine cortisol‐secreting adrenocortical tumours

Hypercortisolism is caused by a cortisol‐secreting adrenocortical tumour (ACT) in approximately 15%‐20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol‐secreting ACTs were included from 40 dogs, of which follow‐up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate‐high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT‐PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour‐transforming gene‐1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor‐1 (SF‐1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets.     

Evaluation of cyclooxygenase-1 and cyclooxygenase-2 expression and the effect of cyclooxygenase inhibitors in canine prostatic carcinoma

Prostatic carcinoma occurs primarily in older castrated male dogs and is typically a fatal disease (most dogs die within few months after the initial diagnosis). Surgery, i.e., total prostatectomy, or radiation therapy is often not pursued due to risks of complications and a high rate of distant metastasis. Cyclooxygenase‐2 (Cox‐2) expression has been documented in several malignancies, including canine prostatic carcinoma. Cox‐2 inhibition has been reported to have preventative effects on several human malignancies and has therapeutic effects on both laboratory and spontaneous tumour models. The purpose of this retrospective study was to evaluate Cox expression and the effects of Cox inhibitors on survival in dogs with prostatic carcinoma. 94.1 and 88.2% of the tumours expressed Cox‐1 and Cox‐2, respectively. Furthermore, dogs treated with Cox inhibitors (piroxicam or carprofen) lived significantly longer than untreated dogs, 6.9 versus 0.7 months (P < 0.0001), suggesting that Cox inhibitors may have an important role in canine prostate cancer therapy.     

Evaluation of canine antimicrobial peptides in infected and noninfected chronic atopic skin

Background – Antimicrobial peptides (AMPs) are small immunomodulatory peptides produced by epithelial and immune cells. β‐Defensins (BDs) and cathelicidins (Caths) are the most studied AMPs. Recently, increased cutaneous expression of AMPs was reported in atopic humans and in beagles with experimentally induced atopy. Hypothesis/Objectives – Our goal was to analyse mRNA expression and protein levels of canine (c)BD1‐like, cBD2‐like/122, cBD3‐like, cBD103 and cCath in healthy and naturally affected atopic dogs, with and without active skin infection, along with their distribution in the epidermis using indirect immunofluorescence. Animals – Skin biopsies were taken from 14 healthy and 11 atopic privately owned dogs. Methods – The mRNA levels of cBD1‐like, cBD2‐like/122, cBD3‐like, cBD103 and cCath were quantified using quantitative real‐time PCR. The protein levels of cBD3‐like and cCath were analysed by relative competitive inhibition enzyme‐linked immunosorbent assay, while the distributions of cBD2‐like/122, cBD3‐like and cCath were detected by indirect immunofluorescence. Results – Dogs with atopic dermatitis had significantly greater mRNA expression of cBD103 (P = 0.04) than control dogs. Furthermore, atopic skin with active infection had a higher cBD103 mRNA expression (P = 0.01) and a lower cBD1‐like mRNA expression (P = 0.04) than atopic skin without infection. No significant differences in protein levels (cBD3‐like and cCath) or epidermal distribution of AMPs (cBD2‐like/122, cBD3‐like and cCath) were seen between healthy and atopic dogs. Conclusions and clinical importance – Expression of cBD103 mRNA was greater, while expression of cBD1‐like mRNA was lower in dogs with atopic dermatitis that had active infections. Work is needed to clarify the biological mechanisms and possible therapeutic options to maintain a healthy canine skin.     

Somatic SNPs of the BRCA2 gene at the fragments encoding RAD51 binding sites of canine mammary tumors

Mammary tumors are the most common tumor type both in women and in female dogs. In women, heritable breast cancers have been linked mutations in the breast cancer susceptibility gene BRCA2 and it contains eight BRC repeats in exon 11 that bind to RAD51. In this study, we investigated the sequence variations of BRC1‐BRC8 and C‐terminus of canine BRCA2 gene. From a total of 64 canine patients with mammary tumors, 31 mammary tumors with benign and malign carcinomas and the 3 normal mammary glands were used for the study. In this study, 19 SNPs of exon 11 of BRCA2 in canine mammary tumors were detected for the first time. The c.2383A>C (T1425P) SNP was found to be the most probable disease‐associated nsSNP. Our findings suggest that T1425P variation in BRC3 to be the most probable disease‐associated nsSNP and may affect RAD51 binding strength.     

Transcriptomic profile reveals molecular events associated to focal adhesion and invasion in canine mammary gland tumour cell lines

The prevalence of cancer in animals has increased significantly over the years. Mammary tumours are the most common neoplasia in dogs, in which around 50% are presented in the malignant form. Hence, the development and characterization of in vitro models for the study of canine tumours are important for the improvement of cancer diagnosis and treatment. Thus, the aim of this study was to characterize cell lines derived from canine mammary gland neoplasias which could be further used for basic and applied oncology research. Samples of canine mammary carcinomas were taken for cell culture and 2 cell lines were established and characterized in terms of cell morphology, tumourigenicity and global gene expression. Both cell lines presented spindle‐shape morphology and shown common malignant features as in vitro invasion potential and expression of epithelial and mesenchymal proteins. Also, we found gene expression patterns between the 2 cell cultures in comparison to the normal mammary gland tissue. Cells from M25 culture showed a higher invasion and in vivo tumourigenic potential, associated to the overexpression of genes involved in focal adhesion and extracellular matrix communication, such as FN1, ITGA8 and THBS2. The phenotypic characterization of these cells along with their global gene expression profile potentially determine new therapeutic targets for mammary tumours.     

Exploring new biomarkers in the tumour microenvironment of canine inflammatory mammary tumours

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (CIMC) are the most aggressive forms of mammary cancer. Current research aims to identify new therapeutic targets. Here, we investigated gene expression levels of biomarkers associated with the inflammatory microenvironment. A total of 32 formalin‐fixed paraffin‐embedded samples of canine mammary carcinoma (CIMC = 26; non‐CIMC = 6) were used and their cDNA subjected to quantitative polymerase chain reaction (qPCR) to establish gene expression levels for mediators commonly implicated in linking carcinogenesis with inflammation. Gene expression differences between CIMC and non‐CIMC types were obtained for cyclooxygenase 2 (COX‐2) (P = 0.004), synuclein gamma (SNCG) (P = 0.006), tribbles 1 (P = 0.025), vascular endothelial growth factor (VEGF) (P = 0.017) and CSF1R (P = 0.045). Among these biomarkers correlations were found, particularly between SNCG and tribbles 1 (r = 0.512, P = 0.001). The efficient metastasis of CIMC is intimately linked to components in the tumour microenvironment. This study suggests that upregulation and correlation of SNCG and tribbles 1 deserves to be further explored.