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

NEUZIL,*,1 TOBIAS WEBER,* ANDREAS SCHRO DER,* MIN LU, JIRI GEORG OSTERMANN,* NINA GELLERT,* GEORGE C. MAYNE, BEATA OLEJNICKA, ` GRE-SALVAYRE, MARTIN STI CHA, ROBERT J. COFFEY, AND ANNE NE ,1 CHRISTIAN WEBER* *Institute for Prevention of Cardiovascular Diseases and Medical Policlinic, Ludwig-MaximiliansUniversity, Munich, Germany; Medical Center North, Vanderbilt University, Nashville, Tennessee, USA; Flinders University of South Australia, Adelaide, South Australia, Australia; Department of Pathology II, University Hospital, Linko ping, Sweden; Biochemistry Department, INSERM, Toulouse, France; and Faculty of Science, Charles University, Prague, Czech Republic
ABSTRACT

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

Vitamin E is an essential micronutrient, important for maintaining the balance between anti- and prooxidant reactions in tissues (1). Recent reports have implicated analogs of vitamin E in inducing apoptosis in several cell lines (2 4). In colon cancer cells expressing mutant p53, Trolox, a water-soluble analog of -tocopherol (-TOH), has been shown to induce p21Waf1/Cip1-dependent apoptosis (2). In a retrovirus-transformed T cell line, apoptosis induced by -tocopheryl succinate (-TOS) involved c-jun
0892-6638/01/0015-0403 FASEB

activation, nuclear binding of AP1, and down-regulation of the proto-oncogene c-myc (3). We have recently shown that -TOS-triggered apoptosis in Jurkat T lymphoma cells involves caspase-3 activation as well as lysosomal and mitochondrial destabilization (4), consistent with participation of mitochondria and the caspase cascade in various mammalian models of apoptosis (5, 6). Thus, the general understanding is that inducers of apoptosis activate the caspase cascade, either directly after recruitment of an initiator caspase to the receptor-associated death domain and/or via relocalization of mitochondrial factors. Executioner caspases then cleave death substrate, leading to the morphological changes typical of apoptosis (7, 8). Such pathways have been characterized for different triggers, including anti-Fas immunoglobulin M (IgM), TRAIL, or anti-cancer drugs (8, 9). Little is known about the role of vitamin E analogs in regulating apoptosis and pathways involved in this process. For instance, -TOH inhibits staurosporintriggered neuronal cell apoptosis by preventing activation of the caspase cascade (10). This may be reconciled with the diverse redox properties of -TOH (1, 11). On the other hand, several reports suggest that the effect of -TOH (or vitamin E analogs) may be due to an involvement in cellular signaling, e.g., by inhibition of protein kinase C (PKC), as shown for various cell types (1214) and functions such as proliferation, adhesion, or inammatory responses (15). Inhibition of PKC by -TOH is mediated by activation of protein phosphatase 2A (PP2A), leading to hypophosphorylation of PKC (16). The role of PKC in apoptosis has been documented (1719). Among the diverse mechanisms proposed, PKC can affect Fas ligand expression (20), phosphorylation, and activation of the anti-apoCorrespondence: Institute for Prevention of Cardiovascular Diseases, Pettenkoferstrasse 9, 80336 Munich, Germany. E-mail: jneuzil@klp.med.uni-muenchen.de or cweber@klp. med.uni-muenchen.de
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ptotic bcl-2 protein (21, 22) or cell cycle transition by modulating checkpoint proteins (23, 24). Despite circumstantial data inferring that vitamin E (analogs) can induce apoptosis in cell lines, this has not been elucidated in detail. In this report, we have characterized pathways involved in -TOS-induced apoptosis and show that the inhibition of PKC and the succinate moiety of -TOS play a pivotal role in efcient induction of apoptosis by this vitamin E analog in several malignant cell lines. Notably, we found that -TOS inhibits tumor cell growth in a murine model of colorectal cancer.

Assessment of apoptosis Apoptosis was assessed by light microscopic evaluation of changes in cytoarchitectural morphology directly or after Giemsa staining (27) or by uorescent microscopy after incubating adherent cells grown on coverslips with 10 M 4,6-diamidino-2-phenylindole dihydrochloride (Calbiochem, San Diego, Calif.). To detect surface-exposed phosphatidyl serine, cells in suspension or on coverslips were labeled with annexin V-FITC (Boehringer Mannheim, Mannheim, Germany) with or without propidium iodide (Sigma), and analyzed by uorescence-activated cell sorting (FACS; BectonDickinson, Rutherford, N.J.) or uorescence microscopy, respectively (28). DNA laddering was carried out as described (4), TUNEL staining of adherent cells was performed using the In situ cell detection kit according to the manufacturers protocol (Boehringer Mannheim). Cell cycle analysis was performed by FACS using the CycleTEST PLUS kit (BectonDickinson) according to the manufacturers instructions. Measurement of caspase-3-like activity To measure caspase-3 activity, cells were lysed, lysates (100 l) were incubated with 1 ml of reaction buffer containing 20 M Ac-DEVD-AMC (Calbiochem), and uorescence of the liberated amino-4-methylcoumarin (AMC) was measured at ex380 nm and em435 nm (29). The caspase inhibitors Ac-DEVD-CHO (50 M) or z-VAD-fmk (25 M; both from Calbiochem) were used in some experiments. Assessment of mitochondrial stability Mitochondrial integrity was studied in Jurkat cells using APO 2.7 monoclonal antibody (mAb) (Coulter Immunochemicals) directed against a 48 kDa mitochondrial protein accessible on apoptosis induction (30). Cells were permeabilized (digitonin), incubated with APO 2.7-PE, and analyzed by FACS. In adherent cells on coverslips, mitochondrial stability was assessed by uorescence microscopy after incubation with 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidoazolylcarbocyanino iodide (JC-1; Molecular Probes, Eugene, Oreg.). Lysosomal stability assays Cells were assessed for lysosomal stability by vital staining using the lysosomotrophic weak base acridine orange (AO; Sigma) (31). Suspension cells and cells adherent on coverslips were incubated with RPMI 1640, containing 10 mM HEPES and 5 g/ml AO, and assessed for AO binding by FACS or uorescence microscopy, respectively. Cytoskeletal integrity analysis Cells grown on coverslips were incubated with phalloidinFITC (Molecular Probes) and inspected by uorescence microscopy. Vinblastine (Calbiochem), causing cytoskeletal disruption by actin depolymerization, was used as a positive control. Western blotting Total cell lysates were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Primary antibodies to the following antigens were used: poly(ADP-ribose)polymerase (PARP; Santa Cruz, Santa Cruz, Calif.); caspase-3 (reactive with the active and inactive form; PharMingen, San Diego, Calif.),
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MATERIALS AND METHODS

Cell culture Human parental, bcl-2-overexpressing or Neo-vector-transfected Jurkat T lymphoma cells (25), primary broblasts, and murine macrophage-like J774 cells were cultured in RPMI 1640 with 10% fetal calf serum (FCS). Rat intestinal epithelial cells (RIE-I), human breast cancer cell line MCF-7 (26), human epithelial BEAS-2B, lung adenocarcinoma A549, and colon cancer cell lines (DKO-1, DKO-3, DKS-5, DKS-8, DLD-1, HCT-15, HCT-116) were cultured in Dulbeccos modied Eagles medium (DMEM) with 10% FCS. Human umbilical vein endothelial cells obtained by a standard procedure were grown in collagen-coated dishes in the endothelial cells medium (PromoCell, Heidelberg, Germany). Rat cardiomyocytes were prepared from 1-day-old rats by a routine procedure and maintained in DMEM supplemented with dialyzed bovine serum and 10 g/ml arabinofuranosyl-cytosine. The murine leukemia NSF/N1.H7 parental cells and transfectants with vector, gain-of-function (S70E), and loss-of-function (S70A) mutants of bcl-2 were maintained in RPMI 1640, 10% FCS, and 10% conditioned media from the interleukin 3-producing WEHI-3 cells (21, 22). Human broblasts were prepared by skin autopsy of a volunteer and used between passages 5 and 10. For experiments, suspension cells were seeded at 0.5 106/ml and adherent cells were used at 60 70% conuence. Cells were treated with -TOS, other vitamin E analogs, cholesteryl hemisuccinate (CHS), and free succinate [all in DMSO; all from Sigma (St. Louis, Mo.) unless indicated otherwise] at concentrations shown, with DMSO vehicle (0.1% v/v), or with anti-Fas IgM [clone CH-11 in phosphate-buffered saline (PBS); Coulter Immunochemicals, Hialeah, Fla.]. Synthesis of -tocopheryl succinate -Tocopherol (Henkel, La Grange, Ill.) dissolved in acetic acid containing zinc powder was mixed with succinyl anhydride at 50% excess of the latter, kept at 130C for 6 h under stirring, extracted with water and diethyl ether-containing hexane, and the -TOS-containing hexane layer was evaporated under vacuum. -TOS was puried from the crude preparation to 98% purity by silica gel chromatography, and its authenticity and purity veried by high-performance liquid chromatography and gas chromatography-mass spectrometry (MS). MS analysis of -TOS revealed a major peak of 516 and fragments corresponding to those of -TOS, and a spectrum 95% identical to its library.
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bcl-2 (Calbiochem or Boehringer Mannheim), PKC (reactive with all PKC isozymes), PKC (Calbiochem). Secondary antibodies (Santa Cruz) and the ECL system (Amersham, Arlington Heights, Ill.) were used for visualization. Measurement of PKC and PP2A activity PKC activity was measured using the colorimetric SpinZyme PKC assay kit with neurogranin peptide as a substrate (Pierce, Rockford, Ill.) according to the manufacturers instructions. Cells (0.5106/ml) were shortly sonicated in the lysis buffer and lysates (10 l) were incubated with the reaction buffer, activation buffer and PKC substrate (5 l each). The mixture was loaded onto SpinZyme afnity membranes, nonphosphorylated peptide was removed by low-speed centrifugation, phosphorylated, bound PKC substrate was eluted, and its absorbance read at 570 nm. Human recombinant PKC (Calbiochem) was used for calibration. In parallel, PP2A activity was measured using a kit supplied by Promega (Madison, Wis.). Cell lysates prepared as for PKC assays were passed through Sephadex-25 spin columns to remove free endogenous phosphate and incubated with reaction buffer containing the phosphopeptide substrate RRA(pT)VA (100 M). The reaction was stopped by the addition of molybdate dye solution and absorbance was read at 600 nm after 30 min. Treatment with PKC or PP2A inhibitors/activators Jurkat cells were treated with the following PKC/PP2A effectors (Calbiochem) at the concentrations indicated: calphostin C (general PKC inhibitor); Go 6976 (specic for Ca2dependent PKC isozymes, i.e., PKC and PKCI); 2,2,3,3,4,4-hexahydroxy-1,1-biphenyl-6,6-dimethanol dimethyl ether (HBDDE; selective inhibitor of PKC and PKC); phorbol-12-myristate-13-acetate (PMA; general PKC activator); sapintoxin D (specic activator of PKC); calyculin A and okadaic acid (both PP2A inhibitors); N-acetyl-Derythro-sphingosine (NAS; PP2A activator). Some cells (PMA, sapintoxin D, calyculin A, okadaic acid) were cotreated with -TOS. Treatment with PKC oligodeoxynucleotides (ODN) and PKC overexpression PKC antisense, reverse antisense or sense, phosphorothioate-modied ODN (MWG-Biotech, Ebersberg, Germany) were synthesized according to published sequences (32) and used essentially as described (33). Jurkat cells were supplemented with 10 M (nal concentration) sense, antisense, or reverse antisense ODN or left untreated (control cells). After 18 h, aliquots for PKC immunoblotting were taken and a second portion of ODN (10 M) and -TOS (30 M) was added. Aliquots for PKC activity were taken 6 h later, apoptosis was assessed, and nal aliquots for PKC immunoblotting were taken 8 and 14 h after -TOS addition. HCT116 cells grown on coverslips were treated with ODNs (7.5 M) and 10 l/ml LipofectAmine (Life Technologies, Grand Island, N.Y.). After 18 h, ODN (7.5 M) and -TOS (50 M) were added. PKC was analyzed by immunouorescence microscopy using PKC mAb and rhodamine-conjugated anti-IgG mAb; apoptosis was assessed by TUNEL. PKC was transiently overexpressed in HCT116 cells using the bovine PKC gene that shows 95% homology with the human PKC gene or its mutant, catalytically nonactive form in the PMT-2 plasmid (34). Briey, cells were supplemented in serum-free DMEM with the PKC plasmid (1 g), pGreen Lantern-1 (1 g), and 10 l LipofectAmine (Life Technologies). After 1.5 h, medium was replaced with fresh complete
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DMEM, cells were incubated for 21 h, and -TOS (50 M) was added. After 24 h, cells were assessed for apoptosis by uorescence microscopy and FACS analysis using annexin V-PE (channel 3) and for transfection efcacy using GFP expression (channel 1). After induction of apoptosis, PKCand mock-transfected HCT116 cells were assessed for caspase-3 activity by FACS analysis after staining with a rabbit polyclonal anti-caspase-3 antibody recognizing the active form of the protease (PharMingen). Fluorescence microscopy Fluorescence microscopy was performed using a Leica DMRBE microscope or a Zeiss LSM 410 inverted confocal laser scanning microscope. Cells were mounted either with PBS (confocal microscopy) or Mowiol (conventional microscopy). The images were taken using the BW or RGB camera supported by the Hamamatsu or SPOT32 software package, respectively, and transferred to Adobe Photoshop or NIH Image software for processing and evaluation. At least three representative images were taken from each sample. Mouse xenografts Colorectal cancer cells HCT116 were used for the xenografts, as described (2). Briey, HCT116 cells (107 cells in 0.2 ml PBS) were injected subcutaneously (s.c.) between the scapulae of each mouse. Once tumors were established, animals received an intraperitoneal (i.p.) dose of 50 l of 200 mM -TOS (100 mg -TOS per kg) dissolved in DMSO or the same volume of DMSO alone every third day. Tumor volumes were estimated by measuring the maximal height, length, and width at the times indicated. At the end of the treatment period, the mice were killed, and tumor sections were prepared by xation in paraformaldehyde and embedding in parafn. The sections were stained for apoptosis using the In situ cell death detection kit (TUNEL-FITC, Boehringer Mannheim) according to the manufacturers protocol and analyzed by uorescence microscopy. Statistics Unless stated otherwise, data are given as mean sd of at least three independent experiments, and images shown are representative pictures of three to six independent experiments.

RESULTS -TOS induces apoptosis in malignant hematopoietic or cancer cells: inhibition by PMA It has been shown that -TOS induces apoptosis in Jurkat cells (4). As some vitamin E derivativesnamely, -TOH can inhibit PKC (11), we elucidated the role of PKC in this process. Figure 1 illustrates that cotreatment with the PKC activator PMA can rescue Jurkat cells from apoptosis induced by -TOS or anti-Fas IgM. The kinetics of apoptosis induction and its inhibition by PMA are shown based on morphological evaluation or lysosomal destabilization (Fig. 1A, B). The antiapoptotic effects of PMA were also assessed by annexin V binding, mitochondrial destabilization as evident by staining with APO2.7 mAb, caspase-3 activation, cleav405

Figure 1. PMA inhibits apoptosis induced by -TOS in hematopoietic cells. Jurkat cells were treated with vehicle (control), -TOS (50 M), anti-Fas IgM (20 ng/ml), or PMA (100 nM) for 12 h unless indicated otherwise; apoptosis was assessed by morphological analysis after Giemsa staining (A) and lysosomal destabilization after staining with AO at different time points (B), annexin V-FITC binding (C), mitochondrial destabilization using the APO2.7-PE mAb (D), caspase-3 activation (E), and PARP cleavage (F). E) Insert: representative FACS histograms of Jurkat cells treated as indicated after staining with a mAb recognizing the active form of caspase-3.

age of the death substrate PARP (Fig. 1CF), or DNA laddering (not shown). The dose-response revealed that full protection requires PMA at 100 nM (Fig. 1F ). -TOS treatment also resulted in cell cycle dysregulation, which was reversed by PMA (not shown). PMA also inhibited PARP cleavage and apoptosis induced by -TOS in REH, THP-1, Mono Mac 6, or K562 cells (not shown), inferring that the anti-apoptotic effects of PMA extend to other hematopoietic cell lines. Next we tested whether this is also true for adherent cells. As apparent by lysosomal leakage (AO staining), mitochondrial destabilization ( JC-1 staining), exposure of phosphatidyl serine (annexin V staining), or
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cytoskeletal disruption (phalloidin staining), -TOS induced apoptosis in human colon cancer HCT116 cells (Fig. 2). As in suspension cells, all changes were largely reversed by cotreatment with PMA (Fig. 2). Mitochondrial morphology and the energetic state were evaluated using the polychromatic dye JC-1, which forms red-orange clusters with high potential (100 mV) mitochondria, whereas low-potential mitochondria show green staining. Similar results were obtained in murine J774 macrophage-like and human BEAS-2B epithelial, MCF-7 breast cancer, and A549 lung adenocarcinoma cells (not shown). Notably, -TOS was proapoptotic in transformed cell lines with high proliferaNEUZIL ET AL.

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Figure 2. -TOS induces apoptosis in a colon cancer cell line but not in primary broblasts. Human primary broblasts or human colon cancer HCT116 cells were treated with -TOS (50 M) with or without PMA (100 nM) for 12 h, reacted with AO for staining of lysosomes, JC-1 for staining of mitochondria, annexin V-FITC for detection of apoptosis, or phalloidin-FITC for staining of polymerized actin. Fluorescent images were recorded during microscopic inspection.

tion rates, but had little effect on normal cells. This is exemplied by a resistance of primary human broblasts to -TOS-induced apoptosis, evident by compartmental integrity and lack of annexin V binding (Fig. 2). Similar observations were made in other nontransformed, primary cell types, including rat intestinal epithelial cells and cardiomyocytes and human endothelial cells (J. Neuzil et al., unpublished results), inferring that proapoptotic effects of -TOS are largely restricted to malignant cells. This may have important implications for therapeutic use of -TOS. PKC and PP2A are regulated during -TOS-induced apoptosis Protective effects of PMA suggest an involvement of PKC activity in regulating apoptosis. In Jurkat cells, -TOS resulted in a dose- and time-dependent inhibition of PKC activity (Fig. 3A, B), which was prevented by PMA. A concentration-dependent decrease in PKC activity correlated with induction of apoptosis by -TOS (Fig. 3A, insert). Inhibition of PKC activity by -TOS and its prevention by PMA were also seen in THP-1, Mono Mac 6, REH, and K562 cells (not shown). Inhibition of PKC by -TOS or anti-Fas IgM was not complete, i.e., residual activity was detected (Fig. 3A, B). Since this infers PKC isotype-specic effects and PKC is regulated by PP2A modulating its phosphorylation state (16), we used multiple agents affecting these enzymes to identify a role of PKC isotypes and PP2A in
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-TOS effects. Table 1 shows PKC and PP2A activities in relation to apoptosis, suggesting an effector-specic link between PP2A activation, PKC inhibition, and apoptosis induction. Figure 3C depicts PARP cleavage in Jurkat cells treated with PKC and PP2A effectors (see Materials and Methods) in the presence of -TOS. The pattern of apoptosis and PKC and PP2A activities indicated that -TOS inhibits PKC via PP2A activation and subsequent hypophosphorylation of PKC, rather than by direct action of -TOS on PKC. This is further supported by in vitro measurements that failed to reveal the effects of -TOS on the activity of human recombinant PKC (not shown). Analysis of PKC distribution in HCT116 cells by immunouorescence microscopy showed that loss of the granular PKC staining pattern after -TOS was restored by PMA (Fig. 3D). Long-term PMA treatment can result in down-regulation of PKC under certain conditions. As assessed by immunoblot analysis, however, exposure of Jurkat cells to 100 nM PMA for 12 h did not result in a discernible decrease in the amount of the PKC protein that was observed after treatment with 1 M PMA, whereas the inhibitory effect of -TOS was exerted at the level of PKC activity rather than expression (not shown). On the other hand, caspase-3 activation leads to PP2A activation during Fas-induced apoptosis (35). However, z-VAD-fmk, a general caspase inhibitor that blocks apoptosis induced by -TOS or Fas, prevented the decrease in PKC activity and increase in PP2A activity in Jurkat cells treated with anti-Fas IgM but not -TOS (not shown, Fig. 3E), ruling out a role of caspases in PP2A activation and PKC inhibition by -TOS and suggesting caspase activation to be downstream of PP2A/PKC dysregulation. Role of PKC and bcl-2 in -TOS-induced apoptosis To provide direct evidence for an involvement of PKC in -TOS-induced apoptosis, Jurkat cells were treated with antisense ODN to PKC. This resulted in reduced levels of PKC protein and activity (inserts Fig. 4A) and in increased apoptosis after exposure to -TOS, as compared to cells treated with or without sense or reverse antisense ODN (Fig. 4A, not shown). Before addition of -TOS, PKC activity was reduced only by antisense ODN (Fig. 4A). The fact that PMA cotreatment did not rescue the antisense ODN pretreated Jurkat cells from -TOS-induced apoptosis suggests that PKC isozymes not affected by the ODN do not play a major role in -TOS-induced apoptosis (cf. Fig. 4A). Treatment with PKC antisense but not sense ODN, reduced expression of PKC and increased sensitivity to -TOS-induced apoptosis in HCT116 cells (Fig. 4B). We next studied the effect of PKC overexpression on resistance to -TOS of HCT116 cells cotransfected with a GFP-encoding vector (Fig. 4C). Cells overexpressing wild-type PKC were less susceptible to -TOSinduced apoptosis than parental cells, whereas overexpression of an inactive PKC mutant had no effect (Fig. 4C, D). Similar effects were found with MCF7 human
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Figure 3. PKC and PP2A activities are involved in -TOS-induced apoptosis. Jurkat cells were treated with vehicle or indicated concentrations of -TOS (M) or anti-Fas IgM (ng/ml), with -TOS (50 M) or anti-Fas IgM (20 ng/ml) in the presence of PMA (100 nM) or PMA alone for 6 h (A), or for indicated periods (B), and PKC activity was determined in whole cell extracts. A) Insert: dose dependence of apoptosis induced by -TOS treatment for 6 h. C) Cleavage of PARP in cells treated with vehicle (Con), -TOS (50 M) alone or in the presence of calyculin A (Cal, 10 nM), sapintoxin D (Sap, 1 M), or okadaic acid (OA, 1 nM) or with Go 6976 (Go , 10 nM), HBDDE (HBD, 50 M), N-acetyl sphingosine (NAS; 10 M), or calphostin C (Calp, 10 M) for 9 h. D) Immunouorescence microscopy shows HCT116 colon cancer cells treated with -TOS (50 M) and/or PMA (100 nM) for 6 h and stained with anti-PKC. E) The activity of PKC and PP2A in Jurkat cells treated for 6 h with -TOS (50 M) or anti-Fas IgM (20 ng/ml) in the absence (Ctrl) or presence (Inh) of the pan-caspase inhibitor z-VAD-fmk at 25 M.

breast cancer cells stably overexpressing PKC (not shown). Cotreatment with PMA resulted in a relatively mild and only partial inhibition of apoptosis in -TOSexposed parental HCT116 cells and cells transfected with mutant PKC, whereas suppression of -TOSinduced apoptosis by PMA was almost complete in cells transfected with wild-type PKC (Fig. 4D). Taken together, these data suggest an increase in basal PKC activity in the cells transfected with wild-type PKC and an increase in PKC activity after PMA, which was most pronounced in the wild-type PKC-transfected cells. Hence, we studied the effect of PKC overexpression on PKC activity and caspase activation in HCT116 cells after -TOS exposure. Indeed, basal PKC activity was increased by overexpression of wild-type PKC, but not affected by overexpression of the inactive PKC mutant (Fig. 4E). Accordingly, the cells transfected with wildtype PKC showed lower caspase-3 activity after -TOS treatment (Fig. 4E). These data lend further support for the anti-apoptotic role of PKC in Jurkat cells and HCT116 cells. Since our data support a role of mitochondria in
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TABLE 1. Effect of PKC/PP2A effectors on enzyme activity and apoptosis in Jurkat cells
Treatmenta PKC activityb PP2A activityc Apoptosisd

Control -TOS -TOS calyculin -TOS sapintoxin -TOS OA Go 6976 HBDDE NAS Calphostin

6.2 1.1 2.3 0.8 5.2 1.0 4.9 1.2 4.2 0.5 2.4 0.7 1.9 0.4 3.1 0.4 2.5 0.3

2.5 0.4 5.8 1.2 1.3 0.5 2.3 0.4 1.1 0.3 2.6 0.5 1.9 0.6 6.8 1.3 2.4 0.6

7.2 4.5 56.3 8.5 28.3 4.5 25.6 3.8 28.9 4.7 75.3 7.9 32.1 5.4 86.5 9.8 91.2 8.1

a JT cells (0.5 106) were exposed to the effectors (calyculin A, PP2A inhibitor, 10 nM; sapintoxin D, activator of PKC, 1 M; OA, inhibitor of PP2A, 1 nM; Go 6976, inhibitor of PKC and PKCI, 10 nM; HBDDE, inhibitor of PKC and PKC, 50 M; NAS, activator of PP2A, 10 M; calphostin C, general inhibitor of PKC, 10 M) in the absence or presence of 50 M -TOS; PKC and PP2A activities were b measured 6 h and apoptosis assessed 12 h later. PKC activity is c expressed in arbitrary units (103) per 10 l cell lysate. PP2A 3 activity is expressed in arbitrary units (10 ) per 10 l cell d lysate. Apoptosis is expressed as percentage of annexin V-FITCpositive cells (mean sd, n 3).

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Figure 4. Effect of PKC antisense ODN and overexpression on -TOS-induced apoptosis. Jurkat cells were treated with vehicle, antisense, or sense PKC ODN (10 M) for 18 h and assessed for apoptosis 0 and 14 h after addition of 30 M -TOS in the absence or presence of 100 nM PMA (A). Inserts show the effect of antisense and sense PKC ODN on PKC and PKC protein levels and activity. Immunouorescence shows PKC expression and sensitivity to apoptosis (TUNEL) in HCT116 colon cancer cells treated with PKC sense or antisense ODN, and exposed to -TOS (30 M) for 12 h (B). C) The efcacy of transient PKC transfection in HCT116 cells is shown by immunouorescence for PKC in comparison to GFP. D) Effects of overexpressing wild-type or mutant PKC in HCT116 cells on sensitivity to apoptosis after treatment with -TOS (30 M) for 12 h with or without 100 nM PMA. E) The effect of transient wild-type or mutant PKC overexpression on PKC activity (before -TOS addition) and caspase-3 activity (9 h after addition of 30 M -TOS).

-TOS-induced apoptosis (4) and protection by PKC, we tested whether susceptibility to apoptosis is affected by the anti-apoptotic mitochondrial protein bcl-2, whose anti-apoptotic activity is regulated by phosphorylation/dephosphorylation on serine 70 by PKC/ PP2A. In Jurkat cells overexpressing bcl-2, the extent of apoptosis induced by -TOS or Fas was considerably lower than in mock-transfected cells (Fig. 5A), as were effects of -TOS or anti-Fas IgM on lysosomal destabilization and PARP cleavage (Fig. 5B, C). This is supVITAMIN E SUCCINATE AND CANCER CELL APOPTOSIS

ported by sensitization of Jurkat cells to -TOS with bcl-2 antisense ODN (T. Weber et al., unpublished results). Since mitochondrial membrane association and thus activity of bcl-2 is regulated PKC/PP2A, we tested the effects of gain-of-function (S70E) and lossof-function (S70A) mutants stably transfected in H7 cells. Cells with loss-of-function bcl-2 were more sensitive and cells with gain-of-function bcl-2 more resistant to -TOS-induced apoptosis (Fig. 5D), inferring that bcl-2 mutation mimicking permanent phosphorylation
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Figure 5. Role of bcl-2 in -TOS-induced apoptosis. Jurkat cells (AC) overexpressing bcl-2 or transfected with empty vector (Neo) were treated with indicated concentrations of -TOS (M) or anti-Fas IgM (ng/ml) in the absence or presence of 100 nM PMA (50 M -TOS or 20 ng/ml anti-Fas IgM). Apoptosis was assessed by morphological evaluation (A) or lysosomal destabilization (B). A) Insert: immunoblot of bcl-2. Cleavage of PARP in cells treated with vehicle (1) or -TOS (50 M) in the absence (2) or presence (3) of PMA (100 nM) for 12 h was analyzed by immunoblotting (C). Parental H7 murine leukemia cells, H7 cells transfected with vector (WT), loss- (S70A) or gain-of-function (S70E) mutants of bcl-2 were treated with indicated concentrations of -TOS for 12 h; apoptosis was assessed by staining with annexin V-FITC (D).

provides protection against -TOS. Thus, these data show an anti-apoptotic role of bcl-2 in -TOS-induced apoptosis and imply its regulation on the level of phosphorylation/dephosphorylation, features affected by -TOS. Structural requirements for -TOS-induced apoptosis In a previous report (4), -TOS but not -TOH was found to induce apoptosis in Jurkat cells, although -TOH can activate PP2A and suppress PKC activity (16). Hence, we tested a role of the succinyl moiety in this process using multiple vitamin E analogs (Scheme 1). At equimolar concentrations, -TOH, -tocopheryl acetate, -TOH, or M--TOH had no discernible effect. -TOS induced apoptosis and lysosomal leakage to 50% of the extent exerted by -TOS alone or to a similar extent as -TOS in the presence of PMA (Fig. 6A, B). Accordingly, PP2A activity was increased and PKC activity decreased by -TOS and -TOH, but not by -TOS (Fig. 6C). CHS, a hydrophobic compound containing a charged succinyl moiety, had moderate
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proapoptotic effects comparable to those of -TOS (Fig. 6A, B), whereas free succinate was without effect at concentrations up to 10 mM (not shown). The changes induced by CHS or -TOS were not prevented by PMA

Scheme 1. Vitamin E analogs used in this study.

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Figure 6. Structural requirements for apoptosis induced by vitamin E analogs. Jurkat cells were treated with agents (50 M) as indicated in absence or presence of 100 nM PMA, and analyzed for annexin V-FITC binding (A) and lysosomal destabilization (B) after 12 h, and for PP2A and PKC activity after 6 h (C). HCT116 cells transiently transfected with PKC were exposed to - or -TOS (50 M) for 12 h and analyzed for annexin V-FITC binding (D).

(Fig. 6A, B). Cotreatment of Jurkat cells with -TOH and CHS resulted in an apparent additive increase in apoptosis and lysosomal leakage comparable to that with -TOS alone and was partially inhibited by PMA (Fig. 6A, B). Consistently, PKC-transfected HCT116 cells showed similar susceptibility to -TOS as controls (Fig. 6D). Together, this indicates that effective induction of apoptosis by -TOS requires both PKC inhibition and the succinyl moiety. Suppression of tumor growth by -TOS We next investigated whether -TOS is also effective against tumor growth in vivo. In an established model of colon cancer (2), nude mice received xenografts derived from the human colon cancer cell line HCT116, which was sensitive to -TOS-induced apoptosis (Fig. 2 and Fig. 4B, F). A comparable sensitivity to -TOS in vitro was observed in other colon cancer cell lines, including HCT-15, DKO-1, DKO-3, DLD-1, DKS-5, or DKS-8 (not shown). The mice were injected with -TOS i.p. each third day, so that its dose was 100
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mg per kg (50 l of 200 mM -TOS in DMSO), and the tumor volume was measured at indicated time points. As shown in Fig. 7A, tumor volume was reduced by 75% after treatment with -TOS compared to negative controls injected with vehicle alone, showing that -TOS can inhibit cancer cell growth in vivo. Moreover, tumors from mice treated with -TOS contained substantially more apoptotic cells, as revealed by TUNEL staining of tumor section (Fig. 7B). Such inhibition of tumor growth and increase in TUNEL-positive cells were not observed in mice injected with equimolar doses of -TOH (J. Neuzil et al., unpublished results). Thus, -TOS is an efcient proapoptotic and antitumorigenic agent in vivo.

DISCUSSION In this report we show that 1) -TOS, an esteried vitamin E analog, induces apoptosis in multiple cancer cell lines but not in primary cells involving lysosomal
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Figure 7. -TOS treatment of nude mice with HCT116 xenografts inhibits tumor growth. Nude mice (8 per treatment) were s.c. injected with 107 HCT116 cells (in 0.2 ml PBS). Once tumors were established, mice received i.p. injections (50 l) of 200 mM -TOS or DMSO as indicated by arrows. Tumor volume was measured at indicated time points, and is expressed as mean relative increase se (A). Initial mean tumor volumes for the two groups were not signicantly different. B) Representative images from tumor sections from control or -TOS-treated mice, stained for TUNEL-positive cells. Asterisks in panel A indicate difference between tumor volumes of control and -TOS-treated animals with P0.01, as calculated using Wilcoxons nonparametric signed rank test.

and mitochondrial destabilization, caspase-3 activation, and PARP cleavage, features shared with apoptosis induced by Fas engagement; 2) maximal apoptosis induced by -TOS requires both inhibition of PKC (due to PP2A activation) and PKC-independent mechanisms mediated by the succinyl moiety; 3) -TOS limits tumor growth in a murine model with human colorectal cancer xenografts. Our data represent a considerable extension of ndings that -TOS induces apoptosis in Jurkat cells and that this process includes destabilization of mitochondria and lysosomes and caspase-3 activation (4). In the current study, we attempted to further dene cellular targets that transmit proapoptotic signals of -TOS. Since -TOS, unlike the free form of vitamin E, does not possess redox activity, we focused on effects of vitamin E analogs not involving redox activity. Several reports indicate that vitamin E inhibits the activity of PKC in various cell lines. Notably, -TOH but not -TOH has been shown to inhibit proliferation of a smooth muscle cell line (12). As the redox activity of -TOH is 50% of that of the -form, it was concluded that -TOH affects cellular proliferation by other properties, e.g., due to inhibition of PKC activity, which was subsequently demonstrated (12). Moreover, the inhibition of PKC has been implicated in the suppression of monocyte adhesion by -TOH (13) and in its protection of vasorelaxation during inammation (14). In parallel to our ndings, inhibitory actions of -TOH appear to be mediated by modulating its phosphorylation state via PP2A activation (16). It has also been shown that due to inhibition of PKC, -TOH but not -TOH or Trolox inhibited monocytic respiratory burst by preventing phosphorylation and translocation of p47phox (36). This may be relevant with respect to
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ndings that PKC can regulate production of reactive oxygen species (ROS) in monocytes (32). Indeed, ROS formation has been involved in initial stages of druginduced and receptor-mediated apoptosis of different cell lines (6, 10, 25, 37, 38), and we have found radical generation during early phases of -TOS-induced apoptosis (J. Neuzil et al., unpublished results). The role of PKC in intracellular signal transduction has been extensively studied. In general, activation of PKC is associated with cell proliferation (39, 40). Conversely, PKC inhibition has been implicated in induction or potentiation of apoptosis (1721, 23, 41, 42). We provide several lines of evidence for a role of PKC activity in -TOS-induced apoptosis: 1) cotreatment with PMA prevented multiple features of apoptosis after exposure to -TOS; 2) treatment of cells with -TOS resulted in a decrease in PKC and an increase in PP2A activity; 3) cells were protected from apoptosis by PKC overexpression and sensitized to apoptosis by PKC antisense ODN. PKC isotypes involved in regulation of apoptosis may differ depending on the cell type. We hence performed experiments with a variety of effector compounds of PKC, indicating a role for inhibition of PKC after PP2A activation. Perhaps the most compelling evidence for participation of this PKC isotype in apoptosis induced by -TOS comes from the experiments in which PKC levels were down-regulated by antisense ODN or up-regulated by overexpression. However, we should point out that although our experiments do strongly implicate this PKC isotype in -TOS-induced apoptosis, it cannot be excluded that other isotypes may also be involved. This follows, for example, results demonstrating an effect of PKC overexpression in the MCF-7 cells on several other PKC isotypes (26). Another approach when studying the role of different PKC isotypes in cellular events is to assess their intracellular partition. Whereas -TOS led to a loss of PKC membrane association in HCT116 but not in Jurkat cells, of the PKC isotypes tested in Jurkat cells treated with -TOS, only PKC translocated to the cytosol, and this could be prevented by PMA (G. Mayne et al., unpublished results). This is consistent with reports that the activity of both PKC and PKC is inhibited during Fas-induced apoptosis in Jurkat cells, but only PKC undergoes cytosolic relocalization, while relocalization of PKC is not obligatory for its regulation (42 44). The role of PKC inhibition in -TOS-induced apoptosis extends to other hematopoietic cell lines. Moreover, PMA or PKC overexpression conferred protection or resistance to -TOS apoptosis in several adherent cell lines: murine macrophage J774 cells and human breast, lung, or colon cancer cells. Thus, PMA clearly exerted anti-apoptotic effects in cell lines tested here. It should be noted, however, that PMA itself has also been found to induce apoptosis in a different cellular context, such as in LNCaP prostate cancer cells (45). In contrast to malignant cell lines, primary nontransformed cells were not sensitive to -TOS-induced
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apoptosis, possibly by establishing anti-apoptotic programs at steady-state density (46). A selective susceptibility to -TOS-induced apoptosis thus may be a universally relevant principle shared by proliferative and/or malignant cells. Since -TOS but not -TOH induced apoptosis, although -TOH can activate PP2A and suppress PKC (16), we characterized the role of the succinyl moiety, showing that inhibition of PKC is insufcient for efcient apoptosis, which required cooperative destabilization of subcellular compartments by the succinyl moiety. This is consistent with a report that CHS can induce apoptosis in breast cancer cells (47). Moreover, -TOS (but not -TOH or -TOA) can inhibit NF-B mobilization in Jurkat or endothelial cells (48, 49), which is crucial for transcription of anti-apoptotic proteins (e.g., the caspase inhibitor iap-1) controlling the apoptotic balance (46). As both pathways required for -TOS-induced apoptosis converge at a mitochondrial level, we studied the effects of bcl-2, whose mitochondrial membrane association and anti-apoptotic action are regulated by its phosphorylation via PKC/PP2A. Affecting the equilibrium by overexpressing gain- and loss-of-function bcl-2 mutants suggested that impaired bcl-2 function may be a central mechanism involved in apoptosis induced by -TOS. Using these cell lines, ceramide has been shown to activate PP2A and inactivate PKC, suppressing anti-apoptotic actions of bcl-2 (50). In line with these ndings, we have observed early ceramide formation in -TOS-treated Jurkat cells (A. Ne ` gre-Salvayre et al., unpublished results). Thus, -TOS may activate PP2A at least in part via the induction of ceramide formation; alternatively, these pathways may operate in parallel. Inhibition of PKC by -TOS via activation of PP2A appears consistent with the general notion that activity of PKC isozymes is regulated positively by their transand autophosphorylation (51, 52) and negatively by dephosphorylation by protein phosphatases, including PP2A, at the autophosphorylation sites of PKC (53). Based on our results, we propose a concept of the proapoptotic actions of -TOS in malignant cells (cf. Scheme 2). The -tocopheryl moiety signals via the PP2A/ PKC pathway, resulting in hypophosphorylation of targets such as bcl-2. The succinyl moiety destabilizes intracellular membranous structures such as lysosomes and/or mitochondria. Together, these pathways cooperate in activation of the caspase cascade to induce effective apoptosis. To test the relevance of our ndings in vivo, we used a nude mouse model with human colon cancer xenografts. Since vitamin E esters (largely acetate) are routinely used in vitamin supplements and are hydrolyzed during the intestinal uptake, -TOS could potentially be used to treat gastrointestinal cancer. Our data show that the suppression of tumor growth by -TOS was either more or similarly effective as recently found for the antioxidants N-acetyl-cysteine or pyrrolidine dithiocarbamate (2). However, as a micronutrient, -TOS may be devoid of side effects. This is further supported by our ndings of very low, if any, toxicity of
VITAMIN E SUCCINATE AND CANCER CELL APOPTOSIS

Scheme 2. Pathways involved in -TOS-induced apoptosis. Due to its hydrophobic nature, -TOS translocates into the cell, where it resides primarily within membranous structures. The -tocopheryl moiety is involved in PP2A activation (pathway 1), which in turn hypophosphorylates and thereby inactivates PKC, which is required for phosphorylation of the anti-apoptotic protein bcl-2 on Ser 70 (pathway 1a), or directly dephosphorylates bcl-2 (pathway 1b). The charged succinyl moiety causes destabilization of intracellular compartments such as lysosomes (pathway 2) and/or mitochondria (alternative pathway 2). Such destabilization of subcellular organelles cooperates with PP2A activation and PKC inhibition, resulting in mitochondrial release of cytochrome c, which forms (with apaf-1 and pro-caspase-9) the apoptosome for release of mature caspase-9, which activates an execution caspase, such as caspase-3, culminating in cell death.

-TOS toward normal, primary cells, including vascular endothelial and intestinal epithelial cells and cardiomyocytes ( J. Neuzil et al., unpublished results), consistent with reports by others (54 56). Taken together, these ndings suggest that this vitamin E analog is a promising anti-cancer drug or adjuvant, and warrants more extensive studies using i.p. or oral application, which are currently under way.
The authors wish to thank Drs. S. J. Korsmeyer for bcl-2overexpressing Jurkat cells, W. S. May for the H7-derived bcl-2 gain- and loss-of-function cell lines, J. Noti for the PKCoverexpressing MCF-7 cells, N. Hrboticky for human primary broblasts, P. J. Parker for the PMT-2 plasmid harboring the bovine PKC gene, and D. Liebler for M--TOH. The APO 2.7 antibody was kindly provided by Coulter Immunochemicals; -TOH was donated by Henkel. The expert technical assistance of T. Lum, I. Svensson, and U. Johansson and fruitful discussions with and critical reading of the manuscript by Drs. L. Andera, U. Brunk, J. Eaton, J .F. Keaney, Jr., and R. Stocker are highly appreciated.

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