Enhanced Cell Growth Inhibition following PTEN Nonviral Gene Transfer Using Polyethylenimine and Photochemical Internalization in Endometrial Cancer Cells

Abstract

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%.

Photochemical Internalization: A New Tool for Drug Delivery

“…** Abbreviations: EGFP -enhanced green fluorescence protein, EGF -epidermal growth factor: TRAIL -tumor necrosis factor-related apoptosis-inducing ligand; PTEN -phosphatase and tensin homolog However, PCI-mediated gene delivery has also been shown to induce the delivery of therapeutic genes, such as the genes encoding HSV-tk (Herpes Simplex Virus thymidine kinase) [33], p53 [34], PTEN [35], TRAIL (unpublished results) and IL-12 (interleukin-12) (unpublished results). Delivery of genes to cells for therapeutic purposes requires in most cases that the DNA is protected from degradation and that the cellular uptake is facilitated (Fig.…”

“…Polylysine (with reporter gene EGFP) TPPS2a, TPPS4, AlPcS2a [28][29][30]85] Polyethyleneimine (PEI) (with reporter genes eGFP, luciferase and therapeutic gene HSV-thymidine kinase) TPPS2a,AlPcS2a [29,33,79] Glucosylated PEI with p53 gene TPPS2a [34,87] Glucosylated PEI with PTEN gene TPPS2a [35] NLS-peptide/plasmid complex complexed to a dendrimer-photosensitizer conjugate Dendrimer phthalocyanine [45] Cationic lipids (with reporter eGFP)* AlPcS2a [88] Targeted Gene Delivery:…”

P53 and PTEN expression contribute to the inhibition of EGFR downstream signaling pathway by cetuximab

“…20,21 Western blot Cells were exposed to epidermal growth factor (EGF, 100 ng ml À1 ) for 5 min (Roche Diagnostics, Penzberg, Germany). Western blots were carried out with total protein extracted from cells as already described using monoclonal antibody directed against PTEN, pPTEN, P53, AKT, phosphorylated-AKT (pAKT), GSK3b, pGSK3b, P70S6K, pP70S6K, ERK1/2, pERK1/2, BAX and tubulin 22 (Table 1).…”

Gene therapy in gynecological cancer

“…Maurice-Duelli and colleagues demonstrated that PTEN gene transfer can be improved by polyethlenimine-mediated photochemical internalization (PEI) in Ishikawa endometrial cell lines with inactivated PTEN. The method caused a 4.2-fold increase in PTEN mRNA expression, with restoration of PTEN protein expression leading to 44% growth inhibition in Ishikawa endometrial cells [62]. Sakurada and colleagues used adenovirus-mediated gene transfer in endometrial lines with absent and wild-type PTEN expression.…”