3,5-Dicaffeoyl-epi-quinic acid inhibits the PMA-stimulated activation and expression of MMP-9 but not MMP-2 via downregulation of MAPK pathway.

Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, are very important gelatinases that are overexpressed during tumor metastasis. Up to date, several MMP inhibitors have been developed from natural sources as well as organic synthesis. In the present study, the MMP-2 and MMP-9 inhibitory effects of 3,5-dicaffeoyl-epi-quinic acid (DCEQA), a caffeoylquinic acid derivative isolated from Atriplex gmelinii, were investigated in phorbol 12-myristate 13-acetate (PMA)-treated human HT1080 fibrosarcoma cells. Gelatin zymography and immunoblotting showed that DCEQA significantly inhibited the PMA-induced activation and expression of MMP-9 but was not able to show any effect against MMP-2. DCEQA treatment was also shown to upregulate the protein expression of tissue inhibitor of MMP-1 along with decreased MMP-9 protein levels. Moreover, the effect of DCEQA on phosphorylation of mitogen activated protein kinases (MAPKs), analyzed by immunoblotting, indicated the DCEQA inhibited the MMP-9 by downregulation of MAPK pathway. Collectively, current results suggested that DCEQA is a potent MMP-9 inhibitor and can be utilized as lead compound for treatment of pathological complications involving enhanced MMP activity such as cancer metastasis.

Dexamethasone inhibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells.

The production of inflammatory mediators by epithelial cells in inflammatory lung diseases may represent an important target for the anti-inflammatory effects of glucocorticoids. Activator protein-1 is a major activator of inflammatory genes and has been proposed as a target for inhibition by glucocorticoids. We have used human pulmonary type-II A549 cells to examine the effect of dexamethasone on the phorbol ester (PMA)/Lipopolysaccharide (LPS) induced pro-inflammatory cytokines and AP-1 factors. A549 cells were treated with and without PMA or LPS or dexamethasone and the cell viability and nitric oxide production was measured by MTT assay and Griess reagent respectively. Expression of pro-inflammatory cytokines and AP-1 factors mRNA were measured using semi quantitative RT-PCR. The PMA/LPS treated cells show significant 2-3 fold increase in the mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-8 and TNF-α), cyclo­oxygenase-2 (COX-2) and specific AP-1 factors (c-Jun, c-Fos and Jun-D). Whereas, pretreatment of cells with dexamethasone significantly inhibited the LPS induced nitric oxide production and PMA/LPS induced mRNAs expression of above pro-inflammatory cytokines, COX-2 and AP-1 factors. Cells treated with dexamethasone alone at both the concentrations inhibit the mRNAs expression of IL-1ß, IL-6 and TNF-α compared to control. Our study reveals that dexamethasone decreased the mRNAs expression of c-Jun and c-Fos available for AP-1 formation suggested that AP-1 is the probable key transcription factor involved in the anti-inflammatory activity of dexamethasone. This may be an important molecular mechanism of steroid action in asthma and other chronic inflammatory lung diseases which may be useful for treatment of lung inflammatory diseases.

Epimedium koreanum Nakai inhibits PMA-induced cancer cell migration and invasion by modulating NF-κB/MMP-9 signaling in monomorphic malignant human glioma cells.

Previously, we showed that the herbal extract EYK (Epimedium koreanum Nakai) can regulate the immune response. Other studies showed that EYK has beneficial effects in human lung cancer, angiogenesis and Alzheimer's disease (AD). However, it remains unknown whether EYK can affect cancer cell migration and invasion in human brain cancer cell lines. In the present study, we found that pre- or post-treatment with EYK inhibited phorbol 12-myristate 13-acetate (PMA)-induced cancer cell migration and invasion in A172 cells, but not in U373MG or T98G cells. Additionally, pre- or post-treatment with PMA followed by EYK decreased MMP-9 activity in A172 cells. Moreover, treatment with a NF-κB inhibitor significantly decreased cell migration in A172 cells pre- or post-treated with EYK and PMA, suggesting that EYK requires NF-κB to alter cancer cell migration. Either pre- or post-treatment with EYK significantly decreased NF-κB nuclear translocation in comparison with PMA treatment. Taken together, our results suggest that EYK suppresses PMA-induced cancer cell migration in monomorphic malignant human glioma cells by downregulating the NF-κB pathway and decreasing MMP-9 activity.

Selenoprotein MsrB1 promotes anti-inflammatory cytokine gene expression in macrophages and controls immune response in vivo.

Post-translational redox modification of methionine residues often triggers a change in protein function. Emerging evidence points to this reversible protein modification being an important regulatory mechanism under various physiological conditions. Reduction of oxidized methionine residues is catalyzed by methionine sulfoxide reductases (Msrs). Here, we show that one of these enzymes, a selenium-containing MsrB1, is highly expressed in immune-activated macrophages and contributes to shaping cellular and organismal immune responses. In particular, lipopolysaccharide (LPS) induces expression of MsrB1, but not other Msrs. Genetic ablation of MsrB1 did not preclude LPS-induced intracellular signaling in macrophages, but resulted in attenuated induction of anti-inflammatory cytokines, such as interleukin (IL)-10 and the IL-1 receptor antagonist. This anomaly was associated with excessive pro-inflammatory cytokine production as well as an increase in acute tissue inflammation in mice. Together, our findings suggest that MsrB1 controls immune responses by promoting anti-inflammatory cytokine expression in macrophages. MsrB1-dependent reduction of oxidized methionine in proteins may be a heretofore unrecognized regulatory event underlying immunity and inflammatory disease, and a novel target for clinical applications.

Taxifolin Activates the Nrf2 Anti-Oxidative Stress Pathway in Mouse Skin Epidermal JB6 P+ Cells through Epigenetic Modifications.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a vital transcription factor that regulates the anti-oxidative defense system. Previous reports suggested that the expression of the Nrf2 gene can be regulated by epigenetic modifications. The potential epigenetic effect of taxifolin (TAX), a potent cancer chemopreventive agent, in skin cancer chemoprotection is unknown. In this study, we investigated how Nrf2 is epigenetically regulated by TAX in JB6 P+ cells. TAX was found to inhibit the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced colony formation of JB6 P+ cells. TAX induced antioxidant response element (ARE)-luciferase activity in HepG2-C8 cells and up-regulated mRNA and protein levels of Nrf2 and its downstream genes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1), in JB6 P+ cells. Furthermore, bisulfite genomic sequencing revealed that TAX treatment reduces the methylation level of the first 15 CpGs sites in the Nrf2 promoter. Western blotting showed that TAX inhibits the expression levels of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) proteins. In summary, our results revealed that TAX can induce expression of Nrf2 and its downstream target genes in JB6 P+ cells by CpG demethylation. These finding suggest that TAX may exhibit a skin cancer preventive effect by activating Nrf2 via an epigenetic pathway.

Topical application of a platelet activating factor receptor agonist suppresses phorbol ester-induced acute and chronic inflammation and has cancer chemopreventive activity in mouse skin.

Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-KitW-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-KitW-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development.

Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke.

Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose-response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34ng/ml for TPA, 0.050mg/ml for the 2R4F, 0.044mg/ml for the Bright cigarette, and 0.060mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P<0.0001), and between the single-tobacco cigarettes and the 2R4F (P=0.0008, 2R4F vs. Burley and P<0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision.

Inhibitory effect of dihydroartemisinin against phorbol ester-induced cyclooxygenase-2 expression in macrophages.

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has recently been shown to possess antitumor activity in various cancer cells. However, the effect of anti-inflammatory potentials of DHA in murine macrophage RAW 264.7 cells has not been studied. The present study investigated the effect of COX-2 and molecular mechanisms by DHA in PMA stimulated RAW 264.7 cells. DHA dose-dependently decreased PMA-induced COX-2 expression and PGE2 production, as well as COX-2 promoter-driven luciferase activity. Additionally, DHA decreased luciferase activity of COX-2 regulation-related transcription factors including NF-κB, AP-1, C/EBP and CREB. DHA also remarkably reduced PMA-induced p65, C/EBPß, c-jun and CREB nuclear translocation. Furthermore, DHA evidently inhibited PMA-induced phosphorylation of AKT and the MAP Kinases, such as ERK, JNK and p38. Taken together, our data indicated that DHA effectively attenuates COX-2 production via down-regulation of AKT and MAPK pathway, revealing partial molecular basis for the anti-inflammatory properties of DHA.

Bioactive components in the fruits of Ziziphus jujuba Mill. against the inflammatory irritant action of Euphorbia plants.

Chinese jujube (also known as Chinese date) is the fruit of Ziziphus jujuba Mill. (Rhamnaceae). As a famous folk medicine, it is used as antidote in traditional Chinese formula, Shi Zao Decoction, to relieve the drastic inflammatory irritant nature of Euphorbia species. The irritant activities may cause serious adverse effects in clinical practices. This study aimed to investigate the active components of Z. jujuba through the inhibitory effects on the inflammatory cells activated by Euphorbia kansui and prostratin, a phorbol ester isolated from Euphorbia fischeriana. Peritoneal macrophage of rat and splenic lymphocyte (splenocyte) of mouse were selected to evaluate these actions in vitro. Nitric oxide (NO) release of macrophage and the proliferation of splenocyte were examined through Griess method and MTT assay. TNF-α, as an important pro-inflammatory cytokines, was detected with enzyme-linked immunosorbent assay (ELISA) method. Six fractions extracted from Z. jujuba were evaluated and fraction F (triterpene acids fraction) was demonstrated to be the most active part, and then, 21 compounds isolated from Z. jujuba were tested at the concentrations range from 1 µg/ml to 100 µg/ml. The results show that 7 compounds of them are likely to be active compounds concerning to their pronounced inhibitory action on the activated inflammatory cells. These effects might be helpful to attenuate the irritant action of Euphorbiaceae plants and protect the gastrointestinal tissue from potent inflammatory injury, which should be beneficial to some diseases, like inflammatory bowel disease.

Synthesis and biological evaluation of the 12,12-dimethyl derivative of Aplog-1, an anti-proliferative analog of tumor-promoting aplysiatoxin.

Aplog-1 is a unique analog of tumor-promoting aplysiatoxin that inhibits tumor-promotion by phorbol diesters and proliferation of tumor cells. While the structural features relevant to the biological activities of Aplog-1 remain to be identified, recent studies by us have suggested that local hydrophobicity around the spiroketal moiety of Aplog-1 is a crucial determinant of its anti-proliferative activity. This hypothesis led us to design 12,12-dimethyl-Aplog-1 (3), in which a hydrophobic geminal dimethyl group is installed proximal to the spiroketal moiety to improve biological potency. As expected, 3 was more effective than Aplog-1 in inhibiting cancer cell growth and binding to protein kinase Cδ, a putative receptor responsible for the biological response of Aplog-1. Moreover, an induction test on Epstein-Barr virus early antigen demonstrated 3 to be a better anti-tumor promoter than Aplog-1. These results indicate that 3 is a superior derivative of Aplog-1, and thus a more promising lead for anti-cancer drugs.

Neuroprotective versus tumorigenic protein kinase C activators.

Protein kinase C (PKC) activators possess potent neurotrophic and neuroprotective activity, thus indicating potential applications in treating neurodegenerative diseases, stroke and traumatic brain injury. Although some activators, such as bryostatin and gnidimacrin, have been tested as antitumor agents, others, such as phorbol esters, are potent tumor promoters. All PKC activators downregulate PKC at high concentrations and long application times. However, tumorigenic activators downregulate certain PKC isozymes, especially PKCdelta, more strongly. Tumorigenic activators possess unique structural features that could account for this difference. At concentrations that minimize PKC downregulation, PKC activators can improve long-term memory, reduce beta-amyloid levels, induce synaptogenesis, promote neuronal repair and inhibit cell proliferation. Intermittent, low concentrations of structurally specific, non-tumorigenic PKC activators, therefore, could offer therapeutic benefit for a variety of neurologic disorders.

A skin irritant phorbol ester from Euphorbia cooperi N E Br.

From the fresh latex of Euphorbia cooperi N E Br was isolated by partition and chromatographic methods, a diterpene ester 12-deoxyphorbol-16-isobutyrate-13-tigliate. The phorbol ester exhibited highly irritant activity on the mouse ear. Since skin irritancy is an indication of possible tumour promotion, the use of this plant as a medicine should be discouraged.

Phorbol ester-induced hairy cell features on chronic lymphocytic leukemia cells in vitro.

Leukemic cells from eight patients with chronic lymphocytic leukemia were isolated and cultured in the continuous presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) at a concentration of 1.6 x 10(-9) M for 4-10 days. Aliquots of cells were then analyzed at intervals of 24-72 hr for changes in morphology, acid phosphatase staining (AP), and expression of two hairy cell-associated surface antigens, HCL1 (CD22, Leu 14) and HCL3 (CD11c, Leu M5). All cases studied showed typical B-CLL phenotype, and only a small proportion of cells expressed CD22 and CD11c (mean 7% and 4.9%, respectively). TPA treatment induced the coexpression of CD22 (mean 49%) and CD11c (mean 48%) and tartrate-resistant acid phosphatase in seven of eight cases. Morphologically, cells in TPA cultures expressed hairy cell features that were evident in light and electron microscopic studies. Collectively these changes indicate that TPA can induce hairy cell features on CLL cells in vitro, suggesting the later maturational stage of HCL compared with CLL.

[Promoting effect of the Chinese medicinal herb, wikstroemia chamaedaphne and tung oil extracts on carcinoma of the uterine cervix induced by HSV-2 or methylcholanthrene (MCA) in mice].

The promoting effect of the Chinese medicinal herb, Wikstroemia chamaedaphne and Tung oil extracts (WC and HHPA) on carcinoma of uterine cervix induced by HSV-2 or MCA in mice was studied. The results showed that WC and HHPA extracts were not carcinogenic themselves. After carcinogen HSV-2 and MCA treatment, WC and HHPA were added separately. The inducing rates by HSV-2 increased from 7.4% to 21.1% and 26.3%, those by MCA increased from 56.5% to 82.8% and 84.4%. There was a significant difference between the combined groups and groups with HSV-2 or MCA only. The experimental results suggest that these two kinds of extracts play a promoting effect on carcinogenesis. The relation between the carcinogenesis of uterine cervix or nasopharynx and WC or HHPA extracts is discussed.

Tumor promotion by phorbol esters in skin: evidence for a memory effect.

By means of a two-stage promotion protocol in mouse epidermis with 12-O-tetradecanoylphorbol-13-acetate as first-stage promoter and 12-O-retinoylphorbol-13-acetate as second-stage promoter, the effects of the former that are critical and obligatory for tumor promotion were shown to be irreversible in nature for at least 8 weeks. The reversibility of tumor promotion was related to the second stage of promotion, reflecting the reversibility of epidermal hyperplasia induced by 12-O-tetradecanoylphorbol-13-acetate.

Studies on the mechanism of skin tumor promotion: evidence for several stages in promotion.

The effects of nonpromoting and weakly promoting diterpenes on skin tumor promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA) were investigated. When phorbol and phorbol 12,13-diacetate (both nonpromoting) were given simultaneously with TPA after 7,12-dimethylbenz[a]-anthracene (DMBA) initiation in female mice, they had no effect on TPA promotion. However, the nonpromoter 4-O-methyl-TPA and the weak promoter mezerein were found to inhibit TPA promotion in a dose-dependent manner when given simultaneously with TPA. Because mezerein was found to be an effective inhibitor of TPA promotion when given simultaneously and because it induces many biological responses similar to those to TPA, the capacity of mezerein to act as an incomplete promoter in a two-stage promotion protocol was also investigated. Twice-weekly applications of 1,2, or 5 mug of TPA for 2 weeks after DMBA initiation produced 0, 0, and 0.5 papilloma per mouse, respectively, at 20 weeks. When the twice-weekly applications of TPA for 2 weeks were followed by twice-weekly treatments with 2 mug of mezerein for 18 weeks, the number of papillomas per mouse was 2.2, 3.5, and 9.0, respectively. Twice-weekly applications of 2 mug of TPA for 2 weeks followed by twice-weekly treatments with 1, 2, or 4 mug of mezerein for 18 weeks produced 2.1, 3.5, and 6.8 papillomas per mouse, respectively, in DMBA-treated mice. Twice-weekly doses as high as 40 mug of 4-O-methyl-TPA were not effective in producing tumors when given after a limited treatment with TPA; however, 4-O-methyl-TPA had weak activity as a first-stage promoter. The results suggest that although mezerein by itself is a weak promoter and mimics TPA in many biochemical and morphological effects it is a potent second-stage promoter in a two-stage promotion regimen.