RESUMO
Hyper-activation of the MAPK and PI3K-AKT pathways is linked to tumour progression in triple-negative breast cancer (TNBC). However, clinically effective predictive markers for drugs targeted against protein kinases involved in these pathways have not been identified. We investigated the ability of MEK and PI3K catalytic activity to predict sensitivity to trametinib and wortmannin in TNBC. MEK and PI3K activities correlated strongly with each other only in cell lines showing wortmannin-specific sensitivity, as shown by a linear regression curve (R = 0.951). Accordingly, we created a new parameter that distinguishes trametinib and wortmannin sensitivity in vitro and in vivo. Our findings suggest that the catalytic activities of MEK and PI3K might predict the response of TNBC to trametinib and wortmannin.
Assuntos
Biocatálise , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Androstadienos/farmacologia , Animais , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Piridonas/farmacologia , Pirimidinonas/farmacologia , Relação Estrutura-Atividade , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , WortmaninaRESUMO
The cellular slime mold Dictyostelium discoideum expresses a gene encoding a 452-amino-acid polypeptide that is 47% identical to Escherichia coli RecA. A recA-deficient E. coli, JE6651, was transformed by pYSN1, which was designed to express the truncated form of the D. discoideum gene, and used in suppression assays. The viability of the transformant, JE6651(pYSN1), increased following UV irradiation or mitomycin C treatment. Phage lambda (red(-) gam(-)), which required RecA activity for DNA packaging, formed plaques on a lawn of JE6651(pYSN1). These results indicate that the gene product has a DNA recombination activity. Fluorescence of D. discoideum protein fused with GFP was detected in mitochondria. The gene disruption mutant was hypersensitive to UV-light (254nm), mitomycin C and H(2)O(2), indicating that D. discoideum recA is important for survival following exposure to DNA damaging agents.
Assuntos
Dictyostelium/enzimologia , Escherichia coli/enzimologia , Mitocôndrias/metabolismo , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Dictyostelium/citologia , Dictyostelium/efeitos dos fármacos , Dictyostelium/genética , Escherichia coli/genética , Peróxido de Hidrogênio/farmacologia , Dados de Sequência Molecular , Mutação/genética , Transporte Proteico , Recombinases Rec A/química , Raios UltravioletaRESUMO
Labeled oligodeoxyribonucleotide bearing fluorescent dyes at both ends and aptamer sequence for adenosine 5'-monophosphate (AMP) was synthesized. Fluorescence spectra of labeled aptamer were not so much different between with and without AMP. This result suggests the binding of AMP didn't cause the global structural change to the aptamer. Therefore, we used short complementary DNA (SCD) as an assistant DNA, which is an unmodified 11mer and have a complementary sequence of 5'-region of the labeled aptamer. In the presence of SCD, the fluorescence intensities decrease with increasing the concentration of AMP compared with a change in absence of SCD.