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. 2009 Nov 6:9:392.
doi: 10.1186/1471-2407-9-392.

A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced apoptosis via modulating both IAPs and NF-kappaB

Yao Dai  1 , Meilan Liu, Wenhua Tang, Yongming Li, Jiqin Lian, Theodore S Lawrence, Liang Xu
Affiliations

A Smac-mimetic sensitizes prostate cancer cells to TRAIL-induced apoptosis via modulating both IAPs and NF-kappaB

Yao Dai et al. BMC Cancer. .

Abstract

Background: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for human cancer therapy, prostate cancer still remains resistant to TRAIL. Both X-linked inhibitor of apoptosis (XIAP) and nuclear factor-kappaB function as key negative regulators of TRAIL signaling. In this study, we evaluated the effect of SH122, a small molecule mimetic of the second mitochondria-derived activator of caspases (Smac), on TRAIL-induced apoptosis in prostate cancer cells.

Methods: The potential of Smac-mimetics to bind XIAP or cIAP-1 was examined by pull-down assay. Cytotoxicity of TRAIL and/or Smac-mimetics was determined by a standard cell growth assay. Silencing of XIAP or cIAP-1 was achieved by transient transfection of short hairpin RNA. Apoptosis was detected by Annexin V-PI staining followed by flow cytometry and by Western Blot analysis of caspases, PARP and Bid. NF-kappaB activation was determined by subcellular fractionation, real time RT-PCR and reporter assay.

Results: SH122, but not its inactive analog, binds to XIAP and cIAP-1. SH122 significantly sensitized prostate cancer cells to TRAIL-mediated cell death. Moreover, SH122 enhanced TRAIL-induced apoptosis via both the death receptor and the mitochondrial pathway. Knockdown of both XIAP and cIAP-1 sensitized cellular response to TRAIL. XIAP-knockdown attenuated sensitivity of SH122 to TRAIL-induced cytotoxicity, confirming that XIAP is an important target for IAP-inhibitor-mediated TRAIL sensitization. SH122 also suppressed TRAIL-induced NF-kappaB activation by preventing cytosolic IkappaB-alpha degradation and RelA nuclear translocation, as well as by suppressing NF-kappaB target gene expression.

Conclusion: These results demonstrate that SH122 sensitizes human prostate cancer cells to TRAIL-induced apoptosis by mimicking Smac and blocking both IAPs and NF-kappaB. Modulating IAPs may represent a promising approach to overcoming TRAIL-resistance in human prostate cancer with constitutively active NF-kappaB signaling.

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Figures

Figure 1
Figure 1
Smac-mimetic compound interacted with XIAP and cIAP-1 in human prostate cancer cells. A. Structure of the Smac-mimetic compound SH122, and its inactive control compounds SH123 and SH110. B. Pull-down assay. Biotin-labeled SH122 (SH122BL, 20 μM) was incubated with whole cell lysates of DU145 cells with or without 10-fold excess of non-labeled SH122, followed by incubation with precleared Streptavidin agarose beads. Eluted beads were employed to Western blot analysis with anti-XIAP or cIAP-1 antibody. Data shown represent one of at least three independent experiments. SH122BL, Biotin-labeled SH122. C. CL1 cell lysate was incubated with 1 μM and 2.5 μM of SH122BL, or 2.5 μM of SH-123BL, with or without 10-fold excess of their unlabeled forms. SH123BL, Biotin-labeled SH123.
Figure 2
Figure 2
SH122 promoted TRAIL-induced cell death in prostate cancer cell line DU145 (A), LNCaP (B) and CL1 (C). Cells (5,000 cells/well in 96-well plates) were treated with different concentrations of TRAIL and SH122, alone or in combination, with SH110 as a negative control. After incubation for 96 h, cells were stained with Cell Counting kit-8 reagent. The optical density of each sample was measured. Data were normalized as described in Materials and Methods. Data were presented as mean ± SD (n = 3).
Figure 3
Figure 3
SH122 enhanced TRAIL-induced apoptosis. DU145 cells were seeded into 6-well plates at a concentration of 2 × 105/ml, and exposed by 2.5, 5 and 10 μM of SH122, with or without TRAIL (50 ng/ml). Eighteen hours after incubation, cells were harvested and processed for Annexin V-FITC and PI staining by flow cytometry. Numbers represented total apoptosis (Annexin V positive cell population). Data represented one of three independent experiments.
Figure 4
Figure 4
SH122 potentiated TRAIL-induced apoptosis by activating both the death-receptor pathway (A) and mitochondrial pathway (B). DU145 cells were treated with 10 and 20 μM of SH122, in the presence or absence of 300 ng/ml of TRAIL, with or without pretreatment with zVAD (2 μM), for 4, 6, and 8 h, respectively. Whole cell lysates (30 μg) were subjected to Western blot analysis. Membranes were probed with antibodies against caspase-8, caspase-3, caspase-9, PARP and Bid. Actin was shown as a loading control.
Figure 5
Figure 5
Downregulation of XIAP or cIAP-1 sensitized TRAIL-induced cell death. DU145 cells were transfected with shRNA of XIAP (shXIAP) (A, B) or cIAP-1 (shcIAP-1) (C, D), or the vector control (shVector), and knockdown effect was measured 48 h after transfection (A, C). Transfected cells were treated with TRAIL, and cytotoxicity was determined by CCK-8 detection kit (B, D). Normalized data were presented as mean ± SD (n = 3).
Figure 6
Figure 6
Downregulation of XIAP attenuated sensitization effect of SH122 on TRAIL-induced cell death. Cells transfected with either XIAP shRNA (A) or the vector control shRNA (B) were treated with serial diluted TRAIL, alone or in combination with 0.1, 1 and 10 μM of SH122, respectively, with 10 μM of SH110 as a negative control. Cytotoxicity was determined by CCK-8 detection kit. Normalized data were presented as mean ± SD (n = 3). Fold of sensitization was calculated by dividing IC50 of the compound-treated group by that of DMSO control.
Figure 7
Figure 7
SH122 suppressed TRAIL-induced NF-κB activation. A. DU145 cells were treated with 300 ng/ml of TRAIL for the indicated time. Cytosol and nuclei subcompartments were fractionated for detection of IκBα and RelA, with Actin and PARP used as markers of cytosolic and nuclear extracts, respectively. Relative expression of IκBα and RelA was shown by dividing band intensity by that of Actin and PARP, respectively. CE, cytosolic extract; NE, nuclear extract. B. After treatment with 300 ng/ml of TRAIL, cells were harvested for quantitative RT-PCR detect NF-κB target genes. Fold increase of gene expression was calculated by dividing the normalized gene expression activity by that of the untreated control. C. Cells were pre-treated with desired compounds for 1 h, and challenged with TRAIL (300 ng/ml) for 40 min. Cytosol and nuclei subcellular compartments were fractionated for detection of IκBα and RelA, respectively. MG132 was used as a positive control for blocking NF-κB. D. After treatment as described in C, cells were harvested for quantitative RT-PCR to detect three NF-κB target genes. E, Time-course of TRAIL-induced NF-κB activation examined by NF-κB luciferase reporter assay. DU145 cells were transiently co-transfected with pNF-κB or pControl together with β-galactosidase plasmid, and then treated with TRAIL for the indicated time. Luciferase and β-galactosidase activities were measured as described in Materials and Methods. F, SH122 inhibited TRAIL-induced NF-κB activation in NF-κB luciferase reporter assay. Transfected DU145 cells were pretreated with SH122 or SH123 for 1 h followed by TRAIL treatment for 4 h. Fold of NF-κB activation was calculated by dividing the normalized luciferase activity by that of the untreated control. Representative results of at least two independent experiments. Columns, mean; bars, SD (n = 3). Con, DMSO vehicle control. ** P < 0.01; * P < 0.05, Student's t-test (n = 3).
Figure 8
Figure 8
Working model of Smac-mimetic IAP-antagonist sensitizing TRAIL-induced apoptosis by suppressing NF-κB. TRAIL triggers apoptosis via both the death-receptor (DR4/DR5) and mitochondrial pathways, by activating initiator caspase-8 and -9, and effector caspase-3. Furthermore, both Bid and PARP are cleaved by caspases, which are typical predictors of TRAIL-mediated apoptosis. A Smac-mimetic effectively blocks IAP (XIAP/cIAP-1) function and facilitates caspase activation. Simultaneously, the Smac-mimetic suppresses TRAIL-induced classical NF-κB activation by preventing IκBα degradation and RelA nuclear translocation. Blockade of NF-κB - XIAP signaling by small molecule Smac-mimetic abolishes counteraction of pro-survival factors on TRAIL-mediated apoptosis.
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References

    1. DiPaola RS, Patel J, Rafi MM. Targeting apoptosis in prostate cancer. Hematol Oncol Clin North Am. 2001;15(3):509–524. doi: 10.1016/S0889-8588(05)70229-X. - DOI - PubMed
    1. Xu L, Frederik P, Pirollo KF, Tang WH, Rait A, Xiang LM, Huang W, Cruz I, Yin Y, Chang EH. Self-assembly of a virus-mimicking nanostructure system for efficient tumor-targeted gene delivery. Hum Gene Ther. 2002;13(3):469–481. doi: 10.1089/10430340252792594. - DOI - PubMed
    1. Bucur O, Ray S, Bucur MC, Almasan A. APO2 ligand/tumor necrosis factor-related apoptosis-inducing ligand in prostate cancer therapy. Front Biosci. 2006;11:1549–1568. doi: 10.2741/1903. - DOI - PubMed
    1. Shankar S, Siddiqui I, Srivastava RK. Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells. Mol Cell Biochem. 2007;304(1-2):273–285. doi: 10.1007/s11010-007-9510-x. - DOI - PubMed
    1. Steele LP, Georgopoulos NT, Southgate J, Selby PJ, Trejdosiewicz LK. Differential susceptibility to TRAIL of normal versus malignant human urothelial cells. Cell Death Differ. 2006;13(9):1564–1576. doi: 10.1038/sj.cdd.4401846. - DOI - PubMed

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