Targeting 14-3-3 protein, difopein induces apoptosis of human glioma cells and suppresses tumor growth in mice

Apoptosis. 2010 Feb;15(2):230-41. doi: 10.1007/s10495-009-0437-4.

Abstract

14-3-3 protein has emerged as critical regulators of diverse cellular responses. Previous studies found that strong 14-3-3 protein expression was observed and associated with tumor genesis and progression in glioma. Here, we further elucidated the role of 14-3-3 protein in apoptosis of human glioma U251 and U87 cells by global inhibition of 14-3-3 functions with a general 14-3-3 antagonist, difopein. In vitro, morphological observation and DNA laddering assay showed that difopein-treated glioma cells displayed outstanding apoptosis characteristics, such as nuclear fragmentation, appearance of membrane-enclosed apoptotic bodies and DNA laddering fragment. Moreover, flow cytometric detection of phosphatidylserine externalization indicated that difopein-induced apoptosis occurred in a time-dependent manner. Interestingly, inhibiting 14-3-3 with small interfere RNA also induce apoptosis of human glioma U251 cells. Furthermore, RT-PCR and western blot assay further substantiated that difopein had strong effects to induce glioma cell apoptosis through down-regulating Bcl-2, up-regulating Bax and activating caspase-9 and caspase-3. In vivo, retroviral vector was constructed and retroviral-mediated transfer of difopein to glioma was implanted in nude mice. Difopein effectively hindered proliferation and triggered apoptosis of tumor cells implanted into nude mice. This work not only reveals a critical role of 14-3-3 in apoptosis suppression in glioma cells, but also identifies and validates 14-3-3 as a potential molecular target for anticancer therapeutic development.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / antagonists & inhibitors*
  • 14-3-3 Proteins / metabolism
  • Animals
  • Apoptosis / drug effects*
  • Cell Cycle / drug effects
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • DNA Fragmentation / drug effects
  • Flow Cytometry
  • Fluorescence
  • Gene Expression Regulation, Neoplastic / drug effects
  • Gene Transfer Techniques
  • Glioma / pathology*
  • Glioma / ultrastructure
  • Humans
  • Mice
  • Proteins / pharmacology*
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / metabolism
  • Retroviridae / genetics
  • Xenograft Model Antitumor Assays*

Substances

  • 14-3-3 Proteins
  • Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Messenger
  • RNA, Small Interfering
  • difopein