To Inhibit 12-O-tetradecanoylphorbol-13-acetate Research Articles

Inhibitory Effects of the Aerial Parts of Epimedium koreanum on TPA-Induced Inflammation and Promotion in Two-Stage Carcinogenesis in Mouse Skin

Cancer prevention by supplements offers the most cost-effective long-term health strategy. Methanol extracts from the aerial parts of Epimediumkoreanum were previously found to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory ear oedema and tumour promotion by TPA in the two-stage mouse skin carcinogenesis model. Four prenyl flavonol glycosides (14) were isolated from the active fraction of this extract, and were identified. The isolated compounds showed inhibitory activity against TPA-induced ear inflammatory ear oedema. The 50% inhibitory dose (ID50) of icariin (1), epimedin A (2), epimedin B (3) and epimedin C (4) for TPA-induced inflammation ranged from 114 to 255 nmol/ear, suggesting greater potency than indomethacin (ID50: 908 nmol/ear), an anti-inflammatory drug. Thus, the epimedium herb may be useful in cancer prevention.

Galangin, a novel dietary flavonoid, attenuates metastatic feature via PKC/ERK signaling pathway in TPA-treated liver cancer HepG2 cells.

BackgroundGalangin (3,5,7-trihydroxyflavone) is a flavonoid compound found in high concentration in lesser galangal. The objective of this study was to investigate the ability of galangin to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced the invasion and metastasis of HepG2 liver cancer cells.ResultsFirst, using a cell-matrix adhesion assay, immunofluorescence assay, transwell-chamber invasion/migration assay, and wound healing assay, we observed that galangin exerted an inhibitory effect on TPA-induced cell adhesion, morphology/actin cytoskeleton arrangement, invasion and migration. Furthermore, the results of gelatin zymography and reverse transcriptase polymerase chain reaction (RT-PCR) assays showed that galangin reduced the TPA-induced enzyme activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in HepG2 cells; moreover, the messenger RNA level was downregulated. We also observed through a Western blotting assay that galangin strongly inhibited the TPA-induced protein expressions of protein kinase Cα (PKCα), protein kinase Cδ (PKCδ), phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), the phospho-inhibitor of kappaBα (phospho-IκBα), c-Fos, c-Jun, and nuclear factor kappa B (NF-κB). Next, galangin dose-dependently inhibited the binding ability of NF-κB and activator protein 1 (AP-1) to MMP-2/MMP-9 promoters, respectively, resulting in the suppression of MMP-2/MMP-9 enzyme activity.ConclusionsThe results revealed that galangin effectively inhibited the TPA-induced invasion and migration of HepG2 cells through a protein kinase C/extracellular signal-regulated kinase (PKC/ERK) pathway. Thus, galangin may have widespread applications in clinical therapy as an anti-metastatic medicament.

Abstract 2244: AKT activation by Pdcd4 knockdown upregulates cyclin D1 expression and promotes cell proliferation

Abstract Programmed Cell Death 4 (Pdcd4) is a novel tumor suppressor that frequently exhibits down-regulated expression in several types of cancer. In colon cancer tissues, expression level of Pdcd4 is continuously down-regulated in the order of normal-adenoma-carcinoma. Over-expression of Pdcd4 has been demonstrated to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation and tumor phenotype. In addition to cultured cells, Pdcd4 transgenic mice that over-express Pdcd4 in the epidermis show significant reductions in 7,12-dimethylbenz(a)anthracene (DMBA)/TPA induced skin papilloma formation and carcinoma incidence. Knock-out of Pdcd4 expression in mice results in induction of lymphomas with frequent metastases to the liver and kidney, and an increase in DMBA/TPA induced skin papilloma formation and carcinoma incidence. These findings suggest that Pdcd4 suppresses carcinogenesis not only at an early stage (tumor promotion), but also at a late stage (tumor progression) in both cultured cells and transgenic mice. Recently, we showed that aerosol delivery of pdcd4 cDNA into a murine lung inhibits cell proliferation and enhances apoptosis in K-ras null mice and AP-1 reporter mice. Moreover, immunohistochemical studies showed that expression of Pdcd4 is inversely correlated with the proliferation marker, Ki-76, in the human tissues of intraductal papillary mucinous carcinoma. However, the mechanisms involved in these processes remain unknown. In this study, we found that knockdown of Pdcd4 promoted cell proliferation and up-regulated cyclin D1 expression. Previously, we demonstrated that Pdcd4 knockdown activated NF-κB dependent transcription. Mutations of NF-κB binding sites on the cyclin D1 promoter attenuated the cyclin D1 promoter activity induced by Pdcd4 knockdown. In addition, knockdown of NF-κB/IαB kinase (IKK) ≤ or IKKα, the kinase regulating NF-κB activation, inhibited cyclin D1 promoter activity and cyclin D1 expression, indicating that up-regulation of cyclin D1 by Pdcd4 knockdown is contributed, at least in part, by NF-κB activation. To investigate the mechanism of how Pdcd4 knockdown activates NF-κB, we found that the level of AKT phosphorylation and AKT kinase activity were increased in the Pdcd4 knockdown cells. Conversely, ectopic expression of Pdcd4 inhibited AKT phosphorylation and cyclin D1 expression, suggesting that Pdcd4 regulates AKT activity and cyclin D1 expression. Furthermore, knockdown of AKT in the Pdcd4 knockdown cells inhibited IKK phosphorylation, NF-κB activation, cyclin D1 promoter activity, and cyclin D1 expression as well as cell proliferation. Taken together, these findings suggest that activation of NF-κB by Pdcd4 knockdown through AKT contributes to the elevated cyclin D1 expression, thus provide new insights into how loss of Pdcd4 expression promotes tumor development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2244. doi:1538-7445.AM2012-2244

Abstract 3096: c-Myc regulates MAP4K1 expression and AP-1 activity in Pdcd4 knock-down cells

Abstract Programmed Cell Death 4 (Pdcd4) is a novel tumor suppressor that frequently exhibits down-regulated expression in several types of cancer. In colon cancer tissues, expression level of Pdcd4 is continuously down-regulated in the order of normal-adenoma-carcinoma. Over-expression of Pdcd4 has been demonstrated to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation and tumor phenotype. In addition to cultured cells, Pdcd4 transgenic mice that over-express Pdcd4 in the epidermis show significant reductions in 7,12-dimethylbenz(a)anthracene (DMBA)/TPA induced skin papilloma formation and carcinoma incidence. Knock-out of Pdcd4 expression in mice resulted in induction of lymphomas with frequent metastases to the liver and kidneys, and an increase in DMBA/TPA induced skin papilloma formation and carcinoma incidence. These findings suggest that Pdcd4 suppresses carcinogenesis not only at an early stage (tumor promotion), but also at a late stage (tumor progression) in both cultured cells and transgenic mice. Recently, we demonstrated that over-expression of Pdcd4 inhibits invasion and MAP4K1 expression in colon tumor cells. Conversely, knock-down of Pdcd4 promotes invasion, activates AP-1 and β-catenin/Tcf dependent transcription, and up-regulates c-Myc expression in colon tumor cells. However, how MAP4K1 expression is regulated by Pdcd4 remains unknown. MAP4K1, a kinase upstream of JNK signaling pathway, is also known as HPK1 (haematopoietic progenitor kinase 1) whose function and expression regulation in solid tumors is not well studied. It has been known that activation of MAP4K1 results in activation of JNK, c-Jun and AP-1 dependent transcription. In the present study, we found that MAP4K1 expression is up-regulated by Pdcd4 knock-down. Concurrently, the phosphorylation of JNK and c-Jun is increased in Pdcd4 knock-down cells. c-Myc is a direct downstream target gene of β-catenin/Tcf dependent transcription and is up-regulated by Pdcd4 knock-down. To investigate the role of c-Myc in regulating MAP4K1 expression, we over-expressed c-Myc in HEK293 cells and found that over-expression of c-Myc induces MAP4K1 expression and activates c-Jun. Using chromatin immunoprecipitation, we demonstrate that c-Myc directly binds to MAP4K1 promoter in Pdcd4 knock-down cells. Down-regulation of c-Myc using c-myc RNAi reverses MAP4K1 expression and c-Jun activation in Pdcd4 knock-down cells. Thus, these results provide an insight into how Pdcd4 regulates MAP4K1 expression and AP-1 dependent transcription. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3096.

Curcumin decreases 12- O -tetradecanoylphorbol-13-acetate-induced protein kinase C translocation to modulate downstream targets in mouse skin

Curcumin has been shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumour promotion and some of the TPA-responsive markers in mouse skin. However, its mechanism of action is not fully elucidated. The present study focuses on understanding the role of protein kinase C (PKC), the major cellular receptor of TPA, in mediating TPA-induced biological responses in mouse skin and subsequently, elucidating the effects of curcumin on PKC and its downstream target molecules. As compared with controls, single topical application of TPA (5 nmol) to skin increased the translocation of PKC from cytosolic to particulate fraction, determined in terms of activity and protein levels. Ro-31- 8220 (PKC inhibitor, 1 nmol) when applied topically, alone or prior to TPA, inhibited PKC activity in both the compartments but did not affect the TPA-induced protein translocation. In contrast, though curcumin (10 mumol) alone did not alter the basal activity/levels, its pre-treatment decreased the TPA-induced translocation of PKC isozymes (alpha, beta, gamma, epsilon, eta), resulting in appropriate alterations in activity. Despite differences in modes of action of Ro-31-8220 (activity inhibition) and curcumin (decreasing translocation) in modulating PKC, their pre-treatment blunted the TPA-induced levels of mitogen-activated protein kinases and transcription factors (c-jun, c-fos and nuclear factor-kappa B) and downstream target proteins associated with cell proliferation (cyclin D1 and ornithine decarboxylase), cell death (Bax and Bcl2), inflammation (cyclooxygenase-2 and prostaglandin E2) and oxidative stress (8-hydroxy-2'-deoxyguanosine) in skin. These results demonstrate the crucial role of PKC in TPA-mediated cellular responses in skin and that curcumin modulates transmembrane signal transduction via PKC to affect TPA-induced biochemical and molecular alterations in mouse skin.

Evaluation of in vivo efficacy of topical formulations containing soybean extract

In the present study it was evaluated the: (i) functional stability of the soybean extract as a raw material and dispersed in two different topical formulations, (ii) skin retention using modified Franz diffusion cells, and (iii) in vivo activity of these formulations to inhibit 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced hydrogen peroxide (H 2O 2) and malondialdehyde (MDA) increases in the skin of hairless mice. The physico-chemical stability was evaluated by pH, globule size and centrifugation test. Furthermore, functional stability was also evaluated by antilipoperoxidative activity. The two topical formulations were stored at 4 °C, 30 °C/60% RH and 40 °C/70% RH for 6 months. The evaluation of the antiperoxidative stability of soybean extract itself and incorporated in formulations did not demonstrate loss of activity by storage at 4 °C/6 months. During 6 months of the study in different storage conditions the formulations 1 and 2 added or not with soybean extract were stable to physico-chemical tests. The effect of antioxidant compounds detected by the inhibition of MDA formation was time-dependent for formulation 2 as detected in the skin retention study. Pretreatment with formulation 1 or 2 significantly diminished TPA-induced H 2O 2 and MDA generation. In conclusion, the present results suggest for the first time that formulations containing soybean extract may be a topical source of antioxidant compounds that decrease oxidative damages of the skin.

Rhein inhibits TPA-induced activator protein-1 activation and cell transformation by blocking the JNK-dependent pathway

Rhein (4,5-Dihydroxyanthraquinone-2-carboxylic acid), a constituent enriched in the rhizome of rhubarb (R. palmatum L. or R. tanguticum Maxim), is a traditional Chinese herb used as a laxative and stomachic drug. In the present study, we investigated the anti-carcinogenesis of rhein by using mouse epidermal cell JB6 line, an in vitro model for elucidating the molecular mechanisms of cancer chemopreventive agents. Rhein is shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 (AP-1) activation in a dose-dependent manner. Signal cascade analysis revealed that rhein inhibits the phosphorylation and abundance of c-Jun protein, c-Jun NH2-terminal kinase (JNK) phosphorylation, but does not inhibit the phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 kinase. Thus, these results provide the first evidence suggesting that rhein inhibits AP-1 activity and cell transformation through the inhibition of a JNK-dependent, ERK- and p38-independent molecular mechanism.

Aloe arborescens extract inhibits TPA-induced ear oedema, putrescine increase and tumour promotion in mouse skin.

The ethyl acetate extract of the acetone-soluble Aloe arborescens fraction was found to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear oedema, putrescine increase and tumour promotion in mouse skin. Chromatographic analyses of this extract revealed that phenolic compounds such as aloenin, barbaloin and isobarbaloin could be useful as cancer chemopreventive agents against tumour promotion.

Bioactive constituents of Thuja occidentalis.

An ethyl acetate-soluble extract of the combined leaves and twigs of Thuja occidentalis was found to inhibit 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase (ODC) in cultured mouse epidermal ME 308 cells. Bioassay-guided fractionation of this extract led to the isolation of six active constituents (1-6), namely, (+)-7-oxo-13-epi-pimara-14,15-dien-18-oic acid (1), (+)-7-oxo-13-epi-pimara-8,15-dien-18-oic acid (2), (+)-isopimaric acid (3), (1S,2S,3R)-(+)-isopicrodeoxypodophyllotoxin (4), (-)-deoxypodophyllotoxin (5), and (-)-deoxypodorhizone (6). Compounds 1 and 4 are new natural products, and their structures and stereochemistry were determined using spectroscopic methods. Compounds 1-6 were evaluated for inhibition of the transformation of murine epidermal JB6 cells, inhibition of ornithine decarboxylase induction with murine epidermal ME 308 cells, and cytotoxic activity against KB cells.

Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds

The anti-tumor promoting activity of a polyphenolic fraction from grape seeds (GSP) was examined in CD-1 mouse skin epidermis. Specifically, the ability of this fraction to inhibit 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion and two markers of promotion in mouse skin, ornithine decarboxylase (ODC) and myeloperoxidase (MPO) activities, was evaluated. Pretreatment of mouse skin with 5, 10, 20 and 30 mg of GSP resulted in a dose-dependent reduction in TPA-induced epidermal ODC activity of 27, 37, 48 and 70%, respectively, compared to controls. In addition, pretreatment of mouse skin with 1, 5, 10 and 20 mg of GSP resulted in a significant 43, 39, 54 and 73% inhibition of MPO activity, respectively, compared to controls. In 7,12-dimethylbenz[a]anthracene (DMBA)-initiated CD-1 mice, biweekly treatment of mouse skin with 5, 10, and 20 mg of GSP 20 min prior to TPA application resulted in a 30, 40, and 60% inhibition of final skin tumor incidence, respectively, compared to controls. In addition, the final number of tumors per mouse in the 5, 10 and 20 mg GSP-treated animals was decreased 63, 51, and 94%, respectively, compared to controls. These studies indicate that GSP possesses anti-tumor promoting activity when applied to CD-1 mouse skin prior to treatment with TPA. The mechanism of this tumor inhibition is due, in part, to a GSP-associated inhibition of TPA-induced epidermal ODC and MPO activities. Thus, GSP warrants further evaluation as a skin cancer chemopreventative agent.

Inhibitory Effect of Poria cocos on 12-O-Tetradecanoylphorbol-13-Acetate‐Induced Ear Oedema and Tumour Promotion in Mouse Skin

The methanol extract from the sclerotium of Poria cocos was found to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumour promotion in two-stage carcinogenesis in mouse skin. From the active fraction of the extract, eight lanostane-type triterpene acids and four 3,4-secolanostane-type triterpene acids were isolated. The isolated compounds showed inhibitory activity against TPA-induced ear inflammatory oedema. The 50% inhibitory dose of pachymic acid, 3-O-acetyl-16α-hydroxytrametenolic acid, dehydropachymic acid, dehydroeburiconic acid, 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid, poricoic acid A and poricoic acid B for TPA-induced inflammation was 17–44 μg/ear, at a grade corresponding to that of hydrocortisone.

Inhibitory Effect of Poria cocos on 12-O-Tetradecanoylphorbol-13-Acetate-Induced Ear Oedema and Tumour Promotion in Mouse Skin

The methanol extract from the sclerotium of Poria cocos was found to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumour promotion in two-stage carcinogenesis in mouse skin. From the active fraction of the extract, eight lanostane-type triterpene acids and four 3,4-secolanostane-type triterpene acids were isolated. The isolated compounds showed inhibitory activity against TPA-induced ear inflammatory oedema. The 50% inhibitory dose of pachymic acid, 3-O-acetyl-16α-hydroxytrametenolic acid, dehydropachymic acid, dehydroeburiconic acid, 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid, poricoic acid A and poricoic acid B for TPA-induced inflammation was 17-44 μg/ear, at a grade corresponding to that of hydrocortisone.

Isolation of inhibitors of TPA-induced mouse ear edema from Hoelen, Poria cocos.

Triterpene carboxylic acids were isolated from the methanol extract of Hoelen, Poria cocos, and found to inhibit 12-O-tetradecanoylphorbol 13-acetate (TAP)-induced mouse ear edema. Their chemical structures were identified as 3 beta,-16 alpha-dihydroxylanosta-7,9(11),24-trien-21-oic acid, 16 alpha-hydroxydehydropachymic acid, 16 alpha-hydroxytrametenolic acid and dehydrotumulosic acid.

A labile hyperphosphorylated c-Fos protein is induced in mouse fibroblast cells treated with a combination of phorbol ester and anti-tumor promoter curcumin.

Curcumin is a potent inhibitor of tumor promotion, and was shown previously to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activity. The c-Fos protein is inducible by TPA and thus is associated with c-Jun to result in an increased AP-1 activity in mouse fibroblast cells. We therefore hypothesized that c-Fos may be one of the targets of curcumin action. In the present study, the effects of curcumin on TPA-induced c-fos mRNA and protein levels were determined by RNA hybridization and western blot analysis, respectively. Curcumin decreases the TPA-induced nuclear abundance of c-Fos protein in spite of the slight super-induction of c-fos mRNA. Upon TPA stimulation, the amount of c-Fos in the quiescent cells increases and reaches maximum at 30 min, and then progressively disappears over a period of 60 min. However, the c-Fos protein seems susceptible to rapid degradation by 45 min if NIH 3T3 cells were treated with TPA in the presence of curcumin. The curcumin-induced hyperphosphorylated forms of c-Fos proteins are significantly more unstable; they entirely disappeared within 40 min after incubation at 37 degrees C. These findings prompted us to suggest that the decrease of c-Fos protein could account for the repressed in vitro DNA binding probably by reducing the Jun/Fos complex formation.

Inhibitory effects of steroidal saponins on 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced 32P-incorporation into phospholipids of HeLa cells and proliferation of human malignant tumor cells.

Certain Lilium plants contain (25S)-spirost-5-ene-3 beta,27-diol glycosides embracing 3-hydroxy-3-methylglutaric acid at the C-27 hydroxy position. One of their derivatives, methyl ester of (25R)-27-O-[(S)-3-hydroxy-3-methylglutaryl]-spirost-5-ene-3 beta,27-diol 3-O-(O-alpha-L-rhamnopyranosyl-(1-->2)-O-[beta-D-glucopyranosyl-(1-->4)] - beta-D-glucopyranoside) was found to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated 32P-incorporation into the phospholipids of human cervical cancer (HeLa) cells and also to inhibit the proliferation of various kinds of human malignant tumor cells, pancreatic cancer (PANC-1), osteosarcoma (OST), human gastric cancer (HGC-27), pheochromocytoma (PC-12) and HeLa cells, in vitro.

Carba-prostacyclin inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation in sensitive murine epidermal JB6 cells.

The ability of carba-prostacyclin (cPGI2), a stable analog of prostacyclin (PGI2), to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 cells was investigated. JB6 cells sensitive (P+) and resistant (P-) to TPA-induced transformation to anchorage-independent growth were plated in soft agar in the presence or absence of cPGI2 for 14-21 days. Transformation frequencies were determined by recording colony numbers. cPGI2 was found to inhibit TPA-induced transformation of P+ cells in a dose-dependent fashion with 1 microM cPGI2 producing approximately 50% inhibition of colonies in soft agar. Our findings are consistent with the hypothesis that TPA-induced transformation in JB6 cell variants is mediated by PGI2 via regulation of adenylate cyclase activity and cAMP accumulation, with resultant inhibition of expression of the transformed phenotype, reflected in anchorage-independent growth.

Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea

An antioxidant fraction of Chinese green tea (green tea antioxidant; GTA), containing several catechins, has been previously shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. In the present study, GTA was shown to have antioxidative activity toward hydrogen peroxide (H2O2) and the superoxide radical (O2-). GTA also prevented oxygen radical and H2O2-induced cytotoxicity and inhibition of intercellular communication in cultured B6C3F1 mouse hepatocytes and human keratinocytes (NHEK cells). GTA (0.05-50 micrograms/ml) prevented the killing of hepatocytes (measured by lactate dehydrogenase release) by paraquat (1-10 mM) and glucose oxidase (0.8-40 micrograms/ml) in a concentration-dependent fashion. GTA (50 micrograms/ml) also prevented the inhibition of hepatocyte intercellular communication by paraquat (5 mM), glucose oxidase (0.8 micrograms/ml), and phenobarbital (500 micrograms/ml). In addition, GTA (50 micrograms/ml) prevented the inhibition of intercellular communication in human keratinocytes by TPA (100 ng/ml). Cytotoxicity and inhibition of intercellular communication, two possible mechanisms by which tumor promoters may produce their promoting effects were therefore prevented by GTA. The inhibition of these two effects of pro-oxidant compounds may suggest a mechanism by which GTA inhibits tumor promotion in vivo.