Benzyl isothiocyanate ameliorates lipid accumulation in 3T3-L1 preadipocytes during adipocyte differentiation

ABSTRACT

Benzyl isothiocyanate (BITC) is an organosulfur compound derived from cruciferous vegetables and papaya seeds. In this study, we investigated the effect of BITC on the lipid accumulation in 3T3-L1 preadipocytes during adipocyte differentiation. The treatment of BITC during the differentiation-inducing stage significantly ameliorated the lipid accumulation, whereas it had no inhibitory effect during the differentiation-maintaining stage. BITC also significantly suppressed the mRNA expression of the adipocyte-specific markers, such as CCAAT/enhancer-binding protein α (C/EBPα), C/EBPβ, C/EBPδ and peroxisome proliferator-activated receptor γ. BITC significantly inhibited the phosphorylation of extracellular signal-regulated kinase phosphorylation, whereas it enhanced that of AMP-activated protein kinase. Furthermore, BITC significantly suppressed the intracellular 2-deoxyglucose uptake as well as glucose transporter 4 expression. These results suggest that inhibition of the adipocyte differentiation and glucose uptake may mainly contribute to the inhibitory effect of BITC on the lipid accumulation in 3T3-L1 preadipocytes.

Abbreviations: PPARγ: peroxisome proliferator-activated receptor γ; CEBP: CCAAT/enhancer-binding protein; GLUT4: glucose transporter 4; AMPK: AMP-activated protein kinase; ERK1/2: extracellular signal-regulated kinase 1/2; MAPK: a mitogen-activated protein kinase; ITCs: isothiocyanates; BITC: benzyl isothiocyanate; FBS: fetal bovine serum; CS: calf serum; AITC: allyl ITC; IBMX: 3-isobutyl-1-methylxanthine; LDH: lactate dehydrogenase; KRH: Krebs-Ringer-Hepes-bicarbonate; 2-DG: 2-deoxy-d-glucose     

Mechanism of oxidative DNA damage induced by carcinogenic allyl isothiocyanate

Several isothiocyanates have been proposed as promising chemopreventive agents for human cancers. However, it has been reported that allyl isothiocyanate exhibit carcinogenic potential, and benzyl isothiocyanate and phenethyl isothiocyanate have tumor-promoting activities. We investigated whether these isothiocyanates could cause DNA damage, using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Allyl isothiocyanate caused Cu(II)-mediated DNA damage and formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) more strongly than benzyl and phenethyl isothiocyanates. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited Cu(II)-mediated DNA damage by these isothiocyanates, suggesting involvement of H(2)O(2) and Cu(I). Isothiocyanates induced DNA damage frequently at thymine and cytosine residues in the presence of Cu(II). A UV-visible spectroscopic study revealed an association between the generation of superoxide and the yield of SH group from isothiocyanates. Furthermore, the yield of 8-oxodG formation was correlated with their superoxide-generating ability. Allyl isothiocyanate significantly induced 8-oxodG formation in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. We conclude that oxidative DNA damage may play important roles in carcinogenic processes induced by allyl isothiocyanate.     

氧化修饰低密度脂蛋白对巨噬细胞一氧化氮合酶基因表达的影响

氧化修饰LDL(OX-LDL)可抑制脂多糖(LPS)诱导的巨噬细胞NO释放, 而正常(N-LDL)和乙酰化LDL(AC-LDL)则没有抑制作用.OX-LDL对NO释放的抑制作用随LDL修饰程度的升高而增强,且具有浓度和时间效应.狭缝杂交结果显示OX-LDL处理可使LPS诱导的巨噬细胞NOS mRNA含量下降,提示OX-LDL对NO释放的抑制作用可能发生在转录水平.     

Reliability of laboratory models in the analysis of TBP2 and other meningococcal antigens

Abstract Lipoarabinomannan derived from the virulent Erdman strain and a rapidly growing, laboratory-attenuated strain of Mycobacterium tuberculosis were evaluated for their ability to modulate the production of nitric oxide (NO) by macrophages activated with IFN-γ or IFN-γ and LPS. It was observed that in macrophages pretreated with 100 μg ml⁻¹ LAM, the NO induced by IFN-γ alone was augmented while the NO induced by IFN-γ and LPS was reduced. LAM was also shown to synergize with IFN-γ in the induction of NO, with AraLAM from the attenuated strain exhibiting greater potency than ManLAM from the Erdman strain. Despite the modulation of NO production, LAM did not affect the IFN-γ-induced macrophage growth inhibition of Francisella tularensis LVS, an organism whose growth inhibition in activated macrophages is dependent upon NO.     

Allyl isothiocyanate (AITC) and phenyl isothiocyanate (PITC) inhibit tumour-specific angiogenesis by downregulating nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) production.

Angiogenesis, a crucial step in the growth and metastasis of cancers, is initiated with vasodilation mediated by nitric oxide (NO). The pro-inflammatory cytokine, tumour necrosis factor-alpha (TNF-alpha), is a mediator of nitric oxide synthesis. We analyzed the effect of allyl isothiocyanate (AITC) and phenyl isothiocyanate (PITC) on serum NO as well as TNF-alpha level during angiogenesis. In vivo antiangiogenic activity was studied using B16F-10 melanoma cell-induced capillary formation in C57BL/6 mice. Intraperitoneal administration of AITC and PITC at a concentration of 25 microg/dose/animal significantly inhibited tumour-directed capillary formation. Treatment of AITC and PITC significantly downregulated serum NO as well as TNF-alpha level in angiogenesis-induced animals compared to untreated control animals. The in vitro antiangiogenic study, using rat aortic ring assay, showed that both AITC and PITC at non-toxic concentrations inhibited the production of proangiogenic factors from B16F-10 melanoma cells which was evident with the inhibition of microvessel outgrowth from aortic rings. Both AITC and PITC significantly inhibited sodium nitroprusside as well as TNF-alpha-induced microvessel outgrowth from rat aortic ring. Administration of AITC and PITC also significantly reduced NO and TNF-alpha production by LPS-stimulated macrophages both in vivo as well as in vitro. Bio-assay using serum of angiogenesis-induced animals and supernatant from LPS-stimulated macrophages clearly confirmed the downregulatory action of AITC and PITC on TNF-alpha production. These results clearly demonstrated that AITC and PITC inhibited tumour-specific angiogenesis by downregulating NO and TNF-alpha production.     

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.     

β-Phenylethyl and 8-methylsulphinyloctyl isothiocyanates, constituents of watercress, suppress LPS induced production of nitric oxide and prostaglandin E2 in RAW 264.7 macrophages

Beta-phenylethyl (PEITC) and 8-methylsulphinyloctyl isothiocyanates (MSO) represent two phytochemical constituents present in watercress Rorripa nasturtium aquaticum, with known chemopreventative properties. In the present investigation, we examined whether PEITC and MSO could modulate the inflammatory response of Raw 264.7 macrophages to bacterial lipopolysaccharide (LPS) by assessment of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Overproduction of both nitric oxide (NO) and prostaglandins (PGE) has been associated with numerous pathological conditions including chronic inflammation and cancer. Our results demonstrate that LPS (1 microg/ml approximately 24 h) induced nitrite and prostaglandin E2 (PGE-2) synthesis in Raw 264.7 cells was attenuated by both isothiocyanates (ITCs) in a concentration-dependent manner. Both PEITC and MSO decreased (iNOS) and (COX-2) protein expression levels leading to reduced secretion of both pro-inflammatory mediators. Interestingly, the reduction in both iNOS and COX-2 expression were associated with the inactivation of nuclear factor-kappaB and stabilization of IkappaBalpha. Taken together our data gives further insight into the possible chemopreventative properties of two dietary derived isothiocyanates from watercress.     

Mortality and behavior in Heterodera glycines juveniles following exposure to isothiocyanate compounds

For this report, we examined the toxic effects of three plant-derived isothiocyanate compounds on second-stage juveniles (J2) of Heterodera glycines. We found significant differences among compounds in the concentration required to affect nematodes, according to mortality and behavioral measurements. The concentrations required to affect behavior were significantly lower than those required for mortality. Both mortality and behavioral measurements were used to investigate whether nematodes in a quiescent state display decreased sensitivity to isothiocyanates compared with actively moving nematodes. Mortality measurements revealed that quiescent nematodes were significantly less sensitive to isothiocyanates than active nematodes. All behavioral measurements following exposure to benzyl- and phenyl isothiocyanate showed significant differences in sensitivity between quiescent and active nematodes. However, significant differences between quiescent and active nematodes were observed in only one of the five behavioral measurements following exposure to allyl isothiocyanate. These results expand the list of plant-derived compounds toxic to H. glycines and illustrate the impact of behavioral quiescence on nematode sensitivity to exogenous toxins.     

Inhibitory effects of flavonol glycosides from Cinnamomum osmophloeum on inflammatory mediators in LPS/IFN-gamma-activated murine macrophages

Four kaempferol glycosides were isolated from the leaves of Cinnamomum osmophloeum Kaneh, a Taiwan endemic tree. These compounds namely, kaempferitrin (1), kaempferol 3-O-beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-7-O-alpha-L-rhamnopyranoside (2), kaempferol 3-O-beta-D-apiofuranosyl-(1-->2)-alpha-L-arabinofuranosyl-7-O-alpha-L-rhamnopyranoside (3), and kaempferol 3-O-beta-D-apiofuranosy-(1-->4)-alpha-L-rhamnopyranosyl-7-O-alpha-L-rhamnopyranoside (4). The structure of compound 2 was determined by spectroscopic analyses and acid hydrolysis. The isolates 1-4 were evaluated as inhibitors of some macrophage functions involved in the inflammatory process. These four compounds inhibited lipopolysaccharide (LPS) and interferon (IFN)-gamma-induced nitric oxide (NO), and cytokines [tumor necrosis factor (TNF)-alpha and interleukin (IL)-12] in a dose-dependent manner. The concentration of 50% inhibition (IC(50)) of NO by compounds 1, 3, 4 were 40, 15, 20microM, respectively. In parallel, these concentrations were approximately in a similar manner to that observed for TNF-alpha and IL-12 production. However, compound 2 inhibited NO and cytokines production by 30% at 100microM concentration. On the other hand, compounds 3 and 4 showed no inhibitory effect on the production of NO from macrophages, when inducible NO synthase was already expressed by the stimulation with LPS and IFN-gamma. Taken together, our results provide evidence that isolates of C. osmophloeum possess an anti-inflammatory potential which constitutes a previously unrecognized biological activity.     

Effect of Synthetic Hydroxy Isothiocyanates on a Bacterial Virus and DNA

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on φK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.     

Effect of a combination of deoxynivalenol and nivalenol on lipopolisaccharide-induced nitric oxide production by mouse macrophages

Deoxynivalenol (DON) and nivalenol (NIV) are trichothecene mycotoxins produced by Fusarium fungi as secondary metabolites. Both compounds have the immunotoxic effects that the productions of inflammatory mediators by activated macrophages is disturbed. Co-contamination with DON and NIV can occur; however, the effects of simultaneous contamination are not well known. The present study investigated the combined effects of DON and NIV on nitric oxide (NO) production by mouse macrophages stimulated with lipopolisaccharide (LPS). The inhibitory effect of DON and NIV on NO release from activated macrophages has already been reported as an appropriate indicator of immunotoxic effect of the both compounds. LPS-induced NO production in macrophages was inhibited by both of these toxins individually in a dose-dependent manner, and toxin mixtures at the same concentration inhibited NO production in the same manner. In addition, there were no unique inhibitory effects on LPS-induced NO production in macrophages in the presence of mixtures of various molar ratios. These results suggest that the combined effects of DON and NIV can be predicted based on addition of each compound alone.     

Electrophysiological and behavioural studies of the biting midge, Culicoides impunctatus Goetghebuer (Diptera, Ceratopogonidae): interactions between some plant-derived repellent compounds and a host-odour attractant, 1-octen-3-ol

Abstract. Electroantennogram (EAG) and y-tube bioassays have been used to demonstrate the repellent properties of five plant compounds with host-seeking parous female Culicoides impunctatus Goetghebuer. The compounds were methyl salicylate and allyl-, butyl-, phenyl- and 2-phenylethyl isothiocyanate. EAG thresholds were 1 times 10^-3 to 1 μg. In the bioassays, maximal repellencies occurred with 1 times 10˜² to lOug. When each compound was combined with 1-octen-3-ol, a confirmed host-odour attractant for C.impunctatus females, additive effects were recorded in EAG assays and in bioassays, all of the compounds either reduced or reversed the attractancy of l-octen-3-ol. Of the isothiocyanates, allyl isothiocyanate was the most potent and when combined with 1-octen-3-ol in field trials, the attractant effect of l-octen-3-ol was reduced.     

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).     

Lipopolysaccharide and interferon-gamma-induced nitric oxide production and protein oxidation in mouse peritoneal macrophages are affected by glutathione peroxidase-1 gene knockout

This study investigated the role of glutathione peroxidase-1 (GPX1) in protein oxidation in peritoneal macrophages. Macrophages isolated from both wild-type (WT) and GPX1 knockout (KO) mice were activated by lipopolysaccharide (LPS, 1 microg/ml) and interferon-gamma (IFN, 10 U/ml for 24 or 48 h in the presence or absence of 1 microM diquat (DQ), 250 microM aminoguanidine (AG, an inhibitor of inducible nitric oxide synthase), and (or) 100 microM diethyldithiocarbamate (DETC, an inhibitor of Cu,Zn-SOD). In the KO macrophages, there was no protein band detected by Western blot with anti-GPX1 antibody and 98% reduction in total GPX activity compared with WT cells. Nitric oxide (NO) synthesis was greatly enhanced after 24 h by GPX1 knockout and DQ, but inhibited by AG or DETC. Protein carbonyl formation in total cell extract was clearly associated with NO synthesis as higher levels of protein carbonyl were detected in activated KO than WT macrophages, and DQ enhanced slightly while AG or DETC virtually blocked its formation. A similarly marginal effect of GPX1 KO was observed on protein nitration. The LPS/IFN/DQ-induced DNA fragmentation was blocked by AG, but not by DETC. Cell viability at 48 h was decreased by the LPS/IFN activation and further reduced by the addition of DQ, but restored by AG. In conclusion, GPX1 affects the NO production in activated peritoneal macrophages and protects these cells against NO-associated protein oxidation.     

Tetracycline up-regulates COX-2 expression and prostaglandin E2 production independent of its effect on nitric oxide

Tetracyclines (doxycycline and minocycline) augmented (one- to twofold) the PGE2 production in human osteoarthritis-affected cartilage (in the presence or absence of cytokines and endotoxin) in ex vivo conditions. Similarly, bovine chondrocytes stimulated with LPS showed (one- to fivefold) an increase in PGE2 accumulation in the presence of doxycycline. This effect was observed at drug concentrations that did not affect nitric oxide (NO) production. In murine macrophages (RAW 264.7) stimulated with LPS, tetracyclines inhibited NO release and increased PGE2 production. Tetracycline(s) and L-N-monomethylarginine (L-NMMA) (NO synthase inhibitor) showed an additive effect on inhibition of NO and PGE2 accumulation, thereby uncoupling the effects of tetracyclines on NO and PGE2 production. The enhancement of PGE2 production in RAW 264.7 cells by tetracyclines was accompanied by the accumulation of both cyclooxygenase (COX)-2 mRNA and cytosolic COX-2 protein. In contrast to tetracyclines, L-NMMA at low concentrations (< or = 100 microM) inhibited the spontaneous release of No in osteoarthritis-affected explants and LPS-stimulated macrophages but had no significant effect on the PGE2 production. At higher concentrations, L-NMMA (500 microM) inhibited NO release but augmented PGE2 production. This study indicates a novel mechanism of action of tetracyclines to augment the expression of COX-2 and PGE2 production, an effect that is independent of endogenous concentration of NO.     

Viral hemorrhagic septicemia virus alters turbot Scophthalmus maximus macrophage nitric oxide production.

The effect of viral hemorrhagic septicemia virus (VHSV) in vitro infection on the nitric oxide (NO) production by turbot Scophthalmus maximus kidney macrophages has been addressed in the past. Previously, we had determined that only a small fraction of turbot possess head kidney macrophages that respond to a single exposure of lipopolysaccharide (LPS) with NO production (LPS-responsive macrophages), whereas macrophage cultures from other individuals were not activated by LPS alone and needed a combination of stimuli to respond (LPS-non-responsive macrophages). In the current work, we examined the effect of VHSV on NO production by macrophages characterized as LPS-responsive macrophages or LPS-non-responsive macrophages. Combinations of LPS and tumor necrosis factor alpha (TNF-alpha) and macrophage-activating factor (MAF) were also used to stimulate the cells for NO production. The effect of VHSV on NO production depends on the response to LPS alone. When a low multiplicity of infection was used (1.78 x 10(-3)), the NO production in response to LPS in LPS-responsive macrophages was significantly decreased. However, LPS-non-responsive macrophage cultures produced NO when a combination of LPS and VHSV was used. In the case of a higher VHSV multiplicity of infection (1.78), no significant change was observed in LPS-non-responsive animals. Combinations of LPS with TNF-alpha, LPS with MAF, and TNF-alpha with MAF were used to induce NO production in LPS-non-responsive macrophages. In all these cases, VHSV suppressed NO production, although at a significant level only when a combination of TNF-alpha and MAF was used for the induction of NO.     

Methyl-β-cyclodextrin potentiates the BITC-induced anti-cancer effect through modulation of the Akt phosphorylation in human colorectal cancer cells

ABSTRACT

Methyl-β-cyclodextrin (MβCD) is an effective agent for the removal of plasma membrane cholesterol. In this study, we investigated the modulating effects of MβCD on the antiproliferation induced by benzyl isothiocyanate (BITC), an ITC compound mainly derived from papaya seeds. We confirmed that MβCD dose-dependently increased the cholesterol level in the medium, possibly through its removal from the plasma membrane of human colorectal cancer cells. The pretreatment with a non-toxic concentration (2.5 mM) of MβCD significantly enhanced the BITC-induced cytotoxicity and apoptosis induction, which was counteracted by the cholesterol supplementation. Although BITC activated the phosphoinositide 3-kinase (PI3K)/Akt pathway, MβCD dose-dependently inhibited the phosphorylation level of Akt. On the contrary, the treatment of MβCD enhanced the phosphorylation of mitogen activated protein kinases, but did not potentiate their BITC-induced phosphorylation. These results suggested that MβCD might potentiate the BITC-induced anti-cancer by cholesterol depletion and thus inhibition of the PI3K/Akt-dependent survival pathway.

Abbreviations: CDs: cyclodextrins; MβCD: methyl-β-cyclodextrin; ITCs: isothiocyanates; BITC: benzyl isothiocyanate; PI3K: phosphoinositide 3-kinase; PDK1: phosphoinositide-dependent kinase-1; MAPK: mitogen activated protein kinase; ERK1/2: extracellular signal-regulated kinase1/2; JNK: c-Jun N-terminal kinase; PI: propidium iodide; FBS: fatal bovine serum; TLC: thin-layer chromatography; PBS(-): phosphate-buffered saline without calcium and magnesium; MEK: MAPK/ERK kinase; PIP2: phosphatidylinositol-4,5-bisphosphate; PIP3: phosphatidylinositol-3,4,5-trisphosphate     

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.