Establishment and characterization of cisplatin-resistant cell lines from canine mammary gland tumors.

The development of cisplatin resistance is one of the major causes of mammary cancer treatment failure, and is associated with changes in Sox4 gene expression. To investigate the characteristic changes that occur in canine mammary gland tumor (CMGT) cells following the development of acquired cisplatin resistance, along with the relationship between these changes and the Sox4 gene. We constructed cisplatin-resistant cell line, CHMpCIS, from the cell line CHMp, which was isolated from the primary lesion of a malignant CMGT. The biological characteristics of these cells were examined by Western blot analysis, Transwell assays, and mammosphere formation assays. Compared to CHMp cells, CHMpCIS cells exhibited elevated cisplatin resistance, apoptotic escape ability, enhanced epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) features, in addition to over-activation of the Wnt/ß-catenin signaling pathway and increased Sox4 protein. In CMGT cases, CMGT tissues (CMGTT) expressed higher levels of Sox4 protein and mRNA compared to adjacent tissues (CAMGTT). We found that these changes were inhibited by silencing of Sox4 expression in CHMpCIS cells. Furthermore, activation of the Wnt/ß-catenin signaling pathway increased Sox4 expression levels through a positive feedback loop. These results suggested that CHMpCIS cells circumvented the damage caused by cisplatin through altering the expression of the Sox4 gene and activating the Wnt/ß-catenin pathway, thereby changing the cellular biological characteristics.

Serum Metabolomics and Ionomics Analysis of Hoof-Deformed Cows Based on LC-MS/MS and ICP-OES/MS.

In order to explore the metabolic and ionic changes of hoof-deformed cows, the serum samples of 10 healthy cows (group C) and 10 hoof-deformed cows (group T) were analyzed by LC-MS/MS and ICP-OES/MS. The pathway enrichment of differential metabolites was analyzed by screening and identifying differential metabolites and ions and using a bioinformatics method. The integration of metabolomics and ionics was analyzed with ggplot2 software in R language, and verified by MRM target metabolomics. The results showed that 127 metabolites were screened by metabolomics, of which 81 were up-regulated (p < 0.05) and 46 were down-regulated (p < 0.05). The results of ICP-OES/MS showed that 13 kinds of ions such as K, Li, and Pb in serum of dairy cows were up-regulated, while 18 kinds of ions such as Al, Cu and Sb were down-regulated. The integrated analysis of metabolomics and ionics found that potassium ions were positively correlated with L-tyrosine, L-proline, thiamine and L-valine. Sodium ions were positively correlated with L-valine and negatively correlated with α-D-glucose. The results of high-throughput target metabolomics showed that the contents of L-proline, L-phenylalanine and L-tryptophan in serum of dairy cows increased significantly, which was consistent with the results of non-target metabolomics. In a word, the metabolism and ion changes in dairy cows with hoof deformation were revealed by metabolomics and ionics.

Aspirin alleviates cisplatin-induced acute kidney injury through the AMPK-PGC-1α signaling pathway.

Cisplatin (CIS) is a widely used clinical chemotherapeutic agent for solid malignancies such as lung, testicular and ovarian cancers, but the development of nephrotoxicity has limited the use of this class of drugs. Some studies have shown that aspirin can reduce cisplatin-induced nephrotoxicity, but the mechanism of protection is not yet clear. By establishing a mouse model of cisplatin-induced acute kidney injury and a mouse model of aspirin combination, we observed a reduction in creatinine, blood urea nitrogen, and tissue damage, thus verifying that aspirin can alleviate cisplatin-induced acute kidney injury in mice. Aspirin was found to have a significant protective effect against cisplatin-induced acute kidney injury, as evidenced by the reduction in levels of ROS, NO, and MDA and the increase in T-AOC, CAT, SOD, and GSH. Furthermore, aspirin was observed to down-regulate the expression of pro-inflammatory factors TNF-α, NF-κB, IL-1ß, and IL-6 mRNA and proteins, increase the expression of BAX and Caspase3 as indicators of apoptosis, decrease the expression of Bcl-2, and improve the reduced expression of mtDNA, ATP content, ATPase activity and mitochondrial respiratory chain complex enzyme-related genes ND1, Atp5b, and SDHD. These findings suggest that the protective effects of aspirin are associated with its anti-inflammatory, antioxidant, anti-apoptotic properties, and its ability to maintain mitochondrial function, as demonstrated by the detection of AMPK-PGC-1α pathway-related genes. The results showed that the reduced expression of p-AMPK and mitochondrial production-related mRNA PGC-1α, NRF1, and TFAM in the kidney tissue of mice in the cisplatin group was alleviated by the effect of aspirin, indicating that aspirin could activate the p-AMPK, regulate mitochondrial production and alleviate cisplatin acute kidney injury through the AMPK-PGC-1α pathway. In summary, certain doses of aspirin protect the body from acute kidney injury by alleviating the cisplatin-induced inflammatory response oxidative stress, mitochondrial dysfunction, and apoptosis. Further studies have shown that the protective effect of aspirin is associated with AMPK-PGC-1α pathway activation.

MicroRNA-124 inhibits canine mammary carcinoma cell proliferation, migration and invasion by targeting CDH2.

Canine mammary carcinoma (CMC) is the most common malignant tumor and the second leading cause of cancer-related mortality of dogs worldwide. MicroRNA-124 (miR-124) is an important tumor suppressor implicated in various aspects of carcinogenesis. However, the roles and mechanisms of miR-124 in CMC development remains to be determined. We used the quantitative real-time polymerase chain reaction (qRT-PCR) assay to evaluate the expression levels of miR-124 in CMC tissues obtained from 20 CMC cases and CHMm and CHMp cells. CMC cell lines were transfected with lipfactormine™2000, and the cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Transwell assay were employed for evaluating the cell invasion and migration, while western blot assay was used to detect the protein changes in epithelial-mesenchymal transition (EMT) and CDH2 protein levels. The relationship between miR-124 and the 3'-untranslated region (3'-UTR) of CDH2 was predicted via bioinformatics analysis and verified by dual-luciferase reporter assay. The results revealed that miR-124 was reduced in CMC tissues and cell lines. Besides, observed high histological grade and tumor metastasis were associated with the down-regulation of miR-124 and up-regulation of CDH2. Functional analyses showed that in vitro transfection of CHMm and CHMp cells with miR-124 mimics inhibited their proliferation, migration, invasion, and EMT; however, transfection with miR-124 inhibitor resulted in the reversed effect. Besides, we showed that miR-124 directly suppressed the expression of CDH2, leading to the inhibition of CHMm cell proliferation and EMT. In conclusion, miR-124 regulates CMC tumor growth and EMT by targeting CDH2, maybe a potential therapeutic strategy against CMC.

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.

Aspirin inhibits proliferation and metastasis of canine mammary gland tumor cells through Wnt signaling axis.

BACKGROUND: Breast cancer is one of the most common, lethal types of neoplasia. The high mortality is largely due to rapid growth and distal metastasis. Epithelial-mesenchymal transition (EMT) and increased activity of matrix metalloproteinases (MMPs), which are related to tumor aggressiveness and progression, are known to be essential processes. Aspirin, a classic anti-inflammatory treatment, has been shown to inhibit the different types of cancer, though its research have yet to be elucidated and remain controversial. METHODS: We set out to elucidate the mechanism of Aspirin against the metastasis of canine mammary gland tumor cells (CMGTs), and provide theoretical support for the rational use of non-steroidal anti-inflammatory drugs. Firstly, we utilized CCK8, wound healing and transwell invasion assays to determine the effect of Aspirin on proliferation and migration of CMGTs. Next, we determined the effect of Aspirin on Wnt/ß-catenin signaling by cellular immunofluorescence and western blot analysis. Finally, Wnt/ß-catenin pathway activating agent (Wnt3a) and inhibitor (FH535) were utilized to further validate the relationship between Aspirin and Wnt/ß-catenin signaling. RESULTS: The results indicated that Aspirin inhibits proliferation and migration of CMGTs in a time-and concentration-dependent manner. Moreover, the inhibitory effect of Aspirin on CMGTs was reduced upon addition of a Wnt pathway activating agent (Wnt3a), while the effect following the addition of Wnt pathway inhibitor (FH535) remained the same as that of Aspirin. CONCLUSIONS: Our study demonstrates that Aspirin can inhibit the proliferation, migration and invasion by inhibiting Wnt/ß-catenin signaling, which suggest it can be a promising tool for deterring metastasis of CMGTs.

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.

Combined detection of CA15-3, CEA, and SF in serum and tissue of canine mammary gland tumor patients.

The purpose of this study is to evaluate the levels and clinical diagnosis value of CA15-3, CEA, and SF in canine mammary gland tumors (CMGTs). In this study, the levels of tissues/serum CA15-3, CEA, and SF in 178 CMGT patients or healthy dogs were determined by ELISA and qRT-PCR assay. CA15-3, CEA, and SF levels of the malignant tumor group were significantly higher than that of the benign tumor group and the healthy control group. In the malignant tumor group, CA15-3 held a sensitivity of 51.8%, a specificity of 93.9%, and an accuracy of 76.8%. The sensitivity, specificity, and accuracy of CEA were 44.6%, 84.1%, and 68.1% respectively. SF held a sensitivity of 62.5%, a specificity of 85.4%, and an accuracy of 76.1%. SF showed the highest sensitivity and CA15-3 showed the highest specificity. The sensitivity, specificity, and accuracy of the combined detection of the three biomarkers in malignant tumor groups were 80.4%, 78.0%, and 80.0%, respectively, therefore combined detection increased sensitivity and accuracy but decreased specificity. In conclusion, the combined detection of serum/tissue markers CA15-3, CEA, and SF may improve the detection sensitivity of CMGTs, providing reference value for clinical application.

LncRNA-42060 Regulates Tamoxifen Sensitivity and Tumor Development via Regulating the miR-204-5p/SOX4 Axis in Canine Mammary Gland Tumor Cells.

Tamoxifen is the drug of choice for endocrine therapy of breast cancer. Its clinical use is limited by the development of drug resistance. There is increasing evidence that long non-coding RNAs (lncRNAs) are associated with tumor drug resistance. Therefore, we established two TAM-resistant cell lines, CHMpTAM and CHMmTAM. The different expression levels of lncRNA and miRNA in CHMmTAM and CHMm were screened by RNA sequencing, and the lncRNA-miRNA interactions were analyzed. LncRNA ENSCAFG42060 (lnc-42060) was found to be significantly upregulated in drug-resistant cells and tumor tissues. Further functional validation revealed that the knockdown of lnc-42060 inhibited proliferation, migration, clone formation, restoration of TAM sensitivity, and reduction of stem cell formation in drug-resistant cells, whereas overexpression of lnc-4206 showed opposite results. Bioinformatics and dual-luciferase reporter gene assays confirmed that lnc-42060 could act as a sponge for miR-204-5p, further regulating SOX4 expression activity and thus influencing tumor cell progression. In conclusion, we screened lncRNAs and miRNAs associated with TAM resistance in canine mammary gland tumor cells for the first time. lnc-42060 served as a novel marker that may be used as an important biomarker for future diagnosis and treatment.