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+, Ca2+-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.

Children's screen time alters the expression of saliva extracellular miR-222 and miR-146a.

An imbalance between energy uptake and energy expenditure is the most important reason for increasing trends in obesity starting from early in life. Extracellular miRNAs are expressed in all bodily fluids and their expression is influenced by a broad range of stimuli. We examined whether screen time, physical activity and BMI are associated with children's salivary extracellular miR-222 and miR-146a expression. In 80 children the extracellular fraction of saliva was obtained by means of differential centrifugation and ultracentrifugation. Expression levels of miR-222 and miR-146a were profiled by qPCR. We studied the association between children's salivary extracellular miRNA expression and screen time, physical activity and BMI using mixed models, while accounting for potential confounders. We found that higher screen time was positively associated with salivary extracellular miR-222 and miR-146a levels. On average, one hour more screen time use per week was associated with a 3.44% higher miR-222 (95% CI: 1.34 to 5.58; p = 0.002) and 1.84% higher miR-146a (95% CI: -0.04 to 3.75; p = 0.055) level in saliva. BMI and physical activity of the child were not significantly associated with either miR-222 or miR-146a. A sedentary behaviour, represented by screen time use in children, is associated with discernible changes in salivary expression of miR-146a and or miR-222. These miRNA targets may emerge attractive candidates to explore the role of these exposures in developmental processes of children's health.

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.