探討MCF-7乳癌細胞中常態堆積之XIAP:p19/p12-Casp7複合體作為I-Lys標靶

Abstract

先前我們合成了一化合物Iodoacetyl-Boc-lysine (I-Lys)並確認其在MCF-7乳癌細胞中，藉由直接活化caspase-7引發細胞凋亡的能力。在這篇論文中，我們試圖用X光結晶學、二維核磁共振以及液態層析質譜儀來確認I-Lys在caspase-7上的結合位置，雖然X光結晶學以及二維核磁共振的方式失敗，我們還是用液態層析質譜儀確認了caspase-7的半胱氨酸246為I-Lys的結合位置，藉由在MCF-7細胞中強迫表達C246S 突變的caspase-7，我們確認了I-Lys在MCF-7細胞中對半胱氨酸246的專一性，我們也藉由staurosporine以及caspase活性測試確認了C246S 突變的caspase-7的酵素仍具活性。而藉由即時測定細胞內caspase-7活性，我們發現I-Lys的加入會立即提升細胞內caspase-7的活性且此活性並非來自procaspase-7活化造成的活化型caspase-7總量上升。XIAP:caspase-7複合體的免疫沉澱證明了I-Lys專一的標定p19/p12-casp7並破壞XIAP: p19/p12-casp7複合體以引發MCF-7的細胞凋亡，在穩定表達D23A突變的caspase-7的MCF-7細胞中，p19/p12-casp7的數量減少指出了p19/p12-casp7在MCF-7中會持續存在。從一些癌症和正常細胞，我們發現XIAP:p19/p12-casp7只有表現在caspase-3不表達之乳癌細胞，給予了在對付caspase-3不表達的乳癌細胞時，以XIAP:p19/p12-casp7為標靶的專一性。最後，藉由在MCF-7細胞中重建caspase-3的表達，我們發現表達caspase-3的MCF-7對I-Lys具抵抗力並因此確定I-Lys對caspase-3不表達之乳癌細胞的專一性。 總括而論，這些研究發現了I-Lys的標的以及在MCF-7細胞中持續堆積的XIAP:p19/p12-casp7複合體，說明了I-Lys在MCF-7細胞中引發細胞凋亡的機制，並且提出一個對臨床上caspase-3表達量下降的乳癌細胞的治療標靶

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.