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Apoptosis. 2017 Jan;22(1):27-40. doi: 10.1007/s10495-016-1299-1.

MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.

Author information

1
Dirección de Infecciones Crónicas y Cáncer. Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, Mexico.
2
Departamento de Genética y Biología Molecular, CINVESTAV-IPN, Av. IPN 2508 Col. San Pedro Zacatenco. C. P. 07360, Mexico City, Mexico.
3
Instituto Nacional de Medicina Genómica, Mexico City, Mexico.
4
Centro Nacional de Investigación Disciplinaria en Parasitología Veterinaria, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Jiutepec, Morelos, Mexico.
5
Departamento de Genética y Biología Molecular, CINVESTAV-IPN, Av. IPN 2508 Col. San Pedro Zacatenco. C. P. 07360, Mexico City, Mexico. vidal@cinvestav.mx.

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.

KEYWORDS:

APO-1; Apoptosis; Autophagy; HPV; HPV16 E6; Phospho-p53 Ser46

PMID:
27766434
DOI:
10.1007/s10495-016-1299-1
[Indexed for MEDLINE]

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