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Intracellular β-Tubulin/Chaperonin Containing TCP1-β Complex Serves as a Novel Chemotherapeutic Target against Drug-Resistant Tumors

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan R.O.C.

Requests for reprints: Po-Huang Liang, Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Taipei, 11529, Taiwan. Phone: 886-2-2785-5696, ext. 6070; Fax: 886-2-2788-9759; E-mail: phliang{at}gate.sinica.edu.tw.

Key Words: tubulin • CCT • anticancer • cell apoptosis • cysteine

In the present study, treatment of HEK-293 cells with the synthetic small molecule N-iodoacetyl-tryptophan (I-Trp) at submicromolar concentrations efficiently induced cell apoptosis as judged from the accumulation of sub-G₀ cells and intracellular DNA fragmentation. Activation of all intracellular caspases, except caspase-1, was detected in I-Trp–treated cells. Proteomic analysis revealed that β-tubulin acted as a specific intracellular target of I-Trp. Protein fingerprinting analysis indicated that the Cys³⁵⁴ residue in the peptide fragment TAVCDIPPR of β-tubulin, which is located at the binding interface with chaperonin containing TCP1-β (CCT-β), was alkylated by I-Trp. Moreover, site-directed mutagenesis of Cys³⁵⁴ (Cys-Ala) abolished the incorporation of I-Trp into β-tubulin, suggesting Cys³⁵⁴ is indeed the targeting site of I-Trp. Immunoprecipitation showed that the β-tubulin/CCT-β complex was constitutively formed but disrupted after treatment with I-Trp. Overexpression of the truncated β-tubulin (T351-S364) or treatment with I-Trp or the synthetic peptide Myr-TAVCDIPPRG caused more severe cell apoptosis in multidrug-resistant MES-SA/Dx5 cancer cells due to higher levels of CCT-β relative to wild-type MES-SA cancer cells. Silencing the expression of CCT-β rendered MES-SA/Dx5 cells less sensitive to I-Trp–induced apoptotic cell death. These findings suggest that the β-tubulin/CCT-β complex may serve as an effective chemotherapeutic target for treating clinical tubulin-binding agent-resistant or CCT-β–overexpressing tumors. [Cancer Res 2009;69(17):6879–88]