RESUMO
Due to the lack of markers (ER, PR, and HER-2/Neu) for the molecular-targeted therapies triple-negative breast cancer (TNBC) is more challenging than other subtypes of breast cancer. Moreover, the conventional chemotherapeutic agents are still the mainstay of most therapeutic protocols and eventually turn into a refractory drug-resistance , hence, more efficient therapeutic regimens are urgently required. The present study aimed to elucidate the effects of PU-H71 combined with DHEA on triple-negative breast cancer cell line MDA-MB-231 and to assess the synergy using the Chou-Talalay method. The combined therapy controlled the expression of an array of antioxidants and metabolizing enzymes, leading to the induction of oxidative stress which in turn induced apoptotic cell death. Our results indicated that the combined treatment with PU-H71 and DHEA exerts a synergistic anti-tumor effect on MDA-MB-231 triple-negative breast cancer cell line.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Benzodioxóis/farmacologia , Desidroepiandrosterona/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Purinas/farmacologia , Apoptose/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Modelos Biológicos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
Recombinant l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS-PAGE. The activity of the purified L.ASNase was enhanced by Mg2+ and inhibited by Zn2+ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately.
Assuntos
Asparaginase/química , Proteínas de Bactérias/química , Expressão Gênica , Pseudomonas aeruginosa/enzimologia , Asparaginase/genética , Proteínas de Bactérias/genética , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por SubstratoRESUMO
l-Asparaginase (EC 3.5.1.1) is an important medical enzyme that catalysis the hydrolysis of l-asparagine to aspartic acid and ammonium. For over four decades l. asparaginase utic agent for the treatment of a variety of lymphoproliferative disorders and lymphoma such as acute lymphoblastic leukemia. In the present study A. terreus full length l. asparaginase gene, 1179bp was optimized for expression in Escherichia coli BL21 (DE3) pLysS. The full length A. terreusl. asparaginase gene encoding a protein of 376 amino acids with estimated molecular weight of 42.0kDa and a theoretical isoelectric point (pI) of 5.0. BLAST and phylogeny analysis revealed that the A. terreusl. asparaginase shared high similarity with other known fungal l. asparaginase (75% homology with A. nomius and 71% with A. nidulans). The recombinant protein was overexpressed in the form of amorphous submicron proteinaceous inclusion bodies upon induction with 1mM IPTG at 37°C for 18h.
