Days of Treatment

FIGURE 6.3 Antimesothelioma effects of a-tocopheryl succinate. (A) Immunocompro-mised mice were injected intraperitoneally (i.p.) with human mesothelioma cells and peritoneal mesotheliomas allowed to established. On week 4 after the animals were injected with the cells, treatment was initiated consisting of i.p. administration of 200 |ll of 200 ||M a-TOS (in DMSO) every third day for more than 20 weeks. Survival was used as a marker for the effect of a-TOS. (Adapted from Reference 16.) (B) Immunocompromised mice were injected subcutaneously with human mesothelioma cells and tumors allowed to reach 100 mm3 before treatment onset. The mice were then injected i.p. with 200 |ll of 200 mM a-TOS (in DMSO) every second day for up to 2 weeks. Tumor size was estimated and volume calculated. (Adapted from Reference 96.)

Recently, Kline's group demonstrated anticancer activity of a novel VE analog against breast cancer and lung metastases alone or in combination with celo-coxib.4344 These data demonstrate the therapeutic promise of a-TOS and are likely to result in clinical application of the agent and design of novel anticancer agents.

A major reason for the anticancer efficacy of VE analogs is their high apo-ptogenic activity. We observed that while a-TOS significantly suppressed experimental colon cancer, a-TOH showed only marginal, nonsignificant effect.37 Analysis of the tumor sections revealed that both agents inhibited proliferation, but only a-TOS induced apoptosis.45,46 Apoptosis as a major mechanism for antitumor activity of VE analogs has also been proposed for its antimelanoma effect39 and may be expected also in mesotheliomas.41 Although multiple modes of action may be expected for VE analogs, induction of apoptosis is the major determinant of their anticancer efficacy.

6.3.3 Selectivity of VE Analogs Increases Their Clinical Application

A paradigm rendering VE analogs clinically applicable is their selectivity for neoplastic cells,1 as shown in many cultured cells.28 45 47-49 The reasons for the selectivity are not fully understood. Some types of normal cells have the propensity to hydrolyze a-TOS into its redox-active counterpart.1 Selective hydrolysis of a-TOS may, at least in some cases, explain the selective toxicity of the agent, since the proapoptotic species a-TOS would then accumulate in nonmalignant cells at levels below the toxicity threshold.

Hydrolysis of a-TOS cannot always explain its selectivity. For example, a-TOS is highly toxic to mesothelioma cells but not to nonmalignant mesothelial cells.4150 However, in this case, the resistant cells do not hydrolyze a-TOS. It is possible that the selectivity is given here by the resistance of the nonmalignant cells to oxidative stress. It has been published that a-TOS provokes generation of ROS in cancer cells.51-53 We observed that mesothelioma cells generated ROS as a fast response to a-TOS while nonmalignant mesothelial cells showed very little ROS accumulation (J. Neuzil, unpublished data). It is possible that anti-oxidant enzymes are more expressed in nonmalignant cells, whereby affording protection from ROS-induced apoptosis. In support of this, it has been published that cells depleted of their mtDNA are resistant to apoptosis,51 53 54 and a recent paper showed that a reason for this was adaptive upregulation of the mitochondrial superoxide dismutase.55 It remains to be shown whether this is a mechanism of resistance of nonmalignant cells to apoptosis induced by a-TOS, although there is a report indicating the importance of lack of efficient antioxidant systems to render cancer cells susceptible to the VE analog.56 A recent report documented a direct correlation between susceptibility of cancer cells to a-TOS and their propensity to respond to this agent by generation of ROS.57 In support for low ROS accumulation as a mode of resistance to a-TOS, we have observed relatively low accumulation of ROS in a-TOS-resistant differentiated neuroblastoma cells when compared to the a-TOS-susceptible parental cells (J. Neuzil, unpublished data).

There is yet another plausible explanation for selectivity of VE analogs for malignant cells. This is based on the physicochemical nature of these compounds that are weak acids and that are deprotonated at physiological pH but largely protonated at the acidic pH of the tumor interstitium. This allows selective faster diffusion of the drug across the plasma membrane of malignant cells. Thus, there are several alternative or parallel mechanisms underlying the selectivity of VE analogs for cancer cells. Because this is one of the most important features of VE analogs, it is important to understand these mechanisms and, consequently, design and synthesize novel agents with higher proapoptotic activity while retaining selectivity.

We have recently studied the effect of a-TOS on neuroblastoma cells and their differentiated counterparts mimicking nonmalignant neurons. While the former were highly susceptible to the VE analog, the latter were relatively resis-tant.58 We found two lines of defense against a-TOS in the differentiated cells. First, the cells, unlike the parental neuroblastomas, did not accumulate ROS. Second, their levels of antiapoptotic Bcl-2 family proteins were elevated, in particular that of Mcl-1. These data clearly suggest that mitochondria are important not only in induction of apoptosis, but also in the selectivity of importance VE analogs for malignant cells.

6.3.4 VE Analogs Overcome Resistance of Mutant Cancer Cells to Apoptosis, Induce the Mitochondrial Apoptotic Pathway, and Cooperate with Immunological Apoptogens

Many cancer cells avoid established therapy by constantly mutating the relevant genes. This is a complicating factor, compromising many successful treatments. Therefore, it is imperative to design novel agents that would overcome these complications. Many anticancer drugs act via induction of apoptosis of malignant cells by causing damage to their genomic DNA, which results in activation of p53 and inhibition of proliferation accompanied by apoptosis. VE analogs show promise for treatment of cancers where such genes are mutated, most likely because they use the mitochondrial apoptogenic route.

It was reported by Coffey's laboratory that Trolox, a water-soluble analog of VE, inhibited proliferation and induced apoptosis in human colon cancer cells by increasing the expression of the cell cycle protein p21 Cip1/Waf1 in a p53-inde-pendent manner.59 This paper also showed that the agent could efficiently inhibit growth of tumors in athymic mice derived from p53- but not p21Cip1/Waf1 -deficient colon cancer cells. It was later observed that a-TOS caused efficient apoptosis in both p53- and p21Cip1/Waf1 -deficient colon cancer cells.38 These findings place VE analogs among potential anticancer drugs capable of being used instead of or as adjuvants to other drugs whose application may be compromised by mutations in the above genes.

Antiapoptotic/pro-survival consequences of other mutations can also be overcome by VE analogs. For example, overexpression of the receptor tyrosine kinase erbB2/HER2 complicates breast cancer treatment.60 The reason is that the auto-activated erbB2 activates Akt, a protein kinase phosphorylating multiple substrates, including IkB kinase (IKK), caspase-9, and Bad, resulting in elevated expression of pro-survival genes and inhibition of proapoptotic proteins.6162 It has been suggested that VE analogs overcome the erbB2-Akt antiapoptotic signaling. Akazawa et al.63 showed that a-tocopheryloxybutyric acid suppressed auto-phosphorylation of erbB2, shutting down the whole signaling pathway, although the mechanism is not clear. We have observed that a-TOS caused comparable apoptosis in both erbB2-low and erbB2-high breast cancer cells by a two-tier mechanism64 that includes cytosolic mobilization of cytochrome c, activating the downstream caspases, as well as that of Smac/Diablo, a protein antagonizing the caspase-inhibitory activity of the inhibition of apoptosis proteins (IAPs) that are elevated due to Akt-dependent activation of nuclear factor-KB (NF-kB).65 66 a-TOS thus induces apoptosis in cells over-expressing erbB2 on at least three levels (Figure 6.4). We are currently investigating the anticancer activity of VE analogs using transgenic mice overexpressing erbB2 in the mammary epithelial cells, resulting in spontaneous formation of breast carcinomas.67

The major apoptogenic pathway induced by VE analogs is linked to mito-chondrial destabilization. This is compatible with mitochondria as a novel target for anticancer drugs.6869 Mitochondrial proapoptotic signaling has been documented in several papers, and involves activation of the sphingomyelinase pathways, generation of ROS, and mitochondrial translocation of Bax.51 52 70-73 Conceivably, the ratio between the mitochondrial pro- and antiapoptotic proteins may determine the overall susceptibility of the cells to VE analogs.

Perhaps more importantly, the mode of proapoptotic signaling of VE analogs makes them candidates for adjuvant therapy, that is, synergizing with apoptogens using a different mode of action. a-TOS has been investigated for sensitization of cancer cell to apoptosis induced by the Fas ligand (FasL) and the TNF-related apoptosis-inducing ligand (TRAIL). Kline's group has shown that the VE analog sensitized cancer cells to FasL by mobilizing the latent cytosolic Fas to the plasma membrane,7475 as also observed for gastric cancer cells.76 77 This may suggest a role of a-TOS in cancer surveillance by boosting the immune anticancer/proap-optotic mechanism.

We have explored the possibility that VE analogs synergize/cooperate with TRAIL. In colon cancer cells, a-TOS synergized with TRAIL in apoptosis induction, by utilizing different, convergent pathways. Cooperation between aTOS and TRAIL was observed in experimental colon cancer.38 In mesothelioma cells, a-TOS synergized with TRAIL by upregulating the TRAIL death receptor-4 (DR4) and DR5.50 This report as well as our recent finding that a-TOS extends survival of mice with experimental mesothelioma41 is important because

Apo Trail Dr4 Apoptosis

FIGURE 6.4 Possible mechanism of apoptosis induction by a-TOS in erbB2/HER2 over-expressing cells. a-TOS causes cytosolic mobilization of cytochrome c (Cyt c) and Smac/Diablo. Cyt c forms a ternary complex with Apaf-1 and pro-caspase-9, resulting in activation of the initiator caspase-9 that, in turn, leads to activation of the effector caspases. Smac/Diablo amplifies this process by suppressing the IAP family proteins that are elevated due to Akt-dependent activation of NF-kB. This suggests that a-TOS induces apoptosis in cells overexpressing erbB2 on at least three levels: inhibition of erbB2 activation, induction of the mitochondrial-linked apoptotic pathway, and relocalization of Smac/Diablo, thereby suppressing the caspase-inhibitory IAP activity. Mechanistically, aTOS induces mitochondrial generation of ROS as well as mitochondrial translocation of Bax. Additional effects of a-TOS include: inhibition of erbB2 and NF-kB activation and suppression of Akt-dependent phosphorylation of Bad (rendering it inactive due to association with the protein 14-3-3) and pro-caspase-9 (suppressing its activity normally leading to activation of effector caspases). These effects of a-TOS not only induce apop-tosis in erbB2-overexpressing cells, but also sensitize them to other apoptogens, as shown here for TRAIL.

mesotheliomas are refractory to treatment, largely due to resistance to apoptosis by established drugs.78 Another mode by which a-TOS can sensitize cancer cells to TRAIL killing is its interference with activation of NF-kB, as shown for Jurkat cells.79 A possible mechanism of inhibition of the pro-survival transcription factor may be caspase-dependent cleavage of its subunit p65 due to activation of the apoptosis machinery.8081

6.3.5 Vitamin E Analogs as Antitumor Agents: Beyond Mitochondria

Although the mitochondrial pathway is central to the apoptogenic action of VE analogs, there are other pathways that may play an important role in the effects of the agents. Kline's group reported regulation of the AP-1 and mitogen-activated protein kinase/extracellular-regulated protein kinase pathways as additional signaling routes for apoptosis induced by a-TOS.82-86 Some but not all papers suggested that a-TOS induces apoptosis by interfering with the tumor necrosis factor-P (TGF-P) signaling, compromising the expression of pro-survival factors.87-89 These findings may be related to the effect of the VE analog on transition of cell through the cell cycle. Several reports showed that the agent caused arrest in G1 or G2. Turley et al.90 presented evidence that a-TOS inhibited proliferation of breast cancer cells by interfering with the cyclin A-E2F restriction point machinery. Yeh's group reported that a-TOS inhibited proliferation of cancer cells by modulation of the cell cycle transition91 as well as cell invasiveness due to inhibition of the activity of matrix metalloproteinase-9.92 These effects may be more specific for prostate cancer cells, as a-TOS has been reported to inhibit the function of the prostate-specific antigen.93

Our results also show inhibitory effects of a-TOS on cell cycle transition, with G1 and/or G2 arrest, depending on the cell type.94-96 The mode of cell cycle inhibition may be related to the effect of a-TOS on its modulators. The G1 arrest of the osteosarcoma cells may be linked to inhibition of the activity of the cyclin A/cyclin-dependent kinase-2 complex, while the levels of the transcription factor E2F1 were enhanced and were followed by phosphorylation of p53.94

Mesothelioma cells undergo a G2 arrest when exposed to low levels of a-TOS.95 96 This is related to disruption of the FGF-FGFR autocrine proliferation signaling loop by selective downregulation of both FRF2 and its receptor FGFR1 in the mesothelioma but not the nonmalignant mesothelial cells.95,96 a-TOS exerts its effect on the transcriptional level, downregulating FGFR1 by inhibiting the activity of E2F1,95 while FGF2 is downregulated via modulating the transcriptional activity of the early-response growth factor-1.96 The selectivity appears related to the low level of ROS generation by the nonmalignant mesothelial cells while their malignant counterparts respond to a-TOS by early generation of high levels of ROS.96

There are reports suggesting the possibility that VE analogs inhibit angio-genesis, which indicates an effect of the agents on tumorigenesis and the meta-static potential. We have observed a reduction in the number of blood vessels in experimental colon cancer xenografts in mice treated with a-TOS (J. Neuzil, unpublished data). Preliminary results also indicate an effect of a-TOS on pro-angiogenic cytokines in endothelial cells, suggesting a proliferation-suppressive activity of the VE analog. This can be reconciled with findings that a-TOS caused downregulation of VEGF.36 Another possibility follows from studies indicating that proliferating endothelial cells are highly susceptible to a-TOS while the confluent arrested cells are resistant.80 This highly intriguing paradigm is supported by comparable results from Hogg's group using a different inducer of apoptosis.97 Thus, a-TOS may specifically target angiogenic endothelial cells while being nonapoptotic toward normal endothelium. Resistance of endothelial cells suspended in G0 can be explained by high levels of expression of the checkpoint proteins p21 WafllC^' and p27Kip1 that prevent reentry of the cells into the cell cycle98,99 and that inhibit caspase activity.100

By virtue of inhibiting angiogenesis, VE analogs would be efficient against cancers irrespective of the frequent mutations in various genes. This area is also worth exploring for novel VE analogs. A recent paper showed a potential anti-angiogenic effect of y-TOH by inhibiting the activity of cyclooxygenase-2,101 thereby suppressing formation of pro-angiogenic eicosanoids.102 It is expected that y-TOS combines an antiangiogenic effect with apoptogenic activity (see Table 6.1).

6.3.6 Pharmacokinetics of VE Analogs — A Potential Secondary Beneficial Bioactivity

A most intriguing aspect of the potential use of VE analogs as anticancer agents follows from their pharmacokinetics. These agents are esters of VE and, therefore, hydrolyzed upon intestinal intake after ingestion.103 To overcome the intestinal hydrolysis step, agents such as a-TOS need to be administered intraperi-toneally. After reaching the circulation, they associate with lipoproteins104 105 that carry them to the neoplastic microvasculature. Once in the tumor blood vessels, a-TOS migrates to the malignant tissue where it induces apoptosis, thereby suppressing growth of the tumor. Because the blood components and the peripheral tissue are a dynamic system, there is constant exchange of hydrophobic molecules. a-TOS is thus gradually moved from the tumor and cleared, bound to remnant lipoproteins, via the liver. Here, a-TOS is hydrolyzed by nonspecific esterases. It is then partially disposed of within the bile, partially resecreted into circulation depending on the level of the saturable a-tocopherol-binding protein (a-TTP).105-108

The paradigm described above is shown in Figure 6.5. It is clear that, based on this scheme, supported by theoretical considerations and experimental data,109110 esters of VE are hydrolyzed in the liver into the redox-active VE that is, in part, returned into circulation. From this, it can be deduced that a-TOS and similar compounds possess at least two bioactivities. In the pro-vitamin form, aTOS suppresses cancer. After conversion to its vitamin form, it acts as a redox-active compound and as an anti-inflammatory agent.111115 Thus, we believe that the novel VE analogs represent agents with thus far unrecognized dual beneficial bioactivity relevant to a variety of highly deleterious pathologies, including cancer and inflammatory diseases.

VITAMIN EFFECT

VE accumulates in circulating lipoproteins, thus enhancing the anti-oxidant/anti-inflammatory defences a-TOS —► (circuí

Mechanism Vitamin Myopathy

PRO-VITAMIN EFFECT

Lipoprotein-associated VES exerts its anti neoplastic effect within tumor microcirculation

FIGURE 6.5 Proposed molecular mechanism for the "double-edge" activity of a-toco-pheryl succinate. Following entry into the bloodstream, the pro-vitamin a-TOS vitamin E succinate (VES) associates with circulating lipoproteins (LP) that carry the agent to the microvasculature of the neoplastic tissue. Here, VES exerts its antiproliferative/proapop-totic activity toward malignant cells that translates into inhibition of tumor growth. Since there is a constant exchange of hydrophobic molecules between the peripheral tissues and the circulation, a-TOS is gradually removed by virtue of remnant lipoproteins that are endocytosed by hepatocytes that possess high levels of nonspecific esterases (NE). Here, the remnants are dissembled and nascent VLDL formed. a-TOH (VE), both the original and that formed by hydrolysis of VES, is partially shuttled to nascent VLDL bound to the saturable tocopherol-transfer protein (TTP), partially excreted in the bile. Nascent VLDL enriched with VE is then resecreted into circulation, endowing the system with additional VE that promotes the antioxidant and anti-inflammatory defenses.

6.4 CONCLUSIONS AND FUTURE DIRECTIONS

The above discussion reveals the exceptional promise of VE analogs as therapeutic agents. There are several features that make these compounds unique: (1) VE analogs induce apoptosis selectively in malignant cells and inhibit tumor growth in experimental models; (2) they overcome resistance to established anticancer drugs due to bypassing mutations in or deletions of critical genes; (3) they synergize with anticancer agents and/or sensitize resistant cells toward them; (4) they cause apoptosis in proliferating endothelial cells, suggesting their anti-angiogenic activity; and (5) they are metabolized into the redox-active VE with secondary beneficial bioactivity.

At this stage, we need more data to understand the exact mechanisms by which these compounds exert their proapoptotic/anticancer activity and, in particular, what makes them selective for malignant cells. Importantly, too, we need to obtain more data from preclinical studies using several experimental models. Thus far, the majority of these data comes from work with athymic mice. Transgenic mice predisposed to develop tumors need to be used because in such animals the role of the immune system, essential for tumor surveillance, is not impaired. It is hoped that, with better understanding of the molecular mechanism underlying the activity of VE analogs, they will be approved for clinical testing. Should they prove successful, the very cheap a-TOS or its derivatives may become drugs of choice against multiple malignancies with a secondary beneficial activity.

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