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.

Figure 1

Immunohistochemical examination of Smac (A, C) and XIAP (B, D) in normal kidney (A, B) and RCC (C, D). In normal kidney, Smac expression was detected in tubular epithelial cells, Bowman capsule cells, and a portion of glomerular cells (A). In tumours, Smac staining was weaker than in normal kidney (C). XIAP expression was restricted to tubular epithelial and several glomerular cells in normal kidney (B).

Figure 2

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.

Figure 3

Western blot analysis of XIAP in Caki1 cells transfected with an XIAP-targeting shRNA vector (A). Figures above the panel indicate normalised expression (%). Clone nos. 14 and 19 with the lowest levels of XIAP were selected for further experiments. The relative viability (MTS assay) of Caki1 parental cells, mock-transfected cells, and clone nos. 14 and 19 treated with the indicated doses of adriamycin, mitomycin C, cisplatin, and docetaxel for 24 h is shown (B).

Figure 4

RT–PCR analysis (A) and western blot analysis (B) for Bcl-2 and IAP family members in the Caki1 parental cells, mock-transfected cells, and clone nos. 14 and 19. β-Actin was used as a control for loading. The messengers of all examined genes were present in the cells and remained unchanged. Apoptosis-related proteins were also not changed except cIAP-1 and cIAP-2, which were slightly increased in mock transfectants and clone nos. 14 and 19, which presumably reflects the effect of cellular stress during transfection and selection process.

Figure 5

Relative viability (MTS assay) of Caki1 parental cells, mock-transfected cells, and clone nos. 14 and 19 treated with the small-molecule Bcl-2 inhibitor HA14-1 for 24 h (A). The same cells treated with either CH11 (500 ng ml⁻¹) Fas-stimulating antibody or a combination of CH11 (500 ng ml⁻¹) and HA14-1 (25 μg ml⁻¹) for 24 h (B). One-way ANOVA with a Tukey post-test to compare all pairs of values was used. (C) Cells untreated (left column) or treated with a combination of CH11 (500 ng ml⁻¹) and HA14-1 (25 μg ml⁻¹) for 24 h (right column) were fixed and stained with PI and further analysed on a flow cytometer to detect sub-G1 population – a late apoptotic event. Figures indicate percentage of cells of sub-G1 population. A drastic increase in sub-G1 population was observed in clone nos. 14 and 19. (D) Western blot of untreated control cells (C) or cells treated with a combination of CH11 (500 ng ml⁻¹) and HA14-1 (25 μg ml⁻¹) for 24 h (T). Membranes were probed with anti-caspase 3 or anti-PARP antibody. The upper panel presents caspase-3 blot after normal exposure showing a decrease in zymogen caspase 3 in clone nos. 14 and 19, and the lower panel shows overexposed blot with clearly observed caspase-3-cleaved fragments in clone nos. 14 and 19 after double treatment. PARP cleavage was also observed, indicating activation of biochemical apoptotic pathways. β-Actin was used as a control for loading.

Figure 5

Relative viability (MTS assay) of Caki1 parental cells, mock-transfected cells, and clone nos. 14 and 19 treated with the small-molecule Bcl-2 inhibitor HA14-1 for 24 h (A). The same cells treated with either CH11 (500 ng ml⁻¹) Fas-stimulating antibody or a combination of CH11 (500 ng ml⁻¹) and HA14-1 (25 μg ml⁻¹) for 24 h (B). One-way ANOVA with a Tukey post-test to compare all pairs of values was used. (C) Cells untreated (left column) or treated with a combination of CH11 (500 ng ml⁻¹) and HA14-1 (25 μg ml⁻¹) for 24 h (right column) were fixed and stained with PI and further analysed on a flow cytometer to detect sub-G1 population – a late apoptotic event. Figures indicate percentage of cells of sub-G1 population. A drastic increase in sub-G1 population was observed in clone nos. 14 and 19. (D) Western blot of untreated control cells (C) or cells treated with a combination of CH11 (500 ng ml⁻¹) and HA14-1 (25 μg ml⁻¹) for 24 h (T). Membranes were probed with anti-caspase 3 or anti-PARP antibody. The upper panel presents caspase-3 blot after normal exposure showing a decrease in zymogen caspase 3 in clone nos. 14 and 19, and the lower panel shows overexposed blot with clearly observed caspase-3-cleaved fragments in clone nos. 14 and 19 after double treatment. PARP cleavage was also observed, indicating activation of biochemical apoptotic pathways. β-Actin was used as a control for loading.

Figure 6

Relative viability (MTS assay) of Caki1 cells treated with indicated concentrations of Smac-Ant peptide (AVPIAQK) alone or in combination with cisplatin (50 μg ml⁻¹) for 24 h (A). The cells were exposed to 200 μM Smac-Ant peptide (AVPIAQK), cisplatin (50 μg ml⁻¹), or a combination of the two agents for 24, 48, and 72 h (B). One-way ANOVA with post-test for linear trend was used to analyse the data.

Figure 7

Phase contrast (left), Hoechst 33342 (middle), and Giemsa (right) photographs of Caki1 cells treated with 200 μM Smac-Ant peptide, 50 μg ml⁻¹ cisplatin, or a combination of the two agents for 24 h (A). (B) Higher magnification of phase contrast (upper panel) and Hoechst 33342 (lower panel) photographs of the same field of Caki1 cells treated with a combination of Smac-Ant peptide (AVPIAQK) (200 μM) and cisplatin (50 μg ml⁻¹). Cells undergo typical apoptotic morphological changes (condensed fragmented nuclei with formation of apoptotic bodies) as indicated by arrows.