31 research outputs found

    In Silico and In Vitro Evaluation of Bevacizumab Biosimilar MB02 as an Antitumor Agent in Canine Mammary Carcinoma

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    Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC.Fil: Cardama, Georgina Alexandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bucci, Paula Lorena. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lemos, Jesus. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Llavona, Candela. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Benavente, Micaela Andrea. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Hellmén, Eva. Gobierno de la Provincia de Buenos Aires. Hospital El Cruce Doctor Nestor Carlos Kirchner. Centro de Medicina Traslacional.; ArgentinaFil: Fara, María Laura. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; ArgentinaFil: Medrano, Eduardo. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; ArgentinaFil: Spitzer, Eduardo. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Fisiopatología. Laboratorio de Endocrinología; ArgentinaFil: Demarco, Ignacio A.. No especifíca;Fil: Sabella, Patricia. No especifíca;Fil: Garona, Juan. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

    Diverse Bone Morphogenetic Protein Expression Profiles and Smad Pathway Activation in Different Phenotypes of Experimental Canine Mammary Tumors

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    BACKGROUND:BMPs are currently receiving attention for their role in tumorigenesis and tumor progression. Currently, most BMP expression studies are performed on carcinomas, and not much is known about the situation in sarcomas. METHODOLOGY/PRINCIPAL FINDINGS:We have investigated the BMP expression profiles and Smad activation in clones from different spontaneous canine mammary tumors. Spindle cell tumor and osteosarcoma clones expressed high levels of BMPs, in particular BMP-2, -4 and -6. Clones from a scirrhous carcinoma expressed much lower BMP levels. The various clones formed different tumor types in nude mice but only clones that expressed high levels of BMP-6 gave bone formation. Phosphorylated Smad-1/5, located in the nucleus, was detected in tumors derived from clones expressing high levels of BMPs, indicating an active BMP signaling pathway and BMP-2 stimulation of mammary tumor cell clones in vitro resulted in activation of the Smad-1/5 pathway. In contrast BMP-2 stimulation did not induce phosphorylation of the non-Smad pathway p38 MAPK. Interestingly, an increased level of the BMP-antagonist chordin-like 1 was detected after BMP stimulation of non-bone forming clones. CONCLUSIONS/SIGNIFICANCE:We conclude that the specific BMP expression repertoire differs substantially between different types of mammary tumors and that BMP-6 expression most probably has a biological role in bone formation of canine mammary tumors

    Complex mammary tumours in the female dog: a review

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    In Silico and In Vitro Evaluation of Bevacizumab Biosimilar MB02 as an Antitumor Agent in Canine Mammary Carcinoma

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    Simple Summary Canine mammary carcinomas (CMC) are associated with poor clinical outcomes and high mortality. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and tumor progression in many solid tumors, including mammary carcinomas. The goal of this work was to establish the therapeutic value of MB02 monoclonal antibody biosimilar to bevacizumab that targets VEGF in CMC. For this purpose, first, we were able to predict in silico that bevacizumab was able to recognize and bind canine VEGF. This was confirmed in vitro using an ELISA-based assay. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These results show a high potential for MB02 as a promising therapeutic agent for the management of CMC. Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC

    The effect of BMP-2 on phosphorylation of Smad-1, Smad-5 and p38 in mammary tumor clones.

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    <p>CMT-U353 H4 (clone 12), CMT-U309 (clone 4) and HTh 74 (positive control for Smad-1/5 phosphorylation) cells were stimulated with BMP-2 (as indicated) in vitro and phosphorylation of Smad-1/5 (P-Smad-1, -5) and p38 (P-p38) was analyzed by Western blot. Immunoblot analysis for total Smad-5, total p38 and β-Actin was performed as loading controls.</p

    Immunohistochemical analysis for BMP-6.

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    <p>BMP-6 staining was performed in tumors generated by clones from the CMT-U309 (spindle cell), CMT-U353 B (osteosarcoma) and CMT-U353 H4 (scirrhous carcinoma) cell lines. (A–B) Spindle cell tumors generated by CMT-U309, clone C6. (A) Spindle cell tumor with an area of formed bone (*). Cells adjacent to the bone area and spindle cells further away from the formed bone showed strong positive cytoplasmic staining (arrows). (B) Spindle cell tumors with cells strongly positive for BMP-6, evenly distributed in the tumors. (C) Osteosarcoma formed by CMT-U353 B, clone 2. BMP-6 was detected in the cell dense border of the osteosarcoma and some cells showed membranous accentuation of the staining (arrow). (D) Spindle cell tumor generated by CMT-U353 B, clone 6. Faint staining for BMP-6 was seen throughout the tumors, with only a few cells with stronger staining and clear cytoplasmic positivity (arrow). (E–F) Spindle cell tumors generated by CMT-U353 H4, clone 6 (E) and 9 (F) were positive for BMP-6. Tumor cells showed cytoplasmic positivity and some cells had membranous accentuation of the staining (arrow).</p

    Immunohistochemical analysis for BMP-2/4.

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    <p>Staining for BMP-2/4 was performed in tumors generated by clones from the CMT-U309 (spindle cell), CMT-U353 B (osteosarcoma) and CMT-U353 H4 (scirrhous carcinoma) cell lines. (A–B) Spindle cell tumors generated by CMT-U309, clone C6. (A) Spindle cell tumor with an area of formed bone (*). Cells adjacent to the bone area and spindle cells further away from the formed bone showed strong positive cytoplasmic staining. (B) Spindle cell tumors with cells strongly positive for BMP-2/4, evenly distributed in the tumors. (C) Osteosarcoma formed by CMT-U353 B, clone 2. BMP-2/4 was detected in the cell dense border of the osteosarcoma as well as in cells adjacent to bone (*). (D) Spindle cell tumor generated by CMT-U353 B, clone 6. Strong staining for BMP-2/4 was seen throughout the tumors. (E–F) Spindle cell tumors generated by CMT-U353 H4, clone 6 (E) and 9 (F) stained less intensely for BMP-2/4.</p

    Tumors from cloned cell lines CMT-U309, CMT-U353 B and CMT-U353 H4 inoculated in mice.

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    <p>Tumors from cloned cell lines CMT-U309, CMT-U353 B and CMT-U353 H4 inoculated in mice.</p

    The antiprogestins mifepristone and onapristone reduce cell proliferation in the canine mammary carcinoma cell line CMT-U27

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    Canine mammary tumours (CMTs) represent nearly half of all tumours in female dogs and some 50% have malignant behaviour. Simple epithelial carcinomas have shorter disease free periods after surgery and a higher reduction of the proliferation index reduction after antiprogestin aglepristone treatment in vivo related to the expression of progesterone receptors (PR). These findings make simple carcinomas good candidates for endocrine therapy. To further explore this possibility, the effects of the antiprogestins mifepristone (RU486) and onapristone (ZK299) on cell viability and PR expression of the canine mammary carcinoma cell line isolated from a simple epithelial carcinoma CMT-U27 were studied. Twenty five percent of CMT-U27 control cells expressed PR. RU486 (p<0.05) and ZK299 (p<0.05) reduced the number of viable cells (WST-8 test) at 24h but only the latter treatment reduced significantly PR expression in viable tumour cells at 24h of incubation. The results suggest that both RU486 and ZK299 induce a decrease in the number of viable CMT-U27 tumour cells with different effects on PR expression. The canine mammary carcinoma cell line CMT-U27 is sensitive to the effects of antiprogestins and may serve to further explore the role of these drugs in canine mammary carcinomas
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