Methyl Gallate Suppresses Canine Mammary Gland Tumors by Inducing Apoptosis and Anti-angiogenesis.

BACKGROUND/AIM: Methyl gallate (MG), a plant phenolic compound, has known anticancer properties. However, its effects on canine mammary gland tumors (CMTs) are unclear. This study evaluated the impact of MG on cell viability, migration, and apoptosis in two CMT cell lines. MATERIALS AND METHODS: CMT-U27 and CF41.mg cells were used. In vitro experiments included MTT and scratch assays, Annexin-V/propidium iodide double staining, immunocytochemistry, and western blot analyses. An in vivo CMT xenograft mouse model was also used to observe the effects of MG on tumor growth and vasculature. Immunohistochemistry was performed to analyze vessel density and apoptosis in tumor tissues. Cell migration and tube formation assays with canine aortic endothelial cells assessed the anti-angiogenic effects of MG. RESULTS: Data showed a significant decrease in cell viability and migration in both CMT cell lines after 24 h exposure to various MG concentrations. MG treatment induced dose-dependent apoptotic cell death and elevated cleaved caspase-3 expression. In vivo experiments confirmed tumor growth suppression 21 days post-treatment with 40 mg/kg MG. Tumor tissues displayed increased cleaved caspase-3 and reduced vessel density. MG also inhibited cell migration and disrupted tube formation in canine endothelial cells. CONCLUSION: MG has potential as an anticancer drug for CMTs by promoting apoptotic cell death and reducing angiogenesis, highlighting its therapeutic promise.

Establishment of canine mammary gland tumor cell lines harboring PI3K/Akt activation as a therapeutic target.

Canine mammary gland tumors (MGT) have a poor prognosis in intact female canines, posing a clinical challenge. This study aimed to establish novel canine mammary cancer cell lines from primary tumors and characterize their cellular and molecular features to find potential therapeutic drugs. The MGT cell lines demonstrated rapid cell proliferation and colony formation in an anchorage-independent manner. Vimentin and α-SMA levels were significantly elevated in MGT cell lines compared to normal canine kidney (MDCK) cells, while CDH1 expression was either significantly lower or not detected at all, based on quantitative real-time PCR (qRT-PCR) analysis. Functional annotation and enrichment analysis revealed that epithelial-mesenchymal transition (EMT) phenotypes and tumor-associated pathways, particularly the PI3K/Akt signaling pathway, were upregulated in MGT cells. BYL719 (Alpelisib), a PI3K inhibitor, was also examined for cytotoxicity on the MGT cell lines. The results show that BYL719 can significantly inhibit the proliferation of MGT cell lines in vitro. Overall, our findings suggest that the MGT cell lines may be valuable for future studies on the development, progression, metastasis, and management of tumors.

Synergistic Anti-Cancer Effects of ERB-041 and Genistein through Estrogen Receptor Suppression-Mediated PI3K/AKT Pathway Downregulation in Canine Mammary Gland Tumor Cells.

Canine-mammary-gland tumors (CMTs) are prevalent in female dogs, with approximately 50% of them being malignant and often presenting as inoperable owing to their size or metastasis. Owing to poor outcomes, effective alternatives to conventional chemotherapy for humans are necessary. Two estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERß), which act in opposition to each other, are involved, and CMT growth involves ERα through the phosphoinositide 3-kinases (PI3K)/AKT pathway. In this study, we aimed to identify the synergistic anti-cancer effects of ERB-041, an ERß agonist, and genistein, an isoflavonoid from soybeans known to have ERß-specific pseudo-estrogenic actions, on CMT-U27 and CF41.Mg CMT cell lines. ERB-041 and genistein synergistically inhibited cell proliferation and increased the number of annexin V-positive cells in both cell lines. Furthermore, we observed a synergistic increase in the Bax/Bcl-2 ratio and cleaved caspase-3 expression. Additionally, cell-cycle arrest occurred through the synergistic regulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). We also found a synergistic decrease in the expression of ERα, and the expression of proteins involved in the PI3K/AKT pathway, including p-PI3K, phosphatase and tensin homolog (PTEN), AKT, and mechanistic target of rapamycin (mTOR). In conclusion, ERB-041 and genistein exhibited a synergistic anticancer effect on CMTs, suggesting that cotreatment with ERB-041 and genistein is a promising treatment for CMTs.

YRNA and tRNA fragments can differentiate benign from malignant canine mammary gland tumors.

Mammary gland tumors (MGT) are the most common tumors in sexually intact female dogs. The functional regulation of miRNAs, a type of noncoding RNAs (ncRNAs), in canine MGT has been extensively investigated. However, the expression of other ncRNAs, such as YRNAs and transfer RNA-derived fragments (tRFs) in canine MGT is unknown. We investigated ncRNAs other than miRNAs from our small RNA project (PRJNA716131) in different canine MGT histologic subtypes. This study included benign tumors (benign mixed tumor, complex adenoma) and malignant tumors (carcinoma in benign tumor and carcinoma with metastasis) samples. Aberrantly expressed ncRNAs were examined by comparisons among MGT subtypes. The relative expression trends were validated in canine MGT tissues, plasma, extracellular vesicles, and MGT cell lines using quantitative reverse transcription PCR. Three aberrantly expressed ncRNAs were identified by comparisons among MGT subtypes. YRNA and tRNA-Gly-GCC distinguished benign mixed tumor from other MGT histologic subtypes, while tRNA-Val differentiated complex adenoma, carcinoma in benign tumors, and carcinoma with metastasis. The ROC curve of the three ncRNAs showed they might be potential biomarkers to discriminate malignant from benign MGT. YRNA and tRFs expression levels were decreased in metastatic compared with primary canine MGT cell lines. To the best of our knowledge, this is the first investigation of YRNA and tRFs in canine MGT. The three identified ncRNAs may be biomarkers for differentiating MGT histologic subtypes. Suggested Reviewers: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporatio.

Anti-cancer effect of palmatine through inhibition of the PI3K/AKT pathway in canine mammary gland tumor CMT-U27 cells.

Canine mammary gland tumors (CMTs) are the most common and lethal cancers in female dogs. Dysregulated phosphoinositide 3-kinases (PI3K)/AKT pathway reportedly was involved in the growth and metastasis of CMTs. However, there are few studies on therapeutic strategies for targeting the PI3K pathway in CMTs. In this study, we aimed to determine whether palmatine, a natural isoquinoline alkaloid with anti-cancer properties, could inhibit the growth of CMTs and whether the inhibitory effect was mediated through the PI3K/AKT pathway. Our in vitro experiments on CMT-U27, a CMT cell line, showed that palmatine reduced cell proliferation and induced cell death. Western blotting results revealed that palmatine decreased the protein expression of PI3K, PTEN, AKT, and mechanistic target of rapamycin in the PI3K/AKT pathway, which was supported by the results of immunocytochemistry. Additionally, palmatine suppressed the migration and tube formation of canine aortic endothelial cells as well as the migration of CMT U27 cells. Our in vivo results showed that palmatine inhibited tumor growth in a CMT-U27 mouse xenograft model. We observed a decreased expression of proteins in the PI3K/AKT pathway in tumor tissues, similar to the in vitro results. Furthermore, palmatine significantly disrupted the tumor vasculature and inhibited metastasis to adjacent lymph nodes. In conclusion, our findings demonstrate that palmatine exerts anti-cancer effects against CMTs by inhibiting PI3K/AKT signaling pathway, suggesting that palmatine has potential as a canine-specific PI3K inhibitor for the treatment of CMTs.

Antitumor Effects of Esculetin, a Natural Coumarin Derivative, against Canine Mammary Gland Tumor Cells by Inducing Cell Cycle Arrest and Apoptosis.

Mammary gland tumors are the most common neoplasms in female dogs, of which 50% are malignant. Esculetin, a coumarin derivative, reportedly induces death in different types of cancer cells. In this study, we explore the anticancer effects of esculetin against CMT-U27 and CF41.mg canine mammary gland tumor cells. Esculetin significantly inhibited the viability and migration of both CMT-U27 and CF41.mg cells in a dose- and time-dependent manner. Flow cytometric analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay revealed increased numbers of annexin-V-positive cells and DNA fragmentation. Furthermore, a cell cycle analysis demonstrated that esculetin blocked the cell progression at the G0/G1 phase and the S phase in CMT-U27 and CF41.mg cells. These results were supported by a Western blot analysis, which revealed upregulated protein expression of cleaved caspase-3, a marker of apoptosis, and downregulated cyclin-dependent kinase 4 and cyclin D1 protein, the cell cycle regulators. In conclusion, this novel study proves that esculetin exerts in vitro antitumor effects by inducing apoptosis and cell cycle arrest in canine mammary gland tumors.

Hypoxia Increases the Proliferative and Metastatic Ability of Canine Mammary Tumor Cells via Up-regulation of TSG-6.

BACKGROUND/AIM: HIF1α-induced hypoxia is a major characteristic of solid tumors that plays an important role in cancer growth, metastasis, and chronic inflammation. Tumor necrosis factor (TNF) stimulated gene (TSG)-6 is a strong regulator of anti-inflammatory pathways, but its role in cancer cells remains unclear. We hypothesized that hypoxia up-regulates TSG-6, thereby increasing the metastatic and growth potential of cancer cells. MATERIALS AND METHODS: Primary and metastatic canine mammary gland tumor (MGT) cell lines (CIPp and CIPm), were transfected with TSG-6 specific siRNA and treated with cobalt chloride (CoCl2) for 48 h to chemically induce a hypoxia environment. The expression of hypoxia-inducible factor-1-alpha (HIF1α) was evaluated by RT-qPCR and western blot analysis. The metastatic ability of cancer cells and cell cycle distribution were assessed with extracellular matrix invasion assays and flow cytometry. RESULTS: HIF1α up-regulation, induced by CoCl2, was significantly inhibited in the TSG-6-knockdown group in both canine MGT cell lines. The change in the expression levels of HIF1α corresponded to the change of invading cells in the TSG-6-knockdown group. TSG-6-knockdown in the hypoxia group showed decreased proliferation, associated with G2/M phase arrest. CONCLUSION: HIF1α expression in hypoxic condition is regulated by TSG-6 expression in canine MGT. TSG-6-knockdown causes down-regulation of HIF1α, thereby reducing the metastatic and proliferative abilities of cancer cells. TSG-6 in canine MGT has a potential as a therapeutic target in anti-cancer therapy.

Establishment and transcriptome characterization of tamoxifen-resistant canine mammary gland tumor cells.

Tamoxifen (TAM) currently is still the drug of choice for endocrine therapy in patients with estrogen receptor positive breast cancer. However, the development of drug resistance not only limits the drug utilization, but also greatly reduces the survival of patients. At the same time, TAM is poorly understood in canine mammary gland tumors. Therefore, it is crucial to find effective methods to reverse drug resistance and prevent the development of drug resistance so as to improve the efficacy of endocrine therapy for breast cancer. Firstly, we successfully established two TAM-resistant canine mammary gland tumor cells lines including TAMp,TAMm by drug concentration gradient plus drug maintenance, and then we confirmed that the resistant cells have stronger proliferation, migration, invasion and cloning ability by CCK8, Wound healing assay, Transwell invasion assay and Clone formation assay. Second, we performed sequencing analysis of TAMm and CHMm and detected a large number of different expression genes, including reported and novel drug-resistant genes, and genes involved in complex biological processes. Finally, we explored the role of the classical Wnt signaling pathway in drug-resistant cells, and immunofluorescence and western blot results showed increased expression of Wnt pathway related genes ß-catenin and P-GSK3ß in drug-resistant cells, indicating abnormal activation of the classical Wnt/ß-catenin pathway This study successfully established two TamR cell lines and assayed its resistance generation in many aspects, which provides a good experimental model and theoretical support for a more comprehensive understanding of the endocrine drug resistance mechanism.

Metformin inhibits the proliferation of canine mammary gland tumor cells through the AMPK/AKT/mTOR signaling pathway in vitro.

As an anti-diabetic drug, metformin has been demonstrated to exhibit antitumor effects. However, the mechanisms involved in decreasing tumor formation, including canine mammary gland tumors (CMGTs), are not well elucidated. The aim of the present study was to evaluate the ability of metformin to induce apoptosis and cell cycle arrest in CMGT cells, as well as identifying the pathways underlying these effects. Cell viability was assessed by Cell Counting Kit-8 analysis following treating with metformin. Subsequently, apoptosis and cell cycle progression were assessed by flow cytometry, and the expression of associated proteins was examined. Expression levels of classical AMP-activated protein kinase (AMPK), protein kinase B (AKT), mechanistic target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were then investigated using western blot analysis. Metformin inhibited the proliferation of CHMm cells in a concentration-dependent manner. Specifically, metformin induced cell cycle arrest in the G0/G1 phases, accompanied by increased expression of p21 and p27, and decreased expression of cyclin D1 and cyclin-dependent kinase 4. Marked levels of apoptosis were observed in CHMm cells alongside the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. Also, the level of Bcl-2 was decreased, and that of Bax was increased. The expression of associated signaling molecules revealed that metformin markedly increased the phosphorylation of AMPK in CHMm cells, and decreased the levels of phosphorylated (p-)AKT, p-mTOR and p-4E-BP1, while Compound C reversed these changes. These findings demonstrated that metformin may be a potential therapeutic agent for CMGTs, acting via the AMPK/AKT/mTOR signaling pathway.

A Preliminary Study of the Cross-Reactivity of Canine MAGE-A with Hominid Monoclonal Antibody 6C1 in Canine Mammary Gland Tumors: An Attractive Target for Cancer Diagnostic, Prognostic and Immunotherapeutic Development in Dogs.

Melanoma-associated antigen-A (MAGE-A), a family of cancer/testis antigens, has been recognized as a potential target molecule for cancer immunotherapy. However, there has been very little information available with regard to this antigen in dogs. This study aimed to investigate the expression of MAGE-A in canine mammary gland tumors (CMTs) using immunohistochemistry and immunoblotting with human monoclonal MAGE-A antibody 6C1. The present study has provided evidence of cross-reactivity of the canine MAGE-A expression with the human MAGE-A antibody in CMTs. The MAGE-A antigens were expressed in moderate- and high-grade malignant CMTs (22.22%, 2/9), but no expression was observed in benign CMTs. The immunohistochemical staining of canine MAGE antigen in CMT cells showed nuclear and nuclear-cytoplasmic expression patterns that may be involved with the mitotic cell division of tumor cells. Molecular weights of the canine MAGE-A antigen presented in this study were approximately 42-62 kDa, which were close to those of other previous studies involving humans and dogs. The findings on this protein in CMTs could supply valuable oncological knowledge for the development of novel diagnostic, prognostic and immunotherapeutic tumor markers in veterinary medicine.

Microsatellite Instability and MMR Genes Abnormalities in Canine Mammary Gland Tumors.

Early diagnosis of mammary gland tumors is a challenging task in animals, especially in unspayed dogs. Hence, this study investigated the role of microsatellite instability (MSI), MMR gene mRNA transcript levels and SNPs of MMR genes in canine mammary gland tumors (CMT). A total of 77 microsatellite (MS) markers in 23 primary CMT were selected from four breeds of dogs. The results revealed that 11 out of 77 MS markers were unstable and showed MSI in all the tumors (at least at one locus), while the other markers were stable. Compared to the other markers, the ABC9TETRA, MEPIA, 9A5, SCNA11 and FJL25 markers showed higher frequencies of instability. All CMT demonstrated MSI, with eight tumors presenting MSI-H. The RT-qPCR results revealed significant upregulation of the mRNA levels of cMSH3, cMLH1, and cPMSI, but downregulation of cMSH2 compared to the levels in the control group. Moreover, single nucleotide polymorphisms (SNPs) were observed in the cMSH2 gene in four exons, i.e., 2, 6, 15, and 16. In conclusion, MSI, overexpression of MMR genes and SNPs in the MMR gene are associated with CMT and could be served as diagnostic biomarkers for CMT in the future.

Association between DLA-DRB1.2 allelic diversity and development of mammary gland tumors in dogs.

BACKGROUND: The major histocompatibility complex (MHC) is the best-characterized genetic region related to resistance/susceptibility to a wide range of infectious and immune-mediated diseases. Evidences suggest that MHC class II genes may play an important role in developing different types of tumors including breast cancer. Canine mammary gland tumors (CMTs) are the most common neoplasms in female dogs. In the current study, the association of canine MHC class II DLA-DRB1.2 genotypes with development of mammary gland tumor profiles in dogs was investigated. DLA-DRB1.2 allelic diversity was determined in 40 dogs (18 CMT cases and 22 controls) using HRM technique and DNA sequencing. Association of the DLA-DRB1.2 genotypes with CMT profiles was expressed as odds ratio (OR). RESULTS: Based on the histopathological typing of tumors, CMT cases were categorized into 4 groups: simple carcinoma, complex carcinoma, carcinoma arising in a benign tumor and special types of carcinoma. A total of eight HRM profiles (A to H) were identified in dogs sampled. The association study revealed a significant correlation between DLA-DRB1.2 genotypes with different CMT profiles. The E genotype was significantly associated with increased risk of carcinoma arising in a benign tumor, and the B genotype represented a positive correlation with complex carcinoma. Significant association was also observed between the heterozygosity of DLA-DRB1.2 genotypes and decreased risk of developing tumor in dogs. CONCLUSIONS: These results provide additional support for the association between DLA-DRB1 genes and development of mammary gland tumors in dogs and could potentially be used for early diagnosis of neoplasia and identifying susceptible dogs.

18F-FDG-PET/CT in Canine Mammary Gland Tumors.

Medical imaging techniques play a central role in clinical oncology, helping to obtain important information about the extent of disease, and plan treatment. Advanced imaging modalities such as Positron Emission Tomography-Computed Tomography (PET/CT), may help in the whole-body staging in a single procedure, although the lesions should be carefully interpreted. PET/CT is becoming commonly used in canine cancer patients, but there is still limited information available on specific tumors such as mammary cancer. We evaluated the utility of fluorine-18 fluorodeoxyglucose (18F-FDG)-PET/CT to detect malignant lesions in eight female dogs with naturally occurring mammary tumors. A whole-body scan was performed prior to surgery, and mammary and non-mammary lesions detected either on PET/CT or during pre-surgical physical exam were resected when possible and submitted for histopathological examination. Multiple mammary lesions involving different mammary glands were detected in 5/8 dogs, for a total of 23 lesions; there were 11 non-mammary-located lesions in 6/8 dogs, three of these were lung or lymph node metastasis. A total of 34 lesions were analyzed: 22 malignant (19 mammary tumors and three metastatic lesions), and 12 benign (four mammary lesions and eight of non-mammary tissues). Glucose uptake by maximum standardized uptake value (SUVmax) was analyzed and correlated with tumor size, and benign vs. malignant pathology. We found that the minimum tumor size needed to distinguish malignant lesions according to the SUVmax was 1.5 cm; benign and malignant lesions <1.5 cm did not differ in glucose uptake (mean SUVmax = 1.1). In addition, a SUVmax value >2 was 100% sensitive for malignancy. Combining these data, lesions >1.5 cm with a SUVmax >2 had a positive predictive value of 100%. Finally, we did not find an association between SUVmax and histologic subtype or grade, which may be present in a larger sample. Thus, 18F-FDG PET/CT is useful for distinguishing malignant from benign lesion but further imaging of dogs with diverse tumors, should establish characteristic SUV value cutoffs for detecting primary and metastatic disease, and distinguishing them from benign lesions.