EGFR-targeted hybrid plasmonic magnetic nanoparticles synergistically induce autophagy and apoptosis in non-small cell lung cancer cells.

Tomohisa Yokoyama,Manish Shanker,Konstantin Sokolov,Justina Tam,Seiji Kondo,Shinji Kuroda,Ailing W Scott,Jack A Roth,Jesse Aaron,Rajagopal Ramesh,Arlene M Correa,Tim Larson,Sujit Basu

doi:10.1371/journal.pone.0025507

Abstract

BackgroundThe epidermal growth factor receptor (EGFR) is overexpressed in 80% of non-small cell lung cancer (NSCLC) and is associated with poor survival. In recent years, EGFR-targeted inhibitors have been tested in the clinic for NSCLC. Despite the emergence of novel therapeutics and their application in cancer therapy, the overall survival rate of lung cancer patients remains 15%. To develop more effective therapies for lung cancer we have combined the anti-EGFR antibody (Clone 225) as a molecular therapeutic with hybrid plasmonic magnetic nanoparticles (NP) and tested on non-small cell lung cancer (NSCLC) cells.Methodology/Principal FindingsCell viability was determined by trypan-blue assay. Cellular protein expression was determined by Western blotting. C225-NPs were detected by electron microscopy and confocal microscopy, and EGFR expression using immunocytochemistry. C225-NP exhibited a strong and selective antitumor effect on EGFR-expressing NSCLC cells by inhibiting EGFR-mediated signal transduction and induced autophagy and apoptosis in tumor cells. Optical images showed specificity of interactions between C225-NP and EGFR-expressing NSCLC cells. No binding of C225-NP was observed for EGFR-null NSCLC cells. C225-NP exhibited higher efficiency in induction of cell killing in comparison with the same amount of free C225 antibody in tumor cells with different levels of EGFR expression. Furthermore, in contrast to C225-NP, free C225 antibody did not induce autophagy in cells. However, the therapeutic efficacy of C225-NP gradually approached the level of free antibodies as the amount of C225 antibody conjugated per nanoparticle was decreased. Finally, attaching C225 to NP was important for producing the enhanced tumor cell killing as addition of mixture of free C225 and NP did not demonstrate the same degree of cell killing activity.Conclusions/SignificanceWe demonstrated for the first time the molecular mechanism of C225-NP induced cytotoxic effects in lung cancer cells that are not characteristic for free molecular therapeutics thus increasing efficacy of therapy against NSCLC.

Highlights

The epidermal growth factor receptor (EGFR) is overexpressed in 80% of non-small cell lung cancer (NSCLC) and is associated with poor survival [1]
We examined EGFR expression in several human NSCLC cell lines that are wild type (H1229), mutated and overexpressed (HCC827, H1975, H3255), amplified (H1819), or null (H520) for EGFR and compared with EGFR expression in normal lung fibroblasts (MRC-9 and WI38) and normal human bronchial epithelial cells (NHBE) by Western blotting. Both total and phosphorylated EGFR expression was detected in all cell lines tested except for H520 cells which are null for EGFR
The phosphorylated EGFR (pEGFR) expression levels varied among the cell lines with high expression detected in HCC827, H1819 and H3255 cells (Figure 1a)

Summary

Introduction

The epidermal growth factor receptor (EGFR) is overexpressed in 80% of NSCLC and is associated with poor survival [1]. MRC-9 but not NHBE cells showed some inhibitory effects when treated with C225-NP and compared with IgG-NP treatment (Figure 1b). Reduced expression of phosphorylated-EGFR, -AKT, -p38MAPK, and p44/42MAPK after C225-NP treatment was observed in HCC827 and H1299 cells when compared to cells treated with control IgG-NP (Figure 2).

Results

Conclusion