Weber et al, 2002; Wu et al, 2002, 2004a; Prasad et al, 2003; Anderson et al, 2004; Hrzenjak et al, 2004; Kline et al, 2004; Stapelberg et al, 2005). Diverse types of malignant cells also show different susceptibilities in response to a-TOS. Neuzil et al, (2001) demonstrated that apoptotic rate induced by exposure to a-TOS at 50 mM for 12 h varied from 30% to 60% in different malignant cells. About 50% of apoptosis was induced by a-TOS treatment at 20 mg/ml (equivalent to 38 mM) for 48 h in MDA-MB-435 human breast cancer cells (Yu et al, 2003). Exposure to a-TOS at 20 mg/ml for 24 and 48 h triggered 14 and 90% of SGC-7901 human stomach cancer cells to undergo apoptosis, respectively, but a-TOH at the same dosage did not show any apoptotic effect (Wu et al, 2002, 2004a). Importantly, a-TOS is not harmful toward normal cells and tissues with apoptotic rate less than 5% (Neuzil et al, 2001). In summary, a-TOS is a potent apoptosis inducer highly selective for malignant cells.

Recently published in vivo results have supported the hypothesis that the non-antioxidant analogs of vitamin E strongly suppress cancer cell growth as well. Such inhibition is observed in athymic mice with tumor xeno-grafts including human neuroblastoma, breast cancer, colon carcinoma, peritoneal mesotheliomas and murine melanoma (Malafa et al, 2000, 2002; Barnett et al, 2002; Weber et al, 2002; Stapelberg et al, 2005) and in the benzo(a)pyrene-induced fore stomach carcinoma in female thymus-bearing mice (Wu et al, 2001) after intraperitoneal administration of a-TOS. Colon and mammary tumor metastases are reduced by a-TOS (Barnett et al, 2002; Lawson et al, 2004). a-TOS may also enhance sensitization of resistant cells to other inducers of apoptosis, such as the immunological TNF-related apoptosis-inducing ligand (Weber et al, 2002). The above results further strengthen and extend the prospects for a-TOS, an efficient apoptosis-triggering agent, as a promising anti-cancer drug.

a-TOS is effective only when given intra-peritoneally, not by oral administration in the in vivo experiments, because the ester bond linking the succinyl moiety to tocopherol is subject to hydrolysis by non-specific esterases upon intestinal uptake of the drug. The recently synthesized ether analogues of vitamin E, a-tocopheryloxybutyrate and 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (a-TEA) have improved this aspect to a great extent. In the two compounds, butyryl and malonyl groups are attached to the Functional Domain of the analog, respectively, via an ether bond that is resistant to esterase-modulated hydrolysis, endowing the compounds with superior stability to their ester counterparts. It has been reported that a-tocopheryloxybutyrate exerts comparable apoptotic activity to that of a-TOS in leukaemia cell lines (Fariss et al, 1994) or even more potency in prostate cancer cells (Wu et al, 2004b). a-TEA exerts similar anti-cancer and apoptogenic properties as does a-TOS in human breast, prostate, colon, lung and endometrium cancer cells, and is more efficient than a-TOS in apoptosis induction in human ovarian and cervical cancer cells and mouse mammary tumor cells regardless of the administration method (Anderson et al, 2004; Lawson et al, 2004). These new vitamin E analogs plus a-TOM and a-TOS may epitomize new approaches to development and establishment of anti-cancer drugs of the broad vitamin E group of compounds.

Taken together, vitamin E analogs with potent apoptogenic activity show efficient anti-cancer activity in vitro and in vivo using experimental animal models. However, the exact mechanisms of triggering apoptosis are still unclear at this stage. Most of the recent advances have shed some light on the characterization of the effector mechanisms. Two such mechanisms, associated with the caspase cascade, have been intensively investigated, viz. the intrinsic or mitochondria-mediated mechanism and the extrinsic or death receptor-mediated mechanism. Some aspects of these signaling pathways, in relation to apoptosis induction in cancer cells by vitamin E analogs, are provided below.

III. Mitochondrial apoptogenic path-ways as a target for pro-apoptotic activity of vitamin E analogs

Mitochondria are membrane-enclosed organelles distributed through the cytosol of most eukaryotic cells. They have an outer membrane that defines their structure and an inner membrane (also known as cristae) that encloses a fluid-filled matrix. The outer membrane contains complexes of integral membrane proteins that form channels through which a variety of molecules and ions move in and out of the mitochondrion. The inner membrane contains 5 complexes of integral membrane proteins: NADH dehydrogenase, succinate dehydrogenase, cytochrome c reductase (also known as the cytochrome b-c₁ complex), cytochrome c oxidase and ATP synthase. Mitochondria are essential for optimal life of most eukaryotic cells by mediating energy generation in the form of ATP. Paradoxically, recent research demonstrated that mitochondria also play an important role in programmed cell death (Green and Kroemer, 2004), and the role of mitochondria has also been demonstrated for apoptosis induced by vitamin E analogs (Neuzil et al, 2004). Thus, vitamin E analogs belong to the class of ‘mitocans’, agents that initiate cell death and potentially suppress cancer by targeting mitochondria (Ralph et al, 2006).

A. Initiation of apoptotic pathways leading to destabilization of mitochondria

How could vitamin E analogs affect mitochondria and trigger the initial apoptotic signals? The first event observed upon exposure of cells to a-TOS is the activation of sphingomyelinase (SMase), an enzyme that converts sphingomyelin, which is a relatively rare constituent of the plasma membrane, to a lipid second messenger ceramide, a well-known strong inducer of apoptosis (Ogretmen and Hannun, 2004). We showed that treatment of Jurkat cells resulted in activation of SMase within 15-30 min and this was not suppressed by a pan-caspase inhibitor, zVAD-fmk, suggesting that SMase is a caspase-in-dependent, possibly a direct target of the vitamin E analog (Weber et al, 2003). It is also plausible that the activation is due to a change in the plasma membrane fluidity upon incorporation of the lipophilic a-TOS, consistent with a recently suggested mechanism (Dimanche-Boitrel et al, 2005). Generation of the lipid second messenger ceramide in cancer cells as a very early response to a-TOS may also provide an explanation for activation of protein phosphatase 2A (PP2A) and the ensuing hypo-phosphorylation of protein kinase C-a in cells exposed to a-TOS, since the drug does not directly target PP2A (Neuzil et al, 2001c). This hypothesis is in agreement with the previous finding that long-chain ceramides are activators of PP2A (Ruvolo et al, 1999).

There is evidence, however, that treatment of cells with a-TOS causes generation of reactive oxygen species (ROS) (Ottino and Duncan, 1997; Kogure et al, 2001, 2002; Weber et al, 2003; Wang et al, 2005; Stapelberg et al, 2005; Swettenham et al, 2005). Generation of radicals appears to be a relatively early event in cells as a response to vitamin E analogs, since we observed substantial accumulation of ROS in Jurkat cells after one hour of a-TOS challenge. The major form of ROS generated by cells in response to a-TOS appears to be superoxide, because addition of superoxide dismutase removed the radicals and also inhibited apoptosis (Kogure et al, 2001; Wang et al, 2005). Moreover, the site of superoxide generation as well as the target of ROS are very likely mitochondria, as suggested by experiments, in which mitochondrially targeted coenzyme Q (Kelso et al, 2001) suppressed radical generation and inhibited apoptosis induced by a-TOS in cancer cells (Alleva et al, 2001; Weber et al, 2003; Wang et al, 2005). Also, it has been reported that a-TOS-induced apoptosis was more pronounced in cancer cells with low efficacy of the antioxidant machinery (Kogure et al, 2002). It is not clear at present, whether the initiation of apoptotic pathways leading to mitochondria-dependent events, is a direct response to the challenge of a-TOS or whether this is mediated via ceramide formation, which, in both cases, results in destabilization of the mitochondrial membrane. This process is either a direct consequence of ROS or is amplified by the oxyradicals, which are generated as a response to a-TOS challenge (Ottino et al, 1997; Kogure et al, 2001).

B. Apoptotic signaling down-stream of mitochondria and their culmination in the commitment phase of apoptosis

While the evidence of the initial triggers in apoptosis induced by vitamin E analogs is not very clear, the events in apoptosis induced by vitamin E analogs down-stream of mitochondria are known in more detail.

Mitochondria are sites of mediators of apoptosis, whose re-localization relays further the up-stream pro-apoptotic signals. In apoptosis induced by vitamin E analogs, such down-stream events following mito-chondrial destabilization comprise mobilization of apoptotic mediators, which include cytochrome c, the apoptosis-inducing factor (AIF) and Smac/Diablo (Neuzil et al, 2004). Cytochrome c, upon cytosolic translocation, forms a ternary complex with Apaf-1 and pro-caspase-9, leading to auto-activation of the initiator caspase-9 with ensuing activation of the effector caspase-3, -6 or -7. At this stage, the cell enters the ‘point of no return’, i.e. the irreversible phase of the apoptotic pathway (Yamamoto et al, 2000; Neuzil et al, 2001a; Weber et al, 2003). It is now clear that this particular pathway is critically important in apoptosis induced by a-TOS in a variety of cancer cells (Neuzil et al, 2004).

Smac/Diablo is an important agonist of the caspase-dependent apoptotic signaling, since it antagonises the caspase-inhibitory members of the family of inhibitors of apoptosis proteins (IAPs), including c-IAP1, c-IAP2 and X-IAP (Du et al, 2000; Verhagen et al, 2002). The expression of IAPs is under control of the transcriptional factor nuclear factor-kB (NFkB), whose activity is inhibited by a-TOS (Erl et al, 1997; Neuzil et al, 2001a; Dalen and Neuzil, 2003). Thus, cytosolic translocation of Smac/Diablo may promote inhibition of the survival pathways in apoptosis induced by a-TOS, which could maximize the apoptogenic potential in resistant cells (Neuzil et al, 2003; Wang et al, 2005).

Another mitochondrial protein amplifying apoptosis in cells exposed to vitamin E analogs is AIF (Weber et al, 2003) that translocates directly into the nuclei, thereby bypassing the caspase activation cascade (Susin et al, 1999). AIF, upon translocation to the nucleus, triggers cleavage of chromatin in a caspase-independent manner (Cande et al, 2002). AIF can thus avoid mutations in the caspase-dependent signaling or situations where IAPs are over-expressed, that can render the cancer cell resistant, and may mediate a-TOS-induced apoptosis in cells resistant to conventional anti-cancer drugs that rely solely on caspase activation (Neuzil et al, 2004).

The mitochondrial pro- and anti-apoptotic proteins, including Bax, Bcl-2, Mcl-1 and Bcl-x_L,are important factors related to mitochondrial apoptotic signaling pathways (Cory et al, 2003). Generation of the mitochondrial permeability transition pore has also been suggested in cells exposed to a-TOS (Yamamoto et al, 2000). It is likely that this is modulated by a cross talk between the mitochondrial pro- and anti-apoptotic proteins (Yamamoto et al, 2000; Weber et al, 2003). Over-expression of Bax sensitized cells to a-TOS-induced apoptosis (Weber et al, 2003; Yu et al, 2003), whereas over-expression of Bcl-2 or Bcl-x_L protected them from the vitamin E analog. This was not observed when truncated proteins lacking the mitochondrial-targeting terminus were used for transfection of the cells (Weber et al, 2003). Similarly, down-regulation of Bcl-2 by anti-sense oligodoxynucleotide treatment sensitized cells to the vitamin E analog (Neuzil et al, 2001b, c; Weber et al, 2003). Finally, transfection with a gain-of-function mutant of Bcl-2 protected from while a loss-of-function mutant of the protein sensitized cancer cells to a-TOS (Neuzil et al, 2001c): in these mutant versions of Bcl-2, serine 70 was replaced with glutamine and alanine, respectively. This observation can be explained by PKC-dependent phosphorylation of S70 that plays a role in mitochondrial docking of Bcl-2 (Ruvolo et al, 1998).

A compelling evidence for mitochondria as major transmitters of apoptotic signaling induced by vitamin E analogs follows from experiments, in which mtDNA-deficient (r°) cells were found to be resistant to a-TOS when compared to their wild-type and revertant counter-parts (Weber et al, 2003; Wang et al, 2005). It has also been observed that transfection of cancer cells with dominant-negative (DN) caspase-9 suppressed apoptosis induced by a-TOS (Weber et al, 2002). We found that cancer cells lacking mtDNA, resistant to apoptosis (Dey et al, 2000), failed to translocate cytochrome c when challenged with a-TOS, unlike the apoptosis-sensitive parental and revertant cells, and this resistance also in-cluded low levels of phosphatidyl serine externalization and caspase-3 activation (Weber et al, 2003). Similar resistance of r° cells has been found for other inducers of apoptosis, such as tumor necrosis factor-a (TNF-a) (Higuchi et al, 1997).

Thus, mitochondria are indisputably the major intra-cellular organelles that relay the initial apoptotic signals down-stream to the stage at which the cell enters the apoptosis commitment stage. It needs to be emphasized though, that other organelles may also be involved in the process of apoptosis induced by vitamin E analogs, such as lysosomes, as shown in the literature (Neuzil et al, 1999, 2002). Notwithstanding, mitochondria are obli-gatory for transmission of the early apoptogenic events in cells, probably amplified by mediators released from organelles like lysosomes or the endoplasmatic reticulum. The major pathways of apoptosis induction by vitamin E analogs are suggested in Figure 2.

IV. Modulation of signaling pathways by vitamin E analogs and its role in apoptosis induction

Although mitochondria play a major role in apoptosis triggered by vitamin E analogs, there are other signaling pathways that parallel and/or amplify the intrinsic apoptogenic pathway (Neuzil et al, 1999; Yamamoto et al, 2000; Weber et al, 2003; Yu et al, 2003). The mitochondrial pathway is initiated by cytosolic trans-location of the mediators cytochrome c (Weber et al, 2003; Wang et al, 2005), AIF (Weber et al, 2003; Neuzil et al, 2001) or Smac/Diablo (Wang et al, 2005), all of which may occur as a response to exposure of cancer cells to vitamin E analogs, as shown primarily for a-TOS. Mobilization of these modulators of apoptosis results in either caspase-dependent (cytochrome c) or caspase-independent apoptosis (AIF), or in secondary modulation of other pathways regulating the ultimate outcome of pro-apoptotic signaling routes (Smac/Diablo). The cytochrome c- and AIF-dependent pathways were discussed in the previous chapter, the role of Smac/Diablo is also covered below in more detail, as well as are signaling pathways modulated by vitamin E analogs in apoptosis induction/amplification in cancer cells.

A. Inhibition of cell cycle progression by vitamin E analogs

Several reports implicated inhibition of the cell cycle progression as a means by which vitamin E analogs may induce apoptosis or inhibit proliferation of cancer cells and/or sensitize them to other anti-cancer drugs. Ni et al, (2003) showed that a-TOS inhibits proliferation of prostate cancer cells by down-regulating expression of several critical cyclins and the cognate cyclin-dependent kinases (CDK), resulting in hypo-phosphorylation of the Rb protein and a G1/S arrest. Cell cycle arrest and apoptosis were also induced by a-TOS in osteosarcoma cells via activation of p53 and reduced expression of the transcription factor E2F1, critical for the G1/S transition (Alleva et al, 2006). Further, exposure of osteosarcoma cells to a-TOS promoted a prolonged arrest at the S/G2 border, sensitizing the cells to methotrexate-induced apoptosis (Alleva et al, 2005). These findings can be reconciled with an earlier report, in which a-TOS suppressed proliferation of breast cancer cells by inhibiting the E2F1-dependent trans-activation via increased binding of cyclin A (Turley et al, 1997).

Apoptosis induction and inhibition of proliferation by a-TOS have been shown for malignant mesothelioma cells (Tomasetti et al, 2004), the latter paradigm being due to selective disruption of the FGF-FGFR autocrine signaling loop, most likely affected by modulation of the E2F1 and egr-1 trans-activation activity (Stapelberg et al, 2004, 2005). These are exciting results, since malignant mesotheliomas cannot be treated at this stage and since we found that a-TOS shows a strong anti-mesothelioma effect in animal models (Tomasetti et al, 2004; Stapelberg et al, 2005).

Thus, proliferation and apoptosis are intimately coupled, and cell cycle modulators can influence both cell division and apoptosis (Vermeulen et al, 2003). The cell cycle is coordinately controlled by CDKs and their cyclin partners, whose levels fluctuate throughout the cell cycle. The pRb pathway plays a central role in cell proliferation by modulating the activity of the transcription factor E2F (Dimova and Dyson, 2005). E2F1 can signal p53 phosphorylation that is coincident with p53 accumulation and apoptosis (Rogoff et al, 2002). The p53 gene is frequently lost or mutated in many cancers, and lack of functional p53 is accompanied by elevated rates of genomic instability, rapid tumor progression and resistance to anti-cancer drugs and radiation (Weller et al, 1998). Alleva et al, (2006) used three human osteosarcoma cell lines, the SAOS and U2OS cells carrying the wild-type p53 gene, and the mutant p53 cell line MG63. They showed that a-TOS markedly inhibited cell proliferation

in MG63 cells without affecting cell growth in both SAOS and U2OS cells. In SAOS cells, a-TOS induced cell accumulation in S/G2 phase coincident with a decrease of cells in G1, which was observed after 24 h of treatment. However, the highest a-TOS concentration was able to induce cell death at prolonged times of drug exposure. The U2OS cell line responded to a-TOS treatment by a transient accumulation of cells in the G1 phase. Higher concentration of a-TOS induced cell death after 48h of treatment, with accumulation of apoptotic cells in sub-G1. In MG63 cells, a-TOS induced cell accumulation in the S/G2 phase, and this was associated with disappearance of cells in G1, similarly as observed in SAOS cells.

In order to evaluate the molecular mechanism involved in the cell cycle arrest caused by a-TOS, expression of the cell cycle regulatory proteins that control the S/G2 progression was examined. Treatment of SAOS and U2OS cells with a-TOS did not affect the expression of cyclin A and cyclin E, which was similar in the un-treated cells for up to 72 h of incubation. Conversely, treatment of MG63 cells with a-TOS caused a reduction in both cyclin A and cyclin E protein levels.

The above results showed that vitamin E analogs, epitomized by a-TOS, exert a potent modulatory activity towards cell cycle progression and that different cell types respond differently to the agent, in particularly as shown by arrest in different stages of cell cycle. Although these differences point to possibly different targets for a-TOS in various cells, the vitamin E analog does affect the cell cycle progression, and this in its own right inhibits cell proliferation resulting in suppression of tumor growth, and/or amplifies the apoptogenic signaling pathways.

B. The c-Jun pathway as a target for apoptosis induced by vitamin E analogues

Several signaling pathways that have been shown to play a role in modulation of apoptotic signaling appear to be affected by vitamin E analogs. Of these the c-Jun pathway has been investigated in more detail due to its tight association with modulation of apoptotic pathways (Liu and Lin, 2005).

The effect of a-TOS on the activity of the c-Jun NH2-terminal kinase (JNK) pathway up-stream components was investigated (Zu et al, 2005). The vitamin E analog markedly increased the level of expression of the Ask1, GADD45, Sek1, and phospho-Sek1 proteins, of which Ask1 and GADD45 are associated with the cell membrane. Consistent with these findings, the phos-phorylated form of JNK was also noticeably increased, although the expression level of total JNK was not affected. Activated Ask1 and GADD45 phosphorylate the Sek1 protein that then leads to phosphorylation of JNK itself. In relation to this effect, the protein Bim, that is normally in the cytosol, tranlocates during apoptosis to the mitochondrial membrane, where it binds to Bcl-2 and Bcl-x_L. Prior to this tranlocation, Bim is phosphorylated by JNK (Kirschnek et al, 2005). Thus, JNK activation leads to antagonization of the anti-apoptotic function of proteins like Bcl-2 and Bcl-x_L, a mechanism permitting cytosolic translocation of mitochondrial mediators of apoptosis.

Activation of c-Jun NH₂-terminal kinase by a-TOS has also been shown for gastric cancer cells (Wu et al. 2004a), and it may amplify the mitochondrial apoptosis signaling pathway, as shown for prostate cancer cells (Zu et al. 2005).

C. Akt, NFkB and other pro-survival pathways as a target for vitamin E analog-induced apoptosis

Of the signaling pathways modulated by vitamin E analogs, some are implicated in high levels of malignancy and resistance of cancers to established drugs. One problem encountered in pathologies like breast carcinomas stems from over-expression of erbB2, a receptor tyrosine kinase proto-oncogene. ErbB2 is a member of the epithelial growth factor receptor super-family and a product of the c-neu gene (Roskoski, 2004; Slamon et al, 1989). This tyrosine kinase-linked trans-membrane protein is over-expressed in >30% breast cancers. The major complication associated with erbB2 over-expression is linked to activation of Akt via the phos-phatidylinositol 3-kinase pathway (Zhou and Hung, 2003; Vivanco and Sawyers, 2002). Akt is a serine/threonine kinase that promotes cellular survival (Dudek et al, 1997). Once activated, Akt exerts anti-apoptotic effects through phos-phorylation of several proteins, including Bad (Datta et al, 1997) or caspase-9 (Cardone et al, 1998). Moreover, Akt causes activation of the transcriptional factor NFkB (Kane et al, 1999) that controls expression of pro-survival genes, such as members of the IAP family (LaCasse et al, 1998). In most non-transformed cells, NFkB complexes (a heterotrimer composed of p50 and p65 subunits bound to an inhibitor subunit IkB) are largely cytoplasmic. Activation of NFkB results in its translocation to the nucleus and binding to promoter regions of specific pro-survival genes, such as those coding for IAPs, the caspase-8 inhibitor FLIP, or the TRAIL decoy receptor DcR1. One possibility by which a-TOS may suppress NFkB-dependent transcription of pro-survival genes is activation of caspase-3 that cleaves the NFkB subunit p65 (Neuzil et al, 2001a), as also documented for growth factor-starved cells (Levkau et al, 1999).

We showed that vitamin E analogs induced apoptosis at comparable levels in mouse and human breast cancer cells, regardless of their erbB2 status. One plausible mechanism is that these agents induce re-localization of Smac/Diablo from mitochondria to the cytosol (Wang et al, 2005), where Smac/Diablo binds to IAPs so that caspase-3 is librated to execute its apoptotic function (Du et al, 2000). In another report, it has been shown that a-tocopheryloxybutyric acid, a compound analogous to a-TOS, induced apoptosis in the erbB2-over-expressing human breast cancer cells MDA-MB-453 by simultaneously inhibiting activation (phosphorylation) of erbB2 and ensuing activation of p38 MAP kinase (Akazawa et al, 2002). Several other papers showed modulation of the MAP kinase pathway by vitamin E analogs as a way by which the agents induce apoptosis. Interestingly, Kline’s group reported that extra-cellular signal-regulated kinases (ERKs) and JNK, but not the p38 MAP kinase, were involved in a-TOS-induced apoptosis in the human breast cancer cells MDA-MB-435 cells, and this activated the down-stream transcription factors c-Jun and ATF-2 (Yu et al, 2001). It is possible that this pathway is targeted by a-TOS in the erbB2-low MDA-MB-435 cells while the erbB2-high MDA-MB-453 cells activate their apoptotic machinery by concerted deregulation of the erbB2/Akt and p38 pathways when challenged with vitamin E analogs.

One of the intriguing targets of vitamin E analogs is the pro-survival transcription factor NFkB (see above). Inhibition of activation of NFkB by a-TOS was first documented in the context of cardiovascular diseases (Erl et al, 1997). One possibility is that the vitamin E analogs trigger apoptosis resulting in activation of caspase-3 that cleaves the obligatory NFkB subunit p65, rendering it inactive (Levkau et al, 1999). We have shown that a-TOS initiates a ‘sub-apoptotic’ phenotype, under which cells activate their effector caspase but do not enter the commitment phase (Neuzil et al. 2001a), probably because this requires efficient activation of specific cyclin-dependent kinases (Harvey et al, 2000). Regardless of the precise mechanism, inhibition of NFkB activation by vitamin E analogs has an anti-survival effect, i.e. is pro-apoptotic, and can also be implicated in adjuvant cancer therapy, such as shown for the T lymphoma Jurkat cells, whose treatment with a-TOS sensitized them to TRAIL-dependent killing (Dalen and Neuzil, 2003).

V. Sensitization of cancer cells to TRAIL by modulation of signaling pathways by vitamin E analogs

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has attracted much interest because of its potential selectively in triggering apoptosis in tumor cells rather than in their normal counterparts (Pitti et al, 1996; Ashkenazi et al, 1999; Shi et al, 2003). TRAIL has been proven relatively safe in “in vivo” studies of rodents and primates compared with other death receptor ligands, TNF and Fas, which induce significant inflammation and tissue injury (Ashkenazi et al, 1999; Walczak et al, 1999). Thus, two unique characteristics of TRAIL have been identified: firstly, TRAIL can selectively induce apoptosis in tumorigenic or transformed cells, but not in normal cells, highlighting its potential application in cancer treatment. Second, in contrast to other member of TNF family, whose expression is tightly regulated and which are often only transiently expressed on activated cells, TRAIL mRNA is expressed continuously in a wide range of tissues (Wiley et al, 1995).

A. TRAIL-induced signaling and apoptosis

TRAIL is a type II membrane protein or secreted in soluble form which binds to its cognate DRs, DR4 and DR5, inducing their trimerization and intracellular recruitment of the adaptor protein Fas-associated death domain (FADD) (Schneider et al, 1997). The death domain (DD), in turn, recruits pro-caspase-8 into a death-inducing signaling complex (DISC) that triggers autocatalytic cleavage and activation of caspase-8, which then leads to activation of effector caspase-3 (type I pathway). Alternatively, the death pathway can be further amplified by involvement of the mitochondrial signaling (type II pathway) (Scaffidi et al, 1998). TRAIL-activated caspase-8 can generate truncated Bid, which triggers the release of cytochrome c from mitochondria, leading to the assembly of the apoptosome (cytochrome c, Apaf-1, pro-caspase-9). Formation of apoptosome activates caspase-9, which then activates effector caspases (Zou et al, 1999). Type I cells exert DISC-activated caspase-8, which activates down-stream effector caspases and triggers execution of apoptosis. However, in the majority of cells (Type II), TRAIL-induced activation of caspase-8 is insufficient to kill without recruiting the mitochondrial apoptotic program.

A number of proteins are involved in regulation of the TRAIL apoptotic pathways. The FLICE-like inhibitory protein (FLIP) contains two DDs that can bind to DDs of FADD and inhibit recruitment of pro-caspase-8 to the DISC) (Irmler et al, 1997). Inhibitor of apoptosis proteins (IAPs) are characterized by the presence of one to three baculoviral IAP repeated (BIR) domains that bind to caspases (Verhagen et al, 2001). Binding of IAPs to caspases can be inhibited by several proteins released from mitochondria. The second mitochondria-derived activator of caspases (Smac, also referred to as Diablo), which directly binds IAPs, is located in the inter-membrane space of mitochondria and is released into the cytosol upon changes in the mitochondrial membrane permeability (Verhangen et al, 2000). Smac/Diablo facilitates apoptosis by liberating caspase-3 or -7 from inhibition mediated by IAPs.

B. Resistance to TRAIL-induced apoptosis

Although TRAIL is a potent anti-cancer agent in pre-clinical models, it is known that some tumor cells possess intrinsic or acquired resistance to TRAIL. Tumor cells can acquire resistance to apoptosis through interference with either intrinsic (Type II pathway) or extrinsic (Type I pathway) apoptotic signaling pathways. Mutation of the pro-apoptotic Bcl-2 family member Bax confers resistance to TRAIL-induced apoptosis in HCT116 cells (LeBlanc et al, 2002). Over-expression of FLIP suppresses DR-induced apoptosis in malignant mesothelioma (MM) cells (Rippo et al, 2004).

Tumor cells may avoid TRAIL-mediated killing by down-regulation of DRs (extrinsic resistance). Besides the importance of the balance of DRs and decoy receptors (DcRs), which lack the functional cytoplasmic death domain, the ratio between DR4 and DR5 plays a role in determining sensitivity to TRAIL. In addition, in order to induce apoptosis, DR4 and DR5 have distinct cross-linking requirements. DR4 equally responds to cross-linked TRAIL (membrane bound) and non cross-linked TRAIL (soluble), whereas DR5 signals only in response to non cross-liked soluble TRAIL (Wajant et al, 2001). It was observed that low expression of DRs on the cell surface is responsible for cellular resistance to TRAIL-induced cyto-toxicity in human colon cancer cells (Jin et al, 2004). Anti-cancer drugs have been shown to sensitize TRAIL receptor-negative cells to TRAIL-mediated apoptosis by inducing expression of DRs on the cell surface (Arizono et al, 2003). Thus, combination of TRAIL with other drugs resulted in a cooperative or synergist effect (LeBlanc et al, 2002; Wang and El-Deiry, 2003).

C. Modulation of TRAIL sensitivity by a-TOS

A synergistic and cooperative effect was observed when TRAIL was combined with a derivative of vitamin E, a-TOS, in malignant mesothelioma (MM) cells and the effect was selective for cancer cells (Tomasetti et al, 2004b). MM is a fatal type of neoplasia with poor thera-peutic prognosis, largely due to resistance to apoptosis. Impaired apoptotic pathways render MM cells rather resistant to TRAIL-induced apoptosis. Sub-lethal doses of a-TOS significantly decreased the high IC₅₀ values for TRAIL by a factor of ~10-100 (Tomasetti et al, 2004b).

The observation that a-TOS and TRAIL synergize in p53^wt MM but not in the p53^null cells suggests a role of p53 in trans-activation of the pro-apoptotic genes involved in drug synergism (Tomasetti et al, 2006). The p53 protein is a key component of the cellular ‘emergency-response’ mechanism (Levin, 1997; Sionov and Haupt, 1999). A variety of stress-associated signals activate p53 that induces growth arrest or apoptosis, thereby eliminating damaged and potentially dangerous cells (Lane, 1992). The p53 apoptotic target genes can be divided into two groups; the first group encodes proteins that act through receptor-mediated signaling, the second group codes for proteins involved in regulation of the apoptotic effector proteins. a-TOS has the propensity to induce apoptosis in a p53-independent manner (Weber et al, 2002). However, at low concentrations the vitamin E analog induces expression and activation of p53. The p53 induction was concomitant with the enhancement of both DR4 and DR5 expression. Notably, such expression of DRs does not occur in the p53^null MM cells. Studies using siRNA directed at p53 revealed that the p53 protein contributes significantly to the expression of TRAIL’s DRs. Thus, p53-dependent up-regulation of DR4 or DR5 is a basis for sensitization of MM cells to TRAIL. Additionally, the presence of a redox environment efficiently contributes to enhanced expression of DR4 and DR5 via p53 when MM cells are treated with the vitamin E analog (Tomasetti et al, 2006). Regulation of activity of many transcription factors by redox modulators was previously described (Sun and Oberley, 1996). Thus, a novel mode of action for a-TOS has been described: reduction of the redox-sensitive amino acid residues on the p53 protein leads to an increase in the efficiency of TRAIL’s DR expression, sensitizing MM cells to the immunological apoptogen.

MM cells express both DR4 and DR5 on the surface and their up-regulation by a-TOS could facilitate activation of caspase-8 and cleavage of Bid. Kinetic analysis of TRAIL-induced signaling revealed a transient activation of caspase-8, which resulted in induction, albeit low, of apoptosis. Caspase-8 activation was less pro-nounced in the presence of TRAIL plus a-TOS. Under this setting, activation of the mitochondria-dependent apoptotic pathway, including Bid cleavage, cytochrome c cytosolic mobilization and, finally, caspase-9 activation, was observed (Tomasetti et al, 2004). Bid cleavage may lead to mitochondrial translocation of Bax, as shown for a-TOS in other cancer models (Weber et al, 2003, Yu et al, 2003). Thus, the elevation of p53 in response to a-TOS could facilitate TRAIL-induced apoptosis by releasing both Bid and Bax from their sequestration by Bcl-x_L, promoting mitochondria-dependent apoptosis (Fig. 2).

A cooperative pro-apoptotic effect of a-TOS with immunological apoptogens has been also observed in breast cancer (Yu et al, 1999) and colon cancer cells and in an animal model (Weber et al, 2002). The former report showed that a-TOS converted Fas-resistant cells to Fas-sensitive ones via mobilization of the Fas receptor from the cytosol to the plasma membrane. Moreover, a-TOS enhanced sensitivity of Jurkat T lymphoma cells to the induction of apoptosis by TRAIL and the effect was not observed in the presence of a-TOH (Dalen and Neuzil, 2003). In this report, a transient NFkB activation was found when the cells were exposed to TRAIL. Control of transcription by NFkB proteins can be of relevance to the function of TRAIL by induction of the anti-apoptotic NFkB-dependent genes that determine cellular suscepti-bility toward apoptosis induction via DR4 and/or DR5. It is known that NFkB controls expression of pro-survival genes, including FLIP (Kreuz et al, 2001) and IAPs (Degli-Esposti et al, 1997). a-TOS, by inhibiting the TRAIL-induced transient NFkB activation, which in turn inhibits expression of pro-survival proteins that confer resistance of cells to TRAIL-induced apoptosis, may have a role in adjuvant therapy of TRAIL-resistant cancers.

In conclusion, there is evidence that a-TOS can be used in combination with immunological inducers of apoptosis. It can also be used alone, since it is expected to sensitize cancer cells to endogenously produced immuno-logical inducers of apoptosis by cells of the immune system, whereby potentiating the natural tumor sur-veillance.

VI. Conclusions and further perspectives

This paper summarizes some of the major mechanisms by which redox-silent vitamin E analogs, epitomized by a-TOS, induce apoptosis in cancer cells. These intriguing agents, as shown primarily for a-TOS, exert their anti-cancer effect by inducing, inhibiting or modulating a variety of cellular processes and intracellular pathways. Some of them are highlighted in this review. All of these activities separately or in combination contribute to the overall efficacy with which vitamin E analogs suppress cancer progression, including the metastatic process. Studies in pre-clinical settings, using experimental animals, clearly emphasize the potential of agents like a-TOS to become wide-spectrum anti-cancer drugs.

Thus far, there is very little, if anything, known about the effect of vitamin E analogs on cancer in case of human patients. One of the problems encountered is the process of administration of the agents. Most analogs of vitamin E with anti-cancer activity are hydrophobic esters that are completely hydrolyzed upon intestinal uptake. Thus, other means have to be used to deliver the drugs to the blood-stream. The intraperitoneal or intravenous injection used in animal experiments is not readily applicable to humans. A plausible delivery of a-TOS and other analogues of vitamin E with anti-cancer activity may be achieved by trans-dermal application. We are pursuing this option in a limited perspective clinical trial with post-chemotherapy malignant mesothelioma patients. The results will be available later in 2006.

We believe that the multifaceted activity and selectivity of vitamin E analogs provide a substantial promise of these agents to become drugs of choice against multiple malignancies. It can be expected that during 2006 or 2007, we will, hopefully, witness the advent of the use of vitamin E analogs, like a-TOS, in clinical experiments, culminating in application of the agent as an anti-cancer drug. This will require a better knowledge of the molecular mechanism underlying the multiple activity of vitamin E analogs, which have been a subject of intensive research within the recent years.

References

Akazawa A, Nishikawa K, Suzuki K, Asano R, Kumadaki I, Satoh H, Hagiwara K, Shin SJ, and Yano T (2002) Induction of apoptosis in a human breast cancer cell over-expressing ErbB-2 receptor by a-tocopheryloxybutyric acid. Jpn J Pharmacol 89, 417-421.

Alleva R, Benassi MS, Tomasetti M, Gellert N, Borghi B, Neuzil J, and Picci P (2006) a-Tocopheryl succinate alters cell cycle distribution sensitising human osteosarcoma cells to methotrexate-induced apoptosis. Cancer Lett 232, 226-253.

AllevaR, Benassi MS, Tomasetti M, Gellert N, Pazzaglia L, Borghi B, Picci P, and Neuzil J (2005) a-Tocopheryl succinate induces cytostasis and apoptosis in osteosarcoma cells: the role of E2F1. Biochem Biophys Res Commun 331, 1515-1521.

Anderson K, Simmons-Menchaca M, Lawson KA, Atkinson J, Sanders BG, and Kline K (2004) Differential response of human ovarian cancer cells to induction of apoptosis by vitamin E succinate and vitamin E analogue, a-TEA. Cancer Res 64, 4263-4269.

Arizono Y, Yoshikawa H, Naganuma H, Hamada Y, Nakajima Y, and Tasaka K (2003) A mechanism of resistance to TRAIL/Apo2L-induced apoptosis of newly established glioma cell line and sensitization to TRAIL by genotoxic agent. Br J Cancer 88, 298-306.

Ashkenazi A, Pai RC, Frong S, Leung S, Lawrence DA, Marsters SA, Blackie C, Chang L, McMurtrey AE, Hebert A, DeForge L, Koumenis IL, Lewis D, Harris L, Bussiere J, Koeppen H, Shahrokh Z, and Schwall RH (1999) Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 104, 155-162.

Bang OS, Park JH, and Kang SS (2001) Activation of PKC but not of ERK is required for vitamin E succinate-induced apoptosis of HL-60 cells. Biochem Biophys Res Commun 288, 789-797.

Barnett KT, Fokum FD, and Malafa MP (2002) Vitamin E succinate inhibits colon cancer liver metastases. J Surg Res 106, 292-298.

Birringer M, Drogan D, and Brigelius-Flohe R (2001) Tocopherols are metabolized in HepG2 cells by side chain w-oxidation and consecutive b-oxidation. Free Radic Biol Med 31, 226-232.

Cande C, Cecconi F, Dessen P, and Kroemer G (2002) Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death? J Cell Sci 115, 4727-4734.

Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, and Reed JC (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318-1321.

Cory S, Huang DC, and Adams JM (2003) The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22, 8590-8607.

Dalen H, and Neuzil J (2003) a-Tocopheryl succinate sensitises T lymphoma cells to TRAIL killing by suppressing NF-kB activation. Br J Cancer 88, 153-158.

Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, and Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 9, 231-241.

Degli-Esposti MA, Dougall WC, Smolak PJ, Waugh JY, Smith CA, and Goodwin RG (1997) The novel receptor TRAIL-R4 induces NF-kB and protects against TRAIL-mediated apoptosis, yet retains and incomplete death domain. Immunity 7, 813-820.

Dey R, and Moraes CT (2000) Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x_L in osteosarcoma cells. J Biol Chem 275, 7087-7094.

Dimanche-Boitrel MT, Meurette O, Rebillard A, and Lacour S (2005) Role of early plasma membrane events in chemotherapy-induced cell death. Drug Resist Updat 8, 5-14.

Dimova DJ, and Dyson NJ (2005) The E2F transcriptional network: old acquaintances with new faces. Oncogene 24, 2810-2826.

Du C, Fang M, Li Y, Li L, and Wang X (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33-42.

Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segal RA, Kaplan DR, and Greenberg ME (1997) Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275, 661-665.

Erl W, Weber C, Wardemann C, and Weber PC (1997) a-Tocopheryl succinate inhibits monocytic cell adhesion to endothelial cells by suppressing NF-kB mobilization. Am J Physiol 273, H634-640.

Fariss MW, Fortuna MB, Everett CK, Smith JD, Trent DF, and Djuric Z (1994) The selective anti-proliferative effects of a-tocopheryl hemisuccinate and cholesteryl hemisuccinate on murine leukemia cells result from the action of the intact compounds. Cancer Res 54, 3346-3351.

Ghobrial IM, Witzig TE, and Adjei AA (2005) Targeting apoptosis pathways in cancer therapy. CA Cancer J Clin 55, 178-194.

Green DR, and Kroemer G (2004) The pathophysiology of mitochondrial cell death. Science 305, 626-629.

Harvey KJ, Lukovic D, and Ucker DS (2000) Caspase-dependent Cdk activity is a requisite effector of apoptotic death events. J Cell Biol 148, 59-72.

Higuchi M, Aggarwal BB, and Yeh ET (1997) Activation of CPP32-like protease in tumor necrosis factor-induced apoptosis is dependent on mitochondrial function. J Clin Invest 99, 1751-1758.

Hrzenjak A, Reicher H, Wintersperger A, Steinecker-Frohnwieser B, Sedlmayr P, Schmidt H, Nakamura T, Malle E, and Sattler W (2004) Inhibition of lung carcinoma cell growth by high density lipoprotein-associated a-tocopheryl-succinate. Cell Mol Life Sci 61, 1520-1531.

Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, Bodmer JL, Schroter M, Burns K, Mattmann C, Rimoldi D, French LE, and Tschopp J (1997) Inhibition of death receptor signals by cellular FLIP. Nature 388, 190-195.

Israel K, Sanders BG, and Kline K (1995) RRR-a-tocopheryl succinate inhibits the proliferation of human prostatic tumor cells with defective cell cycle/differentiation pathways. Nutr Cancer 24, 161-169.

Israel K, Yu W, Sanders BG, and Kline K (2000) Vitamin E succinate induces apoptosis in human prostate cancer cells: role for Fas in vitamin E succinate-triggered apoptosis. Nutr Cancer 36, 90-100.

Jin Z, McDonald ER, Dicker DT, and El-Deiry WS (2004) Deficient TRAIL death receptor transport to the cell surface in human colon cancer cells selected for resistance to TRAIL-induced apoptosis. J Biol Chem 279, 35829-35839.

Kane LP, Shapiro VS, Stokoe D, and Weiss A (1999) Induction of NF-kB by the Akt/PKB kinase. Curr Biol 9, 601-604.

Kirschnek S, Ying S, Fischer SF, Hacker H, Villunger A, Hochrein H, and Hacker G. (2005) Phagocytosis-induced apoptosis in macrophages is mediated by up-regulation and activation of the Bcl-2 homology domain 3-only protein Bim. J Immunol 174, 671-679.

Kline K, Yu W, and Sanders BG (2004) Vitamin E and breast cancer. J Nutr 134, 3458S-3462S.

Kogure K, Hama S, Manabe S, Tokumura A, and Fukuzawa K (2002) High cytotoxicity of a-tocopheryl hemisuccinate to cancer cells is due to failure of their anti-oxidative defense systems. Cancer Lett 186, 151-156.

Kogure K, Morita M, Nakashima S, Hama S, Tokumura A, and Fukuzawa K (2001) Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by a-tocopheryl hemisuccinate. Biochim Biophys Acta 1528, 25-30.

Kreuz S, Siegmund D, Scheurich P, and Wajant H (2001) NF-kB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling. Mol Cell Biol 21, 3964-3973.

Labriola D, and Livingston R (1999) Possible interactions between dietary antioxidants and chemotherapy. Oncology 13, 1003-1008.

LaCasse EC, Baird S, Korneluk RG, and MacKenzie AE (1998) The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 17, 3247-3259.

Lane DP (1992) Cancer. p53, guardian of the genome. Nature 358, 15-16.

Lawson KA, Anderson K, Simmons-Menchaca M, Atkinson J, Sun L, Sanders BG, and Kline K (2004) Comparison of vitamin E derivatives a-TEA and VES in reduction of mouse mammary tumor burden and metastasis. Exp Biol Med 229, 954-963.

LeBlanc H, Lawrence D, Varfolomeev E, Totpal K, Morlan J, Schow P, Fong S, Schwall R, Sinicrop D, and Ashkenazi A (2002) Tumor-cell resistance to death receptor-induced apoptosis through mutational inactivation of the pro-apoptotic Bcl-2 homolog Bax. Nat Med 8, 274-281.

Levine AJ (1997) p53 the cellular gatekeeper for growth and division. Cell 88, 323-331.

Levkau B, Scatena M, Giachelli CM, Ross R, and Raines EW (1999) Apoptosis overrides survival signals through a caspase-mediated dominant-negative NF-kB loop. Nat Cell Biol 1, 227-233.

Liu J and Lin A (2005) Role of JNK activation in apoptosis: a double-edged sword. Cell Res 15-36-42.

Malafa MP, Fokum FD, Mowlavi A, Abusief M, and King M (2002) Vitamin E inhibits melanoma growth in mice. Surgery 131, 85-91.

Malafa MP, and Neitzel LT (2000) Vitamin E succinate promotes breast cancer tumor dormancy. J Surg Res 93, 163-170.

Malila N, Taylor PR, Virtanen MJ, Korhonen P, Huttunen JK, Albanes D, and Virtamo J (2002) Effects of a-tocopherol and b-carotene supplementation on gastric cancer incidence in male smokers (ATBC Study, Finland). Cancer Causes Control 13, 617-623.

Neuzil J (2003) Vitamin E succinate and cancer treatment: a vitamin E prototype for selective antitumour activity. Br J Cancer 89, 1822-1826.

Neuzil J, Svensson I, Weber T, Weber C, and Brunk UT (1999) a-Tocopheryl succinate-induced apoptosis in Jurkat T cells involves caspase-3 activation, and both lysosomal and mitochondrial destabilisation. FEBS Lett 445, 295-300.

Neuzil J, Schröder A, von Hundelshausen P, Zernecke A, Weber T, Gellert N, and Weber C (2001a) Inhibition of inflammatory endothelial responses by a pathway involving caspase activation and p65 cleavage. Biochemistry 40, 4686-4692.

Neuzil J, Tomasetti M, Mellick AS, Alleva R, Salvatore BA, Birringer M, and Fariss MW (2004) Vitamin E analogues: a new class of inducers of apoptosis with selective anti-cancer effects. Curr Cancer Drug Targets 4, 355-372.

Neuzil J, Weber T, Gellert N, and Weber C (2001b) Selective cancer cell killing by a-tocopheryl succinate. Br J Cancer 84, 87-89.

Neuzil J, Weber T, Schroder A, Lu M, Ostermann G, Gellert N, Mayne GC, Olejnicka B, Negre-Salvayre A, Sticha M, Coffey RJ, and Weber C (2001c) Induction of cancer cell apoptosis by a-tocopheryl succinate: molecular pathways and structural requirements. FASEB J 15, 403-415.

Neuzil J, Zhao M, Ostermann G, Sticha M, Gellert N, Weber C, Eaton JW, and Brunk UT (2002) a-Tocopheryl succinate, an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability. Biochem J 362, 709-715.

Ni J, Chen M, Zhang Y, Li R, Huang J, and Yeh S (2003) Vitamin E succinate inhibits human prostate cancer cell growth via modulating cell cycle regulatory machinery. Biochem Biophys Res Commun 300, 357-363.

Ogretmen B, and Hannun YA (2004) Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer 4, 604-616.

Ottino P, and Duncan JR (1997) Effect of a-tocopherol succinate on free radical and lipid peroxidation levels in BL6 melanoma cells. Free Radic Biol Med 22, 1145-1151.

Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, and Ashkenazi A (1996) Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271, 12687-12690.

Prasad KN, Kumar B, Yan WD, Hanson AJ, and Cole WC (2003) a-Tocopheryl succinate, the most effective form of vitamin E for adjuvant cancer treatment: a review. J Am Coll Nutr 22, 108-117.

Ralph SJ, Dong LF, Low P, Lawen A, and Neuzil J (2006) Mitocans: Mitochondria-targeted anti-cancer drugs as improved therapies and related patents. Recent Patent Reviews on Anti-Cancer Drug Discovery (in press).

Rippo MR, Moretti S, Vescovi S, Tomasetti M, Orecchia S, Amici G, Catalano A, and Procopio A (2004) FLIP overexpression inhibits death receptor-induced apoptosis in malignant mesothelial cells. Oncogene 26, 7753-7760.

Rogoff HA, Pickering MT, Debatis ME, Jones S, and Kowalik TF (2002) E2F1 induces phosphorylation of p53 that is coincident with p53 accumulation and apoptosis. Mol Cell Biol 22, 5308-5318.

Roskoski R (2002) The ErbB/HER receptor protein-tyrosine kinases and cancer. Biochem Biophys Res Commun 319, 1-11.

Ruvolo PP, Deng X, Carr BK, and May WS (1998) A functional role for mitochondrial protein kinase C-a in Bcl2 phosphorylation and suppression of apoptosis. J Biol Chem 273, 25436-25442.

Ruvolo PP, Deng X, Ito T, Carr BK, and May WS (1999) Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. J Biol Chem 274, 20296-20300.

Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, and Peter ME (1998) Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17, 1675-1687.

Schneider P, Thome M, BurnsK, Bodmer JL, HofmannK, Kataoka T, Holler N, and Tschopp J (1997) TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-kB Immunity 7, 831-836.

Shi J, Zheng D, Mam K, Tam P, Fan ST, and Xu R (2003) TRAIL: a potential agent for cancer therapy. Curr Mol Med 3, 727-736.

Sionov RV, and Haupt Y (1999) The cellular response to p53: the decision between life and death. Oncogene 18, 6145-6157.

Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, and Ullrich A (1989) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244, 707-712.

Stapelberg M, Gellert N, Swettenham E, Tomasetti M, Witting PK, Procopio A, and Neuzil J (2005) a-Tocopheryl succinate inhibits malignant mesothelioma by disruption of the FGF autocrine signalling loop: Mechanism and the role of oxidative stress. J Biol Chem 280, 25369-25376.

Stapelberg M, Tomasetti M, Gellert, N, Alleva R, Procopio A, and Neuzil J (2004) a-Tocopheryl succinate inhibits proliferation of mesothelioma cells by differential down-regulation of fibroblast growth factor receptors. Biochem Biophys Res Commun 318, 636-641.

Sun SY, Hail N, and Lotan R (2004) Apoptosis as a novel target for cancer chemoprevention. J Natl Cancer Inst 96, 662-672.

Sun Y, and Oberley LW (1996) Redox regulation of transcriptional activators. Free Radic Biol Med 21, 335-348.

Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, and Kroemer G (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441-446.

Swettenham E, Witting PK, Salvatore BA, and Neuzil J (2005) a-Tocopheryl succinate selectively induces apoptosis in neuroblastoma cells: Potential therapy of malignancies of the nervous system? J Neurochem 94, 1448-1456.

Tomasetti M, Andera L, Neuzil J, Procopio A (2006) a-Tocopheryl succinate sensitizes cells to TRAIL apoptosis by inducing p53-dependent expression of DR4/DR5. FEBS Lett (in press).

Tomasetti M, Gellert N, Procopio A, and Neuzil J (2004a) A vitamin E analogue suppresses malignant mesothelioma in a pre-clinical model: A prototype of a future drug against a fatal neoplastic disease? Int J Cancer 109, 641-642.

Tomasetti M, Rippo MR, Alleva R, Moretti S, Andera L, Neuzil J, and Procopio A (2004b) a-Tocopheryl succinate and TRAIL selectively synergise in induction of apoptosis in human malignant mesothelioma cells. Br J Cancer 90, 1644-1653.

Turley JM, Ruscetti FW, Kim SJ, Fu T, Gou FV, and Birchenall-Roberts MC (1997) Vitamin E succinate inhibits proliferation of BT-20 human breast cancer cells: increased binding of cyclin A negatively regulates E2F transactivation activity. Cancer Res 57, 2668-2675.

Verhagen AM, Coulson EJ, and Vaux DL (2001) Inhibitor of apoptosis proteins and their relatives: IAPs and other BIRPs. Genome Biol 2, reviews3009

Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM; Reid GE, Moritz R, Simpson RJ, and Vaux DL (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102, 43-53.

Verhagen AM, and Vaux DL (2002) Cell death regulation by the mammalian IAP antagonist Diablo/Smac. Apoptosis 7, 163-166.

Vermeulen K, Berneman ZN, and Van Bockstaele DR (2003) Cell cycle and apoptosis. Cell Prolif 36, 165-75.

Vivanco I, and Sawyers CL (2002) The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2, 489-501.

Wajant H, Moosmayer D, Wuest T, Bartke T, Gerlach E, Schonherr U, Peters N, Scheurich P, and Pfizenmaier K (2001) Differential activation of TRAIL-R1 and -2 by soluble and membrane TRAIL allows selective surface antigen-directed activation of TRAIL-R2 by a soluble TRAIL derivative. Oncogene 5, 4101-4106.

Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, and Lynch DH (1999) Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 5, 157-63.

Wang S, and El-Deiry WS (2003) Requirement of p53 targets in chemosensitization of colonic carcinoma to death ligand therapy. Proc Natl Acad Sci USA 100, 15095-100.

Wang XF, Witting PK, Salvatore BA, and Neuzil J (2005) a-Tocopheryl succinate induces apoptosis in HER2/erbB2-overexpressing breast cancer cells by signalling via the mitochondrial pathway. Biochem Biophys Res Commun 326, 282-289.

Weber T, Dalen H, Andera L, Nègre-Salvayre A, AugéN, Sticha M, Loret A, Terman A, Witting PK, Higuchi M, Plasilova M, Zivny J, Gellert N, Weber C, and Neuzil J (2003) Mitochondria play a central role in apoptosis induced by a-tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signalling. Biochemistry 42, 4277-4291.

Weber T, Lu M, Andera L, Lahm H, Gellert N, Fariss MW, Korinek V, Sattler W, Ucker DS, Terman A, Schröder A, Erl W, Brunk U, Coffey RJ, Weber C, and Neuzil J (2002) Vitamin E succinate is a potent novel anti-neoplastic agent with high tumor selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2L) in vivo. Clin Cancer Res 8, 863-869.

Weller M (1998) Predicting response to cancer chemotherapy: the role of p53. Cell Tissue Res 292, 435-445.

Wiley SR, Schooley JK, Smolak PJ, Din WS, Huang CP, Nicholl JK Sutherland GR, Smith TD, Rauch C, and Smith CA (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673-82.

Woodson K, Tangrea JA, Barrett MJ, Virtamo J, Taylor PR, and Albanes D (1999) Serum a-tocopherol and subsequent risk of lung cancer among male smokers. J Natl Cancer Inst 91, 1738-1743.

Wu K, Shan YJ, Zhao Y, Yu JW, and Liu BH (2001) Inhibitory effects of RRR-a-tocopheryl succinate on bezo(a)pyrene (B(a)P)-induced forestomach carcinogenesis in female mice. World J Gastroenterol 7, 60-64.

Wu K, Zhao Y, Li GC, and Yu WP (2004a) c-Jun N-terminal kinase is required for vitamin E succinate-induced apoptosis in human gastric cancer cells. World J Gastroenterol 10, 1110-1114.

Wu K, Zhao Y, Liu BH, Li Y, Liu F, and Guo J (2002) RRR-a-tocopheryl succinate inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest. World J Gastroenterol 8, 26-30.

Wu Y, Zu K, Ni J, Yeh S, Kasi D, James NS, Chemler S, and Ip C (2004b) Cellular and molecular effects of a-tocopheryloxybutyrate: lessons for the design of vitamin E analog for cancer prevention. Anticancer Res 24, 3795-3802.

Yamamoto S, Tamai H, Ishisaka R, Kanno T, Arita K, Kobuchi H, and Utsumi K (2000) Mechanism of a-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells. Free Radic Res 33, 407-418.

Yu W, Israel K, Liao QY, Aldaz CM, Sanders BG, and Kline K (1999) Vitamin E succinate (VES) induces fas sensitivity in human breast cancer cells: role for Mr 43,000 Fas in VES-triggered apoptosis. Cancer Res 59, 953-961.

Yu W, Liao QY, Hantash FM, Sanders BG, and Kline K (2001)Activation of extracellular signal-regulated kinase and c-Jun-NH₂-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-a-tocopheryl succinate-induced apoptosis of human breast cancer cells. Cancer Res 61, 6569-6576.

Yu W, Sanders BG, and Kline K (2003) RRR-a-Tocopheryl succinate-induced apoptosis of human breast cancer cells involves Bax translocation to mitochondria. Cancer Res 63, 2483-2491.

Zhou BP, and Hung MC (2003) Dysregulation of cellular signalling by HER2/neu in breast cancer. Semin Oncol 30, 38-48.

Zou H, Li Y, Liu X, and Wang X (1999) An APAF-1 cytochrome c multimeric complex is a functional apoptosome that activates pro-caspase-9. J Biol Chem 274, 11549-11556.

Zu K, Hawthorn L, and Ip C (2005) Up-regulation of c-Jun-NH2-kinase pathway contributes to the induction of mitochondria-mediated apoptosis by a-tocopheryl succinate in human prostate cancer cells. Mol Cancer Ther 4, 43-50.

Review Article

Received: 16 January 2006; Accepted: 31 January 2006; electronically published: February 2006

I. Introduction

References