Jiri Neuzil

Induction of cancer cell apoptosis by α-tocopheryl succinate: molecular pathways and structural requirements

The vitamin E analog α‐tocopheryl succinate (α‐TOS) can induce apoptosis. We show that the proapoptotic activity of α‐TOS in hematopoietic and cancer cell lines involves inhibition of protein kinase C (PKC), since phorbol myristyl acetate prevented α‐TOS‐triggered apoptosis. More selective effectors indicated that α‐TOS reduced PKCα isotype activity by increasing protein phosphatase 2A (PP2A) activity. The role of PKCα inhibition in α‐TOS‐induced apoptosis was confirmed using antisense oligonucleotides or PKCα overexpression. Gain‐ or loss‐of‐function bcl‐2 mutants implied modulation of bcl‐2 activity by PKC/ PP2A as a mitochondrial target of α‐TOS‐induced proapoptotic signals. Structural analogs revealed that α‐tocopheryl and succinyl moieties are both required for maximizing these effects. In mice with colon cancer xenografts, α‐TOS suppressed tumor growth by 80%. This epitomizes cancer cell killing by a pharmacologically relevant compound without known side effects.—Neuzil, J., Weber, T., Schröder, A., Lu, M., Ostermann, G., Gellert, N., Mayne, G. C., Olejnicka, B., Nègre‐Salvayre, A., Stícha, M., Coffey, R. J., Weber, C. Induction of cancer cell apoptosis by α‐tocopheryl succinate: molecular pathways and structural requirements. FASEB J. 15, 403‐415 (2001)

Selective cancer cell killing by α-tocopheryl succinate

We report that α-tocopheryl succinate, a vitamin E analogue with pro-apoptotic properties, selectively kills cells with a malignant or transformed phenotype, i.e. multiple haematopoietic and carcinoma cell lines, while being non-toxic to normal, i.e. primary and non-transformed cells. These findings strongly suggest a potential of this micronutrient in the therapy and/or prevention of cancer without significant side-effects.

Lysosomal involvement in apoptosis.

and morphologically distinct way of eliminating cells. Apoptosis occurs without induction of the inflammatory response that typically accompanies necrotic cell death with its attendant release of toxic and chemotactic material from decomposing cells. Morphological hallmarks of apoptosis include cellular shrinkage, cytoplasmic vacuoles, plasma and nuclear membrane blebbing, nuclear pycnosis and fragmentation. These changes are usually accompanied by leakage to the cytosol of mitochondrial cytochrome c and other apoptosis activating factors with ensuing caspase activation – all within intact plasma membranes. Necrosis, on the contrary, is characterized by cellular swelling, nuclear disintegration, and plasma membrane rupture. A final important distinction between these two modes of cell death is that apoptosis is an active energy requiring process, while necrosis is passive.1–4 Apoptosis is a necessary process during embryonic development and organ remodeling, allowing superfluous cells to form apoptotic bodies which are easily phagocytosed by neighboring cells. This phagocytosis is facilitated by externalization of phosphatidylserine, which normally resides almost exclusively in the inner leaflet of the cell membrane. The exposed phosphatidylserine, either directly or through the opsonic function of annexins, serves as a ligand for scavenger receptors present on the surface of most cells. In this way, apoptotic cells are eliminated in an efficient and orderly manner. Furthermore, there is a certain economy in this process in as much as the apoptotic cells may contain nutrients for neighboring normal cells.5–9 Dysfunction of apoptosis has been invoked in a variety of pathological conditions, such as developmental disorders, cancer, AIDS, atherosclerosis, aging, and autoimmune and neurodegenerative diseases. This explains why the study of apoptosis is today such an important and rapidly growing area of research. A variety of agonists may induce apoptosis. These range from blunt instruments, such as oxidative stress, to more subtle triggers of apoptosis which include agonists that bind to receptors belonging to the TNF-receptor superfamily, such as those for tumor necrosis factor-α, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and the Fas-ligand. In these latter cases, ligation activates binding of pro-caspase-8 to intracellular ‘death domains’, resulting in caspase-8 activation.3,10,11 Through a cascade of events, effector caspases, such as caspases 3 or 7, are ultimately activated and apoptosis ensues. Another pathway of apoptosis involving mitochondria is thought to proceed via a changed balance between antiand pro-apoptotic factors, such as Bcl-2, Bcl-xL, Bax, and Bid, which causes a drop in the mitochondrial transmembrane potential and opens mitochondrial pores allowing cytochrome c and other pro-apoptotic factors, such as apoptosis-inducing factor, to diffuse into the cytosol. Released cytochrome c binds to cytosolic apoptosis activating factor (apaf-1) and pro-caspase-9 to form an apoptosome. This leads to activation of caspase-9 which in turn activates the effector caspases 3 and 7. Granzyme B, which is used by NK cells to kill targets, proteolytically activates effector caspases and/or the pro-apoptotic protein Bid.3,12–14 Most contemporary research on apoptosis focuses on the above mechanisms. Since the pioneering work of Kerr et al. during the early 1970s,1,2 details of this series of events have become clearer and there is now abundant experimental support for most of these mechanisms. Nonetheless, there are aspects of apoptosis that remain mysterious. For a long time, apoptosis and necrosis were considered as fundamentally different as suicide and homicide, each with its own distinct initiating mechanisms. Only recently was it pointed out that one agonist alone may induce either apoptosis or necrosis depending on the magnitude of the insult. This is especially obvious for oxidative stress which in low doses may induce cell proliferation, in

Mitochondrial Genome Acquisition Restores Respiratory Function and Tumorigenic Potential of Cancer Cells without Mitochondrial DNA

We report that tumor cells without mitochondrial DNA (mtDNA) show delayed tumor growth, and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory (super)complexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest pathophysiological processes for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.

α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

α-Tocopheryl succinate (α-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of α-TOS has not been identified. Here, we show that α-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (QP and QD, respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of α-TOS compared to that of UbQ for the QP and QD sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of α-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to α-TOS. We propose that α-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

Molecular mechanism of 'mitocan'-induced apoptosis in cancer cells epitomizes the multiple roles of reactive oxygen species and Bcl-2 family proteins

Mitochondria have emerged recently as effective targets for novel anti‐cancer drugs referred to as ‘mitocans’. We propose that the molecular mechanism of induction of apoptosis by mitocans, as exemplified by the drug α‐tocopheryl succinate, involves generation of reactive oxygen species (ROS). ROS then mediate the formation of disufide bridges between cytosolic Bax monomers, resulting in the formation of mitochondrial outer membrane channels. ROS also cause oxidation of cardiolipin, triggering the release of cytochrome c and its translocation via the activated Bax channels. This model may provide a general mechanism for the action of inducers of apoptosis and anticancer drugs, mitocans, targeting mitochondria via ROS production.

Cosupplementation With Coenzyme Q Prevents the Prooxidant Effect of α-Tocopherol and Increases the Resistance of LDL to Transition Metal–Dependent Oxidation Initiation

There is considerable interest in the ability of antioxidant supplementation, in particular with vitamin E, to attenuate LDL oxidation, a process implicated in atherogenesis. Since vitamin E can also promote LDL lipid peroxidation, we investigated the effects of supplementation with vitamin E alone or in combination with coenzyme Q on the early stages of the oxidation of isolated LDL. Isolated LDL was obtained from healthy subjects before and after in vitro enrichment with vitamin E (D-alpha-tocopherol, alpha-TOH) or dietary supplementation with D-alpha-TOH (1 g/d) and/or coenzyme Q (100 mg/d). LDL oxidation initiation was assessed by measurement of the consumption of alpha-TOH and cholesteryl esters containing polyunsaturated fatty acids and the accumulation of cholesteryl ester hydroperoxides during incubation of LDL in the transition metal-containing Hams F-10 medium in the absence and presence of human monocyte-derived macrophages (MDMs). Native LDL contained 8.5 +/- 2 molecules of alpha-TOH and 0.5 to 0.8 molecules of ubiquinol-10 (CoQ10H2, the reduced form of coenzyme Q) per lipoprotein particle. Incubation of this LDL in Hams F-10 medium resulted in a time-dependent loss of alpha-TOH with concomitant stoichiometric conversion of the major cholesteryl esters to their respective hydroperoxides. MDMs enhanced this process. LDL lipid peroxidation occurred via a radical chain reaction in the presence of alpha-TOH, and the rate of this oxidation decreased on alpha-TOH depletion. In vitro enrichment of LDL with alpha-TOH resulted in an LDL particle containing sixfold to sevenfold more alpha-TOH, and such enriched LDL was more readily oxidized in the absence and presence of MDMs compared with native LDL. In vivo alpha-TOH-deficient LDL, isolated from a patient with familial isolated vitamin E deficiency, was highly resistant to Hams F-10-initiated oxidation, whereas dietary supplementation with vitamin E restored the oxidizability of the patients LDL. Oral supplementation of healthy individuals for 5 days with either alpha-TOH or coenzyme Q increased the LDL levels of alpha-TOH and CoQ10H2 by two to three or three to four times, respectively. alpha-TOH-supplemented LDL was significantly more prone to oxidation, whereas CoQ10H2-enriched LDL was more resistant to oxidation initiation by Hams F-10 medium than native LDL. Cosupplementation with both alpha-TOH and coenzyme Q resulted in LDL with increased levels of alpha-TOH and CoQ10H2, and such LDL was markedly more resistant to initiation of oxidation than native or alpha-TOH-enriched LDL. These results demonstrate that oral supplementation with alpha-TOH alone results in LDL that is more prone to oxidation initiation, whereas cosupplementation with coenzyme Q not only prevents this prooxidant activity of vitamin E but also provides the lipoprotein with increased resistance to oxidation.

Bioenergetic pathways in tumor mitochondria as targets for cancer therapy and the importance of the ROS-induced apoptotic trigger

Mitochondria are emerging as idealized targets for anti-cancer drugs. One reason for this is that although these organelles are inherent to all cells, drugs are being developed that selectively target the mitochondria of malignant cells without adversely affecting those of normal cells. Such anti-cancer drugs destabilize cancer cell mitochondria and these compounds are referred to as mitocans, classified into several groups according to their mode of action and the location or nature of their specific drug targets. Many mitocans selectively interfere with the bioenergetic functions of cancer cell mitochondria, causing major disruptions often associated with ensuing overloads in ROS production leading to the induction of the intrinsic apoptotic pathway. This in-depth review describes the bases for the bioenergetic differences found between normal and cancer cell mitochondria, focussing on those essential changes occurring during malignancy that clinically may provide the most effective targets for mitocan development. A common theme emerging is that mitochondrially mediated ROS activation as a trigger for apoptosis offers a powerful basis for cancer therapy. Continued research in this area is likely to identify increasing numbers of novel agents that should prove highly effective against a variety of cancers with preferential toxicity towards malignant tissue, circumventing tumor resistance to the other more established therapeutic anti-cancer approaches.

Vitamin E analogues as inducers of apoptosis: structure-function relation.

Recent results show that α-tocopheryl succinate (α-TOS) is a proapoptotic agent with antineoplastic activity. As modifications of the vitamin E (VE) molecule may affect its apoptogenic activity, we tested a number of newly synthesised VE analogues using malignant cell lines. Analogues of α-TOS with lower number of methyl substitutions on the aromatic ring were less active than α-TOS. Replacement of the succinyl group with a maleyl group greatly enhanced the activity, while it was lower for the glutaryl esters. Methylation of the free succinyl carboxyl group on α-TOS and δ-TOS completely prevented the apoptogenic activity of the parent compounds. Both Trolox and its succinylated derivative were inactive. α-tocotrienol (α-T3 H) failed to induce apoptosis, while γ-T3 H was apoptogenic, and more so when succinylated. Shortening the aliphatic side chain of γ-T3 by one isoprenyl unit increased its activity. Neither phytyl nor oleyl succinate caused apoptosis. These findings show that modifications of different functional moieties of the VE molecule can enhance apoptogenic activity. It is hoped that these observations will lead to the synthesis of analogues with even higher apoptogenic and, consequently, antineoplastic efficacy.

α‐Tocopheryl succinate‐induced apoptosis in Jurkat T cells involves caspase‐3 activation, and both lysosomal and mitochondrial destabilisation

α‐Tocopheryl succinate (α‐TOS), but not α‐tocopherol, triggered apoptosis in Jurkat T cells. Apoptosis was induced by α‐TOS in a time‐ and concentration‐dependent mode, and signs of apoptosis were visible at concentrations of α‐TOS as low as 30 μM, and within 3–5 h after addition of the ester. Employing a specific fluorogenic substrate, caspase‐3 was found to be activated rapidly in response to α‐TOS at 50 μM. We also found that Jurkat T cells challenged with α‐TOS, when exposed to the lysosomotropic weak base acridine orange, showed decreased lysosomal uptake of the dye. This is suggestive of the involvement of lysosomal destabilisation in apoptosis of the cells. Apoptosis of Jurkat T cells induced with α‐TOS also involved a drop in the mitochondrial membrane potential, although this phenomenon occurred after the initiation of lysosomal rupture. All apoptotic features observed with α‐TOS were very similar to those found when cross‐linking of the Fas receptor triggered apoptosis. These findings are consistent with the recent idea that vitamin E can contribute to elimination of malignant cells by the induction of apoptosis, and can be of (patho)physiological significance.