Inhibition of Chk1 stimulates cytotoxic action of platinum-based drugs and TRAIL combination in human prostate cancer cells.

Checkpoint kinase 1 (Chk1) plays an important role in regulation of the cell cycle, DNA damage response and cell death, and represents an attractive target in anticancer therapy. Small-molecule inhibitors of Chk1 have been intensively investigated either as single agents or in combination with various chemotherapeutic drugs and they can enhance the chemosensitivity of numerous tumor types. Here we newly demonstrate that pharmacological inhibition of Chk1 using potent and selective inhibitor SCH900776, currently profiled in phase II clinical trials, significantly enhances cytotoxic effects of the combination of platinum-based drugs (cisplatin or LA-12) and TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in human prostate cancer cells. The specific role of Chk1 in the drug combination-induced cytotoxicity was confirmed by siRNA-mediated silencing of this kinase. Using RNAi-based methods we also showed the importance of Bak-dependent mitochondrial apoptotic pathway in the combined anticancer action of SCH900776, cisplatin and TRAIL. The triple drug combination-induced cytotoxicity was partially enhanced by siRNA-mediated Mcl-1 silencing. Our findings suggest that targeting Chk1 may be used as an efficient strategy for sensitization of prostate cancer cells to killing action of platinum-based chemotherapeutic drugs and TRAIL.

Switching on/off aryl hydrocarbon receptor and pregnane X receptor activities by chemically modified tryptamines.

Microbial indoles have been demonstrated as selective or dual agonists and ligands of the pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR). However, structural determinants of microbial indoles selectivity towards both receptors remain elusive. Here, we studied the effects of existing and newly synthesized derivatives of indole microbial metabolite tryptamine on the activity of AhR and PXR receptors. We show that the elongation of indolyl-3-alkaneamine chain, indole N-methylation and conversion of indolyl-3-alkaneamines to oleamides resulted in a major increase of PXR activity and in parallel loss of AhR activity. Using reporter gene assays, RT-PCR and TR-FRET techniques, we have characterized in detail the activation of PXR by novel indolyl-3-alkanyl-oleamides, 1-methyltryptamine and 1-methyltryptamine-acetamide. As a proof of concept, we demonstrated anti-inflammatory and epithelial barrier-protective activity of lead derivatives in intestinal Caco-2 cells, employing the measurement of expression of pro-inflammatory chemokines, tight junction genes, trans-epithelial electric resistance TEER, and dextran-FITC permeability assay. In conclusion, we show that a subtle chemical modifications of simple microbial indole metabolite tryptamine, leads to substantial changes in AhR and PXR agonist activities.

Aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) play both distinct and common roles in the regulation of colon homeostasis and intestinal carcinogenesis.

Both aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) belong among key regulators of xenobiotic metabolism in the intestinal tissue. AhR in particular is activated by a wide range of environmental and dietary carcinogens. The data accumulated over the last two decades suggest that both of these transcriptional regulators play a much wider role in the maintenance of gut homeostasis, and that both transcription factors may affect processes linked with intestinal tumorigenesis. Intestinal epithelium is continuously exposed to a wide range of AhR, PXR and dual AhR/PXR ligands formed by intestinal microbiota or originating from diet. Current evidence suggests that specific ligands of both AhR and PXR can protect intestinal epithelium against inflammation and assist in the maintenance of epithelial barrier integrity. AhR, and to a lesser extent also PXR, have been shown to play a protective role against inflammation-induced colon cancer, or, in mouse models employing overactivation of Wnt/ß-catenin signaling. In contrast, other evidence suggests that both receptors may contribute to modulation of transformed colon cell behavior, with a potential to promote cancer progression and/or chemoresistance. The review focuses on both overlapping and separate roles of the two receptors in these processes, and on possible implications of their activity within the context of intestinal tissue.

Zearalenone Does Not Show Genotoxic Effects in the Drosophila melanogaster Wing Spot Test, but It Induces Oxidative Imbalance, Development, and Fecundity Alterations.

Zearalenone (ZEN) is a non-steroidal mycoestrogen produced by the Fusarium genus. ZEN and its metabolites compete with 17-beta estradiol for cytosolic estrogen receptors, causing reproductive alterations in vertebrates. ZEN has also been associated with toxic and genotoxic effects, as well as an increased risk for endometrial adenocarcinomas or hyperplasia, breast cancer, and oxidative damage, although the underlying mechanisms remain unclear. Previous studies have monitored cellular processes through levels of transcripts associated with Phase I Xenobiotic Metabolism (Cyp6g1 and Cyp6a2), oxidative stress (hsp60 and hsp70), apoptosis (hid, grim, and reaper), and DNA damage genes (Dmp53). In this study, we evaluated the survival and genotoxicity of ZEN, as well as its effects on emergence rate and fecundity in Drosophila melanogaster. Additionally, we determined levels of reactive oxygen species (ROS) using the D. melanogaster flare and Oregon R(R)-flare strains, which differ in levels of Cyp450 gene expression. Our results showed that ZEN toxicity did not increase mortality by more than 30%. We tested three ZEN concentrations (100, 200, and 400 µM) and found that none of the concentrations were genotoxic but were cytotoxic. Taking into account that it has previously been demonstrated that ZEN administration increased hsp60 expression levels and apoptosis gene transcripts in both strains, the data agree with an increase in ROS and development and fecundity alterations. Since Drosophila lacks homologous genes for mammalian estrogen receptors alpha and beta, the effects of this mycotoxin can be explained by a mechanism different from estrogenic activity.

Aryl Hydrocarbon Receptor (AhR) Limits the Inflammatory Responses in Human Lung Adenocarcinoma A549 Cells via Interference with NF-κB Signaling.

Apart from its role in the metabolism of carcinogens, the aryl hydrocarbon receptor (AhR) has been suggested to be involved in the control of inflammatory responses within the respiratory tract. However, the mechanisms responsible for this are only partially known. In this study, we used A549 cell line, as a human model of lung alveolar type II (ATII)-like cells, to study the functional role of the AhR in control of inflammatory responses. Using IL-1ß as an inflammation inducer, we found that the induction of cyclooxygenase-2 and secretion of prostaglandins, as well as expression and release of pro-inflammatory cytokines, were significantly higher in the AhR-deficient A549 cells. This was linked with an increased nuclear factor-κB (NF-κB) activity, and significantly enhanced phosphorylation of its regulators, IKKα/ß, and their target IκBα, in the AhR-deficient A549 cells. In line with this, when we mimicked the exposure to a complex mixture of airborne pollutants, using an organic extract of reference diesel exhaust particle mixture, an exacerbated inflammatory response was observed in the AhR-deficient cells, as compared with wild-type A549 cells. Together, the present results indicate that the AhR may act as a negative regulator of the inflammatory response in the A549 model, via a direct modulation of NF-κB signaling. Its role(s) in the control of inflammation within the lung alveoli exposed to airborne pollutants, especially those which simultaneously activate the AhR, thus deserve further attention.

Increased expression of FAK isoforms as potential cancer biomarkers in ovarian cancer.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is expressed in most human cell types (example: Epithelial cells, fibroblasts and endothelial), it serves a key role in the control of cell survival, proliferation and motility. The abnormal expression of FAK has been associated with poor prognosis in cancer, including ovarian cancer. However, although FAK isoforms with specific molecular and functional properties have been characterized, there are a limited number of published studies that examine FAK isoforms in ovarian cancer. The aim of the present study was to analyze the expression level of FAK and its isoforms in ovarian cancer. The expression of FAK kinase and focal adhesion targeting (FAT) domains was determined with immunohistochemistry in healthy ovary, and serous and mucinous cystadenoma, borderline tumor and carcinoma samples. Additionally, the expression of FAK and its isoforms were investigated in three ovarian cancer-derived cell lines with western blotting and reverse transcription-semi-quantitative polymerase chain reaction. An increased expression of FAK kinase domain was determined in serous tumor samples and was associated with advancement of the lesion. FAK kinase domain expression was moderate-to-low in mucinous tumor samples. The expression of the FAK FAT domain in tumor samples was reduced, compared with healthy ovary samples; however, the FAT domain was localized to the cellular nucleus. Expression of alternative transcripts FAK°, FAK28,6 and FAK28 was determined in all three cell lines investigated. In conclusion, FAK kinase and FAT domains are differentially expressed among ovarian tumor types. These results indicated the presence of at least two isoforms of FAK (FAK and the putative FAK-related non-kinase) in tumor tissue, which is supported by the cells producing at least three FAK alternative transcripts. These results may support the use of FAK and its isoforms as biomarkers for ovarian cancer.

Erythropoietin promotes expression of survivin via STAT3 activation and reduces sensitivity to cisplatin in cervical cancer cells.

Erythropoietin (Epo) is used for the treatment of cancer­associated anaemia. However, certain studies have identified that the administration of Epo mediates the acquisition of resistance to cisplatin, which is widely used to treat cervical cancer. Our group previously reported that cervical cancer cells express Epo receptor and that exogenous Epo induces cell proliferation and migration. However, the effect of Epo on cervical cancer cell death mediated by chemotherapeutic agents has not yet been evaluated. Thus, the aim of the present study was to assess the potential effect of Epo on the cytotoxic activity of cisplatin in cervical cancer cells. The effect of Epo was assessed in 3 cervical cancer­derived cell lines. It was observed that pre­incubation with Epo induced a significant reduction of cisplatin­induced apoptosis in vitro and in vivo. Incubation with Epo induced the expression and activation of the transcriptional factor signal transducer and activator of transcription 3 (STAT3), which in turn stimulated the expression and activation of the anti­apoptotic protein survivin. The cytotoxicity of cisplatin was partially restored by treating the cells with MY155, an inhibitor of survivin. Conversely, inhibition of STAT3 activation using sub­lethal doses of WP1066, completely abolished the cytoprotective effect of Epo. These observations indicated that Epo was able to hinder the cytotoxic effect of cisplatin in cervical cancer cells by activating anti­apoptotic responses regulated by STAT3.

Aryl Hydrocarbon Receptor-Dependent Metabolism Plays a Significant Role in Estrogen-Like Effects of Polycyclic Aromatic Hydrocarbons on Cell Proliferation.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that interact in a complex manner with both the aryl hydrocarbon receptor (AhR) and estrogen receptors (ER). Their potential endocrine-disrupting activities may depend on both inhibitory AhR-ER cross-talk and on AhR-dependent metabolic production of estrogenic PAH metabolites. Here, we analyzed the impact of AhR on estrogen-like effects of PAHs, such as benzo[a]pyrene (BaP), in particular, on control of cell cycle progression/cell proliferation. Using AhR knockout variant of estrogen-sensitive human breast cancer MCF-7 cells (MCF-7 AhRKO cells), we observed that the AhR-dependent control of cytochrome P450 family 1 (CYP1) expression played a major role in formation of estrogenic BaP metabolites, most notably 3-OH-BaP, which contributed to the ER-dependent induction of cell cycle progression/cell proliferation. Both BaP metabolism and the BaP-induced S-phase transition/cell proliferation were inhibited in MCF-7 AhRKO cells, whereas these cells remained sensitive towards both endogenous estrogen 17ß-estradiol or hydroxylated BaP metabolites. BaP was found to increase the activity of ER-dependent luciferase reporter gene in wild-type MCF-7 cells; however, unlike its hydroxylated metabolite, BaP failed to stimulate luciferase activity in MCF-7 AhRKO cells. Similarly, estrogen-like effects of other known estrogenic PAHs, such as benz[a]anthracene or 3-methylcholanthrene, were diminished in MCF-7 AhRKO cells. Ectopic expression of human CYP1A1 and CYP1B1 enzymes partly restored both BaP metabolism and its effects on cell proliferation. Taken together, our data suggest that the AhR-dependent metabolism of PAHs contributes significantly to the impact of PAHs on cell proliferation in estrogen-sensitive cells.