TY - JOUR
T1 - Intracellular β-tubulin/chaperonin containing TCP1-β complex serves as a novel chemotherapeutic target against drug-resistant tumors
AU - Lin, Yuan Feng
AU - Tsai, Wen Ping
AU - Liu, Hon Ge
AU - Liang, Po Huang
PY - 2009/9/1
Y1 - 2009/9/1
N2 - 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-G 0 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.
AB - 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-G 0 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.
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U2 - 10.1158/0008-5472.CAN-08-4700
DO - 10.1158/0008-5472.CAN-08-4700
M3 - Article
C2 - 19690144
AN - SCOPUS:70149124085
SN - 0008-5472
VL - 69
SP - 6879
EP - 6888
JO - Cancer Research
JF - Cancer Research
IS - 17
ER -