Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca2+ signalling

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological-or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca(2+)calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction

Xanthohumol attenuates tumour cell-mediated breaching of the lymphendothelial barrier and prevents intravasation and metastasis

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In vitro characterisation of the anti-intravasative properties of the marine product heteronemin

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A systems pharmacology workflow with experimental validation to assess the potential of anakinra for treatment of focal and segmental glomerulosclerosis.

Focal and Segmental Glomerulosclerosis (FSGS) is a severe glomerulopathy that frequently leads to end stage renal disease. Only a subset of patients responds to current therapies, making it important to identify alternative therapeutic options. The interleukin (IL)-1 receptor antagonist anakinra is beneficial in several diseases with renal involvement. Here, we evaluated the potential of anakinra for FSGS treatment. Molecular process models obtained from scientific literature data were used to build FSGS pathology and anakinra mechanism of action models by exploiting information on protein interactions. These molecular models were compared by statistical interference analysis and expert based molecular signature matching. Experimental validation was performed in Adriamycin- and lipopolysaccharide (LPS)-induced nephropathy mouse models. Interference analysis (containing 225 protein coding genes and 8 molecular process segments) of the FSGS molecular pathophysiology model with the drug mechanism of action of anakinra identified a statistically significant overlap with 43 shared molecular features that were enriched in pathways relevant in FSGS, such as plasminogen activating cascade, inflammation and apoptosis. Expert adjudication of molecular signature matching, focusing on molecular process segments did not suggest a high therapeutic potential of anakinra in FSGS. In line with this, experimental validation did not result in altered proteinuria or significant changes in expression of the FSGS-relevant genes COL1A1 and NPHS1. In summary, an integrated bioinformatic and experimental workflow showed that FSGS relevant molecular processes can be significantly affected by anakinra beyond the direct drug target IL-1 receptor type 1 (IL1R1) context but might not counteract central pathophysiology processes in FSGS. Anakinra is therefore not suggested for extended preclinical trials

Apigenin and Luteolin Attenuate the Breaching of MDA-MB231 Breast Cancer Spheroids Through the Lymph Endothelial Barrier in Vitro

Flavonoids, present in fruits, vegetables and traditional medicinal plants, show anticancer effects in experimental systems and are reportedly non-toxic. This is a favorable property for long term strategies for the attenuation of lymph node metastasis, which may effectively improve the prognostic states in breast cancer. Hence, we studied two flavonoids, apigenin and luteolin exhibiting strong bio-activity in various test systems in cancer research and are readily available on the market. This study has further advanced the mechanistic understanding of breast cancer intravasation through the lymphatic barrier. Apigenin and luteolin were tested in a three-dimensional (3-D) assay consisting of MDA-MB231 breast cancer spheroids and immortalized lymph endothelial cell (LEC) monolayers. The 3-D model faithfully resembles the intravasation of breast cancer emboli through the lymphatic vasculature. Western blot analysis, intracellular Ca2+ determination, EROD assay and siRNA transfection revealed insights into mechanisms of intravasation as well as the anti-intravasative outcome of flavonoid action. Both flavonoids suppressed pro-intravasative trigger factors in MDA-MB231 breast cancer cells, specifically MMP1 expression and CYP1A1 activity. A pro-intravasative contribution of FAK expression in LECs was established as FAK supported the retraction of the LEC monolayer upon contact with cancer cells thereby enabling them to cross the endothelial barrier. As mechanistic basis, MMP1 caused the phosphorylation (activation) of FAK at Tyr397 in LECs. Apigenin and luteolin prevented MMP1-induced FAK activation, but not constitutive FAK phosphorylation. Luteolin, unlike apigenin, inhibited MMP1-induced Ca2+ release. Free intracellular Ca2+ is a central signal amplifier triggering LEC retraction through activation of the mobility protein MLC2, thereby enhancing intravasation. FAK activity and Ca2+ levels did not correlate. This implicates that the pro-intravasative contribution of FAK and of Ca2+ release in LECs was independent of each other and explains the better anti-intravasative effects of luteolin in vitro. In specific formulations, flavonoid concentrations causing significant anti-intravasative effects, can certainly be achieved in vivo. As the therapeutic strategy has to be based on permanent flavonoid treatment both the beneficial and adverse effects have to be investigated in future studies.(VLID)470327

Reliable Quantification of Protein Expression and Cellular Localization in Histological Sections

<div><p>In targeted therapy, patient tumors are analyzed for aberrant activations of core cancer pathways, monitored based on biomarker expression, to ensure efficient treatment. Thus, diagnosis and therapeutic decisions are often based on the status of biomarkers determined by immunohistochemistry in combination with other clinical parameters. Standard evaluation of cancer specimen by immunohistochemistry is frequently impeded by its dependence on subjective interpretation, showing considerable intra- and inter-observer variability. To make treatment decisions more reliable, automated image analysis is an attractive possibility to reproducibly quantify biomarker expression in patient tissue samples. We tested whether image analysis could detect subtle differences in protein expression levels. Gene dosage effects generate well-graded expression patterns for most gene-products, which vary by a factor of two between wildtype and haploinsufficient cells lacking one allele. We used conditional mouse models with deletion of the transcription factors <i>Stat5ab</i> in the liver as well <i>Junb</i> deletion in a T-cell lymphoma model. We quantified the expression of total or activated STAT5AB or JUNB protein in normal (<i>Stat5ab^+/+ or JunB^+/+</i>), hemizygous (<i>Stat5ab^+/Δ</i> or <i>JunB^+/Δ</i>) or knockout (<i>Stat5ab^Δ/Δ</i> or <i>JunB^Δ/Δ</i>) settings. Image analysis was able to accurately detect hemizygosity at the protein level. Moreover, nuclear signals were distinguished from cytoplasmic expression and translocation of the transcription factors from the cytoplasm to the nucleus was reliably detected and quantified using image analysis. We demonstrate that image analysis supported pathologists to score nuclear STAT5AB expression levels in immunohistologically stained human hepatocellular patient samples and decreased inter-observer variability.</p></div

Detection and quantification of transcription factor translocation into the nucleus in mouse livers.

<p><b>A</b> IHC of STAT5AB expression in <i>Stat5ab^+/+</i> (n = 3), <i>Stat5ab^+/Δ</i> (n = 3) and <i>Stat5ab^Δ/Δ</i> (n = 3) livers of GH treated and control mice. Size bar: 50 µm. <b>B</b> Scattergram of the analyzed IHC samples used in A. Discrimination of hepatocytes due to their hematoxylin area and mean intensity depicted in gate 4. Image of gated (hepatocytes, red circled) and of non-hepatocytic cells, mainly Kupffer cells (green circled) in an IHC stained liver sample. <b>C</b> Nuclear and cytoplasmic mask of cells as they are recognized by HistoQuest. The bar graph shows the ratios of nuclear versus cytoplasmic STAT5AB expression levels. Error bars are S.D. Student's t-test was performed to demonstrate statistical significance. <b>D</b> Histograms of nuclear/cytoplasmic STAT5AB intensities are shown; <i>Stat5ab^+/+</i> (blue), <i>Stat5ab^+/Δ</i> (red) and <i>Stat5ab^Δ/Δ</i> (green). The corresponding values in the histograms (black values) showed the gradual decrease of the mean intensities. Based on the fact that <i>Stat5ab^Δ/Δ</i> mice have no hepatic STAT5AB their values in the histograms can be set as background intensity. After background subtraction the numbers displayed the expected 2∶1∶0 ratios (red values). <b>E</b> Whiskers-box plots depicting the quantification of STAT5AB positive hepatocytes (cytoplasmic C; nuclear N) from <i>Stat5ab^+/+</i> with and without GH stimulation using the HistoQuest software. The box indicates the interquartile range; the horizontal line in the box depicts the median. Whiskers indicate the data range. Student's t-test was used to demonstrate statistical significance.</p

Apigenin and Luteolin Attenuate the Breaching of MDA-MB231 Breast Cancer Spheroids Through the Lymph Endothelial Barrier in Vitro

Flavonoids, present in fruits, vegetables and traditional medicinal plants, show anticancer effects in experimental systems and are reportedly non-toxic. This is a favorable property for long term strategies for the attenuation of lymph node metastasis, which may effectively improve the prognostic states in breast cancer. Hence, we studied two flavonoids, apigenin and luteolin exhibiting strong bio-activity in various test systems in cancer research and are readily available on the market. This study has further advanced the mechanistic understanding of breast cancer intravasation through the lymphatic barrier. Apigenin and luteolin were tested in a three-dimensional (3-D) assay consisting of MDA-MB231 breast cancer spheroids and immortalized lymph endothelial cell (LEC) monolayers. The 3-D model faithfully resembles the intravasation of breast cancer emboli through the lymphatic vasculature. Western blot analysis, intracellular Ca2+ determination, EROD assay and siRNA transfection revealed insights into mechanisms of intravasation as well as the anti-intravasative outcome of flavonoid action. Both flavonoids suppressed pro-intravasative trigger factors in MDA-MB231 breast cancer cells, specifically MMP1 expression and CYP1A1 activity. A pro-intravasative contribution of FAK expression in LECs was established as FAK supported the retraction of the LEC monolayer upon contact with cancer cells thereby enabling them to cross the endothelial barrier. As mechanistic basis, MMP1 caused the phosphorylation (activation) of FAK at Tyr397 in LECs. Apigenin and luteolin prevented MMP1-induced FAK activation, but not constitutive FAK phosphorylation. Luteolin, unlike apigenin, inhibited MMP1-induced Ca2+ release. Free intracellular Ca2+ is a central signal amplifier triggering LEC retraction through activation of the mobility protein MLC2, thereby enhancing intravasation. FAK activity and Ca2+ levels did not correlate. This implicates that the pro-intravasative contribution of FAK and of Ca2+ release in LECs was independent of each other and explains the better anti-intravasative effects of luteolin in vitro. In specific formulations, flavonoid concentrations causing significant anti-intravasative effects, can certainly be achieved in vivo. As the therapeutic strategy has to be based on permanent flavonoid treatment both the beneficial and adverse effects have to be investigated in future studies

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-kappa B 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-kappa B], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-kappa B] significantly attenuated CCID formation at low mu M concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naive 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, naive- 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