Canine transmissible venereal tumour (CTVT) is a neoplasm transmitted among healthy dogs by direct contact with injured skin and/or mucous tissue. This study aimed to identify the TP53 gene, messenger RNA (mRNA) as well as the expression of p53, Bcl-2 and p63 proteins in histological sections of 13 CTVT samples at different stages of evolution. The in situ hybridization (ISH) and in situ reverse transcriptase polymerase chain reaction (RT-PCR) assays were used, which showed the DNA homologous to TP53 and its respective mRNA in 92.3% of the samples. We detected p53, p63 and Bcl-2 proteins in most of the cell samples in different grades of intensity. In addition, 46% of the samples were in the progressive and 54% in the regression phase. This is the first description of these proteins and a detailed study of their role in CTVT cells needs to be addressed in or to verify how these cells undergo apoptosis.
In this study, derived complex carcinoma (CC) and simple carcinoma (SC) cell lines were established and cultured under two-dimensional (2D) and three-dimensional (3D) conditions. The 3D was performed in six-well AlgiMatrix™ (LifeTechnologies®, Carlsbad, CA, USA) scaffolds, resulting in spheroids sized 50-125 µm for CC and 175-200 µm for SC. Cell viability was demonstrated up to 14 days for both models. Epidermal growth factor receptor (EGFR) was expressed in CC and SC in both systems. However, higher mRNA and protein levels were observed in SC 2D and 3D systems when compared with CC (P < 0.005). The connective tissue modulators, metalloproteinases-1, -2, -9 and -13 (MMPs), relaxin receptors 1 and 2 (RXR1 and RXR2) and E-cadherin (CDH1) were quantitated. All were upregulated similarly when canine mammary tumour (CMT)-derived cell lines were cultured under 3D AlgiMatrix, except CDH1 that was downregulated (P < 0.005). These results are promising towards the used of 3D system to increase a high throughput in vitro canine tumour model.
Canine mammary carcinomas represent an important pathology in small animal clinic, and benign mixed type carcinomas (MC-BMT) are one of the most diagnosed worldwide. The use of chemotherapeutic carboplatin has been one of the new protocols for the treatment of BMT. In this respect, three-dimensional cell culture (3D) represents an alternative in the evaluation of drugs by simulating what occurs in vivo. The present study aimed to verify the effect of carboplatin on 3D culture of cells derived from TMB, in addition to possible changes in cell viability, ballpoint size, apoptosis and Bcl-2/Bax ratio. For this, tumours samples were collected during mastectomy surgery procedure in private veterinary clinics, which were submitted to in vitro culture and part to histopathological analysis. After 28 days of 3D culture, spheroids were documented in both groups (treated and control) and sizes and morphology were compared. The carboplatin interfered in the cell viability by affecting their division and promoting apoptotic events. In the treated group, a higher transcription of Bax and caspase 3 was observed, in addition to low levels of caspases 2, 8 and 9, which was not observed in the control group. We thus suggest that mitochondrial dysfunction plays a critical role in cancer progression and that targeting mitochondrial alterations and mitochondrial retrograde signalling might be a promising strategy for the development of selective anticancer therapy. Thus, it was possible to demonstrate that the results achieved may contribute to the establishment of a new chemotherapy therapy in female dogs with MC-BMT.
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