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

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-Go cells and intracellular DNA fragmentation. Activation of all intracellular caspases, except caspase-1, was detected in I-Trp-treated cells. Proteomic analysis revealed that P-tubulin acted as a specific intracellular target of I-Trp. Protein fingerprinting analysis indicated that the Cys(354) residue in the peptide fragment TAVCDIPPR of beta-tubulin, which is located at the binding interface with chaperonin containing TCP1-beta (CCT-beta), was alkylated by I-Trp. Moreover, site-directed mutagenesis of Cys(354) (Cys-Ala) abolished the incorporation of I-Trp into beta-tubulin, suggesting Cys(354) is indeed the targeting site of I-Trp. Immunoprecipitation showed that the beta-tubulin/CCT-beta complex was constitutively formed but disrupted after treatment with I-Trp. Overexpression of the truncated beta-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-beta relative to wild-type MES-SA cancer cells. Silencing the expression of CCT-beta rendered MES-SA/Dx5 cells less sensitive to I-Trp-induced apoptotic cell death. These findings suggest that the beta-tubulin/CCT-beta complex may serve as an effective chemotherapeutic target for treating clinical tubulin-binding agent-resistant or CCT-beta-overexpressing tumors. [Cancer Res 2009;69(17):6879-88]