Inhibited Phorbol 12-myristate 13-acetate Research Articles

A potassium ion channel is involved in cytokine production by activated human macrophages.

Macrophages play an important role in immune and inflammatory responses, largely through secretion of bioactive molecule such as cytokines. While calcium is known to be an important regulator of this process, less is known about the role of other ions and the ion channels that regulate them. We have previously implicated an outwardly rectifying potassium channel (Kor) in this process and for this reason we have investigated the role of potassium (K+) and K+ channels in the regulation of tumour necrosis factor-alpha (TNF-alpha)and interleukin (IL)-8 production by activated human culture-derived macrophages. The effect of blockade of Kor is to inhibit phorbol myristate acetate (PMA)-induced cytokine production by translational or post-translational mechanisms, an effect that is duplicated by increasing extracellular K+. By contrast, the effects of K+ on LPS-stimulated cells are far more complex and are probably mediated through the change of osmolality and occur largely at the mRNA level. This data directly implicates K+, and its regulation through Kor, in early events following PMA stimulation of these cells.

Hydroxyurea inhibits the transactivation of the HIV-long-terminal repeat (LTR) promoter.

HIV-1 gene expression is regulated by the promoter/enhancer located within the U3 region of the proviral 5' LTR that contains multiple potential cis-acting regulatory sites. Here we describe that the inhibitor of the cellular ribonucleoside reductase, hydroxyurea (HU), inhibited phorbol myristate acetate- or tumour necrosis factor-alpha-induced HIV-1-LTR transactivation in both lymphoid and non-lymphoid cells in a dose-dependent manner within the first 6 h of treatment, with a 50% inhibitory concentration of 0.5 mM. This inhibition was found to be specific for the HIV-1-LTR since transactivation of either an AP-1-dependent promoter or the CD69 gene promoter was not affected by the presence of HU. Moreover, gel-shift assays in 5.1 cells showed that HU prevented the binding of the NF-kappaB to the kappaB sites located in the HIV-1-LTR region, but it did not affect the binding of both the AP-1 and the Sp-1 transcription factors. By Western blots and cell cycle analyses we detected that HU induced a rapid dephosphorylation of the pRB, the product of the retinoblastoma tumour suppressor gene, and the cell cycle arrest was evident after 24 h of treatment. Thus, HU inhibits HIV-1 promoter activity by a novel pathway that implies an inhibition of the NF-kappaB binding to the LTR promoter. The present study suggests that HU may be useful as a potential therapeutic approach for inhibition of HIV-1 replication through different pathways.

Binding Specificity for RACK1 Resides in the V5 Region of βII Protein Kinase C

Identification of selective anchoring proteins responsible for specialized localization of specific signaling proteins has led to the identification of new inhibitors of signal transduction, inhibitors of anchoring protein-ligand interactions. RACK1, the first receptor for activated C kinase identified in our lab, is a selective anchoring protein for betaII protein kinase C (betaIIPKC). We previously found that at least part of the RACK1-binding site resides in the C2 domain of betaIIPKC (Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180-24187). Here we show that the V5 domain also contains part of the RACK1-binding site in betaIIPKC. In neonatal rat cardiac myocytes, the betaIIV5-3 peptide (amino acids 645-650 in betaIIPKC) selectively inhibited phorbol 12-myristate 13-acetate (PMA)-induced translocation of betaIIPKC and not betaIPKC. In addition, the betaIIV5-3 peptide inhibited cardiac myocyte hypertrophy in PMA-treated cells. Interestingly, betaIV5-3 (646-651 in betaIPKC), a selective translocation inhibitor of betaIPKC, also inhibited PMA-induced cardiac myocyte hypertrophy, demonstrating that both betaI- and betaIIPKC are essential for this cardiac function. Therefore, the betaIIV5 domain contains part of the RACK1-binding site in betaIIPKC; a peptide corresponding to this site is a selective inhibitor of betaIIPKC and, hence, enables the identification of betaIIPKC-selective functions.

Protein kinase C-dependent and -independent inhibition of Ca 2+ influx by phorbol ester in rat pancreatic β-cells

Phorbol esters were used to investigate the action of protein kinase C (PKC) on insulin secretion from pancreatic β-cells. Application of 80 nM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, had little effect on glucose (15 mM)-induced insulin secretion from intact rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, PMA significantly reduced the glucose-induced insulin secretion. PMA decreased the level of intracellular Ca 2+ concentration ([Ca 2+] i) elevated by the glucose stimulation when tested in isolated rat β-cells. This inhibitory effect of PMA was not prevented by BIM. PMA inhibited glucose-induced action potentials, and this effect was not prevented by BIM. Further, 4α-phorbol 12,13-didecanoate (4α-PDD), a non-PKC-activating phorbol ester, produced an effect similar to PMA. In the presence of nifedipine, the glucose stimulation produced only depolarization, and PMA applied on top of glucose repolarized the cell. When applied at the resting state, PMA hyperpolarized β-cells with an increase in the membrane conductance. Recorded under the voltage-clamp condition, PMA reduced the magnitude of Ca 2+ currents through L-type Ca 2+ channels. BIM prevented the PMA inhibition of the Ca 2+ currents. These results suggest that activation of PKC maintains glucose-stimulated insulin secretion in pancreatic β-cells, defeating its own inhibition of the Ca 2+ influx through L-type Ca 2+ channels. PKC-independent inhibition of electrical excitability by phorbol esters was also demonstrated.

Peroxisome proliferator-activated receptor and retinoid X receptor ligands inhibit monocyte chemotactic protein-1-directed migration of monocytes

Monocyte chemotactic protein-1 (MCP-1)-directed transendothelial migration of monocytes plays a key role in the development of inflammatory diseases. Infiltration of tissues by monocytes requires degradation of extracellular matrices, a process that involves matrix metalloproteinases. We studied the effects of peroxisome proliferator-activated receptor (PPAR) γ, α, and retinoid X receptor α (RXRα) ligands on MCP-1-directed migration and matrix metalloproteinase expression of a human acute monocytic leukemia cell line (THP-1). PPARγ ligands attenuated MCP-1-induced migration, with 50% inhibition (IC 50) at 2.8 μM for troglitazone and 4.8 μM for rosiglitazone. PPARα ligands WY-14643 (IC 50: 0.9 μM) and 5,8,11,14-eicosatetranoic acid (IC 50: 9.9 μM), and the potent RXRα ligand AGN 4204 (IC 50: 3.6 nM) also blocked monocyte migration. Troglitazone, rosiglitazone, or AGN 4204 inhibited phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 expression. PPARα activators WY-14643 and 5,8,11,14-eicosatetraynoic acid, however, had no inhibitory effect. AGN 4204 increased PMA-induced tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) expression, whereas all PPAR ligands showed no effect. All PPAR and RXRα ligands blocked chemotaxis of THP-1 monocytes in the absence of a matrix barrier. This study demonstrates that activated PPARs and RXRα, block MCP-1-directed monocyte migration, mediated, at least in part, through their effects on matrix metalloproteinase-9 or TIMP-1 production, or chemotaxis.

Histamine Induces Melanogenesis and Morphologic Changes by Protein Kinase A Activation via H2 Receptors in Human Normal Melanocytes

Hyperpigmentation frequently accompanies chronic or acute inflammation. A number of inflammatory mediators have been shown to stimulate melanin synthesis in human melanocytes. Although histamine is ubiquitous as an inflammatory factor, its involvement in pigmentation remains obscure. In this work, we examined the effects of histamine on cultured human melanocytes. Treatment of human melanocytes with 0.1-10 microM histamine evoked morphologic changes and increases in tyrosinase activity. The concomitant increases in melanin content of the histamine-treated melanocytes indicated an elevation of melanin synthesis by tyrosinase activation. These stimulatory effects of histamine were completely inhibited by an H2 antagonist, famotidine, whereas H1 and H3 antagonists had no inhibitory effect whatsoever. In addition, an H2 agonist, dimaprit, induced the same degree of melanogenesis as histamine at concentrations of 0.1-10 microM. We observed an increase in the intracellular cAMP contents of human melanocytes induced by histamine via the H2 receptors. We know that this cAMP accumulation and subsequent protein kinase A activation plays a critical role in histamine-induced melanogenesis, because a specific protein kinase A inhibitor, H-89, completely suppressed these stimulatory effects of histamine, and because dibutylic cAMP, a specific protein kinase A activator, stimulated human melanocytes as potently as histamine. Taken together, we show here that histamine induces melanogenesis of human cultured melanocytes by protein kinase A activation via H2 receptors.

Second-generation perfluorocarbon emulsion blood substitutes.

A novel series of perfluorocarbon (PFC) emulsions, based on perfluorodecalin (C10F18) and stabilised with up to 2.5% (w/v) of lecithin have been produced for evaluation as injectable, temporary respiratory gas-carrying blood substitutes. Some formulations contained 1.0% (w/v) of perfluorodimorpholinopropane (C11F22N2O2) to retard droplet growth through molecular diffusion (Ostwald Ripening). Other emulsions contained novel, amphiphilic fluorinated surfactants, such as, for example, the monocarbamate, C8F17C2H4NHC(O)(CH2CH2O)2Me (designated compound P6), at 0.1% (w/v) to enhance stability. Emulsions were prepared by homogenisation, were steam sterilisable and were stable for > 300 days (25 degrees C). Injection of rats (7.5 ml kg-1 b.w.) with emulsions produced significant (P < 0.05), transient increases in liver and spleen weights. One emulsion inhibited phorbol 12-myristate 13-acetate (PMA)-stimulated, Luminol-enhanced, chemiluminescence of human polymorphonuclear leucocytes (PMNL) in vitro, suggesting possible applications in ischaemic tissues for suppressing PMNL-mediated inflammation. The P6 fluoro-surfactant inhibited spontaneous platelet aggregation in hirudin-anticoagulated human blood in vitro, suggesting possible applications as an anti-thrombotic agent.

Arachidonic acid release induced by LPS in microglial cells

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in inflammation by catalyzing the release of arachidonic acid, a substrate for lipoxygenase and cyclooxygenase enzymes, from membrane phospholipids. In the present study we examined the role of cPLA2 in inflammatory responses through the use of a specific inhibitor of the enzyme, cPLA2, arachidonyl trifluoromethyl ketone (AACOCF3). Interestingly, we observed that AACOCF3 is an inhibitor of chronic but not acute inflammatory responses. Specifically, AACOCF3 inhibited phorbol 12-myristate 13-acetate (PMA)-induced chronic ear edema in mice. Additionally, oral treatment of ovalbumin-sensitized/ovalbumin-challenged BALB/c mice with 20 mg/kg AACOCF3 prevented the development of airway hyper-responsiveness in a model of asthma. Furthermore, AACOCF3 decreased cellular recruitment in the airway lumen and airway inflammation after the ovalbumin challenge. Taken together, these results suggest that a potent and specific chemical inhibitor of cPLA2 may be useful for the treatment of chronic inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma.

Acridone derivatives are selective inhibitors of HIV-1 replication in chronically infected cells

In our extensive screening of anti-HIV-1 agents in chronically infected cell lines, we have found acridone derivatives to be selective inhibitors of HIV-1 replication. Among the acridone derivatives, 1-hydroxy-10-methyl-9,10-dihydroacrid-9-one (RD6-5071) suppressed tumor necrosis factor (TNF)-α-induced HIV-1 expression in the latently infected cell line OM-10.1, U1, and ACH-2. Its 50% effective concentration for HIV-1 p24 antigen production was 2.0 μg/ml in OM-10.1 cells, while its 50% cytotoxic concentration was 18 μg/ml. The compound also inhibited phorbol 12-myristate 13-acetate (PMA)-induced HIV-1 expression in these cell lines. Furthermore, RD6-5071 was inhibitory to HIV-1 replication in acutely infected U937 and peripheral blood mononuclear cells. The compound was found to suppress TNF-α-induced HIV-1 long terminal repeat-driven gene expression. An inhibition assay for protein kinase C (PKC) revealed that RD6-5071 could reduce the enzyme activity. Furthermore, the compound was a moderate inhibitor of PMA-induced nuclear factor κB (NF-κB) activation, as determined by a gel mobility shift analysis. These results suggest that the acridone derivatives suppress HIV-1 replication at the transcriptional level primarily through a mechanism of PKC inhibition.

Somatostatin and its analog lanreotide inhibit the proliferation of dispersed human non-functioning pituitary adenoma cells in vitro.

Somatostatin is a powerful inhibitor of hormone secretion and cell proliferation. Treatment with somatostatin analogs in humans causes a reduction in size and secretory activity of some endocrine tumors, including somatotropic pituitary adenomas. Less studied are the effects of somatostatin agonists on non-functioning pituitary adenomas (NFPAs). In this study we characterized the effects of somatostatin and its analog lanreotide on the proliferation of NFPAs in vitro and the intracellular mechanisms involved. Twenty-three NFPA post-surgical specimens were analyzed for somatostatin receptor (SSTR) expression and 12 of them were cultured in vitro to study somatostatin's effects on cell proliferation, assessed by means of [(3)H]thymidine uptake, and the intracellular signaling. One or more SSTR subtypes were expressed in 90% of the adenomas tested. Somatostatin and lanreotide treatment inhibited phorbol myristate acetate (PMA)-induced cell proliferation. Vanadate pretreatment reversed somatostatin and lanreotide inhibition of PMA-induced DNA synthesis suggesting an involvement of tyrosine phosphatase in this effect. In the only adenoma tested, somatostatin directly induced a tyrosine phosphatase activity. Somatostatin and lanreotide caused also a significant inhibition of voltage-sensitive calcium channel activity induced by 40mmol/l K(+) depolarization in microfluorimetric analysis. These data show that somatostatin and lanreotide inhibit human NFPA cell proliferation in vitro, and suggest that activation of tyrosine phosphatases and inhibition of the activity of voltage-dependent calcium channels may represent intracellular signals mediating this effect.

Tranilast inhibits protein kinase C-dependent signalling pathway linked to angiogenic activities and gene expression of retinal microcapillary endothelial cells.

1. Tranilast, first developed as an anti-allergic drug, has been reported to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis and vasopermeability. To further clarify the inhibitory mechanism, we investigated the effects of tranilast on VEGF binding and subsequent intracellular signalling pathway linked to angiogenic activities and gene expression of bovine retinal microcapillary endothelial cells. 2. Tranilast significantly (P<0.01) inhibited VEGF, basic fibroblast growth factor (bFGF), and hypoxia conditioned media-induced BREC proliferation in a dose dependent manner with IC50's of 22, 82 and 10 microM, respectively. 3. VEGF-induced migration was also inhibited by tranilast in a dose dependent manner, with IC50 of 18 microM, and complete inhibition was observed at 300 microM (P<0.01). Tranilast suppressed VEGF-induced tube formation in a dose dependent manner with maximum (46%) inhibition observed at 300 microM (P<0.05). 4. Tranilast inhibited phorbol myristate acetate (PMA)-dependent stimulation of [3H]-thymidine incorporation and VEGF- and PMA-induced gene expression of integrin alpha v and c-fos in BREC. 5. Tranilast suppressed VEGF- and PMA-stimulated PKC activity in BREC. 6. Tranilast did not affect VEGF binding or VEGF-induced phosphorylation of tyrosine residues of VEGF receptor- and phospholipase Cgamma and their associated proteins. 7. These data suggest that tranilast might prove an effective inhibitor to prevent retinal neovascularization in ischaemic retinal diseases, and that its inhibitory effect might be through suppression of PKC-dependent signal transduction in BREC.

Plant alkaloid tetrandrine downregulates protein kinase C-dependent signaling pathway in T cells

Tetrandrine, a purified traditional Chinese medicinal herb that acts as an immunosuppressant and a Ca 2+ channel blocker, has been clinically used to treat patients with arthritis, silicosis and hypertension. Since T cells play a critical role as autoreactive and pathogenic population in autoimmune diseases, in this study, we examined the immunosuppressive effect of tetrandrine on human peripheral blood T cells. We showed that tetrandrine inhibited phorbol 12-myristate 13-acetate (PMA)+ionomycin-induced T cell proliferation, interleukin-2 secretion and the expression of the T cell activation antigen, CD71. Further investigation of the molecular mechanism demonstrated that tetrandrine inhibited the expression of the protein kinase C-dependent interleukin-2 receptor alpha chain and CD69 but not the expression of the Ca 2+-dependent CD40 ligand and CD69. Interestingly, when tetrandrine and cyclosporin A were added together, significant synergism in the suppression of T cell activation was observed. Moreover, of the several tetrandrine analogues studied, hernandezine was the most potent inhibitor of protein kinase C signaling events. These results also suggest that the protein kinase C-inhibitory capacity of tetrandrine and its analogues may not be associated with their function as Ca 2+ channel blockers. Lastly, we showed that, within therapeutic concentrations, tetrandrine and its analogues could induce cellular apoptosis, which is defective in autoimmune diseases. In conclusion, our findings provide novel information about the molecular mechanism of the immunosuppressive effect of tetrandrine and its analogues in human peripheral blood T cells.

Regulation of noradrenaline release by S-nitroso-cysteine: inhibition in PC12 cells in a cyclic GMP-independent manner

Nitric oxide (NO), including NO free radicals (·NO) and peroxynitrite (OONO −), modulates the release of neurotransmitters from neuronal tissues. Although we reported that S-nitroso-cysteine stimulated noradrenaline release in brain slices, we now show that only S-nitroso-cysteine inhibits noradrenaline release from PC12 cells. S-Nitroso-cysteine inhibited, in a dose-dependent manner (up to 0.6 mM), the Ca 2+-dependent [ 3 H] noradrenaline release induced by ionomycin, adenosine 5′- O-(3-thiotriphosphate), or high K +, from PC12 cells labeled with [ 3 H] noradrenaline. Sodium nitroprusside, S-nitroso- N-acetylpenicillamine, and 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene, which specifically release NO free radicals in neutral buffer, had minimal effects on [ 3 H] noradrenaline release, although they markedly stimulated cyclic GMP accumulation. 3-Morpholinosydonimine, which releases peroxynitrite, had no effect on either [ 3 H] noradrenaline release or cyclic GMP accumulation. S-Nitroso-cysteine inhibited phorbol 12-myristate 13-acetate- and mastoparan (wasp venom toxin)-induced [ 3 H] noradrenaline release. These findings suggest that 1) S-nitroso-cysteine, but not other NO donors, inhibits some common process occurring during noradrenaline release in PC12 cells, 2) neither NO radicals, peroxynitrite, nor cyclic GMP mediate the inhibitory effects of S-nitroso-cysteine in PC12 cells.

Possible involvement of protein kinase c inhibition in the reduction of phorbol ester-induced neutrophil aggregation by magnolol in the rat.

The influence of the plant product magnolol on neutrophil aggregation has been investigated in the rat. Magnolol inhibited phorbol 12-myristate 13-acetate (PMA)-activated rat neutrophil aggregation in a concentration-dependent manner with an IC50 (concentration resulting in 50% inhibition) of 24.2 +/- 1.7 microM. Magnolol suppressed the enzyme activity of neutrophil cytosolic and rat brain protein kinase C (PKC) over the same range of concentrations at which it inhibited the aggregation. Magnolol did not affect PMA-induced cytosolic PKC-alpha and -delta membrane translocation or trypsin-treated rat-brain PKC activity, but attenuated [3H]phorbol 12,13-dibutyrate binding to neutrophil cytosolic PKC. These results suggest that the inhibition of PMA-induced rat neutrophil aggregation by magnolol is probably attributable, at least in part, to the direct suppression of PKC activity through blockade of the regulatory region of PKC.

Antioestrogens enhance tumour necrosis factor receptor 2 (TNF-R2) expression and TNF-R2-mediated proliferation in activated T cells.

In the present study we demonstrate that the non-steroidal antioestrogens toremifene and tamoxifen inhibit mitogen-induced proliferation and up-regulation of tumour necrosis factor (TNF) receptor family molecules on peripheral blood T cells. In activated T cells, however, toremifene and tamoxifen increase the surface expression of tumour necrosis factor receptor 2 (TNF-R2). This up-regulation is functionally important as TNF-R2-mediated proliferation is significantly enhanced in antioestrogen-treated activated T cells. The regulation of TNF-R2 expression in activated T cells seems to involve the c-Jun amino terminal kinase (JNK) pathway, as activation of JNK with anisomycin down-regulates TNF-R2. In activated T cells toremifene clearly inhibits phorbol 12-myristate 13-acetate (PMA)-induced JNK activity, suggesting that the JNK pathway may also be involved in the up-regulation of TNF-R2 expression by antioestrogens. Taken together, the enhancement of TNF-R2 expression and TNF-R2-mediated proliferation in activated T cells represents a novel feature for the effects of antioestrogens. The inhibitory effects of toremifene on the JNK pathway demonstrates that antioestrogens can influence not only cell growth, but also a variety of other cellular responses by inhibiting protein kinase C (PKC).

The regulation of superoxide generation and nitric oxide synthesis by C‐reactive protein

Activated macrophages utilize both reactive oxygen intermediates and reactive oxynitrogen intermediates for defence against microbes. However, simultaneous generation of superoxide (O- 2;) and nitric oxide (NO) could be harmful to host cells due to the production of peroxynitrite, nitrogen dioxide and hydroxyl radicals. Therefore, the regulation of the production of these molecules is critical to host survival. During periods of inflammation or infection, the level of serum C-reactive protein (CRP) increases in many species. Human and rat CRP have been shown to bind and interact with phagocytic cells. Since many of the interactions of CRP involve the binding to the phosphocholine ligand, we studied the role of CRP in O- 2; and NO generation through the modulation of phosphatidylcholine (PC) metabolism in macrophages. This study has shown that, while rat CRP inhibited phorbol myristate acetate- (PMA) induced release of O- 2; by rat macrophages, CRP-treated macrophages released NO in a time- and dose-dependent manner. CRP increased inducible nitric oxide synthase (iNOS) enzyme as well as iNOS mRNA levels in rat macrophages. Tricyclodecan-9-yl-xanthogenate (D609), an inhibitor to PC phospholipase C (PC-PLC), suppressed iNOS induction but enhanced PMA-induced release of O- 2;. These data indicate that an increased level of CRP during periods of inflammation may result in differential regulation of macrophage NADPH oxidase and iNOS activity. Increased hepatic synthesis of CRP may contribute to the mechanism by which phagocytic cells avoid simultaneous O- 2; and NO synthesis, and this could possibly be mediated through the regulation of PC-PLC.

Inhibitory effect of calcium-binding protein regucalcin on protein kinase C activity in rat renal cortex cytosol.

The effect of regucalcin, a calcium-binding protein, on Ca2+-and phospholipid-dependent protein kinase (protein kinase C) activity in the cytosol of rat kidney cortex was investigated. With increasing concentrations of Ca2+, phosphatidylserine or dioctanoylglycerol in the reaction mixture, regucalcin (10[-8] M) caused a remarkable inhibition of protein kinase C activity. Regucalcin did not have a significant effect on protein kinase C activity in the presence of phosphatidylserine or dioctanoylglycerol without Ca2+ addition. Moreover, regucalcin significantly inhibited phorbol 12-myristate 13-acetate (PMA)-increased protein kinase C activity. Meanwhile, staurosporine (10[-9] M) caused a significant inhibition of protein kinase C activity. This inhibition was further enhanced by regucalcin addition. Regucalcin itself did not have protein kinase activity in either the presence or the absence of both Ca2+ and phospholipids. These results clearly indicate that regucalcin has an inhibitory effect on protein kinase C activity in the cytosol of rat kidney cortex. This inhibitory effect may be partly due to the regucalcin-induced Ca2+ binding.

Inhibition of tissue factor gene activation in cultured endothelial cells by curcumin. Suppression of activation of transcription factors Egr-1, AP-1, and NF-kappa B.

Binding of plasma factor VII(a) to tissue factor (TF) initiates the coagulation cascade. In health, TF is not expressed in endothelial cells. However, endothelial cells express TF in response to lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF alpha), and other biological stimuli. TF expression by endothelial cells is implicated in thrombotic disorders in patients with a variety of clinical disorders. In the present study, we demonstrate that curcumin (diferulolylmethane), a known anticarcinogenic and anti-inflammatory agent, inhibited phorbol 12-myristate 13-acetate (PMA), LPS, TNF alpha, and thrombin-induced TF activity and TF gene transcription in human endothelial cells. The present data show that curcumin prevented the activation of c-Rel/p65, which is essential for TF gene activation in endothelial cells, by impairing the proteolytic degradation inhibitor protein, I kappa B alpha. The data also show that curcumin downregulated AP-1 binding activity. The present studies are the first to demonstrate that PMA, but not LPS, TNF alpha, and thrombin, induced Egr-1 binding to the second serum-responsive region (SRR-2) of TF promoter and that curcumin inhibited the PMA-induced Egr-1 binding to SRR-2. Overall, the data suggest that the anticarcinogenic and anti-inflammatory properties of curcumin may be related to its ability to inhibit cellular gene expression regulated by transcription factors NF-kappa B, AP-1, and Egr-1.

Early response gene signalling in bryostatin-stimulated primary B chronic lymphocytic leukaemia cells in vitro

The protein kinase C activator bryostatin induces differentiation and antagonizes the effects of tumour-promoting phorbol esters in a number of different cell types. We show here that bryostatin preferentially inhibits phorbol 12-myristate 13-acetate (PMA)-induced proliferation compared with differentiation in a number of different B chronic lymphocytic leukaemia (BCLL) cell populations examined. By using a panel of 11 early-response gene probes in Northern hybridization analysis, we found that the profile of genes induced in response to bryostatin and PMA was qualitatively similar and displayed comparable sensitivities to inhibition with the serine-threonine kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine hydrochloride (H7), consistent with common signalling through protein kinase C. However, the nuclear oncogene. c-myc, which was induced strongly in response to PMA treatment, was only marginally up-regulated by bryostatin. In addition, bryostatin selectively inhibited the magnitude of PMA-responsive induction of c-myc, to a degree commensurate with its antagonistic effects seen at the biological level. Finally, an anti-sense oligonucleotide blockade of c-myc inhibited PMA-induced proliferation but not the differentiation of BCLL cells, implicating this nuclear oncogene as an important determinant distinguishing PMA from bryostatin-coupled biological responses and also as a candidate third-messenger effector target for the anti-tumour effects of bryostatin.

Cannabinoid Inhibition of Adenylate Cyclase-mediated Signal Transduction and Interleukin 2 (IL-2) Expression in the Murine T-cell Line, EL4.IL-2

Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.