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

J Neurooncol. 2007 Nov;85(2):159-70. doi: 10.1007/s11060-007-9402-7. Epub 2007 May 30.


Object: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents, Phytogenic / administration & dosage
  • Antineoplastic Combined Chemotherapy Protocols / pharmacology*
  • Brain Neoplasms / drug therapy*
  • Brain Neoplasms / enzymology
  • Cell Differentiation / drug effects
  • Cell Line, Tumor
  • Cyclin-Dependent Kinase Inhibitor p21 / drug effects*
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism
  • DNA Topoisomerases, Type II / drug effects*
  • DNA Topoisomerases, Type II / metabolism
  • Drug Synergism
  • Enzyme Inhibitors / pharmacology*
  • Etoposide / administration & dosage
  • Gene Expression Regulation, Neoplastic / drug effects
  • Glioblastoma / drug therapy*
  • Glioblastoma / enzymology
  • Histone Deacetylase Inhibitors
  • Histones / drug effects
  • Histones / metabolism
  • Humans
  • Isoenzymes
  • Valproic Acid / pharmacology*


  • Antineoplastic Agents, Phytogenic
  • Cyclin-Dependent Kinase Inhibitor p21
  • Enzyme Inhibitors
  • Histone Deacetylase Inhibitors
  • Histones
  • Isoenzymes
  • Valproic Acid
  • Etoposide
  • DNA Topoisomerases, Type II