In vitro inhibition of breast cancer spheroid-induced lymphendothelial defects resembling intravasation into the lymphatic vasculature by acetohexamide, isoxsuprine, nifedipin and proadifen

Abstract

Background:As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration.Methods:A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called ‘circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis.Results:Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation.Conclusion:The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.

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

“…In contrast, at low xanthohumol concentrations (5 lM), another CCID-forming mechanism was affected that was more active in MCF-7 than in MDA-MB231 cells. We propose that this mechanism involves the activity of CYP1A1 since CYPs may contribute to CCID formation (Kretschy et al 2013). CYP1A1 plays a role in breast cancer metastasis (Jiang et al 2007), and therefore, its function in CCID formation needs to be investigated in more detail.…”

“…This phenomenon was also observed when CYP was inhibited by proadifen resulting in increased levels of the COX metabolite prostaglandin E2 (Kozak et al 2000, COX also uses arachidonic acid as substrate). Consequently, the inhibition of CYP1A1 by xanthohumol or proadifen (Kretschy et al 2013) prevents the consumption of arachidonic acid by CYP, and hence, higher substrate levels are available for ALOX12/15, which in turn produce more 12(S)-HETE as it was observed after treatment with xanthohumol (Fig. 4a).…”

“…At higher concentration (25 lM), xanthohumol inhibited CYP1A1 also in MDA-MB231 cells, yet less efficiently, and this was in accordance with the weak CCID inhibitory effect of xanthohumol in this cell line. Also proadifen, which is a specific inhibitor of CYPs, attenuated CCID formation of LECs (Kretschy et al 2013). ALOX12/15 and CYPs use arachidonic acid as their main substrate for the synthesis of their metabolites.…”

The germacranolide sesquiterpene lactone neurolenin B of the medicinal plant Neurolaena lobata (L.) R.Br. ex Cass inhibits NPM/ALK-driven cell expansion and NF-κB-driven tumour intravasation

“…The concentration of 12(S)-HETE in the cellular supernatant was measured with minor modifications as described previously Kretschy et al 2013) using the 12(S)-HETE enzyme immunoassay kit (EIA, # ADI-900-050; Enzo Life Sciences, Lausen, Switzerland) and measuring absorbance with a Wallac 1420 Victor 2 multilabel plate reader (Perkin Elmer Life and Analytical Sciences).…”

“…8b). To investigate whether the NF-κB-inhibitory property of neurolenin B affects the intravasation of tumour emboli through the lymphendothelial barrier, we used a validated three-dimensional model consisting of MCF7 tumour cell spheroids that are co-cultivated with lymphendothelial cell (LEC) monolayers Kretschy et al 2013;Viola et al 2013b). Neurolenin B significantly inhibited the formation of "circular chemorepellent induced defects" (CCIDs) within the LEC monolayer, which are areas through which tumours transmigrate and colonise lymph nodes (Fig.…”