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.
14-3-3 Difopein Apoptosis Glioma Gene therapy
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Supported by the National Natural Science Foundation of China (39970752) and Scientific and Technological Project of ShaanXi Province (2008K09-09). We gratefully acknowledge Michael Brenner (university of Alabama in Birmingham, USA) for providing pGfa2-LacZ plasmid, Wei Zhang (Biochemistry department in our university) for providing retroviral vector pLncx, and Haian Fu (Department of Pharmacology in Emory University, USA) for providing pSCM138 plasmid. We thank Jielai Xia’s (statistics department in our university) help in statistical analysis. We thank for Xiaoyan Chen (immune department in our university) assistance with flow cytometry, Angang Yang (immune department in our university) and Haian Fu for their comments on the manuscript. We thank Juan Li, Guo Geng, Wei Lin, Wei Zhang, LuHua Zhang, Yuhai Zhang and Jinxiang Chen for their helps in the process of major revision. And we also thank Junli Huo and Wanjuan Yang for material preparing for this research.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Morrison DK (2009) The 14–3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development. Trends Cell Biol 19:16–23CrossRefPubMedGoogle Scholar
Lee JA, Park JE, Lee DH, Park SG, Myung PK, Park BC, Cho S (2008) G1 to S phase transition protein 1 induces apoptosis signal-regulating kinase 1 activation by dissociating 14–3-3 from ASK1. Oncogene 27:1297–1305CrossRefPubMedGoogle Scholar
Nishino TG, Miyazaki M, Hoshino H, Miwa Y, Horinouchi S, Yoshida M (2008) 14–3-3 regulates the nuclear import of class IIa histone deacetylases. Biochem Biophys Res Commun 377:852–856CrossRefPubMedGoogle Scholar
Obsilova V, Silhan J, Boura E, Teisinger J, Obsil T (2008) 14-3-3 Proteins: a family of versatile molecular regulators. Physiol Res 57(Suppl 3):S11–S21Google Scholar
Cao WD, Zhang X, Zhang JN, Yang ZJ, Zhen HN, Cheng G, Li B, Gao D (2006) Immunocytochemical detection of 14–3-3 in primary nervous system tumors. J Neurooncol 77:125–130CrossRefPubMedGoogle Scholar
Rapp UR, Fischer A, Rennefahrt UE, Hekman M, Albert S (2007) BAD association with membranes is regulated by Raf kinases and association with 14–3-3 proteins. Adv Enzyme Regul 47:281–285CrossRefPubMedGoogle Scholar
Cao WD, Zhang X, Zhang JN (2007) 14–3-3 protein and glioma prognosis. Chin J Exp Surgery (in Chinese) 24:377Google Scholar
Tzivion G, Gupta VS, Kaplun L, Balan V (2006) 14-3-3 Proteins as potential oncogenes. Semin Cancer Biol 16:203–213Google Scholar
Wang B, Yang H, Liu YC, Jelinek T, Zhang L, Ruoslahti E, Fu H (1999) Isolation of high-affinity peptide antagonists of 14–3-3 proteins by phage display. Biochemistry 38:12499–12504CrossRefPubMedGoogle Scholar
Zamorano A, Lamas M, Vergara P, Naranjo JR, Segovia J (2003) Transcriptionally mediated gene targeting of gas1 to glioma cells elicits growth arrest and apoptosis. J Neurosci Res 71:256–263CrossRefPubMedGoogle Scholar
Cortez N, Trejo F, Vergara P, Segovia J (2000) Primary astrocytes retrovirally transduced with a tyrosine hydroxylase transgene driven by a glial-specific promoter elicit behavioral recovery in experimental parkinsonism. J Neurosci Res 59:39–46CrossRefPubMedGoogle Scholar
Jin N, Chen W, Blazar BR, Ramakrishnan S, Vallera DA (2002) Gene therapy of murine solid tumors with T cells transduced with a retroviral vascular endothelial growth factor–immunotoxin target gene. Hum Gene Ther 13:497–508CrossRefPubMedGoogle Scholar
de Moraes MM, Dinguirard N, Franco GR, Yoshino TP (2009) Phenotypic screen of early-developing larvae of the blood fluke, schistosoma mansoni, using RNA Interference. PLoS Negl Trop Dis 3:e502CrossRefGoogle Scholar