Reactivation of the p53 pathway by MDM2 inhibitor Nutlin-3: Application in photochemical internalisation (PCI)

Abstract

With approximately 50% of tumours bearing mutations in the TP53 gene, the tumour suppressor p53 is one of the most frequently altered proteins in human cancer. In tumours retaining wild-type p53, other aberrations including overexpression of MDM2 may impair the function of the p53 pathway. MDM2 regulates p53 by inhibiting p53-mediated transcriptional activity and promoting its degradation. Inhibiting the MDM2-p53 interaction with small molecule antagonist Nutlin-3 is an attractive approach to reactivate p53 in tumours overexpressing MDM2. Photochemical internalisation (PCI) is a novel technology for the release of therapeutic macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles that upon activation by light induces release of the endocytosed macromolecules. In the current study, the p53Wt MDM2Ampl osteosarcoma cell line SJSA-1 was treated with a combination of Nutlin-3 and PCI of bleomycin or saporin to study the impact of Nutlin-3 on the p53 pathway. Treatment with PCI of bleomycin and Nutlin-3 resulted in increased expression of p53 and the p53 target genes MDM2, p21 and Bax. PCI of bleomycin in combination with Nutlin-3 also induced cell cycle arrest in the G1/S checkpoint, but did not have a substantial impact on the induction of apoptosis. PCI of bleomycin and saporin both in presence and absence of Nutlin-3 inhibited cell growth, but the differences were negligible. These results indicate that Nutlin-3 in the concentration given in this study activates p53 leading to cell cycle arrest, while the effect on apoptosis remain ambiguous. The current study suggests that inhibition of the p53-MDM2 interaction with Nutlin-3 may be a promising strategy for improving tumour response to PCI in MDM2Ampl malignancies retaining wild-type p53.