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The apoptosis-triggering properties of vitamin E succinate (VES, RRR-alpha-tocopheryl succinate) for human LNCaP and PC-3 prostate carcinoma cells and normal PrEC human prostate epithelial cells were investigated. LNCaP and PC-3 cells were sensitive to VES-induced apoptosis, with 100% and 60% of cells undergoing apoptosis after three days of treatment with 10 micrograms of VES/ml, respectively. PrEC cells were resistant to VES-induced apoptosis. Treatment of prostate cells with agonistic anti-Fas antibody triggered apoptosis in approximately 50% of PC-3 cells within 48 hours, whereas LNCaP and PrEC cells were resistant. Prostate cells simultaneously treated with VES and agonistic anti-Fas antibodies revealed 1) no effect on PrEC cells, 2) an additive effect on Fas-sensitive PC-3 cells, and 3) a synergistic effect on LNCaP cells. VES treatment of LNCaP cells caused depletion of cytosolic 43-kDa Fas, enhanced membrane levels of 43-kDa Fas, and induced Fas sensitivity. PC-3 cells expressed high levels of membrane 43-kDa Fas that were enhanced by VES treatments. Fas ligand expression by LNCaP cells was enhanced by VES treatments. In summary, VES triggers apoptosis in human prostate carcinoma cells but not normal prostate cells in vitro, and VES modulates Fas signaling.

N-(4 hydroxyphenyl)retinamide (4-HPR), a synthetic derivative of all-trans-retinoic acid, induces DNA synthesis arrest and apoptosis in human breast cancer cells in a dose- and time-dependent manner. MDA-MB-435 cells treated with 3 microM 4-HPR exhibited 58% and 75% DNA synthesis arrest after 1 and 2 days of treatment and 31%, 39%, 48%, and 56% apoptosis after 3, 4, 5, and 6 days of treatment, respectively. Conditioned media from 4-HPR-treated MDA-MB-435 cells contained 63 and 57 pg of active transforming growth factor-beta (TGF-beta) per 10(6) cells after 1 and 2 days of treatment, whereas conditioned media from control cells contained only 9 pg/10(6) cells. TGF-beta involvement in 4-HPR-induced apoptosis, but not DNA synthesis arrest, in MDA-MB-435 cells was demonstrated by 1) blockage of 4-HPR-induced apoptosis by 66-75% after treatment of cells with neutralizing antibodies to TGF-beta s, 2) blockage of 4-HPR-induced apoptosis by 64-67% after transient transfection of cells with antisense oligomers to TGF-beta 1 or TGF-beta type II receptor, 3) blockage of 4-HPR-induced apoptosis by approximately 50% after inhibition of latent TGF-beta activation, and 4) demonstration that human breast cancer cells (T47D) defective in TGF-beta signaling were refractive to 4-HPR-induced apoptosis. These data indicate that 4-HPR is a potent activator of TGF-beta and that TGF-beta participates in 4-HPR-induced apoptosis of human breast cancer cells.

The apoptosis-inducing properties of RRR-alpha-, beta-, gamma-, and delta-tocopherols, alpha-, gamma-, and delta-tocotrienols, RRR-alpha-tocopheryl acetate (vitamin E acetate), and RRR-alpha-tocopheryl succinate (vitamin E succinate) were investigated in estrogen-responsive MCF7 and estrogen-nonresponsive MDA-MB-435 human breast cancer cell lines in culture. Apoptosis was characterized by two criteria: 1) morphology of 4,6-diamidino-2-phenylindole-stained cells and oligonucleosomal DNA laddering. Vitamin E succinate, a known inducer of apoptosis in several cell lines, including human breast cancer cells, served as a positive control. The estrogen-responsive MCF7 cells were more susceptible than the estrogen-nonresponsive MDA-MB-435 cells, with concentrations for half-maximal response for tocotrienols (alpha, gamma, and delta) and RRR-delta-tocopherol of 14, 15, 7, and 97 micrograms/ml, respectively. The tocotrienols (alpha, gamma, and delta) and RRR-delta-tocopherol induced MDA-MB-435 cells to undergo apoptosis, with concentrations for half-maximal response of 176, 28, 13, and 145 micrograms/ml, respectively. With the exception of RRR-delta-tocopherol, the tocopherols (alpha, beta, and gamma) and the acetate derivative of RRR-alpha-tocopherol (RRR-alpha-tocopheryl acetate) were ineffective in induction of apoptosis in both cell lines when tested within the range of their solubility, i.e., 10-200 micrograms/ml. In summary, these studies demonstrate that naturally occurring tocotrienols and RRR-delta-tocopherol are effective apoptotic inducers for human breast cancer cells.

MDA-MB-435 human breast cancer cells treated with 10 micrograms/ml of RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) for one, two, three, and four days exhibit 9%, 19%, 51%, and 73% apoptotic cells, respectively. Likewise, cells cultured for one, two, and three days with conditioned media (CM) obtained from MDA-MB-435 cells treated with VES exhibit 10%, 36%, and 74% apoptosis, respectively. A quantitative luciferase-based assay showed CM from VES-treated cells collected at 24 and 48 hours after treatment initiation to contain 75 and 32 pg of active transforming growth factor-beta (TGF-beta), respectively, per 10(6) cells. Although purified TGF-beta 1 is not an effective apoptotic agent for MDA-MD-435 cells, cotreatment of the cells for three days with suboptimal levels of VES (2.5 and 5 micrograms/ml) + 10 ng/ml of purified TGF-beta 1 enhanced apoptosis by 66% and 68%, respectively. Interference of the TGF-beta-signaling pathway by transient transfection of MDA-MB-435 cells with antisense oligomers to TGF-beta type II receptor (TGF-beta R-II) blocked VES-induced apoptosis. Likewise, addition of neutralizing antibodies to TGF-beta 1 or to all three mammalian isoforms of TGF-beta (TGF-beta 1, -beta 2, -beta 3) blocked VES- and CM-induced apoptosis. Furthermore, inhibitors of TGF-beta conversion from an inactive latent form to a biologically active form inhibited VES-induced apoptosis. In summary, the ability to reduce apoptosis by blocking TGF-beta or the TGF-beta receptor-signaling pathway with antisense oligomers or ligand-neutralizing antibodies or prevention of activation of TGF-beta indicates a role for TGF-beta signaling in VES-induced apoptosis.

RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

The RRR-alpha-tocopheryl succinate form of vitamin E [vitamin E succinate (VES)] inhibits the proliferation of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells in a dose-dependent manner, blocks the cells in the G2/M cell cycle phase, and induces the cells to undergo apoptosis. Apoptosis was documented by demonstrating changes that are characteristic of this type of cell death, including morphological analyses of chromatin condensation by 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) staining using scanning confocal and traditional fluorescent microscopy; flow cytometry analyses of propidium iodide-labeled DNA showing fragmented DNA as a pre-G1 peak; two-color flow cytometry analyses of intact cells labeled first by the TUNEL procedure (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end-labeled DNA stained with fluorescein isothiocyanate-labeled avidin) and then by propidium iodide demonstrating fragmented DNA; and electrophoresis of DNA showing a DNA ladder created by internucleosomal DNA fragmentation. The percentage of apoptotic cells was determined by DAPI staining and showed 11%, 27%, and 49% of cells to be apoptotic after treatment with 10 micrograms/ml VES for one, two, and three days, respectively. Analyses of mRNA levels of genes that have been implicated in the apoptotic process, namely, bcl-2, c-myc, and c-jun, revealed no change in bcl-2, decreases in c-myc mRNA levels after 36 hours of treatment, and increases in c-jun mRNA levels within four hours after treatment. Western immunoblotting analyses of protein levels for the transcription factors c-Myc and c-Jun showed normal levels of c-Myc at early time points and decreased levels at 24 and 48 hours after treatment. c-Jun increased as early as 6 hours after treatment and returned to lower (yet still elevated over control) levels by 48 hours. To determine possible functional consequences of increased c-Jun expression, gel electrophoretic mobility assays were conducted that showed increased AP-1 binding at 24 and 48 hours after treatment. These data show that VES induces apoptosis in reticuloendotheliosis virus-transformed lymphoid cells and suggest that decreases of c-Myc protein and increases of c-Jun protein and DNA binding capacity may be playing a role in VES-mediated events leading to apoptosis in this cell type.

RRR-alpha-tocopheryl succinate (VES) was studied for effects on murine EL-4 cell proliferation and production of interleukin-2 (IL-2) and transforming growth factor-beta (TGF-beta). VES was biphasic in its actions: 0.1 microgram/ml enhanced EL-4 cell proliferation, whereas 10-20 microgram/ml inhibited cellular proliferation. Cell-conditioned media (CM) from EL-4 cells treated with 0.2 ng/ml phorbol myristate acetate (PMA) + 0.1 microgram/ml VES contained increased amounts of IL-2, as determined by the murine cytotoxic T cell IL-2-dependent CTLL-2 bioassay. VES at 0.1 microgram/ml or 0.1 microgram/ml VES + 0.2 ng/ml PMA induced the expression of IL-2 mRNA by EL-4 cells three to nine hours after treatment. CM from EL-4 cells treated with VES at 10-20 microgram/ml exhibited potent antiproliferative activity when tested in the TGF-beta-responsive mink lung cell (Mv1Lu) bioassay and showed reduced inhibitory effects when tested on TGF-beta receptor-negative mink lung (DRA-27) cells. CM from control-treated EL-4 cells exhibited no antiproliferative activity. The VES-induced antiproliferative activity was characterized as TGF-beta by neutralization analyses and immunoprecipitation of metabolically labeled proteins with TGF-beta-specific reagents. VES treatment of EL-4 cells had no effect on TGF-beta 1 mRNA expression while downregulating TGF-beta 3 mRNA expression. In summary, these studies showed that 0.1 microgram/ml VES enhanced cellular proliferation, in part, via increased IL-2 production, whereas 10-20 micrograms/ml VES inhibited cellular proliferation, in part, via the secretion of biologically active TGF-beta.

The proliferation of MDA-MB-435 human breast cancer cells was inhibited by RRR-alpha-tocopheryl succinate (vitamin E succinate, VES). Conditioned media (CM) from VES growth-inhibited cells contained potent antiproliferative activity, part of which is contributed by transforming growth factor-beta (TGF-beta) isoforms. Antibody neutralization analysis, employing TGF-beta isoform-specific antibody reagents, showed that TGF-beta 1, -beta 2, and -beta 3 were present in the CM from VES-treated cells. Culturing MDA-MB-435 cells with VES did not alter the levels of constitutively expressed 2.4-kb TGF-beta 1, 3.0- and 4.0-kb TGF-beta 2, or 1.2- and 3.5-kb TGF-beta 3 mRNA transcripts. Inhibition of DNA synthesis by MDA-MB-435 cells was increased by combinations of suboptimal levels of VES and purified TGF-beta 1. VES-treated MDA-MB-435 cells exhibited enhanced binding of radiolabeled TGF-beta 1, and Western immunoblotting analyses showed that VES treatment enhanced TGF-beta type II receptor protein expression. TGF-beta type I receptor protein levels were not modified by VES treatments. Although the mRNA transcript for the 5.5-kb TGF-beta type II receptor was upregulated after four hours of treatment with VES, this treatment did not modify the 6.5-kb TGF-beta type I or the 6.5-kb TGF-beta type II receptor mRNAs. Results demonstrate that biologically active TGF-beta 1, -beta 2, -beta 3 and levels of TGF-beta type II receptor expressed by human breast cancer cells are enhanced by VES treatment.

The RRR-alpha-tocopheryl succinate form of vitamin E, referred to as vitamin E succinate (VES), inhibits the proliferation of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells in a dose-dependent manner in vitro. Analyses of conditioned medium (CM) from VES growth-inhibited cells revealed a potent antiproliferative activity. Characterization of the antiproliferative activity as transforming growth factor-beta (TGF-beta) was established by 1) growth inhibition of TGF-beta-responsive Mv1Lu mink lung and murine CTLL-2 cell lines, 2) a combination of physical characteristics including heat stability, acid stability, and Bio-Gel P-60 column chromatography elution profile, 3) neutralization of the antiproliferative activity by antibodies specific for TGF-beta, and 4) immunoprecipitation of metabolically labeled TGF-beta in CM from VES-treated C4-1 cells by use of TGF-beta-specific antibodies. Northern blot analyses of total cellular RNA revealed that VES does not alter the levels of constitutively expressed TGF-beta isoform-specific mRNAs; namely, VES does not alter the levels of the 3.9- and 4.1-kb TGF-beta 2 mRNAs, the 3.0-kb TGF-beta 3 mRNA, or the 2.5-, 2.7-, and 1.7-kb TGF-beta 4 mRNAs. The data show that VES inhibits C4-1 cell proliferation and induces the cells to produce and secrete active forms of TGF-beta, suggesting that one mechanism whereby VES inhibits C4-1 cell proliferation may be via the TGF-beta pathway for cellular growth control.

The RRR-alpha-tocopheryl succinate derivative of vitamin E, referred to as vitamin E succinate (VES), inhibits the proliferation of three metastatic human prostatic cancer cell lines, LNCaP, PC-3, and DU-145. LNCaP is a lymph node-derived androgen-sensitive prostate cell line; these cells are defective for response to transforming growth factor-beta (TGF-beta) but are normal for cell cycle-related tumor suppressor genes: p53 and retinoblastoma (Rb). PC-3 is a bone marrow-derived androgen-insensitive prostate cell line; these cells are defective for both p53 alleles but normal for both Rb alleles. DU-145 is a brain-derived androgen-insensitive prostate cell line; these cells are defective for both p53 and both Rb alleles. VES at 5, 10, and 20 micrograms/ml inhibited DNA synthesis in the three cell lines in a dose-dependent manner. Purified TGF-beta 1 at 1 ng/ml inhibited DNA synthesis of PC-3 cells within 24-72 hours and DU-145 cells at 72 hours but did not inhibit DNA synthesis of LNCaP cells. Previous studies in our laboratory showed that VES growth-inhibited tumor cells secrete biologically active antiproliferative factor TGF-beta s, suggesting that VES's mechanism of growth inhibition may involve the TGF-beta system of growth control.(ABSTRACT TRUNCATED AT 250 WORDS)

HL-60 cells, growing as single cells in suspension, exhibit marked cell-cell adhesion when treated for 24 hours with 10 micrograms/ml RRR-alpha-tocopheryl succinate, also called vitamin E succinate (VES). VES-induced cell-cell adhesion is dependent on divalent cations and a functional cytoskeleton and is protein mediated. Cell adhesion molecules CD11a/CD18, CD11b/CD18, CD29, and CD54 do not appear to be mediating VES-induced cell adhesion. HL-60 cells treated with VES adhere to fibronectin-coated plastic and secrete elevated levels of fibronectin. A 72-kDa fibronectin-binding membrane molecule was detected on VES-treated HL-60 cells, and antibodies to fibronectin were shown to inhibit VES-induced cell aggregation. VES induction of HL-60 cell-cell adhesion is proposed to result from increased amounts of extracellular fibronectin binding to VES-induced cell surface fibronectin-binding molecules.

Vitamin E is thought to enhance immunity by increasing interleukin-1 (IL-1) production and by downregulating prostaglandin E2 (PGE2) synthesis. In an effort to understand the mechanism(s) whereby the form of vitamin E known as RRR-alpha-tocopheryl succinate [also called vitamin E succinate (VES)] ameliorates retrovirus-induced immune dysfunctions, peritoneal exudate cells (PECs) derived from normal chickens and avian and murine macrophage cell lines were used as in vitro model systems to test the effects of VES treatments on PGE2 and IL-1 production. Supernatants from PECs that were exposed to avian erythroblastosis virus (AEV) for 45 minutes exhibited a 256% increase in PGE2 levels compared with supernatants from replica cultures of PECs not exposed to AEV. Pretreatment of PECs with VES before exposure to AEV maintained PGE2 levels at normal control levels. VES treatment enhanced IL-1 production by avian (HD11) and murine (P388D1) macrophage cells, respectively. Supernatants from VES-treated HD11- and P388D1-stimulated cells contained IL-1 activity 196% and 385%, respectively, greater than that observed with supernatants from untreated control cells. On the basis of these studies, downregulation of retrovirus-induced PGE2 production and/or upregulation of IL-1 production by VES are potential mechanisms for VES amelioration of retrovirus-induced immune suppression.

The effect of RRR-alpha-tocopheryl succinate (VES) on lectin-induced chicken T cell proliferation was investigated. The T cell mitogens concanavalin A and phytohemagglutinin induce chicken thymic and splenic T cell proliferation. Addition of VES to the in vitro cultures inhibited T cell proliferation in a dose-dependent manner. Addition of VES to spleen cell cultures at different times after mitogen stimulation also suppressed T cell mitogenesis, suggesting that VES is not mediating its antiproliferative effects by interfering with ligand (mitogen)-receptor binding or early ligand-bound receptor-signaling events. Three lines of evidence suggest that the growth-inhibitory properties of VES are unique and may not involve antioxidant properties. 1) Three other forms of vitamin E, dl-alpha-tocopherol, d-alpha-tocopherol, and d-alpha-tocopherol acetate, do not inhibit the proliferation of mitogen-stimulated chicken spleen cells. 2) Spleen cells were treated with an inhibitor of nonspecific esterases to prevent the conversion of VES, which does not exhibit antioxidant properties to d-alpha-tocopherol, a lipid-soluble antioxidant. Treatment of spleen cells with the inhibitor did not affect VES's growth-inhibitory properties. 3) Trolox, a water-soluble vitamin E analogue with potent antioxidant properties and two lipid-soluble antioxidants, butylated hydroxyanisole and butylated hydroxytoluene, did not inhibit mitogen-induced T cell proliferation. Attempts to reverse VES's antiproliferative effects by addition of exogenous interleukin-2 or addition of sodium selenite, an enhancer of interleukin-2 receptors, failed. Acetylsalicylic acid had no effect on VES's inhibition of mitogen-activated T cell proliferation. These studies support the role of VES as a growth inhibitor of lectin-activated normal T cells in chickens.

The RRR-alpha-tocopheryl succinate form of vitamin E inhibits the proliferation of estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines in a dose-dependent manner in vitro. Analyses of cell-conditioned medium from RRR-alpha-tocopheryl succinate growth-inhibited cells revealed the presence of a potent antiproliferative activity. Characterization of the antiproliferative activity as transforming growth factor-beta (TGF-beta) was established by 1) growth inhibition of the TGF-beta-responsive Mv1Lu-CCL-64 mink lung and murine CTLL-2 cell lines, 2) combination of physical characteristics including heat stability, acid stability, and Bio-Gel P-60 column chromatography elution profile, and 3) neutralization of the antiproliferative activity in the conditioned media by antibodies specific for TGF-beta.

HL-60 human promyelocytic leukemia cells can be induced to differentiate to granulocytes by retinoic acid and dimethyl sulfoxide or monocyte-macrophages by phorbol esters and 1,25-dihydroxyvitamin D3. These studies show that RRR-alpha-tocopheryl succinate (TS) inhibits HL-60 cell proliferation and induces the HL-60 cells to differentiate toward a functionally deficient macrophage-like cell. TS at (15 micrograms/ml) was found to suppress HL-60 cell proliferation by 63% and 89% at 24 and 48 hours, respectively. This suppression of proliferation, however, is not permanent and requires the presence of TS. HL-60 cells treated for 48 hours with TS (15 micrograms/ml) were found to be blocked in the G2/M phase of the cell cycle. HL-60 cells blocked in the G2/M cell cycle phase by TS expressed normal levels of the transferrin receptor. TS-treated HL-60 cells exhibited binucleated morphological appearance; however, the cells did not exhibit chemotaxis, phagocytosis, or changes in the expression of the cell surface markers, CD11a and CD18. However, HL-60 cells treated for 48 hours with TS (15 micrograms/ml) could be stimulated to produce superoxide radicals and exhibited nonspecific esterase activity, two characteristics of macrophages. These results suggest a role for TS as an antitumor proliferative agent and as a modifier of human leukemia cell differentiation.

RRR-alpha-tocopheryl succinate was demonstrated to be a potent in vitro modulator of retrovirus-induced immune abnormalities. Spleen cells from avian erythroblastosis virus (AEV)-infected chickens exhibit suppressed T cell mitogen-induced proliferative responses and elevated levels of suppressor T cell activity. In vitro addition of RRR-alpha-tocopheryl succinate resulted in amelioration of these abnormalities. Antioxidants including Trolox (a water-soluble analogue of RRR-alpha-tocopherol with antioxidant properties) and a combination of butylated hydroxyanisole and butylated hydroxytoluene were able to restore immune functions to levels similar to those achieved with RRR-alpha-tocopheryl succinate treatment. Aspirin, an irreversible inhibitor of cyclooxygenase activity, was capable of ameliorating some of the AEV-induced immune dysfunctions. These studies suggest a role for the antioxidant functions of RRR-alpha-tocopheryl succinate in modulation of retrovirus-induced immune abnormalities.

Vitamin E succinate inhibited proliferation of C4#1 cells, an established avian retrovirus [reticuloendotheliosis virus (REV)]-transformed immature lymphoid tumor cell line, in a dose-dependent manner. The cytostatic effects of vitamin E succinate were reversible in that treated cells regained their ability to divide after vitamin E succinate removal. Possible mechanism(s) for the antiproliferative actions of vitamin E succinate were investigated. Analyses of C4#1 cell surface membrane antigen profiles and morphology indicated that vitamin E succinate was not inducing differentiation of the tumor cells to a more mature, differentiated, nonproliferative state. Five antioxidants, including a synthetic analogue of vitamin E, Trolox, as well as the active vitamin form, DL-alpha-tocopherol, were incapable of inhibiting C4#1 tumor cell growth, indicating that a mechanism of action other than or in addition to functions as an antioxidant may be operating. Cell cycle analyses suggested that C4#1 tumor cells treated with vitamin E succinate were blocked in the G0G1/early S phases of the cell cycle. Tumor growth arrested by vitamin E succinate did not affect the expression of the REV-encoded oncogene, v-rel, at either the RNA or protein level. These studies demonstrated that vitamin E, in the form of vitamin E succinate, inhibited the growth of retrovirus-transformed tumor cells in vitro and suggested that the antiproliferative effects of vitamin E succinate did not involve antioxidant properties but rather, as yet, unidentified mechanisms leading to cell cycle blockage.