Antiproliferative and antimetastatic characterization of an exo-heterocyclic androstane derivative against human breast cancer cell lines.

Cancer in general, and specifically gynaecological neoplasms, represents a major public health issue worldwide. Based on the effect of sex hormones on breast tumorigenesis and prognosis, as well as on the development of breast cancer metastases, modification of the steroid skeleton is a hotspot of research for novel anticancer agents. Numerous recent studies support that minor modifications of the androstane skeleton yield potent antiproliferative and antimetastatic drug candidates. The aim of the present study was to assess the antitumor and antimetastatic properties, as well as the mechanism of action of a D-ring-modified exo-heterocyclic androstadiene derivative named 17APAD. The test compound was found to be highly selective towards human breast cancer-derived cell lines (MCF-7, T47D, MDA-MB-361, MDA-MB-231) compared to non-cancerous fibroblast cells (NIH/3T3), and exerted superior effect compared to the clinically applied reference drug cisplatin. Changes in MCF-7- and MDA-MB-231 cell morphology and membrane integrity induced by the test substance were assessed by fluorescent double staining. Cell cycle disturbances were analyzed by flow cytometry, and concentration-dependent alterations were detected on breast cancer cell lines. Mitochondrial apoptosis induced by the test compound was demonstrated by JC-1 staining. Inhibitory effects on metastasis formation, including the inhibition of migration, invasion and intravasation were investigated in 2D and 3D models. Significant anti-migratory and anti-invasive effects on MCF-7 and MDA-MB-231 cells were detected after 24 h exposure in 2D wound healing and Boyden-chamber assays. The anti-intravasative properties of 17APAD were evident after 4 h of incubation in a co-culture 3D circular chemorepellent-induced defects (CCID) assay, and the level of inhibition at concentrations ≥2 µM was comparable to that exerted by the focal adhesion kinase inhibitor defactinib. Single cell mass cytometry revealed that chemosensitive subpopulations of MDA-MB-231 cells engaged to apoptosis were less positive for EGFR, CD274, and CD326, while the percentage of cells positive for GLUT1, MCT4, Pan-Keratin, CD66(a,c,e), Galectin-3 and TMEM45A increased in response to 17APAD treatment. Finally, the novel androstane analogue 17APAD had an outstanding inhibitory effect on tumour growth in the 4T1 orthotopic murine breast cancer model in vivo after 2 weeks of intraperitoneal administration. These findings support that substitution of the androsta-5,16-diene framework with a N-containing heterocyclic moiety at C17 position yields a molecular entity rational to be considered for design and synthesis of novel, effective antitumor agents, and 17APAD is worth further investigation as a promising anticancer drug candidate.

Feed­back loops integrating RELA, SOX18 and FAK mediate the break­down of the lymph­endothelial barrier that is triggered by 12(S)­HETE.

Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre­metastatic niches, leading to their colonisation. 'S' stereoisomer 12S­hydroxy­5Z,8Z,10E,14Z­eicosatetraenoic 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)­HETE­treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v­rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY­box 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 transcription­quantitative 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 Bay11­7082, extended the mono­culture experiments to co­culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent­induced 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 FAK­inhibitor defactinib and the NF­κB inhibitor Bay11­7082 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 pro­metastatic signalling cascade. Therefore, targeting the endothelial­specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.