RÉSUMÉ
Caffeic acid phenethyl ester (CAPE), an active ingredient of honeybee propolis, has been identified as having anti-inflammatory, anti-viral and anti-cancer properties. Since the deficiency of gap junctional intercellular communication (GJIC) has been shown to be a characteristic of most cancer cells, this study was designed to test the hypothesis that the anti-carcinogenic activity of CAPE might be related to its ability to restore GJIC in tumorigenic GJIC-deficient cells (WB-ras2 cells). The results showed that CAPE restored GJIC, phosphorylation of connexin 43 (Cx43) and its normal localization on the plasma membrane in WB-ras2 cells after 3 days at 5 microg/ml concentration. Additionally, CAPE inhibited growth in soft agar and decreased the protein level of p21(ras). The results are consistent with the hypothesis that the anti-cancer mechanism of CAPE may be mediated by its ability to restore GJIC.
Sujet(s)
Anticarcinogènes/pharmacologie , Acides caféiques/pharmacologie , Communication cellulaire/effets des médicaments et des substances chimiques , Transformation cellulaire néoplasique/effets des médicaments et des substances chimiques , Jonctions communicantes/effets des médicaments et des substances chimiques , Gènes ras/effets des médicaments et des substances chimiques , Foie/effets des médicaments et des substances chimiques , Alcool phénéthylique/analogues et dérivés , Alcool phénéthylique/pharmacologie , Animaux , Anticarcinogènes/toxicité , Acides caféiques/toxicité , Adhérence cellulaire/physiologie , Communication cellulaire/physiologie , Division cellulaire/effets des médicaments et des substances chimiques , Division cellulaire/physiologie , Lignée de cellules transformées , Transformation cellulaire néoplasique/génétique , Transformation cellulaire néoplasique/anatomopathologie , Connexine 43/biosynthèse , Connexine 43/métabolisme , Cellules épithéliales/cytologie , Cellules épithéliales/effets des médicaments et des substances chimiques , Cellules épithéliales/métabolisme , Jonctions communicantes/physiologie , Expression des gènes/effets des médicaments et des substances chimiques , Gènes ras/physiologie , Foie/cytologie , Foie/métabolisme , Protéine oncogène p21(ras)/biosynthèse , Protéine oncogène p21(ras)/génétique , Alcool phénéthylique/toxicité , Phosphorylation/effets des médicaments et des substances chimiques , Rats , Rats de lignée F344 , RT-PCRRÉSUMÉ
Epidemiological studies consistently indicate that consumption of fruits and vegetables lowers cancer risk in humans and suggest that certain dietary constituents may be effective in preventing colon cancer. Plant-derived phenolic compounds manifest many beneficial effects and can potentially inhibit several stages of carcinogenesis in vivo. In this study, we investigated the efficacy of several plant-derived phenolics, including caffeic acid phenethyl ester (CAPE), curcumin, quercetin and rutin, for the prevention of tumors in C57BL/6J-Min/+ (Min/+) mice. These animals bear a germline mutation in the Apc gene and spontaneously develop numerous intestinal adenomas by 15 weeks of age. At a dietary level of 0.15%, CAPE decreased tumor formation in Min/+ mice by 63%. Curcumin induced a similar tumor inhibition. Quercetin and rutin, however, both failed to alter tumor formation at dietary levels of 2%. Examination of intestinal tissue from the treated animals showed that tumor prevention by CAPE and curcumin was associated with increased enterocyte apoptosis and proliferation. CAPE and curcumin also decreased expression of the oncoprotein beta-catenin in the enterocytes of the Min/+ mouse, an observation previously associated with an antitumor effect. These data place the plant phenolics CAPE and curcumin among a growing list of anti-inflammatory agents that suppress Apc-associated intestinal carcinogenesis.
Sujet(s)
Polypose adénomateuse colique/anatomopathologie , Tumeurs de l'intestin/prévention et contrôle , Phénols/pharmacologie , Plantes/composition chimique , Transactivateurs , Polypose adénomateuse colique/métabolisme , Animaux , Division cellulaire/effets des médicaments et des substances chimiques , Protéines du cytosquelette/métabolisme , Modèles animaux de maladie humaine , Entérocytes/cytologie , Entérocytes/effets des médicaments et des substances chimiques , Souris , Souris de lignée C57BL , bêta-CaténineRÉSUMÉ
The CC chemokine, monocyte chemotactic protein, 1 (MCP-1) functions as a major chemoattractant for T-cells and monocytes by interacting with the seven-transmembrane G protein-coupled receptor CCR2. To identify which residues of MCP-1 contribute to signaling though CCR2, we mutated all the surface-exposed residues to alanine and other amino acids and made some selective large changes at the amino terminus. We then characterized the impact of these mutations on three postreceptor pathways involving inhibition of cAMP synthesis, stimulation of cytosolic calcium influx, and chemotaxis. The results highlight several important features of the signaling process and the correlation between binding and signaling: The amino terminus of MCP-1 is essential as truncation of residues 2-8 ([1+9-76]hMCP-1) results in a protein that cannot stimulate chemotaxis. However, the exact peptide sequence may be unimportant as individual alanine mutations or simultaneous replacement of residues 3-6 with alanine had little effect. Y13 is also important and must be a large nonpolar residue for chemotaxis to occur. Interestingly, both Y13 and [1+9-76]hMCP-1 are high-affinity binders and thus affinity of these mutants is not correlated with ability to promote chemotaxis. For the other surface residues there is a strong correlation between binding affinity and agonist potency in all three signaling pathways. Perhaps the most interesting observation is that although Y13A and [1+9-76]hMCP are antagonists of chemotaxis, they are agonists of pathways involving inhibition of cAMP synthesis and, in the case of Y13A, calcium influx. These results demonstrate that these two well-known signaling events are not sufficient to drive chemotaxis. Furthermore, it suggests that specific molecular features of MCP-1 induce different conformations in CCR2 that are coupled to separate postreceptor pathways. Therefore, by judicious design of antagonists, it should be possible to trap CCR2 in conformational states that are unable to stimulate all of the pathways required for chemotaxis.
Sujet(s)
Acides aminés/physiologie , Chimiokine CCL2/physiologie , Récepteurs aux chimiokines/physiologie , Récepteurs aux cytokines/physiologie , Transduction du signal , Acides aminés/isolement et purification , Sites de fixation/génétique , Calcium/antagonistes et inhibiteurs , Calcium/métabolisme , Lignée cellulaire , Membrane cellulaire/génétique , Membrane cellulaire/physiologie , Inhibition de la migration cellulaire , Chimiokine CCL2/agonistes , Chimiokine CCL2/génétique , AMP cyclique/antagonistes et inhibiteurs , Humains , Fragments peptidiques/génétique , Fragments peptidiques/métabolisme , Fragments peptidiques/physiologie , Structure secondaire des protéines/génétique , Structure tertiaire des protéines/génétique , Récepteurs CCR2 , Récepteurs aux chimiokines/métabolisme , Récepteurs aux cytokines/métabolisme , Transduction du signal/génétique , Tyrosine/génétique , Tyrosine/physiologieRÉSUMÉ
The CC chemokine, MCP-1, has been identified as a major chemoattractant for T cells and monocytes, and plays a significant role in the pathology of inflammatory diseases. To identify the regions of MCP-1 that contact its receptor, CCR2, we substituted all surface-exposed residues with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrophobicity, or aromaticity at specific positions. The binding affinity of each mutant for CCR2 was assayed with THP-1 and CCR2-transfected CHL cells. The majority of point mutations had no effect. Residues at the N-terminus of the protein, known to be crucial for signaling, contribute less than a factor of 10 to the binding affinity. However, two clusters of primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 A hydrophobic groove, reduced the level of binding by 15-100-fold. A peptide fragment encompassing residues 13-35 recapitulated some of the mutational data derived from the intact protein. It exhibited modest binding as a linear peptide and dramatically improved affinity when the region which adopts a single turn of a 3(10)-helix in the protein, which includes R24, was constrained by a disulfide bond. Additional constraints at the ends of the peptide, corresponding to the disulfide between the first and third cysteines in MCP-1, yielded further improvements in affinity. Together, these data suggest a model in which a large surface area of MCP-1 contacts the receptor, and the accumulation of a number of weak interactions results in the 35 pM affinity observed for the wild-type (WT) protein. The receptor binding site of MCP-1 also is significantly different from the binding sites of RANTES and IL-8, providing insight into the issue of receptor specificity. It was previously shown that the N-terminus of CCR2 is critical for binding MCP-1 [Monteclaro, F. S., and Charo, I. F. (1996) J. Biol. Chem. 271, 19084-92; Monteclaro, F. S., and Charo, I. F. (1997) J. Biol. Chem. 272, 23186-90]. Point mutations of six acidic residues in this region of the receptor were made to test their role in ligand binding. This identified D25 and D27 of the DYDY motif as being important. On the basis of our data, we propose a model in which the receptor N-terminus lies along the hydrophobic groove in an extended fashion, placing the DYDY motif near the basic cluster involving R24 and K49 of MCP-1. This in turn orients the signaling residues (Y13 and the N-terminus) for productive interaction with the receptor.
Sujet(s)
Chimiokine CCL2/composition chimique , Chimiokine CCL2/métabolisme , Récepteurs aux chimiokines/métabolisme , Récepteurs aux cytokines/métabolisme , Séquence d'acides aminés , Animaux , Sites de fixation/génétique , Lignée cellulaire , Chimiokine CCL2/génétique , Cricetinae , Cricetulus , Humains , Ligands , Modèles moléculaires , Données de séquences moléculaires , Mutagenèse dirigée , Résonance magnétique nucléaire biomoléculaire , Fragments peptidiques/composition chimique , Fragments peptidiques/génétique , Fragments peptidiques/métabolisme , Récepteurs CCR2 , Récepteurs aux chimiokines/composition chimique , Récepteurs aux cytokines/composition chimiqueRÉSUMÉ
We investigated the mechanisms by which caffeic acid phenethyl ester (CAPE), a phenolic antioxidant, inhibited the stimulation of prostaglandin (PG) synthesis in cultured human oral epithelial cells and in an animal model of acute inflammation. Treatment of cells with CAPE (2.5 microg/ml) suppressed phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) and calcium ionophore (A23187)-mediated induction of PGE2 synthesis. This relatively low concentration of CAPE did not affect amounts of cyclooxygenase (COX) enzymes. CAPE nonselectively inhibited the activities of baculovirus-expressed hCOX-1 and hCOX-2 enzymes. TPA- and A23187-stimulated release of arachidonic acid from membrane phospholipids was also suppressed by CAPE (4-8 microg/ml). Higher concentrations of CAPE (10-20 microg/ml) suppressed the induction of COX-2 mRNA and protein mediated by TPA. Transient transfections using human COX-2 promoter deletion constructs were performed; the effects of TPA and CAPE were localized to a 124-bp region of the COX-2 promoter. In the rat carrageenan air pouch model of inflammation, CAPE (10-100 mg/kg) caused dose-dependent suppression of PG synthesis. Amounts of COX-2 in the pouch were markedly suppressed by 100 mg/kg CAPE but were unaffected by indomethacin. These data are important for understanding the anticancer and anti-inflammatory properties of CAPE.
Sujet(s)
Anti-inflammatoires non stéroïdiens/pharmacologie , Anticarcinogènes/pharmacologie , Acides caféiques/pharmacologie , Inhibiteurs des cyclooxygénases/pharmacologie , Inflammation/génétique , Isoenzymes/biosynthèse , Muqueuse de la bouche/cytologie , Alcool phénéthylique/analogues et dérivés , Régions promotrices (génétique)/effets des médicaments et des substances chimiques , Prostaglandin-endoperoxide synthases/biosynthèse , Air , Animaux , Acides arachidoniques/métabolisme , A-23187/antagonistes et inhibiteurs , A-23187/pharmacologie , Carcinome épidermoïde/anatomopathologie , Carragénane/toxicité , Membrane cellulaire/effets des médicaments et des substances chimiques , Membrane cellulaire/métabolisme , Cyclooxygenase 2 , Inhibiteurs de la cyclooxygénase 2 , Dinoprostone/biosynthèse , Activation enzymatique/effets des médicaments et des substances chimiques , Induction enzymatique/effets des médicaments et des substances chimiques , Cellules épithéliales/effets des médicaments et des substances chimiques , Cellules épithéliales/enzymologie , Vecteurs génétiques/génétique , Humains , Indométacine/pharmacologie , Inflammation/induit chimiquement , Inflammation/métabolisme , Ionophores/antagonistes et inhibiteurs , Ionophores/pharmacologie , Isoenzymes/génétique , Mâle , Lipides membranaires/métabolisme , Protéines membranaires , Nucleopolyhedrovirus/génétique , Alcool phénéthylique/pharmacologie , Phospholipides/métabolisme , Prostaglandin-endoperoxide synthases/génétique , Rats , Rats de lignée LEW , Protéines de fusion recombinantes/biosynthèse , 12-Myristate-13-acétate de phorbol/antagonistes et inhibiteurs , 12-Myristate-13-acétate de phorbol/pharmacologie , Transfection , Cellules cancéreuses en cultureRÉSUMÉ
To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, [1+9-76]MCP-1 (MCP-1 lacking residues 2-8), antagonizes wild type MCP-1 by competitive inhibition, or by a dominant negative mechanism wherein heterodimers of MCP-1 and [1+9-76]MCP-1 bind to the receptor but are signaling incompetent. Consistent with the finding that MCP-1 can bind and activate the receptor as a monomer, we demonstrate that binding of MCP-1 in the presence of [1+9-76]MCP-1 over a range of concentrations of both ligands fits well to a simple model in which monomeric [1+9-76]MCP-1 functions as a competitive inhibitor of monomeric MCP-1. These results are crucial for elucidating the molecular details of receptor binding and activation, for interpreting mutagenesis data, for understanding how antagonistic chemokine variants function, and for the design of receptor antagonists.
Sujet(s)
Chimiokine CCL2/métabolisme , Récepteurs aux chimiokines , Récepteurs aux cytokines/métabolisme , Dimérisation , Disulfures/composition chimique , Humains , Spectroscopie par résonance magnétique , Liaison aux protéines , Récepteurs CCR2 , Récepteurs aux cytokines/antagonistes et inhibiteurs , Protéines recombinantes/métabolismeRÉSUMÉ
Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant derived from the propolis of honeybee hives. CAPE was shown to inhibit the formation of intracellular hydrogen peroxide and oxidized bases in DNA of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated HeLa cells and was also found to induce a redox change that correlated with differential growth effects in transformed cells but not the nontumorigenic parental ones. Mediated via the electrophile or human antioxidant response element (hARE), induction of the expression of NAD(P)H quinone oxidoreductase (NQO1) and glutathione S-transferase Ya subunit genes by certain phenolic antioxidants has been correlated with the chemopreventive properties of these agents. Here, we determined by Northern analysis that CAPE treatment of hepatoma cells stimulates NQO1 gene expression in cultured human hepatoma cells (HepG2), and we characterized the effects of CAPE treatment on the expression of a reporter gene either containing or lacking the hARE or carrying a mutant version of this element in rodent hepatoma (Hepa-1) transfectants. A dose-dependent transactivation of human hARE-mediated chloramphenicol acetyltransferase (cat) gene expression was observed upon treatments of the Hepa-1 transfectants with TPA, a known inducer, as well as with CAPE. The combined treatments resulted in an apparent additive stimulation of the reporter expression. To learn whether this activation of cat gene expression was effected by protein kinase C in CAPE-treated cells, a comparison was made of cat gene activity after addition of calphostin, a protein kinase C inhibitor. Calphostin reduced the cat gene induction by TPA but not by CAPE, suggesting that stimulation of gene expression in this system by these agents proceeds via distinct mechanisms. Band-shift experiments to examine binding of transactivator proteins from nuclear extracts of treated and untreated cells to a hARE DNA probe showed that TPA exposure increased the binding level. In contrast, binding of factors to this probe was inhibited after either in vivo treatment of cells with CAPE or in vitro addition of this compound to the nuclear extract. In view of the clear stimulation by CAPE of gene expression mediated by hARE, possible explanations of this result are discussed.
Sujet(s)
Antioxydants/pharmacologie , Acides caféiques/pharmacologie , Alcool phénéthylique/analogues et dérivés , Quinone reductases/génétique , ADN/métabolisme , Humains , Alcool phénéthylique/pharmacologie , 12-Myristate-13-acétate de phorbol/pharmacologie , Facteur de transcription AP-1/métabolismeRÉSUMÉ
The effects of topical applications of very low doses of curcumin (the major yellow pigment in turmeric and the Indian food curry) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidation of DNA bases in the epidermis and on tumor promotion in mouse skin were investigated. CD-1 mice were treated topically with 200 nmol of 7,12-dimethylbenz[a]anthracene followed one week later by 5 nmol of TPA alone or together with 1, 10, 100 or 3000 nmol of curcumin twice a week for 20 weeks. Curcumin-mediated effects on TPA-induced formation of the oxidized DNA base 5-hydroxymethyl-2'-deoxyuridine (HMdU) and tumor formation were determined. All dose levels of curcumin inhibited the mean values of TPA-induced HMdU formation in epidermal DNA (62-77% inhibition), but only the two highest doses of curcumin strongly inhibited TPA-induced tumor promotion (62-79% inhibition of tumors per mouse and tumor volume per mouse). In a second experiment, topical application of 20 or 100 nmol (but not 10 nmol) of curcumin together with 5 nmol TPA twice a week for 18 weeks markedly inhibited TPA-induced tumor promotion. Curcumin had a strong inhibitory effect on DNA and RNA synthesis (IC50 = 0.5-1 microM) in cultured HeLa cells, but there was little or no effect on protein synthesis.
Sujet(s)
Antinéoplasiques/pharmacologie , Cancérogènes/toxicité , Curcumine/pharmacologie , ADN/effets des médicaments et des substances chimiques , Tumeurs cutanées/prévention et contrôle , 12-Myristate-13-acétate de phorbol/toxicité , 7,12-Diméthyl-benzo[a]anthracène/toxicité , Administration par voie topique , Animaux , Antinéoplasiques/administration et posologie , Curcumine/administration et posologie , Relation dose-effet des médicaments , Tests de criblage d'agents antitumoraux , Femelle , Cellules HeLa , Humains , Souris , Tumeurs expérimentales/induit chimiquement , Tumeurs expérimentales/prévention et contrôle , Oxydoréduction , Tumeurs cutanées/induit chimiquementRÉSUMÉ
Caffeic acid phenethyl ester (CAPE), an active component of propolis from bee hives, exerts a plethora of biological changes in diverse systems. These include antimitogenic, anticarcinogenic, anti-inflammatory and immunomodulatory responses. CAPE directly induces programmed cell death (apoptosis) in type 5 adenovirus (Ad5)-transformed cloned rat embryo fibroblast cells, wt3A. To identify the gene and protein expression changes induced by CAPE in wt3A cells we used a strategy involving in vitro translation of mRNAs followed by high resolution two-dimensional (2D) gel electrophoresis. This approach results in the detection of 745 spots, including 172 displaying differences in expression upon exposure to CAPE. A high proportion of spots show profound changes in spot intensity (42 spots with increased and 27 spots with decreased intensity) following CAPE treatment. These studies provide a basis for comparing these changes to known protein patterns of various cell populations with an ultimate aim of identifying families of polypeptides responsible for the up- and down-regulation of cellular proteins during CAPE-induced apoptosis. Specific newly appearing or completely disappearing spots (52 and 51 molecular species, respectively) will be used to attempt to identify and retrieve their cDNA counterparts from an ordered cDNA library. These approaches represent a novel strategy for cloning genes associated with and potentially mediating apoptosis.
RÉSUMÉ
Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antiinflammatory, and immunomodulatory properties. The molecular basis for these diverse properties is not known. Since the role of the nuclear factor NF-kappa B in these responses has been documented, we examined the effect of CAPE on this transcription factor. Our results show that the activation of NF-kappa B by tumor necrosis factor (TNF) is completely blocked by CAPE in a dose- and time-dependent manner. Besides TNF, CAPE also inhibited NF-kappa B activation induced by other inflammatory agents including phorbol ester, ceramide, hydrogen peroxide, and okadaic acid. Since the reducing agents reversed the inhibitory effect of CAPE, it suggests the role of critical sulfhydryl groups in NF-kappa B activation. CAPE prevented the translocation of the p65 subunit of NF-kappa B to the nucleus and had no significant effect on TNF-induced I kappa B alpha degradation, but did delay I kappa B alpha resynthesis. The effect of CAPE on inhibition of NF-kappa B binding to the DNA was specific, in as much as binding of other transcription factors including AP-1, Oct-1, and TFIID to their DNA were not affected. When various synthetic structural analogues of CAPE were examined, it was found that a bicyclic, rotationally constrained, 5,6-dihydroxy form was superactive, whereas 6,7-dihydroxy variant was least active. Thus, overall our results demonstrate that CAPE is a potent and a specific inhibitor of NF-kappa B activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities.
Sujet(s)
Acides caféiques/pharmacologie , Protéines I-kappa B , Facteur de transcription NF-kappa B/antagonistes et inhibiteurs , Alcool phénéthylique/analogues et dérivés , Séquence nucléotidique , Acides caféiques/composition chimique , Lignée cellulaire , Céramides/pharmacologie , Protéines de liaison à l'ADN/métabolisme , Histiocytes/cytologie , Humains , Données de séquences moléculaires , Inhibiteur alpha de NF-KappaB , Acide okadaïque/pharmacologie , Alcool phénéthylique/composition chimique , Alcool phénéthylique/pharmacologie , Phosphorylation/effets des médicaments et des substances chimiques , Liaison aux protéines , Relation structure-activité , 12-Myristate-13-acétate de phorbol/pharmacologie , Facteur de nécrose tumorale alpha/pharmacologieRÉSUMÉ
Topical application of caffeic acid phenethyl ester (CAPE), a constituent of the propolis of honeybee hives, to the backs of CD-1 mice previously initiated with 7,12-dimethylbenz[a]anthracene (DMBA) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion and the formation of 5-hydroxymethyl-2'-deoxyuridine (HMdU) in epidermal DNA. Topical application of 5 nmol TPA twice weekly for 20 weeks to mice previously initiated with 200 nmol of DMBA resulted in 18.8 skin papillomas per mouse. Topical application of 1, 10, 100 or 3000 nmol of CAPE together with 5 nmol of TPA twice a week for 20 weeks inhibited the number of skin papillomas per mouse by 24, 30, 45 or 70%, respectively, and tumor size per mouse was decreased by 42, 66, 53 or 74%, respectively. Topical application of 5 nmol of TPA twice weekly for 20 weeks to mice previously initiated with DMBA produced an average of 12.6 HMdU residues per 10(4) normal bases in epidermal DNA. Topical application of 1, 10, 100 or 3000 nmol of CAPE with 5 nmol of TPA twice weekly for 20 weeks to DMBA-initiated mice decreased the level of HMdU in epidermal DNA by 40-93%. The in vitro addition of 1.25, 2.5, 5, 10 or 20 microM CAPE to cultured HeLa cells inhibited the synthesis of DNA by 32, 44, 66, 79 or 95%, respectively, the synthesis of RNA was inhibited by 39, 43, 58, 64 or 75%, respectively, and the synthesis of protein was inhibited by 29, 30, 37, 32 or 47%, respectively. The results indicate a potent inhibitory effect of CAPE on TPA-induced tumor promotion and TPA-induced formation of HMdU in DNA of mouse skin as well as an inhibitory effect of CAPE on the synthesis of DNA, RNA and protein in culture HeLa cells.
Sujet(s)
Acides caféiques/pharmacologie , ADN/biosynthèse , Alcool phénéthylique/analogues et dérivés , Biosynthèse des protéines , ARN/biosynthèse , Tumeurs cutanées/prévention et contrôle , 12-Myristate-13-acétate de phorbol/pharmacologie , 7,12-Diméthyl-benzo[a]anthracène , Animaux , Anticarcinogènes/pharmacologie , Cancérogènes/pharmacologie , Altération de l'ADN , Femelle , Cellules HeLa , Humains , Souris , Stress oxydatif , Papillome/induit chimiquement , Papillome/prévention et contrôle , Alcool phénéthylique/pharmacologie , Tumeurs cutanées/induit chimiquement , 12-Myristate-13-acétate de phorbol/antagonistes et inhibiteursRÉSUMÉ
Efficient replication of HIV-1 requires integration of a DNA copy of the viral genome into a chromosome of the host cell. Integration is catalyzed by the viral integrase, and we have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE, 2), and curcumin confer inhibitory activity against HIV-1 integrase. We now extend these findings by performing a comprehensive structure-activity relationship using CAPE analogues. Approximately 30 compounds have been prepared as HIV integrase inhibitors based on the structural lead provided by CAPE, which has previously been shown to exhibit an IC50 value of 7 microM in our integration assay. These analogues were designed to examine specific features of the parent CAPE structure which may be important for activity. Among the features examined for their effects on inhibitory potency were ring substitution, side chain length and composition, and phenyl ring conformational orientation. In an assay which measured the combined effect of two sequential steps, dinucleotide cleavage and strand transfer, several analogues have IC50 values for 3'-processing and strand transfer lower than those of CAPE. Inhibition of strand transfer was assayed using both blunt-ended and "precleaved" DNA substrates. Disintegration using an integrase mutant lacking the N-terminal zinc finger and C-terminal DNA-binding domains was also inhibited by these analogues, suggesting that the binding site for these compounds resides in the central catalytic core. Several CAPE analogues were also tested for selective activity against transformed cells. Taken together, these results suggest that the development of novel antiviral agents for the treatment of acquired immune deficiency syndrome can be based upon inhibition of HIV-1 integrase.
Sujet(s)
Antiviraux/synthèse chimique , Acides caféiques/composition chimique , DNA nucleotidyltransferases/antagonistes et inhibiteurs , Antienzymes/synthèse chimique , Alcool phénéthylique/analogues et dérivés , Animaux , Apoptose/effets des médicaments et des substances chimiques , Séquence nucléotidique , Sites de fixation , Acides caféiques/pharmacologie , Lignée de cellules transformées , ADN/composition chimique , ADN/métabolisme , VIH (Virus de l'Immunodéficience Humaine)/effets des médicaments et des substances chimiques , Humains , Hydroxylation , Integrases , Spectroscopie par résonance magnétique , Conformation moléculaire , Données de séquences moléculaires , Structure moléculaire , Alcool phénéthylique/composition chimique , Alcool phénéthylique/pharmacologie , Rats , Relation structure-activité , Cellules cancéreuses en culture , Doigts de zincRÉSUMÉ
Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of bee hives, was shown previously to block tumor promoter- and carcinogen-generated oxidative processes in several assays and to engender differential toxicity to some transformed cells. To study the mechanisms of CAPE-induced differential cytotoxicity, nontumorigenic rat embryo fibroblasts (CREF) and adenovirus (type 5)-transformed CREF cells (Wt3A) were used. As shown by nucleosomal-length DNA degradation, morphological alterations by electron microscopy, in situ labeling of 3'-OH ends, and the appearance of a hypodiploid cell population by bivariant flow cytometry, cell death induced by CAPE in the transformed Wt3A cells was apoptosis. Under the same CAPE treatment conditions, CREF cells transiently growth arrested. Both CREF and Wt3A cells were radioresistant, suggesting deficiencies in the proteins controlling the G1 checkpoint. To explore possible mechanisms of CAPE-induced apoptosis, it was determined whether CAPE-induced toxicity was influenced by the redox state of the cells. Depletion of cellular glutathione (GSH) with buthionine sulfoximine before CAPE treatment caused CREF sensitive to CAPE-induced cell death. GSH levels were also determined in CAPE-treated CREF and Wt3A cells. The GSH level in the CREF cells was unaffected by CAPE, whereas the Wt3A cells showed a significant reduction. When the GSH levels were increased in Wt3A cells by treatment with the reducing agent, N-acetyl-cysteine before CAPE treatment, the Wt3A cells were partially rescued. Furthermore, Bcl2, which protects cells from oxidative stress, had a protective effect against CAPE-induced apoptosis in Wt3A cells. Finally, the sensitivity of Wt3A cells to a known oxidant, hydrogen peroxide (H2O2), was examined. Wt3A cells were killed by H2O2-induced apoptosis, whereas CREF cells remained resistant. When Wt3A cells were treated with catalase, a cellular enzyme that inactivates H2O2, CAPE-induced apoptosis in Wt3A cells was reduced, further proving that Wt3A cells were more sensitive than CREF cells to oxidative stress. These results suggest that CAPE can modulate the redox state of cells. Sensitivity of cells to CAPE-induced cell death may be determined by the loss of normal redox state regulation in transformed cells.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Acides caféiques/pharmacologie , Transformation cellulaire néoplasique/anatomopathologie , Alcool phénéthylique/analogues et dérivés , Acétylcystéine/pharmacologie , Animaux , Catalase/métabolisme , Cycle cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire , Altération de l'ADN/effets des médicaments et des substances chimiques , Glutathion/métabolisme , Peroxyde d'hydrogène/pharmacologie , Oxydoréduction , Alcool phénéthylique/pharmacologie , Protéines proto-oncogènes/physiologie , Protéines proto-oncogènes c-bcl-2 , Rats , Rats de lignée F344RÉSUMÉ
Two-dimensional proton NMR and energy minimization computations have been employed to characterize the conformations of the N-(deoxyguanosin-8-yl)aminofluorene adduct [(AF)G] positioned opposite deoxyguanosine in one, and opposite deoxyinosine in another DNA undecamer duplex in aqueous solution. The two oligomer duplexes used in this study are d[C1-C2-A3-T4-C5-(AF)G6-C7-T8-A9-C10-C11].[G12-G13-T14 -A15-G16-X17-G18- A19-T20-G21-G22], where X17 was deoxyinosine in one duplex and deoxyguanosine in another. The exchangeable and nonexchangeable protons of the DNA are well resolved and narrow in the NMR spectra of the duplexes, and the base and sugar nucleic acid protons were assigned by NOESY and COSY data sets. All nine of the nonexchangeable aminofluorene ring protons were also assigned for the duplex that has deoxyinosine across from the modification site, and the (AF)G-I structure was employed to model the (AF)G-G one. The NOE distance restraints establish that the glycosidic torsion angle at (AF)G6 is syn. All other glycosidic torsion angles are anti, Watson-Crick type A.T and G-C base pairing is intact throughout the duplex except at the site of modification, and the helix maintains an overall B-DNA conformation. The syn orientation at the (AF)G6 places the aminofluorene ring in the B-DNA minor groove in a conformation similar to that found previously when the (AF)G was positioned opposite deoxyadenosine [Norman et al. (1989) Biochemistry 28, 7462-7476].
Sujet(s)
N-Fluorén-2-yl-acétamide/analogues et dérivés , Adduits à l'ADN/composition chimique , Désoxyguanosine/analogues et dérivés , N-Fluorén-2-yl-acétamide/composition chimique , N-Fluorén-2-yl-acétamide/métabolisme , Composition en bases nucléiques , Séquence nucléotidique , Adduits à l'ADN/métabolisme , Désoxyguanosine/composition chimique , Désoxyguanosine/métabolisme , Inosine/analogues et dérivés , Inosine/composition chimique , Inosine/métabolisme , Données de séquences moléculaires , Conformation d'acide nucléique , Hétéroduplexes d'acides nucléiques/composition chimique , ProtonsRÉSUMÉ
Caffeic acid phenethyl ester (CAPE) was evaluated for its potential in regulating keratinocyte proliferation. CAPE inhibited the proliferation of SV40 transformed keratinocytes (Z114) in a concentration- and time-dependent manner. Inhibition by CAPE was seen with 0.5 to 5.0 micrograms/ml at 48 h. Cell toxicity was observed at 10 micrograms/ml by changes in morphology and decreased viability. Pretreatment of Z114 cells with CAPE significantly prevented the full induction of ornithine decarboxylase (ODC) by epidermal growth factor (EGF) in a concentration- and time-dependent manner. Inhibition was observed with a concentration of CAPE as low as 1 microgram/ml, and complete inhibition of ODC induction by EGF occurred at 5 micrograms/ml. Northern analysis showed that treatment of cells with CAPE for 24 h suppressed EGF induction of ODC gene expression. Incubation of Z114 cells with CAPE for 24 h resulted in a concentration-dependent decrease in EGF binding and a 30% reduction in the EGF induced autophosphorylation of the EGF receptor. CAPE decreased both membranous and cytosolic PKC activity in a concentration- and time-dependent manner. Because significant inhibition of keratinocyte proliferation occurred at concentrations of CAPE that interfered with PKC activity and EGF signal transduction but did not cause overt toxicity, CAPE may prove useful for the treatment of hyperproliferative skin diseases.
Sujet(s)
Acides caféiques/toxicité , Division cellulaire/effets des médicaments et des substances chimiques , Facteur de croissance épidermique/pharmacologie , Kératinocytes/effets des médicaments et des substances chimiques , Ornithine decarboxylase/effets des médicaments et des substances chimiques , Ornithine decarboxylase/métabolisme , Alcool phénéthylique/analogues et dérivés , Sites de fixation , Technique de Northern , Technique de Western , Membrane cellulaire/effets des médicaments et des substances chimiques , Membrane cellulaire/métabolisme , Cytosol/enzymologie , Relation dose-effet des médicaments , Récepteurs ErbB/métabolisme , Humains , Mâle , Ornithine decarboxylase/biosynthèse , Alcool phénéthylique/toxicité , Protéine kinase C/métabolisme , Transduction du signal/effets des médicaments et des substances chimiques , Facteurs tempsRÉSUMÉ
The active component of the honeybee hive product propolis, caffeic acid phenethyl ester (CAPE), induces a selective growth suppressive and toxic effect toward cloned rat embryo fibroblast cells transformed by adenovirus type 5 (Ad5) or the Ad5 E1A transforming gene versus untransformed cloned rat embryo fibroblast cells (Z-z. Su et al., Mol. Carcinog., 4: 231-242, 1991). The present study was conducted to determine whether CAPE-induced growth suppression/toxicity was a direct result of expression of the Ad5 E1A and E1B transforming genes or a consequence of the action of these genes resulting in the transformed state. For this investigation we used somatic cell hybrids and 5-azacytidine-treated Ad5-transformed rat embryo cells that display different stages of expression of the transformed phenotype. This series of cell lines has permitted us to determine whether expression of the transformed state and the stage of transformation progression regulates CAPE sensitivity. Evidence is presented indicating that sensitivity to CAPE is directly determined by the state of expression of the transformed progression phenotype, as opposed to simply the expression of the Ad5 E1A and E1B transforming genes. These results provide further evidence that CAPE may represent a unique compound that can specifically target progressed transformed cells for growth suppression and toxicity. An understanding of the mechanism underlying this selective effect of CAPE could result in the identification of important biochemical pathways mediating cellular transformation and progression of the transformed state.
Sujet(s)
Adénovirus humains/génétique , Acides caféiques/toxicité , Division cellulaire/effets des médicaments et des substances chimiques , Transformation cellulaire néoplasique , Cytotoxines/toxicité , Alcool phénéthylique/analogues et dérivés , Protéines E1A d'adénovirus/génétique , Protéines E1B d'adénovirus/génétique , Animaux , Lignée de cellules transformées , Cellules cultivées , Clones cellulaires , Embryon de mammifère , Expression des gènes/effets des médicaments et des substances chimiques , Alcool phénéthylique/toxicité , ARN messager/effets des médicaments et des substances chimiques , ARN messager/métabolisme , Rats , Rat Sprague-Dawley , Facteurs temps , Transcription génétique/effets des médicaments et des substances chimiquesRÉSUMÉ
Three experiments using 20 microM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C-->T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a-1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by 32P-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 x 10(-6), the adduct levels after a 1-h exposure of UA21 cells to 20 microM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural determinations of (deoxyguanosin-8-yl)-aminofluorene; the carcinogen is in the B-DNA minor groove and its adopts a syn conformation mispaired with an anti A. The implications of this conformational distortion in DNA structure for damage recognition by cellular repair enzymes are discussed.
Sujet(s)
Cancérogènes/toxicité , Réparation de l'ADN , Désoxyguanosine/analogues et dérivés , Imidazoles/métabolisme , Imidazoles/toxicité , Mutagènes/toxicité , Pyridines/toxicité , Dihydrofolate reductase/génétique , Animaux , Séquence nucléotidique , Cellules CHO , Cricetinae , Cricetulus , ADN/métabolisme , Désoxyguanosine/métabolisme , Modèles moléculaires , Données de séquences moléculaires , Tests de mutagénicité , Conformation d'acide nucléiqueRÉSUMÉ
Because high concentrations of IL-8 are found in the sputum of cystic fibrosis patients, we hypothesized that Pseudomonas aeruginosa (PA) induces the production of IL-8 in airway epithelial cells and in monocytes. Therefore, we incubated the supernatant from PA culture with human transformed bronchial epithelial cells (16-HBE) or with monocytes. The culture medium of 16-HBE cells that had been incubated with PA supernatant for 6 h had chemotactic activity that was inhibited by an antibody to human IL-8. The PA supernatant induced IL-8 production by primary bronchial epithelial cells, by 16-HBE cells, and by monocytes. After incubation with PA supernatant, 16-HBE cells showed a marked increase in the levels of IL-8 gene expression. The PA product responsible for IL-8 production resisted freezing, boiling, and proteolysis. This product was not lipid extractable and was present in a 1-kD filtrate. We conclude that a small molecular mass product of PA stimulates IL-8 production by 16-HBE cells and by monocytes, and that the chemotactic activity produced by 16-HBE cells after exposure to PA is due principally to IL-8.
Sujet(s)
Bronches/métabolisme , Chimiotaxie des leucocytes , Interleukine-8/biosynthèse , Monocytes/physiologie , Granulocytes neutrophiles/physiologie , Pseudomonas aeruginosa/physiologie , Technique de Northern , Lignée cellulaire , Lignée de cellules transformées , Cellules cultivées , Milieux de culture , Épithélium/métabolisme , Humains , Pseudomonas aeruginosa/classification , Pseudomonas aeruginosa/isolement et purification , ARN messager/analyse , ARN messager/biosynthèse , Spécificité d'espèceRÉSUMÉ
Caffeic acid phenethyl ester (CAPE) displays enhanced growth suppressive and toxic effects toward cloned rat embryo fibroblast (CREF) cells transformed by adenovirus type 5 (Ad5) or the Ad5 E1A transforming gene versus untransformed CREF cells (Su et al, Mol Carcinogen 4: 231-242, 1991). The present study was conducted to determine if transformation of CREF cells with additional oncogenes rendered these cells sensitive to the antiproliferative effect of CAPE. Additionally, studies were conducted to determine if reversion of the transformed phenotype could modify CAPE sensitivity. CAPE displayed increased growth suppressive activity toward CREF cells transformed by a number of oncogenes, including Ha-ras, v-src, v-raf, human papillomavirus type 18 (HPV-18) and human papillomavirus type 51 (HPV-51). Employing Ha-ras-transformed CREF (Ha-ras) and Ha-ras-transformed CREF cells overexpressing the Krev-1 suppressor gene (Ha-ras/Krev-1), evidence for a direct relationship between expression of the transformed phenotype and CAPE sensitivity was demonstrated. Ha-ras/Krev-1 cells displaying a suppression of the transformed phenotype exhibited increased resistance to CAPE-induced growth suppression versus Ha-ras cells, whereas Ha-ras/Krev-1 cells escaping transformation-suppression following in vivo growth in nude mice displayed enhanced sensitivity to growth-suppression induced by CAPE. Similarly, mutant Ad5 (H5hr1)-transformed and v-src-transformed CREF cells displaying a stable reversion in transformation also displayed a reduced sensitivity to CAPE versus their transformed counterparts. These observations indicate a direct relationship between expression of the transformed phenotype and CAPE sensitivity. Elucidation of the mechanism by which CAPE selectively inhibits growth of transformed cells should provide important insights into the critical molecular changes mediating expression of the transformed state and could help identify cellular targets for cancer therapy.
RÉSUMÉ
12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated oxidative stress in HeLa cells and its inhibition were studied by fluorometric measurement of H2O2 and by 3H-postlabeling of the oxidized bases 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU). TPA treatment (10 fmol/cell) caused approximately 7-fold increase in H2O2 levels (0.1 nmol TPA/ml), and 5-10-fold increase in 8-OHdG and HMdU (10 nmol TPA/ml). Naturally occurring compounds [caffeic acid phenethyl ester (CAPE), (-).epigallocatechin gallate (EGCG), penta-O-galloyl-beta-D-glucose (PGG) and sarcophytol A (Sarp A)] and the anticancer drug tamoxifen (TAM) were tested as potential chemopreventive agents. These agents dose-dependently inhibited TPA-induced H2O2, 8-OHdG and HMdU. The doses required for a 50% decrease in H2O2 were approximately 2.5 microM for TAM; 5 microM for CAPE, EGCG and PGG; and 75 microM for Sarp A. TAM and PGG (10 microM), EGCG (25 microM), and CAPE (50 microM) abolished TPA-mediated H2O2 production, even below the normal cellular levels. TAM (2.5-20 microM) decreased TPA-mediated HMdU and 8-OHdG formation 2-29 times. Maximum inhibition occurred at 20 microM TAM, which caused an approximately 95% decline in HMdU and 8-OHdG. CAPE was effective at 0.5-50 microM. CAPE (25 microM) decreased 8-OHdG 95%, and HMdU 58%, while Sarp A (250 microM) reduced 8-OHdG by 93% and HMdU by 78%. EGCG (1-25 microM) and PGG (1-10 microM) inhibited of 8-OHdG and HMdU dose-dependently. However, higher doses (50 and 100 microM) decreased the efficacy of that inhibition. Of those agents tested, TAM appears to be the most and Sarp A the least effective. Our results point to these 5 compounds as being potential chemopreventive agents, which at very low doses decrease the tumor promoter-mediated oxidative processes.