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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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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.
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fig5: 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.

Mentions: To explore the effect of a constitutional decrease in XIAP on cell survival and susceptibility to apoptosis, we generated stable Caki1 clones of shRNA expressing vector targeting XIAP (TA0025-4) as well as a control vector as described in Materials and Methods. In the two clones selected for further experiments, XIAP protein was suppressed by 79.4% in clone no. 14 and by 86% in clone no. 19 (Figure 3A). The stable transfectants and mock clone did not demonstrate any morphological difference from the parental cells. We studied sensitivity of the cells to conventional anticancer drugs, which are considered to exert their effect through induction of apoptosis and are widely used in urology to treat bladder, testicular, and prostate cancer. Unexpectedly, all the cells, including parental cells and mock clones, showed equal sensitivity to adriamycin, mitomycin C, cisplatin, and docetaxel (Figure 3B). As there is redundancy in the IAP family and other members can be reciprocally upregulated to substitute for XIAP, we performed RT–PCR and western blotting for apoptosis-related genes. The messengers of Bcl-2 and IAP family members examined were present and did not show drastic changes among the parental cells, mock transfectants, and clone nos. 14 and 19 (Figure 4A). Furthermore, to check for protein levels, we performed western blot analysis. The levels of Bcl-2 family members were unchanged but those of c-IAP1 and c-IAP2 (Figure 4B) increased slightly in mock cells and transfectants. This was not a specific modulation due to downregulation of XIAP, as it was also observed in mock cells. Rather, it can be explained by the effect of cellular stress due to transfection and selection process. The knockdown of XIAP by RNAi sensitised the cells to HA14-1, a small-molecule Bcl-2 inhibitor (Figure 5A), but this effect was moderate and observed only at a single concentration of HA14-1 (25 μg ml−1). At this dose, the overall P-value was less than 0.0001. P-values between each pair of the cells calculated with the post-test are presented in Figure 5A. As XIAP inhibition sensitised chronic lymphocytic leukaemia cells (Kater et al, 2005) and RCC cells (Mizutani et al, 2007) to Fas-induced apoptosis, we treated the parental cells, mock transfectants, and clone nos. 14 and 19 with CH11 IgM Fas-stimulating monoclonal antibody (Figure 5B). Overall, the viability of clone nos. 14 and 19 was significantly lower than that of the parental cells or mock transfectants (P<0.0001). P-values between each pair of the cells calculated with the post-tests are presented in Figure 5B. Double treatment with CH11 and HA14-1 resulted in a significant (P<0.0001) decrease in the viability of clone nos. 14 and 19 (Figure 5B), and a synergistic effect of the two compounds was observed in parental cells, mock transfectants, and clone no. 19. As the decrease in relative viability can be attributed to decreased cell proliferation, nonspecific toxicity, and non-apoptotic cell death, we performed additional experiments to clear this point. There was no difference in BrdU incorporation, which is a marker of cell proliferation, between untreated and treated cells (data not shown). The proportion of annexin V-positive cells, which is an early apoptosis marker, increased in double-treated cells from 5.0% (parental) and 3.05% (mock no. 5) to 6.03% (clone no. 14) and 9.41% (clone no. 19). Furthermore, there was a drastic increase in sub-G1 population after PI staining in clone nos. 14 (21.59%) and 19 (29.73%) compared to parental (8.01%) and mock transfectants (12.12%) (Figure 5C). This was accompanied with PARP and caspase-3 cleavage (Figure 5D, the upper panel presents caspase-3 blot after normal exposure demonstrating consumption of the proform of the protein and the lower panel shows overexposed blot with clearly observed caspase-3-cleaved fragments in clone nos. 14 and 19 after double treatment), indicating activation of biochemical apoptotic pathways. These data confirm the hypothesis that the decrease in viability of clone nos. 14 and 19 was due to acceleration of apoptosis.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: 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.
Mentions: To explore the effect of a constitutional decrease in XIAP on cell survival and susceptibility to apoptosis, we generated stable Caki1 clones of shRNA expressing vector targeting XIAP (TA0025-4) as well as a control vector as described in Materials and Methods. In the two clones selected for further experiments, XIAP protein was suppressed by 79.4% in clone no. 14 and by 86% in clone no. 19 (Figure 3A). The stable transfectants and mock clone did not demonstrate any morphological difference from the parental cells. We studied sensitivity of the cells to conventional anticancer drugs, which are considered to exert their effect through induction of apoptosis and are widely used in urology to treat bladder, testicular, and prostate cancer. Unexpectedly, all the cells, including parental cells and mock clones, showed equal sensitivity to adriamycin, mitomycin C, cisplatin, and docetaxel (Figure 3B). As there is redundancy in the IAP family and other members can be reciprocally upregulated to substitute for XIAP, we performed RT–PCR and western blotting for apoptosis-related genes. The messengers of Bcl-2 and IAP family members examined were present and did not show drastic changes among the parental cells, mock transfectants, and clone nos. 14 and 19 (Figure 4A). Furthermore, to check for protein levels, we performed western blot analysis. The levels of Bcl-2 family members were unchanged but those of c-IAP1 and c-IAP2 (Figure 4B) increased slightly in mock cells and transfectants. This was not a specific modulation due to downregulation of XIAP, as it was also observed in mock cells. Rather, it can be explained by the effect of cellular stress due to transfection and selection process. The knockdown of XIAP by RNAi sensitised the cells to HA14-1, a small-molecule Bcl-2 inhibitor (Figure 5A), but this effect was moderate and observed only at a single concentration of HA14-1 (25 μg ml−1). At this dose, the overall P-value was less than 0.0001. P-values between each pair of the cells calculated with the post-test are presented in Figure 5A. As XIAP inhibition sensitised chronic lymphocytic leukaemia cells (Kater et al, 2005) and RCC cells (Mizutani et al, 2007) to Fas-induced apoptosis, we treated the parental cells, mock transfectants, and clone nos. 14 and 19 with CH11 IgM Fas-stimulating monoclonal antibody (Figure 5B). Overall, the viability of clone nos. 14 and 19 was significantly lower than that of the parental cells or mock transfectants (P<0.0001). P-values between each pair of the cells calculated with the post-tests are presented in Figure 5B. Double treatment with CH11 and HA14-1 resulted in a significant (P<0.0001) decrease in the viability of clone nos. 14 and 19 (Figure 5B), and a synergistic effect of the two compounds was observed in parental cells, mock transfectants, and clone no. 19. As the decrease in relative viability can be attributed to decreased cell proliferation, nonspecific toxicity, and non-apoptotic cell death, we performed additional experiments to clear this point. There was no difference in BrdU incorporation, which is a marker of cell proliferation, between untreated and treated cells (data not shown). The proportion of annexin V-positive cells, which is an early apoptosis marker, increased in double-treated cells from 5.0% (parental) and 3.05% (mock no. 5) to 6.03% (clone no. 14) and 9.41% (clone no. 19). Furthermore, there was a drastic increase in sub-G1 population after PI staining in clone nos. 14 (21.59%) and 19 (29.73%) compared to parental (8.01%) and mock transfectants (12.12%) (Figure 5C). This was accompanied with PARP and caspase-3 cleavage (Figure 5D, the upper panel presents caspase-3 blot after normal exposure demonstrating consumption of the proform of the protein and the lower panel shows overexposed blot with clearly observed caspase-3-cleaved fragments in clone nos. 14 and 19 after double treatment), indicating activation of biochemical apoptotic pathways. These data confirm the hypothesis that the decrease in viability of clone nos. 14 and 19 was due to acceleration of apoptosis.

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