Non-cancerous Breast Epithelial Cells Research Articles

Abstract 197: The GPR52-MCAM axis as a novel regulatory mechanism of breast cancer cell clustering and metastatic potential

Abstract Background: Breast cancer is the most diagnosed cancer worldwide and is the second leading cause of cancer death among women in the United States. Despite breakthroughs in targeted therapies for breast cancer, the 5-year survival rate for women with metastatic breast cancer is 30%. There is a strong rationale to explore the therapeutic potential of G-protein coupled receptors (GPCRs), which comprise the largest class of proteins successfully targeted by FDA-approved drugs. Importantly, at least one-third of GPCRs are “orphans” and remain largely uncharacterized. One of these orphan GPCRs is GPR52. To date, there is no literature on the role of GPR52 in any cancer type, but we find from publicly available clinical data that low mRNA expression of GPR52 in breast tumors correlates with reduced overall survival (hazard ratio=0.67 [0.53-0.83], logrank P= 0.00035) and that GPR52 mRNA levels tend to be lower in tumor metastases compared to primary tumor (n>80, p=4.45e-17). Therefore, we hypothesize that loss of GPR52 may support breast cancer metastasis. Results: We used CRISPR-Cas9 to knockout (KO) GPR52 in triple-negative breast cancer cell lines MDA-MB-468 and MDA-MB-231, and in the non-cancerous breast epithelial cell line MCF10A, and confirmed the generation of frameshift mutations by Sanger sequencing. Interestingly, we observed that GPR52 KO cells clustered together in vitro in 2D and hypothesized that this characteristic may allow GPR52 KO breast cancer cells to increase their metastatic potential. We conducted invasion assays though Matrigel and found that GPR52 KO cells were more likely to invade as sheets or clusters. To determine the in vivo impact of GPR52 loss, we injected MDA-MB-468 GPR52 KO cells in zebrafish (Danio rerio) two days post-fertilization and observed that GPR52 loss led to increased metastatic foci and total cancer surface area thirty hours post-injection (n≥12, p<0.05). We determined from RNA-sequencing (n=3, p<.0001) and confirmed with western blotting that the melanoma cell adhesion molecule (MCAM) is upregulated in the MCF10A and MDA-MB-468 GPR52 KO cells. Increased mRNA expression of MCAM has been associated with reduced overall survival in breast cancer patients (hazard ratio=1.33 [1.05-1.62], logrank P= 0.02) Conclusion: Knockout of GPR52 from breast cancer cells promotes increased cell clustering in vitro and metastasis in zebrafish. These data support the investigation of GPR52 as a biomarker of cancer aggression and a potential therapeutic target for breast cancer patients whose tumors express GPR52. Citation Format: Sarah Z. Hanif, CheukMan C. Au, Ingrid Torregroza, Bhavneet Bhinder, Lukas Dow, Olivier Elemento, Todd Evans, Kristy A. Brown. The GPR52-MCAM axis as a novel regulatory mechanism of breast cancer cell clustering and metastatic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 197.

Surface Proteome of Extracellular Vesicles and Correlation Analysis Reveal Breast Cancer Biomarkers

Breast cancer (BC) is the second most frequently diagnosed cancer and accounts for approximately 25% of new cancer cases in Canadian women. Using biomarkers as a less-invasive BC diagnostic method is currently under investigation but is not ready for practical application in clinical settings. During the last decade, extracellular vesicles (EVs) have emerged as a promising source of biomarkers because they contain cancer-derived proteins, RNAs, and metabolites. In this study, EV proteins from small EVs (sEVs) and medium EVs (mEVs) were isolated from BC MDA-MB-231 and MCF7 and non-cancerous breast epithelial MCF10A cell lines and then analyzed by two approaches: global proteomic analysis and enrichment of EV surface proteins by Sulfo-NHS-SS-Biotin labeling. From the first approach, proteomic profiling identified 2459 proteins, which were subjected to comparative analysis and correlation network analysis. Twelve potential biomarker proteins were identified based on cell line-specific expression and filtered by their predicted co-localization with known EV marker proteins, CD63, CD9, and CD81. This approach resulted in the identification of 11 proteins, four of which were further investigated by Western blot analysis. The presence of transmembrane serine protease matriptase (ST14), claudin-3 (CLDN3), and integrin alpha-7 (ITGA7) in each cell line was validated by Western blot, revealing that ST14 and CLDN3 may be further explored as potential EV biomarkers for BC. The surface labeling approach enriched proteins that were not identified using the first approach. Ten potential BC biomarkers (Glutathione S-transferase P1 (GSTP1), Elongation factor 2 (EEF2), DEAD/H box RNA helicase (DDX10), progesterone receptor (PGR), Ras-related C3 botulinum toxin substrate 2 (RAC2), Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), Aconitase 2 (ACO2), UTP20 small subunit processome component (UTP20), NEDD4 binding protein 2 (N4BP2), Programmed cell death 6 (PDCD6)) were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. In total, 846 surface proteins were identified from the second approach, of which 11 were already known as BC markers. This study supports the proposition that Evs are a rich source of known and novel biomarkers that may be used for non-invasive detection of BC. Furthermore, the presented datasets could be further explored for the identification of potential biomarkers in BC.

Phytochemicals from Pterocarpus angolensis DC and Their Cytotoxic Activities against Breast Cancer Cells.

Pterocarpus anglonesis DC is an indigenous medicinal plant belonging to the Pterocarpus genus of the Fabaceae family. It is used to treat stomach problems, headaches, mouth ulcers, malaria, blackwater fever, gonorrhea, ringworm, diarrhea, heavy menstruation, and breast milk stimulation. Column chromatography of the stem bark extracts resulted in the isolation of eight compounds, which included friedelan-3-one (1), 3α-hydroxyfriedel-2-one (2), 3-hydroxyfriedel-3-en-2-one (3), lup-20(29)-en-3-ol (4), Stigmasta-5-22-dien-3-ol (5), 4-O-methylangolensis (6), (3β)-3-acetoxyolean-12-en-28-oic acid (7), and tetradecyl (E)-ferulate (8). The structures were established based on NMR, IR, and MS spectroscopic analyses. Triple-negative breast cancer (HCC70), hormone receptor-positive breast cancer (MCF-7), and non-cancerous mammary epithelial cell lines (MCF-12A) were used to test the compounds' cytotoxicity. Overall, the compounds showed either no toxicity or very low toxicity to all three cell lines tested, except for the moderate toxicity displayed by lupeol (4) towards the non-cancerous MCF-12A cells, with an IC50 value of 36.60 μM. Compound (3β)-3-acetoxyolean-12-en-28-oic acid (7) was more toxic towards hormone-responsive (MCF-7) breast cancer cells than either triple-negative breast cancer (HCC70) or non-cancerous breast epithelial (MCF-12A) cells (IC50 values of 83.06 vs. 146.80 and 143.00 μM, respectively).

Luminescent Heterobimetallic PtII-AuI Complexes Bearing N-Heterocyclic Carbenes (NHCs) as Potent Anticancer Agents.

This study aims to probe into new series of heterobimetallic PtII-AuI complexes with a general formula of [Pt(p-MeC6H4)(dfppy)(μ-dppm)Au(NHC)]OTf, NHC = IPr, 2; IMes, 3; dfppy = 2-(2,4-difluorophenyl)pyridinate; dppm = 1,1-bis(diphenylphosphino)methane, which are the resultant of the reaction between [Pt(p-MeC6H4)(dfppy)(κ1-dppm)], 1, with [AuCl(NHC)], NHC = IPr, B; IMes, C, in the presence of [Ag(OTf)]. In the heterobimetallic complexes, the dppm ligand is settled between both metals as an unsymmetrical bridging ligand. Several techniques are employed to characterize the resulting compounds. Moreover, the photophysical properties of the complexes are investigated by means of UV-vis and photoluminescence spectroscopy. Furthermore, the experimental study is enriched by ab initio calculations (density functional theory (DFT) and time-dependent DFT (TD-DFT)) to assess the role of Pt and Au moieties in the observed optical properties. It is revealed that 1-3 is luminescent in the solid state and solution at different temperatures. In addition, the achieved results indicate the emissive properties of 1-3 are originated from a mixed 3IL/3MLCT excited state with major contribution of intraligand charge transfer (dfppy). A comparative study is conducted into the cytotoxic activities of starting materials and 1-3 against different human cancer cell lines such as the pancreas (MIA-PaCa2), breast (MDA-MB-231), cervix (HeLa), and noncancerous breast epithelial cell line (MCF-10A). The achieved results suggest the heterobimetallic PtII-AuI species as optimal compounds that signify the existence of cooperative and synergistic effects in their structures. The complex 3 is considered as the most cytotoxic compound with the maximum selectivity index in our screened complex series. Moreover, it is disclosed that 3 effectively causes cell death by inducing apoptosis in MIA-PaCa2 cells. Furthermore, the finding results by fluorescent cell microscopy manifest cytoplasmic staining of 3 rather than nucleus.

In vitro evaluation of anticancer, antiviral, and antioxidant properties of an aqueous methanolic extract of Rhus typhina L. leaves

In recent decades, natural products of plant origin have predominated among the sources of new therapeutic agents. Plants, which are intensively used in folk medicine, are the focus of particular interest, and being subjected to in-depth analyses for the assessment of their pharmacological properties. The objective of the present work was to evaluate the in vitro anticancer, antiviral, and antioxidant properties of a crude aqueous methanolic extract from the leaves of Rhus typhina L., which is a plant species with broad applications in traditional phytotherapy. The anticancer capacity of the extract was assessed on two human breast cancer cell lines (MCF7 and MDA-MB-231) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay, and its cell growth inhibitory effect on cancer cells was compared with its action on a non-cancerous breast epithelial cell line (MCF10A). Moreover, the in vitro antiviral activity of the plant extract was studied against viral strains of three taxonomic groups: herpes simplex virus type 1 (HSV-1), coxsackievirus B1 (CVB1), and influenza A virus (IAV/H3N2) using a cytopathic effect inhibition test, extract cytotoxicity evaluation through the neutral red uptake assay, a virucidal assay, a viral attachment assay, and cell pre-treatment with the extract. In turn, a radical scavenging assay with 2,2-diphenyl-1-picrylhydrazyl (DPPH) was applied to explore the antioxidant potential of R. typhina leaf extract. Results indicated that the plant extract possessed antiproliferative activity against the tested cell lines which was stronger toward cancer cells. A modest inhibitory effect on IAV/H3N2 replication was detected; as well as on an effect on herpes virions and on the adsorption of the HSV-1 and IAV/H3N2 strains; and a protective effect on uninfected cells before HSV-1 contact. The leaf extract of R. typhina also exhibited a strong free radical scavenging activity. The results of the present work demonstrated the pharmacological potentials of the plant extract, which warrants further and more detailed study in the future.

Copper-Plumbagin Complex Produces Potent Anticancer Effects by Depolymerizing Microtubules and Inducing Reactive Oxygen Species and DNA Damage.

Here, we have synthesized a copper complex of plumbagin (Cu-PLN) and investigated its antiproliferative activities in different cancer cells. The crystal structure of Cu-PLN showed that the complex was square planar with a binding stoichiometry of 1:2 (Cu/Plumbagin). Cu-PLN inhibited the proliferation of human cervical carcinoma (HeLa), human breast cancer (MCF-7), and murine melanoma (B16F10) cells with half-maximal inhibitory concentrations (IC50) of 0.85 ± 0.05, 2.3 ± 0.1, and 1.1 ± 0.1 μM, respectively. Plumbagin inhibited the proliferation of HeLa, MCF-7, and B16F10 cells with IC50 of 7 ± 0.1, 8.2 ± 0.2, and 6.2 ± 0.4 μM, respectively, showing that Cu-PLN is a stronger antiproliferative agent than plumbagin. Interestingly, Cu-PLN showed much stronger toxicity against breast carcinoma and skin melanoma cells than noncancerous breast epithelial and skin fibroblast cells, indicating its specific cytotoxicity toward cancer cells. A short exposure of Cu-PLN triggered microtubule disassembly in cultured cancer cells, and the complex also inhibited the polymerization of purified tubulin much more strongly than plumbagin. Furthermore, Cu-PLN inhibited the binding of colchicine to tubulin. In addition to microtubule depolymerization, the antiproliferative mechanism of Cu-PLN involved induction of reactive oxygen species, reduction of the mitochondrial membrane potential, and DNA damage. Moreover, the cytotoxic effects of Cu-PLN reduced significantly in cells pre-treated with N-acetyl cysteine, suggesting that reactive oxygen species generation is crucial in Cu-PLN's mode of action. Thus, the complexation of plumbagin with copper yields a promising antitumor agent having a stronger antiproliferative activity than cisplatin, a widely used anticancer drug.

Wnt-pathway inhibitors with selective activity against triple-negative breast cancer: From thienopyrimidine to quinazoline inhibitors.

The Wnt-pathway has a critical role in development and tissue homeostasis and has attracted increased attention to develop anticancer drugs due to its aberrant activation in many cancers. In this study, we identified a novel small molecule series with a thienopyrimidine scaffold acting as a downstream inhibitor of the β-catenin-dependent Wnt-pathway. This novel chemotype was investigated using Wnt-dependent triple-negative breast cancer (TNBC) cell lines. Structure activity relationship (SAR) exploration led to identification of low micromolar compounds such as 5a, 5d, 5e and a novel series with quinazoline scaffold such as 9d. Further investigation showed translation of activity to inhibit cancer survival of HCC1395 and MDA-MB-468 TNBC cell lines without affecting a non-cancerous breast epithelial cell line MCF10a. This anti-proliferative effect was synergistic to docetaxel treatment. Collectively, we identified novel chemotypes acting as a downstream inhibitor of β-catenin-dependent Wnt-pathway that could expand therapeutic options to manage TNBC.

Development and characterisation of mouse monoclonal antibody against ‘neonatal’ Nav1.5

‘Neonatal’ Nav1.5 (nNav1.5) is a potent tumour metastasis marker found especially in aggressive human breast cancer cells in vitro, in tumour tissues of in vivo metastatic animal models and in patients positive for lymph-node metastasis. Its expression has been recently described in human brain neuroblastoma and astrocytoma. However, a thorough understanding of nNav1.5’s role in cancers has been limited by the lack of specific antibodies against it. Here, a mouse monoclonal antibody, 4H8 mAb-nNav1.5, was obtained and characterised concerning its efficacy in detecting nNav1.5 using indirect ELISA, surface plasmon resonance (SPR), Western blotting and immunofluorescence microscopy. 4H8 mAb-nNav1.5 was selected from a panel of hybridoma clones raised against nNav1.5 specific peptide (15 mers). The antibody exhibited linear association against nNav1.5 specific-linear peptide in indirect ELISA and was supported by SPR. The antibody also demonstrated strong immunoreactivity in immunofluorescence imaging of nNav1.5-abundant cells, human and mouse aggressive breast cancer cells, MDA-MB-231 and 4T1, respectively, which was not observed in nNav1.5-deficient cells, human less aggressive breast cancer cells, MCF-7 and non-cancerous breast epithelial cells, MCF-10A. This study demonstrates the initial description of 4H8 mAb-nNav1.5, which could serve as a beneficial tool to enhance future studies on nNav1.5 expression and function in cancers.

NNav1.5 expression is associated with glutamate level in breast cancer cells

BackgroundGlutamate and voltage-gated sodium channels, both have been the target of intense investigation for its involvement in carcinogenesis and progression of malignant disease. Breast cancer with increased level of glutamate often metastasize to other organs (especially bone), whilst re-expression of ‘neonatal’ Nav1.5, nNav1.5 in breast cancer is known to promote cell invasion in vitro, metastasis in vivo and positive lymph node metastasis in patients.MethodsIn this study, the role of nNav1.5 in regulating glutamate level in human breast cancer cells was examined using pharmacological approach (VGSCs specific blocker, TTX, glutamate release inhibitor, riluzole and siRNA-nNav1.5). Effect of these agents were evaluated based on endogenous and exogenous glutamate concentration using glutamate fluorometric assay, mRNA expression of nNav1.5 using qPCR and finally, invasion using 3D culture assay.ResultsEndogenous and exogenous glutamate levels were significantly higher in aggressive human breast cancer cells, MDA-MB-231 cells compared to less aggressive human breast cancer cells, MCF-7 and non-cancerous human breast epithelial cells, MCF-10A. Treatment with TTX to MDA-MB-231 cells resulted in significant reduction of endogenous and exogenous glutamate levels corresponded with significant suppression of cell invasion. Subsequently, downregulation of nNav1.5 gene was observed in TTX-treated cells.ConclusionsAn interesting link between nNav1.5 expression and glutamate level in aggressive breast cancer cells was detected and requires further investigation.

Arene-ruthenium(II) complexes with tetracyclic oxime derivatives: synthesis, structure and antiproliferative activity against human breast cancer cells

Six new arene-ruthenium(II) complexes containing two different η6-arene ligands – benzene and hexamethylbenzene, with indenoquinoxalinone oxime analogues (11H-indeno[1,2-b]quinoxalin-11-one oxime, 6H-indeno[1,2-b]pyrido[3,2-e]pyrazin-6-one oxime and 6–(hydroxyimino)indolo[2,1-b]quinazolin-12(6H)-one oxime (tryptanthrin-6-oxime)) as N,N’- chelating ligands are reported. The complexes were characterized by elemental analysis, IR, UV–VIS, 1H and 13C NMR spectroscopy and by single-crystal X-ray structure analysis. Complexes adopt a half-sandwich «piano-stool» geometry with oxime ligands in anionic form, the ruthenium(II) center coordinates one arene, one N,N-bidentate oximate and one chloride ligand. The computational analysis of non-covalent intermolecular interactions revealed weak attraction between the oxime oxygen atoms and the nearest hydrogen atoms of the oxime and the arene ligands. The cytotoxic activity of the complexes was evaluated against human breast cancer cell line MCF-7 and cisplatin-resistant human breast cancer cell line MCF-7CR as well as non-cancerous human breast epithelial cell line MCF10A. The cytotoxicity tests show micromolar IC50 values and tryptanthrin-6-oxime hexamethylbenzene-ruthenium(II) complex was found to exhibit the best antiproliferative activity among the studied compounds against the MCF-7 and MCF-7CR cell lines (IC50 9.0 ± 4.4 and 8.9 ± 1.5 µM, respectively).

Group VIII Metal Carbonyl Cluster-Boronic Acid Conjugates: Cytotoxicity and Mode of Action Studies.

A set of metal carbonyl cluster-boronic acid conjugates of the group VIII metals (Fe, Ru, and Os) were synthesized and their antiproliferative effects measured against two breast cancer cell lines (MCF-7 and MDA-MB-231) and a noncancerous breast epithelial (MCF-10A) cell line. The cytotoxicity followed the order Ru > Os > Fe for the MDA-MB-231 cells, although the latter two exhibited similar cytotoxicity against MCF-7 and MCF-10A cells. The osmium species {Os3(CO)10(μ-H)[μ-SC6H4-p-B(OH)2]} (2) could be chemically oxidized to its hydroxy analogue [Os3(CO)10(μ-H)(μ-SC6H4-p-OH)] (2-OH), which showed comparable cytotoxicity. Mode of action studies pointed to an apoptotic pathway for cell death.

Leptin Mediates Obesity-Induced DNA Damage in BRCA1 Breast Epithelial Cells

Abstract Background: Obesity is a risk factor for breast cancer development in women who carry a mutation in the DNA repair enzymes BRCA1 or BRCA2. Previously, we found that obesity was positively associated with DNA damage in breast epithelium from BRCA mutation carriers. Furthermore, factors secreted by obese breast adipose tissue stimulated DNA damage in BRCA mutant breast epithelial cells, suggesting a cross-talk between breast epithelial cells and the adipose tissue that surrounds them. We hypothesized that leptin, a hormone secreted in abundance by obese adipose tissue, may be a driver of DNA damage and/or decrease capacity for DNA repair in breast epithelial cells. If true, this would provide a molecular target for intervention to reduce the risk of tumor formation in this high-risk population of women. Methods: RNA-seq followed by Ingenuity Pathway Analysis (IPA) was conducted on primary breast epithelial organoids isolated from lean and obese BRCA mutation carriers. Breast adipose tissue obtained from lean and obese women were cultured as explants for 24 hours to produce lean and obese conditioned media (CM). The effect of leptin on DNA damage was assessed in a non-cancerous breast epithelial cell line (MCF10A) carrying a heterozygous BRCA1 mutation. Immunofluorescence staining of the DNA damage marker ƴH2AX was carried out after treatment with leptin (100ng-800ng), CM, or CM+leptin antibody, used to neutralize leptin. To test whether leptin affects DNA repair capacity, BRCA1+/- MCF10A cells were treated with leptin or vehicle and then irradiated (1Gy) to induce DNA damage. Resolution of damage was quantified at 0, 0.5, 4, 12, and 24 hrs post-irradiation. Results: IPA analysis identified leptin signaling as significantly upregulated in breast epithelial organoids from obese women compared with lean women. Both obese CM and leptin treatment induced DNA damage in BRCA1+/- MCF10A cells while lean CM did not have this effect. Neutralizing leptin in obese CM was sufficient to inhibit obese CM-mediated induction of DNA damage. No significant difference was observed between leptin or vehicle treatments on DNA repair capacity after irradiation of BRCA+/- MCF10A cells. Conclusions: These data identify leptin, an adipose-derived hormone, as a novel driver of DNA damage in breast epithelial cells. To date, no studies have elucidated the molecular mechanisms that explain the increased penetrance of breast cancer in obese BRCA mutation carriers compared to lean BRCA mutation carriers. This work suggests that leptin may be a mediator of the link between obesity and breast cancer development in this population. Further studies are warranted to determine if targeting the leptin signaling axis will be an effective risk reduction strategy in BRCA mutation carriers who have excess adiposity.

Facile green synthesis of ZnO-RGO nanocomposites with enhanced anticancer efficacy.

Drug resistance and inability to distinguish between cancerous and non-cancerous cells are important obstacles in the treatment of cancer. Zinc oxide nanoparticles (ZnO NPs) is now emerging as a crucial material to challenge this global issue due to its tunable properties. Developing an effective, inexpensive, and eco-friendly method in order to tailor the properties of ZnO NPs with enhanced anticancer efficacy is still challenging. For the first time, we reported a facile, inexpensive, and eco-friendly approach for green synthesis of ZnO-reduced graphene oxide nanocomposites (ZnO-RGO NCs) using garlic clove extract. Garlic has been playing one of the most important dietary and medicinal roles for humans since centuries. We aimed to minimize the use of toxic chemicals and enhance the anticancer potential of ZnO-RGO NCs with minimum side effects to normal cells. Aqueous extract of garlic clove was used as reducing and stabilizing agent for green synthesis of ZnO-RGO NCs from the zinc nitrate and graphene oxide (GO) precursors. A potential mechanism of ZnO-RGO NCs synthesis with garlic clove extract was also proposed. Preparation of pure ZnO NPs and ZnO-RGO NCs was confirmed by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and dynamic light scattering (DLS). The in vitro study showed that ZnO-RGO NCs induce two-fold higher cytotoxicity in human breast cancer (MCF7) and human colorectal cancer (HCT116) cells as compared to pure ZnO NPs. Besides, biocompatibility of ZnO-RGO NCs in non-cancerous human normal breast (MCF10A) and normal colon epithelial (NCM460) cells was higher than those of pure ZnO NPs. This work highlighted a facile and inexpensive green approach for the preparation of ZnO-RGO NCs with enhanced anticancer activity and improved biocompatibility.

Screening of a series of 3,5-disubstituted 1,2,4-thiadiazoles for selectivity of cytotoxic action to cancer cells

New 3,5-disubstituted-1,2,4-thiadiazoles were synthesized and together with a series of their analogs obtained earlier were tested for cytotoxic activity to breast adenocarcinoma MCF-7 and lung carcinoma A 549 cells. The selectivity of cytotoxic action was determined in comparison to etiologically noncancerous breast epithelial cells MCF-10A and lung fibroblasts VA13. From five- to six-fold selectivity of cytotoxic action was revealed for 3,4-dichlorophenyl-{3-[2-(2-morpholin-4-ylethylamino)propyl]-[1,2,4]thiadiazol-5-yl}amine and (2-{5-[(4-diethylami-nobenzyl)pyridin-3-ylmethylamino]-[1,2,4]thiadiazol-3-yl}-1-methylvinyl)-(2,2,6,6-tetra-methylpiperidin-4-yl)amine in the pairs A 549/VA13 and MCF-7/MCF-10A, respectively. Some structure—property relationships for the compounds under study were discussed.

Effect of extremely low frequency electromagnetic field parameters on the proliferation of human breast cancer

ABSTRACT Extremely low-frequency electromagnetic field (ELF-EMF) exposures influence many biological systems. These effects are mainly related to the intensity, duration, frequency, and pattern of the ELF-EMF. Our intent was to characterize the effect of specific pulsed electromagnetic fields on the in vitro proliferation of MCF-7 adenocarcinoma and MDA-MB-231 breast cancer cell lines and one non-cancerous M10 breast epithelial cell line. The following four important parameters of ELF-EMF were examined: frequencies (7.83 ± 0.3, 23.49 ± 0.3, and 39.15 ± 0.3 Hz), flux density (0.5 and 1 mT), exposure duration (12, 24, and 48 h), and the exposure methodology (continuous exposure versus switching exposure). The viability of MDA-MB-231 cells exposed to the optimized ELF-EMF pattern (7.83 ± 0.3 Hz, 1 mT, and 6 h switching exposure) was 40.1%. By contrast, the optimized ELF-EMF parameters that were most cytotoxic to breast cancer MDA-MB-231 cells were not damaging to normal M10 cells. In vitro studies also showed that exposure of MDA-MB-231 cells to the optimized ELF-EMF pattern promoted Ca2+ influx and resulted in apoptosis. These data confirm that exposure to this specific ELF-EMF pattern can influence cellular processes and inhibit cancer cell growth. The specific ELF-EMF pattern determined in this study may provide a potential anti-cancer treatment in the future.

SiRNA Knockdown of REDD1 Facilitates Aspirin-Mediated Dephosphorylation of mTORC1 Target 4E-BP1 in MDA-MB-468 Human Breast Cancer Cell Line

BackgroundMutations within genes encoding components of the PI3K/AKT/mTOR (phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin) signaling axis frequently activate the pathway in breast cancer, making it an attractive therapeutic target. Inhibition of mTORC1 (mechanistic target of rapamycin complex 1) activity upon aspirin treatment has been reported in breast cancer cells harboring PI3KCA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation and is considered to account for anticancer action.MethodsMDA-MB-468 (harbors mutated PTEN (phosphatase and TENsin homolog)), MCF-7 (PI3KCA-mutated), MDA-MB-231 (no PI3K pathway mutations) cancer cell lines and MCF10A non-cancerous breast epithelial cells were employed for the assessment of modulation of mTORC1 signaling by aspirin. Targeted amplicon-based next-generation sequencing using the Ion Torrent technology was carried out to determine gene expression changes following drug treatment. Western blot was performed to analyze the expression and phosphorylation of proteins. Knockdown by siRNA approach was applied to assess the role of REDD1/DDIT4 (DNA damage-inducible transcript 4) in mTORC1 inhibition by aspirin.ResultsWe show a decline in phosphorylation of mTORC1 downstream substrate 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) in response to treatment with aspirin and its metabolite salicylic acid in MDA-MB-468, MCF-7, MDA-MB-231, and MCF10A cell lines. We further demonstrate a novel molecular response to aspirin in breast cancer cells. Specifically, we found that aspirin and salicylic acid increase the expression of REDD1 protein, that is known for its suppressive function towards mTORC1. Unexpectedly, we observed that siRNA knockdown of REDD1 expression facilitated aspirin-mediated suppression of mTORC1 downstream substrate 4E-BP1 phosphorylation in the MDA-MB-468 cell line. REDD1 downregulation slightly encouraged reduction in 4E-BP1 phosphorylation by aspirin in MCF-7 cells but did not elicit a reproducible effect in the MDA-MB-231 cell line. siRNA knockdown of REDD1 did not affect the expression of phosphorylated form of 4E-BP1 following aspirin treatment in MCF10A non-cancerous breast epithelial cells.ConclusionThe current findings suggest that REDD1 downregulation might improve the anticancer activity of aspirin in a subset of breast tumors.

Synthesis and analysis of silver\u2013copper alloy nanoparticles of different ratios manifest anticancer activity in breast cancer cells

BackgroundBreast cancer is therapeutically very challenging to treat as it has the main four known genetic alterations, which result in the existence of several phenotypes leading to the difference in the mode of therapy and with poor outcome. Metallic nanoparticles of silver or copper have been studied previously as anticancer agents in breast cancer and other types of cancers. However, the anticancer effect of silver–copper alloy nanoparticles (AgCu-NP) is not studied in breast cancer. In this study, we aim to synthesize silver nanoparticles (Ag-NP), or copper nanoparticles (Cu-NP), and AgCu-NP and evaluate their toxicity in breast cancer and healthy breast cells.ResultsWe synthesized sodium citrate and mercapto-propionic acid (MPA-3) capped water-soluble metallic nanoparticles of Ag-NP or Cu-NP and an alloy of three different combinations of AgCu-NP. High-resolution transmission electron microscopy characterization of nanoparticles revealed the spherical shape nanoparticles of varied sizes, furthermore dynamic light scattering characterization was performed, which investigated the hydrodynamic size and stability in phosphate buffer solution. Energy-dispersive X-ray spectroscopy (EDS) measurements were obtained from the transmission electron microscope to study the composition of alloy nanoparticles and the distribution pattern of silver and copper in the alloy nanoparticles. We measured the toxicity of nanoparticles to breast cancer MCF-7 cell line by MTT assay and compared the toxic effect with non-cancerous breast epithelial cells MCF-10A. Our data showed that Ag-NP or Cu-NP have no effect on cancer cells or healthy cells, except Ag-NP at 20 µg/ml were toxic to cancer cells. However, AgCu-NP were significantly toxic to MCF-7 cells at 10 µg/ml concentration, while as AgCu-NP have no toxic effect on healthy cells. Furthermore, we observed the cell death pathway by the apoptosis marker Annexin-V which showed non-significant results, while the exposure of AgCu-NP in MCF-7 cells leads to toxicity and also caused significant increase in MMP-9 level, which suggests the cell death may be associated with other pathways such as autophagy and oxidative stress related.ConclusionThe data suggest that the AgCu-NP alloy imposes preferential toxicity in breast cancer MCF-7 cells and thus could be exploited as a new candidate for further anticancer investigation

Cytotoxicity of triterpenoids from Clerodendrum glabrum against triple negative breast cancer cells in vitro

Clerodendrum glabrum is an indigenous medicinal plant that is used to treat cough, cold, sore throat and chest complaints. The stem bark of Clerodendrum glabrum afforded four tritepenoids namely, 3β-olean-12-en-3-yl palmitate (β-amyrin palmitate), (1), 3β‑hydroxy‑5-glutinene (glutinol), (2), 3β-lup-20(29)-en-3-palmitate (Lupeol-3-palmitate), (3), 3β-lup-20(29)-en-3-ol (lupeol) (4) and one common phytosterol (stigmasterol) (5). The structures were established on the basis of their spectroscopic analysis. The compounds were screened for cytotoxicity against the HCC70 triple negative breast cancer (TNBC), MCF-7 hormone receptor positive breast cancer and MCF-12A non-cancerous mammary epithelial cell lines. Interestingly, none of the compounds were toxic towards hormone receptor positive breast cancer cells, while displaying varying toxicity against the TNBC and non-cancerous breast epithelial cells. In particular, lupeol-3-palmitate (47.6 ± 1.50 μM) and glutinol (26.9 ± 1.30 μM) displayed the greatest inhibitory activity against the HCC70 cell line, with the former being selectively toxic to HCC70 and not MCF-12A non-cancerous cells.

Co-localization of Interleukin-1α and Annexin A2 at the plasma membrane in response to oxidative stress

Interleukin-1α (IL-1α) and Annexin A2 (AnxA2) are pleiotropic molecules with both intracellular and extracellular roles. They share several characteristics including unconventional secretion aided by S100 proteins, anchoring of the externalized proteins at the outer surface of the plasma membrane and response to oxidative stress. Although IL-1α and AnxA2 have been implicated in a variety of biological processes, including cancer, little is known about the mechanisms of their cellular release. In the present study, employing the non-cancerous breast epithelial MCF10A cells, we demonstrate that IL-1α and AnxA2 establish a close association in response to oxidative stress. Stress conditions lead to translocation of both proteins towards lamellipodia rich in vimentin and association of full-length IL-1α and Tyr23 phosphorylated AnxA2 with the plasma membrane at peripheral sites depleted of F-actin. Notably, membrane-associated IL-1α and AnxA2 preferentially localize to the outer edges of the MCF10A cell islands, suggesting that the two proteins participate in the communication of these epithelial cells with their neighboring cells. Similarly, in U2OS osteosarcoma cell line both endogenous IL-1α and transiently produced IL-1α/EGFP associate with the plasma membrane. While benign MFC10A cells present membrane-associated IL-1α and AnxA2 at the edges of their cell islands, the aggressive cancerous U2OS cells communicate in such manner also with distant cells.

Nanoparticle-Mediated Co-Delivery of Notch-1 Antibodies and ABT-737 as a Potent Treatment Strategy for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) accounts for nearly one-quarter of all breast cancer cases, but effective targeted therapies for this disease remain elusive because TNBC cells lack expression of the three most common receptors seen on other subtypes of breast cancer. Here, we exploit TNBC cells' overexpression of Notch-1 receptors and Bcl-2 anti-apoptotic proteins to provide an effective targeted therapy. Prior studies have shown that the small molecule drug ABT-737, which inhibits Bcl-2 to reinstate apoptotic signaling, is a promising candidate for TNBC therapy. However, ABT-737 is poorly soluble in aqueous conditions, and its orally bioavailable derivative causes severe thrombocytopenia. To enable targeted delivery of ABT-737 to TNBC and enhance its therapeutic efficacy, we encapsulated the drug in poly(lactic-co-glycolic acid) nanoparticles (NPs) that were functionalized with Notch-1 antibodies to produce N1-ABT-NPs. The antibodies in this NP platform enable both TNBC cell-specific binding and suppression of Notch signaling within TNBC cells by locking the Notch-1 receptors in a ligand unresponsive state. This Notch inhibition potentiates the effect of ABT-737 by up-regulating Noxa, resulting in effective killing of TNBC cells. We present the results of in vitro studies that demonstrate N1-ABT-NPs can preferentially bind TNBC cells versus noncancerous breast epithelial cells to effectively regulate Bcl-2 and Notch signaling to induce cell death. Further, we show that N1-ABT-NPs can accumulate in subcutaneous TNBC xenograft tumors in mice following systemic administration to reduce tumor burden and extend animal survival. Together, these findings demonstrate that NP-mediated co-delivery of Notch-1 antibodies and ABT-737 is a potent treatment strategy for TNBC that may improve patient outcomes with further development and implementation.