Difopein, a high affinity inhibitor of 14-3-3 proteins



The peptide Difopein, designed, isolated and identified by Haian Fu, is a high affinity inhibitor of 14-3-3 proteins. And when expressed in COS-7 and various cancer cells, difopein induces apoptosis, which may pave a way for a more effective treatment in human glioma.

Pharmacologic action

Difopein is the dimeric version of R18 peptide that binds to 14-3-3 proteins with high affinity. It could inhibit 14-3-3-ligand interactions, competitively. Besides, it blocks the ability of 14-3-3 to bind to target proteins such as Raf-1, Bad, ASK1 and exoenzyme S. Furthermore, when expressed in COS-7 and various cancer cells, difopein induces apoptosis.


In vitro, difopein effectively suppressed the growth of xenograft tumor. 14-3-3 has been shown to be a potential molecular target for the development of anticancer therapies. The general inhibition of 14-3-3 is sufficient to improve their sensitivity to apoptosis and to inhibit the growth of established tumors. Moreover, the results of morphological observation and DNA laddering assay showed that in vitro difopein-treated glioma cells displayed outstanding apoptosis characteristics, such as the appearance of membrane-enclosed apoptotic bodies, nuclear fragmentation and DNA laddering fragment. Besides, difopein had strong effects to induce glioma cell apoptosis through down-regulating Bcl-2, up-regulating Bax and activating caspase-9 and caspase-3, which was substantiated by RT-PCR and western blot assay. Furthermore, it was suggested that difopein-induced apoptosis occurred in a time-dependent manner in the flow cytometric detection of phosphatidylserine externalization. And inhibiting 14-3-3 with small interfere RNA also induced apoptosis of human glioma U251 cells. The experimental results showed that in nude mice in vivo, retroviral vector was constructed and retroviral-mediated transfer of difopein to glioma was implanted. It was confirmed that difopein effectively hindered the proliferation and triggered the apoptosis of tumor cells implanted into nude mice. The critical role of 14-3-3 in apoptosis suppression in glioma cells was revealed.

Pharmacokinetics and metabolism

The apoptosis of human glioma cells treated with difopein was confirmed by a number of different approaches so as to confirm the role of 14-3-3 in apoptosis suppression. After AO/EB staining, the apoptotic U251 cells were scarce when observed under CSLM in three groups, namely, difopein-treated group (Group A), empty vector group (Group B) and blank control group (Group C). The experimental results showed that with time delaying (24–48 h), apoptotic cells gradually increased in Group A. Besides, more late apoptotic cells were observed 48 hours after transfection in Group A.


W. Cao, X. Yang, J. Zhou, Z. Teng, L. Cao, X. Zhang and Z. Fei. Targeting 14-3-3 protein, difopein induces apoptosis of human glioma cells and suppresses tumor growth in mice. Apoptosis, 2010, 15(2), 230-241.

S. C. Masters, R. R. Subramanian, A. Truong, H. Yang, K. Fujii and H. Zhang. Survival-promoting functions of 14-3-3 proteins. Biochem. Soc. Transactions, 2002, 30(4), 360.

S. C. Masters and H. Fu. 14-3-3 proteins mediate an essential anti-apoptotic signal. J. Biol. Chem., 2001, 276(48), 45193-45200.

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