Enhancing the apoptotic effect of IL-24/mda-7 on the human hepatic stellate cell through RGD peptide modification

Abstract

ABSTRACTInduction of apoptosis or quiescent hepatic stellate cells (HSCs) can be an attractive molecular strategy due to the importance of activation of HSCs during hepatic fibrogenesis. Interleukin-24/melanoma differentiation-associated gene-7 (IL-24/mda-7) is a cytokine that has attracted a great deal of attention in the tumor killing as well as pathophysiology of the diseases. In this study, the Pro-apoptotic and senescence inductive properties of IL-24/mda-7 were assessed in human-derived HSCs. Three plasmids expressing natural mda-7, peptide modified version, mda-7-RGD genes beside a recombinant IL-24 protein, were added or transfected into activated LX-2 cells. Cell viability and the amount of apoptosis were analyzed using MTT and Annexin V staining method, respectively. Hence, the expression levels of apoptotic genes and PPARγ in different groups were also compared by real-time PCR analysis. Furthermore, the senescence effect of IL-24/mda-7 by a β-galactosidase (SA-β-gal) senescence assay, was evaluated. The viability assessment showed that pmda-7-RGD had the most significant growth inhibitory effect when compared to the control group, pcDNA3.1 (P = 0.0002). The apoptosis analysis also revealed a significant impact of different mda-7 forms in apoptosis induction. The measuring of cell senescence also indicated that IL-24/mda-7 in plasmid and protein forms exhibited a senescence inductive activity as determined by an increase in PPARγ gene expression and beta-galctosidase activity. In conclusion, our findings demonstrated that both endogenous and soluble forms of IL-24/mda-7 induced apoptosis and senescence in activated LX-2 cells and more importantly, fusion of RGD peptide to this cytokine enhanced these activities. So, RGD-modified IL-24/mda-7 could be a suitable candidate for further molecular therapy of fibrosis.