Introduction

Cancer can be partially attributed to defects in the regulation of apoptotic cell death. Failure to trigger the cellular suicide program not only predisposes to development of malignancies but also increases the resistance of tumor to anticancer drugs. Human neuroblastoma (NB), the second most common solid tumor in children, exhibits a clinically and biologically heterogeneous behavior, in part attributed to differential regulation of apoptosis (Nicholson, 2000). Whereas localized tumors in young infants often spontaneously regress or mature in response to treatment, the outcome of advanced staged NB with MYCN amplification remains poor and has not improved in recent years (Brodeur et al., 1997; Brodeur, 2003).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has generated considerable interest as a potential tumor-specific therapeutic for highly resistant malignancies since it selectively induces apoptosis in many transformed cells but not in normal cells (Ashkenazi and Dixit, 1998). TRAIL is a type II transmembrane protein that induces apoptosis via the extrinsic or death receptor pathway by interaction with two receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Stimulation of death receptors results in receptor aggregation and recruitment of the adaptator molecule FADD and procaspase-8, forming the death-inducing signalling complex (DISC). Recruitment of caspase-8 to the DISC results in its autocatalytic activation, a process that directly initiates a downstream caspase cascade and cell death in type I cells. In type II cells, caspase-8 activates the mitochondrial or intrinsic pathway through cleavage of the Bcl-2 family member Bid (Scaffidi et al., 1998, 1999). Caspase-8 is thus involved in the amplification of the apoptotic process by activation of the mitochondrial signalling pathway (Kroemer et al., 1998). Another amplification of the apoptotic signal described involves the caspase-3-mediated activation of caspase-8 (Slee et al., 1999; Von Haefen et al., 2003). Both pathways are regulated at different levels. The cellular Fas-associated death domain-like interleukin-1-converting enzyme-inhibitory protein, long form (FLIP_L) is a structural homologue of caspase-8, devoid of protease activity, that acts as a potent inhibitor of death-receptor-mediated apoptosis (Thome et al., 1997). On the other hand, the intrinsic pathway is regulated by Bcl-2 family members that have pro- and antiapoptotic regulatory functions. Translocation from the mitochondria of proapoptotic proteins such as Bax or Bak results in the release of the apoptogenic mediators cytochrome c and Smac/Diablo in the cytosol and is blocked by Bcl-2 and Bcl-x_L (Green and Reed, 1998). The activities of caspases can be inhibited by a conserved family of inhibitors of apoptosis proteins (IAPs). The X-linked IAP (XIAP) specifically interacts with and inhibits caspase-3/-7, a blockage relieved and antagonized by Smac/Diablo (Igney and Krammer, 2002). Survivin, a protein that is overexpressed in several tumors is another member of IAPs that prevents cell death by interacting with the apoptosome (Pitti et al., 1996; Altieri, 2003).

The resistance to TRAIL-mediated cell death in various tumors has been attributed to deregulation of a number of signalling molecules (French and Tschopp, 1999; Jaattela, 1999; Igney and Krammer, 2002), including inactivation of CASP8 (Hopkins-Donaldson et al., 2000; Teitz et al., 2000; Eggert et al., 2001), CASP10 genes expression (Harada et al., 2002; Park et al., 2002), downregulation of TRAIL-receptors expression (Strand et al., 1996; Lee et al., 1999; Shin et al., 2001), overexpression of antiapoptotic molecules such as c-FLIP (Irmler et al., 1997; Thome et al., 1997; Mizutani et al., 1999), Bcl-2 (Reed et al., 1991; Brambilla et al., 1996), or survivin (Altieri, 2003).

Our previous studies have shown that highly malignant and invasive neuroblastoma cell lines (N-type) were resistant to TRAIL-mediated cell death, whereas TRAIL induced apoptosis in the more differentiated and noninvasive NB cell lines (S-type). Our group as well as others have shown that the majority of high-stage NB tumors or invasive N-type cell lines downregulated the expression of the initiator caspase-8 by a mechanism of gene hypermethylation and/or allelic deletion (Hopkins-Donaldson et al., 2000; Teitz et al., 2000; Eggert et al., 2001). Since sensitivity to TRAIL could be partially restored in N-type cells after demethylation, we proposed that caspase-8 silencing in NB was responsible for the observed resistance to TRAIL-mediated apoptosis. However, the moderate sensitivity to TRAIL exhibited by NB cells as compared with other cell types suggested the existence of additional repressive mechanisms involved in TRAIL resistance in these cells. In addition, the particular contribution of caspase-10 in TRAIL resistance has not been explored in caspase-8 silenced cells.

Several recent studies have reported that the metabolic inhibitor cycloheximide (CHX), -IFN or subtoxic concentrations of chemotherapeutic drugs could sensitize various tumor cell types to TRAIL-induced cell death (Nagane et al., 2001; Wajant et al., 2002), although the precise mechanisms of such synergism remain controversial.

The objectives of this study were therefore to identify the precise contribution of caspase-8 in the resistance of malignant neuroblastoma cells to TRAIL-induced death by restoring its expression and function in NB cells. We next explored the molecular mechanisms responsible for the sensitization to TRAIL-induced death by CHX or chemotherapeutic drugs in cells with constitutive or restored caspase-8 expression. The identification and better understanding of putative target pathways are essential to develop new proapoptotic therapeutic approaches and to overcome caspase silencing in highly aggressive tumors such as neuroblastoma.

Results

Complemented IGR-N91-C8 cells express physiological levels of caspase-8

In order to analyse the contribution of caspase-8 silencing in the resistance to TRAIL-induced apoptosis in N-type NB cells, we restored a stable caspase-8 expression in the caspase-8 and caspase-10-negative MYCN amplified cell line IGR-N91. The IGR-N91 cells were either infected with a bicistronic retroviral vector encoding for the GFP and the caspase-8a genes (IGR-N91-C8), or with the GFP control vector (IGR-N91-M). The GFP-positive cells were selected by fluorescence-activated cell sorting (FACS) as bulk populations, and several clones of IGR-N91-C8 cells were isolated. The expression level of the caspase-8 protein in the different clones was analysed by immunoblotting and compared with that of the caspase-8/-10-positive NB cell line SH-EP. As shown in Figure 1a, the amount of caspase-8 protein expressed in the IGR-N91-C8 clones was variable, with clones 10, 11, 19 and 21 expressing higher level of caspase-8 protein than clones 4, 12 and 13. Nevertheless, the expression level of caspase-8 in the IGR-N91-C8 clones was comparable to that of SH-EP control cells, indicating that caspase-8 was expressed to a physiological level in the complemented cells (Figure 1a). As expected, the IGR-N91-M cells remained deficient in caspase-8 and -10 whereas the IGR-N91-C8 cells were still deficient in caspase-10, while SH-EP expressed both caspases. All cells expressed a similar level of caspase-3 (Figure1a and data not shown).

Figure 1.

(a) Restoration of caspase-8 expression in IGR-N91-C8 cells. Whole cell extracts from the bulk population of IGR-N91-C8 cells (bulk), IGR-N91-C8 clones (number 4, 10, 11, 12, 13, 19 and 21), the positive control SH-EP cells or the negative control IGR-N91-M cells were analysed by immunoblotting for the presence of caspase-8 and -3. Equal amount of protein loading was confirmed by staining the membrane with Ponceau red. (b) Restoration of TRAIL sensitivity of IGR-N91-C8 cells. Cell viability was measured in IGR-N91-M, IGR-N91-C8 cells (bulk) and IGR-N91-C8 clones (4, 10, 11, 12, 13, 19 and 21) without (black) or with 48 h stimulation with TRAIL (white). Mean values of two representative experiments are shown. (c) Cell death induced by TRAIL in caspase-8 complemented cells is caspases dependent. IGR-N91-C8-21 cells were pretreated 30 min with the caspase inhibitors zIETD-fmk (dots), zDEVD-fmk (horizontal lines), zLEHD-fmk (hatched) or zVAD-fmk (white), or without inhibitor (black) and either stimulated or not with TRAIL during 16 h. A representative experiment is shown

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IGR-N91-C8 cells are fully sensitive to TRAIL-induced cell death

The caspase-8 complemented IGR-N91-C8 cells were analysed for their sensitivity to TRAIL-induced cell death by cell proliferation assay. Results demonstrate that the IGR-N91-C8 cells became sensitive to TRAIL, whereas the control IGR-N91-M cells remained resistant (Figure 1b). Indeed, while the IGR-N91-C8 bulk population of cells displayed a viability of 88% after TRAIL treatment, three IGR-N91-C8 clones were moderately sensitive to TRAIL (between 55 and 80% of viability). In addition, four clones displayed a very high response to TRAIL (between 7.5–23% of viability) (Figure 1b). Interestingly, the clones with the highest level of caspase-8 expression were also the most sensitive to TRAIL-mediated cytotoxicity. The cell death induced by TRAIL in the caspase-8 complemented cells was protected by the caspase-8, -3 and -9 protease inhibitors (zIETD-fmk, zDEVD-fmk and zLEHD-fmk, respectively) or by the pan-caspase inhibitor zVAD-fmk (Figure 1c). It was recently reported that difference in TRAIL receptor expression could account for variable TRAIL sensitivity in NB cells (Yang and Thiele, 2003). We therefore asked whether different level of cell surface expression of TRAIL receptors could account for the difference in sensitivity to TRAIL of the IGR-N91-C8 clones in addition to various caspase-8 expression levels. However, no difference in cell surface expression of TRAIL-R1 and TRAIL-R2 between the different IGR-N91-C8 clones was revealed by FACS analyses (data not shown).

TRAIL triggers the extrinsic and the intrinsic pathway in the complemented IGR-N91-C8 cells

TRAIL-induced caspases activation was investigated in the highly sensitive clone IGR-N91-C8-21. After 8 h of treatment with TRAIL, the exogenous caspase-8a was activated by proteolytic cleavage as shown by the disappearance of the inactive form of caspase-8 and the appearance of the cleaved intermediate (43 kDa) and the active caspase-8 (18 kDa) fragments (Figure 2a). The active form of caspase-9 (37 kDa) also appeared after TRAIL treatment, while the procaspase-9 was cleaved. The downstream caspases-3 and -7 as well as the proapoptotic Bcl-2 family member protein Bid were also cleaved (Figure 2a). As expected, no cleavage was observed in IGR-N91-M control cells (Figure 2a). Caspase activation was then confirmed by measurement of the caspase-3-like activity in IGR-N91-C8-21 cells. Indeed, the DEVD-pNA substrate was hydrolysed by cell lysates of TRAIL-treated cells compared to untreated cells. As control, caspase-3 activity was blocked by addition of the caspase-3 inhibitor zDEVD-fmk (Figure 2b). These results indicate that the exogenous caspase-8a is necessary and sufficient to engage the caspase cascade activation in response to TRAIL triggering in the NB cell line IGR-N91. This also indicates that caspase-10 is not essential for TRAIL signalling in IGR-N91 cells. The cleavage of Bid and caspase-9 induced by TRAIL and cell death protection by zLEHD-fmk suggests that the mitochondrial apoptotic pathway is also activated. To confirm this, the release of Smac/Diablo from the mitochondria was measured after 4 h of TRAIL treatment. The results show that the amount of Smac/Diablo in the cytosolic fraction was increased, whereas it was reduced in the mitochondrial fraction (Figure 2c). This confirms that TRAIL induces the activation of the mitochondrial pathway in the IGR-N91-C8 cells.

Figure 2.

(a) Cleavage of caspases was induced by TRAIL in IGR-N91-C8 cells. IGR-N91-M control cells and IGR-N91-C8 clone 21 cells were grown 8 h with or without TRAIL. Whole cell extracts were analysed by immunoblotting for the cleavage of caspase-8, -3, -7 and –9, and Bid. Anticaspase-8 antibody detects the inactive form (caspase-8), the cleaved intermediate (43 kDa) and the active form (18 kDa). Anticaspase-9 antibody detects both the inactive form (caspase-9) and the active form (37 kDa). -Actin was used as loading control. (b) Caspase-3 activity was induced in IGR-N91-C8 cells after TRAIL treatment. Hydrolysis of DEVD-pNA was measured in IGR-N91-C8-21 cells unstimulated or treated with TRAIL for 8 h. The caspase-3-like activities of induced cells, relative to unstimulated cells are indicated. The caspase-3 inhibitor DEVD-fmk was used as control to inhibit hydrolysis of DEVD-pNA. (c) TRAIL induces the activation of the mitochondrial pathways in caspase-8 complemented cells. Cytosolic (60 g) and mitochondrial (10 g) extracts isolated from IGR-N91-C8-21 cells treated (+) or not (-) with TRAIL for 4 h were analysed by immunoblotting for the presence of Smac/Diablo. Equal amount of protein loading was confirmed by staining the membrane with Ponceau red

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NB cell lines can be highly sensitized to TRAIL by chemotherapeutic drugs or CHX

In the second part of this study, we explored the ability of CHX or chemotherapeutic drugs to sensitize caspase-8-expressing NB cell lines to TRAIL. Indeed, the SH-EP cell line are slightly sensitive to TRAIL while some clones of IGR-N91-C8 cells are moderately TRAIL sensitive. The cytotoxic activity of TRAIL in combination with CHX or the drugs DOX, etoposide, cisplatin and taxol was measured in SH-EP and IGR-N91-C8-12 cells. As shown in Figure 3a, TRAIL-mediated cell death was highly enhanced in SH-EP cells by cotreatment with CHX, DOX, cisplatin, etoposide or taxol. The IGR-N91-C8-12 cells could be strongly sensitized to TRAIL-induced cell death by DOX, etoposide and cisplatin, but weakly by CHX and taxol (Figure 3a).

Figure 3.

(a) CHX and chemotherapeutic drugs strongly sensitize neuroblastoma cells to TRAIL-mediated cell death. SH-EP and IGR-N91-C8-12 cells were untreated (no) or treated with CHX or DOX or etoposide (Etop) or cisplatin (Cispl) or taxol in the absence (white) or in the presence of TRAIL (black) for 48 h. (b) Sensitization by CHX and drugs is dependent on an intact TRAIL-receptor pathway. SH-EP-FADD-DN and IGR-N91-M cells were treated as described in (a). Mean values of two representative experiments are shown in (a) and (b)

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The contribution of the TRAIL receptor pathway was analysed using SH-EP cells overexpressing a FADD dominant-negative protein (FADD-DN), lacking the death effector domain (DED) required for caspase-8 recruitment to the DISC. SH-EP-FADD-DN cells were completely resistant to TRAIL and could not be resensitized to TRAIL by CHX or by any of the drug tested (Figure 3b). In addition, the caspase-8-negative IGR-N91-M cells could not be sensitized to TRAIL by CHX, DOX and taxol. Although the IGR-N91-M cells were weakly sensitized by etoposide and cisplatin (Figure 3b), the effect remained marginal when compared to IGR-N91-C8-12 cells (Figure 3a), indicating that the synergistic effect essentially involved caspase-8 activation. These results show that TRAIL sensitization by CHX and drugs is dependent on the integrity of the TRAIL-receptor pathway.

Mechanism of TRAIL sensitization by CHX and drugs requires caspase-8 and is caspases-dependent

The involvement of caspases in sensitization to TRAIL was analysed using caspases inhibitors. SH-EP cells were strongly protected by the caspase-8 (zIETD-fmk), the caspase-3/-7 (zDEVD-fmk) and the caspase-9 (zLEHD-fmk) protease inhibitors or by the pan-caspase inhibitor zVAD-fmk, indicating that the enhanced cell death by the combined treatments was caspase-dependent (Figure 4a, b). Although caspase inhibitors are not absolutely specific for a defined caspase, the protection observed with the caspase-8 inhibitor zIETD-fmk indicated that the sensitization to TRAIL by CHX or the drugs involved caspase-8, while the protection obtained by the caspase-9 inhibitor z-LEHD-fmk suggests that it activates the mitochondrial apoptotic pathway.

Figure 4.

(a, b)Enhanced cell death induced by TRAIL and cotreatment with CHX or drugs is caspase-dependent. (a) SH-EP cells were uninduced (no) or induced for 48 h by TRAIL, CHX or DOX, without caspase inhibitors (black), or with zIETD-fmk (dots), zDEVD-fmk (horizontal lines), zLEHD-fmk (hatched) or zVAD-fmk (white) (b) SH-EP cells were induced for 48 h by TRAIL, etoposide (Etop), cisplatin (Cispl,) or taxol, without caspase inhibitors (black), or with zIETD-fmk (dots) or zVAD-fmk (white). Mean values of representative experiments are shown in (a) and (b). (c) and (d). CHX and DOX increase TRAIL-induced caspase activation. (c) SH-EP, IGR-N91-M and IGR-N91-C8-12 cells were unstimulated (-) or treated with TRAIL (T), CHX (C) or DOX (D) for 8 h. Cell lysates were analysed by immunoblotting for the cleavage of caspase-8, caspase-10, caspase-3, caspase-7, caspase-9 and Bid. -Actin was used as loading control. (d) Hydrolysis of DEVD-pNA was measured in SH-EP cells unstimulated (-) or treated with TRAIL (T), CHX (C) or DOX (D) for 8 h. The caspase-3-like activities of induced cells, relative to unstimulated cells are indicated. The caspase-3 inhibitor zDEVD-fmk inhibits hydrolysis of DEVD-pNA induced by combined treatment

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The molecular mechanisms of sensitization to TRAIL-induced apoptosis by CHX or subtoxic concentration of drugs were further investigated. Caspases activation by proteolytic cleavage was analysed by immunoblotting in SH-EP and IGR-N91-C8-12 cells. Both CHX and DOX increased the ability of TRAIL to induce the initiator caspase-8, -10 and -9 cleavages in SH-EP cells, compared to TRAIL alone (Figure 4c). In IGR-N91-C8-12, caspase-8 and -9 cleavages were only weakly increased by the combined treatments. Cleavage of the effector caspases-3 and -7 was highly enhanced by cotreatment with TRAIL and CHX or DOX compared to TRAIL alone in both SH-EP and IGR-N91-C8-12 cells (Figure 4c). The cleavage of Bid was also increased by combined treatment, compared to TRAIL alone in particular in SH-EP cells (Figure 4c). The increased activation of caspases cascade induced by combined treatment is caspase-8 dependent since no cleavage of caspase was observed in the caspase-8- deficient IGR-N91-M control cells (Figure 4c). Importantly, neither subtoxic concentration of CHX nor DOX alone had any effect on the expression level of the analysed proteins nor on caspases activation (Figure 4c). In addition, the increased activation of caspases induced by co-treatment with TRAIL and CHX or DOX was demonstrated by measurement of the caspase-3-like activities in SH-EP cells. The relative caspase-3-like activities of cells treated with both TRAIL and CHX or DOX was higher compared to cells treated with TRAIL alone (Figure 4d). No hydrolysis of DEVD-pNA was measured in lysates from cells treated with CHX or DOX alone. As control, caspase-3 activity was blocked by addition of the caspase-3 inhibitor zDEVD-fmk (Figure 4d). These results are in accordance with the caspase-3 cleavages shown by immunoblotting in Figure 4c.

Mechanisms of TRAIL sensitization involves the mitochondrial pathway

Apoptosis induced by cotreatment with TRAIL and CHX or DOX increased Bid and caspase-9 cleavage. It was caspase-9 dependent since cell death was reduced by the caspase-9 inhibitor (Figure 4a). This suggests that the sensitization by CHX or drugs to TRAIL-induced apoptosis also affects the activation of the mitochondrial signalling pathway. The disruption of the mitochondrial transmembrane potential (m) was therefore measured after combined treatment with TRAIL and CHX or drugs in the SH-EP cell line using the fluorescent dye JC-1. The loss of m induced by TRAIL (49% of cells) was increased by cotreatment with CHX, DOX, etoposide, taxol or cisplatin (91, 88, 85, 87 and 82% of cells, respectively) (Figure 5a). The disruption of m was highly prevented by the caspase-8 inhibitor zIETD-fmk and the pan-caspase inhibitor zVAD-fmk in cells treated with TRAIL and CHX (20 and 12%, respectively) or with TRAIL and DOX (25 and 11%, respectively) (Figure 5a). This shows that the disruption of the m induced by TRAIL is strongly enhanced by drugs and CHX and highly dependent on caspase-8 activation. The release of Smac/Diablo from the mitochondria was measured in SH-EP cells induced by TRAIL and/or CHX. The results show that the amount of Smac/Diablo in the mitochondrial fraction decreased when cells were cotreated with TRAIL and CHX as compared to each treatment alone (Figure 5b).

Figure 5.

CHX and drugs increased TRAIL-induced activation of the mitochondrial signalling pathway in a caspases-dependent manner. (a) SH-EP cells were untreated (no) or treated for 15 h with TRAIL (T), CHX (C), DOX (D), etoposide (Etop), cisplatine (Cispl) or taxol. Cells were pretreated 30 min with the protease inhibitors zIETD-fmk or zVAD-fmk. The loss of m was measured with the fluorescent dye JC-1. The percentages of cells with low m are indicated. (b) Mitochondrial (10 g) extracts isolated from SH-EP cells unstimulated (-) or treated with TRAIL (T) and/or CHX (C) for 4 h were analysed by immunoblotting for the presence of Smac/Diablo. Equal amount of protein loading was confirmed by staining the membrane with Ponceau red

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Sensitization by drugs involves the increase of cell surface TRAIL-R2 receptor expression

We further investigated the mechanisms by which CHX or chemotherapeutic drugs sensitize NB cells to TRAIL-induced apoptosis and measured the cell surface expression of TRAIL-R1 and TRAIL-R2 receptors and the decoy receptor TRAIL-R3 by flow cytometry. The cell surface expression of TRAIL-R2 was increased by subtoxic concentration of DOX, etoposide, cisplatin and taxol (6.1-, 3.8-, 2.8- and 2.1-fold induction, respectively), with DOX inducing the strongest expression of TRAIL-R2 in SH-EP cells. In contrast, TRAIL-R1 and the decoy receptor TRAIL-R3 remained barely detectable after drug induction. Upregulation of TRAIL-R2 was also observed in the IGR-N91-C8 cells. In contrast, no change in caspase-3, -7 -8, -9, -10, Bid or FADD protein expression could be detected by immunoblotting in SH-EP and IGR-N91 cells after 24 h of treatment with subtoxic concentration of drugs, except a marginal increase in caspase-10 expression induced by DOX in SH-EP cells (data not shown).

Combined treatment with TRAIL and drugs increase the cleavage of antiapoptotic proteins

We explored if the CHX or DOX could affect the stability of some antiapoptotic proteins. CHX was shown to reduce the amount of short-lived antiapoptotic proteins such as XIAP or RIP (Zhang et al., 1999; Fulda et al., 2000). Our data show that survivin was not affected in SH-EP and in IGR-N91-C8-12 cells treated either by CHX or by DOX in the presence or in the absence of TRAIL (Figure 6a). In contrast, the antiapoptotic proteins Bcl-x_L and XIAP were cleaved by TRAIL treatment in IGR-N91-C8 cells (clones 12 and 21) and by cotreatment with CHX or DOX in SH-EP cells (Figure 6a). RIP was also cleaved in SH-EP and IGR-N91-C8-12 by combined treatments, whereas it was cleaved in IGR-N91-C8-21 after induction by TRAIL alone (Figure 6a). No cleavage of Bcl-x_L, XIAP or RIP was found in the caspase-8 negative IGR-N91-M cells, suggesting that the cleavage of these antiapoptotic proteins is dependent on the activation of the caspases cascade (Figure 6a). To confirm this hypothesis, IGR-N91-C8-21 cells were induced by TRAIL in the presence of the caspase-3/-7 or caspase-9 inhibitors, and cleavage of caspases and antiapoptotic proteins was analysed by immunoblotting. The results show that both caspases inhibitors strongly reduced the cleavage of the antiapoptotic proteins Bcl-x_L, XIAP and RIP whereas Bcl-2 remained stable (Figure 6b). As expected, both caspase inhibitors prevented activation of caspases-3 and -7 (Figure 6b). Surprisingly, caspase-8 and Bid cleavage were also protected by these inhibitors, indicating that caspase-8 and Bid were most likely also activated by effector caspases via an amplification loop (Figure 6b). The observation that caspase-9 is protected from cleavage by the caspase-3/-7 protease inhibitor zDEVD-fmk also suggests the existence of an amplification loop mechanism.

Figure 6.

TRAIL induces caspases-dependent cleavage of antiapoptotic proteins in NB cell lines. (a) SH-EP, IGR-N91-M, IGR-N91-C8-12 and IGR-N91-C8-21 cells were unstimulated (-) or treated with TRAIL (T), CHX (C) or DOX (D) during 8 h. Cell lysates were analysed by immunoblotting for the presence of the antiapoptotic proteins survivin, Bcl-x_L, XIAP and RIP. -Actin was used as loading control. (b) IGR-N91-C8-21 cells were unstimulated (-) or treated for 8 h with TRAIL (+) in the absence (-) or in the presence (+) of the caspase-3/-7 inhibitor zDEVD-fmk or the caspase-9 inhibitor zLEHD-fmk. Cell lysates were analysed by immunoblotting for the cleavage of caspase-8, -3, -7 and 9, Bid, Bcl-x_L, XIAP, RIP and Bcl-2. Equal amount of protein loading was confirmed by staining the membrane with Ponceau red

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Discussion

Other groups and ours have shown that caspase-8 was frequently inactivated by a combination of methylation and allelic deletion in NB with MYCN amplification. We proposed that this phenomenon played an essential role in resistance to TRAIL-induced cell death in N-type NB cell lines, whereas drugs could efficiently induce cell death in the absence of caspase-8 in NB cells (Hopkins-Donaldson et al., 2002). However, accumulating evidence suggests that additional intrinsic (FLIP, antiapoptotic proteins) and extrinsic (TRAIL receptors and decoy receptor molecules) signalling molecules can contribute to TRAIL resistance in childhood and adult cancers (Igney and Krammer, 2002; Salvesen and Duckett, 2002). Like caspase-8, caspase-10 maps to chromosome 2q33, a frequent LOH region (Park et al., 2002). Whereas both caspases have been recently shown to be cosilenced in childhood tumors (Harada et al., 2002), the role of caspase-10 in the death receptor signalling pathway remains largely controversial.

In order to precisely define the contribution of caspase-8 in TRAIL-induced cell death, we attempted to restore active caspase-8 expression in the caspases-8 and -10-deficient IGR-N91 NB cell line. Retroviral infection of the IGR-N91 cells with the caspase-8a isoform resulted in a caspase-8 expression level similar to that of a constitutively expressing caspase-8 NB cell line (SH-EP) and was sufficient to fully restore TRAIL sensitivity. Moreover, cells expressing a higher amount of caspase-8 were the most sensitive to TRAIL, whereas the difference in sensitivity to TRAIL could not be explained by difference in cell surface expression of TRAIL-R1 and TRAIL-R2. This demonstrates that caspase-8 is necessary and sufficient to restore TRAIL response in the IGR-N91 NB cell line. However the difficulty in inducing functional caspase-8 expression in NB cell lines suggests the existence of other inhibitory mechanism resulting in TRAIL resistance. This also indicates that caspase-10 is not essential for TRAIL signalling, although it was cleaved in the caspase-8/-10-positive SH-EP cells. The caspase-8-restored cells were able to undergo apoptosis in response to TRAIL in a caspase-dependent manner, since they could be protected from death by caspases inhibitors. In addition, the complete downstream signalling cascade, including Bid cleavage was engaged in response to TRAIL triggering. The drop of mitochondrial transmembrane potential (m) and release of Smac/Diablo from the mitochondria further confirmed the ability of the corrected cells to engage both signalling pathways in response to TRAIL.

We concluded that caspase-8 silencing was solely responsible for the resistance to TRAIL-mediated apoptosis in the N-type IGR-N91 NB cell line. In addition, these data represent the first report revealing that caspase-10 is not essential for the complete proceeding of neuroblastoma cells via intrinsic and extrinsic death pathways. Whether caspase-10 has a redundant function, can substitute for caspase-8 or plays a role in other death induction signals, is currently explored.

Different studies have demonstrated that subtoxic concentration of chemotherapeutic drugs can sensitize tumor cell lines to TRAIL-induced apoptosis (Nagane et al., 2001; Wajant et al., 2002). The enhanced cytotoxic effect of chemotherapeutic drugs and TRAIL was also demonstrated in multidrug-resistant cell lines (Mizutani et al., 1999; Jazirehi et al., 2001). Indeed, we show here that SH-EP cells can be sensitized to TRAIL-induced cell death by etoposide, even though these cells are resistant to etoposide (data not shown) (Rodriguez-Lopez et al., 2001). The molecular mechanisms of the synergistic action of TRAIL and drugs are poorly understood. One mechanism proposed is the transcriptional induction of TRAIL-R1 (DR4) and TRAIL-R2 (DR5) in certain tumor cell lines (Wu et al., 1997; Sheikh et al., 1998; Gibson et al., 2000), while no induction of TRAIL-R1 and -R2 was found in other systems (Lacour et al., 2003). Cytotoxic drugs were also shown to increase the expression of apoptotic proteins such as caspase-8, -2 and -3, FADD and Bax in colon carcinoma cells (Micheau et al., 1999). Caspase-8 mRNA expression was also increased by etoposide in epithelial cells (Gibson et al., 2000). More recently, it was shown that cytotoxic drugs sensitize colon cancer cells to TRAIL by enhancing DISC assembly (Lacour et al., 2003).

Here, we show that chemotherapeutic drugs upregulate the expression of TRAIL-R2 but not TRAIL-R1 at the cell surface of neuroblastoma cell lines. In contrast, no change in caspase-8, -10, -9, -3, -7, Bid or FADD protein expression was induced by subtoxic concentration of drugs in SH-EP and IGR-N91 cells, except for a marginal increase in caspase-10 expression induced by DOX in SH-EP cells. This indicates that TRAIL-sensitization in NB cells does not occur through increased expression of proapoptotic molecules, but might in part result from an enhanced expression of TRAIL-R2 receptor, leading to increased amounts of caspase-8 recruited at the DISC, thus enhancing the apoptotic signal. We further observed that CHX and drugs sensitized NB cells to TRAIL by enhancing the caspase activation cascade. Indeed, as compared to the effect of TRAIL alone, important increased activation of caspase-8, -3, -7 and -9 as well as Bid appeared by proteolytic cleavage with the combination of TRAIL and DOX or CHX, whereas no caspase activation occurred using either drugs or CHX alone. Thus, in accordance with observations made in colon carcinoma cells (Lacour et al., 2001) or breast carcinoma (Keane et al., 1999), the increased cell death involves caspase activation. The activation of Bid and the enhanced drop of m demonstrated that the combined use of TRAIL and drugs also triggered the mitochondrial apoptotic pathway in NB cells, suggesting that amplification of the apoptotic signal could be provided by the concomitant use of both intrinsic and extrinsic pathways. Interestingly, the protection observed by zIETD-fmk from cell death induced by DOX, cisplatin and taxol alone in SH-EP cells (Figure 4a, b) indicated the existence of an amplification loop for the activation of caspase-8 after drug treatment. Indeed, caspase-8 was shown to be activated by caspase-3 (Keane et al., 1999). This most likely resulted from the activation of the mitochondrial pathway as it was recently described for taxol in B lymphoid BJAB cells (Von Haefen et al., 2003).

Another mechanism that could explain the sensitization to TRAIL-induced apoptosis by drugs and CHX is the modulation of the intracellular level of apoptosis inhibitors such as Bcl-2, Bcl-x_L or inhibitor of apoptosis proteins such as XIAP, cIAP1/2 or survivin (Igney and Krammer, 2002). XIAP and RIP expression were shown to decrease upon exposure to Actinomycin D or CHX in SH-EP cells or melanoma cells (Fulda et al., 2000; Zhang et al., 1999). Here, in contrast, we did not observe any change in the expression level of XIAP, RIP, survivin or Bcl-x_L following CHX treatment. In contrast, we showed that the antiapoptotic proteins XIAP and Bcl-x_L were cleaved after combined treatment with TRAIL and CHX or DOX, as well as by TRAIL alone in highly sensitive NB cells. We also observed that proteolytic cleavage of XIAP and Bcl-x_L was prevented by the caspase-3/7 or the caspase-9 inhibitors, suggesting that their cleavage was dependent on caspases activity. XIAP inhibits apoptosis by directly interacting with caspase-3/-7 and -9 (Deveraux et al., 1998, 1999). Moreover, XIAP was previously shown to be cleaved following induction of apoptosis by Fas, staurosporin, etoposide or TRAIL (Deveraux et al., 1998, 1999; Johnson et al., 2000; Zhang et al., 2001). The antiapoptotic Bcl-x_L protein was also shown to be converted into a potent proapoptotic factor after its cleavage by caspases (Clem et al., 1998). This indicates that inactivation of the antiapoptotic proteins Bcl-x_L and XIAP by proteolytic cleavage may increase the response of NB cells to TRAIL-induced apoptosis and might represent one of the important and new mechanisms of sensitization of TRAIL-induced death by drugs and CHX in NB.

RIP was described as a death domain kinase in TNF-R1 signalling, mediating activation of NF-B which exerts an antiapoptotic function (Stanger et al., 1995). RIP was shown to be cleaved by caspase-8 during TNF-induced apoptosis resulting in the blockage of TNF-induced NF-B activation and up-regulated sensitivity to death receptor signalling (Lin et al., 1999; Kim et al., 2000; Martinon et al., 2000). Although RIP was not found to interact with TRAIL receptors (Kischkel et al., 2000), caspase-8-dependent release of RIP fragment in response to TNF already suggested that cleavage-mediated inactivation of RIP was an important process in the regulation of TNF and other death receptor-induced apoptosis (Kischkel et al., 2000). Moreover, cells deficient in RIP displayed increased sensitivity to TNF/CHX, FAS and TRAIL indicating that RIP has a protective effect in apoptosis (Lin et al., 1999). Here, we show that the amount of RIP protein is reduced by cotreatment with TRAIL and CHX or DOX in cells weakly sensitive to TRAIL or by TRAIL alone in highly sensitive cells. As caspase inhibitors protected RIP from cleavage induced by TRAIL treatment, the reduction and inactivation of RIP occurred by caspase cleavage and is likely to mediate the observed TRAIL/drugs synergy.

In conclusion, NB cells expressing an active caspase-8 can be further sensitized to TRAIL-induced apoptosis by cotreatment with subtoxic doses of CHX or chemotherapeutic drugs. We propose that the synergistic effect of combined treatments first lowers the signalling threshold required for TRAIL-mediated apoptosis, by enhancing the level of TRAIL-R2 expression. Simultaneously, by increasing the activation of caspases, the combination of TRAIL and drugs accelerates the crosstalk between the extrinsic and intrinsic pathways via an amplification loop: enhanced caspase-8-mediated Bid cleavage is followed by increased formation of the apoptosome and caspase-3 activity that further activates caspase-8. Finally, the apoptotic signal is amplified by the caspase-dependent inactivation of antiapoptotic molecules such as XIAP, Bcl-x_L and RIP.

Our results indicate that the highly malignant character of caspase-8 and -10 deficient NB tumors might originate from their inability to undergo apoptosis as triggered by death receptor signalling. In order to be efficient, TRAIL-related apoptosis inducing therapy will have to be developed in combination with anticancer drugs and caspase-8-inducing treatments, since caspase-8 silencing appears to be the principal limiting factor for TRAIL-induced cell death in human neuroblastoma.

Materials and methods

Cell culture and reagents

The S-type neuroblastoma cells SH-EP and SH-EP FADD-DN (Hopkins-Donaldson et al., 2002) and N-type NB cells IGR-N91 (Vassal et al., 1997) were grown in RPMI medium supplemented with 10% of FCS, 200 g/ml gentamicin (Essex Chemie AG). Cells were incubated with 100 ng/ml of soluble recombinant TRAIL (a gift from J Tschopp) and 1 g/ml of crosslinking mouse anti-Flag Ab M2 (Sigma). SH-EP cells were treated with 1 g/ml of cycloheximide, 0.2 g/ml of doxorubicin, 5 g/ml of cisplatin, 5 g/ml of etoposide and 10 nM of taxol (Sigma). IGR-N91-C8 and IGR-N91-M cells were treated with 0.1 g/ml of cycloheximide, 0.1 g/ml of doxorubicin, 5 g/ml of cisplatin, 10 g/ml of etoposide and 5 nM of taxol. Cells were pretreated 30 min with caspase inhibitors zVAD-fmk (100 M, Bachem), zIETD-fmk, zDEVD-fmk and zLEHD-fmk (50 M, R&D systems) before TRAIL or drug treatments.

Plasmids

The pMIGR1 vector (Pear et al., 1998) is a bicistronic retroviral vector containing an IRES upstream of the enhanced green fluorescent protein (eGFP) gene. Caspase-8a/MIGR vector was constructed by subcloning the BamHI–SalI fragment of caspase-8a cDNA (a gift from O Micheau) into the BglII and XhoI site of pMIGR.

Retroviral infection

IGR-N91 cells were infected with the caspase-8a retroviral pMIGR construct as previously described (Soneoka et al., 1995; Benedict et al., 2000).

Cell viability assays

Cells (10⁵/well in 96-well-plates; 100 l) were plated 24 h before treatment and incubated with TRAIL or drugs for 48 h. Assays were performed in quadruplicates. Viability was measured using the MTS/PMS cell proliferation kit from Promega according to the manufacturer's instructions. Percentage of cell viability as compared to untreated controls was calculated.

Protein extracts

Whole cell extracts were prepared as already described (Hopkins-Donaldson et al., 2000). Cytosolic and mitochondrial extracts were prepared by washing the cells twice in ice-cold PBS. The cell pellet was then resuspended in 4 PCV of ice-cold buffer A (250 mM sucrose, 20 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF) and a cocktail of protease inhibitor (CompleteTM, Roche) and incubated 15 min on ice. Cells were homogenized by passing through a G25 needle for 30 strokes and centrifuged 5 min at 1000 g at 4°C. The supernatant was spun 30 min at 20 000 g at 4°C. The pellet representing the mitochondrial fraction was suspended in buffer B (20 mM Tris pH 7.5, 100 mM Nacl, 1% Triton, 1 mM DTT, 1 mM PMSF) and a cocktail of protease inhibitor (CompleteTM, Roche)). The supernatant was centrifuged at 20 000 g 30 min at 4°C, this final supernatant representing the cytosolic extract.

Immunoblotting

Protein extracts (30–50 g) were loaded on 12% SDS–PAGE and transferred on nitrocellulose membranes. Blots were saturated with 5% skim milk, 0.1% Tween-20 in TBS and revealed using mouse monoclonal antibodies to detect caspase-8 (MBL), caspase-10 (MBL), FADD, caspase-3, XIAP, RIP, caspase-7 (BD Pharmingen), -actin (Sigma). Polyclonal rabbit antibodies were used to detect caspase-9 (Cell Signaling), Bid, Bcl-x_L (BD Transduction Laboratories), survivin (R&D systems), Smac/Diablo (Calbiochem), Trail-R2 (ProSci Inc.). Binding of the first antibody was revealed by incubation with either goat anti-mouse IgG (Jackson ImmunoResearch) or goat anti-rabbit IgG (Nordic Immunological Laboratories). Bound antibodies were detected using the Lumi-light Western blotting substrate (Roche) according to the manufacturer's instructions.

Caspase-3 activity

Caspase-3-like protease activity were measured using the caspase-3 colorimetric protease assay kit from MBL. Cytosolic lysates were prepared 8 h after TRAIL, CHX or DOX treatment according to the manufacturer's instructions. A measure of 100 g (IGR-N91-C8-21) or 200 g (SH-EP) of protein extracts were incubated with 200 M of DEVD-pNA substrate for 2 h at 37°C. Cell lysates were incubated with 10 M of caspase-3 inhibitor (DEVD-fmk) for 30 min before addition of the substrate for control experiments. Hydrolysed pNA was detected using a microtiter plate reader at 405 nm. Background absorbance from cell lysates and buffers were substracted from the absorbance of induced and unstimulated samples before calculation of relative caspase-3 activities.

Analysis of mitochondrial transmembrane potential

The drop of mitochondrial membrane potential m was measured by staining the cells with the fluorescent dye JC-1 (Sokol et al., 2001) according to the manufacturer's protocol (Calbiochem). Loss of m resulting in reduction of red aggregates was measured by flow cytometry using the FL2 channel (550–650 nm) (FACScan, Becton Dickinson). Results are given in percentage of cells with low m compared to untreated controls.

Flow cytometry

Cells were washed in FACS buffer (RPMI, 10% FCS, 2 mM EDTA) and stained with mouse monoclonal antibodies anti-TRAIL-R1, -TRAIL-R2 and -TRAIL-R3 (Alexis), followed by goat secondary antibodies conjugated with FITC (Caltag laboratory) or PE (Serotec) and analysed by FACScan (Beckton Dickinson).

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Acknowledgements

We thank O Micheau and J Tschopp for helpful advice as well as for providing us with TRAIL, the caspase-8a cDNA sequence and plasmids. We also thank D Cefaï for collaboration in the retroviral infections and P Batar for helping us with the FACS analyses.

This work was supported by grants from the Swiss Cancer League (to NG, KFS 1086-09-2000), from the Swiss National Scientific Foundation (to NG, 3100-067918.02) and from the FORCE foundation.