Effects of garcinol and its derivatives on intestinal cell growth: Inhibitory effects and autoxidation-dependent growth-stimulatory effects

Garcinol, a polyisoprenylated benzophenone, from the Garcinia indica fruit rind, has been suggested to be an anti-inflammatory and anti-cancer agent. To explore the possible use of this redox-sensitive compound as a colon cancer preventive agent, we investigated the effects of garcinol and its oxidative derivatives, cambogin, garcim-1, and garcim-2, on the growth of HT-29 and HCT-116 colon cancer cells, as well as IEC-6 and INT-407 normal immortalized intestinal cells. Garcinol and its derivatives showed potent growth-inhibitory effects on all intestinal cells, showing IC50 of 3.2-21.4 microM after a 3-day treatment. Garcim-1 exhibited the strongest effect with IC50 of 3.2-5.9 microM. Garcinol was more effective in inhibiting growth of cancer cells than that of normal immortalized cells. Flow-cytometric analysis showed increased sub-G1 cells by treatment with garcinol and cambogin. Induction of apoptosis by garcinol and cambogin (2-10 microM) was also observed based on caspase-3 activation and enhanced annexin V staining. The inhibitory effect of garcinol on cell growth was much more pronounced in the absence of fetal bovine serum (FBS), decreasing IC50 to 1.5 from 11.8 microM in 72-h incubations and to 3 from 38 microM in 24-h incubations, possibly due to the binding of garcinol to FBS, which markedly reduced cellular levels of garcinol. Under these conditions, redox reactions seem not to be involved in the inhibition. In contrast to the inhibitory effect, low concentrations (<1 microM) of garcinol and cambogin stimulated the growth of both normal and cancer cells by 10-100%, and the activity seemed to be mediated by reactive oxygen species. In the presence of superoxide dismutase/catalase or N-acetyl cysteine, low concentrations of garcinol (<1 microM) decreased cell growth. Garcinol (0.5-1 microM) also increased the phosphorylation of extracellular signal-related kinase 1/2 and AKT and the level of survivin, and the effects were abolished in the presence of superoxide dismutase/catalase. Our results indicate that garcinol and its derivatives can inhibit intestinal cell growth, but low concentrations of garcinol can stimulate cell growth. It remains to be determined whether the currently observed stimulatory and inhibitory effects of garcinol on colon cell growth occur in vivo.     

Minocycline inhibits apoptotic cell death via attenuation of TNF-alpha expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co-cultures

We attempted to ascertain the neuroprotective effects and mechanisms of minocycline in inflammatory-mediated neurotoxicity using primary neuron/glia co-cultures treated with lipopolysaccharide (LPS). Neuronal cell death was induced by treatment with LPS for 48 h, and the cell damage was assessed using lactate dehydrogenase (LDH) assays and by counting microtubule-associated protein-2 (MAP-2) positive cells. Through terminal transferase deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-staining and by measuring caspase-3 activity, we found that LPS-induced neuronal cell death was mediated by apoptosis. We determined that pre-treatment with minocycline significantly inhibited LPS-induced neuronal cell death. In addition, LPS induced inducible nitric oxide synthase (iNOS) expression significantly, resulting in nitric oxide (NO) production within glial cells, but not in neurons. Both nitric oxide synthase (NOS) inhibitors (N(G)-monomethyl-L-arginine monoacetate (L-NMMA) and S-methylisothiourea sulfate (SMT)) and minocycline inhibited iNOS expression and NO release, and increased neuronal survival in neuron/glia co-cultures. Pre-treatment with minocycline significantly inhibited the rapid and extensive production of tumor necrosis factor-alpha (TNF-alpha) mediated by LPS in glial cells. We also determined that the signaling cascade of LPS-mediated iNOS induction and NO production was mediated by TNF-alpha by using neutralizing antibodies to TNF-alpha. Consequently, our results show that the neuroprotective effect of minocycline is associated with inhibition of iNOS induction and NO production in glial cells, which is mediated by the LPS-induced production of TNF-alpha.     

Oltipraz inhibits inducible nitric oxide synthase in vitro and inhibits nitric oxide production in activated microglial cells

The clinically relevant drug oltipraz (OPZ) has previously been shown to inhibit cytochrome P450 enzymes [Chem. Res. Toxicol. 13 (2000) 245]. The current study reveals that OPZ is also able to inhibit *NO formation by purified inducible nitric oxide synthase (iNOS) but not by neuronal nitric oxide synthase in hemoglobin assays. The inhibition of iNOS by OPZ is reversible and competitive with an IC(50) of 5.9 microM and Ki of 0.6 microM. In murine BV-2 microglial cells, an immortalized cell line that produces *NO in response to lipopolysaccharide (LPS), OPZ is able to block the formation of nitrite in LPS treated cells. The inhibitory effect of OPZ on LPS treated cells is not due to cell toxicity. Finally, treatment of cells with OPZ does not induce or suppress expression of iNOS protein as shown by Western blot analysis.     

Paradoxical effects of two oximes on nitric oxide production by purified NO synthases, in cell culture and in animals.

We have studied the impact of two novel compounds TO-85 (2,6-di-(alpha-aziridino-alpha-hydroxyiminomethyl)pyridine and TO-133 (bis-(diaziridinoglyoximato)copper), designed as NO donors, on nitrite production by cell cultures, NO production in rat tissues and their ability to inhibit purified NO synthases (NOS). Both substances induced considerable increase of nitrite production in cell cultures. When NO production was assayed in rat organs by means of ESR using Fe(DETC) as a spin trap the anticipated NO-increasing activity of TO-85 was observed only in kidneys; the NO level increasing almost 10-fold. Treatment of rats with TO-133, decreased the NO concentration in brain cortex, cerebellum and liver. When the drugs were administered to animals with high level of iNOS expression induced by LPS, TO-85 did not significantly modify the LPS-induced NO production; administration of TO-133 caused a significant decrease of NO production in blood, brain cortex and cerebellum. Only high concentrations of TO-85 were capable of inhibiting iNOS (IC50=7 mM), the substance inhibited eNOS at lower concentrations (IC50=250 microM). Inhibitory activities of TO-85 on nNOS were dependent on BH4 concentrations, suggesting eventual competition of TO-85 with BH4 when the substance interacts with nNOS. TO-133 reduced eNOS activity with IC50=200 microM, nNOS activity with IC50=200 microM, iNOS activity was not much affected by this substance. Thus, the two tested compounds manifest opposite effects on NO production by purified enzymes and in cell culture. The pattern of the NO synthesis modification in a living animal appears to be even more complex. Our results stress the importance of direct measurements of NO in the tissues using the ESR method.     

Inducible nitric oxide synthase inhibitors of Chinese herbs. Part 2: naturally occurring furanocoumarins

Inducible nitric oxide synthase (iNOS)-dependent production of nitric oxide (NO) plays an important role in inflammation. The effects of various naturally occurring furanocoumarins on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells were evaluated in vitro. The results showed that angelicin, pimpinellin, sphondin, byakangelicol, oxypeucedanin, oxypeucedanin hydrate, xanthotoxin, and cnidilin are potential NO production inhibitors, and their IC50 values for inhibition of nitrite production were 19.5, 15.6, 9.8, 16.9, 16.8, 15.8, 16.6, and 17.7 microg/mL, respectively. Distinct structure-activity relationships were also revealed for the NO production inhibitory activities of these furanocoumarins. Activities of the angelicin type such as pimpinellin and sphondin were more potent than those of the psoralen type. Presence of a methoxy at the C6 position in the angelicin type seemed to be essential to augment the activity. Western blot analysis demonstrated that only sphondin dose-dependently inhibited the expression of the iNOS protein at 2.5-20 microg/mL. However, iNOS enzyme activity was stimulated with LPS for 12 h and sphondin was administered (20 microg/mL) for 24 h, which did not reasonably inhibit iNOS enzyme activity. L-NAME (100 microM), a known specific inhibitor of iNOS, was employed as a positive control with the same protocol and showed more than 50% inhibition activity. The results demonstrate that the NO production inhibitory activity of sphondin is due to the effect of iNOS expression, but not by direct inhibition of iNOS enzyme activity. Thus, sphondin may act as a potent inhibitor of NO production under tissue-damaging inflammatory conditions.     

Garcinol modulates tyrosine phosphorylation of FAK and subsequently induces apoptosis through down-regulation of Src, ERK, and Akt survival signaling in human colon cancer cells

Garcinol, from the fruit rind of Garcinia indica and other species, has been reported to suppress colonic aberrant crypt foci (ACF) formation in rats. In this study, we investigate the beneficial effects of tumor prevention by garcinol on the human colorectal cancer cell line, HT-29. Focal adhesion kinase (FAK) is the major signaling mediator of integrin-mediated cell-matrix contact-regulated cellular proliferation, migration, and apoptosis in adherent cells. Results of Matrigel analysis show that exposure of HT-29 cells to 10 microM garcinol inhibited cell invasion, and decreased the dose-dependent tyrosine phosphorylation of FAK. We further demonstrate by Western blot analysis that garcinol inhibited activation of the Src, MAPK/ERK, and PI3K/Akt signaling pathways. To investigate whether the loss of integrin-mediated cell-matrix contact can induce apoptosis, we demonstrate that garcinol induced it in HT-29 cells. The apoptotic dose of garcinol (20 microM) changed the ratio of the anti-apoptotic Bcl-2 and proapoptotic BAX proteins within 12 h, which correlated with a release of cytochrome c from the mitochondria to the cytosol, and with PARP cleavage. Additionally, we demonstrate that a decreasing MMP-7 protein level in HT-29 cells results in sensitization to garcinol. Garcinol also significantly inhibited the expression of MMP-7 in IL-1beta-induced HT-29 cells. These results suggest that garcinol reduces cell invasion and survival through the inhibition of FAK's downstream signaling.     

Anti-inflammatory flavonoids and pterocarpanoid from Crotalaria pallida and C. assamica

One new isoflavone, 5,7,4'-trihydroxy-2'-methoxyisoflavone (3) and seven, and four known compounds were isolated from the barks of Crotalaria pallida and the seeds of C. assamica, respectively. The known compounds, apigenin (1) and 2'-hydroxygenistein (2), isolated from C. pallida, showed significant concentration-dependent inhibitory effects on the release of beta-glucuronidase and lysozyme from rat neutrophils in response to formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB) with IC(50) values of 2.8+/-0.1 and 17.7+/-1.9, and 5.9+/-1.4 and 9.7+/-3.5 microM, respectively. The known compounds, daidzein (4) and 2'-hydroxydaidzein (6), isolated from C. pallida, inhibited of the release of lysozyme and beta-glucuronidase from rat neutrophils in response to fMLP/CB with IC(50) values of 26.3+/-5.5 and 13.7+/-2.6 microM, respectively. Compounds 1 and 4 also showed significant concentration-dependent inhibitory effects on superoxide anion generation in rat neutrophils stimulated with fMLP/CB with IC(50) values of 3.4+/-0.3 and 25.1+/-5.0 microM, respectively. Compounds 1 and 5, previously isolated from C. pallida, showed the inhibition of NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and LPS/interferon-gamma (IFN-gamma)-stimulated N9 microglial cells with IC(50) values of 10.7+/-0.1 and 13.9+/-1.1 microM, respectively. Flavonoids, suppressed chemical mediators in inflammatory cells, may have value in treatment and prevention of central and peripheral inflammatory diseases associated with excess production of chemical mediators.     

Protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in rat cortical neuron-glia cultures

We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron-glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-gamma-induced neurotoxicity in rat cortical neuron-glia cultures with an EC(50) value of 0.09 microM. Radicicol inhibited the LPS/IFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-gamma-induced release of tumor necrosis factor-alpha (TNF-alpha) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-alpha was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in neuron-glia cultures is mediated via the inhibition of TNF-alpha release, as well as the suppression of iNOS expression in microglia.     

Inhibitory effect of curcumin on nitric oxide production from lipopolysaccharide-activated primary microglia

Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the modulatory effect of curcumin on the functional activation of primary microglial cells, brain mononuclear phagocytes causing the neuronal damage, largely remains unknown. The current study examined whether curcumin influenced NO production in rat primary microglia and investigated its underlying signaling pathways. Curcumin decreased NO production in LPS-stimulated microglial cells in a dose-dependent manner, with an IC(50) value of 3.7 microM. It also suppressed both mRNA and protein levels of inducible nitric oxide synthase (iNOS), indicating that this drug may affect iNOS gene expression process. Indeed, curcumin altered biochemical patterns induced by LPS such as phosphorylation of all mitogen-activated protein kinases (MAPKs), and DNA binding activities of nuclear factor-kappaB (NF-kappaB) and activator protein (AP)-1, assessed by reporter gene assay. By analysis of inhibitory features of specific MAPK inhibitors, a series of signaling cascades including c-Jun N-terminal kinase (JNK), p38 and NF-kappaB was found to play a critical role in curcumin-mediated NO inhibition in microglial cells. The current results suggest that curcumin is a promising agent for the prevention and treatment of both NO and microglial cell-mediated neurodegenerative disorders.     

Effect of naturally occurring organosulfur compounds on nitric oxide production in lipopolysaccharide-activated macrophages

Excessive nitric oxide (NO) production is involved in cellular injury and possibly in the multistage process of carcinogenesis. In this study, we investigated the effect of organosulfur compounds (S-allyl cysteine, allyl sulfide, diallyl disulfide, allyl isothiocyanate, phenyl isothiocyanate, and benzyl isothiocyanate) that are found in allium or cruciferous vegetables on NO production in J774.1 macrophages activated with lipopolysaccharide (LPS). Diallyl disulfide, allyl, phenyl, and benzyl isothiocyanates inhibited NO production, as evaluated by nitrite formation at 25 microM. Allyl and benzyl isothiocyanates, the most active of the six organosulfur compounds, exhibited dose-dependent inhibition and had IC(50) values of 1.6 and 2.7 microM, respectively. Western blot analysis suggested that suppression of the induction of inducible NO synthase (iNOS) expression is responsible for the inhibition of NO production by allyl and benzyl isothiocyanates. In contrast, these isothiocyanates increased LPS-stimulated tumor necrosis factor alpha (TNF-alpha) release, suggesting their selective action on genes activated by LPS. Our results demonstrate that certain organosulfur compounds inhibit NO synthesis in LPS-activated macrophages, and the inhibitory effect may be a significant component of their anticarcinogenic activity.     

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-gamma.

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC(50) of 10.7 microM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[(14)C]arginine to L-[(14)C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.     

Synergistic activation of lipopolysaccharide-stimulated glial cells by propofol

Despite the extensive use of propofol in general anesthetic procedures, the effects of propofol on glial cell were not completely understood. In lipopolysaccharide (LPS)-stimulated rat primary astrocytes and BV2 microglial cell lines, co-treatment of propofol synergistically induced inflammatory activation as evidenced by the increased production of NO, ROS and expression of iNOS, MMP-9 and several cytokines. Propofol augmented the activation of JNK and p38 MAPKs induced by LPS and the synergistic activation of glial cells by propofol was prevented by pretreatment of JNK and p38 inhibitors. When we treated BV2 cell culture supernatants treated with LPS plus propofol on cultured rat primary neuron, it induced a significant neuronal cell death. The results suggest that the repeated use of propofol in immunologically challenged situation may induce glial activation in brain.     

Expression of glucose transporter GLUT3 by endotoxin in cultured rat astrocytes: the role of nitric oxide

The induction of nitric oxide (NO) synthase in astrocytes by endotoxin and/or cytokine treatment is associated with increased glucose consumption and glycolysis, but the mechanism whereby this phenomenon occurs remains obscure. In this work, we have addressed this issue and found that incubation of cultured rat astrocytes with lipopolysaccharide (LPS; 1 microg/mL) for 24 h increased the level of constitutively expressed GLUT1 glucose transporter mRNA, and triggered GLUT3 mRNA expression, which was absent in normal astrocytes. The occurrence of GLUT3 protein after LPS treatment was corroborated by western blotting and immunocytochemistry. A 4-h incubation of astrocytes in the absence of glucose, or under an oxygen-poor (3%) atmosphere also resulted in GLUT3 mRNA overexpression. Experiments performed with 2-deoxy-D-[U-14C]glucose (at 0.1 mM of D-glucose) confirmed that LPS (0.1-10 microg/mL) dose-dependently increased the rate of glucose uptake (by a factor of 1.6 at 1 microg/mL of LPS), which was paralleled with the increase in NO synthesis. Furthermore, blockade of NO synthase with 2-amino-5,6-dihydro-6-methyl-(4H)-1,3-thiazine (AMT; 50 microM) partially (by 45%) prevented the LPS-mediated increase in glucose uptake. Finally, incubation of astrocytes with the NO donor 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA; 100 microM) increased by a factor of 1.4 the rate of glucose uptake. We conclude that the increase in GLUT3-driven glucose uptake in astrocytes would have a neuroprotective role under conditions in which NO formation is combined with hypoglycaemia, such as in brain ischemia.     

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1

The present study focuses on the effect of various naturally occurring flavonoids (apigenin, galangin, morin, naringenin, quercetin, and silymarin) on nitric oxide (NO) and prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS) in the macrophage cell line J774A.1. Moreover, we evaluated flavonoid modulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzyme expression by western blot analysis. Apigenin and quercetin (0.5-50 microM) were the most potent inhibitors of NO production and this effect was concentration-dependent and significant at 5 and 50 microM. These data were consistent with the modulation of iNOS enzyme expression. A similar pattern was observed considering the inhibitory effect of flavonoids on LPS-induced PGE2 release and COX-2 expression. Quercetin, galangin, apigenin, and naringenin markedly decreased PGE2 release and COX-2 expression in a concentration-dependent manner. This study suggests that inhibition of iNOS and COX-2 expression by flavonoids may be one of the mechanisms responsible for their anti-inflammatory effects.     

Inhibitory effects of sesquiterpenes from bay leaf on nitric oxide production in lipopolysaccharide-activated macrophages: structure requirement and role of heat shock protein induction

The methanolic extract from the leaves of Laurus nobilis (bay leaf, laurel) was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. Through bioassay-guided separation, fourteen known sesquiterpenes were isolated from the active fraction and were examined for ability to inhibit the NO production. Seven sesquiterpene lactones (costunolide, dehydrocostus lactone, eremanthine, zaluzanin C, magnolialide, santamarine and spirafolide) potently inhibited LPS-induced NO production (IC50 = 1.2 approximately 3.8 microM). Other sesquiterpene constituents also showed the inhibitory activity (IC50 > or = 21 microM), but their inhibitory activities were less than those of sesquiterpene lactones. Alpha-methylene-gamma-butyrolactone also showed inhibitory activity (IC50 = 9.6 microM), while mokko lactone and watsonol A etc., reductants of the alpha-methylene-gamma-butyrolactone moiety by NaBH4 or DIBAL, and a 2-mercaptoethanol adduct of dehydrocostus lactone showed little activity (IC50 > or = 18 microM). These results indicated that the alpha-methylene-gamma-butyrolactone moiety is important for the activity. Furthermore, costunolide and dehydrocostus lactone inhibited inducible nitric oxide synthase (iNOS) induction in accordance with induction of heat shock protein 72 (HSP 72). These results suggested that, as one of their mechanisms of action, sesquiterpene lactones induce HSP 72 thereby preventing nuclear factor-kappaB activation followed by iNOS induction.     

Inhibition of LPS-induced NO production by the oleogum resin of Commiphora wightii and its constituents

Three new (1-3) and five known compounds (4-8) were isolated from the oleogum resin of Commiphora wightii (Arnott.) Bhanol. Their structures were elucidated by spectroscopic and chemical methods. The MeOH extract and the EtOAc-sol. fraction were found to demonstrate significant inhibition of NO formation in lipopolysaccharide (LPS)-activated murine macrophages J774.1 in vitro (IC(50) values of 16.4 and 12.8 microg/ml, respectively). When compared with curcumin (IC(50) value of 12.3 microM), Z- and E-Guggulsterones (4 and 5, respectively) were the most potent inhibitors of NO production (IC(50) values of 1.1 and 3.3 microM, respectively), followed by myrrhanol A (7) and myrrhanone A (8) (IC(50) values of 21.1 and 42.3 microM, respectively). Guggulsterone-M (1) and its didehydro derivative (2) were weak inhibitors, while guggulsterols I (6) and Y (3) were inactive (IC(50) >500 microM).