Mitochondrially delivered anti-cancer compounds
RE047300 ยท 2019-03-19
Assignee
Inventors
Cpc classification
C07D311/72
CHEMISTRY; METALLURGY
A61K47/551
HUMAN NECESSITIES
C07F9/65522
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07F9/655
CHEMISTRY; METALLURGY
A61K47/55
HUMAN NECESSITIES
Abstract
This invention relates to anti-cancer compounds and to methods for treating or preventing cancer. In one aspect the invention concerns mitochondrially delivered pro-oxidant anti-cancer compounds that generate reactive oxygen species and induce apoptosis of cancerous cells. The delivery moiety can be a lipophilic cation and the pro-oxidant moiety can be a pro-oxidant vitamin E analogue, such as -tocopheryl succinate, -tocopheryl maleate, -tocopheryl maleyl amide, or 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (-tocopheryloxyacetic acid).
Claims
1. A compound .[.for inducing death of a cancerous cell, said compound comprising: a pro-oxidant moiety for (i) generating reactive oxygen species within mitochondria of the cancerous cell, (ii) inducing apoptosis of the cancerous cell, and (iii) interacting with mitochondrial complex II of the cancerous cell, wherein the pro-oxidant moiety comprises a vitamin E analogue comprising a functional domain at position C6 of a substituted chromanol ring that is capable of interacting with a Qp ubiquinone binding site of mitochondrial complex II, a domain that includes the substituted chromanol ring, and a hydrophobic domain comprising an at least seven-carbon long aliphatic chain; and a delivery moiety for delivering the pro-oxidant moiety to the mitochondria of the cancerous cell and for correctly positioning the functional domain for interaction with the Qp ubiquinone binding site, wherein the delivery moiety comprises a delocalized lipophilic triphenylphosphonium cation and said vitamin E analogue is.]. selected from the group consisting of .[.-tocopheryl succinate, -tocopheryl maleate, -tocopheryl maleyl amide, and 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (-tocopheryloxyacetic acid).]. .Iadd. ##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024## triphenylphosphonium tagged 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (triphenylphosphonium tagged -tocopheryloxyacetic acid), and a derivative of any of the foregoing; wherein said derivative has a functional domain substituted at position C6 of the chromanol ring of the compound that comprises a functional domain group selected from the group consisting of an ether, ester, amide, dissociated carboxylic acid, and hydrophilic head group comprising a dissociated acid, a charged ammonium group, or both; and a physiologically acceptable salt of any of the foregoing.Iaddend..
.[.2. The compound of claim 1, wherein the vitamin E analogue is -tocopheryl succinate (-TOS)..].
.[.3. A pharmaceutical or veterinary composition comprising the compound of claim 1, or a physiologically acceptable salt thereof, and a physiologically acceptable carrier..].
.[.4. A method of inducing the death of a cancerous cell, said method comprising the step of administering to said cancerous cell or a subject having said cancerous cell a therapeutically effective amount of the compound according to claim 1..].
.[.5. The compound of claim 1, wherein the functional domain is selected from the group consisting of an ether, ester and amide substituted at position C6 of the chromanol ring..].
.[.6. The compound of claim 1, wherein the aliphatic chain comprises an 11 carbon long aliphatic chain..].
.[.7. The pharmaceutical or veterinary composition of claim 3, wherein the pro-oxidant moiety is a vitamin E analogue selected from the group consisting of -tocopheryl succinate, -tocopheryl maleate and -tocopheryl maleyl amide, and the carrier is a transdermally applicable cream..].
.[.8. The pharmaceutical or veterinary composition of claim 3, wherein the pro-oxidant moiety is -tocopheryloxyacetic acid and the carrier is suited for oral administration..].
.Iadd.9. The compound of claim 1, wherein the compound is MitoVE.sub.7S or a physiologically acceptable salt thereof..Iaddend.
.Iadd.10. The compound of claim 1, wherein the compound is MitoVE.sub.9S or a physiologically acceptable salt thereof..Iaddend.
.Iadd.11. The compound of claim 1, wherein the compound is MitoVE.sub.11S or a physiologically acceptable salt thereof..Iaddend.
.Iadd.12. The compound of claim 1, wherein the compound is MitoVE.sub.11AE or a physiologically acceptable salt thereof..Iaddend.
.Iadd.13. The compound of claim 1, wherein the compound is TPP--TOS or a physiologically acceptable salt thereof..Iaddend.
.Iadd.14. A pharmaceutical or veterinary composition comprising the compound of claim 1, or a physiologically acceptable salt thereof, and a physiologically acceptable carrier..Iaddend.
.Iadd.15. The pharmaceutical or veterinary composition of claim 14, wherein the carrier is a cream..Iaddend.
.Iadd.16. The pharmaceutical or veterinary composition of claim 14, wherein the carrier is suited for oral administration..Iaddend.
.Iadd.17. A method of inducing death of a cancerous cell, said method comprising the step of administering to said cancerous cell or a subject having said cancerous cell a therapeutically effective amount of the compound according to claim 1, or a physiologically acceptable salt thereof..Iaddend.
.Iadd.18. The method of claim 17, wherein said method is used to treat cancer in the subject..Iaddend.
.Iadd.19. The compound of claim 1, wherein the compound is triphenylphosphonium tagged 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (triphenylphosphonium tagged -tocopheryloxyacetic acid) or a physiologically acceptable salt thereof..Iaddend.
.Iadd.20. The compound of claim 1, wherein the derivative is a maleate derivative of MitoVE.sub.11S or a physiologically acceptable salt thereof..Iaddend.
.Iadd.21. The compound of claim 1, wherein the derivative is a maleyl amide derivative of MitoVE.sub.11S or a physiologically acceptable salt thereof..Iaddend.
.Iadd.22. The compound of claim 1, wherein the derivative is a maleate derivative of TPP--TOS or a physiologically acceptable salt thereof..Iaddend.
.Iadd.23. The compound of claim 1, wherein the derivative is a maleyl amide derivative of TPP--TOS or a physiologically acceptable salt thereof..Iaddend.
.Iadd.24. A compound of Formula (I), or a physiologically acceptable salt thereof: ##STR00025## wherein the compound is selected from the group consisting of ##STR00026## ##STR00027## ##STR00028## ##STR00029## wherein R.sup.1 comprises a hydrophilic head group comprising a dissociated acid, an ammonium group, or both, and is linked to the chromanol ring through an ester bond, an amide bond, or an ether bond..Iaddend.
.Iadd.25. A compound of Formula (I), or a physiologically acceptable salt thereof: ##STR00030## wherein the compound is selected from the group consisting of ##STR00031## ##STR00032## ##STR00033## ##STR00034## wherein R.sup.1 comprises a carboxylate or ammonium group, and is linked to the chromanol ring through an ester bond, an amide bond, or an ether bond..Iaddend.
Description
DESCRIPTION OF THE FIGURES
(1)
(2) The compounds used were -TOH, -TOS, VE.sub.11S (hereafter referred to as VES), Mito VE.sub.3S, Mito VE.sub.3S, Mito VE.sub.7S, Mito VE.sub.9S and Mito VE.sub.11S (hereafter referred to as MitoVES), Mito VE.sub.11AE (hereafter referred to as Mito VEAE), Mito VE.sub.11F [not shown] (hereafter referred to as MitoVEF), MitoVE.sub.11M [not shown] (hereafter referred to as MitoVEM), and VES4TPP. -TOH and -TOS were obtained from Sigma, VES, MitoVES, MitoVEAE, MitoVEF, MitoVEM and VES4TPP were synthesised as described in the section entitled General Materials and Methods.
(3)
(4) Jurkat (A, G), HCT-116 (B), MCF-7 (C), MDA-MB-453 (D), and Meso-2 cells (E) were exposed to MitoVES at concentrations 1-50 M (A-F), or to VES4TPP or -TOS at 50 M (B). At the times shown, the cells were harvested and assessed for apoptosis. Panel H shows the A014578 or 40% confluency for the times shown and assessed for apoptosis. Panel J shows TEM of control Jurkat cells or the cells exposed to 50 M -TOS or 5 M MitoVES (upper panels at lower magnification and lower panels at higher magnification). Apoptosis data shown are derived from three independent experiments and are presented as mean valuesS.D., the micrographs are representative images of at least three independent experiments.
(5)
(6) 3AProduction of oxygen radicals induced by MitoVES measured by electron paramagnetic resonance spectroscopy (EPR) (left hand panel) as, well as by using the fluorescent dye indicator DMPO (right hand panel).
(7) 3BParental, Bax.sup.-/- and Bax.sup.-/- Jurkat cells were exposed to -TOS (T) at 50 M and MitoVES (M) 5 M, and apoptosis assessed.
(8)
(9) 4ART-PCR, of Ras-transformed B1, B9 and B 10 Chinese hamster lung fibroblast cell lines, for hSDHC and chSDHC mRNA expression.
(10) 4BWestern blotting analysis of B1, B9 and B10 Chinese hamster lung fibroblast cell lines with relevant antibodies to detect protein levels of expression as shown. The results are from clonally selected Ras transformed B1.sub.Ras and B9.sub.Ras-SDHC sub lines, positive for Ras and GFP fusion protein expression and comparison is made with samples from the parental non-transformed B1, B9 and B10 cells.
(11) 4CAfter 24 h of treatment with Complex II targeting drugs TTFA or MitoVES, Ras transformed B1 and B9 cells were assessed for apoptosis using the Annexin V binding method.
(12)
(13) Space filling model of Complex II with MitoVES (identified as a stick figure structure) produced using AutoDock (Morris G M, Goodsell D S, Halliday R S, Huey R, Hart W E, Belew R K, Olson A J (1998) Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function, J Comp Chem 19: 1639-1662) and Astex Viewer (Hartshorn M J (2002) AstexViewer: An aid for structure-based drug design. J Computer Aided Mol Des 16: 871-881). The surface of Chain B (iron-sulfur protein) of Complex II and the surfaces of Chains C and D (the transmembrane section) are shown, but Chain A (flavoprotein-succinate reductase) is not shown.
(14)
(15) Parental EAhy926 cells were seeded in 24-well plate so that they would acquire after an overnight recuperation 50% or 100% confluency, while their .sup.0 counterparts were seeded at 50% confluency. The proliferating and confluent cells were then exposed to MitoVES at concentrations and for times shown, and assessed for apoptosis level by the Annexin V-binding method (A) and for ROS accumulation using the fluorescent probe DHE (B). The inset in panel A shows the cell cycle distribution in proliferating and confluent EAhy926 cells. Panel C shows ROS accumulation in proliferating and confluent EAhy926 cells using EPR spectroscopy.
(16)
(17) Wound-healing (A-F) and tube-forming activity (G, H) were assessed using the EC EAhy926 cells. For wound-healing activity, the cells were seeded in 35-mm Petri dishes and allowed to reach 100% confluency. The injury was then performed, resulting in a denuded gap of 0.4-0.5 mm. Wound-healing activity in control cells and cells supplemented with 1, 5 or 10 M MitoVES was assessed on the basis of proliferation and migration of cells into the denuded zone using a light microscope equipped with a grid and a digital camera (A). Panel B shows morphology of the cells in zones of arrested ECs 20 h after the injury for the control culture and from cells exposed to 10 M MitoVES. In panel C, representative images of injured control cells and cells treated with 10 M MitoVES at different times are presented. Panel D shows the healing rate for the different conditions derived from the slopes of the individual curves in panel A. In panel E, the level of apoptosis is shown at 20 h after the injury for control cultures and for cells exposed to MitoVES. EAhy926 cells were seeded at 10.sup.5 per well in Matrigel-coated 24-well plates and allowed for 24 h to form tubes at the absence or presence of MitoVES. Complete capillaries connecting points of individual polygons (see General Materials and Methods) were counted per the field of 0.16 mm.sup.2 and took as a measure of the tube-forming activity (F). Panel G shows representative images of the Matrigel cultures of control cells and cells treated for 20 h with 5 M MitoVES. At 20 h of treatment, the cells were retrieved from the Matrigel and assessed for apoptosis using the Annexin V binding method (H). Parental EAhy926 cells and their .sup.0 counterparts were seeded in Petri dishes at confluency, injured as described above, and the wound-healing activity assessed in the absence or presence of 10 M MitoVES (MVES) (I; see symbols above panel L). The healing rate was estimated from the slopes in panel I and plotted in mmol/h (J). At 20 h, the cells were assessed for the level of apoptosis (K). Panel L documents tube-forming activity in Matrigel of the .sup.0 EAhy 926 cells in the absence or presence of 5 M MitoVES. Data shown are derived from three independent experiments and are presented as mean valuesS.D., the micrographs are representative images of at least three independent experiments.
(18)
(19) Adherent MCF7 cells and the corresponding mammosphere cells (MS) were cultured and assessed for morphological changes by microscopy (A) and for expression levels of markers as indicators of sternness by RT-PCR (B) and flow cytometry (C), including the expression of CD44 and CD24 (D). The adherent MCF7 cells (E) and the MS cells (F) were treated with 5 M MitoVES, 50 M -TOS or 10 M parthenolide and assessed for their sensitivity to drug induced cell death. Panel G shows the initial levels of cell death by apoptosis in MS cells exposed to MitoVES and panel H shows the histogram analysis of the MS cells exposed to 5 M MitoVES for 3 h and then analysed for annexin V-FITC binding. Adherent MCF7 cells (I) and MS cells (J) were treated for 3 h with -TOS (50 M) or MitoVES homologues containing different lengths in the aliphatic side chain (5 M each, either 11, 7 or 5 carbon chain in length) and the treated cells assessed for production of reactive oxygen species (ROS) by flow cytometry.
(20)
(21) The transgenic FVB/N c-neu female mice with spontaneous small breast carcinomas were treated with MitoVES at 3 mol per mouse per dose and the tumour volume was assessed by repeated ultrasound imaging, monitoring tumour growth over several weeks.
EXAMPLES
(22) In order that this invention may be better understood, the following examples are set fourth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any manner.
General Materials and Methods
(23) Cell Culture.
(24) The following cell lines used in this study were obtained from ATCC, unless specified otherwise: Human T lymphoma cells Jurkat, human mesothelioma cells Meso-I, MM-BI, Ist-Mes, Ist-Mes-2 (Pass, H. I. et al. Characteristics of nine newly derived mesothelioma cell lines. Ann. Thorac. Surg. 59, 835-844 (1995)), human breast cancer cells MCF-7 (erbB2-low) and MDA-MB-453 (erbB2-high), human colorectal cells HCT-116, human hepatocarcinoma cells Huh-7, mouse mesothelioma cells AE17 (Jackaman, C. et al. IL-2 intratumoral immunotherapy enhances CD8+ T cells that mediate destruction of tumor cells and tumor-associated vasculature: a novel mechanism for IL-2. J. Immunol. 171, 505150-505163 (2003), human non-malignant mesothelial cells Met-5A, rat ventricular myocyte-like cells HL-1 (Claycomb, W. C. et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc. Natl. Acad. Sci. USA 95, 2979-2984 (2001)) and H9c2, and the human endothelial-like cells EAhy926 (Edgell, C. J., McDonald, C. C. & Graham, J. B. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc. Natl. Acad. Sci. USA 80, 3734-3737 (1983)). Jurkat cells were grown in the RPMI medium while DMEM was used for other malignant cell lines and for Met-5A cells, supplemented with 10% FCS and antibiotics. HL-1 cells, maintained in fibronectin/gelatine-coated dishes, were grown in the Claycomb medium supplemented with noradrenalin (Claycomb, W. C. et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc. Natl. Acad. Sci. USA 95, 2979-2984 (2001)), EAhy926 cells were grown in complete DMEM supplemented with HAT (Edgell, C. J., McDonald, C. C. & Graham, J. B. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc. Natl. Acad. Sci. USA 80, 3734-3737 (1983)). Cells deficient in mtDNA (.sup.0 phenotype) were prepared as detailed in Weber, T. et al., (Mitochondria play a central role in apoptosis induced by -tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signaling. Biochemistry 42, 4277-4291 (2003)). Acquisition of the .sup.0 phenotype was confirmed by lack of expression by the cells of the mtDNA-encoded cytochrome c oxidase subunit II (COXII not shown). Chinese hamster lung fibroblasts deficient in complex I (B10 cells) (See, B. B. et al. Molecular remedy of complex I defects: rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells. Proc. Natl. Acad. Sci. USA 95, 9167-9171 (1998)) and complex II (CybL.sup.-/-; B9 cells) (Oostveen, F. G., Au, H. C., Meijer, P. J. & Scheffler, I. E. A Chinese hamster mutant cell line with a defect in the integral membrane protein CII-3 of complex II of the mitochondrial electron transport chain. J. Biol. Chem. 270: 26104-26108 (1995)) as well as the parental cells (B1 cells) (Oostveen, F. G., Au, H. C., Meijer, P. J. & Scheffler, I. E. A Chinese hamster mutant cell line with a defect in the integral membrane protein CII-3 of complex II of the mitochondrial electron transport chain. J. Biol. Chem. 270: 26104-26108 (1995)) were grown in DMEM with 10% FCS, antibiotics, 10 mg/ml glucose and non-essential aminoacids.
(25) Synthesis of VE Analogs.
(26) The following compounds (most of which are shown in
(27) Assessment of IC.sub.50, Apoptosis and Mitochondrial Potential.
(28) Toxicity of -TOS, VE.sub.11S (VES), MitoVE.sub.3S, MitoVE.sub.5S, MitoVE.sub.7S, MitoVE.sub.9S, MitoVE.sub.11S (MitoVES), MitoVE.sub.11AE (MitoVEAE), MitoVE.sub.11F (MitoVEF), MitoVE.sub.11M (MitoVEM), and VES4TPP towards cancer cells was assessed on the basis of IC.sub.50, as detailed in Turanek, J., et al. ((2008). Liposomal formulation of vitamin E analogs as an efficient and selective anti-cancer treatment. Clin. Cancer Res. (submitted)). Apoptosis was assessed using the Annexin V method (Weber, T. et al. Mitochondria play a central role in apoptosis induced by -tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signaling. Biochemistry 42, 4277-4291 (2003)) and dissipation of the mitochondrial inner transmembrane potential was estimated using the polychromatic probe JC-1 (Molecular Probes) (Weber, T. et al. Mitochondria play a central role in apoptosis induced by -tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro-apoptotic signaling. Biochemistry 42, 4277-4291 (2003)).
(29) Evaluation of Cell Proliferation, and Cell Cycle Distribution.
(30) Cell proliferation was determined, using an ELISA colorimetric kit (Roche) to determine the number of cells in S phase of the cell cycle, based on DNA incorporation of 5-bromo-2-deoxyuridine (BrdUrd) using the manufacturer's protocol. For cell cycle analysis, cells were plated in 24-well plates so that they reached 50%, 70%, and 100% confluency after 24-h recuperation. Cells were then harvested and resuspended in buffer containing sodium citrate (1%), Triton X-100 (0.1%), RNase A (0.05 g/mL), and propidium iodide at 5 g/mL, incubated in the dark for 30 min at 4 C. and analyzed by flow cytometry.
(31) Assessment of SDH Activity.
(32) A time course for the reduction of the complex II substrate 2,6-dichlorophenol indophenol (DCIP) by the mitochondrial preparations was followed by measuring the absorbance at 600 nm in 1 cm cuvettes containing a 1 ml reaction volume (.sub.600=2110.sup.3 M.sup.1 cm.sup.1). The reaction components included NADH, 0.5 mM; succinate, 5 mM; KCN, 10 mM; DCIP, 50 M; phenazine methosulphate (PMS), 50 M. For each assay point, 0.5 mg sample protein was used and -TOS was added at either 100 or 300 M as indicated. The change in absorbance of DCPIP was measured using a spectrophotometer (UVIKON XL, Secomam) and replicate samples were assayed (n=3). When measuring the complex I (NADH dehydrogenase activity), PMS was omitted. For the control reactions without -TOS, the diluent DMSO was added so that its final concentration was <0.1% (v/v).
(33) ROS Accumulation Assessment.
(34) Cellular ROS were detected indirectly by flow cytometry and directly by electron paramagnetic resonance (EPR) spectroscopy, following treatment of cells with -TOS as indicated in the Legend to Figures. In some experiments, the cells were pre-treated for 1 h with 2 M mitochondrially targeted coenzyme Q (MitoQ) (Kelso, G. F., et al. Selective targeting of a redox-active ubiquinone to mitochondria within cells: antioxidant and antiapoptotic properties. J. Biol. Chem. 276, 4588-45896 (2001)) or co-incubated with superoxide dismutase (SOD; Sigma S4636; EC 1.15.1.1) at 750 units per ml. For indirect evaluation, cells were treated with -TOS and reacted with dihydrodichlorofluorescein diacetate (DCF; Molecular Probes) for 30 min, and scored by flow cytometry for cells with high fluorescence, which was evaluated on the bases on increase in mean fluorescence intensity. EPR spectroscopy analysis of ROS generation was based on the use of the radical trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Sigma). In brief, cells were plated in T25 flasks and allowed to reach 60-70% confluency (510.sup.6 cells per flask). Cells were washed, overlayed with the PSS medium (Thomas, S. R., Chen, K., & Keaney, J. F. Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway. J Biol. Chem. 277, 6017-6024 (2002)) and incubated with 50 M -TOS 5 min after addition of 10 mM DMPO. Analyses of DMPO adducts were performed with samples taken from the cell suspension as well as the cell-conditioned medium transferred into a quartz flat cell (Wilmad). The quartz cell was then placed into the cavity of the Bruker EMX bench-top spectrometer set at 293 K with the following spectrometer parameters: field sweep 10 mT, microwave power 20 mW, microwave frequency 100 kHz, modulation amplitude, 0.1 mT, sweep time 83.9 s. The detection limit of the stable nitroxide (TEMPO) under identical conditions was 50 nM.
(35) Cell Transfections.
(36) B9 cells were transfected using the Topo pCR3.1 Uni plasmid harbouring the CybL gene (Slane B G, Aykin-Burns N, Smith B J, Kalen A L, Goswami P C, Domann F E, Spitz D R. (2006). Mutation of succinate dehydrogenase subunit C results in increased oxidative stress, and genomic instability. Cancer Res 66: 7615-7620) and selected as described (Weber T, Dalen H, Andera L, Negre-Salvayre A, Auge N, Sticha M et al. (2003). Mitochondria play a central role in apoptosis induced by -tocopheryl succinate, an agent with anticancer activity. Biochemistry 42: 4277-4291). Stably transfected and siRNAtreated cells were assessed for SDH activity and SDHC expression. Western blotting was performed as described (Wang X F, Birringer M, Dong L F, Veprek P, Low P, Swettenham E et al. (2007). A peptide adduct of vitamin E succinate targets breast cancer cells with high erbB2 expression. Cancer Res 67: 3337-3344) using anti-SDHC immunoglobulin G (IgG) (clone 3E2; Novus Biologicals) with anti--actin IgG (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) as a loading control. RT-PCR was performed using a standard protocol. The published human CybL (Slane B G, Aykin-Burns N, Smith B J, Kalen A L, Goswami P C, Domann F E, Spitz D R. (2006). Mutation of succinate dehydrogenase subunit C results in increased oxidative stress, and genomic instability. Cancer Res 66: 7615-7620) and Chinese hamster glyceraldehyde 3-phosphate dehydrogenase primers (Sever et al., 2004) were used.
(37) Assessment of Angiogenesis In Vitro.
(38) To assess the effect of -TOS on the wound-healing activity of the EAhy926 cells, the cells were seeded in 3.5 mm Petri dishes and allowed to reach complete confluence. Using a sterile yellow pipette tip, the monolayer of the cells was wounded by removal of cells, generating a denuded area of 0.4-0.5 mm across. Regrowth of cells (wound healing) in the presence of -TOS or -TEA was assessed by following the kinetics of narrowing the denuded gap in the microscope equipped with a grid in the eyepiece and healing expressed as the rate of filling the gap as the rate of healing in m/h.
(39) For the tube-forming activity of EAhy926 cells, formation of capillary-like structures in a 3-dimensional setting was assessed, essentially as described elsewhere (Albini, A., et al Inhibition of angiogenesis and vascular tumor growth by interferon-producing cells: A gene therapy approach. Am. J. Pathol. 156, 1381-1393 (2000)). In brief, 300 l of cold Matrigel (BD Biosciences) was transferred using cold tips into each well in a 24-well plate. After solidification in the incubator, the surface of Matrigel was gently overlayed by a suspension of EAhy926 cells trypsinized from a proliferating culture, so that 200 l of the complete cell media including the HAT supplement with 510.sup.5 cells was added to each well. After 1-2 h in the incubator, the polygonal structures, made by a network of EAhy926 capillaries, established. The cells were treated by addition of MitoVES, added to the cell suspension just before it was transferred to the wells or after the tubes were established. Tube-forming activity was estimated by counting the number of complete capillaries interconnecting individual points of the polygonal structures in a selected field in a light microscope. Three fields in the central area of the well were chosen randomly in every well. The number of such capillaries in control cultures was considered 100%. For treated cultures, the number of complete capillaries was counted at various times after the onset of the experiment to obtain the kinetics of inhibition of tube-forming activity of EAhy926 cells.
(40) Transmission Electron Microscopy.
(41) Cultures to be subjected to transmission electron microscopy were prepared as previously described (Weber, T. et al. Mitochondria play a central role in apoptosis induced by -tocopheryl succinate, an agent with anticancer activity. Comparison with receptor-mediated pro -apoptotic signaling. Biochemistry 42, 4277-4291 (2003)). Briefly, cultures of Jurkat cells were fixed by adding 2% glutaraldehyde (Agar Scientic, Essex, UK) in 0.1 M sucrose-sodium cacodylate-HCl buffer (pH 7.2; Sigma, St Louis, Mo., USA) and post-fixed in osmium (Johnson Matthey Chemicals, Roystone, UK). Thereafter, cells were pelleted in 2% agar prior to dehydration, staining with uranyl acetate (Sigma), dehydration and embedding in Epon-812 (Fluka A G, Buchs, Switzerland). Thin sections of cured blocks were cut with a diamond knife (DIATOME, Bienne, Switzerland), stained with lead-citrate (Sigma), examined and photographed in a JEOL 1230-EX electron microscope (Tokyo, Japan) at 100 kV.
(42) Molecular ModelingComplex II and MitoVES.
(43) The crystal structure of mitochondrial respiratory membrane protein Complex II from porcine heart was obtained from the Brookhaven Protein Databank (code 1ZOY) (Sun F, Huo X, ZhaiY, Wang A, Xu J, Su D, Bartlam M, Rao Z. 2005. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121:1043-1057). The Complex contains four proteins. Three subunits in this Complex, the iron-sulfur protein (Chain B), the large (Chain C) and small (Chain D) trans-membrane proteins are involved in the binding to UbQ. A BLAST search from the NCBI website revealed that the sequence identity between porcine and human Complex II is very high, 97% for the iron-sulfur protein, 90% for the large trans-membrane protein and 94% for the small trans-membrane protein.
(44) The protein structure was prepared for docking using AutoDock Tools (Sanner M F (1999) Python: a programming language for software integration and development, J Mol Graphic Mod 17: 57-61) with the heteroatoms being removed first. Polar hydrogens were added to the structure and Kollman United Atom charges were used for the protein atoms. UbQ5 was built from the crystal structure coordinates of the bound UbQ (1ZOY) using InsightII (Accelrys, 2001). MitoVE.sub.11S was built from the crystal structure MOPHLB01 retrieved from the Cambridge Structural Database (Allen F H (2002) The Cambridge Structural Database: a quarter of a million crystal structures and rising. Acta Crystallogr B 58(Pt 3 Pt 1):380-388) by a sub-structure search for the ring system of -TOS, again using InsightIl. Both ligands were then prepared for docking by AutoDock Tools, which included merging non-polar hydrogens, assigning Gasteiger charges and defining the rotatable bonds.
(45) Docking was performed using the Lamarckian Genetic Algorithm as implemented in Autodock 3.0.5. (Morris G M, Goodsell D S, Halliday R S, Huey R, Hart W E, Belew R K, Olson A J (1998) Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. J Comp Chem 19: 1639-1662); two docking grids were prepared. Both were 126126126 points with a grid pacing of 0.375 , with the first centred on Tyr173 (Chain B) in the Q.sub.P site and the second centered on Trp134 (Chain D) in the Q.sub.D site. Default parameters were used except for the following, which were increased due to the relatively high number of rotatable bonds present in the ligands of interest (UbQ5=16, -TOS=17): ga_run=250, ga_pop_size=250, ga_num_evals=10,000,000. Also, the parameter rmstol was increased to 2.5, to produce more manageable clusters during the analysis phase of the calculation. Each docking calculation took just over 49 h using a 2 GHz G5 PowerPC Macintosh. Analysis of results was performed using scripts provided with AutoDock and docked structures were visualized using Astex Viewer (Hartshorn M J (2002) AstexViewer: An aid for structure-based drug design. J Computer Aided Mol Des 16: 871-881).
Example 1
MitoVES Selectively Kill Cancerous Cells
(46)
(47) IC.sub.50 values of -TOS, VES4TPP and MitoVE.sub.11S (labelled MitoVES) for different malignant and non-malignant cells are shown in Table V below.
(48) TABLE-US-00005 TABLE V Cell type.sup.a -TOS VES4TPP MitoVES Jurkat .sup.18 3.sup.b 21 5 0.48 0.1 MM-BI 26 4 n.d. 1.4 0.3 Meso-2 29 5 28 6 2.4 0.5 Ist-Mes 24 5 n.d. 2.2 0.3 Ist-Mes-2 21 3 n.d. 1.1 0.25 MCF-7 22 4 19 3 1.9 0.5 MDA-MB-453 28 5 n.d. 3.3 0.7 HCT-116 31 6 n.d. 2.8 0.8 AE-17 33 5 n.d. 3.1 0.7 H9c2 >100 >100 54 8 HL-1 >100 >100 48 6 Met5A 69 8 n.d. 21 4 EAhy926.sup.c >100 n.d. 32 6 9.5 2.7 n.d. 0.7 0.2 .sup.aJurkat cells were treated at 0.5 10.sup.6 per ml, other cell lines (except for EAhy926 cells) were treated at ~60% confluency. .sup.bThe IC.sub.50 values are derived from viability curves using the MTT viability assay and are expressed mol/l. .sup.cThe EAhy926 cells were treated at 100% confluency (top lines) or at ~50% confluency (bottom lines).
(49) IC.sub.50 values for various other MitoVES analogs and compounds are shown in Table VI below.
(50) TABLE-US-00006 TABLE VI Analog IC.sub.50 -TOH.sup.a >100.sup.b -TOS 18 3 VE3S ND VE5S ND VE7S ND VE11S 17.1 4.2 MitoVE3S ND MitoVE5S ND MitoVE7S ND MitoVE11S 0.48 0.1 VES4TPP 21 5 .sup.aJurkat cells were treated at 0.5 10.sup.6 per ml and exposed to the analogs shown, added as EtOH solution. .sup.bThe IC.sub.50 values are derived from viability curves using the MTT viability assay and are expressed in mol/l.
(51) The apoptosis assays of
(52) The micrographs of -TOS- and MitoVE.sub.11S-treated Jurkat cells in panel J reveal typical hallmark signs of apoptosis.
(53) Interestingly, the apoptosis assay of
(54) It can also be gleaned from the apoptosis assay of
(55) The results of Tables V and VI highlight the much greater potency of MitoVES in killing a range of cancer cell types, including lymphoma, mesothelioma, breast cancer and colon cancer, but not normal cells. In addition, the results suggest that chain length of the MitoVES is important probably for accessing down into the UbQ binding complex II site. The length of the carbon chain separating the TPP moiety from the TOS moiety is critical for activity and most likely reflects the ability of the TOS group to become inserted into the mitochondrial membrane deep enough to enter the UbQ sites on complex II.
Example 2
MitoVES Causes Apoptosis in Proliferating but not Arrested Endothelial Cells
(56) The results of
Example 3
MitoVES Inhibits Angiogenesis In Vitro
(57) The wound-healing, tube-forming and apoptosis assays of
(58) As with -TOS, MitoVE.sub.11S has a very potent anti-angiogenic activity in targeting and killing proliferating endothelial cells. However, MitoVE.sub.11S shows a surprising 5-fold greater potency as an antiangiogenic drug. For comparable levels of effects on the same endothelial cell types, about 5-10 micromolar MitoVE.sub.11S compared to about 25-50 micromolar -TOS is needed. (The -TOS level was taken from Lan-Feng Dong, Emma Swettenham, Johanna Eliasson, Xiu-Fang Wang, Mikhal Gold, Yasmine Medunic, Marina Stantic, Pauline Low, Lubomir Prochazka, Paul K. Witting, Jaroslav Turanek, Emmanuel T. Akporiaye, Stephen J. Ralph, and Jiri Neuzil, Vitamin E Analogues Inhibit Angiogenesis by Selective Induction of Apoptosis in Proliferating Endothelial Cells: The Role of Oxidative Stress, Cancer Res 2007; 67: (24). Dec. 15, 2007.)
Example 4
Complex II Genetically Deficient Mutant Cancer Cell Line is not Responsive to MitoVES
(59) Previous studies by the present inventors have shown that -TOS induces ROS by interfering with the UbQ sites in the respiratory chain of Complex II. This example shows that a Complex II genetically deficient mutant cancer cell line is not responsive to MitoVES, thus confirming that MitoVES (MitoVE.sub.11S) induces ROS by interfering with the UbQ sites in the respiratory chain of Complex II.
(60) The effects of MitoVE.sub.11S treatment of a parental (B1), Complex II defective (B9) and Complex I-defective mutant (B10) Chinese hamster lung fibroblast cell lines were compared. In addition, a v-Harvey Ras expressing plasmid vector was used to transform those cell lines to the state of malignancy. Thus, the B1, B9 and B10 cell lines were transformed by stable transfection with GFP-H-Ras using the pEGFP-C3-H-Ras plasmid (Baysal B E, Ferrell R E, Willett-Brozick J E, Lawrence E C, Myssiorek D, Bosch A, van der Mey A, Taschner P E, Rubinstein W S, Myers E N, Richard C W 3rd, Cornelisse C J, Devilee P, Devlin B. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science. 2000 Feb. 4; 287(5454):848-51). Complex II in the Ras-transformed B9 cell line was reconstituted by transfecting with human (h) CybL (Albayrak T, Scherhammer V, Schoenfeld N, Braziulis E, Mund T, Bauer M K, Scheffler I E, Grimm S. The tumor suppressor cybL, a component of the respiratory chain, mediates apoptosis induction. Mol Biol Cell. 2003 August; 14(8):3082-96) cloned into the pEFIRES-Puro plasmid. RT-PCR documented the absence of the Chinese Hamster SDHCmRNA expression in the B9 derived cell line and the presence of hSDHC protein in B9.sub.SDHC expressing transformant cells (see
(61) In
Example 5
MitoVES Targets the UbQ-Binding Pockets on Complex II
(62) To rationalize the results that MitoVES interacts with Complex II via the UbQ-binding sites, the present inventors undertook a molecular modeling study using AutoDock (Morris G M, Goodsell D S, Halliday R S, Huey R, Hart W E, Belew R K, Olson A J (1998) Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. J Comp Chem 19: 1639-1662). The crystal structure of porcine heart mitochondrial CII has been reported (Sun F, Huo X, ZhaiY, Wang A, Xu J, Su D, Bartlam M, Rao Z. 2005. Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121:1043-1057). It exhibits a high sequence identity with human CII (95-97% for the individual subunits), therefore the present inventors used this structure (1ZOY) and the related structure (1ZP0) with the inhibitor TTFA bound as the basis for modeling study. As shown in
(63) The predicted position of MitoVE.sub.11S is shown as the sticks and the translucent grey box is drawn to indicate approximately where the membrane bilayer would exist. The model identifies the position proposed for MitoVE.sub.11S binding and docking into the Qp ubiquinone site of Complex II with the triphenylphosphonium ion protruding out on the membrane surface sitting in the mitochondrial matrix near the base of the structure of the SDH enzyme head group outside.
Example 6
MitoVES is an Efficient Drug for Killing Breast Cancer Stem Cells
(64) A recent important discovery in cancer therapy has been that cancer stem cells exist which can be the source for repopulating a tumour. This also highlights the difficulty with treating cancers, because any treatment that kills the bulk of a tumour, but leaves the stem cells alive in the body will fail because the tumour will regrow (Lou and Dean, Oncogene 2007). Cancer stem cells are also highly significant targets because they are commonly resistant to therapy (O'Brien et al. 2008, Li et al., 2008) and are drug resistant, expressing the Multi Drug Resistance (MDR)/ABC transporter glycoproteins on their cell membranes, involved in preventing chemotherapeutic drugs from accumulating in the cancer stem cells. The cancer stem cells have also been found to be radiation resistant because they have greater DNA damage repair capacity (Neuzil et al., 2007 BBRC; Eyler and Ricj, 2008).
(65) Cancer stem cells have been enriched by selective methods including purification based on their propensity to exclude dyes such as Hoechst 33342 providing a side population of cells when gated by fluorescence activated cell sorting (Patrawala L et al 2005, Wu and Alman 2008). Another means for enriching for cancer stem cell populations involves the growth in culture of spheroids from tumour cells. This method has been shown to enrich for higher percentages of tumour initiating cells within such cultures (Grimshaw et al., 2008) with many of the properties of cancer stem cells, including radiation resistance (Phillips T M et al., 2007). Based on analyses of gene expression and protein marker expression on the cancer stem cell enriched populations, these cell types have become more characterised. Among the marker genes and proteins expressed by cancer stem cells are increased levels of the Notch/wnt/beta-catenin signalling pathway, ABC transporters, CD133 high, CD44 high and low levels of CD24 surface markers. These cells, based on their marker expression also correlate closely with the more basal tumour cell phenotypes isolated from aggressively lethal malignancies and analysed by gene expression profiling (for example, see Sorlie T et al., PNAS, 2001; review in Sotiriou and Pusztai, 2008).
(66) Given our understanding of the properties of cancer stem cells or tumour initiating cells, it becomes imperative that a drug is found that targets these cell types and kills them, thereby preventing tumour regrowth. Preferably, this drug is selective and does not significantly affect normal stem cells or other normal cell types. One such drug that has been described is parthenolide, a sesquiterpene lactone derived from the Feverfew plant, which has been found to selectively kill leukemic stem cells (Guzman M L et al., 2005). Another similar drug was the compound 4-benzyl, 2-methyl, 1,2,4-thiadiazolidine, 3,5 dione (TDZD-8, Guzman, M L et al, 2007). However, these compounds proved not to be very effective against other types of cancer such as solid tumours.
(67) The present inventors made the surprising discovery that MitoVES was a highly effective drug for killing populations of the human breast cancer cell line, MCF7, enriched for cancer stem cells by growth as mammospheres (MS) in culture. The results in
(68) The inventors also found that MitoVES was a highly effective anti-cancer drug in vivo. For this purpose, the transgenic FVB/N c-neu mice that form spontaneous ductal breast carcinomas due to high level of expression of the oncogene, HER2 were used as a cancer model. The inventors found that MitoVES treatment completely blocked progression of breast cancer tumours arising in these animals (
(69) The present inventors have shown that untargeted mitocans can be modified by addition of a cationic group that presumably anchors them in the luminal leaflet of the inner mitochondrial membrane, thus maximizing their activity. This is epitomized by mitochondrially targeted analogs of vitamin E that show a considerably higher apoptogenic effect against cancer cells including tumor-initiating cells, and anti-cancer activity compared to their untargeted counterparts, whilst maintaining their selectivity for cancer cells.
(70) In particular, the experimental examples indicate that:
(71) 1) MitoVES (MitoVE.sub.11S) is surprisingly much greater in potency than -TOS, up to 50 fold more active on cancer cells in killing them selectively than -TOS;
(72) 2) MitoVES will, as a result of 1), be much less toxic on normal cells with the improved specificity;
(73) 3) MitoVES is more specific in inducing the mitochondrial pathway for cell death than -TOS;
(74) 4) As with -TOS, MitoVES has a very potent anti-angiogenic activity in targeting and killing proliferating endothelial cells, but MitoVES (MitoVES) shows a surprising 5-fold greater potency as an antiangiogenic drug than -TOS; and
(75) 5) MitoVES is likely to have very broad application in cancer therapy, in view it killing mesothelioma, breast cancer, colon cancer, lymphoma cell lines and cancer stem cells.
(76) The foregoing embodiments are illustrative only of the principles of the invention, and various modifications and changes will readily occur to those skilled in the art. The invention is capable of being practiced and carried out in various ways and in other embodiments. It is also to be understood that the terminology employed herein is for the purpose of description and should not be regarded as limiting.
(77) The term comprise and variants of the term such as comprises or comprising are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.
(78) Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia or elsewhere.
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