AHR/CYP1A1 interplay triggers lymphatic barrier breaching in breast cancer spheroids by inducing 12(S)-HETE synthesis.

A causal link between overexpression of aryl hydrocarbon receptor (AHR) and its target cytochrome P450 1A1 (CYP1A1) and metastatic outgrowth of various cancer entities has been established. Nevertheless, the mechanism how AHR/CYP1A1 support metastasis formation is still little understood. In vitro we discovered a potential mechanism facilitating tumour dissemination based on the production of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Utilising a three-dimensional lymph endothelial cell (LEC) monolayer & MDA-MB231 breast cancer cell spheroid co-culture model in combination with knock-down approach allowed elucidation of the molecular/biochemical basis of AHR/CYP1A1-induced tumour breaching through the LEC barrier. Enzyme immunoassay evidenced the potential of recombinant CYP1A1 to synthesise 12(S)-HETE in vitro and qPCR and Western blotting measured gene and protein expression in specific experimental settings. In detail, AHR induced CYP1A1 expression and 12(S)-HETE secretion in tumour spheroids, which caused LEC junction retraction thereby forming large discontinuities allowing transmigration of the tumour. This was enforced by the activating AHR ligand 6-formylindolo (3,3-b)carbazole (FICZ), or inhibited by the AHR antagonist 3,3'-diindolylmethane (DIM) as well as by siRNA against AHR and CYP1A1. AHR and NF-κB were negatively cross talking and therefore, the inhibition of AHR (but not CYP1A1) induced RELA, RELB, NFKB1, NFKB2 and the NF-κB target MMP1, which itself promotes tumour intravasation by a mechanism that is different from 12(S)-HETE. Conversely, the inhibition of NFKB2 induced AHR, CYP1A1 and 12(S)-HETE synthesis. The approved clinical drugs guanfacine and vinpocetine, which inhibit CYP1A1 and NF-κB, respectively, significantly inhibited LEC barrier breaching in vitro indicating an option to reduce metastatic dissemination.

Bioguided isolation of (9Z)-octadec-9-enoic acid from Phellodendron amurense Rupr. and identification of fatty acids as PTP1B inhibitors.

The therapy of type-2 diabetes mellitus is one of the major challenges of our age. A possible strategy to prevent the progression of this disease is the inhibition of protein tyrosine phosphatase 1B (PTP1B), a major negative regulator in the insulin and leptin signalling pathway. Phellodendri amurensis cortex is a well-known Asian herbal drug traditionally used as antiphlogistic, antibacterial, and anti-inflammatory agent, and its efficacy against diabetes-related symptoms is reported as well. However, information regarding active principle(s) or the molecular mode of action was scarce. By bioguided isolation using an IN VITRO enzyme assay with human recombinant PTP1B, (9 Z)-octadec-9-enoic acid (oleic acid) could be identified as a major PTP1B inhibitor in the bark of Phellodendron amurENSE Rupr. (Rutaceae); it showed an IC50 value of 6.2 µM. Consistent with this inhibition of PTP1B, oleic acid was capable of enhancing insulin signalling in wild-type, but not PTP1B knockout fibroblasts. By testing a series of other fatty acids of different chain length and degree of saturation, their general PTP1B-inhibitory potential in the micromolar range was observed. More pronounced effects were associated with a longer carbon backbone and saturation of the double bonds. Therefore, our work provides first scientific evidences for the antidiabetic properties of P. amurense via a new target, effects which seem to be explainable by oleic acid. The discovery of a PTP1B inhibition by many fatty acids also adds a novel facet to the pharmacological properties of a class of compounds that is found in many food items in considerable amount and triggers speculation over their possible involvement in the feedback regulation of cellular fatty acid synthesis.

Ikarugamycin induces DNA damage, intracellular calcium increase, p38 MAP kinase activation and apoptosis in HL-60 human promyelocytic leukemia cells.

Ikarugamycin (IKA) is an antibiotic with strong antiprotozoal and cytotoxic activity. The purpose of our work was to provide insight into the mechanism of action characterizing the cytotoxic effect of IKA in HL-60 leukemia cells in order to evaluate its potential as an antineoplastic agent. Cell viability was reduced in response to IKA (IC(50) of 221.3nM), while the amount of HL-60 cells with a subdiploid DNA content increased significantly after 24h. Apoptotic cell death was confirmed by the cleavage of caspase-9, -8 and -3 using immunoblotting. Single cell gel electrophoresis pointed to an early genotoxic effect. Monitoring of intracellular calcium ([Ca(2+)](i)) levels by flow cytometric analysis of Fluo-3-AM fluorescence indicated an increase in cytosolic calcium that correlated with the cleavage of caspases. In addition, IKA triggered the activation of p38 MAP kinase which was partly dependent on elevated [Ca(2+)](i) concentrations and contributed to caspase activation. The data demonstrate that IKA induced apoptosis in HL-60 cells through genotoxicity and caspase activation which was in part correlated to an increase in intracellular calcium levels and activation of p38 MAP kinase.

Intravasation of SW620 colon cancer cell spheroids through the blood endothelial barrier is inhibited by clinical drugs and flavonoids in vitro.

Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called "circular chemorepellent induced defects" (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-κB in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-κB], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-κB] significantly attenuated CCID formation at low µM concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naïve SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified) genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naïve- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo.