Tumor Promoter 12-O-tetradecanoylphorbol-13-acetate Research Articles

Alterations in ornithine decarboxylase activity in the rat mammary gland after different periods of 50 Hz magnetic field exposure

In a series of experiments with the chemical carcinogen DMBA (7, 12-dimethyl[a]anthracene), we recently found that exposure of female Sprague-Dawley rats in 50 Hz magnetic fields (MF) in the microtesla range significantly facilitates the development and growth of mammary tumors. One possible explanation for this finding would be enhanced proliferation of breast epithelial stem cells by MF exposure, thereby increasing the sensitivity of these cells to chemical carcinogens. In line with this possibility, we previously determined that 50 Hz, 50 microT MF exposure induces increases in ornithine decarboxylase (ODC), i.e., a key enzyme in cell proliferation, in the mammary gland of female Sprague-Dawley rats. In the present study, we examined the time course of this effect, by using different periods of exposure to a 50 Hz, 100 microT MF. Furthermore, we determined ODC in different mammary complexes of the rat mammary gland to evaluate whether differences in response to MF exist over the anterior-posterior extension of this organ. Exposure of young female Sprague-Dawley rats induced marked increases in ODC in the mammary gland that were similar to ODC increases seen in "positive control" experiments with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). However, this effect of MF critically depended on the duration of MF exposure, with no effect, or at least no consistent effect, for short (<1 week) or long (8 weeks and above) exposure periods, but a robust and reproducible enhancing effect on ODC activity after 2 weeks of exposure. Furthermore, we found that the effect of MF exposure depends on the part of the mammary complexes examined, the cranial thoracic (or cervical) complexes being particularly sensitive to ODC alterations in response to MF. This is in line with recent DMBA experiments of our group in which MF-induced increases in tumor development and growth were predominantly seen in this large cranial/cervical part of the mammary gland. The most likely explanation for the observed ODC changes after MF exposure is the "melatonin hypothesis," although other cellular and molecular effects of MF might be involved as well.

Identification of coumarins from lemon fruit (Citrus limon) as inhibitors of in vitro tumor promotion and superoxide and nitric oxide generation.

Three coumarins were isolated as siginificant inhibitors of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus (EBV) activation in Raji cells from the peel of lemon fruit. They were identified as 8-geranyloxypsolaren (LE-1), 5-geranyloxypsolaren (bergamottin, LE-2), and 5-geranyloxy-7-methoxycoumarin (LE-3), respectively, by spectroscopic analysis. Three isolates had no potential O(2)-scavenging and markedly suppressed TPA-induced superoxide (O(2)(-)) generation in differentiated human promyelocytic HL-60 cells. Furthermore, LE-1 and LE-3 reduced both lypopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-induced nitric oxide (NO) generation in mouse macrophage RAW 264.7 cells. Similarly, they were found to be NO generation inhibitors rather than scavengers by measuring the extracellular L-citrulline levels. The occurrence of these coumarins in a lemon fruit was abundant in the flavedo of the peel based on quantitative analysis using high-performance liquid chromatography (HPLC). The present study suggests that the coumarins in lemon fruit are promising chemopreventive agents by inhibiting radical generation.

Inhibitory effects of monoacylated 2- O-β-galactosylglycerols on Epstein–Barr virus activation: the significant role of the hexanoyl chain

Three series of monoacyl-2- O-β- d-galactosylglycerols bearing an acyl chain of varying length, from C 4 to C 10, were studied due to their antitumor promoting effects on the activation of the Epstein–Barr virus early antigen (EBV-EA), such activation being induced by the tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA). This study indicates that it is more the length of the acyl chain that is important for the activity, six carbon atoms resulting in maximum effect, rather than the position of the ester function and the nature of the sugar (galactose or glucose).

The in vitro effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on Sertoli cell morphology.

The objective of the present study was to examine the effects of the well-known tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the morphology of cultured Sertoli cells from immature rats. The cells were cultured for 5 days and the TPA was added at the end of the culture period for 8 h at a concentration of 10-7 M. Viability tests showed that controls as well as TPA-treated cells remained viable during the culture period and no deleterious effects were observed as a result. Application of computerized morphometry at both light and electron microscopic levels revealed that TPA caused important changes in cell morphology in vitro. Statistical analysis of the results indicated that compared to the controls, Sertoli cells treated with TPA exhibited fewer astrocytic-type cytoplasmic extensions and a smaller size. Our results support the conclusion that the tumor promoter TPA, when applied to immature Sertoli cells in vitro, causes significant morphological alterations.

Cancer chemopreventive agents (antitumor-promoters) from Ajuga decumbens.

Sixteen compounds (1-16) isolated from the flowering whole plant of Ajuga decumbens have been tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) induction by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), as a primary screening test for antitumor-promoters (potential cancer chemopreventive agents). Five compounds (6, 9, and 12-14) showed strong inhibitory effects on EBV-EA induction. Of these active compounds, two major constituents of this plant, cyasterone (6) and 8-acetylharpagide (13), showed potent antitumor-promoting activities on a mouse-skin in vivo two-stage carcinogenesis procedure, using 7, 12-dimethylbenz[a]anthracene as initiator and TPA as promoter. Further, compound 13 also exhibited potent chemopreventive activity in a mouse pulmonary tumor model.

Altered expression of interleukin‐1 receptor antagonist in different stages of mouse skin carcinogenesis

Interleukin-1 receptor antagonist (IL-1Ra) is an endogenous inhibitor of interleukin-1. The expression of IL-1Ra and interleukin-1alpha (IL-1alpha) was measured in murine epidermis after treatment with tumor promoters and in tumor cell lines. A single treatment with three different tumor promoters (12-O-tetradecanoylphorbol-13-acetate (TPA), anthralin, and thapsigargin) induced IL-1Ra mRNA with different kinetics in mouse skin. The expression of IL-1Ra mRNA also was induced by TPA and IL-1alpha in a dose-related and time-dependent manner in cultured mouse keratinocytes. Expression of IL-1Ra mRNA peaked 6 h after treatment. Both IL-1Ra and IL-1alpha protein and IL-1Ra and IL-1alpha mRNA were measured in various keratinocyte tumor cell lines (C50, MT1/2, HEL30, JWF2, CH72, and BPCC2). The expression of IL-1alpha was increased in papilloma and squamous cell carcinoma cell lines. IL-1Ra protein also was increased in nontumorigenic and papilloma cell lines; however, the expression was dramatically reduced in some carcinoma cell lines. Finally, we detected IL-1alpha and IL-1Ra protein in mouse skin tumors by western blot analysis, and localization was assessed by immunohistochemical analysis. Positive staining for both IL-1alpha and IL-1Ra was observed in the cytoplasm and was most prominent in the suprabasal layer. Although IL-1Ra protein increased in papillomas and carcinomas, IL-1alpha protein was not significantly increased above basal level in most tumors.

Increased resistance of GPx-1 transgenic mice to tumor promoter-induced loss of glutathione peroxidase activity in skin.

Reactive oxygen species formation is strongly suspected to play a role in multistep carcinogenesis, notably in tumor promotion. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces peroxide production, oxidative damage to DNA and inflammation in mouse skin. TPA is also known to cause a decrease in the activity of several antioxidant enzymes including glutathione peroxidases (GPx). The observation that several anti-oxidants can inhibit TPA-mediated tumor promotion suggests that a decline in GPx activity could contribute to tumor promotion. We report here the effects of TPA on GPx activity in the skin of transgenic GPx mice that contain human GPx-1 transgenes under the regulation of a metallothionein IIA promoter. As expected, no significant difference in basal level of skin GPx activity was detected in the 3 lines of tg-MT-GPx mice investigated compared with non-transgenic controls. A single topical application of TPA induced gradually, over 20 hr, a small but detectable increase in GPx mRNA and protein levels in skin of non-transgenic mice and a contrasting decrease in both selenium-dependent and selenium-independent GPx activity. The extent of GPx induction was more pronounced in transgenic mice, and in contrast with non-transgenic mice, no significant loss of GPx activity was observed in the TPA-treated skin of these mice. Transgenic mice may, therefore, offer a novel model suitable to assess the role of GPx-1 in skin carcinogenesis, without the potential disadvantage of abnormally high levels of GPx activity produced constitutively in other transgenic models.

Effect of Dietary Conjugated Linoleic Acid on Phorbol Ester-Induced PGE2 Production and Hyperplasia in Mouse Epidermis

Conjugated linoleic acid (CLA) is a chemoprotective fatty acid that inhibits phorbol ester-induced skin tumor promotion in mice. The goal of the present study was to determine potential chemoprotective mechanisms through which CLA may be acting. Mice were fed diets containing 0.0%, 0.5%, 1.0%, or 1.5% CLA (by wt) for six weeks. The epidermis was evaluated for fatty acid composition, vascular permeability, prostaglandin E2; (PGE2) production, hyperplasia, ornithine decarboxylase activity, and c-myc mRNA accumulation. Fatty acid analysis of mouse epidermis demonstrated a dose-dependent increase of CLA incorporation into phospholipids and neutral lipids. In mice topically treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), dietary CLA (1.5%) significantly (p < 0.05) reduced PGE2; synthesis (2-fold). Additionally, CLA lowered accumulation of c-myc mRNA, a gene commonly associated with regulating cell cycle components involved in cellular proliferation, although this trend was not significant. Vascular permeability was unaffected by dietary CLA. These data suggest that dietary CLA modulates TPA-induced tumor promotion through a mechanism involving PGE2 production; however, dietary CLA had a moderate effect on c-myc mRNA levels and little effect on TPA-induced hyperplasia and ornithine decarboxylase activity.

Glucocorticoid Mediation of Dietary Energy Restriction Inhibition of Mouse Skin Carcinogenesis

Dietary energy restriction (DER) inhibits carcinogenesis in numerous animal models. DER is a potent and reproducible inhibitor of two-stage mouse skin carcinogenesis when administered during the promotion phase. Previous research demonstrated that adrenalectomy abolished cancer prevention by food restriction. Several lines of evidence suggest that glucocorticoid elevation in the DER mouse mediates the prevention of skin cancer. Our research tested the hypothesis that elevated glucocorticoid hormone activates the glucocorticoid receptor (GR) and that this activated receptor interferes with the activator protein-1 (AP-1) transcription factor. Induction of AP-1 by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is essential to tumor promotion. We have been unable to demonstrate elevated activated GR in the epidermis of the DER mouse, perhaps because only indirect strategies have been possible with the use of epidermis from DER mice. However, DER blocked the induction of AP-1 and c-jun, a constituent protein of AP-1, in the epidermis of mice. Current studies are focused on the inhibition of signaling down the MAP-1/Raf-1 kinase pathway that leads to induction of constituent proteins of AP-1, including c-Jun. Although several pathways lead to the induction of AP-1 transcriptional activity, the MAP-1/Raf-1 pathway can be activated by protein kinase C (PKC); previous studies from our laboratory demonstrated an inhibition of PKC activity and a reduction in selected isoforms of PKC in the epidermis of the DER mouse. Our current working hypothesis is that elevated glucocorticoid hormone in the DER mouse reduces the amount and activity of PKC isoforms important in the activation of MAP-1/Raf-1 kinase pathway. We propose that this results in attenuation in the induction of the AP-1 transcription factor by TPA. Because AP-1 induction by TPA is obligatory for mouse skin promotion, we propose this as an essential component of the mechanism of DER prevention of mouse skin carcinogenesis.

TGF-β3, but Not TGF-β1, Protects Keratinocytes against 12-O-Tetradecanoylphorbol-13-acetate-induced Cell Deathin Vitro and in Vivo

We have examined the role that individual TGF-beta isoforms, and in particular TGF-beta3, play in control of epidermal homeostasis. Mice with a knockout mutation of the TGF-beta3 gene die a few hours after birth. A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the skin of these mice. Grafted skin of mice with a disruption of the TGF-beta3 gene developed similarly to grafts of wild type and TGF-beta1 knockout animals. However, a strikingly different response was observed after acute treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). When exposed to TPA, the grafted skin of wild type and TGF-beta1 knockout mice underwent a hyperplastic response similar to that of normal mouse skin. In marked contrast, TPA treatment of TGF-beta3 knockout grafts induced widespread areas of keratinocyte cell death. Analysis of cultured keratinocytes treated with purified TGF-beta isoforms revealed that TGF-beta3 plays a direct and specific function in protecting keratinocytes against TPA-induced cell death. The protective function of TGF-beta3 on TPA-induced cell death was not because of general suppression of the signaling pathways triggered by this agent, as ERK1/2 activation occurred to a similar if not greater extent in TGF-beta3-treated versus control keratinocytes. Instead, TGF-beta3 treatment led to a significant reduction in TPA-induced c-Jun N-terminal kinase activity, which was associated and possibly explained by specific counteracting effects of TGF-beta3 on TPA-induced disruption of keratinocyte focal adhesions.

Proteolysis Negatively Regulates Agonist-Stimulated Arachidonic Acid Metabolism

Phenylmethylsulphonyl fluoride, lactacystin (a selective inhibitor of the proteasome) and the peptide aldehydes carbobenzoxyleucylleucylnorvalinal and carbobenzoxyleucylleucylleucinal amplify the production of prostacyclin in rat liver cells incubated for 6 h with the tumour promoter 12- O-tetradecanoylphorbol-13-acetate (TPA) and the TPA-type tumour promoters teleocidin and aplysiatoxin. Such stimulation is not dependent upon the simultaneous presence of the inhibitor and TPA. Preincubation of the cells with TPA followed by addition of the inhibitor or preincubation with the inhibitor followed by addition of TPA results in amplified prostacyclin production. Phenylmethylsulphonyl fluoride, lactacystin, and carbobenzoxyleucylleucylnorvaline also enhance prostacyclin production after incubation with interleukin-1β and transforming growth factor-α. The Ca 2+ chelator ethyleneglycol- O, O′-bis(2-aminoethyl)- N, N, N′, N′-tetraacetic acid inhibits the phenylmethylsulphonyl fluoride–TPA or lactacystin–TPA amplifications. Cells, treated with phenylmethylsulphonyl fluoride, TPA, interleukin-1β, lactacystin or the peptide aldehydes exhibit increased prostaglandin endoperoxide G/H synthase activity. The increased activities as well as the constitutive prostaglandin endoperoxide G/H synthase activity are inhibited by a selective prostaglandin endoperoxide G/H synthase-2 inhibitor, 1-[2-(4-fluorophenyl)cyclopenten-1-yl]-4-(methysulphonyl)-benzene, with an IC 50 of approximately 0.5 μM. These results demonstrate that the C-9 rat liver cells express prostaglandin endoperoxide G/H synthase-2 constitutively and express induced prostaglandin endoperoxide G/H synthase-2. Inhibition of proteolytic activity amplifies agonist-stimulated arachidonic acid metabolism in these cells.

Hydroquinones cause specific mutations and lead to cellular transformation and in vivo tumorigenesis.

Benzo(a)pyrene and benzene are human carcinogens. The metabolic activation of these compounds into ultimate mutagenic and carcinogenic metabolites is prerequisite for their carcinogenic effects. In this report, the mutagenicity and carcinogenicity of hydroquinones of benzo(a)pyrene and benzene was investigated to address two important questions: (1) do hydroquinones contribute to benzo(a)pyrene and benzene carcinogenicity; and (2) how safe is it to increase the levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), a key enzyme in the generation of hydroquinone. The supF tRNA of the plasmid pSP189 was used as the mutational target in a cell-free and Chinese hamster ovary (CHO) cell system to study hydroquinone mutagenicity. RNA and protein-free pSP189 DNA was incubated in a cell-free system with benzo(a)pyrene-3,6-quinone and purified NQO1 or with benzoquinone hydroquinone to generate adducted pSP189 DNA. The adducted pSP189 DNA was transfected in human embryonic kidney cells Ad293. In the CHO cell system, monolayer cultures of CHO cells and CHO cells overexpressing NQO1 or P450 reductase were transfected with pSP189 vector DNA, treated with benzo(a)pyrene-3,6-quinone. The adducted and replicated pSP189 DNA was rescued from transfected Ad293 (cell-free system) and CHO cells (CHO cell system), digested with the restriction enzyme Dpn1 to remove unreplicated DNA followed by transformation in Escherichia coli MBM7070. The mutant colonies [white/pale blue on 5-bromo-4-chloro-3-indolyl beta-D-galactoside/isopropyl beta-D-thiogalactoside (X-gal/IPTG) plates] were selected, regrown and analysed by DNA sequencing. Mutagenesis experiments demonstrated that hydroquinones cause sequence-specific frameshift mutations involving deletion of a single cytosine from the DNA sequence 5'-172-CCCCC176-3' or a single guanosine from the complementary strand sequence 5'-GGGGG-3' in the supF tRNA gene. This mutation was specific to the hydroquinones, as it was not observed with quinones and other components of the redox cycling (semiquinones and reactive oxygen species). Exposure of BALBc/3T3 cells to hydroquinones resulted in cellular transformation leading to the loss of contact inhibition and regulation of cell growth. The transformation efficiency of BALBc/3T3 cells exposed to hydroquinones was significantly increased by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), indicating that hydroquinones are excellent initiators that require additional co-carcinogens or promoters to exert an effect. The hydroquinone + TPA as well as hydroquinone-transformed BALBc/3T3 cells, when injected s.c. in severe combined immunodeficient (SCID) mice, produced tumours at 100% frequency. These results establish that hydroquinones lead to mutagenicity and carcinogenicity.

Cancer chemopreventive activity of synthetic colorants used in foods, pharmaceuticals and cosmetic preparations

In continuation with our studies to uncover cancer chemopreventive effects of non-toxic natural colorants and other products of biologic and synthetic origin, we tested several Food and Drug Administration-approved synthetic colorants for antitumor promoting potential by the in vitro Epstein–Barr virus early antigen activation in Raji cells in response to the tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA). Among 29 such colorants used in foods, pharmaceuticals and cosmetics and evaluated in vitro, six of the 10 most effective had an azo group. Three structurally unrelated colorants tested in this assay were also studied in vivo for chemoprevention of 7,12-dimethylbenz[ a]anthracene (DMBA)-induced TPA-promoted mouse skin carcinogenesis. The results indicate that tartrazine, indigo carmine and erythrosine are potent inhibitors of skin tumor promotion in mice treated with DMBA and TPA.

Heterologous Desensitization in Neoplastic Thyroid Cells: Influence of the Phospholipase C Signal Transduction System on the Thyrotropin–adenylate Cyclase Signal Transduction System

Desensitization is defined as a decreased functional response after continuous or repetitive stimulation of a receptor with its agonist. Thyrotropin (TSH) increases cAMP levels in normal and neoplastic thyroid tissue. The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) activates protein kinase C (PKC). The aim was to determine whether TPA induces heterologous desensitization of the TSH-adenylate cyclase (AC) signal transduction system. Three human thyroid neoplasms in culture for 6 months or longer (one papillary carcinoma, one Hurthle cell carcinoma, one follicular adenoma) were incubated with TSH (10 mU/ml) and TPA (1.6 x 10(-8) M) separately and together for various time periods (from 10 minutes to 24 hours). The mixture was subsequently incubated for 30 minutes with TSH. TPA alone had no effect on cAMP levels, but co-incubation of TPA and TSH caused a significant reduction in cAMP response when compared to the cAMP response that resulted after stimulation with only TSH (p < 0.001). cAMP levels in response to TSH decreased by 31%, 44%, and 57% after preincubation with TSH for 10 minutes, 4 hours, and 24 hours, respectively (p < 0.01; ANOVA). Co-incubation of cells with TPA and staurosporine (10 ng/ml), a PKC inhibitor, prevented the effect of TPA on desensitization at 10 minutes and blunted the effect at 4 hours. This is the first demonstration in human neoplastic thyroid cells that TPA induced heterologous desensitization of the cAMP response to TSH. This TPA-induced effect appears to involve PKC activation, as it can be blocked by staurosporine.

Identification and characterization of several forms of phospholipase A2 in mouse epidermal keratinocytes

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the release of arachidonic acid (AA) from mouse keratinocytes. A distinct difference was observed between the fatty acid release profile elicited by TPA and other stimuli. These findings led to the investigation of keratinocyte phospholipase A2 (PLA2), which catalyzes the release of sn-2 fatty acids from membrane phospholipids and regulates the production of eicosanoids. We characterized and identified several forms of PLA2 in mouse keratinocytes, a cytosolic or cPLA2 and two secretory or sPLA2s in the membrane. The PLA2 in keratinocyte cytosol is sensitive to heating and acid treatment, while resistant to reducing reagent. The PLA2 in keratinocyte membrane is resistant to heating and acid treatment, while sensitive to reducing reagent. These characteristics suggested the presence of a cPLA2 and at least one type of sPLA2. Inhibitor data further confirmed the identities of these PLA2s. The cPLA2 was activated by TPA, and appeared to be responsible for the majority of the specific release of AA observed in mouse keratinocytes treated with TPA. The calcium ionophore A23187, and 4α-TPA did not elicit the selectivity towards AA observed with TPA. The release of linoleic acid (LA) and oleic acid (OA) from A23187- and 4α-TPA-treated keratinocytes suggests activation of sPLA2. These activities may be due to the existence of both type I and type II sPLA2, as both were identified by polymerase chain reactions. In conclusion, keratinocytes express several forms of phospholipase A2 that differ in their substrate specificities and mechanisms of activation, resulting in distinct agonist-specific fatty acid release profiles.—LiStiles, B., H-H. Lo, and S. M. Fischer. Identification and characterization of several forms of phospholipase A2 in mouse epidermal keratinocytes.

Inhibition of gap junctional intercellular communication by tumor promoters in connexin43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C.

In this study, we investigated whether the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), inhibited gap junctional intercellular communication (GJIC) in a cell-specific or connexin-specific manner and whether protein kinase C was involved. To do this, we used highly communicating WB-F344 rat liver epithelial cells, which express connexin43 as their predominant gap junction protein, WB-aB1 cells, which are a GJIC-incompetent mutant line of WB-F344 cells and that express connexin43, WB-a/32-10 cells, which are a highly communicating derivative of WB-aB1 cells generated by stable transduction with a connexin32 retroviral expression vector, and primary cultured rat hepatocytes, which express conexin32 predominantly. Treatment of WB-F344 and WB-a/32-10 cells, but not hepatocytes, with TPA inhibited GJIC (assayed by Lucifer Yellow dye microinjection). This inhibition involved protein kinase C because (i) inhibition was prevented by co-treatment of the cells with a specific protein kinase C inhibitor, bis-indolylmaleimide, and (ii) treatment with TPA for 24 h had no effect on dye-coupling in agreement with the downregulation of protein kinase C. TPA also caused the internalization of Cx43-containing gap junctions and the formation of a hyperphosphorylated form of Cx43, Cx43-P3, in WB-F344 cells only, but TPA had no effect on Cx32-containing gap junctions or protein mobility. In contrast, PB inhibited GJIC only in hepatocytes and DDT inhibited GJIC in all three types of cells; bis-indolylmaleimide did not block the effects of either agent. These results indicate that the inhibitory actions of TPA and PB on GJIC are cell-specific rather than connexin-specific and that TPA inhibits connexin43 and connexin32-mediated GJIC through a protein kinase C-dependent mechanism.

Anti-tumor-promoting activity of majonoside-R2 from Vietnamese ginseng, Panax vietnamensis Ha et Grushv. (I).

Seven saponins (1-7) isolated from the rhizomes and roots of Panax vietnamensis were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells as a primary screening test for anti-tumor-promoters (cancer chemopreventive agents). The ocotillol-type saponin, majonoside-R2 (2), which is the major and characteristic constituent of this plant, exhibited a significant inhibitory effect on EBV-EA activation. Furthermore, the cell cycle analysis of 2 on Raji cells was also examined and strong inhibition was observed on the effect of the cell cycle induced by TPA. Compound 2 showed potent anti-tumor-promoting activity in two-stage carcinogenesis tests of mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA or fumonisin B1 as a promoter. Consequently, these results suggest that majonoside-R2 (2) could be a valuable chemopreventive agent against chemical carcinogenesis.

Adrenergic-mediated connexin43 phosphorylation in the ocular ciliary epithelium

PURPOSE. Gap junctions provide metabolic cooperativity between the nonpigmented and pigmented cells of the ciliary epithelium. Connexin43 is the major protein of these junctions. To learn whether the phosphorylation state of this gap junction is sensitive to adrenergic mediators, we exposed isolated intact ciliary epithelia to agonists of the G-protein receptor-coupled system and analyzed the phosphorylation state of connexin43 by western blot. METHODS. The double layer of intact ciliary epithelia was isolated and exposed to isoproterenol, forskolin, and the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA). The phosphorylation state of connexin43 was analyzed by western blot, using a monoclonal antibody that recognized both the phosphorylated and dephosphorylated connexin43. An upward shift in electrophoretic mobility confirmed the presence of a phosphate group. RESULTS. Connexin43 phosphorylation was rapidly induced by each of these agonists. One µM isoproterenol and 5 µM forskolin induced phosphorylation of connexin43, as did 16 nanomolar TPA. The effect of isoproterenol was partially blocked by 1 µM timolol. Addition of a phosphatase after forskolin treatment reversed the effect of forskolin. Control explant tissue not treated with these agents exhibited a slower but definite phosphorylation of connexin43. CONCLUSIONS. Phosphorylation of ciliary epithelial connexin43 is sensitive to modulators of the cAMP system as well as an agent that activates PKC. Isolated intact ciliary epithelia phosphorylate connexin43 by endogenous mechanisms, most likely as a protective response to stress.

Anti-tumor-Promoting Activity of Diterpenes from Excoecaria agallocha.

To search for possible anti-tumor-promoters, we carried out the primary screening of seventeen diterpenes (1-17) isolated from the resinous wood of Excoecaria agallocha (Euphorbiaceae) using an in vitro synergistic assay system. Of these diterpenes, ent-16-hydroxy-3-oxo-13-epi-manoyloxide (5), (13R,14R)-ent-8alpha,13;14,15-diepoxy-13-epi- labda-3beta-ol (8) ent-3beta-hydroxy-15-beyeren-2-one (10) and ent-15-hydroxy-labda-8(17),13E-dien-3-one (14) exhibited significant inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, 10 exhibited remarkable anti-tumor-promoting activity in vivo on a two-stage carcinogenesis test of mouse tumor using 7,12-dimethylbenz[a]-anthracene (DMBA) as an initiator and TPA as a promoter.

Inhibitory effects of fatty acid monoesters of 2- O- β- d-glucosylglycerol on Epstein–Barr virus activation

In a screening for cancer chemopreventing agents several glycosylglycerols were found to be active. In order to optimize the anti-tumor activity of this class of compounds, a series of 1- O-acyl-2- O- β- d-glucopyranosyl- sn-glycerols differing in the acyl chain length, which varied from C 4 to C 18, were examined for their in vitro anti-tumor promoting effects on Epstein–Barr virus early antigen (EBV-BA) activation induced by the tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA). Among the compounds tested, the monohexanoyl derivative was the most active and, noteworthy, the most potent compound of the glycosylglycerol series hitherto known.