Studying on the constitutively deposited XIAP:p19/p12-casp7 complexes in MCF-7 breast cancer cells as a target for I-Lys
Date Issued
2011
Date
2011
Author(s)
Dong, Jhih-Jhong
Abstract
Previously, from an iodoacetamide-based compound library, iodoacetyl-Boc-lysine (I-Lys) was identified to induce cell apoptosis in MCF-7 breast cancer cell line by directly activating caspase-7. In this thesis, I attempted to use X-ray crystallography, 2D NMR, and LC-mass to identify the binding site of I-Lys on caspase-7. We identify Cys246 residue as a targeting site of I-Lys in caspase-7 by LC-MS although the studies of X-ray crystallography and 2D NMR failed. Then we showed the targeting specificity of I-Lys on Cys246 in vivo by enforcedly expressing caspase-7 C246S mutation in MCF-7 cells. We also showed that caspase-7 C246S mutant still possess the activity by using staurosporine and caspase activity assay. By determining real-time caspase-7 activity in cells, we found that I-Lys immediately elevates intracellular caspase-7 activity without increaseing the active-form caspase-7 level by procaspase-7 activation/processing in MCF-7 cells . Immunoprecipitation of XIAP:caspase-7 complex proved that I-Lys specifically targets p19/p12-casp7 to induce apoptosis of MCF-7 cells by disrupting the XIAP: p19/p12-casp7 complex. The reduction of p19/p12-casp7 level in MCF-7 cells stably expressing caspase-7 D23A mutant indicates that the production of p19/p12-casp7 constitutively occur in MCF-7 cells. From a panel of cancerous and normal cells, we found exclusive deposition of XIAP:p19/p12-casp7 complex in caspase-3null breast tumors, conferring the specificity of I-Lys in killing those tumors over other caspase-3-expressing cells in vitro and in vivo. Finally, by reconstituting caspase-3 expression into MCF-7 cells, we found that MCF-7/CASP3 cells are resistant to I-Lys and therefore confirmed the specificity of I-Lys to caspase-3null breast tumors.
Overall, these studies find out the target of I-Lys and the constitutively deposited XIAP:p19/p12-casp7 complexes in MCF-7 cells, elucidate the mechanism of I-Lys to induce cell apoptosis in MCF-7 cells and to suggest a target of cancer treatment for caspase-3 down regulated breast tumors in clinical.
Subjects
breast cancer
caspase-7
XIAP
MCF-7
I-Lys
SDGs
Type
thesis
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