Effect of simvastatin on cell proliferation and Ras activation in canine tumour cells

Statins are inhibitors of the mevalonate cascade that is responsible for cholesterol biosynthesis and the formation of intermediate metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) used in the prenylation of proteins. Although statins are widely used in the treatment of hypercholesterolemia, recent studies suggest that they also inhibit proliferation of tumour cells by reducing prenylation of small GTP‐binding proteins, such as, Ras. This study aimed to evaluate the effect of simvastatin on cell proliferation and Ras activation in various canine tumour cell lines, including hemangiosarcoma (HSA), melanoma, and lymphoma cell lines. Simvastatin inhibited cell proliferation of all cell lines tested in a concentration‐ and time‐dependent manner, but the susceptibilities were different amongst the cell lines. Simvastatin induced apoptotic cell death via activation of caspase‐3 and cell cycle arrest. The cytotoxic effects of simvastatin were attenuated by GGPP and FPP. Simvastatin decreased the amount of prenylated Ras and GTP‐bound Ras in HSA and melanoma cell lines, but not in lymphoma cell lines. These results indicate that simvastatin induces cytotoxic effects through the depletion of GGPP and FPP in a variety of canine tumour cells, whereas multiple mechanisms are involved in the effects. Further study is required to elucidate the underlying mechanisms of simvastatin‐induced cytotoxic effects in a variety of canine tumour cells.     

Oncolytic reovirus synergizes with chemotherapeutic agents to promote cell death in canine mammary gland tumor

The oncolytic effects of reovirus in various cancers have been proven in many clinical trials in human medicine. Oncolytic virotherapy using reovirus for canine cancers is being developed in our laboratory. The objective of this study was to examine the synergistic anti-cancer effects of a combination of reovirus and low doses of various chemotherapeutic agents on mammary gland tumors (MGTs) in dogs. The first part of this study demonstrated the efficacy of reovirus in canine MGTs in vitro and in vivo. Reovirus alone exerted significant cell death by means of caspase-dependent apoptosis in canine MGT cell lines. A single injection of reovirus impeded growth of canine MGT tumors in xenografted mice, but was insufficient to induce complete tumor regression. The second part of this study highlighted the anti-tumor effects of reovirus in combination with low doses of paclitaxel, carboplatin, gemcitabine, or toceranib. Enhanced synergistic activity was observed in the MGT cell line treated concomitantly with reovirus and in all the chemotherapeutic agents except toceranib. In addition, combining reovirus with paclitaxel or gemcitabine at half dosage of half maximal inhibitory concentration (IC₅₀) enhanced cytotoxicity by activating caspase 3. Our data suggest that the combination of reovirus and low dose chemotherapeutic agents provides an attractive option in canine cancer therapy.     

Erythropoietin receptor expression in canine mammary tumor: an immunohistochemical study

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of the erythropoiesis. Recently, it has been demonstrated that EPO and its receptor (EPOR) are expressed in several neoplastic cell lines and solid tumors. Furthermore, in vitro and in vivo studies have shown that EPO could promote human breast carcinoma growth by means of the binding with its receptor, although a clear function for EPO in this setting has not been yet established. While the human medical literature has been accumulating strong evidence on EPO's role in oncogenesis, to date, there are no veterinary reports focusing on such an issue. The aim of the present study was to investigate the immunohistochemical expression of EPOR in canine mammary gland dysplastic and neoplastic lesions. Our results show a weak to moderate EPOR expression in dysplastic glands, being immunoreactivity enhanced as the lesion shows an increasing malignant pattern. On the basis of these findings, this study describes, for the first time, the evidence for EPOR expression in canine mammary gland tumor and suggests a feasible EPO's role for canine mammary tumor progression.     

Comparative study of anti-oncogenic microRNA-145 in canine and human malignant melanoma

MicroRNA-145 (miRNA-145; miR-145) is aberrantly expressed in most of human cancers and plays a significant role in carcinogenesis and cancer progression. In the current study, we focused on how miR-145 plays a role in canine and human malignant melanomas. MiR-145 was significantly downregulated in canine malignant melanoma tissues and canine melanoma cell lines, as well as human melanoma cell lines tested. The ectopic expression of miR-145 showed a significant growth inhibition in both canine and human melanoma cells tested, and the effect was achieved partly by suppressing c-MYC in canine melanoma LMeC and in human melanoma A2058 and Mewo cells. At the same time, a suppressive tendency on cell migration in canine melanoma KMeC cells and significant suppression of cell migration in human melanoma A2058 cells by suppressing FASCIN1 were also found. These findings suggest that miR-145 acts as a tumor suppressor in both canine and human malignant melanomas.     

Expression of a tumor-associated antigen, RCAS1, in canine mammary tumors

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1), one of novel cancer cell-surface antigens, is strongly expressed in invasive cancers. RCAS1 inhibits the in vitro growth of lymphocytes such as T cells and natural killer (NK) cells, and induces apoptotic cell death. We investigated the expression of RCAS1 in canine mammary tumor cell lines and tumor cells by immunohistochemistry, and also in situ deoxyribonucleic acid (DNA) fragmentation in tumor-infiltrating lymphocytes (TILs) by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. All canine mammary tumor cell lines expressed RCAS1 at both the messenger ribonucleic acid (mRNA) and protein level. Immunohistochemically, RCAS1 was negative in 100% of normal mammary glands, but was expressed in 100% of malignant tumors examined. In most malignant mammary tumors, RCAS1 was localized in the cytoplasm with no polarity of expression. In benign mammary tumors, it was detected on the luminal surface of the tumor cell. RCAS1 expression or localization was significantly correlated with malignancy. In situ DNA fragmentation of CD3-positive TILs was observed in RCAS1-expressing tumors. RCAS1-expressing tumors, indicating a possible induction of apoptotic cell death in TILs through RCAS1 expression. These observations suggest that RCAS1 probably plays an important role in tumor progression and escape from immune surveillance in canine mammary tumors.     

Fractal dimension of canine mammary gland epithelial tumors on cytologic smears

BACKGROUND: Fractal geometry is a tool that can be used for describing, modeling, analyzing, and processing irregular and complex figures. Past investigations in medicine have revealed that fractal analysis could also be applied in tumor pathology to characterize irregular boundaries of the nuclei of tumor cells. OBJECTIVE: The aim of this study was to define whether the fractal dimension parameter could be used on cytologic specimens to differentiate benign from malignant canine mammary gland epithelial tumors. METHODS: The fractal dimension of nuclear surface was determined by computer-assisted morphometry on Hemacolor-stained cytologic smears obtained by fine needle aspiration of normal canine mammary gland epithelial cells, and cells in mammary adenomas, tubulopapillary carcinomas, solid carcinomas, and anaplastic carcinomas. Data were analyzed by Mann-Whitney U test. RESULTS: Significant differences (P <.001) were observed in mean fractal dimension among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for the fractal dimension between solid carcinomas and anaplastic carcinomas). CONCLUSION: The morphometric parameter, fractal dimension, could help in the diagnostic discrimination between benign and malignant canine mammary gland epithelial tumors on cytologic specimens.     

Mast cell tumor destruction by deionized water

In a controlled study, malignant murine P815 mastocytoma cells exposed in vitro to distilled and deionized water died as a result of progressive swelling, degranulation, and membrane rupture. A 90% mean cell death occurred when cells obtained directly from culture were exposed to deionized water for 2 minutes. Of 6 cryopreserved malignant murine cell lines, which included Cloudman S91 melanoma, CMT-93 rectum carcinoma, MMT-06052 mammary carcinoma, and S-180 Sarcoma, only P815 mastocytoma and YAC-1 lymphoma were significantly (P less than 0.05) affected by hypotonic shock; Cloudman S91 melanoma cells were the most resistant. Mastocytoma cells were selectively killed by hypotonic solution, and lymphoma cells were also killed by isotonic saline solution. Local mast cell tumor (MCT) recurrence and percentage survival were evaluated in 12 cats (21 MCT) and 54 dogs (85 MCT) subjected to surgery alone or local infiltration of deionized water as an adjunct to surgery. Of all 16 incompletely excised MCT in cats, there was no local recurrence following injection. Four mast cell tumors (2 cats) regressed after being injected in situ. In dogs with clinical stage-I MCT, local recurrence was detected in 50% (5/10), but with injection after incomplete excision, local MCT recurrence was significantly (P less than 0.05) less (6.6%, 1/15). Percentage recurrence was significantly (P less than 0.05) less and survival significantly greater when incompletely excised grade-II MCT were injected. Mean follow-up period after surgery in cats and dogs was 35 and 23.4 months, respectively.     

Immunohistochemical staining for S100 protein in the diagnosis of canine amelanotic melanoma

Formalin-fixed, paraffin-embedded tissue samples of canine amelanotic melanomas and normal canine tissues were studied immunohistochemically for the presence of S100 protein. Use of the avidin-biotin complex procedure demonstrated variable amounts of S100 protein in the tumor cell cytoplasm and nuclei in 26 of 31 tumors. S100 protein was not observed in some other common canine skin tumors stained by the avidin-biotin complex technique. These were a mast cell tumor, fibrosarcoma, mammary gland adenocarcinoma, histiocytoma, transmissible venereal tumor, and a thyroid gland adenocarcinoma. Among normal tissues the presence of S100 protein was demonstrated in chondrocytes in the trachea, myoepithelial cells in the breast, melanocytes in the skin, some sweat glands and ducts in the skin, stellate cells in the pituitary, and interdigitating reticulum cells in the lymph node and in Peyer's patches. These results indicate that the avidin-biotin complex procedure for demonstrating S100 protein is a useful diagnostic tool in the diagnosis of canine amelanotic melanoma.     

Relationship between retinoic acid receptor alpha gene expression and growth-inhibitory effect of all-trans retinoic acid on canine tumor cells

Retinoids show antitumor effects on human acute promyelocytic leukemia and other tumors via retinoid receptors. In dogs, the role of retinoid receptors in inhibiting tumor development remains unclear. To evaluate the correlation between the degree of expression of retinoic acid receptor alpha (RARalpha) mRNA and the antiproliferative effects of all-trans retinoic acid (ATRA) treatments, expression analysis of RARalpha mRNA and cell growth inhibition assay were performed on 17 established canine tumor cell lines, including 6 mammary gland tumor (MGT) cell lines, 3 osteosarcoma cell lines, 5 melanoma cell lines, and 3 mast cell tumor (MCT) cell lines. Among the cell lines investigated, all 3 MCT cell lines showed high expression of RARalpha, and the most effective cell growth inhibition was observed in ATRA-treated MCT cell lines. However, remarkable antiproliferative effects of ATRA treatments were not observed on other tumor cell lines with moderate or low RARalpha mRNA expression. As a result of the relationship between RARalpha mRNA expression and ATRA treatment with regression analysis, statistically significant correlation was suggested. Furthermore, real-time quantitative polymerase chain reaction analysis of RARalpha was performed on MCT tissue samples of dogs with spontaneous disease, and 5 of 9 tissues showed high expression. These results suggest that ATRA may be an effective antitumor agent for MCT in dogs, and that prior measurement of expression of RARalpha mRNA may be a good indicator of the effectiveness of ATRA treatment.     

Computerized morphometry of mean nuclear diameter and nuclear roundness in canine mammary gland tumors on cytologic smears

BACKGROUND: The use of computer-based image analysis systems in veterinary oncology has increased. Computerized morphometry is a part of image analysis that describes geometric figures of cellular structures in any dimension. Most investigators have performed morphometric analysis on histologic specimens. Computer-assisted nuclear cytomorphometry can provide important preoperative information on neoplastic lesions in animals. OBJECTIVES: The aim of this study was to define whether the morphometric parameters of mean nuclear diameter and nuclear roundness could be used to differentiate benign from malignant canine mammary gland tumors on cytologic specimens. METHODS: Mean nuclear diameter and nuclear roundness were determined by computer-assisted morphometry of epithelial cells in Hemacolor-stained cytologic smears from normal canine mammary gland (n = 7) and from canine mammary adenomas (n = 8), tubulopapillary carcinomas (n = 9), and solid carcinomas (n = 6). Data were analyzed by the Mann-Whitney U test. RESULTS: Significant differences (P <.001) were found in mean nuclear diameter and nuclear roundness among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for nuclear roundness between tubulopapillary carcinomas and solid carcinomas). CONCLUSIONS: The morphometric parameters of mean nuclear diameter and nuclear roundness can be used in the preoperative differentiation of benign from malignant canine mammary gland tumors.     

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.     

一例犬乳腺导管乳头状癌的诊断与治疗

犬乳腺导管乳头状癌是犬乳腺肿瘤的一种,为恶性肿瘤,多发于中老年未绝育母犬。临床症状为乳腺出现肿块,大小不等,生长快速,肿瘤可以在一个或多个腺体中发展。在晚期转移的情况下,癌变会扩散到淋巴结,肺,结肠,肾脏,有时还会扩散到骨骼,及早施行手术通常预后良好。本文对一例8岁母犬的乳腺导管内乳头状癌病例的临床诊断与手术治疗情况作详细介绍,以期为犬乳腺肿瘤的临床诊治提供参考。     

Expression and significance of p53, rb,p21/waf-1, p16/ink-4a,and PTEN tumor suppressors in canine melanoma

The role of tumor suppressor genes in the pathogenesis of canine melanoma is incompletely understood. The genes encoding the tumor suppressors p53, Rb, p21 (waf-1), p16 (ink-4a), and PTEN have been postulated to contribute to the pathogenesis of melanoma in humans and experimental animal models. To assess whether inactivation of these genes similarly contributes to the origin and progression of canine melanoma, we examined their expression in seven distinct canine melanoma cell lines and in 31 retrospective samples (representing 29 dogs) of spontaneous canine melanoma. Various patterns suggestive of loss of tumor suppressor function emerged in these cell lines. The most frequently observed abnormality was loss or significant reduction of p16 expression in six of seven cell lines and in 21 of 26 tumor samples. Loss or significant reduction of PTEN expression was seen in four of seven cell lines and in 13 of 27 tumor samples. Although p53 was detectable in all the cell lines and in 24 of 30 tumors, exclusion of p53 from the nuclear compartment was observed in each of the cell lines and in 18 of 25 tumor samples. These results indicate that loss of function of these tumor suppressor proteins is a common occurrence that may contribute to the origin of canine melanoma. In our sample population, abnormalities in the expression or localization of one or more tumor suppressor proteins occurred with similar frequency in malignant and benign tumors; thus, additional work is necessary to determine how these proteins may impact disease progression and response to therapy.     

Glucocorticoid sensitivity depends on expression levels of glucocorticoid receptors in canine neoplastic mast cells

Glucocorticoid (GC) administration with or without other chemotherapeutic reagents is a commonly used option in the treatment of mast cell malignancies. However, the responsiveness of mast cell tumors to GC treatment varies in individuals, and the regulatory mechanisms determining the GC sensitivity of malignant mast cells remain unclear. Since the expression of the GC receptor (GR) has been reported to be associated with GC sensitivity in human neoplastic lymphocytes, we attempted to investigate the relationship between GR levels and GC sensitivity by using neoplastic mast cells derived from canine mast cell tumors (MCTs). To elucidate the regulatory mechanisms involved in GC responsiveness, we analyzed various canine MCT cell lines and tissue samples from dogs with MCT. While the proliferation of canine MCT cells was suppressed by the addition of GC to the culture, we found that MCT cells derived from humans and rodents, as well as canine lymphoma cells, responded poorly to GC. However, there were also some variations in responsiveness to GC treatment among canine MCT cell lines used in this study. Using real-time polymerase chain reaction and Western blot analysis, we elucidated the relationship between GR expression and responsiveness to GC in canine MCT cells. Furthermore, to assess the involvement of GR expression in GC sensitivity in vivo, clinical investigations were conducted on dogs with cutaneous MCT. Written informed consent was obtained from owners, and the affected dogs were treated with prednisolone (0.5-2.0 mg kg(-1)day(-1), administered orally) 1 or 2 weeks prior to the surgical removal of the tumors. Tumor volume was measured according to WHO criteria both before and after prednisolone treatment, and the GC sensitivity of each MCT was determined on the basis of the reduction in tumor volume. Of the 15 dogs with MCT, 11 responded to treatment with prednisolone completely or partially, whereas 4 dogs showed no response. Examination of clinical samples obtained by surgical removal revealed that GR expression levels were significantly lower in GC-resistant MCT tissues than in GC-sensitive MCT tissues. Thus, these results strongly indicate that GR expression may contribute to GC sensitivity in canine MCT.     

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.     

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.     

PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

Immunohistochemistry with keratin,vimentin, desmin,and α‐smooth muscle actin monoclonal antibodies in canine mammary gland: Malignant mammary tumours

Summary

Ten malignant canine mammary gland tumours and five metastases from three of these tumours were studied immunohistochemically with monoclonal antibodies (MoAbs) directed against different human keratin types (K), α‐smooth muscle actin, vimentin, and desmin.

In all tumours the neoplastic epithelium was rather homogeneously labelled with the keratin MoAbs RCK 102 (K 5 and 8) and CAM 5.2 (K 8). The adenocarcinomas (n=5), the solid carcinomas (n=2), and the carcinosarcoma (n=1) showed heterogeneous labelling with the MoAbs specific for luminal cell antigens in the normal canine mammary gland, i.e., K 18, K 7 and K 19 MoAbs. These cells were also immunoreactive with K 4 and K 10 MoAbs. The spindle cell carcinomas (n=2), however, did not react with these MoAbs.

All tumours except one adenocarcinoma were characterized by the absence of immunoreactive labelling with the α‐smooth muscle actin MoAb. In the solid carcinomas this was associated with the absence of labelling with one or both basal cell specific keratin MoAbs, i.e., 8.7 (K 14 and 17) and RCK 107 (K 14), respectively. In contrast, the other malignant tumours showed marked labelling of neoplastic epithelium with these MoAbs. Another remarkable finding was the labelling of a limited to moderate number of neoplastic epithelial cells with the vimentin MoAb. The presence of such labelling patterns in canine mammary gland tumours may be indicative of malignancy. Metastatic tumour tissues had a labelling pattern largely similar to that of the primary tumour, although also loss of reactivity for some keratin MoAbs was seen.