Identification of Phenotype and Evaluation of Cancer Therapeutic Drugs in Canine Melanoma Cells
Date Issued
2014
Date
2014
Author(s)
Lu, Lu-Ping
Abstract
Malignant melanoma is most common oral malignancy in dogs. It is a fatal disease along with local invasiveness and frequent metastasis. The poor response has been observed with all treatments including surgical removal, chemotherapy and radiation therapy. Thus, developing novel therapeutic approach for canine malignant melanoma is necessary. In this study, we established a drug-screening platform and used that platform to evaluate small molecule inhibitor, Dasatinib, and novel alkylating agents for therapy of canine melanoma. First of all, various phenotypes of eight canine melanoma cell lines were examined in this study. Mutation analysis of specific target genes KIT, NRAS and BRAF were done by PCR/DNA sequencing and KIT T1736C mutation causing KIT L579P change was detected in UCDK9M3, KMeC and C1 cells, while NRAS C181A mutation causing NRAS Q61K change was found in UCDK9M5 cell. Moreover, several target proteins expressions were investigated. Expressions of S100 and KIT were observed in all of the canine melanoma cell lines. KIT downstream protein NRAS was identified in several cell lines with different expression levels. However, no phospho-ERK and phospho-AKT were identified in any cell lines by “cell array”. Secondly, in cell viability assay, different melanoma cells revealed different response when treated with therapeutic drugs, in which Dasatinib and Bo-1978 effectively decreased the cell viability. Thirdly, cell cycle analysis and Annexin V apoptosis assay revealed that the treatment of Dasatinib causes a G1 cell cycle arrest and induction of apoptosis and also the treatment of BO-1978 and BO-2094 caused a G2/M cell cycle arrest and induction of apoptosis. At last, in SCID mice xenograft model of UCDK9M5, KMeC and CM01 cells, the treatment of BO-1978 significantly inhibited the growth of UCDK9M5 and CM01. In this study, we identified the phenotypes of eight melanoma cells and evaluated target drug Dasatinib and novel alkylating agents. Our data suggest that Dasatinib and BO-1978 may potentially be selected as a candidate for therapy of canine melanoma.
Subjects
Canine melanoma
DNA point mutation
Immunocytochemistry
Novel alkylating agent
SDGs
Type
thesis
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