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Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis.

Bilim V, Yuuki K, Itoi T, Muto A, Kato T, Nagaoka A, Motoyama T, Tomita Y - Br. J. Cancer (2008)

Bottom Line: 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).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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Yamagata University School of Medicine, Iida-nishi 2-2-2, Yamagata 990-9585, Japan.

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.

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Western blot analysis of XIAP and Smac expression in paired tissue samples from normal kidney and RCC (A). Smac levels were lower in RCC than in normal kidney. Expression of XIAP and Smac in a panel of RCC cell lines (B). Both molecules were ubiquitously expressed, with the highest levels of XIAP in Caki1 cells. β-Actin was used as a control for loading.
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fig2: Western blot analysis of XIAP and Smac expression in paired tissue samples from normal kidney and RCC (A). Smac levels were lower in RCC than in normal kidney. Expression of XIAP and Smac in a panel of RCC cell lines (B). Both molecules were ubiquitously expressed, with the highest levels of XIAP in Caki1 cells. β-Actin was used as a control for loading.

Mentions: The presence of both proteins was confirmed by western blotting of selected pairs of normal–tumour tissue samples (Figure 2A; data not shown) and Smac levels were lower in tumour tissues than in paired normal kidneys. XIAP protein was detected in all RCC cell lines examined and the expression differed slightly among the cells, with the highest level in Caki1 cells (Figure 2B). A Smac-specific band was also detected in all cell lines, with the highest level of expression in KRC/Y and the lowest in A498 (Figure 2B).


Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis.

Bilim V, Yuuki K, Itoi T, Muto A, Kato T, Nagaoka A, Motoyama T, Tomita Y - Br. J. Cancer (2008)

Western blot analysis of XIAP and Smac expression in paired tissue samples from normal kidney and RCC (A). Smac levels were lower in RCC than in normal kidney. Expression of XIAP and Smac in a panel of RCC cell lines (B). Both molecules were ubiquitously expressed, with the highest levels of XIAP in Caki1 cells. β-Actin was used as a control for loading.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266840&req=5

fig2: Western blot analysis of XIAP and Smac expression in paired tissue samples from normal kidney and RCC (A). Smac levels were lower in RCC than in normal kidney. Expression of XIAP and Smac in a panel of RCC cell lines (B). Both molecules were ubiquitously expressed, with the highest levels of XIAP in Caki1 cells. β-Actin was used as a control for loading.
Mentions: The presence of both proteins was confirmed by western blotting of selected pairs of normal–tumour tissue samples (Figure 2A; data not shown) and Smac levels were lower in tumour tissues than in paired normal kidneys. XIAP protein was detected in all RCC cell lines examined and the expression differed slightly among the cells, with the highest level in Caki1 cells (Figure 2B). A Smac-specific band was also detected in all cell lines, with the highest level of expression in KRC/Y and the lowest in A498 (Figure 2B).

Bottom Line: 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).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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Yamagata University School of Medicine, Iida-nishi 2-2-2, Yamagata 990-9585, Japan.

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.

Show MeSH
Related in: MedlinePlus