Induction of Caspase-Independent Programmed Cell Death by ...

Nutrition and Cancer, 61(6), 864–874 Copyright © 2009, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635580903285130

Induction of Caspase-Independent Programmed Cell Death by Vitamin E Natural Homologs and Synthetic Derivatives Constantina Constantinou Yasoo Health Ltd., Nicosia, Cyprus

John Anthony Hyatt Yasoo Health Inc., Johnson City, Tennessee, and East Tennessee State University, Johnson City, Tennessee, USA

Panayiota S. Vraka University of Cyprus, Nicosia, Cyprus

Andreas Papas and Konstantinos A. Papas Yasoo Health Inc., Johnson City, Tennessee, USA

Constantinos Neophytou and Vicky Hadjivassiliou Yasoo Health Ltd., Nicosia, Cyprus

Andreas I. Constantinou University of Cyprus, Nicosia, Cyprus

Current observations in the literature suggest that vitamin E may be a suitable candidate for cancer chemotherapy. To investigate this further, we examined the ability of the vitamin E natural homologs [α-, β-, γ -, δ-tocopherols (α-TOC, β-TOC, γ -TOC, δTOC) and α-, β-, γ -, δ-tocotrienols (α-TT, β-TT, γ -TT, δ-TT)] and their corresponding succinate synthetic derivatives [α-, β-, γ -, δ-tocopheryl succinates and α-, β-, γ -, δ-tocotrienyl succinates (αTS, β-TS, γ -TS, δ-TS)] to induce cell death in AR– (DU145 and PC3) and AR+ (LNCaP) prostate cancer cell lines. The most effective of all the natural homologs of vitamin E was determined to be δ-TT, whereas δ-TS was the most potent of all the natural and synthetic compounds of vitamin E examined. Both γ -TT and δ-TT induced caspase activity selectively in AR+ LNCaP cells, suggesting a possible role for AR for the activation of caspase-dependent programmed cell death (CD-PCD). More important, however, γ TT, δ-TT, γ -TS, and δ-TS activated dominant caspase–independent programmed cell death (CI-PCD) in all prostate cancer cell lines examined. Thus, vitamin E homologs and synthetic derivatives may find applications in the treatment of prostate tumors that are resistant to caspase-activating therapeutic agents.

Submitted 3 May 2009; accepted in final form 13 August 2009. Address correspondence to Andreas I. Constantinou, Laboratory of Cancer Biology and Chemoprevention and, Department of Biological Sciences, Faculty of Pure and Applied Sciences, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus. Fax: 00-35722-892881. E-mail: [email protected]

INTRODUCTION Prostate cancer is the most common cancer in males, and it is only second to lung cancer with respect to mortality (1). Studies that have been performed using cancer cell lines, animal models, and clinical trials have shown that vitamin E has a possible role in the chemotherapy of prostate cancer (2–7). Vitamin E is an important micronutrient consisting of 8 homologs with strong antioxidant activities: 4 tocopherols (TOC; α-, β-, γ - and δTOC) and 4 tocotrienols (TT; α-, β-, γ -, and δ-TT; Fig. 1A and 1B) (8,9). Recent studies have shown that the anticancer activities of vitamin E homologs can be attributed to their ability to induce apoptosis (10–19). However, the efficacy and mechanism of apoptosis modulated by the 8 homologs had not been evaluated previously in a single study. Although α-TOC is capable of inducing cell cycle block, several studies have shown that this homolog is not a strong proapoptotic inducer (6–7, 10). The role of β-TOC in the induction of programmed cell death (PCD) has not been investigated thoroughly due to the low abundance of this compound in natural sources (11,12). γ -TOC and δ-TOC are both capable of inducing apoptosis (4–6). The induction of apoptosis by γ -TOC is dependent on the cellular microenvironment (4–6). On the other hand, even though the proapoptotic potency of δ-TOC has not been thoroughly investigated, this homolog has been shown to be effective in all carcinogenic cell lines tested (13, 14). Generally, the order of apoptotic efficiency

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In the past few years, most research has focused on structural variations of vitamin E with the aim to improve the proapoptotic potency of these agents. The compounds developed in this manner were shown to be efficient against a variety of malignancies (22). The most studied member of these compounds is α-tocopheryl succinate (α-TOS). α-TOS has an ester-linked, succinic acid moiety attached to the position-6 oxygen atom of the phenolic ring of the chroman head (Fig. 1C). The conversion of α-TOC to α-TOS greatly improves its anticancer action in tumorigenic cell lines and animal models while causing no toxicity in normal cells (23–25). However, one major disadvantage of α-TOS is that oral administration of this compound may not be effective due to the hydrolysis of the ester linkage by cellular esterases of the intestinal tract yielding α-TOC and succinic acid, neither of which exhibits anticancer properties (23–25). Interestingly, there is very limited evidence in the literature regarding the proapoptotic activities of the three other succinate derivatives of the tocopherols (i.e., β-TOS, γ -TOS, and δ-TOS) (22) or the more potent tocotrienols (i.e., α-TS, β-TS, γ -TS, and δ-TS) (22,26). In search of natural homologs and/or synthetic derivatives of vitamin E with strong antitumorigenic potency, we examined the death promoting properties of the 8 natural forms of vitamin E (α-TOC, β-TOC, γ -TOC, δ-TOC and α-TT, β-TT, γ -TT, δ-TT) and their succinate synthetic derivatives (α-TOS, β-TOS, γ -TOS, δ-TOS and α-TS, β-TS, γ -TS, δ-TS) in androgen receptor (AR)– (DU145 and PC-3) and AR+ (LNCaP) prostate cancer cell lines (Table 1, Fig. 1A–1D). To our knowledge, this is the first time that the efficacy and mechanism of apoptosis modulated by the 8 vitamin E homologs have been TABLE 1 List of vitamin E homologs and synthetic derivatives Category of Vitamin E Tocopherols FIG. 1. The structures of vitamin E natural homologs and synthetic derivatives examined. The structures of A: tocopherols, B: tocotrienols, C: tocopheryl succinate derivatives, and D: tocotrienyl succinate derivatives.

of the three vitamin E tocopherols is considered to be δ-TOC > γ -TOC > α-TOC (14). Tocotrienols, generally, display a greater antitumor activity than tocopherols without affecting normal cell growth and viability (13,15–18). Overall, the apoptotic potency of tocotrienols is considered to be as follows: δ-TT> γ -TT> α-TT (19). Similarly to β-TOC, the role of β-TT in apoptosis has not been investigated because it is present at very low levels in natural sources, thereby making its extraction and use in research very difficult (20). Although there is 1 report for total synthesis of β-TT, the synthetic process is considered difficult (21).

Tocotrienols

Tocopheryl succinates

Tocotrienyl succinates

Acronym Natural α-tocopherol β-tocopherol γ -tocopherol δ-tocopherol α-tocotrienol β-tocotrienol γ -tocotrienol δ-tocotrienol Synthetic α-tocopheryl succinate β-tocopheryl succinate γ -tocopheryl succinate δ-tocopheryl succinate α-tocotrienyl succinate β-tocotrienyl succinate γ -tocotrienyl succinate δ-tocotrienyl succinate

α-TOC β-TOC γ -TOC δ-TOC α-TT β-TT γ -TT δ-TT α-TOS β-TOS γ -TOS δ-TOS α-TS β-TS γ -TS δ-TS

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evaluated in a single study. Furthermore, it is also the first time that the tocopheryl- and tocotrienyl-succinate derivatives have been synthesized and their apoptotic properties have been determined and compared to those of vitamin E homologs. Overall, our results have shown the following: 1) δ-TT is the most effective of all the natural homologs of vitamin E, 2) δ-TS is the most potent of all the natural and synthetic compounds of vitamin E investigated, 3) γ -TT and δ-TT induce low caspase activity selectively in LNCaP cells, and 4) γ -TT, δ-TT, γ -TS, and δ-TS activate dominant caspase-independent programmed cell death (CI-PCD) in all prostate cancer cell lines examined. The latter finding is noteworthy since compounds triggering CI-PCD could find medical applications in the adjuvant treatment of cancers with resistance to agents inducing apoptosis via activation of pathways of caspase-dependent programmed cell death (CD-PCD). MATERIALS AND METHODS Reagents DMEM, fetal bovine serum, antibiotic/antimycotic, and trypsin used in cell culture were purchased from Gibco, Invitrogen (Carlsbad, CA). The caspase inhibitor benzyloxycarbonylVal-Ala-Asp-fluoromerthyletone (z.VAD.fmk) was purchased from Calbiochem (Darmstadt, Germany). The Caspase8/FLICE Fluorometric Protease Assay was purchased from Biosource International Inc. (Camarillo, CA), and the Caspase3/CPP32 Fluorogenic substrate was obtained from Kamiya (Seattle, WA). All other reagents were purchased from Sigma (St. Louis, MO). Commercial Vitamin E Natural Homologs and Synthetic Derivatives The compounds listed on Table 1 were purchased, isolated, or synthesized as described in this section (for structures of compounds, see Fig. 1). d-α-tocopherol (α-TOC), and d-δtocopherol (δ-TOC) were obtained from commercial sources (Sigma or ADM) in minimum 95% purity. d-γ -tocopherol (γ TOC) was purchased from Eisai Corp (Tokyo, Japan) in >95% purity. Small samples of d-β-tocopherol (β-TOC) and d-βtocotrienol (β-TT) were generously donated by Tama Biochemical Co., Ltd. (Tokyo, Japan) d-α-tocopheryl succinate (α-TOS) was purchased from Sigma. Isolation of Natural Homologs of Vitamin E Isolation of d-α-tocotrienol (α-TT) and d-γ -tocotrienol (γ TT). α-TT and γ -TT were isolated from approximately 50% total tocol-containing concentrate of palm oil (Tocomin-50R ) obtained from Carotech, Inc. (Ipoh, Malaysia). This concentrate contained approximately 20% γ -TT, 11% α-TT, and a total of 19% other tocopherols and tocotrienols. The remainder of the concentrate was largely comprised of triglycerides, fatty acids, fatty alcohols, carotenoids, and sterols. Approxi-

mately 60-g samples of Tocomin-50 were chromatographed on open columns containing 1.5 kg of silica gel. The chromatography was followed using thin-layer silica gel chromatography (read using UV fluorescence and p-anisaldehyde spray reagent) and proton NMR spectroscopy of concentrated fractions. Elution was with a gradient from pure hexanes to 12% acetone in hexanes using approximately 5 to 8 l increments containing, respectively, 0.5, 1.0, 2.0, 4.0, 7.0, and 12% acetone in hexanes. Tocotrienol-containing fractions (7% acetone and above) were stripped of solvent and rechromatographed on silica gel in the same way to give two fractions that contained approximately 60–75% of α-TT and γ -TT, respectively. To remove nontocol impurities, which co-eluted with the two desired tocotrienols, the enriched fractions were acetylated. The fractions enriched in α-TT and γ -TT were each stripped of solvent on the rotovap, redissolved in 20 ml each of pyridine, and treated with 10 ml of acetic anhydride. After stirring overnight at room temperature, the two acetylation mixtures were quenched by addition of a few ml of water followed by stirring for 2 h. The reaction mixtures were then poured into 500 ml of water, extracted with ethyl acetate, and the ethyl acetate layers washed with 5% HCl (to remove pyridine), brine, and dried over sodium sulfate. The ethyl acetate was then removed on the rotovap, and the resulting two samples (d-α- and d-γ -tocotrienyl acetates) each were carefully chromatographed on silica gel using flash-column techniques, 0–8% acetone in hexane elution. This afforded samples (approximately 5–7 g) of the two acetates, which were judged to be about 85–90% pure by proton NMR spectroscopy. The purified acetates were reconverted to free tocotrienols by transesterification in anhydrous methanol containing a catalytic amount of potassium carbonate (reflux, 1 h). The reaction mixtures were stripped to about 5 ml on the rotovap, diluted with water, neutralized with dilute HCl, and extracted with ethyl acetate. The organic phases were washed with brine, dried over sodium sulfate, and stripped on the rotovap. The resulting two tocotrienols were flash-chromatographed on silica gel (acetone-hexane gradient elution) to give about 3.5 g each of > 95% pure (NMR, HPLC) α-TT and γ -TT as pale yellow viscous oils. The compounds had IR and NMR spectra in agreement with literature values. Isolation of d-δ-tocotrienol (δ-TT). Multigram quantities of δ-TT were obtained in 95% purity using the methods described above for α-TT and γ -TT, the principle difference being that the starting feedstock was a d-δ-tocotrienol-rich fraction of annatto oil (DeltagoldR 50) obtained from American River Nutrition (Hadley, MA). Synthesis of Vitamin E Derivatives Synthesis of d-β-, γ -, δ-tocopheryl- (β/γ /δ/-TOC) and dα-, β-, γ -, δ-tocotrienyl succinates (α/β/γ /δ/-TT). The tocopheryl and tocotrienyl hydrogen succinates are well-known compounds, described previously (27–29). For our research, the 7 commercially unavailable tocol succinates were all prepared by the same method, described as follows. A solution of 0.50 g


(0.00126 mole) of > 95% pure β/γ /δ/-TOC or α/β/γ /δ/-TT in 10 ml of anhydrous pyridine was stirred at 20◦ C under nitrogen atmosphere. There was added a catalytic quantity (ca. 50 mg) of 4-dimethylaminopyridine and 1g (0.010 mole) of succinic anhydride. The reaction mixture was stirred for 20 h, at which time TLC analysis indicated consumption of the starting tocopherol/tocotrienol and formation of a single polar product. There was added to the mixture about 1g of water, and stirring was continued for 2 h to insure hydrolysis of the excess succinic anhydride employed. The mixture was then poured into 250 ml of water and extracted with ethyl acetate. The extract was washed with 5% aq. HCl (to remove pyridine) and with brine. The solution was dried over anhydrous sodium sulfate and stripped of solvent on the rotovap to give a crude product as pale yellow viscous syrup, completely homogeneous by TLC and HPLC. Final traces of solvent were removed by storing the compound on a high-vacuum line at 22◦ C for 7 days. All succinates had the expected NMR and IR spectra. Cell Lines and Culture Conditions DU-145, PC-3, and LNCaP cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). DU145 (AR–), PC3 (AR–), and LNCaP (AR+) cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic. The cells were maintained at 37◦ C and passaged 2 to 3 times/wk. Crystal Violet Staining A total of 1 × 104 DU-145, PC3, or LNCaP cells were seeded per well of a 96-well plate and incubated for 24 h. At the end of 24 h, the growth medium was removed and replaced with fresh DMEM or DMEM supplemented with the various vitamin E compounds and synthetic derivatives (10, 20, 40, or 100 µM) and/or 20 µM z.VAD.fmk, and the plates were incubated for the time periods described in the figure legends. At the end of each incubation period, the medium was removed, and 100 µl of 10% formalin was added in each well for 5 min. Subsequently, formalin was removed; the wells were washed with PBS and incubated with 100 µl of 0.2% crystal violet for 10 min. The wells were then washed with distilled water several times to ensure removal of the dye, and the plates were allowed to dry at room temperature. After drying, 100 µl of acetic acid was added per well, and the plates were incubated at room temperature for 5 min. At the end of the incubation period, 100 µl of acetic acid were added again to each well, and the plates were immediately read on a microplate reader at 620 nm. The absorbance obtained in untreated control cells for each of the three cell lines and time points (24 h, 48 h, 72 h) was considered as 100% viability. The IC50 value (i.e., the concentration of each compound at which 50% of cell death was observed) for the different cell lines, and time points were calculated using Prism software version 5.0 (Graphpad, San Diego, CA). Whenever 50% of cell death was not achieved with up to 100 µM, the IC50 value was designated as >100.

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DAPI Staining Circular TC-treated cover slips were placed in the bottom of each well of a 24-well plate. A total of 1 × 105 of DU-145, PC-3, or LNCaP cells were seeded in each well and incubated for 24 h. At the end of 24 h, the growth medium was removed and replaced with fresh DMEM or DMEM supplemented with 20 µM of the appropriate vitamin E homolog or synthetic derivative. Following an incubation of 24 h, the medium was removed, and the cells were incubated for 5 min with DAPI stain (1 µg/ml). Each cover slip was then removed, covered with glycerol, and the morphology of the cells’ nuclei was observed using a fluorescence microscope (Leica, Wetzlar, Germany) at excitation wavelength 350 nm. Caspase Assays Caspase-8 activity assay. Caspase-8 activity assays were performed using the Caspase 8/FLICE Fluorometric Protease assay kit (Biosource International Inc., Camarillo, CA). The cells in 10 mm2 plates were treated with the vitamin E natural compounds, synthetic derivatives, or etoposide as described in the figure legends, in the presence or absence of 20 µM of the caspase inhibitor z.VAD.fmk (Calbiochem, Darmstadt, Germany). At the end of the incubation period, the cells were washed with PBS, the PBS was removed, and the cells were lysed with 150 µl of cell lysis buffer (provided in Caspase 8 Apotarget kit) per plate. The plates were placed on ice, and cells were allowed to lyse for 10 min. The cells were mixed in well with pipette to ensure lysis. The lysate was transferred into a fresh eppendorf, and the latter was spun for 5 min at 11,000 rpm at 4◦ C. The supernatant was removed and placed into a new eppendorf, leaving the pellet behind. The lysates were stored at –70◦ C until needed for the performance of the assay. When needed, the samples were thawed and used in a Bradford assay to determine protein concentration. Subsequently, 120 µg of each sample was added per well of a 96-well plate. The DTT was then added to the provided reaction buffer mix (DTT final concentration: 10 mM). Fifty µl of reaction buffer mix was then added per sample in a well; 5 µl of 1 mM of the caspase substrate IETD-AFC was then added per well and incubated at 37◦ C for 1 to 2 h. At the end of the incubation period, the 96-well plate was read on a fluorescence reader (excitation 400 nm, emission 505, slit width 15). Caspase-3 activity assay. Caspase-3 activity assays were performed using the Kamiya caspase 3 fluorogenic substrate (Kamiya, Seattle, WA). The cells in 10 mm2 plates were treated with the vitamin E natural compounds or synthetic derivatives as described in the figure legends in the presence or absence of the caspase inhibitor z.VAD.fmk (Calbiochem). At the end of the incubation period, the cells were washed with PBS, the PBS was removed, and the cells were lysed with 150 µl of cell lysis buffer (provided in Caspase 8 Apotarget kit) per plate. The plates were placed on ice, and cells were allowed to lyse for 10 min. The cells were mixed in well with pipette to ensure lysis. The lysate was transferred into a fresh eppendorf, and the latter was spun

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for 5 min at 11,000 rpm at 4◦ C. The supernatant was removed and placed into a new eppendorf, leaving the pellet behind. The lysates were stored at –70◦ C until needed for the performance of the assay. When needed, the samples were thawed and used in a Bradford assay to determine protein concentration. Subsequently, 40 to 80 µg of each sample was added per well of a 96-well plate. The Assay Buffer Mix was then prepared by adding 1 ml Caspase Assay Fluorometric Buffer (2.4 g Hepes, 20 g sucrose, 0.2 g CHAPS, dissolved in 200 ml water and pH 7.4 with NaOH) per 1 µl Caspase 3 substrate (AC-DEVD-AFC, used at a final concentration of 2.5 µM) per 10 µl of 1 M DTT (final concentration 10 mM). Two hundred µl of assay buffer was then added per sample in a well, and the plate was covered in foil and incubated at 37◦ C for 1 to 3 h. At the end of the time points, the plate was read on plate reader (excitation 400 nm, emission 505, slit width 15).

Statistical Analyses Statistical analyses were conducted using Prism software version 5.0 (Graphpad, San Diego, CA).

RESULTS Preparation of Vitamin E Homologs and Synthetic Derivatives The 8 homologs of vitamin E (α-TOC, β-TOC, γ -TOC, δTOC and α-TT, β-TT, γ -TT, δ-TT) and their succinate synthetic derivatives (α-TOS, β-TOS, γ -TOS, δ-TOS and α-TS, β-TS, γ -TS, δ-TS; Table 1, Fig. 1) were prepared as described in Materials and Methods for a subsequent evaluation and comparison of their death promoting potencies. Antiproliferative Effects of Vitamin E Homologs and Synthetic Derivatives Using the crystal violet cell proliferation assay, cell viability was monitored with a range of concentrations between 0 and 100 µM for the 8 vitamin E homologs and 8 synthetic derivatives shown on Table 1 and Fig. 1 as described in Materials and Methods. The results were used to determine the IC50 values (Table 2). Tocotrienols were generally more effective than tocopherols. δ-TT was the most effective of the natural vitamin E homologs, with IC50 values of 20 µM, 25 µM, and 11 µM following a 72-h incubation in DU145, PC3, and LNCaP cells,

TABLE 2 The IC50 values (µM) of vitamin E homologs and synthetic derivatives in DU145, PC3, and LNCaP cells DU145

Tocopherols α-TOC β-TOC γ -TOC δ-TOC Tocopherol succinates α-TOS β-TOS γ -TOS δ-TOS Tocotrienols α-TT β-TT γ -TT δ-TT Tocotrienol succinates α-TS β-TS γ -TS δ-TS

PC3

LNCaP

24 h

48 h

72 h

24 h

48 h

72 h

24 h

48 h

72 h

>100 >100 >100 >100

>100 >100 >100 >100

>100 >100 >100 >100

>100 >100 >100 >100

>100 >100 >100 89

>100 >100 >100 89

>100 >100 >100 62

>100 >100 >100 43

>100 >100 >100 56

>100 >100 >100 60

>100 >100 >100 51

>100 68 >100 56

>100 >100 >100 64

>100 >100 >100 22

57 91 >100 37

17 >100 8 10

40 >100 7 12

60 95 38 16

>100 60 34 20

>100 53 30 27

>100 52 35 20

>100 59 33 32

>100 48 24 25

>100 44 23 25

>100 70 21 26

>100 52 21 19

>100 29 20 11

>100 60 36 20

>100 56 19 11

>100 47 36 15

>100 72 41 20

>100 68 31 18

>100 55 28 28

>100 >100 21 10

>100 >100 15 9

>100 86 11 8

DU145, PC3, and LNCaP cells were incubated in 96 well plates with the vitamin E homologs and synthetic derivatives (range of concentration 0–100 µM) for a period of 24–72 hours. At the end of the incubation period the cells were stained with crystal violet and the IC50 values were determined as described in the Materials and Methods section. The values are the means of three determinations from three independent experiments. Error γ -TT> β-TT> α-TT) (19). Furthermore, the addition of succinic acid on the hydroxyl group of α-TOC and its conversion to α-TOS increases its proapoptotic potency, suggesting a possible role for the addition of succinic acid on other homologs of vitamin E (23–25). This study presents the first time that the efficacy and mechanism of apoptosis modulated by the 8 vitamin E homologs have been evaluated in a single study. Furthermore, it is also the first time that the tocopheryl and tocotrienyl—succinate derivatives of the 8 homologs—have been synthesized and their apoptotic


properties have been determined and compared to those of vitamin E homologs. Our results have shown that δ-TT is the most potent homolog, and δ-TS is the most potent synthetic derivative of vitamin E in the induction of cell death in prostate cancer cells (Table 2). We propose that δ-TS benefits from having 1) at its basis δ-TT, the most potent of all 8 homologs of vitamin E; and 2) its hydroxyl group being modified by the addition of succinic acid. Interestingly, the increased death promoting potency of δ-TS compared to δ-TT could not be attributed to the induction of higher levels of caspase activity. In fact, the conversion of δ-TT to δ-TS completely eliminated the activation of caspase-3 observed in LNCaP cells by the former agent, strongly suggesting that the higher killing potency of δ-TS is mediated via CI-PCD pathways (Table 4). It has been previously proposed that γ -TT and δ-TT are capable of inducing caspase activation and therefore CD-PCD in several cell lines (17,31). For example, γ -TT has been reported to activate caspase-8 and caspase-3 in neoplastic mammary epithelial cells and caspase-8, caspase-9, and caspase-3 in Hep3B hepatoma cells (17,31). Contrary to these reports, in this study, we found that neither γ -TT nor δ-TT produce significant activation of caspase-8 and caspase-3 in DU145 and PC3 cells at concentrations and time points that kill 50% of the cells. Interestingly, under the same treatment conditions in LNCaP cells, these agents are effective, producing substantial activation of caspase-8 and/or caspase-3 (Fig. 3, Table 4). The results suggest that the tocotrienol-induced caspase activation is selective. The caspase activation by γ -TT and δ-TT only in AR+ LNCaP but not in AR– DU145 and PC3 cells may suggest a possible involvement of the androgen receptor. However, to prove this notion, further experiments should be performed in isogenic cell lines expressing or not expressing the receptor. Alternatively, the differences in sensitivity of the three prostate cancer cells may be due to differences in the biovailability and transport proteins of vitamin E, for example, α-tocopherol associated protein (TAP), scavenger receptor class B type I, α -tocopherol transfer protein (TTP), and ATP-binding cassette transporter A1 (12). The results of this study provide strong support to two previous reports that have suggested the involvement of caspaseindependent pathways in the induction of apoptosis by vitamin E homologs (32,33) and help identify CI-PCD as the main mode of cell killing by γ -TT and δ-TT. Jiang et al. (32) reported that although γ -TOC activates caspase-9, caspase-3 and caspase-7 in LNCaP cells, apoptosis could not be completely reversed by the pancaspase inhibitor (z.VAD.fmk), indicating the involvement of an alternative caspase-independent pathway. A similar observation was made in MDA-MB-231 cells treated with γ -TT in which morphological features of apoptosis became evident accompanied by mitochondrial disruption and release of cytochrome c in the absence of poly-(ADP-ribose)-polymerase cleavage, suggesting the lack of involvement of caspases in the induction of apoptosis (33). These results are consistent with our hypothesis that the tocotrienols γ -TT and δ-TT kill prostate cancer cells by activating caspase-independent pathways of PCD.

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Of special interest is the observation that γ -TT and δ-TT induce cell death synergistically only in AR– PC-3 and DU-145 cell lines and in the absence of increased caspase activity (Table 3, Fig. 3). One possible explanation of this observation is that these compounds may be activating two different caspaseindependent pathways that synergistically produce higher levels of cell death. The synergistic death-promoting potencies of the two tocotrienols could find practical applications if the combination of γ -TT and δ-TT were used in the treatment of androgen independent prostate cancer. The activation of synergistic pathways of cell death by the two compounds could ensure better eradication of tumor cells and lower levels of tumor resistance often associated with classical chemotherapy. Also, this combination would avoid the problem of hydrolyzability often associated with synthetic derivatives such as α-TOS (23–25). Similar to the synergistic effects of γ -TT and δ-TT in the induction of cell death (Table 3), the increased levels of cell death induced by the synthetic derivative δ-TS relative to δ-TT (Table 2) could also not be attributed to increased caspase activity (Fig. 3, Table 4). Furthermore, the inability of the caspase inhibitor z.VAD.fmk to recover the loss of cell viability induced by γ TT, δ-TT, γ -TS, and δ-TS (Fig. 4) provides additional support to our hypothesis that the main mode of apoptotic cell death induced by vitamin E homologs and their synthetic derivatives is mediated via a caspase-independent pathway (CI-PCD). Although the exact pathways of CI-PCD have not been unraveled, it is known that the mitochondrion is the main organelle orchestrating the series of events leading to CI-PCD (34,35). The proapoptotic proteins Bax and Bid, the cathepsins (released by lysosomes), the calpains and mainly AIF have been shown to have a central role in the induction of CI-PCD (36–40). Even though at present, the exact pathways activated by vitamin E homologs and their derivatives in prostate cancer cells remain unknown, our data strongly suggest that these agents trigger CI-PCD dominant pathways. The basis of future research should be to develop vitamin E derivatives based on the most potent tocotrienols instead of the inactive homolog α-TOC. One such promising compound is the synthetic derivative δ-TS identified in this study. Our results suggest that δ-TS is more effective than the popular αTOS against prostate cancer. Consequently, the identification of the signalling pathways and in particular, those involved in CI-PCD regulated by δ-TS, is of vital significance. Future experiments should focus on investigating the possible synergistic potency of the effective tocotrienols or tocotrienol synthetic derivatives such as δ-TS in combinations with chemotherapeutic agents commonly used in the clinic. In the future, we should also progress with a more rational design of even better synthetic derivatives of vitamin E. The design of another δ-TT-based molecule should address the issue of hydrolyzability. The production of a nonhydrolyzable molecule based on δ-TT is expected to have an even greater antitumorigenic potency. The ability of γ -TT, δ-TT, and δ-TS documented in this study to activate CI-PCD may prove to be useful in the adjuvant

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treatment of prostate cancers that are resistant to traditionally used chemotherapeutic agents whose mode of action involves the activation of caspases. ACKNOWLEDGMENTS Research in our laboratories is supported by the Cyprus Research Promotion Foundation (grant-KINHTIKOTHTA /0506/06, grant ϒEIA /0104/06, and grant SYNERGIA /0505/20). REFERENCES 1. Jemal A, Siegel R, Ward E, Murra T, Xu J, et al.: Cancer statistics, 2006. CA Cancer J Clin 56, 106–130, 2006. 2. Shun MC, Yu W, Gapor A, Parsons R, Atkinson J, et al.: Pro-apoptotic mechanisms of action of a novel vitamin E analog (α-TEA) and a naturally occurring form of vitamin E (δ-tocotrienol) in MDA-MB-435 human breast cancer cells. Nutr Cancer 48, 95–105, 2004. 3. Limpens J, Schroder FH, de Ridder CM, Wildhagen MF, Obermuller-Jeviv UC, et al.: Combined lycopene and vitamin E treatment suppresses the growth of PC-346C human prostate cancer cells in nude mice. J Nutr 136, 1287–1293, 2006. 4. Campbell SE, Stone WL, Lee S, Whaley S, Yang H, et al.: Comparative effects of RRR-α and RRR-γ -tocopherol on proliferation and apoptosis in human colon cancer cell lines. BMC Cancer 6, 13, 2006. 5. Min J, Guo J, Zhao F, and Cai D: Effect of apoptosis induced by different vitamin E homologous analogues in human hepatoma cells (HepG2). Wei Sheng Yan Jiu 32, 343–345, 2003. 6. McIntyre BS, Briski KP, Gapor A, and Sylvester PW: Anti-proliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells. Proc Soc Exp Biol Med 224, 292–301, 2000. 7. Pastori M, Pfander H, and Boscoboinik D: Lycopene in association with alpha-tocopherol inhibits at physiological concentrations proliferation of prostate carcinoma cells. Biochem Biophys Res Commun 250, 582–585, 1998. 8. Burton GW and Traber MG: Vitamin E: antioxidant activity, biokinetics, and bioavailability. Annu Rev Nutr 10, 357–382, 1990. 9. Kamal-Eldin A and Appelqvist LA: The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids 31, 671–701, 1996. 10. Boscoboinik D, Chatelain E, Bartoli GM, and Azzi A: Molecular basis of alpha-tocopherol inhibition of smooth muscle cell proliferation in vitro. EXS 62, 164–177, 1992. 11. Speek A, Schrijver J, and Schreurs W: Vitamin E composition of some seed oils as determined by high-performance liquid chromatography with fluorescence detection. J Food Sci 50, 121–124, 1985. 12. Tan B: Palm carotenoids, tocopherols, and tocotrienols. J Am Oil Chem Soc 66, 770–776, 1989. 13. Yu W, Simmons-Menchaca M, Gapor A, Sanders BG, and Kline K: Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols. Nutr Cancer 33, 26–32, 1999. 14. Min J, Guo J, Zhao F, and Cai D: Effect of apoptosis induced by different vitamin E homologous analogues in human hepatoma cells (HepG2). Wei Sheng Yan Jiu 32, 343–345, 2003. 15. Srivastava JK and Gupta S: Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells. Biochem Biophys Res Commun 346, 447–453, 2006. 16. Sylvester PW and Shah S: Intracellular mechanisms mediating tocotrienolinduced apoptosis in neoplastic mammary epithelial cells. Asia Pac J Clin Nutr 14, 366–373, 2005. 17. Sakai M, Okabe M, Tachibana H, and Yamada K: Apoptosis induction by gamma-tocotrienol in human hepatoma Hep3B cells. J Nutr Biochem 17, 672–676, 2006. 18. Kumar KS, Raghavan M, Hieber K, Ege C, Mog S, et al.: Preferential radiation sensitization of prostate cancer in nude mice by

19.

20. 21. 22.

23.

24.

25.

26.

27. 28.

29. 30.

31.

32.

33.

34.

35.

36. 37.

38. 39.

40.

nutraceutical antioxidant gamma-tocotrienol. Life Sci 78, 2099–2104, 2006. He L, Mo H, Hadisusilo S, Qureshi AA, and Elson CE: Isoprenoids suppress the growth of murine B16 melanomas in vitro and in vivo. J Nutr 127, 668– 674, 1997. Tan B: Appropriate spectrum vitamin E and new perspectives on desmethyl tocopherols and tocotrienols. JANA 8, 35–43, 2005. Couladouros E, Moutsos V, Lampropoulou M, Little J, and Hyatt J: J Org Chem 72, 6735–6741, 2007. Birringer M, EyTina JH, Salvatore BA, and Neuzil J: Vitamin E analogues as inducers of apoptosis: structure–function relation. Br J Cancer 88, 1948– 1955, 2003. Lawson KA, Anderson K, Simmons-Menchaca M, Atkinson J, Sun L, et al.: Novel vitamin E analogue decreases syngeneic mouse mammary tumor burden and reduces lung metastasis. Mol Cancer Ther 2, 437–444, 2003. Fariss MW, Nicholls-Grzemski FA, Tirmenstein MA, and Zhang JG: Enhanced antioxidant and cytoprotective abilities of vitamin E succinate is associated with a rapid uptake advantage in rat hepatocytes and mitochondria. Free Radic Biol Med 31, 530–541, 2001. Yu W, Liao QY, Hantash FM, Sanders BG, and Kline K: Activation of extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-α-tocopheryl succinate-induced apoptosis of human breast cancer cells. Cancer Res 61, 6569–6576, 2001. Vraka PS, Drouza C, Rikkou MP, Odysseos AD, and Keramidas AD: Synthesis and study of the cancer cell growth inhibitory properties of alpha-, gamma-tocopheryl and gamma-tocotrienyl 2-phenylselenyl succinates. Bioorg Med Chem 14, 2684–2696, 2006. Cawley J and Stern M: U.S. Patent 2,680,749 (to Eastman Kodak), 1951. Goh S, Hew N, Ong A, Choo Y, and Brumby S: Tocotrienols from palm oil: electron spin resonance spectra of tocotrienoxyl radicals. J Am Oil Chem Soc 67, 250–254, 1990. Hyatt J: U.S. Patent 5,869,704 (to Eastman Chem. Co.), 1999. Jia L, Yu W, Wang P, Sanders BG, Kline K: In vivo and in vitro studies of anticancer actions of α-TEA for human prostate cancer cells. Prostate 68, 849–860, 2008. Sylvester PW, Shah SJ, and Samant GV: Intracellular signaling mechanisms mediating the antiproliferative and apoptotic effects of gamma-tocotrienol in neoplastic mammary epithelial cells. J Plant Physiol 162, 803–810, 2005. Jiang Q, Wong J, and Ames BN: Gamma-tocopherol induces apoptosis in androgen-responsive LNCaP prostate cancer cells via caspase-dependent and independent mechanisms. Ann NY Acad Sci 1031, 399–400, 2004. Takahashi K and Loo G: Disruption of mitochondria during tocotrienolinduced apoptosis in MDA-MB-231 human breast cancer cells. Biochem Pharmacol 67, 315–324, 2004. Mathiasen IS, Lademann U, and J¨aa¨ ttel¨a M: Apoptosis induced by vitamin D compounds in breast cancer cells is inhibited by Bcl-2 but does not involve known caspases or p53. Cancer Res 59, 4848–4856, 1999. Rudolf E, Rudolf K, and Cervinka M: Selenium activates p53 and p38 pathways and induces caspase-independent cell death in cervical cancer cells. Cell Biol Toxicol. 24, 123–141, 2008. Okuno S, Shimizu S, Ito T, Nomura M, and Hamada E: Bcl-2 prevents caspase-independent cell death. J Biol Chem 273, 34272–34277, 1998. Xiang J, Chao DT, and Korsmeyer SJ: BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc Natl Acad Sci USA 93, 14559–14563, 1996. Brunk UT, Neuzil J, and Eaton J: Lysosomal involvement in apoptosis. Redox Report 6, 91–97, 2001. Neuzil J, Zhao M, Ostermann G, Sticha M, and Gellert N: Alpha-tocopheryl succinate, an agent with in vivo anti-tumor activity, induces apoptosis by causing lysosomal instability. Biochem J 362, 709–715, 2002. Lorenzo HK and Susin SA: Therapeutic potential of AIF-mediated caspaseindependent programmed cell death. Drug Resist Updates 10, 235–255, 2007.