Canine Mammary Tumor Cell Line Research Articles

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.

Cytotoxic effects of crotoxin from Crotalus durissus terrificus snake in canine mammary tumor cell lines.

Mammary gland tumors are the most prevalent neoplasm in intact female dogs, and they are good natural models to study comparative oncology. Most canine mammary malignancies, as in women, are commonly refractory to conventional therapies and demand continuous new therapeutic approaches. Crotalus durissus terrificus, also called rattlesnake, has more than 60 different proteins in its venom with multiple pharmaceutical uses, such as antitumor, antiviral, and antimicrobial action. Crotoxin, a potent β-neurotoxin formed by the junction of two subunits, a basic subunit (CB-PLA2) and an acidic subunit (crotapotin), has already been reported to have anticancer properties in different types of cancers. In this work, we describe the cytotoxic potential of crotoxin and its subunits compared to doxorubicin (drug of choice) in two canine mammary carcinoma cell lines. Crotoxin, CB-PLA2, crotalic venom, and doxorubicin decreased cell viability and the ability to migrate in a dose-dependent manner, and crotapotin did not present an antitumoral effect. For all compounds, the predominant cell death mechanism was apoptosis. In addition, crotoxin did not show toxicity in normal canine mammary gland cells. Therefore, this work showed that crotoxin and CB-PLA2 had cytotoxic activity, migration inhibition, and pro-apoptotic potential in canine mammary gland carcinoma cell lines, making their possible use in cancer research.

Matrine Targets BTF3 to Inhibit the Growth of Canine Mammary Tumor Cells.

The canine mammary tumor model is more suitable for studying human breast cancer, and the safety concentrations of matrine and the biotin-labeled matrine probe were determined in canine primary mammary epithelial cells, and then selected canine mammary tumor cell lines CHMm and CHMp were incubated with matrine, and cell viability was detected by CCK-8. The biotin-labeled matrine probe was used to pull-down the targets of matrine in canine mammary tumor cells, and the targets were screened in combination with activity-based protein profiling (ABPP) and Genecards database, and verified by qPCR and western blot. The results showed that the maximum non-cytotoxic concentrations of matrine and biotin-labeled matrine probe in canine primary mammary epithelial cells were 250 μg/mL and 500 μg/mL, respectively. Matrine and biotin-labeled matrine probe had a proliferation inhibitory effect time-dependently on CHMm and CHMp cells within a safe concentration range, and induced autophagy in cells. Then BTF3 targets were obtained by applying ABPP and Genecards screening. Cellular thermal shift assay (CETSA) findings indicated that matrine could increase the heat stability of BTF3 protein. Pull-down employing biotin-labeled matrine probe with CHMm and CHMp cell lysates revealed that BTF3 protein was detected in the biotin-labeled matrine probe group and that BTF3 protein was significantly decreased by the addition of matrine. The qPCR and western blot findings of CHMm and CHMp cells treated with matrine revealed that matrine decreased the expression of the BTF3 gene and protein with the extension of the action time, and the impact was more substantial at the protein level, respectively.

Antitumor Effect of Berberine Analogs in a Canine Mammary Tumor Cell Line and in Zebrafish Reporters via Wnt/β-Catenin and Hippo Pathways.

The heterogeneous nature of human breast cancer (HBC) can still lead to therapy inefficacy and high lethality, and new therapeutics as well as new spontaneous animal models are needed to benefit translational HBC research. Dogs are primarily investigated since they spontaneously develop tumors that share many features with human cancers. In recent years, different natural phytochemicals including berberine, a plant alkaloid, have been reported to have antiproliferative activity in vitro in human cancers and rodent animal models. In this study, we report the antiproliferative activity and mechanism of action of berberine, its active metabolite berberrubine, and eight analogs, on a canine mammary carcinoma cell line and in transgenic zebrafish models. We demonstrate both in vitro and in vivo the significant effects of specific analogs on cell viability via the induction of apoptosis, also identifying their role in inhibiting the Wnt/β-catenin pathway and activating the Hippo signals with a downstream reduction in CTGF expression. In particular, the berberine analogs NAX035 and NAX057 show the highest therapeutic efficacy, deserving further analyses to elucidate their mechanism of action more in detail, and in vivo studies on spontaneous neoplastic diseases are needed, aiming at improving veterinary treatments of cancer as well as translational cancer research.

Anticancer effect of superoxide dismutase on canine mammary gland tumour in vitro.

Reactive oxygen species (ROS) have been shown to promote tumour growth and metastasis in human cell lines. The superoxide anion (•O2 - ) is produced during ROS formation and is involved in tumour cell signalling. Superoxide dismutase (SOD) has been applied to canine mammary gland tumours to investigate its antitumour effects in vitro. Cell proliferation, cell cycle cell migration assays, reverse transcription-quantitative polymerase chain reaction, and western blot analysis were performed to determine the effects of SOD on canine mammary tumour cell line. SOD treatment resulted in anti-proliferative effects and mediated cell cycle arrest in the canine mammary gland tumour cell lines (CIPp and CIPm). It also downregulated the expression of N-cadherin and Vimentin. The results confirmed that SOD inhibits tumour cell proliferation and migration, thus supporting the potential applications of SOD as a chemotherapeutic agent for canine mammary gland tumours.

Anticancer effects of alpelisib on PIK3CA-mutated canine mammary tumor cell lines.

Canine mammary tumors (CMTs) are commonly observed in old and unspayed female dogs. Recently, dogs have been increasingly spaying at a young age to prevent mammary tumors. These CMTs require extensive local excision and exhibit a high probability of metastasis to the regional lymph nodes and lungs during malignancy. However, the molecular and biological mechanisms underlying CMT development have not been fully elucidated, and research in this area is limited. Therefore, in this study, we established new CMT cell lines by isolating cells from tumor tissues and investigated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), a target for human breast cancer. PIK3CA mutations were observed at a similar loci as in the human PIK3CA gene in half of all canine samples. Furthermore, we investigated whether alpelisib, a PIK3CA inhibitor approved by the U.S. Food and Drug Administration for human breast cancer treatment, along with fulvestrant, is effective for CMT treatment. Alpelisib exerted stronger anticancer effects on cell lines with PIK3CA mutations than on the wild-type cell lines. In conclusion, we established new CMT cell lines with PIK3CA mutations and confirmed the efficacy of alpelisib for CMT treatment in vitro.

Therapeutic trial of fluvastatin in a cell line xenograft model of canine mammary gland cancer.

The Hippo signalling pathway is involved in breast cancer and canine mammary tumour (CMT). This study sought to evaluate the efficacy of fluvastatin on the Hippo pathway and its main effectors, YAP and TAZ, in vivo in a murine CMT cell line xenograft model. On treatment day 1, mice were divided into four groups: vehicle, fluvastatin, doxorubicin or a combination therapy. Tumour volumes were monitored with callipers and tissues harvested on day 28th of treatment. Histopathological examination of tumour tissues and major organs was performed as well as tumour evaluation of necrosis, apoptosis, cellular proliferation, expression of YAP, TAZ and the mRNA levels of four of their target genes (CTGF, CYR61, ANKRD1 and RHAMM2). Results showed a statistically significant variation in tumour volumes only for the combination therapy and final tumour weight only for the doxorubicin group compared to control. There was no significant difference in tumour necrosis, expression of CC3, ki-67, YAP and TAZ measured by immunohistochemistry and in the mRNA levels of the target genes. Unexpectedly, lung metastases were found in the control group (9) and not in the fluvastatin treated group (7). In addition, mass spectrometry-based quantification of fluvastatin reveals concentrations comparable to levels reported to exert therapeutic effects. This study shows that fluvastatin tumours concentration reached therapeutic levels without having an effect on the hippo pathway or various tumour parameters. Interestingly, only the control group had lung metastases. This study is the first to explore the repurposing of statins for cancer treatment in veterinary medicine.

Macrophage induces anti-cancer drug resistance in canine mammary gland tumor spheroid

Tumor-associated macrophages (TAMs) play an important role in the tumor microenvironment by producing cytokines and growth factors. Furthermore, TAMs play multifunctional roles in tumor progression, immune regulation, metastasis, angiogenesis, and chemoresistance. Hypoxia in the tumor microenvironment induces tumor-supporting transformation of TAMs, which enhances tumor malignancy through developing anti-cancer resistance, for example. In this study, a hybrid spheroid model of canine mammary gland tumor (MGT) cell lines (CIPp and CIPm) and canine macrophages (DH82) was established. The effects of hypoxia induced by the spheroid culture system on the anti-cancer drug resistance of canine MGT cells were investigated. A hybrid spheroid was created using an ultralow-adhesion plate. The interactions between canine MGT cells and DH82 were investigated using a co-culture method. When co-cultured with DH82, cell viability and expression levels of tumor growth factors and multi-drug resistance genes were increased in canine MGT cells under doxorubicin. Additionally, doxorubicin-induced apoptosis and G2/M cell cycle arrest were attenuated in canine MGT cells co-cultured with DH82. In conclusion, the hybrid spheroid model established in this study reflects the hypoxic TME, allowing DH82 to induce anti-cancer drug resistance in canine MGT cells.

Mind the Curve: Dose-Response Fitting Biases the Synergy Scores across Software Used for Chemotherapy Combination Studies.

Drug combinations are increasingly studied in the field of anticancer agents. Mathematical models, such as Loewe, Bliss, and HSA, are used to interpret drug combinations, while informatics tools help cancer researchers identify the most effective combinations. However, the different algorithms each software uses lead to results that do not always correlate. This study compared the performance of Combenefit (Ver. 2.021) and SynergyFinder (Ver. 3.6) in analyzing drug synergy by studying combinations involving non-steroidal analgesics (celecoxib and indomethacin) and antitumor drugs (carboplatin, gemcitabine, and vinorelbine) on two canine mammary tumor cell lines. The drugs were characterized, their optimal concentration-response ranges were determined, and nine concentrations of each drug were used to make combination matrices. Viability data were analyzed under the HSA, Loewe, and Bliss models. Celecoxib-based combinations showed the most consistent synergistic effect among software and reference models. Combination heatmaps revealed that Combenefit gave stronger synergy signals, while SynergyFinder produced better concentration-response fitting. When the average values of the combination matrices were compared, some combinations shifted from synergistic to antagonistic due to differences in the curve fitting. We also used a simulated dataset to normalize each software's synergy scores, finding that Combenefit tends to increase the distance between synergistic and antagonistic combinations. We conclude that concentration-response data fitting biases the direction of the combination (synergistic or antagonistic). In contrast, the scoring from each software increases the differences among synergistic or antagonistic combinations in Combenefit when compared to SynergyFinder. We strongly recommend using multiple reference models and reporting complete data analysis for synergy claiming in combination studies.

Anticancer Effects of Alpelisib on PIK3CA-mutated Canine Mammary Tumor Cell Lines

Anticancer Effects of Alpelisib on PIK3CA-mutated Canine Mammary Tumor Cell Lines

Establishment and characterization of a multi-drug resistant cell line for canine mammary tumors.

Canine mammary tumors are the most common tumor disease of female dogs, and adjuvant chemotherapy often results in multi-drug resistance. Currently, the mechanisms underlying the development of tumor multi-drug resistance are unclear. The translation of research applications that can be used to effectively overcome tumor resistance is similarly hampered. Therefore, it is urgent to construct multi-drug resistance models of canine mammary tumors that can be used for research, to explore the mechanisms and means of overcoming resistance. In this study, the canine triple negative breast cancer cell line CMT-7364 was induced to develop multidrug resistance using doxorubicin by high-dose drug pulse method. The drug resistance and the expression of drug transport pumps of the cells was verified by CCK8 assay, immunoblotting, qPCR and immunofluorescence. Next, we used scratch assay and Transwell invasion assay to compare the migration and invasion abilities of the two cell lines and examined the expression of EMT-related proteins in both using immunoblotting. The differences of transcriptome between parental and drug-resistant cell lines were detected by RNA-seq sequencing. Finally, mouse xenograft models of drug-resistant and parental cell lines were constructed to evaluate the tumorigenic ability. After more than 50 generations of continuous passages stimulated by high-dose drug pulse method, the morphology of drug-resistant cell line CMT-7364/R tended to be mesenchymal-like and heterogeneous under light microscopy compared with the parental cell line CMT-7364/S, and developed resistance to doxorubicin and other commonly used chemotherapeutic drugs. In CMT-7364/R, BCRP was expressed at higher levels at both transcriptional and protein levels, while P-glycoprotein was not significantly different. Secondly, the migration and invasion ability of CMT-7364/R was significantly enhanced, with decreased expression of E-cadherin and increased expression of vimentin and mucin 1-N terminus. Finally, mouse xenograft models were constructed, while there was no significant difference in the volume of masses formed at 21 days. In summary, by using the canine mammary tumor cell line CMT-7364/S as the parental cell line, we successfully constructed a multidrug-resistant CMT-7364/R with high-dose drug pulse methods. Compared to its parental cell line, CMT-7364/R has decreased growth rate, overexpression of BCRP and increased migration and invasion ability due to EMT. The results of this study showed that CMT-7364/R might serve as a model for future studies on tumor drug resistance.

Canine Mammary Tumor Cell Lines Derived from Metastatic Foci Show Increased RAD51 Expression but Diminished Radioresistance via p21 Inhibition.

Due to the high incidence of mammary tumors in dogs, it is important to elucidate the pathogenesis of these tumors in veterinary medicine. Radiation therapy is often used to treat mammary tumors that target DNA lesions. RAD51 is a key molecule that repairs DNA damage via homologous recombination. We examined the relationship between RAD51 expression and radiosensitivity in mammary tumor cell lines. CHMp and CHMm from the same individual were selected based on the differences in RAD51 expression. The radiosensitivity of both cell lines was examined using MTT and scratch assays; CHMm, which has high RAD51 expression, showed higher sensitivity to radiation than CHMp. However, the nuclear focus of RAD51 during DNA repair was formed normally in CHMp, but not in most of CHMm. Since irradiation resulted in the suppression of cell cycle progression in CHMp, the expression of p21, a cell cycle regulatory factor, was detected in CHMp after 15 Gy irradiation but not in CHMm. These results indicate that functional expression is more important than the quantitative expression of RAD51 in canine mammary tumor cells in response to DNA damage.

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

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. 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. 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. 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.

Characterization and function of extracellular vesicles in a canine mammary tumour cell line: Ultracentrifugation versus size exclusion chromatography.

Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles involved in many biological processes such as tumour progression. For years, ultracentrifugation (UC) has been considered the gold standard for EV isolation but limited purity and integrity allowed the diffusion of alternative techniques. In this study, EVs were isolated from a canine mammary tumour cell line using UC and size exclusion chromatography (SEC) and analysed for size and concentration by nanoparticle tracking analysis (NTA) and for protein expression by western blot (WB). EV autocrine effect on cell proliferation, migration and invasiveness was then evaluated in vitro. In all samples, particles were in the EV size range (50-1000 nm), with a higher concentration in UC than in SEC samples (1011 and 1010 particles/ml respectively), and expressed EV markers (Alix, CD9). Functional assays did not show statistically significant difference among conditions, but EV treatment slightly increased cell proliferation and invasiveness and treatment with SEC-isolated EVs slightly enhanced cell migration compared to UC-isolated EVs. In conclusion, the main differences between the two isolation techniques are the quantity of the final EV-product and slight differences on EV functionality, which should be further explored to better highlight the real autocrine effect of tumoral EVs.

Defucosylated mouse‑dog chimeric anti‑HER2 monoclonal antibody exerts antitumor activities in mouse xenograft models of canine tumors.

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti-HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2-positive breast and gastric cancer. An anti-HER2 mAb, H2Mab-77 (mouse IgG1, kappa) was previously developed. In the present study, a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding-affinity [a dissociation constant (KD): 7.5×10−10 M, as determined by flow cytometric analysis] for dog HER2-overexpressed CHO-K1 (CHO/dHER2) cells. H77Bf highly exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for CHO/dHER2 cells by canine mononuclear cells and complement, respectively. Moreover, administration of H77Bf significantly suppressed the development of CHO/dHER2 ×enograft tumor in mice compared with the control dog IgG. H77Bf also possesses a high binding-affinity (KD: 7.2×10−10 M) for a canine mammary gland tumor cell line (SNP), and showed high ADCC and CDC activities for SNP cells. Intraperitoneal administration of H77Bf in mouse xenograft models of SNP significantly suppressed the development of SNP xenograft tumors compared with the control dog IgG. These results indicated that H77Bf exerts antitumor activities against dHER2-positive canine cancers, and could be valuable treatment regimen for canine cancers.

Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Exerted Antitumor Activities in Mouse Xenograft Models of Canine Mammary Gland Tumor.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody, clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed Chinese hamster ovary-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, the reactivity of 134-mG2a-f against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f highly exerted ADCC and CDC for SNP. The administration of 134-mG2a-f significantly suppressed the SNP xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine mammary gland tumors, and could be valuable as part of an antibody treatment regimen for them.

Establishment of a new cell line of canine inflammatory mammary cancer: IMC-118.

Canine inflammatory mammary cancer (IMC) has long been regarded as an attractive animal model for research into human inflammatory breast cancer (IBC), Although some canine mammary tumour cell lines corresponding to human mammary cancer cell lines have been established, there is still a need to supplement the canine mammary tumour cell bank. The goal of this study was to create a new type of IMC cell line. The primary tumour, IMC-118, was identified as IMC by pathology examination. Immunohistochemistry analysis revealed negative immunoreactivity to oestrogen receptor (ER), but positive immunoreactivity to progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2). Immunofluorescence (IF) analysis revealed that the IMC-118 cell line from this primary tumour was negative for ER but positive for PR and HER-2, and was also positive for epithelial and mesenchymal cell markers. This cell line was cultured stably for more than 50 passages and grew well after cryopreservation. In vivo, tumour masses and metastases in the lungs were discovered after inoculating the IMC-118 cells into the nude mice model. As a result, a novel canine IMC cell line, IMC-118, was effectively established, and could be employed as a promising model for immunotherapy and epithelial-mesenchymal transition mechanism of IMC research in both dogs and humans.

Antitumor Activities in Mouse Xenograft Models of Canine Mammary Gland Tumor by Defucosylated Mouse-Dog Chimeric Anti-Epidermal Growth Factor Receptor Antibody (E134Bf).

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf), and the reactivity of E134Bf against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry. Furthermore, E134Bf highly exerted ADCC and CDC for SNP cells. The administration of E134Bf with canine mononuclear cells significantly suppressed the SNP xenograft growth. These results suggest that E134Bf exerts antitumor effects against dEGFR-expressing canine mammary gland tumors and could be valuable as part of an antibody treatment regimen for them.

In vitro anticancer effects of alpelisib against PIK3CA‑mutated canine hemangiosarcoma cell lines.

Hemangiosarcoma (HSA) is a malignant neoplasm that occurs in humans and canines with a poor prognosis owing to metastatic spread, despite effective treatment. The frequency of spontaneous HSA development is higher in canines than in humans. Therefore, canine HSA is a useful model of intractable human disease, which requires early detection and an effective therapeutic strategy. A high frequency of the p110α phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations is detected in a comprehensive genome-wide analysis of canine cases of HSA. The present cloned the full-length cDNA of canine PIK3CA and identified a mutation in codon 1047 from canine cases of HSA and cell lines that were established from these. The enforced expression of the 1047th histidine residue (H1047)R or L mutants of canine PIK3CA in HeLa cells enhanced epidermal growth factor receptor (EGFR) signaling via Akt phosphorylation. PIK3CA mutant canine HSA cell lines exhibited the hyperphosphorylation of Akt upon EGF stimulation as well. Alpelisib, a molecular targeted drug against PIK3CA activating mutations, exerted a significant antitumor effect in canine PIK3CA-mutated HSA cell lines. By contrast, it had no significant effect on canine mammary gland tumor cell lines harboring PIK3CA mutations. On the whole, the findings of the present study suggest that alpelisib may be highly effective against PIK3CA mutations that occur frequently in canine HSA.

Effects of Tyrosine Kinase Inhibitor-masitinib Mesylate on Canine Mammary Tumour Cell Lines.

IntroductionMasitinib mesylate, a selective tyrosine kinase inhibitor of the c-KIT receptor, is used for the treatment of mast cell tumours in dogs. Masitinib has previously been investigated in various cancers; however, its potential anticancer effect in canine mammary tumours (CMTs) is unknown. In the present paper, we investigated the antiproliferative effect of masitinib in CMT cells and its possible mechanisms of action.Material and MethodsThe effect of masitinib on the proliferation of CMT-U27 and CMT-U309 cells was assessed by MTT assay and DNA fragmentation. Flow cytometric analysis was used to measure the effect of masitinib on apoptosis and the cell cycle. Additionally, vascular endothelial growth factor levels (VEGF) were measured, and the proliferation marker Ki-67 was visualised in immunocytochemical stainings in CMT cells.ResultsTreatment with masitinib inhibited the proliferation of CMT cells in a concentration-dependent manner. Maximal apoptotic activity and DNA fragmentation were observed at approximately IC50 of masitinib in both cell lines. In addition, cell cycle distribution was altered and VEGF levels and Ki-67 proliferation indices were decreased in masitinib-treated cells in comparison with control cells.ConclusionIn this study, masitinib suppressed cell proliferation concomitantly via induction of apoptosis and cell cycle arrest by decreasing VEGF levels and the Ki-67 proliferation index in CMT-U27 and CMT-U309 cells in vitro, suggesting its potential as a therapeutic tool in the clinical setting of mammary cancer treatment in dogs.