title: Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis.
abstract: Renal cell carcinoma (RCC) is known to be resistant to chemo- and radiotherapy due to a high apoptotic threshold. Smac and XIAP (X-linked inhibitor of apoptosis protein) proteins were detected in all RCC cell lines and tissue samples examined. We modulated the function of XIAP, either through its constitutional downregulation with an shRNA vector or by applying a Smac-mimicking peptide. Among RCC cell lines, Caki1 expresses the highest levels of XIAP. We transfected Caki1 with XIAP-targeting shRNA vector and generated stable clones. XIAP was knocked down by RNA interference in clone no. 14 by 81.6% and in clone no. 19 by 85.3%. Compared to the parental and mock-transfected cells, neither clone was more sensitive to conventional chemotherapeutic agents, but both clones were more susceptible to Fas stimulation (P<0.0001) and to pharmacological Bcl-2 inhibition (P<0.0001), as well as to a combination of the two (P<0.0001). Mature Smac binds to XIAP via the N-terminal residues, disrupting its interaction with caspases and promoting their activity. We determined that exposure of Caki1 cells to Smac-N7 peptide (AVPIAQK) resulted in a slight but significant decrease in viability (P=0.0031) and potentiated cisplatin's effect (P=0.0027). In contrast with point targeting of XIAP by shRNA, Smac-N7 peptide is active against several IAP (inhibitor of apoptosis protein) family members, which can explain its role in sensitising cells to cisplatin. Our results suggest that multiple targeting of both Bcl-2 and XIAP or, alternatively, of several IAP family members by the Smac-N7 peptide is a potent way to overcome resistance of RCC to apoptosis-triggering treatment modalities, and might be a new tool for molecular targeted therapy.
authors.affiliation: Department of Urology, Yamagata University School of Medicine, Iida-nishi 2-2-2, Yamagata 990-9585, Japan.
authors.forename: V
authors.initials: V
authors.lastname: Bilim
authors.forename: K
authors.initials: K
authors.lastname: Yuuki
authors.forename: T
authors.initials: T
authors.lastname: Itoi
authors.forename: A
authors.initials: A
authors.lastname: Muto
authors.forename: T
authors.initials: T
authors.lastname: Kato
authors.forename: A
authors.initials: A
authors.lastname: Nagaoka
authors.forename: T
authors.initials: T
authors.lastname: Motoyama
authors.forename: Y
authors.initials: Y
authors.lastname: Tomita
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002292:Carcinoma, Renal Cell / Q000188:drug therapy* / Q000473:pathology; D045744:Cell Line, Tumor; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000494:pharmacology; D007680:Kidney Neoplasms / Q000188:drug therapy* / Q000473:pathology; D024101:Mitochondrial Proteins / Q000032:analysis / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000032:analysis / Q000037:antagonists & inhibitors*
chemical_list: D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D010446:Peptide Fragments; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human