Green tea polyphenol EGCG suppresses cigarette smoke condensate-induced NF-kappaB activation in normal human bronchial epithelial cells.

Cigarette smoke is a powerful inducer of inflammatory responses resulting in disruption of major cellular pathways with transcriptional and genomic alterations driving the cells towards carcinogenesis. Cell culture and animal model studies indicate that (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent anti-inflammatory and antiproliferative activity capable of selectively inhibiting cell growth and inducing apoptosis in cancer cells without adversely affecting normal cells. Here, we demonstrate that EGCG pretreatment (20-80 microM) of normal human bronchial epithelial cells (NHBE) resulted in significant inhibition of cigarette smoke condensate (CSC)-induced cell proliferation. Nuclear factor-kappaB (NF-kappaB) controls the transcription of genes involved in immune and inflammatory responses. In most cells, NF-kappaB prevents apoptosis by mediating cell survival signals. Pretreatment of NHBE cells with EGCG suppressed CSC-induced phosphorylation of IkappaBalpha, and activation and nuclear translocation of NF-kappaB/p65. NHBE cells transfected with a luciferase reporter plasmid containing an NF-kappaB-inducible promoter sequence showed an increased reporter activity after CSC exposure that was specifically inhibited by EGCG pretreatment. Immunoblot analysis showed that pretreatment of NHBE cells with EGCG resulted in a significant downregulation of NF-kappaB-regulated proteins cyclin D1, MMP-9, IL-8 and iNOS. EGCG pretreatment further inhibited CSC-induced phosphorylation of ERK1/2, JNK and p38 MAPKs and resulted in a decreased expression of PI3K, AKT and mTOR signaling molecules. Taken together, our data indicate that EGCG can suppress NF-kappaB activation as well as other pro-survival pathways such as PI3K/AKT/mTOR and MAPKs in NHBE cells, which may contribute to its ability to suppress inflammation, proliferation and angiogenesis induced by cigarette smoke.

Stabilization of prolactin receptor in breast cancer cells.

The role of the hormone prolactin (PRL) in the pathogenesis of breast cancer is mediated by its cognate receptor (PRLr). Ubiquitin-dependent degradation of the PRLr that negatively regulates PRL signaling is triggered by PRL-mediated phosphorylation of PRLr on Ser349 followed by the recruitment of the beta-transducin repeats-containing protein (beta-TrCP) ubiquitin-protein isopeptide ligase. We report here for the first time that interaction between PRLr and beta-TrCP is less efficient in human breast cancer cells than in non-tumorigenic human mammary epithelial cells. Furthermore, we demonstrate that both PRLr degradation and PRLr phosphorylation on Ser349 are impaired in breast tumor cells and tissues, an observation that directly correlates with enhanced expression of the PRLr in malignant breast epithelium. These findings represent a novel mechanism through which altered PRLr stability may directly influence the pathogenesis of breast cancer.

Increased expression of p50-NF-kappaB and constitutive activation of NF-kappaB transcription factors during mouse skin carcinogenesis.

To elucidate the possible role of NF-kappaB in mouse skin carcinogenesis we studied the expression of p50 (NF-kappaB1), p52 (NF-kappaB2), p65 (RelA) and IkappaB-alpha inhibitor as well as kappaB-binding activity in adult SENCAR mouse skin, skin papillomas, and squamous cell carcinomas (SCC) generated by a two-stage carcinogenesis protocol. We found that in normal epidermis all of the above proteins were mostly expressed in the cytoplasm of basal cells. Western blot analysis revealed a dramatic increase of p50 and p52 expression in mouse skin tumors starting from the middle stage of promotion. We also found that the level of IkappaB-alpha protein in many late papillomas and SCC was lower than in normal epidermis. Results of EMSA showed an increase in kappaB-binding activity in mouse skin tumors and suggested that p50 is the major component of constitutive kappaB-binding complexes in normal epidermis and in tumors. It has been shown that nuclear IkappaB protein Bcl-3 is able to increase p50/p50 homodimer binding to the different kappaB sites in mouse thymocytes. Our finding on Bcl-3 overexpression in late papillomas and SCC could explain the selective increase of p50-related kappaB-binding in mouse skin tumors. Thus, our results strongly suggest the important role of p50 in skin carcinogenesis.

Inducibility of various cytochrome P450 isozymes by phenobarbital and some other xenobiotics in Drosophila melanogaster.

The inducibility of cytochrome P450 isozymes has been investigated in the Drosophila melanogaster insecticide susceptible (Oregon R) and insecticide resistant (91R) strains. Both the level and induction kinetics of 7-ethoxycoumarin O-deethylase activity were stimulated by phenobarbital (PB) to a lower extent than that of aryl hydrocarbon hydroxylase in the Oregon R strain. The basal level of the cytochrome P450-linked activities in insecticide resistant flies was higher than that noted in susceptible ones. However, treatment with PB has increased levels of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities more in susceptible flies than in resistant ones. In contrast to PB, the polycyclic aromatic hydrocarbon benzo[a]pyrene induced both activities in 91R flies to a greater extent than in Oregon R ones. The potent PB-like inducer in mice but not in rats 1.4-bis[2-(dichloropyridyloxy)]-benzene failed to induce the cytochrome P450 system in D. melanogaster, when triphenyldioxane (PB-like inducer in rats but not in mice) markedly affected this system in a PB-like manner. The SDS-PAGE followed by immunoblotting analysis using monoclonal antibodies 13-2e and 8-1d have shown that the level of the 56,000 and 54,000 Da insecticide resistance-related forms has increased in the susceptible strain by PB and some other PB like inducers. The relationship between these isozymes appearance and 7-ethoxycoumarin O-deethylase activity has been discussed.

Xenobiotic-metabolizing enzymes and benzo[a]pyrene metabolism in the benzo[a]pyrene-sensitive mutant strain of Drosophila simulans.

Basal levels of aryl hydrocarbon hydroxylase, epoxide hydrolase and glutathione S-transferase enzyme activities, cytochrome P-450 content and inducibility of enzymes with phenobarbital were found to be similar in the microsomes of D. simulans mutant strain 364yv, which is sensitive to the toxic and mutagenic effects of benzo[a]pyrene (BP), and of the wild resistant Turku strain. In contrast, increases in the rate of BP turnover per molecule of cytochrome P-450, intensity of the hemoprotein band with apparent molecular weight 56,000 and the yield of BP 7,8-dihydrodiol and 9,10-dihydrodiol occurred only in microsomes of BP-pretreated 364yv flies but not of Turku ones. It is likely that BP induces an aberrant form of cytochrome P-450 in 364yv flies with a rare mutation in one of the P-450 regulating genes.

A Drosophila simulans mutant strain sensitive to benzo[a]pyrene and 2-acetylaminofluorene.

We have identified a Drosophila simulans mutant, 364 yu, that is sensitive to the toxic effects of the procarcinogens B(a)P and 2-AAF. Heterozygotes obtained by crossing it to the wild resistant Turku strain (female 364 yu x male Turku) were more sensitive than heterozygotes obtained from the reciprocal cross (female Turku x male 364 yu) to both the toxic and the mutagenic effects of B(a)P in Drosophila tests that measured lethality and the induction of somatic mosaicism, respectively. The non-carcinogens pyrene, B(e)P and 4-AAF were only weakly toxic and non-mutagenic. In the Ames test B(a)P activation with S15 fractions prepared from the homogenates of Drosophila larvae and imagoes of the 364 yu strain, as well as of the more resistant D. melanogaster y ++/+ w sn3 heterozygotes, did not significantly increase the number of S. typhimurium TA100 revertants even following pretreatment with inducers of microsomal monooxygenases (B(a)P, PCB, PB). As for 2-AAF, a certain increase was observed following only PB, but not B(a)P pretreatment. Possible mechanisms of B(a)P and 2-AAF sensitivity of the 364 yu strain, and perspectives on using it for monitoring genotoxic environmental pollutants, are discussed.

Overexpression of mRNA-binding protein CRD-BP in malignant melanomas.

Wnt/beta-catenin signaling pathway plays an important role in embryogenesis, stem cell maintenance, tumorigenesis and aging. Here, we show that RNA-binding protein, coding region determinant-binding protein (CRD-BP) (a transcriptional target of Wnt signaling pathway), is highly expressed in primary human malignant melanomas and melanoma cell lines with activated Wnt/beta-catenin signaling pathway. Upregulation of CRD-BP is associated with an elevated level of beta-TrCP1 ubiquitin ligase receptor and activation of nuclear transcriptional factors-kappa B (NF-kappaB) signaling. Knockdown of CRD-BP inhibits NF-kappaB activity, induces apoptosis, and suppresses proliferation and tumorigenic properties of melanoma cells.

The effect of the cytochrome P-450 system inducers on the development of Drosophila melanogaster.

D. melanogaster development was markedly retarded and its survival decreased by larvae treatment with compounds being strong inducers of the cytochrome P-450 2B in mammals--phenobarbital (PB*), perfluorodecaline (PFD), transtilbene oxide (TSO), and triphenyldioxane (TPD). At the same time, the weak inducer hexobarbital or the selective cytochrome P-450 inducer in mice but not in rats 1,4-bis[2-(dichloropyridyl-oxy)]-benzene (DPB) did not affect the larvae development. The cytochrome P-450 1A1 inducers benzo(a)anthracene (BA) and beta-naphtoflavone (BNF) were also not effective. The toxicity of phenobarbital was shown to be decreased by the cytochrome P-450 inhibitor piperonyl butoxide by adding 20-hydroxyecdysone or by treatment with aminophylline--the indirect enhancer of ecdysone production in the larval prothoracic gland. The hypothesis of the moulting hormone degradation as the cause of elevated larvae mortality resulting from the induced high mixed function oxidase activity has been discussed.

The expression of insecticide resistance-related cytochrome P450 forms is regulated by molting hormone in Drosophila melanogaster.

The expression and enzymatic activities of insecticide resistance-related cytochrome P450B are increased by the treatment with 20-hydroxyecdysone (20HE) in D. melanogaster Oregon R flies. We have explored the role of this hormone in the maintenance of P450B basal expression. Arrest of ecdysone synthesis led to a decrease in CYP6A2 mRNA level, as well as in P450B expression and activities. This effect occurred both in insecticide susceptible (ecd1) and resistant (IRED) strains carrying the temperature-sensitive ecd mutation. The role of the 20HE in the regulation of cytochrome P450-mediated insecticide resistance has been proposed.

Resistance of transformed mouse keratinocytes to growth inhibition by glucocorticoids.

Glucocorticoid hormones are strong inhibitors of normal keratinocyte proliferation, but established mouse skin papillomas and carcinomas become resistant to these hormones. The biological effect of glucocorticoids is mediated through a highly specific glucocorticoid receptor (GR). To study the possible mechanisms of glucocorticoid resistance of transformed mouse keratinocytes, we evaluated GR expression and function in non-tumorigenic (3PC), papilloma-producing (MT1/2 and P1/17), and squamous cell carcinoma-producing (Ca3/7 and Ca8/29) keratinocyte cell lines and analyzed the DNA sequence of GR in glucocorticoid-sensitive and glucocorticoid-resistant keratinocytes. All transformed keratinocyte cell lines studied appeared to be completely resistant to the growth inhibition by the glucocorticoid fluocinolone acetonide (FA), whereas the untransformed cell line 3PC was very sensitive to FA. Despite the glucocorticoid resistance, all the tumorigenic keratinocyte cell lines expressed high levels of GR mRNA and protein. Southern blot analysis and direct sequencing of the DNA-binding domain of the GR gene revealed no significant changes in GR gene structure in transformed keratinocytes. To test the functional capability of GR, we compared the effect of FA on the expression of glucocorticoid-responsive genes. FA strongly induced metallothionein 1 expression in 3PC cells, slightly induced metallothionein 1 expression in P1/17 and Ca3/7 cells, and did not affect its expression in MT1/2 and Ca8/29 cells. These data suggest that resistance to the growth inhibition of glucocorticoids is an important feature of tumorigenic keratinocyte cell lines. It is likely that this hormone-resistant phenotype is a result of alteration of GR function but not of GR expression or gene structure.

Induction of beta-transducin repeat-containing protein by JNK signaling and its role in the activation of NF-kappaB.

Activation of Jun N-kinase (JNK) and NF-kappaB transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-kappaB inhibitor (IkappaB) by respective stress-inducible kinases (IKK) is a key event in NF-kappaB activation. beta-TrCP F-box protein mediates ubiquitination of phosphorylated IkappaB via recruitment of SCF(beta-TrCP)-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IkappaB results in activation of the NF-kappaB pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of beta-TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKKbeta) also leads to accumulation of beta-TrCP. Transcription of the beta-TrCP gene is not required for JNK-mediated induction of beta-TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-kappaB was observed. The mechanisms of beta-TrCP induction via stress and its role in NF-kappaB activation are discussed.

Wnt/beta-catenin signaling induces the expression and activity of betaTrCP ubiquitin ligase receptor.

Beta-transducing repeat-containing protein (betaTrCP) targets the ubiquitination and subsequent degradation of both beta-catenin and IkappaB, thereby playing an important role in beta-catenin/Tcf and NF-kappaB-dependent signaling. Here evidence is presented that beta-catenin/Tcf signaling elevates the expression of betaTrCP mRNA and protein in a Tcf-dependent manner, which does not require betaTrCP transcription. Induction of betaTrCP expression by the beta-catenin/Tcf pathway results in an accelerated degradation of the wild-type beta-catenin, suggesting that the negative feedback loop regulation may control the beta-catenin/Tcf pathway. This signaling also upregulated NF-kappaB transactivation without affecting the activity of IkappaB kinase, thereby establishing that the maintenance of the betaTrCP level is important for coordination between beta-catenin/Tcf and NF-kappaB signaling.