Targeting 14-3-3 protein, difopein induces apoptosis of human glioma cells and suppresses tumor growth in mice.
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.
Cao
Weidong
W
Institute of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Changle Western Road 17#, 710032 Xi'an, Shaanxi Province, People's Republic of China.
Yang
Xiaoliang
X
Zhou
Jie
J
Teng
Zenghui
Z
Cao
Lei
L
Zhang
Xiang
X
Fei
Zhou
Z
D048948:14-3-3 Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D005434:Flow Cytometry; D005453:Fluorescence; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D018014:Gene Transfer Techniques; D005910:Glioma / Q000473:pathology* / Q000648:ultrastructure; D006801:Humans; D051379:Mice; D011506:Proteins / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
D048948:14-3-3 Proteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C443131:difopein