Nutlin-3a Nanodisks Induce p53 Stabilization and Apoptosis in a Subset of Cultured Glioblastoma Cells.

Krishnamoorthy A,Witkowski A,Ryan Ro

doi:10.4172/2157-7439.1000454

Krishnamoorthy A, Witkowski A + Show 1 more

Open Access

https://doi.org/10.4172/2157-7439.1000454

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Abstract

Nanodisks (ND) are ternary complexes of phospholipid, one or more hydrophobic bioactive agents and an apolipoprotein scaffold. These nanoscale assemblies are organized as a disk-shaped lipid bilayer whose perimeter is stabilized by an apolipoprotein scaffold. Solubilization of hydrophobic bioactive agents is achieved by their integration into the ND lipid milieu. When the cis-imidazoline, nutlin-3a, was incubated with phosphatidylcholine and apolipoprotein A-I, it was conferred with aqueous solubility as judged by spectroscopic analysis. Nondenaturing polyacrylamide gel electrophoresis yielded evidence of a homogeneous population of ND particles ~9 nm in diameter. Gel filtration chromatography experiments revealed the association of nutlin-3a with ND is reversible. Biological activity of nutlin-3a ND was examined in three distinct glioblastoma cell lines, U87MG, SF763 and SF767. Incubation of U87MG cells with nutlin-3a ND induced concentration-dependent cell growth arrest and apoptosis. SF763 cells demonstrated modest cell growth arrest only at high concentrations of nutlin-3a ND and no apoptosis. SF767 cells were unaffected by nutlin-3a ND. Immunoblot analysis revealed nutlin-3a ND induced time-dependent stabilization of the master tumor suppressor, p53, and up regulation of the E3 ubiquitin ligase, murine double minute 2 in U87MG cells, but not the other glioma cell lines. The nanoscale size of the formulation particles, their facile assembly and nutlin-3a solubilization capability suggest ND represent a potentially useful vehicle for in vivo administration of this anti-tumor agent

Highlights

Healthy cells maintain low levels of the tumor suppressor protein, p53 through the action of the E3 ubiquitin ligase, murine double minute 2 (MDM2)
In an effort to confer water solubility, nutlin-3a was incorporated into ND, particles previously shown to function as a transport vehicle for hydrophobic bioactive compounds [27]
Nutlin-3a was originally identified based on its ability to induce apoptosis and cell cycle arrest by activating upstream regulators of the tumor suppressor protein, p53 [17]

Summary

Introduction

Healthy cells maintain low levels of the tumor suppressor protein, p53 through the action of the E3 ubiquitin ligase, murine double minute 2 (MDM2). In response to cellular stress (e.g. oncogene activation, genotoxic drugs or disruption of rRNA synthesis by chemotherapeutic drugs), p14ARF and/or NPM redistribute to the nucleoplasm where they function as inhibitors of MDM2 ubiquitin ligase activity [2,3,4,5]. P53 levels rise, promoting transactivation of genes involved in cell cycle arrest and DNA damage repair [6]. An important gene target of p53 is MDM2 itself, creating an auto-regulatory feedback loop that limits the duration of the stress response [7,8,9]. Given its central role in the regulation of cell fate, many cancers are caused by mutations in the p53 pathway [8,11]

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