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The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L), a member of the TNF superfamily, is a recently discovered potent inducer of apoptosis produced by cells of the immune system (Wiley et al, 1995). TRAIL transmits its proapoptosis signal via crosslinking its cognate receptors, death receptor-4 (DR4), also called TRAIL receptor-1 (TRAIL-R1) and DR5 (TRAIL-R2) (Pan et al, 1997b; Schneider et al, 1997a; Sheridan et al, 1997). These receptors recruit and activate the proximal caspase-8, which in turn activates the effector caspases, an event culminating in cell death (Muhlenbeck et al, 1998; Bodmer et al, 2000; Hopkins-Donaldson et al, 2000). The interaction between TRAIL receptors and the proximal caspase is likely mediated by a protein containing the Fas-associated death domain (FADD) (Kischkel et al, 2000; Sprick et al, 2000) and/or via a GTP-binding adaptor protein (Miyazaki and Reed, 2001). While mitochondrial signalling in TRAIL-induced apoptosis has been postulated in some reports (Thomas et al, 2000; Munshi et al, 2001), it may not be involved at all in some cases (Walczak et al, 2000; Keogh et al, 2000), or may act rather as an amplification loop (Suliman et al, 2001; Alleva et al, 2001).
Two additional receptors for TRAIL have been identified, decoy receptor-1 (DcR-1) (also known as TRAIL-R3) and DcR-2 (TRAIL-R4) (Pan et al, 1997a; Schneider et al, 1997a,1997b; Sheridan et al, 1997; Ashkenazi and Dixit, 1999). These receptors bind TRAIL but fail to transmit its apoptosis-inducing signal downstream, thereby acting as competitive inhibitors of TRAIL apoptosis (Ashkenazi and Dixit, 1999). While the decoy receptors appear unique to the TRAIL system, apoptosis induced by this ligand can also be suppressed by inhibition of caspase-8 (FLICE) activity, via induction of the FLICE-inhibitory protein (FLIP) (Schneider et al, 1997b). The fact that normal cells, compared to malignant cells, appear to overexpress DcR-1 and DcR-2 and/or FLIP suggest that TRAIL-induced apoptosis may be selective for cancer cells (Bonavida et al, 1999; Kim et al, 2000), making TRAIL attractive as a potential anticancer agent (Nagane et al, 2001). Furthermore, recent studies indicate that anticancer chemotherapeutics can sensitise cells to killing by immunological agents, including TRAIL, by upregulating the cognate death receptors and/or overcoming TRAIL resistance (Bonavida et al, 1999; Nagane et al, 2001; Nimmanapalli et al, 2001; Matsuzaki et al, 2001).
We and others have found that certain analogues of vitamin E, in particular α-tocopheryl succinate (α-TOS), are potent inducers of apoptosis in a variety of cells (Fariss et al, 1994; Neuzil et al, 1999,2001d; Yu et al, 1999,2001), and that this action appears to be specific for malignant cells (Neuzil et al, 2001c). Several reports suggest that vitamin E analogues sensitise cancer cells to killing by agents like Fas (Yu et al, 1999) or 5-fluorouracil (Chinery et al, 1997). As α-TOS is an inhibitor of activation of the nuclear factor-κB (NF-αB) (Suzuki and Packer, 1993; Erl et al, 1997; Neuzil et al, 2001b) and because activation of NF-κB has been shown to negatively modulate TRAIL-dependent apoptosis in multiple cancer cells (Bernard et al, 2001; Franco et al, 2001; Oya et al, 2001; Kreuz et al, 2001), we have investigated whether this vitamin E analogue might sensitise malignant cells to TRAIL killing via an NF-κB inhibitory activity. In this report, we show that α-TOS inhibits NF-κB activation in Jurkat T lymphoma cells and that this amplifies their susceptibility to TRAIL.
Jurkat T lymphoma cells were maintained in RPMI-1640 medium supplemented with 10% FCS and antibiotics. The cells were regularly split when reaching a density of 1.5×106ml−1, and used for experiments at 0.5×106ml−1. Cells were treated with α-tocopherol (α-TOH) or α-tocopheryl succinate (α-TOS) (both from Sigma) at 25 or 50μM, 10μM hydrogen peroxide (Fluka) or 40ngml−1 recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) prepared as described elsewhere (Alleva et al, 2001; Plasilova et al, 2002). In brief, the extracellular part of human TRAIL (AA 95-281), obtained by PCR from the HPB T cell line cDNA library, was subcloned into pBSK, sequenced and further subcloned into the His-tagged reading frame of pET15b. The protein was expressed in Escherichia coli and purified using the TALON (Clontech) and SP-Sepharose columns. In some cases, cells were treated with the proteasome inhibitor MG132 (Calbiochem) at 0.5 or 1μM, or tumour necrosis factor-α (TNF-α; PharMingen) at 100Uml–1.
Apoptosis was routinely assessed by the annexin V-binding method, which is based on the affinity of annexin V for phosphatidylserine externalised to the outer leaflet of the plasma membrane early in the course of opoptosis. In brief, cells were harvested by centrifugation, washed with PBS, spun down again, and resuspended in the binding buffer (10mM Hepes/NaOH, 140mM NaCl and 25mM CaCl2, pH 7.4). Cells were then incubated with 2μl of annexin V-FITC (PharMingen) for 20min at room temperature, and analysed by flourescence-assisted cell sorting (FACS; Becton Dickinson). Activation of caspase-3 was estimated by incubating cells with an anticaspase-3 IgG (PharMingen) that recognises the activated form, followed by incubation with FITC-conjugated secondary antibody. Fluorescence intensity of the cells was assessed by FACS (Neuzil et al, 2001b).
Activation of NF-κB was estimated using the Trans-AM kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's protocol. In brief 5–10×106 cells were treated as specified, and lysed using the buffer provided by the manufacturer. The lysates were then transferred into wells containing the immobilised NF-κB (p65) consensus sequence, and incubated for 1h at 37°C. The wells were washed and the bound p65 protein was detected by horseradish peroxidase (HRP)-dependent staining following incubation with anti-p65 lgG and secondary HRP-conjugated secondary lgG. The level of absorbance at 450nm, assessed in a microplate reader, reflected the level of bound p65.
For TEM, Jurkat cells were grown in complete RPMI medium at 0.5×106ml−1, and treated for 12h with 40ngml−1 rhTRAIL or buffer alone (control cells). The cells (107) were briefly rinsed with PBS, centrifuged, fixed overnight in 2% glutaraldehyde, and postfixed with 1% OsO4 for 1h. Both fixatives were made up in 0.1M cacodylate buffer supplemented with 0.1M sucrose (pH 7.2, 300mOsmol) and applied at room temperature. After standard dehydration in ascending concentrations of ethanol, the cells were embedded in Epon-812 monomer and polymerised. Ultrathin sections were cut with a diamond knife mounted in a Reichart ultramicrotome, contrasted with uranyl acetate and lead citrate, and examined in a Jeol 1200 EX transmission electron microscope operated at 80kV (Brunk et al, 1995).
The aim of the present study was to determine whether the semisynthetic vitamin E analogue, α-TOS, could enhance the sensitivity of Jurkat T lymphoma cells to the induction of apoptosis by the immunological agent TRAIL. For initiation of apoptosis, we used rhTRAIL that was expressed in bacterial cells. As shown in Figures 1 and and2Figure2, our rhTRAIL preparation caused massive apoptosis in Jurkat cells, as documented by both morphological changes evaluated by TEM, and by PS externalisation and caspase-3 activation. Figure 2 also demonstrates that apoptosis induction was saturable with regard to the rhTRAIL used, with rhTRAIL being maximally effective at ca 20ngml−1.
We next investigated whether preincubation with α-TOS might sensitise Jurkat cells to TRAIL. As shown in Figure 3, α-TOS–but not α-TOH–rendered the cells more susceptible to TRAIL-induced apoptosis at a concentration at which the vitamin E analogue itself did not cause substantial cell death. To determine whether this potentiation of TRAIL killing might invlove inhibition, by α-TOS, NF-κB activation, the cells were pretreated with the proteasome inhibitor, MG132, or with TNF-α, a potent activator of NF-κB. Figure 3 shows that preincubation with MG132 sensitised cells to TRAIL, as did α-TOS, and that MG132 itself did not cause apoptosis. On the contrary, pretreatment with TNF-α increased resistance of the cells to TRAIL, consistent with the idea that activation of NF-κB may be antiapoptotic (Bernard et al, 2001; Franco et al, 2001). Finally, we used hydrogen peroxide as a negative control. At a low concentration (10μM) that does not interfere with NF-κB activation (see below), hydrogen peroxide did not induce substantial apoptosis nor did it sensitise cells to TRAIL (Figure 3).
More direct studies of NF-κB activation revealed that Jurkat cells exposed to rhTRAIL did activate NF-κB. Figure 4 shows a substantial activation of NF-κB, 30min following addition of rhTRAIL to the cells, although this activation was less pronounced than that caused by treatment with the strong NF-κB activator, TNF-α. This activation was transient and lasted for about 1h, after which it declined. Pretreatment with α-TOS or MG132 abolished the initial NF-κB activation observed with TRAIL alone. Once again, hydrogen peroxide at 10μM had no effect on NF-κB, either alone or in combination with TRAIL (Figure 4).
We demonstrate in this communication that vitamin E succinate, but not vitamin E itself, potentiates killing of Jurkat T lymphoma cells by the immunological inducer of apoptosis, TRAIL. These data are consistent with, and further extend, the earlier observations that α-TOS promotes apoptosis caused by a variety of agonists. This is true, for example, of Fas-dependent killing of breast (Yu et al, 1999) and prostate cancer cells (Israel et al, 2000). In these instances, the vitamin E analogue sensitised the cells to Fas ligand by causing plasma membrane translocation of Fas. Furthermore, α-TOS also promotes TRAIL-induced apoptosis in colon cancer cells, apparently by modulating different, converging signalling pathways, thereby maximising the apoptotic potential of the cells (Weber et al, 2002). Importantly, this cooperation was also reflected in the inhibition of colon cancer in an animal model (Weber et al, 2002).
There are several possible mechanisms by which α-TOS may sensitise leukemic cells towards TRAIL killing. TRAIL crosslinks two cognate, death-signalling receptors. One of these, DR4 (TRAIL-R1), has been shown to transiently activate NF-κB. This leads to an initial expression of survival signals including the inhibitor of apoptosis protein (IAP) family members (Degli-Esposti et al, 1997; Schneider et al, 1997b; Bernard et al, 2001). Perhaps by this mechanism, activation of NF-κB can protect leukemic cells from apoptotic killing (Jeremias et al, 1998). Activation of NF-κB also leads to upregulation of the caspase-8 inhibitor, cFLIP (Kreuz et al, 2001). Jurkat cells express both DR4 and DR5, although the level of expression of the former receptor is lower than that of the latter (JN et al, unpublished). In spite of this, the level of DR4 expression appears to be sufficient to activate NF-κB upon exposure of the cells to TRAIL (this report). We hypothesised that inhibition of NF-κB activation–likely responsible for the lag phase in apoptosis induction by TRAIL in Jurkat cells– could be inhibited by α-TOS. In support of this, preincubation of the cells with α-TOS suppressed TNF-α-dependent NF-κB activation (cf. Fig. 4). The exact mode of suppression of NF-κB activation by α-TOS is not yet clear but there are several possibilities. For example, activation of NF-κB might be suppressed by α-TOS by affecting degradation of the inhibitory subunit, IκB. Indeed, cleavage of, or mutations in, IκB can accentuate apoptosis (Jeremias et al, 1998; Keane et al, 2000) and a recent report documents a caspase-dependent cleavage of IκB in TRAIL-resistant cells, thereby sensitising them to killing by TRAIL (Kim et al, 2002).
The concept that α-TOS can inhibit NF-κB activation is not new (cf. Erl et al, 1997), but the precise structural requirements are not fully known. It is clear, however, that α-TOH, the redox-active counterpart of α-TOS, fails to exert such activity (Erl et al, 1997; Neuzil et al, 2001b). One possibility is suggested by the observation that α-TOS activates caspases that cleave the NF-κB subunit p65, while not killing the cells (Neuzil et al, 2001b), probably via a mitochondria-dependent pathway (Neuzil et al, 2001d; Weber et al, 2002). We have earlier suggested that under certain circumstances, α-TOS can cause ‘subapoptotic’ signalling that may lead to activation of early apoptotic events while not bringing the cell into the execution phase of apoptosis, a possibility also suggested by others (Harvey et al, 2000). Such a mechanism may underlie the inhibitory activity of α-TOS towards activation of NF-κB in Jurkat cells, thereby sensitsing the cells to killing by TRAIL.
α-TOS is not just another example of an inducer of apoptosis that senstises cells to TRAIL killing, a principle that has been published in multiple reports (see, e.g. Bonavida et al, 1999; Nagane et al, 2001). Unlike many chemotherapeutic agents, α-TOS appears to be selective for malignant cells (Neuzil et al, 2001c,2001d; Weber et al, 2001). α-TOS, which has proapoptotic activity in vitro and antineoplastic effects in vivo (Neuzil et al, 2001d; Malafa et al, 2002; Weber et al, 2002), is carried within the bloodstream by circulating lipoproteins (Pussinen et al, 2000), which are cleared in the liver. Here, α-TOS is hydrolysed to α-TOH, at least some of which is released into the circulation, thereby boosting the antioxidant defence system (Neuzil et al, 2001a). Because both α-TOS and TRAIL are relatively nontoxic to normal cells (Bonavida et al, 1999; Jo et al, 2000; Kim et al, 2000; Nagane et al, 2001; Neuzil et al, 2001c,2001d; Nesterov et al, 2002; Weber et al, 2002), the two agents, that is, α-TOS and TRAIL, would seem to represent an exciting partnership of potentially high therapeutic relevance.
In conclusion, we have shown that α-TOS potentiates TRAIL-induced apoptosis in Jurkat T lymphoma cells by inhibiting transient activation of the transcription factor NF-κB. In practical terms, this finding could be utilised for devising strategies of treatment for potentially fatal disorders like lymphomas or carcinomas on two levels: first, by coadministration of α-TOS and TRAIL; second, by administration of α-TOS alone, as the agent could be expected to sensitise cancer cells to endogenously produced TRAIL, thereby potentiating the immune defences against neoplasia. This principle may be especially useful for suppressing cancer involving malignant cells with a high expression of DR4. Further exploration of these possibilities may lead to an effective approach to the treatment of malignancies.
We thank Dr L Andera for providing rhTRAIL, and Drs L Andera, UT Brunk and JW Eaton for a critical reading of the manuscript. The technical assistance of AMS Austarheim and TF Gulbransen is highly appreciated. JN was partially supported by the University of Linköping Grant 83081030 and a grant from the Dust Diseases Board of Australia. HD was supported by the Norwegian Research Council Grant 129557/310.