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...

Tumor progression may be driven by a small subpopulation of cancer stem cells (CSCs characterized by CD44+/CD24− phenotype). We investigated the influence of a newly developed thienopyridine anticancer compound (3-amino-5-oxo-N-naphthyl-5,6,7, 8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide, 1) on the growth, survival and glycophenotype (CD15s and GM3 containing neuraminic acid substituted with acetyl residue, NeuAc) of breast and prostate cancer stem/progenitor-like cell population. MDA-MB-231 and Du-145 cells were incubated with compound 1 alone or in combination with paclitaxel. The cellular metabolic activity was determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. The type of cell death induced by 48-h treatment was assessed using a combination of Annexin-V-FITC and propidium iodide staining. Flow cytometric analysis was performed to detect the percentage of CD44+/CD24− cells, and GM3 and CD15s positive CSCs, as well as the expression of GM3 and CD15s per one CSC, in both cell lines. Compound 1 produces a dose- and time-dependent cytotoxicity, mediated mainly by apoptosis in breast cancer cells, and slightly (2.3%) but statistically significant lowering breast CSC subpopulation. GM3 expression per one breast CSC was increased, and the percentage of prostate GM3+ CSC subpopulation was decreased in cells treated with compound 1 compared with non-treated cells. The percentage of CD15s+ CSCs was lower in both cell lines after treatment with compound 1. Considering that triple-negative breast cancers are characterized by an increased percentage of breast CSCs and knowing their association with an increased risk of metastasis and mortality, compound 1 is a potentially effective drug for triple-negative breast cancer treatment.

Purpose: An increase in resting motor threshold (RMT), prolonged cortical silent period duration (CSP), and reduced short-latency afferent inhibition (SAI), confirmed with previous transcranial magnetic stimulation (TMS), suggest decreased cortical excitability in obstructive sleep apnea syndrome (OSAS). The present study included MRI of OSAS patients for navigated TMS assessment of the RMT, as an index of the threshold for corticospinal activation at rest, and SAI as an index of cholinergic neurotransmission. We hypothesize to confirm findings on SAI and RMT with adding precision in the targeting of motor cortex in OSAS. Subjects and Methods: After acquiring head MRIs for 17 severe right-handed OSAS and 12 healthy subjects, the motor cortex was mapped with nTMS to assess the RMT and SAI, with motor evoked potentials (MEPs) recorded from the abductor-pollicis brevis (APB) muscle. The 120%RMT intensity was used for the SAI by a paired-pulse paradigm in which the electrical stimulation to the median nerve is followed by magnetic stimulation of the motor cortex at inter-stimulus intervals (ISIs) of 18-28 ms (ISIs 18-28). The SAI control condition included a recording of MEPs without peripheral stimulation. Latency and amplitude of MEP at RMT at 120%RMT for eleven different at ISIs 18-28 were analyzed. Results: The study showed a significantly lower percentage deviation of MEP amplitude at ISIs (18-28ms) from the control condition between OSAS and healthy subjects (U=44.0, p=0.01). The intensity of stimulation at RMT was significantly higher in OSAS subjects (U=55.0, p=0.04*). Correlation analysis showed that BMI significantly negatively correlated (ρ=−0.47) with MEP amplitude percentage deviation in OSAS patients. Conclusion: The nTMS study results in increased RMT, and reduced cortical afferent inhibition in OSAS patients for SAI at ISIs 18-28 , confirming previous findings of impaired cortical afferent inhibition in OSAS. Future nTMS studies are desirable to elucidate the role of RMT and SAI in diagnostics and treatment of OSAS, and to elucidate the usefulness of nTMS in OSAS research.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.