A New Benzo[6,7]oxepino[3,2-b] Pyridine Derivative Induces Apoptosis in Canine Mammary Cancer Cell Lines

Simple Summary Canine mammary cancer (CMC) is the most prevalent neoplasm in intact female dogs, with approximately 50% of these cases diagnosed as malignant. Various chemotherapeutic agents have been applied in CMC treatment, but these agents come with notable disadvantages and limited information on efficacy. The benzo[6,7]oxepino[3,2-b] pyridine (BZOP) derivative, 2-((2-methylpyridin-3-yl)oxy)benzaldehyde (MPOBA), exhibited potent anticancer activity against a human colorectal cancer cell line. However, the anticancer effects and molecular mechanisms of MPOBA on CMC have not been elucidated. This study investigated the effects of MPOBA on proliferation, migration, and apoptosis and investigated its potential mechanism in two CMC cell lines, REM134 canine mammary carcinoma and CMGT071020 canine mammary tubulopapillary carcinoma. Furthermore, an in vitro cytotoxicity test was also evaluated in the Madin-Darby canine kidney (MDCK) cell line. The results revealed that MPOBA significantly inhibited proliferation and migration and induced apoptosis in CMC cell lines. In addition, MPOBA showed lower cytotoxicity in MDCK cells. According to the gene expression analysis, the mRNA expression levels of TP53 tumor suppressor (TP53), Bcl-2 associated X (BAX), and B-cell lymphoma-2 (BCL-2) in both CMC cells were significantly altered in the treatment groups compared to the control. These results suggested that MPOBA may have induced apoptosis in both CMC cell lines. This effect may be mediated through the downregulation of BCL-2 and upregulation of BAX gene expressions. These findings indicated that MPOBA may serve as an emerging candidate agent for CMC treatment. However, mRNA expression levels may not be directly proportional to protein expression levels. Therefore, additional studies, including apoptosis-related protein analysis and apoptosis marker assays, are required to confirm the major apoptosis signaling pathway regulated by MPOBA.Abstract CMC is the most frequently diagnosed cancer and one of the leading causes of death in non-spayed female dogs. Exploring novel therapeutic agents is necessary to increase the survival rate of dogs with CMC. MPOBA is a BZOP derivative that has a significant anticancer effect in a human cell line. The main goal of this study was to investigate the anticancer properties of MPOBA against two CMC cell lines (REM134 and CMGT071020) using a 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, a wound healing assay, a transwell migration assay, an Annexin V-FITC apoptosis assay with a flow cytometry analysis, a mRNA expression analysis using quantitative real-time PCR (qRT-PCR), and an immunohistochemistry (IHC). According to the accumulated studies, MPOBA caused significant concentration- and time-dependent reductions in cell proliferation and cell migration and induced apoptosis in both CMC cell lines. In gene expression analysis, nine canine genes, including TP53, BCL-2, BAX, epidermal growth factor receptor (EGFR), snail transcription factor (SNAIL), snail-related zinc-finger transcription factor (SLUG), TWIST, E-cadherin, and N-cadherin, were investigated. The mRNA expression results revealed that MPOBA induced upregulation of TP53 and overexpression of the pro-apoptotic gene BAX, together with an inhibition of BCL-2. Moreover, MPOBA also suppressed the mRNA expression levels of SNAIL, EGFR, and N-cadherin and induced upregulation of E-cadherin, crucial genes related to the epithelial-to-mesenchymal transition (EMT). However, there was no significant difference in the IHC results of the expression patterns of vimentin (VT) and cytokeratin (CK) between MPOBA-treated and control CMC cells. In conclusion, the results of the present study suggested that MPOBA exhibited significant anticancer activity by inducing apoptosis in both CMCs via upregulation of TP53 and BAX and downregulation of BCL-2 relative mRNA expression. MPOBA may prove to be a potential candidate drug to be further investigated as a therapeutic agent for CMC.