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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

TRAIL exhibits potent anti-tumor activity on systemic administration in mice. Because of its proven in vivo efficacy, TRAIL may serve as a novel anti-neoplastic drug. However, approximately half of the tumor cell lines tested so far are TRAIL resistant, and potential toxic side effects of certain recombinant forms of TRAIL on human hepatocytes have been described. Pretreatment with the proteasome inhibitor MG132 and PS-341 rendered TRAIL-resistant hepatocellular carcinoma (HCC) cell lines but not primary human hepatocytes sensitive for TRAIL-induced apoptosis. We investigated the different levels of possible MG132-induced interference with resistance to apoptotic signal transduction. Although proteasome inhibition efficiently suppressed nuclear factor-kappaB (NF-κB) activity, specific suppression of NF-κB by mutIκBα failed to sensitize TRAIL-resistant cell lines for TRAIL-induced apoptosis. In contrast to the previously reported mechanism of sensitization by 5-fluorouracil (5-FU), cellular FLICE-inhibitory protein (cFLIP)L and cFLIPS were markedly upregulated in the TRAIL death inducing signaling complex (DISC) by proteasome inhibitor pretreatment. Compared with 5-FU pretreatment, caspase-8 was more efficiently recruited to the DISC in MG132 pretreated cells despite the presence of fewer death receptors and more cFLIP in the DISC. But downregulation of cFLIP by short interference RNA (siRNA) further sensitized the HCC cell lines. In conclusion, these results show that otherwise chemotherapy-resistant tumor cells can be sensitized for TRAIL-induced apoptosis at the DISC level in the presence of high levels of cFLIP, which suggests the existence of an additional factor that modulates the interaction of FADD and the TRAIL death receptors. Of clinical relevance, proteasome inhibitors sensitize HCC cells but not primary human hepatocytes for TRAIL-induced apoptosis. (HEPATOLOGY 2005.)

Impaired apoptosis has been implicated in tumor formation and progression.1 Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), also called Apo-2 ligand, has been shown to kill various tumor cell lines in vitro and in vivo without being toxic to mice and non-human primates.2, 3 Some conflicting results have been published about the sensitivity of primary human hepatocytes. Some TRAIL preparations seem to be toxic, but others are not.4–6 Although 50% of tumor cell lines were TRAIL-resistant,3 the combination of TRAIL and chemotherapeutic drugs synergistically suppressed tumor growth in vitro and in severe combined immunodeficient mice without significant toxic effects.2

TRAIL induces apoptosis upon binding to TRAIL-R1 and TRAIL-R21, 7 because of receptor crosslinking, which in turn leads to the formation of a death-inducing signaling complex (DISC).8 Via Fas-associated death domain protein (FADD), caspase-8 and caspase-10 are recruited to and activated at the TRAIL DISC.9, 10 Ectopic expression of cellular FLICE-inhibitory protein (cFLIP)L and cFLIPS inhibited TRAIL-induced apoptosis by inhibition of the DISC.11 Specific downregulation of cFLIP by short interference RNA (siRNA) could sensitize hepatocellular carcinoma (HCC) cells for TRAIL-induced apoptosis.12 However, cFLIP expression levels did not directly correlate with TRAIL sensitivity.13, 14 Thus, the role of the TRAIL DISC and cFLIP in sensitization for TRAIL is still unclear.

Hepatocellular carcinoma is one of the most common carcinomas worldwide. Therapeutic options are very limited because of the chemotherapy resistance of this tumor type. A number of studies have shown that cotreatment with chemotherapeutic agents or irradiation resulted in sensitization of TRAIL-resistant tumor cell lines for TRAIL.12, 15, 16

Proteasome inhibitors are a new class of chemotherapeutic drugs with great therapeutic potential.17 The ubiquitin-proteasome pathway is a key regulator of essential cellular processes such as cell cycle, signal transduction, and apoptosis.18 Proteasomal targets include the nuclear factor-kappaB (NF-κB)/IκB system, p53, and inhibitor of apoptosis proteins (IAPs).19 Recently it has been suggested that proteasome inhibitors sensitize for TRAIL-induced apoptosis by NF-κB–dependent mechanisms in some cell types but independently of NF-κB in others.20–22 Therefore, the mechanisms leading to TRAIL sensitivity are controversial and might differ substantially in different cell types. Sensitization of carcinoma cells to TRAIL-induced apoptosis by proteasome inhibitors may provide a more efficient tumor treatment, provided that combinatorial treatment is not toxic to normal cells.

Our data show that, although primary human hepatocytes remained resistant for TRAIL-induced apoptosis, HCC cells could be sensitized with the proteasome inhibitors MG132 and PS-341. This sensitization of HCC cells was independent of NF-κB. We could show that sensitization is mediated through enhanced DISC formation with markedly increased caspase-8 activation even in the presence of increased cFLIP levels.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell Lines.

The human HCC cell lines HepG2, HepG2-TR, and Hep3b were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL, Karlsruhe, Germany) containing 10% fetal calf serum (Gibco-BRL). The HepG2-TR cells were generated as described previously12 and selected for TRAIL resistance.

Isolation and Culture of Primary Human Hepatocytes.

Primary human hepatocytes were isolated from healthy liver tissue obtained from patients undergoing partial liver resection and cultivated as described in detail by Schulze-Bergkamen et al.23 The isolation procedure was approved by the Ethics Committee, Medical Faculty, University of Heidelberg. All experiments were performed in fetal calf serum–free maintenance medium.

Antibodies and Reagents.

The following monoclonal antibodies were used: FADD/MORT1 from Transduction Laboratories (San Diego, CA), anti-caspase-8 C15 and the monoclonal antibody anti-cFLIP NF6 from Alexis (San Diego, CA), caspase-3 (CPP32) and BID (#2002) from Transduction Laboratories and Cell Signaling Technology (Beverly, MA). Leucine Zipper (LZ)-TRAIL was produced as described.10 For FACS analysis of the different TRAIL receptors, the following antibodies were used: HS101, HS201, HS301, and HS402 (mIgG1, Alexis). Anti–TRAIL-R1 polyclonal rabbit Ab 210-730-C100 and anti–TRAIL-R2 polyclonal goat Ab 210-743-R100 (Alexis) were used for Western blot. Horseradish peroxidase–conjugated goat anti-mouse IgG1, IgG2a, and IgG2b, anti-rabbit and anti-goat polyclonal antibodies were obtained from Southern Biotechnology Associates (Birmingham, AL). All other chemicals used were of analytical grade and purchased from Merck (Darmstadt, Germany) or Sigma Chemical Co. (St. Louis, MO).

FACS Analysis.

HCC cell lines were detached from the plates by EDTA, washed and incubated with monoclonal antibodies against the four surface-expressed TRAIL receptors and control mIgG1, followed by biotinylated secondary goat anti-mouse antibodies (Southern Biotechnology Associates) and Streptavidin-PE (Pharmingen, Hamburg, Germany).

Quantification of Cell Death.

As a direct measurement of apoptotic cell death, DNA fragmentation was quantified as described.24 Apoptotic cells were identified by their subdiploid DNA content using a flow cytometer. Cell viability was determined by MTT assay as described.12 The percentage of viable cells was calculated: 100× (absorption of treated cells − absorption of TX100 lysed cells)/(absorption of medium treated cells − absorption of TX100 lysed cells).

Transfection of Cell Lines and Plasmid Constructs.

All cell lines were transiently transfected by Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer's instruction. The following expression plasmids coding for a spectrin–green fluorescence protein (GFP) fusion protein25 together with an expression plasmid coding for the respective component such as NF-κB–inducing kinase (NIK) or mutIκBα,26 or together with empty control vector at a ratio of 3 to 1 were used. The Luciferase reporter plasmids containing 4 copies of NF-κB consensus binding sites were constructed previously.27 Luciferase activity was determined using the luciferase assay substrate (Promega Corp., Heidelberg, Germany) with a Duolumat LB9507 luminometer (Berthold, Germany).

Preparation of Cell Lysates.

Hepatocellular carcinoma cell lines were detached from the plates, washed with phosphate-buffered saline (PBS), and lysed in 30 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 10% glycerol, 1% Triton X-100 supplemented with Complete protease inhibitors (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. After 30 minutes incubation on ice, the lysates were centrifuged at 15,000g at 4°C. In the case of lysate preparation for ligand affinity precipitations, an intermediate centrifugation step (600g for 15 minutes at 4°C) was added after lysis to remove nuclei.

Western Blot Analysis.

Western blot analysis of post-nuclear supernatants or ligand affinity precipitates were performed as described.12 Protein concentration was determined by the bicinchoninic acid (BCA) method (Pierce, Rockford, IL). Proteins were separated on 4-12% NuPage Bis-Tris gradient gels (Novex, San Diego, CA) in 3-(N-Morpholino) propane sulfonic (MOPS) buffer according to the manufacturer's instructions. For stripping, blots were incubated in 50 mmol/L glycine HCl pH 2,3 for 20 minutes at room temperature.

Ligand Affinity Precipitation.

We performed ligand affinity precipitations using biotinylated leucine zipper TRAIL (Bio-LZ-TRAIL) in combination with Neutravidin beads (Pierce). Cells were incubated for 30 minutes at 37°C in the presence of 1 μg/mL Bio-LZ-TRAIL or, for the unstimulated control, in the absence of Bio-LZ-TRAIL. For the precipitation of the non-stimulated receptors, Bio-LZ-TRAIL at an end concentration of 1 μg/mL was added to the lysates prepared from non-stimulated cells. Precipitates were prepared as described in detail by Ganten et al.12

RNA Interference.

Oligonucleotides encoding short hairpin RNA targeting cFLIP were selected according to Zhang et al.28 The specific target sequence for cFLIP (acc. no.: U97074) was nucleotides 909-929 and for the control we used a non-functional siRNA: nucleotides 651-671. Sequences were cloned into pSUPER.gfp/neo (Oligoengine, Seattle, WA) according to the manufacturer's instructions. Cells were then transfected with Fugene 6 (Roche) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were incubated with LZ-TRAIL. Kill of GFP-positive cells was determined by propidium iodide (PI) uptake and analyzed by FACScan cytometer (Becton Dickinson, Heidelberg, Germany).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

TRAIL Induces Apoptosis in Human HCC Lines After Sensitization With the Proteasome Inhibitors MG132 and PS-341 Whereas Primary Human Hepatocytes Remain TRAIL Resistant.

We previously described 3 cell lines that exhibit a pronounced difference in TRAIL sensitivity.12 Hep3b and HepG2-TR cells are TRAIL-resistant, HepG2 cells are TRAIL-sensitive (Fig. 1A). To test whether the proteasome inhibitors MG132 and PS-341 influence TRAIL sensitivity, we incubated Hep3b, HepG2-TR, and primary human hepatocytes after pretreatment with subtoxic concentrations of MG132, PS-341, and 5-fluorouracil (5-FU) with different concentrations of TRAIL (Fig. 1B-C). In approximately 80% of HepG2-TR (data not shown) and Hep3b cells treated with MG132 or PS-341, and approximately 50% of the cells treated with 5-FU (Fig. 1B), apoptosis was induced by TRAIL after 24 hours. TRAIL-resistant primary human hepatocytes could not be sensitized by MG132, PS-341 or 5-FU for LZ-TRAIL–induced apoptosis (Fig. 1C), but were highly sensitive for LZ-CD95L (Fig. 1D). These findings clearly indicate that different mechanisms exist with regard to (i) sensitization for TRAIL between normal and transformed hepatocytes and (ii) sensitivity of normal hepatocytes to CD95L and TRAIL.

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Figure 1. TRAIL induces apoptosis in the human hepatocellular carcinoma cell lines after sensitization with MG132 while primary human hepatocytes remain TRAIL-resistant. (A) Different hepatocellular carcinoma cell lines were treated with 12-1000 ng/mL LZ-TRAIL and analyzed after 24 hours for subdiploid DNA content. (B) Hep3b were treated with different concentrations of LZ-TRAIL for 24 hours in the presence of 50 nmol/L PS-341 (filled square), 25 nmol/L PS-341 (open square), 1 μmol/L MG132 (filled triangle), 0.5 μmol/L MG132 (open triangle), 100 μg/mL 5-FU (filled rhombus), 50 μg/mL 5-FU (open rhombus), or medium (black circle). Apoptosis was determined as in A; the mean of two independent experiments is shown. (C) Primary human hepatocytes were treated with different concentrations of PS-341, MG132, or 5-FU in the absence and presence of LZ-TRAIL. (D) Primary human hepatocytes were treated with different concentrations of LZ-CD95L. After 24 hours, cell survival was analyzed by MTT assay. Data are shown as percent viability of control with standard deviation (three wells for each condition). One representative result out of at least three independent experiments is shown. LZ-TRAIL, leucine zipper tumor necrosis factor–related apoptosis-inducing ligand; 5-FU, 5-fluorouracil.

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Inhibition of NF-κB Is Not Sufficient for Sensitisation of TRAIL-Resistant HCC Cell Lines.

NF-κB activity is reported to regulate sensitivity of cells to TRAIL-induced apoptosis.22, 29 We therefore determined the level of NF-κB activity in HCC cell lines in the presence and absence of MG132 (Fig. 2A-B). In Hep3b and HepG2-TR cells, NF-κB activity was reduced 10-fold and 5-fold, respectively, by MG132 treatment. However, MG132, in addition to the well-documented blockage of NF-κB, also acts in a pleiotropic fashion.30 To determine whether blockage of NF-κB by MG132 is sufficient for the observed TRAIL sensitization, we specifically inactivated NF-κB by using a mutated form of the NF-κB inhibitor IκBα (mutIκBα). We transiently transfected the two TRAIL-resistant HCC cell lines with an expression plasmid encoding for mutIκBα. Even though mutIκBα suppressed NF-κB activity at a level similar to the proteasome inhibitor MG132 (Fig. 2A-B), neither Hep3b nor HepG2-TR cells were sensitized for TRAIL-induced apoptosis (Fig. 2C-D). We thus conclude that specific suppression of NF-κB activity is not sufficient to sensitize TRAIL-resistant HCC cell lines.

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Figure 2. NF-κB activity can be efficiently blocked by proteasome inhibition and mutIκBα, but specific NF-κB inhibition is not sufficient for sensitization of TRAIL-resistant cell lines. Cells were transfected with NF-κB reporter gene and either treated with MG132 or cotransfected with mutIκBα. After 24 hours, cells were lysed and luciferase activity was measured in HepG2-TR (A) and Hep3b (B) cells. Error bars indicate mean of 3 independent experiments. Data are shown as % NF-κB activity relative to the respective medium–treated cells. HepG2-TR (C) and Hep3b (D) were cotransfected with EGFPspec and mutIκBα (triangle) or EGFPspec alone (circle), and subsequently incubated with 2 different concentrations of TRAIL. As a control EGFPspec-transfected cells were cotreated with MG132 (square) and TRAIL. After 24 hours, GFP-positive cells were analyzed for subdiploid DNA content. Error bars indicate mean of 3 independent experiments. NF-κB, nuclear factor-kappaB; mutIκBα, mutated form of the NF-κB inhibitor IκBα; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand.

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TRAIL Receptor Surface Expression in TRAIL-Resistant Cell Lines Changes Upon Proteasome Inhibition But Not Upon Specific Inhibition of NF-κB Activity.

NF-κB activity has been suggested to regulate TRAIL-R1 and -R2 levels on HeLa cells,22 although it does not influence TRAIL receptor expression levels in keratinocytes.21 We therefore examined the surface expression of the different TRAIL receptors on TRAIL-resistant HCC lines, both before and after treatment with MG132 and after specific downregulation of NF-κB activity by transfection with mutIκBα (Fig. 3). Neither Hep3b nor HepG2-TR cells changed the surface expression levels of any of the TRAIL receptors upon transfection with mutIκBα. In marked contrast, treatment with the proteasome inhibitor MG132 resulted in upregulation of TRAIL-R1, -R2, and -R4 on the surface of both TRAIL-resistant cell lines. The surface expression of TRAIL-R3 remained unchanged.

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Figure 3. Surface expression of TRAIL receptors in TRAIL-resistant cell lines HepG2-TR and Hep3b before and after inhibition of NF-κB activity by mutIκBα and proteasome inhibition. Surface expression of TRAIL-R1 to TRAIL-R4 of EGFPspec-positive cells were performed 16 hours after transfection of mutIκBα or treatment with MG132 in comparison to an isotype-matched control mIgG1 monoclonal antibody (dashed line). TRAIL receptor expression of cells transfected with EGFPspec alone are shown as shaded area, EGFPspec and mutIκBα as black solid line and EGFPspec and MG132 (1 μmol/L) as gray solid line. To exclude nonspecific staining of dead cells, only 7-AAD–negative cells were counted. Staining of isotype-matched control mIgG1 monoclonal antibody was comparable with or without incubation with MG132 (data not shown). One representative result out of at least 3 independent experiments is shown. TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; NF-κB, nuclear factor-kappaB; mutIκBα, mutated form of the NF-κB inhibitor IκBα.

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Activation of NF-κB in TRAIL-Sensitive HepG2 Cells Does Not Affect TRAIL Sensitivity.

We determined that specific inhibition of NF-κB did not sensitize for TRAIL-induced apoptosis. However, the activation of NF-κB can result in inhibition of TRAIL-induced apoptosis.31 Therefore, we examined whether induction of NF-κB activity in TRAIL-sensitive HepG2 cells may confer resistance of these cells to TRAIL-induced apoptosis (Fig. 4). To induce NF-κB, we ectopically expressed NIK in HepG2 cells and subsequently determined NF-κB activity and TRAIL sensitivity. The kinase NIK specifically activates NF-κB by phosphorylation-induced activation of the IKK kinases. Transfection of HepG2 cells with NIK resulted in an approximately 10-fold increase in NF-κB activity (Fig. 4A). However, TRAIL-induced apoptosis was not reduced in NIK-expressing HepG2 cells (Fig. 4B) nor did it change the TRAIL receptor expression as it has been reported22 (data not shown). These findings show that neither specific inhibition nor activation of NF-κB is sufficient to regulate TRAIL-induced apoptosis in HCC.

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Figure 4. Activation of NF-κB in TRAIL-sensitive HepG2 cells does not affect TRAIL sensitivity or TRAIL receptor expression. HepG2 cells were transfected with NF-κB reporter gene alone (black bar) or cotransfected with NIK (gray bar) (A). After 24 hours, cells were lysed and luciferase activity was measured. Error bars indicate mean of 3 independent experiments. Data are shown as fold NF-κB activity relative to medium-treated cells. HepG2 were transfected with EGFPspec and NIK or EGFPspec alone and subsequently incubated with 2 different concentrations of LZ-TRAIL (B). EGFPspec-positive cells were analyzed after 8 hours for subdiploid DNA content. Error bars indicate mean of 3 independent experiments. (C) Surface expression of TRAIL-R1 to TRAIL-R4 of EGFPspec-positive cells were performed 16 hours after transfection of NIK or treatment with MG132 in comparison with an isotype-matched control mIgG1 monoclonal antibody (dashed line). TRAIL receptor expression of cells transfected with EGFPspec alone are shown as shaded area, EGFPspec and NIK as black solid line, and EGFPspec and MG132 (1 μmol/L) as gray solid line. To exclude nonspecific staining of dead cells, only 7-AAD–negative cells were counted. Staining of isotype-matched control mIgG1 monoclonal antibodies was comparable with or without incubation with MG132 (data not shown). One representative result out of at least 3 independent experiments is shown. LZ-TRAIL, leucine zipper tumor necrosis factor–related apoptosis-inducing ligand; NF-κB, nuclear factor-kappaB; NIK, NF-κB-inducing kinase.

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Increased Caspase-8 Activation After Treatment of TRAIL-Resistant Cells With MG132 and TRAIL.

So far, our findings pointed toward a mechanism of sensitization of HCC cell lines that takes place at the level of DISC formation. To investigate changes at the intracellular level, we stimulated Hep3b cells (Fig. 5) and HepG2-TR cells (data not shown) with TRAIL for different periods in the presence and absence of MG132 and subsequently analyzed cell lysates for caspase-8, BID, and caspase-3 cleavage. In Hep3b cells treated with MG132 and TRAIL, caspase-8 cleavage was detected after 15 minutes, peaking after 1 to 2 hours. BID and Caspase-3 were cleaved simultaneously, starting at 30 minutes, preceded by caspase-8 cleavage, indicating that the increase of caspase-8 cleavage is not a secondary event attributable to the mitochondrial amplification loop. In marked contrast, only minimal caspase-8 cleavage and no cleavage of caspase-3 or PARP could be detected in Hep3b cells treated with TRAIL in the absence of MG132. Similar results were obtained for HepG2-TR cells (data not shown). Thus, in these TRAIL-resistant HCC cells, neither caspase-8 nor caspase-3 is substantially activated, whereas in keratinocytes caspase-8 but not caspase-3 was activated.21 These results point toward a novel mechanism of MG132-induced TRAIL-sensitization in these HCC cells.

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Figure 5. Increased caspase-8 cleavage after treatment of TRAIL-resistant cells with MG132. Hep3b cells with or without pretreatment with 1 μmol/L MG132 for 12 hours were treated with 1 μg/mL LZ-TRAIL. At the indicated times, cells were lysed and 20 μg protein was analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent Western blot. Subsequently, the same blot was incubated with antibodies for caspase-8, BID, caspase-3, PARP, and anti-ERK1 to control for equal protein loading. One representative result out of at least 3 independent experiments is shown. TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; LZ, leucine zipper.

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Enhanced DISC Formation in MG132-Treated TRAIL-Sensitized Hepatocellular Carcinoma Cells.

The early caspase-8 cleavage observed following TRAIL stimulation of MG132-pretreated cells suggested that an early event during TRAIL-induced apoptosis is influenced by proteasome inhibition. To investigate possible MG132-induced changes in the formation or composition of the TRAIL DISC, we performed an analysis of the native DISC of Hep3b cells (data not shown) and HepG2-TR cells with and without MG132 pretreatment (Fig. 6A). As a control, cells were pretreated with 5-FU, a chemotherapeutic agent that has been recently shown to sensitize for TRAIL-mediated apoptosis by downregulation of cFLIP and thereby enhanced caspase-8 activation at the DISC.12 To control for differential expression of caspase-8, FADD/MORT1, and cFLIP, lysates were analyzed for the respective proteins (Fig. 6B). MG132 pretreatment resulted in enhanced DISC formation in HepG2-TR cells (Fig. 6A). MG132 treatment enhanced TRAIL-R1 and TRAIL-R2 recruitment to the DISC, which correlated with increased caspase-8 recruitment and activation at the DISC.

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Figure 6. Enhanced DISC formation in MG132-treated TRAIL-sensitized hepatocellular carcinoma cells. HepG2-TR cells were pretreated with 5-FU and MG132 for 12 hours and either stimulated with 1 μg/mL biotinylated TRAIL for 15 minutes (+) or left unstimulated (−) before cell lysis. TRAIL was added to the lysates in the unstimulated control at a final concentration of 1 μg/mL. Resulting protein complexes bound to biotinylated TRAIL were analyzed for the presence of TRAIL-R1, TRAIL-R2, caspase-8, FADD/MORT1, and cFLIP by SDS-PAGE and Western blot after affinity precipitation with Neutravidin beads (A). To control for differential expression of caspase-8, FADD/MORT1, and cFLIP, lysates were analyzed for the respective proteins and ERK for equal loading by Western blot (B). One representative result out of at least 3 independent experiments is shown. DISC, death-inducing signaling complex; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; FADD, Fas-associated death domain protein; cFLIP, cellular FLICE-inhibitory protein.

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However, the decisive finding becomes apparent when the TRAIL DISC precipitates of 5-FU–pretreated cells are compared with the TRAIL DISC precipitates of MG132–pretreated cells. FADD and the TRAIL death receptors are thought to directly interact through their respective death domains. Thus, enhanced receptor recruitment should result in a proportional increase of FADD recruitment, as FADD levels in the lysates changed on neither MG132 nor 5-FU pretreatment (Fig. 6B). However, although 5-FU pretreatment resulted in a strong upregulation of TRAIL death receptor recruitment, FADD recruitment was only moderately increased. In contrast, pretreatment with MG132 resulted in a similar increase of recruited TRAIL death receptors, yet it accomplished a tremendous increase in FADD recruitment. Interestingly, although MG132 sensitized for TRAIL-induced apoptosis, more cFLIPS and cFLIPL were recruited to the TRAIL DISC after treatment with MG132 (Fig. 6A). Despite the increase in recruitment, cFLIPS and cFLIPL are not able to prevent caspase-8 activation. Similar results were obtained for Hep3b cells (data not shown). Thus, proteasome inhibition results in an enhanced TRAIL DISC formation that becomes apparent by greatly enhanced recruitment of FADD and caspase-8 in a situation in which receptor surface expression is only moderately elevated.

Specific Downregulation of cFLIP by Short Interference RNA Further Sensitizes MG132 Pretreated Hep3b Cells for TRAIL-Induced Apoptosis.

A dual function of cFLIP has been recently described, in which upregulation of cFLIPL can indeed sensitize for CD95-induced apoptosis.14, 32 Thus, enhanced cFLIP recruitment to the DISC might be responsible for the observed sensitization for TRAIL. To test whether downregulation of cFLIP in cells treated with MG132 might be able to decrease TRAIL sensitization, we transfected Hep3b cells with a cFLIP-targeting siRNA vector33 and pretreated the cells with MG132 before TRAIL incubation. Transfection of Hep3b cells with the cFLIP-targeting siRNA vector resulted in downregulation of cFLIP (Fig. 7A) and an additional increase in TRAIL-induced apoptosis of approximately 20% in the presence or absence of the proteasome inhibitor (Fig. 7B). Comparable results were obtained for HepG2TR (data not shown). Thus, upregulation of cFLIPL by proteasome inhibition is not responsible for TRAIL sensitization of resistant tumor cells.

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Figure 7. Specific downregulation of cFLIP by siRNA further sensitizes MG132 pretreated Hep3b cells for TRAIL-induced apoptosis. (A) Hep3b cells were transfected with a vector encoding for GFP and cFLIP siRNA or control siRNA. After 48 hours, cells were sorted and GFP-positive cells were lysed and analyzed for cFLIP and β-actin expression by Western blot analysis. (B) Hep3b cells were transfected with a vector encoding for GFP and cFLIP siRNA (filled symbols) or control siRNA (open symbols). After 48 hours, cells were incubated with LZ-TRAIL (1 μg/mL) with (circles) or without (squares) 0.5 μmol/L MG132, and GFP-positive cells were analyzed by FACS after 6 hours. Three independent experiments with similar results have been performed. cFLIP, cellular FLICE-inhibitory protein; siRNA, short interference RNA; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; GFP, green fluorescent protein; LZ, leucine zipper.

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Upregulation of TRAIL-R1 and TRAIL-R2 Is Not Essential for Sensitization to TRAIL-Induced Apoptosis by MG132 and 5-FU.

Because both 5-FU and MG132 treatment led to an increase in expression of TRAIL-R1 and TRAIL–R2, we examined whether the upregulation of the death-inducing TRAIL receptors was necessary for sensitization to TRAIL-induced apoptosis.

If the TRAIL-receptor upregulation by MG132 or 5-FU were necessary for the sensitizing effect, then the binding of additional TRAIL (added after washing steps) to newly surface-expressed TRAIL-receptors would be necessary for apoptosis induction. TRAIL-resistant Hep3b cells were incubated for 30 minutes with TRAIL to allow for the occupation of all membrane-bound receptors. Subsequently, unbound TRAIL was removed by thoroughly washing the cells 5 times before they were incubated with MG132 or 5-FU in the presence or absence of additional TRAIL throughout the following 23.5 hours (Fig. 8).

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Figure 8. Upregulation of the death-inducing TRAIL receptors is not essential for sensitization of TRAIL-resistant hepatoma cell lines. Hep3b cells were incubated with LZ-TRAIL at 1 μg/mL for 30 minutes and then washed 5 times with medium. Cells were then cultured either in medium containing 1 μmol/L MG132 in the absence (open bars) or in the presence of LZ-TRAIL at 1 μg/mL (black bars). Control cells were washed 5 times without any addition of TRAIL (gray bars). After 24 hours, cells were analyzed for subdiploid DNA content. This experiment has been done in triplicates. One of 3 experiments with comparable results is shown. LZ-TRAIL, leucine zipper tumor necrosis factor–related apoptosis-inducing ligand.

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Interestingly, only small differences in TRAIL-induced apoptosis were observed between MG132- or 5-FU-sensitized cells when unbound TRAIL was removed and not replaced and MG132-sensitized cells that were further incubated in the presence of TRAIL. Comparable results were obtained for HepG2-TR cells (data not shown). Thus, upregulation of the apoptosis-inducing TRAIL receptors by MG132 or 5-FU treatment is not necessary for sensitization. Instead, these findings point toward a mechanism of sensitization that takes place at the level of DISC formation.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the current study, we investigated the mechanisms of sensitization of HCC cells and primary human hepatocytes to TRAIL-induced apoptosis by proteasome inhibition. Proteasome inhibitors are a new class of chemotherapeutics with great clinical potential in the treatment of different tumor entities.34, 35 Regarding the sensitization for TRAIL-induced apoptosis by suppression of NF-κB activity, cell line–specific differences seem to exist.20–22 We found that MG132 treatment suppressed the NF-κB activity in different HCC cell lines. However, specific suppression of NF-κB by mutIκBα to a level similar to that achieved by MG132 treatment did not sensitize TRAIL-resistant HCC lines nor was a 10-fold activation of NF-κB by NIK able to confer resistance to a TRAIL-sensitive HCC cell line. These findings indicate that the activation status of NF-κB is not sufficient to determine the fate of a cell with respect to TRAIL-induced apoptosis in HCC cells (Figs. 2 and 4). These data are in contrast to data obtained by Kim et al.36 However, this difference in outcome may be attributable to a nearly complete NF-κB suppression obtained by adenoviral transfer of mutIκBα by Kim et al., which may allow for sensitization, whereas the suppression of NF-κB obtained by transfection with mutIκBα to a level comparable to that obtained by MG-132 treatment did not result in sensitization of HCC cells to TRAIL-induced apoptosis.

After MG132 pretreatment, rapid and complete caspase-8 cleavage was induced by stimulation with TRAIL. Simultaneously with caspase-8 cleavage, BID- and complete caspase-3 cleavage occurred. Thus, the mechanism of proteasome inhibition–induced TRAIL sensitization in HCC cells differs from that recently identified in keratinocytes, in which caspase-8 activation was displayed in the absence of proteasome inhibitors.21 Even though we showed enhanced caspase-8 cleavage after MG132 pretreatment at a very early time, we could not rule out the subsequent onset of a secondary amplification loop through the mitochondria. However, immunoprecipitation indicated a strongly increased TRAIL DISC after MG132 pretreatment, which is responsible for the massive and early caspase-8 cleavage. The protein cFLIP had been shown to act as an inhibitor of TRAIL-induced apoptosis in other cellular systems.11, 37 High overexpression of cFLIPL or cFLIPs resulted in inhibition of the CD95 DISC38 and inhibition of caspase-8 processing.39 Conversely, moderate elevation of cFLIPL could in fact enhance CD95-induced apoptosis through its protease-like domain.14 Recently cFLIPL has been shown to be a 10-fold better activator of caspase-8 than caspase-8 itself.40 In the HCC cell lines tested here, the expression level of cFLIPs in the cytosol was markedly upregulated by MG132 (Fig. 6B). This increase in cFLIPs could also be observed at the DISC level together with enhanced cFLIPL recruitment after sensitization of TRAIL-resistant HCC cells with MG132. However, downregulation of cFLIP by siRNA resulted in marked sensitization of the HCC cells in the absence or presence of proteasome inhibition. Therefore, a proapoptotic function of cFLIP in this system seems unlikely.

Caspase-8 and FADD were much better recruited to the TRAIL DISC after proteasome inhibitor pretreatment than with 5-FU pretreatment, despite less precipitated TRAIL receptors and increased cFLIP levels. Further studies are necessary to search for novel regulatory DISC components. Recently it has been suggested that the caspase-8 and caspase-10 associated RING protein (CARP) 1 and 2 function as ubiquitin ligase for death effector domain proteins associated with the DISC.41 The paper suggests that CARP 1 and 2 silence death receptor signaling by suppressing the activity of caspase-8 and -10 via ubiquitination processes and might contribute to the ubiquitin-mediated proteolysis of caspases under certain circumstances. As shown in Fig. 6 in the HCC cells, neither in the lysates nor in the DISC analyses bands of ubiquitinated proteins are present after MG132 treatment. In addition, in contrast to cFLIP, TRAIL-R1, and TRAIL-R2, the proteins FADD and caspase-8 were neither up- nor downregulated by proteasome inhibition. However, McDonald and El-Deiry41 could not detect higher expression of caspase-8 and -10 after silencing of CARP 1 and 2. Nevertheless, silencing of CARP 1 and 2 sensitized cells to chemotherapeutics, TRAIL, CD95L, and mainly to TNFα-induced apoptosis, although it did not alter the DISC composition. Interestingly, unlike CD95L and TRAIL, TNFα does not form a stable DISC. These data suggest—in line with our results—that CARP proteins act mainly downstream of the DISC.

Taken together, our findings show that the proteasome inhibitors MG132 and PS-341 effectively sensitize HCC independently of NF-κB suppression. MG132 treatment upregulates the apoptosis-inducing TRAIL receptors, but this upregulation is insufficient to explain MG132-mediated sensitization. Strikingly, this sensitization of HCC cells was not paralleled by TRAIL sensitization of primary human hepatocytes by MG132, PS-341, or 5-FU. Resistance of primary human hepatocytes to TRAIL together with the lack of sensitization for TRAIL-induced apoptosis in contrast to HCC cells points toward the possible therapeutic use of TRAIL in combination with proteasome inhibitors for the treatment of HCC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Jutta Mohr, Bärbel Moos, and Eva Rieser for excellent technical assistance, and Peter H. Krammer, W. Stremmel, and Martin R. Sprick for helpful discussions and careful reading of the manuscript; and Martina Müller-Schilling and Henning Schulze-Bergkamen for preparation of primary human hepatocytes.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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