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 (Po0.0001) and to pharmacological Bcl-2 inhibition (Po0.0001), as well as to a combination of the two (Po0.0001). 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 ¼ 0.0031) and potentiated cisplatin's effect (P ¼ 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.

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma

“…Increased expression of Bcl-2 and XIAP anti-apoptotic molecules, NF-kB target genes has an important function in renal cancer cell survival (Tomita et al, 1996;Maruyama et al, 2006;Bilim et al, 2008). As GSK-3b has a positive role in expression of certain NF-kB-regulated genes (Hoeflich et al, 2000;Ougolkov et al, 2005Ougolkov et al, , 2007, we investigated whether inhibition of GSK-3 affects NF-kBmediated expression of Bcl-2 and XIAP in renal cancer cells.…”

“…For estimation of cell proliferation BrdU cell proliferation assay (Calbiochem) was applied according to the manufacturer's instructions as described earlier (Bilim et al, 2008). For detection of apoptotic morphology, cells were cultured in Lab-Tek Chambers (Nunc Inc., Naperville, IL, USA), treated with AR-A014418.…”

“…Cell viability was detected with a colorimetric assay, the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) using tetrazolium compound according to the manufacturer's instructions as described earlier (Bilim et al, 2008). For estimation of cell proliferation BrdU cell proliferation assay (Calbiochem) was applied according to the manufacturer's instructions as described earlier (Bilim et al, 2008).…”

“…Immunohistochemical staining was performed as described earlier (Bilim et al, 2008). Two different sections from each tumour were examined by immunohistochemistry.…”

“…Increased expression of Bcl-2 and XIAP anti-apoptotic molecules, NF-kB target genes, has an important function in renal cancer cell survival and chemoresistance (Bilim et al, 2008) and resistance to immunotherapy (Maruyama et al, 2006). Previous studies suggest a positive role for GSK-3b in the regulation of NF-kB activity (Hoeflich et al, 2000;Ougolkov et al, 2005Ougolkov et al, , 2007.…”