RESUMO
Protein transduction therapy using poly-arginine can deliver the bioactive p53 protein into cancer cells and inhibits the proliferation of the cells. However, one disadvantage of such therapy is the short intracellular half-life of the delivered protein. Here, we generated mutant proteins in which multiple lysine residues in the C-terminal were substituted by arginines. The mutant proteins were effectively delivered in glioma cells and were resistant to Mdm2-mediated ubiquitination. Moreover, the mutant proteins displayed higher transcription regulatory activity and powerful inhibition of the proliferation of glioma cells. These results suggest that ubiquitination-resistant p53 protein therapy may become a new effective cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/terapia , Genes p53 , Terapia Genética/métodos , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/química , Adenoviridae/genética , Western Blotting , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Genes Reporter/genética , Glioma/patologia , Glioma/terapia , Humanos , Marcação In Situ das Extremidades Cortadas , Mutagênese Sítio-Dirigida , Peptídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transcrição Gênica , Ubiquitina/metabolismoRESUMO
Protein transduction therapy is a newly developing method that allows proteins, peptides, and biologically active compounds to penetrate across the plasma membrane by being fused with cell-penetrating peptides such as polyarginine. Polyarginine-fused p53 protein penetrates across the plasma membrane of cancer cells and inhibits the growth of the cells. However, the protein is often entrapped inside macropinosomes in the cytoplasm. Therefore, high dose concentrations of the protein are needed for it to function effectively. To overcome this problem, in the present study, polyarginine-fused p53 was linked with the NH(2)-terminal domain of influenza virus hemagglutinin-2 subunit (HA2), which is a pH-dependent fusogenic peptide that induces the lysis of membranes at low pH levels. The protein was capable of efficiently translocating into the nucleus of glioma cells and induced p21(WAF1) transcriptional activity more effectively than did polyarginine-fused p53 protein. Moreover, low concentrations of the protein significantly inhibited the growth of cancer cells. These results suggest that protein transduction therapy using polyarginine and HA2 may be useful as a method for cancer therapy.