No abstract

BACKGROUND:Human epidermal growth factor receptor (HER) downstream signaling kinases have important effects on tumor response to anti-HER monoclonal antibodies and tyrosine kinase inhibitors. We validated an assay that uses phosphoprotein arrays for measurement of HER downstream signaling functionality in breast carcinomas.

The development of new vectors to deliver DNA into cells for therapy of cancers or genetic diseases has been a major area of research for many years. However, the clinical application of this technology requires the development of efficient, reliable and sterile vectors enabling the transfer of genes in vivo. Non viral, polymer or lipid-based vectors offer a new impetus to gene therapy because they are less toxic than viral vectors (no endogenous recombination, fewer immunological reactions, easy production and delivery of large-sized plasmid).The aim of this study is to develop a new tool for DNA delivery composed of methacrylic polymeric (Eudragit ® RS and RL) nanoparticles. These nanoparticles were prepared by two meth- was between 2 and 4%. Nanoparticles prepared by nanoprecipitation were slightly more efficient than nanoparticles prepared from a double emulsion. Particle size was not an important factor for transfection, since no significant difference was observed with size between 50 and 350 nm. We showed that Eudragit ® RS and RL nanoparticles could introduce the transgene into different types of cells, but were generally less effective than the lipofectamine control.

Cetuximab (Erbitux) is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody whose activity is related to the inhibition of EGFR downstream signaling pathways. P53 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) have been reported to control the functionality of PI3K/AKT signaling. In this study we evaluated whether reintroducing P53 using non-viral gene transfer enhances PTEN-mediated inhibition of PI3K/AKT signaling by cetuximab in PC3 prostate adenocarcinoma cell line bearing p53 and pten mutations. Signaling phosphoproteins expression was analyzed using Bio-Plex phosphoprotein array and western blot. Apoptosis induction was evaluated from BAX expression, caspase-3 activation and DNA fragmentation analyses. The results presented show that p53 and pten gene transfer additionally mediated cell growth inhibition and apoptosis induction by restoral of signaling functionality, which enabled the control of PI3K/AKT and MAPKinase signaling pathways by cetuximab in PC3 cells. These results highlight the interest of the analysis of signaling phosphoproteins expression as molecular predictive markers for response to cetuximab and show that p53 and pten mutations could be key determinants of cell response to cetuximab through the functional impact of these mutations on cell signaling.

PTEN is a tumor suppressor gene mapped on chromosome 10q23.3 and encodes a dual specificity phosphatase. PTEN has major implication in PI3 kinase (PI3K) signal transduction pathway and negatively controls PI3 phosphorylation. It has been reported to be implicated in cell cycle progression and cell death control through inhibition of PI3K-Akt signal transduction pathway and in the control of cell migration and spreading through its interaction with focal adhesion kinase. Somatic mutations of PTEN are frequently detected in several cancer types including brain, prostate and endometrium with more than 30% of tumor tissue specimens bearing PTEN mutations and/or deletions. Because of its high frequency of mutations and its important function as tumor suppressor gene, PTEN is a good candidate for gene therapy. Inducible expression of PTEN has been also reported. In cancer cells bearing PTEN abnormalities, the reversion of PTEN function by external gene transfer becomes more and more investigated in cancer treatment research. Several technologies including the photochemical internalization (PCI) and aiming at improving the transfection efficiency have been reported. PCI is an innovative procedure based on light-induced delivery of macromolecules such as DNA, proteins and other therapeutic molecules from endocytic vesicles to the cytosol of target cells. PCI has been reported to enhance the gene delivery potential of viral and nonviral vectors. The present study was designed to evaluate the influence of photochemical internalization on polyethylenimine (PEI)-mediated PTEN gene transfer and its effects on the cellular viability in Ishikawa endometrial cancer cells bearing PTEN abnormalities. PCI was found to significantly (P < 0.01) enhance PTEN mRNA expression (4.2 fold increase). Subsequently, following PEI-mediated PTEN gene transfer, the restoration of the PTEN protein expression was observed. As a consequence, significant cell growth inhibition (44%) was observed in Ishikawa endometrial cells. Using PCI for PEI-mediated PTEN gene transfer was found to further enhance PTEN mRNA and protein expression as well as PTEN-related cell growth inhibition reaching 89%.