title: MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.
abstract: The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.
authors.affiliation: Dirección de Infecciones Crónicas y Cáncer. Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, Mexico.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Lagunas-Martínez
authors.affiliation: Departamento de Genética y Biología Molecular, CINVESTAV-IPN, Av. IPN 2508 Col. San Pedro Zacatenco. C. P. 07360, Mexico City, Mexico.
authors.forename: Enrique
authors.initials: E
authors.lastname: García-Villa
authors.affiliation: Dirección de Infecciones Crónicas y Cáncer. Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, Mexico.
authors.forename: Magaly
authors.initials: M
authors.lastname: Arellano-Gaytán
authors.affiliation: Dirección de Infecciones Crónicas y Cáncer. Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, Mexico.
authors.forename: Carla O
authors.initials: CO
authors.lastname: Contreras-Ochoa
authors.affiliation: Instituto Nacional de Medicina Genómica, Mexico City, Mexico.
authors.forename: Jisela
authors.initials: J
authors.lastname: Dimas-González
authors.affiliation: Centro Nacional de Investigación Disciplinaria en Parasitología Veterinaria, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Jiutepec, Morelos, Mexico.
authors.forename: María E
authors.initials: ME
authors.lastname: López-Arellano
authors.affiliation: Dirección de Infecciones Crónicas y Cáncer. Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, Mexico.
authors.forename: Vicente
authors.initials: V
authors.lastname: Madrid-Marina
authors.affiliation: Departamento de Genética y Biología Molecular, CINVESTAV-IPN, Av. IPN 2508 Col. San Pedro Zacatenco. C. P. 07360, Mexico City, Mexico. vidal@cinvestav.mx.
authors.forename: Patricio
authors.initials: P
authors.lastname: Gariglio
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects; D052160:Benzothiazoles / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D005260:Female; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000821:virology; D007976:Leupeptins / Q000494:pharmacology; D009856:Oncogene Proteins, Viral / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D012097:Repressor Proteins / Q000235:genetics; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000821:virology; D051028:bcl-2-Associated X Protein / Q000235:genetics; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism
chemical_list: D000906:Antibodies; C494826:BAX protein, human; D052160:Benzothiazoles; D002352:Carrier Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; C068651:E6 protein, Human papillomavirus type 16; D006360:Heat-Shock Proteins; D007976:Leupeptins; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; C495901:TP53 protein, human; C429767:TP53INP1 protein, human; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D014050:Toluene; C121565:pifithrin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: APO-1; Apoptosis; Autophagy; HPV; HPV16 E6; Phospho-p53 Ser46