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.
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.
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.