Valproic acid induces p21 and topoisomerase-II (α/β) expression and synergistically enhances etoposide cytotoxicity in human glioblastoma cell lines

Abstract

Etoposide, a topoisomerase-II inhibitor promotes DNA damage and apoptosis of cancer cells. In this study, we have examined the ability of the histone deacetylase inhibitor, valproic acid (VPA) to modulate gene expression and sensitize glioblastoma cell lines to the cytotoxic effects of etoposide in vitro. The effect of VPA and etoposide alone or a combination of the two drugs on the growth of three different glioblastoma cell lines (U87, LN18, and U251) were measured by MTT assays. Drug treated cells were analyzed for their cell cycle profile, gene expression, differentiation status, and induction of apoptosis by flow-cytometry, western blotting, immunofluorescence assays, and caspase activity measurements. We observed that while VPA and etoposide independently inhibited the growth of U87, U251, and LN18 cells, exposure of tumor cells to both drugs significantly enhanced the cytotoxicity of etoposide in all cell lines. VPA promoted a G(1) accumulation of U87, while an increase in the G(2)/M population of U251 and LN18 cells was observed upon exposure to the drug. Treatment with etoposide resulted in a G(2)/M arrest of U87, U251, and LN18 cells, whereas, exposure to both drugs increased the fraction of cells with a G2/M and sub-G1 DNA content. Further, VPA and not etoposide, promoted acetylation of histone H4 and induced the expression of the cyclin-dependent kinase inhibitor (CDKI), p21/WAF1. VPA also up-regulated the expression of the alpha and beta isoforms of topoisomerase-II, as well as the glial differentiation marker, glial fibrillary acidic protein. Finally, a significant increase in caspase-3 activity and apoptosis was observed in the presence of both VPA and etoposide compared to either agent alone. Our study demonstrates that VPA sensitizes U87, U251, and LN18 cells to the cytotoxic effects of etoposide in vitro by inducing differentiation and up-regulating the expression of p21/WAF1 and both isoforms of topoisomerase-II.