Glycosphingolipid expression differs between human breast cancer stem cells (CSC) and cancer non-stem cells (non-CSC). We performed studies of viability, type of cell death, cancer stem cell percent and glycosphingolipid expression on CSC and non-CSC after treatment of MDA-MB-231 and MDA-MB-453 triple-negative breast cancer cells with a newly developed thienopyridine anticancer compound (3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide, 1). Compound 1 was cytotoxic for both breast cancer cell lines and the majority of cells died by treatment-induced apoptosis. The percent of cancer stem cells and number of formed mammospheres was significantly lower. Glycosphingolipids IV 6 Neu5Ac-nLc 4 Cer and GalNAc-GM1b (IV 3 Neu5Ac-Gg5Cer) not reported previously, were identified in both CSCs and non-CSCs. IV 6 Neu5Ac-nLc 4 Cer had increased expression in both CSCs and non-CSCs of both cell lines after the treatment with 1, while GM3 (II 3 Neu5Ac-LacCer) had increased expression only on both cell subpopulations in MDA-MB-231 cell line. GalNAc-GM1b, Gb 4 Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) and GM2 (II 3 Neu5Ac-GalNAcβ1-4Galβ1-4Glcβ1-1Cer) were increased only in CSCs of both cell lines while GD3 was decreased in CSC of MDA-MB-231 cell line. Due to its effect in reducing the percentage of cancer stem cells and number of mammospheres, and its influence upon several glycosphingolipid expressions, it can be concluded that compound 1 deserves attention as a potential new drug for triple-negative breast cancer therapy. The thieno[2,3-b]pyridines were initially discovered as potential inhibitors of phospholipase C (PLC) isoforms by virtual high throughput screen (vHTS) 1. Recently, we described glycoconjugate GM3 and CD15s expression in MDA-MB-231 triple negative breast cancer stem cell subpopulation cultured with 3-amino-5-oxo-N-naphthyl-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide, which was developed as a putative PLC inhibitor. A close structural analogue of 3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide, or compound 1 2 was chosen for this study due to its enhanced potency against the MDA-MB-231 cell line and its mechanism of action has been investigated 3,4. Due to their ability to self-renew and to regenerate the primary tumour phenotypic heterogeneity, cancer stem cells are important therapeutical targets 5. CSCs are defined with their CD44 + /CD24 − or CD133 + phenotype 6. It is believed that CSCs are involved in therapy resistance in various cancers, including triple-negative breast cancers, i.e., breast cancers that do not express the genes for estrogen receptor, progesterone receptor and the human epidermal growth factor receptor-2 7. Glycosphingolipids (GSLs), consisting of a hydrophobic ceramide and hydrophilic carbohydrate residues, are an important component of cell plasma membranes. They regulate numerous cellular processes like adhesion, proliferation, apoptosis, recognition, modulation of signal transduct...

Glucosinolates (GSLs) from Sysimbrium officinale and S. orientale were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS. Eight GSLs were identified in S. officinale, including Val-derived (glucoputranjivin) and Trp-derived (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin) as the major ones followed by Leu-derived (Isobutyl GSL), Ile-derived (glucocochlearin) and Phe/Tyr-derived (glucosinalbin). Different S. orientale plant parts contained six GSLs, with Met-derived (progoitrin, epiprogoitrin, and gluconapin) and homoPhe-derived (gluconasturtiin) as the major ones, followed by glucosinalbin and neoglucobrassicin. GSL breakdown products obtained by hydrodistillation (HD) and microwave-assisted distillation from S. officinale, as well as isopropyl isothiocyanate, as the major volatile in both isolates, were tested for their cytotoxic activity using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Generally, all volatile isolates showed similar activity toward the three cancer cell lines. The best activity was shown by isopropyl isothiocyanate at a concentration of 100 µg/mL after 72 h of incubation, with 53.18% for MDA-MB-231, 56.61% for A549, and 60.02% for the T24 cell line.

The adhesion of cancer cells to vascular endothelium is a critical process in hematogenous metastasis and might be similar to the recruitment of leukocytes at the site of inflammation. It is mediated by E-selectin and its ligands, of which the most stereospecific is a glycoconjugate sialyl Lewis x (CD15s), which may be expressed as an oligosaccharide branch of the CD44 glycoprotein, as well as a self-contained glycosphingolipid. It is also known that increased sialylation of glycoconjugates is a feature of malignant cells. The aim of the study was to analyse the effect of a novel thieno[2,3-b]pyridine, compound 1, in MDA-MB-231 triple-negative breast cancer cells (TNBCs) upon CD15s and CD44 expression in different cell subpopulations using flow cytometry. CD15s expression was compared between mesenchymal-like cancer stem cells (CSC, CD44+CD24−), epithelial cells without CD44 (CD44−CD24+ and CD44−CD24−), and CD44+CD24+ cells that exhibit mesenchymal and epithelial features. In addition, expression of CD44 in CD15s+CSC and CD15s−CSC was determined. Compound 1 significantly decreased the percentage of CD15s+CSC, CD15s+CD44+CD24+, and CD15s+CD44− subpopulations, as well as the expression of CD15s in CD44+CD24+ and CD44− cells, and therefore shows potential as a treatment for TNBC.

Due to the role of cancer stem cells (CSCs) in tumor resistance and glycosphingolipid (GSL) involvement in tumor pathogenesis, we investigated the effect of a newly synthesized compound (3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide 1 on the percentage of CSCs and the expression of six GSLs on CSCs and non-CSCs on breast cancer cell lines (MDA-MB-231 and MCF-7). We also investigated the effect of 1 on the metabolic profile of these cell lines. The MTT assay was used for cytotoxicity determination. Apoptosis and expression of GSLs were assessed by flow cytometry. A GC–MS-coupled system was used for the separation and identification of metabolites. Compound 1 was cytotoxic for both cell lines, and the majority of cells died by treatment-induced apoptosis. The percentage of CSCs was significantly lower in the MDA-MB-231 cell line. Treatment with 1 caused a decrease of CSC IV6Neu5Ac-nLc4Cer+ MDA-MB-231 cells. In the MCF-7 cell line, the percentage of GalNAc-GM1b+ CSCs was increased, while the expression of Gg3Cer was decreased in both CSC and non-CSC. Twenty-one metabolites were identified by metabolic profiling. The major impact of the treatment was in glycolysis/gluconeogenesis, pyruvate and inositol metabolism. Compound 1 exhibited higher potency in MBA-MB-231 cells, and it deserves further examination.

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