Science et thérapie du cancer

High-level Elevation of Transgene Expression via the Joint Effect of Trichostatin A and D-Glucose in a Malignant Pleural Mesothelioma-derived Cell Line Resistant to Adenovirus Vector-based Transgene Expression

Abstract

Junko Mori*, Yujiro Arao, Tomoyuki Honda and Hiromi Kumon

Objective: Malignant Pleural Mesothelioma (MPM), a locally invasive tumor, is treated with a combination of surgical, radiation, and medical therapies, but the treatment efficacy remains insufficient. Gene therapy for MPM has been attempted to improve the prognosis of the disease, but has not yet reached a practical level. One of the barriers to MPM gene therapy is the resistance of MPM cells to transgene expression. Therefore, the purpose of this study was to find techniques to achieve high-level transgene expression in MPM cells resistant to transgene expression.

Methods: We evaluated two MPM cell lines, NCI-H28 (H28) and NCI-H2052 (H2052), for their resistance to transgene expression, and then examined whether transgene expression in the resistant H2052 cells was enhanced by treating the cells with D-glucose, an extracellular signalregulated kinase 1/2 inhibitor LY3214996 (LY), and a histone deacetylase inhibitor trichostatin A (TSA), which have been reported to increase transgene expression. Cellular transgene expression was evaluated by a reporter gene assay in which a human cytomegalovirus immediate early (CMV) promoter-controlled Enhanced Green Fluorescent Protein (EGFP) gene was inserted into the cells using a human adenovirus (ADV) vector. The extent of EGFP gene expression was examined by fluorometric assay, fluorescence microscopy, and EGFP quantification by ELISA.

Results: The fluorescence intensity in H2052 cells was only 13.9% of that in H28 cells. D-glucose treatment of H2052 cells after transduction (posttreatment) increased the fluorescence intensity in H2052 cells by only 1.9-fold. LY treatment of H2052 cells before transduction (pretreatment) increased the fluorescence intensity in the cells by only 1.7-fold. TSA pretreatment increased the fluorescence intensity in H2052 cells by as much as 6.9-fold, to almost the same level as that in H28 cells. When the TSA-pretreated H2052 cells were posttreated with D-glucose, the fluorescence intensity in H2052 cells was enhanced to 400% of that in H28 cells. The elevated EGFP gene expression by the joint effect of TSA and D-glucose was also confirmed by fluorescence microscopy and EGFP quantification by ELISA.

Conclusion: It was suggested that TSA pretreatment could remove the resistance to transgene expression of MPM cells and the joint effect of TSA and D-glucose could highly enhance transgene expression in the resistant MPM cells.