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.

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.

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 (ERβ), 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 ERβ agonist, and genistein, an isoflavonoid from soybeans known to have ERβ-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.

Methyl gallate is a phenolic compound mainly found in medicinal plants. It has been reported to its anticancer activity in various tumors. In this study, we aimed to demonstrate the antitumor effect of methyl gallate in the melanoma mouse model and B16F10 cells. Our results showed that methyl gallate decreased cell viability and induced apoptosis by increasing the expression of cleaved caspase3 in B16F10 cells and prevented cell migration and tube formation in human umbilical vein endothelial cells. In B16F10 cell-inoculated mice, methyl gallate not only decreased tumor volume by 30% but also significantly reduced tumor vessel density and pericyte coverage. Moreover, methyl gallate diminished by close to 50% the expression of cytokeratin and LYVE-1 in mouse right inguinal lymph nodes, indicating that methyl gallate could suppress metastasis. In conclusion, this study suggests that methyl gallate inhibits tumor development by inducing apoptosis and blocking tumor angiogenesis and metastasis and might be considered a therapeutic agent for melanoma.

Canine C-reactive protein (cCRP) is one of the major positive acute phase proteins in dogs and is commonly measured to detect and monitor systemic inflammation as well as the efficacy of treatment. Traditional methods for testing cCPR, including enzyme-linked immunosorbent assay (ELISA), have some drawbacks, such as a long time for diagnosis and the requirement of well-equipped laboratories. Therefore, there is a need for a rapid and precise diagnostic test for cCRP at point-of-care. This study assessed the accuracy, precision, and validated clinical effectiveness of a diagnostic test based on fluorescent lateral flow immunoassay to detect cCRP. For the standard cCRP concentration ranging from 0 to 200 μg/mL, the cCRP diagnostic test showed strong linearity with R² of 0.9977 (p < 0.001), and both inter- and intra-assay CVs were <14%. The limit of detection and limit of quantitation were found to be 4.0 μg/mL and 5.0 μg/mL, respectively. The cCRP serum concentration was evaluated in 21 client-owned dogs and the results were compared to a previously validated ELISA. The Pearson Correlation Coefficient between the diagnostic test kit and ELISA was 0.942 [95% confidence interval: 0.859 to 0.976, p < 0.001], and the Bland–Altman plot indicated a bias of 26.82% [95% limits of agreement: −56.03 to 109.67], indicating a significant correlation and the agreement between the data from the cCRP diagnostic test and ELISA. In conclusion, the fluorescent immunoassay based diagnostic test is a suitable option for rapidly and precisely detecting cCRP in dogs, providing a convenient alternative to traditional methods for diagnosing acute inflammation.