Enhancement of Nitric Oxide and Superoxide Generations by α-Tocopheryl Succinate and Its Apoptotic and Anticancer Effects

Tocopheryl succinate (TS), a succinyl ester of alpha-tocopherol (alpha-T), has been reported to have various biological activities. In this communication, we review the current findings about TS including our recent studies of its effects on nitric oxide (NO) and superoxide (O2-) generations implicated in cancer and atherosclerosis. First, we investigated the effect of TS on NO production in vascular smooth muscle cells (VSMC) under atherosclerosis-like conditions using lipopolysaccharide (LPS) and interferon-gamma (IFN). TS enhanced LPS/IFN-dependent NO production, but alpha-T itself did not. The enhancement by TS of NO production was inhibited by alpha-T but not by antioxidants such as ascorbic acid and 2[3]-t-butyl-4-hydroxyanisole (BHA). TS enhanced the amount of protein kinase Calpha (PKCalpha) in VSMC, and PKC inhibitors inhibited TS-enhanced NO production, suggesting that the enhancing effect of TS on NO production is caused by up-regulation of PKC. Second, we found that TS induced apoptosis in VSMC associated with increase in O2- generation via NADPH-dependent oxidase. We further observed that a mouse breast cancer cell line C127I was more susceptible for TS-induced apoptosis than a mouse breast normal cell line NmuMG, and that superoxide dismutase, alpha-T, and BHA inhibited TS-caused morphological cell damage in C127I. From these results, O2- itself and/or other reactive oxygen species are assumed to associate with TS-induced cell toxicity, and antioxidative defense systems are supposed to be lowered in cancer cells. Finally, we found that intravenous injection of TS vesicles completely inhibited the growth of melanoma cells B16-F1 inoculated on the back of hairless mice and enhanced their survival time.     

Structural characteristic of terminal dicarboxylic moiety required for apoptogenic activity of alpha-tocopheryl esters

alpha-Tocopheryl succinate (TS) is known to induce apoptosis in various cells and has attracted attention as a chemotherapeutic agent. Recently, we reported the structural significance of the terminal dicarboxylic moiety for the action of TS [J. Nutr. Sci. Vitaminol. 49 (2003) 310-314]. In this study, to determine details of the relationship between the structure and the function of the terminal ester moiety of alpha-tocopherol (alpha-T), we synthesized four novel esters, alpha-tocopheryl oxalate (TO), alpha-tocopheryl malonate (TM), alpha-tocopheryl pimelate (TP) and alpha-tocopheryl succinate ethyl ester (TSE), and compared their apoptogenic activities with those of TS, alpha-T, gamma-tocopherol (gamma-T) and two commercially available alpha-T derivatives, alpha-tocopheryl nicotinate (TN) and alpha-tocopheryl acetate (TA), in vascular smooth muscle cells and a mouse breast cancer cell line C127I. TO and TM in addition to TS, but not the others, induced apoptosis in both cells. Particularly, TO was the most potent of all alpha-T derivatives used. The addition of exogenous superoxide dismutase (SOD) significantly prevented the apoptosis induced by TM as well as that by TS as reported previously, but did not affect TO-induced apoptosis. These results suggest that O(2)(-) generated exogenously participates in TM-induced apoptosis but not in TO-induced apoptosis. The difference in their apoptotic effects is attributed to structural properties of the terminal dicarboxylic moiety, which has an inflexible plane conformation in TO, while it is highly flexible in TM and TS.     

Differential regulation of microglial NO production by protein kinase C inhibitors

Nitric oxide (NO) produced by microglia has been implicated in the pathogenesis of various central nervous system diseases; however, the intracellular signal pathways for the production of NO are not well known. Protein kinase C (PKC) plays a key role in a variety of signal transduction processes. To elucidate how PKC regulates microglial NO production, we examined the effects of PKC inhibitors on lipopolysaccharide (LPS)-stimulated NO production by primary cultured rat microglia. Staurosporine, a non-selective PKC inhibitor, increased LPS-induced production of NO at 0.1-10 nM range of concentration. Protein kinase A (PKA) inhibitor, H89, did not affect LPS-induced NO production, suggesting that staurosporine effect is not mediated by inhibition of PKA. However, other two PKC inhibitors, whose specificities for PKC isoforms were different, G?6976 and Ro-32-0432, exhibited different effects on NO production from staurosporine; the former inhibited and the latter showed no effect. Interestingly, an activator of PKC, phorbol 12-myristate 13-acetate (PMA) also increased LPS-induced production of NO at 1-10 nM range of concentration, suggesting that prolonged incubation with PMA caused down-regulation of PKC. These results indicate that the inhibition or down-regulation of some PKC isoforms causes the enhancement of NO production. The different effects of PKC inhibitors on the NO production suggest that the different PKC isoforms play different roles in regulation of NO production in microglia.     

Nitric oxide production in murine spleen cells: role of interferons and prostaglandin E(2) in the generation of cytotoxic activity

The production of nitric oxide (NO) was measured in cultures of spleen cells stimulated by lipopolysaccharide (LPS), IL-2 or LPS + IL-2. We observed that NO synthesis is increased by IFN-gamma but inhibited by IFN-alpha/beta. This is not the case when IL-2 is present in the cultures, since interferons play a minor role in the regulation of the NO production. When IL-2 and LPS were associated in the cultures, the IFN-alpha/beta role seems more important than that of IFN-gamma. PGE(2) inhibits NO production in LPS supplemented cultures but has a slight effect in the presence of IL-2 and no effect with IL-2 + LPS. 3-isoButyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, induces a decrease of IFN production. In the presence of H-7, an inhibitor of protein kinase C (PKC), NO production is reduced when the cultures are supplemented by LPS or IL-2 but not when IL-2 and LPS are both added. H-7 also reduced IFN production. In the presence of N(G)-monomethyl-L-arginine (N-MMA), an inhibitor of NO synthesis, IFN production was increased, with no change in the cytotoxic activity. Hence, interferons regulate NO production by mouse spleen cells and, in return, NO modulates the generation of IFN.     

Role of protein kinase C in induction of gene expression and inhibition of cell proliferation by interferon alpha.

Recent studies have suggested that protein kinase C (PKC) may be involved in the mechanism of signal transduction by which members of the interferon (IFN) family regulate gene expression and cell phenotype. We have investigated the role of PKC in the control of cell growth and gene expression by IFN alpha in Daudi cells. Treatment of these cells with two analogues of staurosporine, which are potent inhibitors of PKC, completely blocked the induction by IFN alpha of the mRNA for 2',5'-oligoadenylate synthetase and the 6-16 gene. These compounds also inhibited cell proliferation and thymidine incorporation in this system. In contrast, the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) did not significantly inhibit the induction of these genes by IFN alpha and had no effect on Daudi cell growth or thymidine incorporation in the presence or absence of IFN alpha. No effect of IFN alpha on total PKC activity could be observed, and there were no significant changes in the overall levels of individual PKC isoforms or their mRNA following IFN alpha treatment. In contrast, treatment of Daudi cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, which also inhibits cell proliferation, strongly down-regulated PKC. These data suggest that the activity of a PKC species, or a closely related enzyme, may be required both for continued cell proliferation and the response to IFN alpha in Daudi cells, but that IFN-induced growth inhibition does not involve overall down-regulation or change in activity of PKC.     

Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by alpha-tocopheryl hemisuccinate

We investigated the mechanism of cell toxicity of alpha-tocopheryl hemisuccinate (TS). TS concentration- and time-dependently induced the lactate dehydrogenase release and DNA fragmentation of rat vascular smooth muscle cells (VSMC). Exogenous addition of superoxide dismutase, but not catalase, significantly inhibited the cell toxicity of TS. The NADPH-dependent oxidase activity of VSMC was stimulated by TS treatment. The cell toxicity of TS was inhibited by NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. Consequently, TS-induced apoptosis of VSMC was suggested to be caused by exogenous O(2)(-) generated via the oxidase system activated with TS.     

TNF-α、IL-1β及LPS对血管平滑肌细胞一氧化氮合酶基因表达的影响及作用机制研究

通过ＲＮＡ印迹分析和亚硝酸盐含量测定检查ＴＮＦ－α、ＩＬ－１β和ＬＰＳ对大鼠血管平滑肌细胞（ＶＳＭＣ）诱导型一氧化氮合酶（ｉＮＯＳ）基因表达及ＮＯ生成的影响．结果表明,ＴＮＦ－α、ＩＬ－１β和ＬＰＳ均能显著诱导ＶＳＭＣｉＮＯＳ基因表达和促进ＮＯ生成,其作用强度与浓度和作用时间有关;双因素（ＴＮＦ－α＋ＬＰＳ,ＬＰＳ＋ＩＬ－１β）对诱导ｉＮＯＳ基因表达及ＮＯ生成产生协同作用．ＰｏｌｙｍｙｘｉｎＢ和地塞米松可部分抑制ＴＮＦ－α对ｉＮＯＳ基因表达的诱导作用及ＮＯ生成     

Interferon-gamma inhibits proliferation of rat vascular smooth muscle cells by nitric oxide generation.

Interferon (IFN)-gamma inhibited the proliferation of rat vascular smooth muscle cells (VSMC) and increased the cyclic GMP (cGMP) concentration in the cells. The dose dependencies of the two effects were similar (IC50 = 4 U/ml for the anti-proliferation and EC50 = 3 U/ml for cGMP formation) and the effect of IFN-gamma was enhanced by tumor necrosis factor-alpha treatment. Furthermore, NG-nitro-L-arginine, a nitric oxide (NO) synthase inhibitor, inhibited both activities induced by IFN-gamma. These findings show that the anti-proliferation and cGMP formation are closely related and that IFN-gamma inhibits the proliferation of rat VSMC by generation of NO through the induction of an NO synthase.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Studies with protein kinase C inhibitors presently available cannot elucidate the role of protein kinase C in the activation of NADPH oxidase

The effects of various protein kinase C (PKC) inhibitors on NADPH oxidase (NO) activation by the phorbol ester PMA and by the chemotactic peptide FMLP were studied. H-7 reduced the effects of both stimuli in human neutrophils (HN) and HL-60 cells by 13-63%. Polymyxin B did not inhibit NO activation by PMA and FMLP in HN and reduced the effects of both stimuli in HL-60 cells by 27-55%. Retinal and retinoic acid enhanced the effects of PMA and FMLP in HL-60 cells and of FMLP in HN up to 4.5-fold. In contrast, retinoic acid inhibited the effect of PMA in HN. In the presence of cytochalasin B, retinal inhibited the effect of FMLP in HN, whereas retinoic acid inhibited NO activation by FMLP in both cell types. The dual PKC/calmodulin inhibitors trifluoperazine and W-7 abolished NO activation by PMA and FMLP in HN and HL-60 cells. Thus, the effects of PKC inhibitors on NO activation exhibit (1) cell type specificity, (2) stimulus dependency and (3) no correlation with in vitro inhibition of PKC. Our results suggest that studies with PKC inhibitors presently available cannot clarify the role of PKC in NO activation.     

Inhibitory effect of vitamin E on proinflammatory cytokines-and endotoxin-induced nitric oxide release in alveolar macrophages

Nitric oxide is thought to be an important modulator of various functions in normal and inflamed airways. In the present study, we evaluated the effects of high vitamin E (250 mg and 1250 mg alpha-tocopheryl acetate (TA)/kg diet/10 days) on nitric oxide (NO(.)) release by alveolar macrophages (AMs) in response to lipopolysaccharide (LPS), interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF-alpha). LPS and IL-1beta treatment (1-10 microg/ml) enhanced NO(.) release in AMs from control animals fed on 50 mg vitamin E/kg diet in a concentration dependent manner. However, this enhancement of NO(.) was attenuated in the AMs of animals fed with 250 mg or 1250 mg vitamin E/kg diet. TNF-alpha had no effect in eliciting the release of NO(.) in AMs obtained either from control or from hyper vitamin E fed animals. Further, LPS (1-10 microg/ml) enhanced the inducible nitric oxide synthase (iNOS) activity of AMs of control group and TA-fed animals almost to equal extent. Similarly, LPS-induced formation of N-nitrosamine (N-nitroso-L-[(14)C]-proline) in AMs of control and TA-supplemented animals were not different statistically. On the other hand, in vitro addition of vitamin E (200 microM) in AMs of control animals, when triggered with 10 microg LPS/ml, caused a significant decrease in N-nitroso-L-[(14)C]-proline formation. It seems that high doses of TA in diet may play a role in reducing the lipopolysaccharide and proinflammatory cytokines-induced NO(.)-mediated damage by AMs.     

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.     

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, these data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways. J. Cell. Physiol. 172:69–78, 1997. © 1997 Wiley-Liss, Inc.     

2-aminopurine inhibits lipopolysaccharide-induced nitric oxide production by preventing IFN-beta production

2-aminopurine (2-AP) is widely used as a specific inhibitor for double stranded-RNA dependent protein kinase (PKR). Here we report that 2-AP can inhibit lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production through the prevention of interferon (IFN)-beta production. 2-AP significantly inhibited NO production in LPS-stimulated RAW 264 murine macrophage cells. 2-AP also reduced the expression of IFN-beta and IFN-inducible genes, such as IFN-gamma-inducible protein (IP)-10 and immune-responsive gene (IRG)-1, and the inducible type of NO synthase (iNOS) mRNA in response to LPS. The addition of exogenous IFN-beta restored 2-AP-inhibited NO production in response to LPS. On the other hand, there was only partial inhibition by 2-AP of nuclear factor (NF)-kappaB activation, IL-6 mRNA expression and tumor necrosis factor (TNF)-alpha production. These results suggested that 2-AP inhibited LPS-induced IFN-beta production by preventing Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta (TRIF)-dependent signaling rather than myeloid differentiation factor (MyD) 88-dependent signaling, resulting in the inhibition of NO production.     

The direct action of estrone on vascular tissue involves genomic and non-genomic actions

A two step model mechanism of steroid action has been recently postulated. In this study, we test the hypothesis that, the biochemical action of estrone (E(1)) on vascular tissue could be performed via genomic and non-genomic actions. Rat aortic rings or vascular smooth muscle cell cultures (VSMC) were used to test the effect of the hormone on nitric oxide (NO) production, protein kinases activities and cell proliferation. Our data showed that estrone increased NO synthesis between 30 s and 20 min treatment, and this stimulatory effect was dependent on MAPK cascade activation, since it was prevented in the presence of a MAPK inhibitor (PD98059). Using a phosphorylation assay, we also showed that E(1) significantly increased MAPK activity. The effect of the hormone on PKC activity was measured in concentrations and time course studies. Direct treatment of rat aortic homogenates with E(1) significantly enhanced PKC activity (1-10 fold increase, p<0.01) at all concentrations (1; 10; 50 nM) and time tested (1-10 min). We demonstrated that 24 h of E(1) treatment markedly increased VSMC proliferation (53% above control), and this effect was suppressed by a PKC inhibitor. The rapid and the long term effects of the hormone were completely suppressed in the presence of an estradiol receptor antagonist (ICI 182780). In summary, we provided evidence that, the steroid exerts both non-genomic and genomic actions, the former associated with MAPK kinase dependent on NO production, and the latter related with induction of VSMC proliferation involving PKC pathway activation.     

Sentulic acid isolated from Sandoricum koetjape Merr attenuates lipopolysaccharide and interferon gamma co-stimulated nitric oxide production in murine macrophage RAW264 cells

A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.