Sonoporation by microbubbles as gene therapy approach against liver cancer

Abstract

Introduction: An innovative method, known as sonoporation, was used to induce the expression of silenced genes, such as (but not restricted to) TRAIL and p53, in liver cancer cells (HepG2). The principal aim of the present study was the re-activation of silenced apoptotic pathways in liver cancer models, by using diagnostic synovial microbubble as plasmid gene delivery tools in combination with epigenetic treatments.

Material and methods: HepG2 cells were used as a liver cancer model. Microbubbles (Sonovue^®) were chosen as gene deliver system in combination with the sonoporation approach. Plasmid pEGFP-TRAIL and pEGFP-p53 were selected and propagated in Escherichia coli grown in LB broth, in order to obtain the necessary amount.

Results: Sonoporation was induced by using transducer (Sonitron 2000) and, among the several conditions tested, 3 MHz, 51% Duty Cycle, and 5 W/cm2, 30 s resulted as the best parameters. Data collected showed a dose dependent effect in terms of output energy. A transfection efficacy of 30 - 50% was achieved and recombinant gene expression induced apoptotic effects. In order to increase efficacy, we used the histone deacetylase inhibitor (HDACi, entinostat) MS-275, able to activate TRAIL and thus inducing a stronger pro-apoptotic effect in combination with TRAIL-gene re-expression.

Conclusion: For the first time, it was shown the possibility to induce the exogenous expression of the pro-apoptotic gene TRAIL and p53 in a liver cancer HepG2 cells via a sonoporation procedure. The epigenetic treatment using HDACi was able to increase the pro-apoptotic effects of the gene therapy.

Keywords: gene therapy; liver cancer; microbubbles; sonoporation; ultrasound.

Figure 1. Standard box-whisker plots of p53 and TRAIL expression in healthy and cancer tissues

(A) The green boxes are representative of healthy tissues, meanwhile the red boxes are for the cancers. The notches on the sides of the boxes (B).

Figure 2. Protein (GFP) construct with several combinations in term o MHz, Duty Cycle and W/cm2 were tested

Efficacy of sonoporation was reported as relative GFP-fluorescence in histogram graph (A). FACS analysis of Pre-G1 phase (B).

Figure 3

Structure of Entinostat (A). Western-blot analysis of Caspase 9 cleavage activation mediated in HepG2 cell line treated with Entinostat (MS-275) (B). The respective % of cells expressing the recombinant proteins are reported ad histograms in the panel (C).