|Home | About | Journals | Submit | Contact Us | Français|
X-linked inhibitor of apoptosis (XIAP), the most potent member of the inhibitor of apoptosis protein (IAP) family of endogenous caspase inhibitors, blocks the initiation and execution phases of the apoptotic cascade. As such, XIAP represents an attractive target for treating apoptosis-resistant forms of cancer. Here, we demonstrate that treatment with the membrane-permeable zinc chelator, N,N,N’,N’,-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces a rapid depletion of XIAP at the posttranslational level in human PC-3 prostate cancer cells and several non-prostate cell lines. The depletion of XIAP is selective, as TPEN has no effect on the expression of other zinc-binding members of the IAP family including cIAP1, cIAP2, and survivin. The down-regulation of XIAP in TPEN-treated cells occurs via proteasome-and caspase-independent mechanisms and is completely prevented by the serine protease inhibitor, Pefabloc. Finally, our studies demonstrate that TPEN promotes activation of caspases-3 and -9 and sensitizes PC-3 prostate cancer cells to TRAIL-mediated apoptosis. Taken together, our findings indicate that zinc-chelating agents may be used to sensitize malignant cells to established cytotoxic agents via down-regulation of XIAP.
Inhibitor of apoptosis proteins (IAPs) are a family of caspase inhibitors that selectively bind and inhibit caspases at both the initiation phase and the execution phase of apoptosis 1. All IAPs contain 1–3 baculoviral IAP repeat (BIR) motifs. Each BIR domain folds into a functionally independent structure that binds a zinc ion. Additionally, many IAPs contain another zinc-binding motif, the RING domain, that has E3 ubiquitin ligase activity 2.
Of all members of the IAP family, XIAP has received the most interest, possibly because XIAP is the only member of this family able to directly inhibit both the initiation and execution phases of the caspase cascade 3. The BIR2 domain of XIAP binds and inhibits caspases-3 and -7. Overexpression of cDNA corresponding to the BIR2 domain inhibits apoptosis from both death receptor and mitochondrial pathway stimuli, consistent with its ability to inhibit effector caspases. 4,5. The BIR3 domain of XIAP binds and inhibits caspase-9, an apical caspase in the mitochondrial arm of the apoptotic pathway 5,6. Overexpression of cDNA corresponding to the BIR3 domain inhibits apoptosis in response to stimuli of the mitochondrial pathway of caspase activation, such as Bax, but not stimuli of the death receptor pathway 4. XIAP levels are elevated in many cancer cell lines and suppression of XIAP protein levels sensitizes cancer cells to chemotherapeutic agents 3.
Here, we demonstrate that treatment of PC-3 prostate cancer cells with the zinc specific chelator, TPEN induces rapid and selective depletion of XIAP at posttranslational level. XIAP depletion coincides with increased activation of caspases-3 and -9 and sensitization of PC-3 cells to apoptosis in response to subsequent treatment with TRAIL.
IAP family members suppress cell death by inhibiting caspase activity. The zinc-binding BIR domains are responsible for this inhibitory function. Therefore, we examined the impact of the intracellular zinc chelation on the expression of IAP family members. As seen in Figure 1a, treatment with TPEN induces a rapid decrease of XIAP protein levels in human PC-3 prostate cancer cells. Our experiments show that the inhibition of XIAP expression is selective, as TPEN had no effect on the levels of other zinc-containing members of IAP family, namely cIAP1, cIAP2 and survivin (Fig. 1a). Notably, a decrease in XIAP protein level was detectable at an extracellular TPEN concentration as low as 6μM and was dose dependent (Fig. 1b).
To validate our observation that zinc chelation induces a rapid decrease of XIAP, we constructed an expression vector encoding a doubly tagged XIAP modified to incorporate both an amino-terminal HA-tag and a carboxy-terminal FLAG-tag in the same protein. PC-3 cells were transfected with the construct followed by incubation with or without TPEN for 2 hours. As demonstrated in Figure 1c, the presence of tagged XIAP protein was detectable only in untreated cells. To discriminate whether TPEN promotes XIAP degradation or prevents its synthesis, we examined the half-life of XIAP protein using [35S]methionine pulse-chase analysis. Results of these experiments clearly demonstrate that exposure of PC-3 cells to TPEN markedly decreases XIAP protein half-life (Fig. 1d). To test the possibility that TPEN might induce down-regulation of XIAP at the mRNA level, we examined the levels of XIAP mRNA in PC-3 cells incubated with or without TPEN. As shown in Figure 1e, XIAP mRNA levels were not decreased in TPEN-treated cells. Therefore, zinc chelation induces depletion of XIAP specifically at the protein level. To determine whether down-regulation of XIAP expression is a reversible process, we added equimolar concentration of ZnSO4 to the cells pre-treated with TPEN. The results presented in Figure 1f demonstrate that the addition of zinc restores XIAP expression in TPEN-treated cells.
To investigate whether TPEN-induced XIAP down-regulation was mediated specifically via zinc depletion, PC-3 cells were exposed to TPEN (8μM) with the addition of equimolar concentrations of physiologically relevant metal ions (Fig. 2a). Addition of zinc and copper but not other metals completely blocked TPEN-induced XIAP degradation. Although TPEN has been used as a zinc-specific chelator in multiple studies 7-9, it has a much higher affinity for copper (Kd = 3 × 10-20 M) than for zinc (Kd = 2.6 × 10- M) 10. This suggests that copper effectively competes with zinc for binding to TPEN and, therefore, “neutralizes” its activity with respect to XIAP down-regulation. In RPMI medium supplemented with 10% FBS, copper and zinc levels are 0.25 and 4.2μM respectively 11. The results of our experiments demonstrating that a noticeable effect upon XIAP expression was detected only when TPEN was used at concentrations greater than 4μM (Fig. 1b) support the concept that TPEN-mediated reduction of the XIAP expression indeed was mediated primarily by chelation of zinc and not other physiologically relevant metals.
Next, we tested the ability of TPEN to modulate XIAP expression in non-prostate cell lines. Figure 2b demonstrates that incubation with TPEN decreased XIAP levels in all tested cell lines, including cervical, colon, ovarian, leukemia and breast cancer cells.
A potential mechanism explaining the TPEN-induced decrease of XIAP at the protein level without a reduction in XIAP mRNA may be its increased degradation by the ubiquitin-proteasome or caspase-mediated pathways 12,13. To address this hypothesis, we pre-incubated PC-3 cells with the proteasome inhibitor MG-132. The results presented in Figure 3a show that MG-132 did not block the ability of TPEN to reduce the protein levels of XIAP, although MG-132 completely prevented TNF-α-induced degradation of IκBα. We have reported previously that caspase-3, -8 and -9 were rapidly activated in T lymphocytes treated with TPEN 9. For this reason, we analyzed whether inhibition of caspase activity in TPEN treated cells prevents XIAP degradation. The inhibition of caspase activity with the pan-caspase inhibitor Z-VAD-FMK failed to prevent TPEN-mediated degradation of XIAP, although the addition of Z-VAD-FMK completely blocked cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP) in PC-3 cells simultaneously treated with TRAIL and methylseleninic acid (Fig. 3b). XIAP is a bona fide substrate of Omi/HtrA2 protease, which mediates XIAP cleavage under various stress conditions 14,15. With this in mind, we treated cells with Omi’s specific inhibitor, Ucf-101 14. This also failed to prevent XIAP depletion (Fig. 3c). Similar results were obtained with ALLM, an effective calpain and cathepsin inhibitor 16; chloroquine, a lysosomal protease inhibitor 17; and CA-074 Me, a cathepsin B inhibitor 18 (Fig. 3d). To examine the potential contribution of other classes of proteases in TPEN mediated XIAP degradation, PC-3 cells were pre-incubated with cell-permeable inhibitors specific for serine proteases (Pefabloc), cysteine proteases (E64d), and aspartic proteases (pepstatin A). As demonstrated in Figure 3e, only the serine protease inhibitor, Pefabloc, was able to prevent XIAP depletion in TPEN-treated cells.
The potential sensitivity of various domains of XIAP to TPEN-mediated degradation was evaluated by testing the ability of truncated versions of the protein to undergo the degradation after addition of TPEN to cells transfected with appropriate constructs containing an amino-terminal HA-tag. As shown in Figure 4, the amino-terminal portion of XIAP containing the three BIR domains and lacking the RING finger domain was degraded in the presence of TPEN, as were the BIR1-2 and full length XIAP constructs. In addition, a truncation mutant lacking all BIR domains and containing the RING finger domain (ΔBIR) was also capable of undergoing the TPEN-induced degradation. In contrast, the BIR2-3 protein was resistant to TPEN-mediated cleavage. These results indicate that the BIR2-3 construct likely lacks the specific motif required for XIAP cleavage.
XIAP is probably the only bona fide inhibitor that selectively binds and inhibits caspases-3, -7, and -9 1. Given that, we examined whether TPEN-mediated down-regulation of XIAP coincides with increased proteolytic processing via caspases-3 and -9 and cleavage of intracellular caspase-3 substrate PARP in cells treated with TRAIL. When PC-3 cells were treated with TPEN alone, the level of proteolytic activation of caspases-3 and -9 remained consistently low throughout a 3-hour incubation (Fig 5a). Although treatment of PC-3 cells with TRAIL alone was capable of inducing noticeable levels of caspase-3 and -9 processing, PARP cleavage was undetectable in cells treated with TRAIL only (Fig. 5a). Importantly, in mammals, XIAP binds exclusively to activated, processed forms of caspases-3, -7 and -9 and does not interact with their zymogenic forms 19,20. These findings can explain the lack of PARP cleavage in the presence of processed caspases-3 and -9 in cells treated with TRAIL alone. In contrast, when PC-3 cells were treated with TPEN and TRAIL simultaneously, a clear effect on the processing of caspases-3 and -9 and PARP cleavage was detected (Fig. 5a). To confirm that TPEN-mediated XIAP deficiency contributes to increased activity of caspase family members in cells treated with TRAIL, caspase-3-like activity was examined in cell lysates from PC-3 cells treated with various combinations of TPEN and TRAIL using fluorogenic substrates DEVD-AMC. As expected, treatment with either TPEN or TRAIL alone failed to induce significant levels of caspase-3-like activity, whereas such activity was easily detectable in PC-3 cells simultaneously treated with both TPEN and TRAIL (Fig. 5b).
Given that suppression of XIAP activity/expression sensitizes cells to cytotoxic agents 3,21, the pro-apoptotic effect of TPEN in combination with TRAIL was evaluated in PC-3 cells. Treatment with TRAIL alone had no significant effect on cell death in PC-3 cells, as prostate cancer cells are generally resistant to TRAIL-mediated killing. Treatment with TPEN alone also did not induce a significant degree of apoptosis. However, concomitant treatment with TRAIL and TPEN resulted in high levels of DNA fragmentation as early as 4.5 hours after treatment initiation (Fig. 5c). Furthermore, as demonstrated in Figure 6, direct small interfering RNA-mediated knockdown of XIAP resulted in the up-regulation of caspase-3-like activity and TRAIL sensitization suggesting that knockdown of XIAP by itself was sufficient to reverse resistance to TRAIL-mediated apoptosis in PC-3 cells. Yet, the results of our experiments presented in Figure 6c demonstrate that the level of apoptosis induced by TRAIL in the presence of TPEN was higher than the level of apoptosis induced by TRAIL in cells with XIAP knockdown. This may be due to the ability of TPEN to trigger more profound XIAP depletion compared with shRNA or due to a potential effect of TPEN on other critical components of the apoptotic pathway.
The present study provides the first evidence that intracellular zinc chelation induces rapid depletion of XIAP in tumor cells of various origins at the posttranslational level. Figure 7 schematically illustrates the potential mechanism of tumor cell sensitization to TRAIL via TPEN-induced down-regulation of XIAP.
The therapeutic potential of XIAP suppression in cancer treatment has encouraged research to identify and characterize mechanisms that regulate XIAP expression as well as antagonists that block XIAP function. The inhibition or down-regulation of XIAP in cancer cells lowers the apoptotic threshold, thereby inducing cell death and/or enhancing the cytotoxic action of chemotherapeutic agents. Indeed, XIAP antagonist 1396-34 sensitized PC-3 and DU-145 prostate cancer cells to chemotherapeutic agents and TRAIL 22. Moreover, XIAP antisense enhanced pro-apoptotic TRAIL potency by 12- to 13-fold in DU-145 prostate cancer cells 23 demonstrating that knock-down of XIAP itself was sufficient to reverse TRAIL resistance.
Zinc, in addition to its catalytic role in more than 300 zinc metalloenzymes, is a known inhibitor of enzymes in general. Removal of zinc from an inhibitory zinc-specific enzymatic site results in a marked increase of enzyme activity 24. For example, human tissue kallikreins, a subgroup of extracellular serine proteases including prostate specific antigen (PSA), are inactivated by reversible binding of zinc (reviewed in 25). Such findings provide a rationale for our observation that the addition of zinc chelators triggers serine protease dependent depletion of XIAP. Nevertheless, the exact mechanism underlying the depletion of XIAP under zinc-deficient conditions remains to be elucidated. A possibility also exists that the TPEN-mediated pro-apoptotic effect is not exclusively due to XIAP depletion. Further studies are needed to fully explore the potential mechanisms of action of zinc chelating agents.
Zinc is not the only metal ion influencing XIAP function. Elevated copper levels result in a conformational change in XIAP, which accelerates its degradation and significantly decreases its ability to inhibit caspase-3 in HEK 293 cells 26. In contrast to our observation that zinc chelation induces specific degradation of XIAP (Fig. 1a), studies by Mufti et al. show that intracellular copper accumulation also affects other IAPs such as Op-IAP and cIAP2. Mufti et al. also report that copper induced no significant changes in XIAP mRNA expression 26. This finding is in agreement with our data, in that XIAP mRNA levels were not decreased in TPEN-treated cells (Fig. 1e) indicating that both copper and TPEN can modulate XIAP expression at the posttranscriptional level. Furthermore, it has been suggested that direct binding of copper to cysteine residues within XIAP induces a conformational change that is associated with decreased stability 26. An intriguing possibility exists that in addition to direct binding of copper to XIAP, it can also substitute for zinc and thus, promote instability of XIAP. Indeed, the ability of various metals, including copper and cadmium, to substitute zinc in zinc finger domains is well established 27,28.
Recent studies suggest that the presence or development of resistance may ultimately limit the use of small-molecule XIAP inhibitors 3. It is unknown whether malignant cells acquire mutations in the BIR domains of XIAP and whether these mutations affect XIAP’s function or its response to inhibitors. It is also unknown whether mutations in XIAP can develop after treatment with small-molecule XIAP inhibitors similar to the development of mutations in ABL kinase after treatment with the inhibitor Gleevec 3,29. Therefore, agents able to completely deplete XIAP in malignant cells might be superior to small-molecule XIAP inhibitors. Taken together, our findings indicate that zinc-chelating molecules, capable of reducing XIAP expression, in combination with other treatment modalities may be beneficial in treating apoptosis resistant tumors.
Cell lines were obtained from ATCC (Rockville, MD) and maintained in RPMI 1640 medium (Bio-Whittaker, Walkersville, MD) supplemented with 10% FCS (Hyclone, Logan, UT), gentamicin (50 mg/L), sodium pyruvate (1 mM) and non-essential amino acids (0.1 mM).
Antibodies to cIAP2, survivin, PARP, HA-tag and IκBα were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to cIAP1, XIAP, caspase-3 and caspase-9 were obtained from Cell Signaling Technology (Beverly, MA). Ucf-101, Chloroquine, CA-074 ME and ALLM were purchased from Calbiochem (San Diego, CA). Antibodies to FLAG-tag, actin, TPEN, and TNF-α were obtained from Sigma (St. Louis, MO). Z-VAD-FMK, TRAIL, CD95 antibody, DEVD-AMC and MG132 were obtained from Biomol (Plymouth Meeting, PA). TNF-α was obtained from Sigma (St. Louis, MO). pSM2 vector encoding XIAP shRNAmir was obtained from Open Biosystems (Huntsville, AL).
Cells were lysed in a boiling SDS buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2% SDS) for 10 minutes. SDS-PAGE and Western blotting were performed as described previously 9.
To create the HA-XIAP-FLAG pEBB expression vector, the insert was obtained by PCR with 5’atcttg ggatcc ATGACTTTTAACAGTTTTGAAGG-3’ (forward) and 5’-atcttg atcgat tta ctt atc gtc gtc atc ctt gta atcAGACATAAAAATTTTTTGCTT-3’ (reverse) specific primers (restriction sites underlined) using pEBB-HA-XIAP vector 30 as a template with subsequent cloning into BamH1/Cla1 sites of the pEBB-HA plasmid. pEBB-HA-XIAP vector and XIAP truncation mutants were a kind gift from Dr. C. S. Duckett (University of Michigan Medical School, Ann Arbor, MI). Transfections were performed using TransIT-Prostate transfection kit (Mirus Bio, Madison, WI).
Half-life of XIAP protein was examined as described previously 31.
Total RNA was isolated from cells using MINI RNA isolation II Kit (Zymo-Research, Orange, CA) and purified using DNA-Free RNA Kit (Zymo-Research). Reverse transcription (RT) of 1μg RNA was subsequently carried out using 200 units of SuperScriptIII reverse transcriptase (Invitrogen, Carlsbad, CA) and then amplified as follows: 94°C for 3 min for one cycle; 94°C for 20 sec, 58°C for 30 sec and 72°C for 1min for 28 cycles followed by 5 min cycle at 72°C using 1 unit of HotTaq DNA-polymerase (Eppendorf, Westbury, NY), 125μM each of dATP, dTTP, dCTP and dGTP, 50pM of 5’-CGAAGTGAATCTGATGCTGTGAG-3’ (forward) and 5’-GTCTTCACTGGGCTTCCAATC-3’ (reverse) primers, 1X HotMaster Taq buffer (Eppendorf) and 1μl of cDNA from RT reaction. The GAPDH gene was used as a control for all RT reaction. The primer pair specific for GAPDH was 5’-ATGGGGAAGGTGAAGGTC-3’ (forward) and 5’-TCAGGCATTGCTGATGATCTT-3’ (reverse).
Caspase-3-like activity was measured using fluorometric tetrapeptide substrate DEVD-AMC. The assay was performed in 96-well plates by incubating 50 μg of cell lysates with 100 μl of reaction buffer (100 mmol/L HEPES, pH 7.5, 20% vol/vol glycerol, 5 mmol/L DTT, and 0.5 mmol/L EDTA) containing 200 μM DEVD-AMC. Release of 7-amino-4-methyl-coumarin (AMC) was monitored after 15 minutes of incubation at 37°C on a microplate fluorimeter with excitation and emission wavelengths of 380 and 460 nm respectively.
DNA fragmentation was detected using APO-BRDU kit (The Phoenix Flow Systems, Inc., San Diego, CA).
This work was supported in part by National Institutes of Health Grant RO1 CA108890 (to V.M.K.).