Role of XIAP in inhibiting cisplatin-induced caspase activation in non-small cell lung cancer cells: a small molecule Smac mimic sensitizes for chemotherapy-induced apoptosis by enhancing caspase-3 activation.

X-linked IAP (XIAP) suppresses apoptosis by binding to initiator caspase-9 and effector caspases-3 and -7. Smac/DIABLO that is released from mitochondria during apoptosis can relieve its inhibitory activity. Here we investigated the role of XIAP in the previously found obstruction of chemotherapy-induced caspase-9 activation in non-small cell lung cancer (NSCLC) cells. Endogenously expressed XIAP bound active forms of both caspase-9 and caspase-3. However, downregulation of XIAP using shRNA or disruption of XIAP/caspase-9 interaction using a small molecule Smac mimic were unable to significantly induce caspase-9 activity, indicating that despite a strong binding potential of XIAP to caspase-9 it is not a major determinant in blocking caspase-9 in NSCLC cells. Although unable to revert caspase-9 blockage, the Smac mimic was able to enhance cisplatin-induced apoptosis, which was accompanied by increased caspase-3 activity. Additionally, a more detailed analysis of caspase activation in response to cisplatin indicated a reverse order of activation, whereby caspase-3 cleaved caspase-9 yielding an inactive form. Our findings indicate that the use of small molecule Smac mimic, when combined with an apoptotic trigger, may have therapeutic potential for the treatment of NSCLC.

[1]  Xiaodong Wang,et al.  A Small Molecule Smac Mimic Potentiates TRAIL- and TNFα-Mediated Cell Death , 2004, Science.

[2]  S. Agrawal,et al.  Loss of XIAP protein expression by RNAi and antisense approaches sensitizes cancer cells to functionally diverse chemotherapeutics , 2004, Oncogene.

[3]  G. Salvesen,et al.  XIAP inhibits caspase‐3 and ‐7 using two binding sites: evolutionarily conserved mechanism of IAPs , 2005, The EMBO journal.

[4]  G. Salvesen,et al.  Neutralization of Smac/Diablo by Inhibitors of Apoptosis (IAPs) , 2004, Journal of Biological Chemistry.

[5]  C. Thompson,et al.  Apoptosis in the pathogenesis and treatment of disease , 1995, Science.

[6]  J C Reed,et al.  Caspase-9 Can Be Activated without Proteolytic Processing* , 1999, The Journal of Biological Chemistry.

[7]  N. Thornberry.  Caspases: key mediators of apoptosis. , 1998, Chemistry & biology.

[8]  G. Giaccone,et al.  Assessment of IAP (inhibitor of apoptosis) proteins as predictors of response to chemotherapy in advanced non-small-cell lung cancer patients. , 2001, Annals of oncology : official journal of the European Society for Medical Oncology.

[9]  Young Chul Park,et al.  Structural Basis of Caspase Inhibition by XIAP Differential Roles of the Linker versus the BIR Domain , 2001, Cell.

[10]  G. Gores,et al.  Synthetic Smac/DIABLO Peptides Enhance the Effects of Chemotherapeutic Agents by Binding XIAP and cIAP1 in Situ * , 2002, The Journal of Biological Chemistry.

[11]  D. Vaux,et al.  Alterations in the apoptotic machinery and their potential role in anticancer drug resistance , 2003, Oncogene.

[12]  Yves Pommier,et al.  Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks , 2004, Oncogene.

[13]  B. Dörken,et al.  Smac induces cytochrome c release and apoptosis independently from Bax/Bcl-xL in a strictly caspase-3-dependent manner in human carcinoma cells , 2004, Oncogene.

[14]  John Calvin Reed,et al.  The c‐IAP‐1 and c‐IAP‐2 proteins are direct inhibitors of specific caspases , 1997, The EMBO journal.

[15]  E. Kohmura,et al.  Synthetic Smac peptide enhances the effect of etoposide-induced apoptosis in human glioblastoma cell lines , 2006, Journal of Neuro-Oncology.

[16]  Geng Wu,et al.  Structural basis of IAP recognition by Smac/DIABLO , 2000, Nature.

[17]  G. Giaccone,et al.  TUCAN/CARDINAL/CARD8 and apoptosis resistance in non-small cell lung cancer cells , 2006, BMC Cancer.

[18]  Boris Zhivotovsky,et al.  DNA damage-induced apoptosis , 2004, Oncogene.

[19]  Emad S. Alnemri,et al.  A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis , 2001, Nature.

[20]  J. Liu,et al.  Dysfunctional apoptosome activation in ovarian cancer: implications for chemoresistance. , 2002, Cancer research.

[21]  Takashi Tsuruo,et al.  Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide. , 2003, Cancer research.

[22]  D. Hanahan,et al.  The Hallmarks of Cancer , 2000, Cell.

[23]  G. Giaccone,et al.  Expression and localization of inhibitor of apoptosis proteins in normal human tissues. , 2006, Human pathology.

[24]  G. Giaccone,et al.  Apoptosis: target of cancer therapy. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[25]  G. Salvesen,et al.  The Human Anti-apoptotic Proteins cIAP1 and cIAP2 Bind but Do Not Inhibit Caspases* , 2006, Journal of Biological Chemistry.

[26]  Xiaodong Wang,et al.  Structural and biochemical basis of apoptotic activation by Smac/DIABLO , 2000, Nature.

[27]  G. Peters,et al.  Drug-induced apoptosis in lung cnacer cells is not mediated by the Fas/FasL (CD95/APO1) signaling pathway. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[28]  M. Hengartner.  The biochemistry of apoptosis , 2000, Nature.

[29]  X. Liu,et al.  An APAF-1·Cytochrome c Multimeric Complex Is a Functional Apoptosome That Activates Procaspase-9* , 1999, The Journal of Biological Chemistry.

[30]  S. Srinivasula,et al.  Mechanism of XIAP-mediated inhibition of caspase-9. , 2003, Molecular cell.

[31]  W. Gerald,et al.  Inactivation of the apoptosis effector Apaf-1 in malignant melanoma , 2001, Nature.

[32]  Emad S. Alnemri,et al.  Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner , 1999, The Journal of cell biology.

[33]  G. Salvesen,et al.  Apoptosis: IAP proteins: blocking the road to death's door , 2002, Nature Reviews Molecular Cell Biology.

[34]  S. Srinivasula,et al.  Role of Smac in human leukaemic cell apoptosis and proliferation , 2003, Oncogene.

[35]  D. Schrump.  Genomic surgery for lung cancer. , 2004, The Journal of surgical research.

[36]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[37]  G. Salvesen,et al.  Caspase activation: the induced-proximity model. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[38]  G. Salvesen,et al.  Mechanisms of caspase activation. , 2003, Current opinion in cell biology.

[39]  S. Srinivasula,et al.  Structural Basis of Caspase-7 Inhibition by XIAP , 2001, Cell.

[40]  Yigong Shi,et al.  Caspases, IAPs and Smac/DIABLO: mechanisms from structural biology. , 2004, Trends in biochemical sciences.

[41]  Yi Sun,et al.  A small molecule Smac-mimic compound induces apoptosis and sensitizes TRAIL- and etoposide-induced apoptosis in breast cancer cells , 2005, Oncogene.

[42]  F. Brescia,et al.  Lung cancer--a philosophical, ethical, and personal perspective. , 2001, Critical reviews in oncology/hematology.

[43]  C. Duckett.  IAP proteins: sticking it to Smac. , 2005, The Biochemical journal.

[44]  Yigong Shi.  Caspase activation, inhibition, and reactivation: A mechanistic view , 2004, Protein science : a publication of the Protein Society.

[45]  Seamus J. Martin,et al.  Analysis of the composition, assembly kinetics and activity of native Apaf‐1 apoptosomes , 2004, The EMBO journal.

[46]  C. Ferreira,et al.  Correspondence re: L. Yang et al., Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide. Cancer Res., 63: 831-837, 2003. , 2003, Cancer research.

[47]  James A. Wells,et al.  Direct activation of the apoptosis machinery as a mechanism to target cancer cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[48]  John Calvin Reed.  Apoptosis-targeted therapies for cancer. , 2003, Cancer cell.

[49]  J C Reed,et al.  IAP family proteins--suppressors of apoptosis. , 1999, Genes & development.

[50]  Stephen W. Fesik,et al.  NMR Structure and Mutagenesis of the Third Bir Domain of the Inhibitor of Apoptosis Protein XIAP* , 2000, The Journal of Biological Chemistry.

[51]  J C Reed,et al.  IAPs block apoptotic events induced by caspase‐8 and cytochrome c by direct inhibition of distinct caspases , 1998, The EMBO journal.

[52]  Felicitas Genze,et al.  Inhibition of clonogenic tumor growth: a novel function of Smac contributing to its antitumor activity , 2005, Oncogene.

[53]  S H Kaufmann,et al.  Mammalian caspases: structure, activation, substrates, and functions during apoptosis. , 1999, Annual review of biochemistry.

[54]  R. Liddington,et al.  Structural Basis for the Inhibition of Caspase-3 by XIAP , 2001, Cell.