Canine Mammary Carcinoma Cell Lines Research Articles

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.

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.

Newcastle disease virus LaSota strain induces apoptosis and activates the TNFα/NF-κB pathway in canine mammary carcinoma cells.

Spontaneous canine mammary carcinomas (CMCs) have been widely considered a good research model for human breast cancers, which brings much attention to CMCs. In recent years, the oncolytic effect of Newcastle disease virus (NDV) on cancer cells has been widely studied, but its effect on CMCs is still unclear. This study aims to investigate the oncolytic effect of NDV LaSota strain on canine mammary carcinoma cell line (CMT-U27) in vivo and in vitro. The in vitro cytotoxicity and immunocytochemistry experiments showed that NDV selectively replicated in CMT-U27 cells, and inhibited cell proliferation and migration but not in MDCK cells. KEGG analysis of transcriptome sequencing indicated the importance of the TNFα and NF-κB signalling pathways in the anti-tumour effect of NDV. Subsequently, the significantly increased expression of TNFα, p65, phospho-p65, caspase-8, caspase-3 and cleaved-PARP proteins in the NDV group suggested that NDV induced CMT-U27 cells apoptosis by activating the caspase-8/caspase-3 pathway and the TNFα/NF-κB signalling pathway. Nude mice tumour-bearing experiments showed that NDV could significantly decrease the growth rate of CMC in vivo. In conclusion, our study demonstrates the effective oncolytic effects of NDV on CMT-U27 cells in vivo and in vitro, and suggests NDV as a promising candidate for oncolytic therapy.

Pretreatment with tadalafil attenuates cardiotoxicity induced by combretastatin A4 disodium phosphate in rats.

Combretastatin A4 disodium phosphate (CA4DP) is a prodrug of combretastatin A4 (CA4), a microtubule-disassembling agent that exhibits antitumor effects by inhibiting tumor cell proliferation and inducing morphological changes and apoptosis in vascular endothelial cells in tumors. However, cardiotoxicity induced by ischemia and hypertension is a severe adverse event. In this study, we focused on the fact that phosphodiesterase (PDE) 5 inhibitors dilate the heart and peripheral blood vessels and aimed to investigate whether co-administration of tadalafil, a PDE5 inhibitor, can attenuate cardiotoxicity without altering the antitumor effect of CA4DP. To investigate cardiotoxicity, CA4DP and/or tadalafil were administered to rats, and blood pressure, echocardiography, histopathology, and cGMP concentration in the myocardium were examined. Administration of CA4DP increased systolic blood pressure, decreased cardiac function, lowered cGMP levels in the myocardium, and led to necrosis of myocardial cells. Co-administration of tadalafil attenuated these CA4DP-induced changes. To investigate the antitumor effect, canine mammary carcinoma cell lines (CHMp-13a) and human umbilical vein endothelial cells were cultured with CA4 and/or tadalafil, and cell proliferation and endothelial vascular tube disruption were examined. CHMp-13a cells were transplanted into nude mice and treated with CA4DP and/or tadalafil. CA4-induced inhibition of cell proliferation and disruption of the endothelial vascular tube were not affected by co-treatment with tadalafil, and the antitumor effects of CA4DP in xenograft mice were not reduced by co-administration of tadalafil. These results revealed that myocardial damage induced by CA4DP was attenuated by co-administration of tadalafil while maintaining antitumor efficacy.

Molecular Characterization of CF33 Canine Cell Line and Evaluation of Its Ability to Respond against Infective Stressors in Sight of Anticancer Approaches.

Simple SummaryCanine mammary cancer is very common and has many similarities with human breast cancer. Risk factors, physiological and pathological behaviors, and the clinical course in dogs are very similar to humans. Several molecular similarities have also been reported, such as overexpression of EGF, proliferation markers, metalloproteinase and cyclooxygenase, TP53 mutations, and CXCR4/SDF1 axis activation. These common characteristics make these breast tumors resistant to conventional therapies. It is therefore necessary to study therapeutic alternatives. Cell lines could be helpful to test in vitro immunomodulant anti-cancer therapies, allowing a reduction of laboratory animals’ involvement in the preliminary tests and achieving results in a shorter time. Although the canine mammary carcinoma cell line CF33 has been widely used in many studies on dog mammary cancer, characterization of its gene expression profile and of the influence of infective stressors of this cell line is poor. Our study shows the interaction of CF33 and Salmonella Typhimurium (ST) as an infective stressor, indicating that these cells may represent an in vitro model for assessing novel therapeutic approaches using bacteria.Spontaneous mammary tumors are the most frequent neoplasms in bitches and show similarities with human breast cancer in risk factors, clinical course, and histopathology. The poor prognosis of some cancer subtypes, both in human and dog, demands more effective therapeutic approaches. A possible strategy is the new anticancer therapy based on immune response modulation through bacteria or their derivatives on canine mammary carcinoma cell lines. The aim of the present study was to analyze the CF33 cell line in terms of basal expression of immune innate genes, CXCR4 expression, and interaction with infectious stressors. Our results highlight that CF33 maintains gene expression parameters typical of mammary cancer, and provides the basal gene expression of CF33, which is characterized by overexpression of CXCR4, CD44, RAD51, LY96, and a non-continuous expression of TP53 and PTEN. No mutations appeared in the CXCR4 gene until the 58th passage; this may represent important information for studying the CXCR4 pathway as a therapeutic target. Moreover, the CF33 cell line was shown to be able to interact with Salmonella Typhimurium (ST) (an infective stressor), indicating that these cells could be used as an in vitro model for developing innovative therapeutic approaches involving bacteria.

Establishment of stable expression of firefly luciferase and EGFP in a canine inflammatory mammary carcinoma cell line and tumor-bearing model in nude mice.

Canine inflammatory mammary carcinoma (CIMC) is a type of canine malignant mammary tumor with a poor prognosis and high mortality. We transduced firefly luciferase and enhanced green fluorescent protein (EGFP) into CHMp, a CIMC cell line, and established CHMp-Luc-EGFP cells. We investigated the characteristics of this cell line in vitro and in vivo. CHMp-Luc-EGFP was passaged continuously 75 times, with stable expression of luciferase and EGFP. Compared with the wild-type, CHMp-Luc-EGFP had similar proliferation, metastasis, histopathology characteristics, and expression of E-cadherin, N-cadherin, and Ki-67. A tumor-bearing model was established by implantation of CHMp-Luc-EGFP cells, and the dynamic changes of tumors were visualized and quantified using the IVIS imaging system. In summary, the cell line we established could reflect the biological characteristics of CHMp cells, visualize the tumor progression in vivo, and provide a powerful tool for the study of CIMC.

Establishment of a New Canine Inflammatory Mammary Carcinoma Cell Line and Analysis of its Cystine-glutamate Transporter Subunit Expression.

IntroductionInflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan.Material and MethodsThe patient was a female toy poodle presenting with an inflamed mammary gland, which was diagnosed as IMC. The cell line was established from a tissue biopsy. Surface antigen marker (CD24 and CD44) expression was determined. Cystine/glutamate antiporter (xCT) expression was determined by Western blotting, flow cytometry and fluorescence immunostaining, and sulfasalazine was administered to ascertain if it suppressed xCT expression. Stem cell marker (Nanog, Sox2, Myc and Klf4) expression and aldehyde dehydrogenase (ALDH) activity were also investigated.ResultsThe cultured cells showed xCT, and its suppression showed downregulation of stem cell markers and ALDH activity. Stable cell proliferation was verified.ConclusionA new canine IMC-derived cell line was established. In the future, we aim to study the effect of xCT on the maintenance of cancer stem cell properties in canine tumours, and propose a new therapeutic method for the treatment of canine IMC by targeting xCT.

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.

Exosomes Derived from Radioresistant Breast Cancer Cells Promote Therapeutic Resistance in Naïve Recipient Cells.

Radiation resistance is a significant challenge in the treatment of breast cancer in humans. Human breast cancer is commonly treated with surgery and adjuvant chemotherapy/radiotherapy, but recurrence and metastasis upon the development of therapy resistance results in treatment failure. Exosomes are extracellular vesicles secreted by most cell types and contain biologically active cargo that, when transferred to recipient cells, can influence the cells’ genome and proteome. We propose that exosomes secreted by radioresistant (RR) cells may be able to disseminate the RR phenotype throughout the tumour. Here, we isolated exosomes from the human breast cancer cell line, MDA-MB-231, and the canine mammary carcinoma cell line, REM134, and their RR counterparts to investigate the effects of exosomes derived from RR cells on non-RR recipient cells. Canine mammary cancer cells lines have previously been shown to be excellent translational models of human breast cancer. This is consistent with our current data showing that exosomes derived from RR cells can increase cell viability and colony formation in naïve recipient cells and increase chemotherapy and radiotherapy resistance, in both species. These results are consistent in cancer stem cell and non-cancer stem cell populations. Significantly, exosomes derived from RR cells increased the tumoursphere-forming ability of recipient cells compared to exosomes derived from non-RR cells. Our results show that exosomes are potential mediators of radiation resistance that could be therapeutically targeted.

Canine Natural Killer Cell-Derived Exosomes Exhibit Antitumor Activity in a Mouse Model of Canine Mammary Tumor.

Natural killer (NK) cells are key immune cells engaged in fighting infection and malignant transformation. In this study, we found that canine NK cell-derived exosomes (NK-exosomes) separated from activated cytotoxic NK cell supernatants express specific markers including CD63, CD81, Alix, HSP70, TSG101, Perforin 1, and Granzyme B. We examined the antitumor effects of NK-exosomes in an experimental murine mammary tumor model using REM134 canine mammary carcinoma cell line. We observed changes in tumor size, tumor initiation, progression, and recurrence-related markers in the control, tumor group, and NK-exosome-treated tumor group. We found that the tumor size in the NK-exosome-treated tumor group decreased compared with that of the tumor group in the REM134-driven tumorigenic mouse model. We observed significant changes including the expression of tumorigenesis-related markers, such as B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1), vascular endothelial growth factor (VEGF), matrix metallopeptidase-3 (MMP-3), interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), multidrug resistance protein (MDR), tumor suppressor protein p53 (p53), proliferating cell nuclear antigen (PCNA), and the apoptotic markers, B cell lymphoma-2 associated X (Bax) and B cell lymphoma-extra large (Bcl-xL) belonging to the Bcl-2 family, in the tumor group compared with those in the control group. The expression of CD133, a potent cancer stem cell marker, was significantly higher than that of the control. By contrast, the NK-exosome-treated tumor group exhibited a significant reduction in Bmi-1, MMP-3, IL-1β, IL-6, TNF-α, Bax, Bcl-xL, and PCNA expression compared with that in the tumor group. Furthermore, the expression of CD133, which mediates tumorigenesis, was significantly decreased in the NK-exosome-treated tumor group compared with that in the tumor group. These findings indicate that canine NK-exosomes represent a promising therapeutic tool against canine solid tumors, including mammary carcinoma.

Involvement of Nestin in the Progression of Canine Mammary Carcinoma

Nestin, a class VI intermediate filament protein, is known to be expressed in various types of human neoplasms, including breast cancer, and is associated with their progression. However, its expression and role in canine mammary tumors remain unknown. We analyzed nestin expression in canine mammary tumors using in situ hybridization and immunohistochemistry. We also investigated its role in a canine mammary carcinoma cell line using RNA interference. Nestin expression was not observed in luminal epithelial cells of any of the 62 cases of benign mammary lesions examined, although myoepithelial cells showed its expression in most cases. In 16/50 (32%) primary mammary carcinomas and 6/15 (40%) metastases of mammary carcinomas, cytoplasmic nestin expression was detected in luminal epithelial cells. In luminal cells of primary mammary carcinomas, its expression was positively related to several pathological parameters that indicate high-grade malignancy, including histological grading (P < .01), vascular/lymphatic invasion (P < .01), Ki-67 index (P < .01), and metastasis (P < .05). Immunohistochemistry revealed that nestin expression was related to vimentin expression in mammary carcinomas (P < .01). This relationship was confirmed using reverse transcription-quantitative polymerase chain reaction using 9 cell lines derived from canine mammary carcinoma (P < .01). Finally, nestin knockdown in canine mammary carcinoma cells using small interfering RNA inhibited cell proliferation and migration based on WST-8, Boyden chamber, and cell-tracking assays. These findings suggest that nestin may at least partially mediate these behaviors of canine mammary carcinoma cells.

Oncolytic effect of Newcastle disease virus is attributed to interferon regulation in canine mammary cancer cell lines.

Canine mammary carcinoma (CMC) is one of the major health threats in dogs. The oncolytic virotherapy is a promising strategy to treat canine as well as human cancer patients with non-pathogenic replicating viruses. Here, we evaluated the antitumor activity of one lentogenic, non-lytic Newcastle disease virus (NDV) LaSota strain expressing GFP (NDV-GFP) on five different CMCs and one non-tumorigenic cell line, regarding cell viability, cell death, selectivity index, morphology, global and target gene expression analysis. As evidenced by the selectivity index, all CMC cell lines were more susceptible to NDV-GFP in comparison with the non-tumorigenic cells (~3.1× to ~78.7×). In addition, the oncolytic effect of NDV-GFP was more evident in more malignant CMC cells. Also, we observed an inverse association of the IFN pathway expression and the susceptibility to NDV. The downregulated genes in NDV-GFP-sensitive cells were functionally enriched for antiviral mechanisms by interferon and immune system pathways, demonstrating that these mechanisms are the most prominent for oncolysis by NDV. To our knowledge, this is the first description of oncolysis by an NDV strain in canine mammary cancer cells. We also demonstrated specific molecular pathways related to NDV susceptibility in these cancer cells, opening the possibility to use NDV as a therapeutic-targeted option for more malignant CMCs. Therefore, these results urge for more studies using oncolytic NDVs, especially considering genetic editing to improve efficacy in dogs.

Establishment and Characterization of Canine Mammary Gland Carcinoma Cell Lines With Vasculogenic Mimicry Ability in vitro and in vivo.

Mammary tumors affect intact and elderly female dogs, and almost 50% of these cases are malignant. Cell culture offers a promising preclinical model to study this disease and creates the opportunity to deposit cell lines at a cell bank to allow greater assay reproducibility and more reliable validation of the results. Another important aspect is the possibility of establishing models and improving our understanding of tumor characteristics, such as vasculogenic mimicry. Because of the importance of cancer cell lines in preclinical models, the present study established and characterized primary cell lines from canine mammary gland tumors. Cell cultures were evaluated for morphology, phenotype, vasculogenic mimicry (VM), and tumorigenicity abilities. We collected 17 primary mammary carcinoma and three metastases and obtained satisfactory results from 10 samples. The cells were transplanted to a xenograft model. All cell lines exhibited a spindle-shaped or polygonal morphology and expressed concomitant pancytokeratin and cytokeratin 8/18. Four cell lines had vasculogenic mimicry ability in vitro, and two cell lines showed in vivo tumorigenicity and VM in the xenotransplanted tumor. Cellular characterization will help create a database to increase our knowledge of mammary carcinomas in dogs, including studies of tumor behavior and the identification of new therapeutic targets.

Protease-Activated Receptor-2 Is Associated With Adverse Outcomes in Canine Mammary Carcinoma

Protease-activated receptor-2 (PAR2) is a G protein-coupled receptor that is activated by serine proteases. In humans, PAR2 is highly expressed in various cancers, including breast cancer, and is associated with cancer progression and metastasis. However, the expression and roles of PAR2 in canine mammary carcinoma remain unclear. The purpose of this study was to examine the expression of PAR2 in canine mammary carcinoma, the association between PAR2 expression and clinical characteristics, and the role of PAR2 in the metastatic phenotypes of tumor cells. Mammary carcinoma from 31 dogs and 10 normal mammary glands were included in this study, and used for immunohistochemical analysis of PAR2 expression. Normal mammary glands did not express PAR2. In contrast, mammary carcinomas showed PAR2 immunoreactivity in the cytoplasm, and its expression level varied between specimens from negative to strongly positive. The overall survival of dogs with high PAR2 expression was shorter than that of dogs with low PAR2 expression. Moreover, PAR2 expression level was associated with the presence of lymph node involvement, advanced clinical stage, and high histopathological grade. In vitro analyses revealed that a PAR2 agonist accelerated cell migration and invasion in a canine mammary carcinoma cell line. In addition, the PAR2 agonist induced epithelial-mesenchymal transition and actin polymerization. These results suggest that PAR2 expression plays a role in tumor progression and clinical outcomes in canine mammary carcinoma.

Effects of doxorubicin associated with amniotic membrane stem cells in the treatment of canine inflammatory breast carcinoma (IPC-366) cells

BackgroundTumours in mammary glands represent the most common neoplasia in bitches, as in humans. This high incidence results in part from the stimulation of sex hormones on these glands. Among mammary tumours, inflammatory carcinoma is the most aggressive, presenting a poor prognosis to surgical treatment and chemotherapy. One of the most widely used chemotherapy drugs for breast cancer treatment is doxorubicin (DOXO). Alternative therapies have been introduced in order to assist in these treatments; studies on treatments using stem cells have emerged, since they have anti-inflammatory and immunomodulatory properties. The aim of this study was to evaluate the effects of DOXO and canine amniotic membrane stem cells (AMCs) on the triple-negative canine inflammatory mammary carcinoma cell line IPC-366.MethodsFour experimental groups were analysed: a control group without treatment; Group I with DOXO, Group II with AMC and Group III with an association of DOXO and AMCs. We performed the MTT assay with DOXO in order to select the best concentration for the experiments. The growth curve was performed with all groups (I-III) in order to verify the potential of treatments to reduce the growth of IPC-366. For the cell cycle, all groups (I-III) were tested using propidium iodide. While in the flow cytometry, antibodies to progesterone receptor (PR), estrogen receptor (ER), PCNA, VEGF, IL-10 and TGF-β1 were used. For steroidogenic pathway hormones, an ELISA assay was performed.ResultsThe results showed that cells treated with 10 µg/mL DOXO showed a 71.64% reduction in cellular growth after 72 h of treatment. Reductions in the expression of VEGF and PCNA-3 were observed by flow cytometry in all treatments when compared to the control. The intracellular levels of ERs were also significantly increased in Group III (4.67% vs. 27.1%). Regarding to the levels of steroid hormones, significant increases in the levels of estradiol (E2) and estrone sulphate (S04E1) were observed in Groups I and III. On the other hand, Group II did not show differences in steroid hormone levels in relation to the control. We conclude that the association of DOXO with AMCs (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC-366 triple-negative cells.ConclusionsThis treatment promoted ER positive expression, suggesting that the accumulated oestrogen conducted these cells to a synergistic state, rendering these tumour cells responsive to ERs and susceptible to new hormonal cancer therapies.

Cytotoxic effects of canine nk cells on canine mammary tumor cells

Background & Aim Natural killer (NK) cells play a crucial role in immune responses against various pathogens. NK cells are innate lymphocytes that rapidly provide defenses against tumors and viral infections allowing pathogen elimination or limiting viral spread. Their fast responses mainly rely on the expression of multiple germ-line encoded activating receptors among which natural cytotoxic receptors (NCRs) and NKG2D play the most relevant role in the recognition and killing of infected cells. NK cells have been characterized in a variety of mammals, but canine NK cells remain incompletely characterized. Mammary tumors account for ∼30% of all tumors in the female canine and approximately 50% of the tumors are malignant. Canine mammary gland tumor cell lines are excellent experimental models for identification of cancer-associated genes and understanding cancer biology and progression. In this study, we isolated canine peripheral blood-derived NK cells and defined their immunophenotype and cytotoxic effects on canine mammary tumor cells. In this study, we compared characteristics of canine NK cells against two canine mammary carcinoma cell lines, REM134 and CF41.Mg. Methods, Results & Conclusion Canine NK cells are proliferated and activated in CD5 negative population under specific culture condition. We peripheral blood mononuclear cells (PBMCs) were isolated from female beagle dogs (n=3). Subsequently, CD5 negative cells were isolated by immunomagnetic separation from PBMCs. For activation and expansion of NK cells, CD5 negative cells and co-cultured K562 cells in the presence of IL-2, IL-15, and IL-21 for 21 days. We found that expression markers of activated NK cells such as NKp30, NKp44, NKp46, NKG2D, CD244, perforin, granzyme B, and tumor necrosis factor alpha were highly upregulated. In addition, we found upregulated production of interferon gamma of activated NK cells against target cells such as REM134 and CF41.Mg. Also, we observed that cytotoxicity of activated NK cells against target cells more sensitively reacted to CF41.Mg than REM134. Taken together, we recommend the development of an experimental application of CF41Mg, which has not been reported in canine mammary carcinoma research.

Cytotoxicity of natural killer cells on canine mammary carcinoma cells

Natural killer (NK) cells play have a crucial role in the early phase of immune responses against various pathogens. We compared characteristics of canine NK cells against two canine mammary carcinoma cell lines, REM134 and CF41.Mg. REM134 showed higher expression of progesterone receptor, proliferative cell nuclear antigen, Ki67, multiple drug resistance, Bmi-1, c-myc, E-cadherin, and human epidermal growth factor receptor type-2 than that of CF41.Mg. For specific expansion and activation of NK cells, we isolated CD5 negative cells from canine peripheral blood mononuclear cells and co-cultured K562 cells in the presence of interleukin (IL)-2, IL-15, and IL-21 for 21 days. As a result, we found that expression markers of activated NK cells such as NKp30, NKp44, NKp46, NKG2D, CD244, perforin, granzyme B, and tumor necrosis factor alpha were highly upregulated. In addition, we found there was upregulated production of interferon gamma of activated NK cells against target cells such as REM134 and CF41.Mg. Specifically, we observed that cytotoxicity of NK cells against target cells was more sensitively reacted to CF41.Mg than REM134. Based on the results of this study, we recommend the development of an experimental application of CF41Mg, which has not been reported in canine mammary carcinoma research. Keywords: canine, mammary carcinoma, natural killer cells, interferon gamma, cytotoxicity

Trypanosoma cruzi calreticulin: In vitro modulation of key immunogenic markers of both canine tumors and relevant immune competent cells.

Recombinant calreticulin from Trypanosoma cruzi (rTcCalr), the parasite responsible for Chagas' disease, binds to Canine Transmissible Venereal Tumor (CTVT) cells from primary cultures and to a canine mammary carcinoma cell line. A Complement-binding assay indicated that interaction of the first component C1q with these tumor cells operated independently of the rTcCalr-presence. This apparent independence could be explained by the important structural similarities that exist among rTcCarl, endogenous normal canine and/or mutated calreticulins present in several types of cancer. In phagocytosis assays, tumor cells treated with rTcCalr were readily engulfed by macrophages and, co-cultured with DCs, accelerated their maturation. In addition, DCs maturation, induced by tumor cells co-cultured with rTcCalr, activated T cells more efficiently than DCs, treated or not with LPS. In an apparent paradox, a decrease in MHC Class I expression was observed when these tumor cells were co-cultivated with rTcCalr. This decrease may be related to a down regulation signaling promoting the rescue of MHC I. Possibly, these in vitro assays may be valid correlates of in vivo sceneries. Based on these results, we propose that rTcCalr improves in vitro the immunogenicity of two widely different tumor cell lines, thus suggesting that the interesting properties of rTcCalr to boost immune responses warrant future studies.

Aldoxorubicin-loaded nanofibers are cytotoxic for canine mammary carcinoma and osteosarcoma cell lines in vitro: A short communication

Chemotherapeutic drugs are given parenterally to treat various canine tumors. A limitation of parenteral administration is low drug penetration into the tumor, which reduces tumoricidal activity. Various drug carriers have been used to enhance tumor delivery, including albumin, liposomes and nanoparticles. A novel peptide-based nanofiber precursor (NFP) has been developed that is designed to take advantage of the leaky tumor neovasculature to promote drug delivery after parenteral administration. In this study, we loaded aldoxorubicin, an albumin-bound prodrug version of doxorubicin, onto NFP and tested the in vitro cytotoxicity in canine mammary carcinoma (CMT12, CMT25) and osteosarcoma (HMPOS, D-17, Abrams) cell lines. The half maximal inhibitory concentration (IC50) was determined with a luminescence-based cell viability assay. The IC50 for aldoxorubicin-loaded NFP was lower than free aldoxorubicin or doxorubicin in all cell lines, whereas non-drug loaded NFP had no cytotoxic effects. There were differences in IC50 between the osteosarcoma lines, with lower and higher IC50 for HMPOS and D-17 cells, respectively, with all drugs (aldoxorubicin-loaded NFP, free aldoxorubicin or free doxorubicin). Our results indicate that drug-loaded NFPs are cytotoxic for various canine mammary carcinoma and osteosarcoma cell lines in vitro and hold promise as a mechanism for enhancing delivery of chemotherapeutic agents to canine tumors.

Alkanna Tinctoria (L.) Tausch Kök Ekstraktlarının Antimikrobiyal Aktivitesi ve Sitotoksisitesi

Alkanna tinctoria (L.) Tausch produced naphthoquinones known as DNA-topoisomerases inhibitors has been used as the traditional therapeutic agent, especially wound healing in Turkey. The antimicrobial activity against 9 clinical microorganisms and cytotoxicity on canine mammary carcinoma cell line (CMT-U27) of Alkanna tinctoria root extracts (methanol, ethanol and acetonitrile) were researched by using MTT analysis. Alkanet root extracts showed antimicrobial activity against 2 (Proteus spp. and S. haemolyticus) out of 9 microorganisms. In vitro study towards CMT-U27 cancer cells, acetonitrile root extract at 100 µg/mL concentration showed strong and significant anti-proliferative effect. Our results are manifested that the Alkanet acetonitrile root extract can be evaluated as potential therapeutic agent, immediately after identification of bio-active metabolits in extract.