We investigated the effect of 21 flavonoids in a three-dimensional in vitro system for their ability to inhibit gap formation by MCF-7 breast cancer spheroids in monolayers of lymphendothelial cells. Different representatives of the classes of flavones, flavonols, and flavanones were tested in the circular chemorepellent-induced defects (CCID)-assay. Bay11-7082, a known inhibitor of CCID formation served as the positive control. This study provides the first comparison of the potential of flavonoids to suppress features influencing the intravasation of MCF-7 breast cancer cells aggregates through the lymph endothelial barrier. The most significant effects were seen after incubation with the flavones luteolin, chrysin, and apigenin. Additional hydroxylation or methoxylation in positions 6 or 8, as expected, resulted in decreased activity. The tested flavanones remained without or low efficacy.
Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre-metastatic niches, leading to their colonisation. 'S' stereoisomer 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(S)-HETE] is secreted by a variety of cancer cell types and has been indicated to open up these barriers. In the present study, another aspect of the endothelial unlocking mechanism was elucidated. This was achieved by investigating 12(S)-HETE-treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY-box transcription factor 18 (SOX18), prospero homeobox 1 (PROX1) and focal adhesion kinase (FAK). These key players of LEC retraction, which is a prerequisite for cancer cell transit into vasculature, were analysed using western blot analysis, reverse transcription-quantitative PCR and transfection with small interfering (si)RNA. The silencing of a combination of these signalling and executing molecules using siRNA, or pharmacological inhibition with defactinib and Bay11-7082, extended the mono-culture experiments to co-culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent-induced defect' assay. 12(S)-HETE was indicated to induce the upregulation of the RELA/SOX18 feedback loop causing the subsequent phosphorylation of FAK, which fed back to RELA/SOX18. Therefore, 12(S)-HETE was demonstrated to be associated with circuits involving RELA, SOX18 and FAK, which transduced signals causing the retraction of LECs. The FAK-inhibitor defactinib and the NF-κB inhibitor Bay11-7082 attenuated LEC retraction additively, which was similar to the suppression of FAK and PROX1 (the target of SOX18) by the transfection of respective siRNAs. FAK is an effector molecule at the distal end of a pro-metastatic signalling cascade. Therefore, targeting the endothelial-specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.
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