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Docosahexaenoic acid (DHA) and linoleic acid (LA) have modulatory effects on breast cancer (BC) cell lines. We aimed to investigate the effects of DHA, LA alone, in combination, and in the presence of paclitaxel on the expression of five microRNAs involved in the pathology of BC in MDA-MB-231 cell line.MDA-MB-231 cells were treated with either DHA or LA or in combination in the presence/absence of paclitaxel (Taxol). Total RNA was extracted and cDNA synthesized from the cells before and after treatment. The expression levels of miR-30, miR-106b, miR-20, miR-126, and miR-194 were determined by quantitative real-time PCR (qPCR).Treatment of MDA-MB-231 cells with DHA modulated the gene expression of miR-30 (increased by 7.74-fold (p 0.0001), miR-194 (decreased by 11-fold (p 0.0001)), miR-106b (increased by 2.64-fold (p = 0.0004), miR-126 (decreased by 50-fold (p 0.0001)), and miR-20 (decreased by 4-fold (p 0.0001)). Additionally, treatment of MDA-MB-231 cells with LA modulated the gene expression of miR-30 (increased by 2.38-fold (p = 0.0001)), miR-194 (decreased by 100-fold (p 0.0001)), miR-106b (decreased by 10-fold (p 0.0001)). The combined DHA/LA treatment of MDA-MB-231 cells showed regulatory effect on the expression of studied microRNAs in which decreased the expression of miR-30 (5.5-fold (p 0.0001)), miR-194 (11-fold (p 0.0001)), miR-20 (3.5-fold (p = 0.0006)), and increased the expression of miR-106b (9.78-fold (p 0.0001)).Modulation of the expression levels of BC-involved microRNAs could be one of the possible mechanisms of action through which DHA and LA may exert their biologic effects on MDA-MB-231 cell line.

Aim: It was aimed to investigate the effect of miR-34a-5p and miR-145-5p on breast cancer cell line MDA-MB-231 and to determine the expressionof target genes of these microRNAs (miRNAs).Materials and Methods: Firstly, literature search and in silico analysis were performed to detect possible target genes of miR-34a-5p and miR-145-5p, which are known to be tumor suppressors. Mimic miR-34a-5p and miR-145-5p were transfected to the breast cancer cell line MDA-MB-231.Deregulated genes were investigated by the quantitative real-time polymerase chain reaction compared to control cells. Also, the effect of thesemiRNAs on proliferation was determined using the Water Soluble Tetrazolium Salt-8 method. Finally, the expressions of epithelial mesenchymaltransition (EMT) markers, which are known to be important in the metastatic process, are examined.Results: The proliferation of the miR-34a-5p or miR-145-5p transfected cells decreased compared to the control groups. The expression of E2Ftranscription factor 1 (E2F1) (p=0.009), mitogen activated protein kinase 1 (MEK1) (p=0.001) and cyclin dependent kinase 4 (CDK4) (p=0.005)genes, which were among the genes targeted by miR-34a-5p, were significantly reduced. EMT markers were significantly changed in miR-34a-5ptransfected cells (E-Cad increase p=0.01; Vimentin decrease p=0.008). Kruppel-like factor 4 (KLF4) (p=0.007) targeted miR-145-5p were significantlyreduced and EMT markers were significantly changed in miR-145-5p transfected cells (E-Cad increase p=0.0005; Vimentin decrease p=0.006).Conclusion: miR-34a-5p and miR-145-5p may have an impact on the breast cancer cell line MDA-MB-231 proliferation and EMT mechanism. Atthe same time, according to our study results, it was revealed that E2F1, MEK1 and CDK4 genes, whose expression level decreased after transfectionof mimic miR-34a-5p, could be targeted by miR-34a-5p in breast cancer, and that the expression level of KLF4, which decreased as a result of mimicmiR-145-5p transfection, could be the target.

Background: A large number of Euphorbia species have been evaluated for anticancer effects; however, their anticancer mechanisms have not been established up to now. Objective: : The present study aimed to evaluate the effects of Euphorbia microsciadia (E. microsciadia) Boiss on the modulation of micro (mi) RNAs in MDA-MB-231 cell line. Methods: As the first step, the inhibitory concentration of hydroalcoholic extract of E. microsciadia on MDA-MB-231 cells was examined using the MTT assay, bypassing 24 and 48h from seeding. The real-time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) was also utilized to determine Let-7, miR-15, miR-16, miR-29, miR-151, miR-155, miR-21, miR-146b, miR-181b, miR-221, miR-222, miR-21, and miR-146b expressions in MDA-MB-231 cells, by passing 24 and 48h from treating with the extract of E. microsciadia. Results: The results reveal the cytotoxic effects of E. microsciadia on MDA-MB-231 cell line in a dose-dependent manner. The half maximal Inhibitory Concentrations (IC50) were also equal to 275 and 240μg/ml for E. microsciadia, by passing 24 and 48h from the treatment, respectively. Furthermore, it was confirmed that, E. microsciadia had augmented the expression levels of Let-7, miR-15, miR-16, miR-29, and miR-34a, which lead to an increase in apoptosis. Conclusion: E. microsciadia could modulate some miRNAs involved in cell cycle arrest and apoptosis in MDA-MB-231 cell line. Accordingly, targeting miRNAs by E. microsciadia can open some newer avenues for breast cancer therapy.

A biosurfactant producing bacterium was identified as Pseudomonas aeruginosa DNM50 based on molecular characterization (NCBI accession no. MK351591). Structural characterization using MALDI-TOF revealed the presence of 12 different congeners of rhamnolipid such as Rha-C8-C8:1, Rha-C10-C8:1, Rha-C10-C10, Rha-C10-C12:1, Rha-C16:1, Rha-C16, Rha-C17:1, Rha-Rha-C10:1-C10:1, Rha-Rha-C10-C12, Rha-Rha-C10-C8, Rha-Rha-C10-C8:1, and Rha-Rha-C8-C8. The radical scavenging activity of rhamnolipid (DNM50RL) was determined by 2, 3-diphenyl-1-picrylhydrazyl (DPPH) assay which showed an IC50 value of 101.8 μg/ ml. The cytotoxic activity was investigated against MDA-MB-231 breast cancer cell line by MTT (4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay which showed a very low IC50 of 0.05 μg/ ml at 72 h of treatment. Further, its activity was confirmed by resazurin and trypan blue assay with IC50 values of 0.01 μg/ml and 0.64 μg/ ml at 72 h of treatment, respectively. Thus, the DNM50RL would play a vital role in the treatment of breast cancer targeting inhibition of p38MAPK.

The paper investigates anticancer effects of methanol extracts of invasive plant species Robinia pseudoacacia L. (RpE) and Amorpha fruticosa L. (AfE) on breast cancer MDA-MB-231 and healthy MRC-5 cells. The anticancer activity was evaluated through examination of cytotoxic effects, anti-invasive potential and impact on redox status in comparative analysis using their chemical composition. According to the IC50 values, the investigated plants had no significant cytotoxic effects either on healthy cell line MRC-5 or on MDA-MB-231 cancer cells, but they showed great anti-invasive potential by suppressing all investigated parameters of tumor invasion and metastases (Matrix Metalloproteinases (MMP), protein concentration and MMP-9, C-X-C Motif Chemokine Ligand 12 (CXCL-12), Vascular Endothelial Growth Factor (VEGF-A) and Hypoxia-Inducible Factor (HIF-1α) gene expression) in MDA-MB-231 cells. Based on their remarkable anti-invasive potential, RpE and AfE are suitable for use as potential supplements in anticancer therapy or as nutritional food supplements.

Breast cancer is the second highest cause of cancer related deaths for women in developed countries. Breast cancer patients with distant metastasis at the time of diagnosis have an estimated 5-year relative survival rate of 26% as compared to a 99% survival rate of patients who have localized tumors. Evidence suggests that collagens play a role in enhancing the metastatic capability of breast cancer cells. Short chain collagen, collagen X, is encoded by the collagen type x alpha 1 chain (COL10A1) gene and is normally expressed exclusively by hypertrophic chondrocytes during endochondral ossification. Recently, COL10A1 gene expression has been found to be overexpressed in various tumor types, including breast tumors. It is hypothesized that an increase in COL10A1 expression may play a role in breast cancer metastasis. The goal of our project was to evaluate the role of collagen X in breast cancer metastasis using the MDA-MB-231 breast cancer cell line. Stable cell lines were generated to express either GFP only (MDA-VEC) or GFP tagged COL10A1 (MDA-COL). GFP and COL10A1 transcript and protein levels were examined to confirm overexpression of collagen X and transwell assays were used to determine changes in the invasive capability of the cells. Cells overexpressing collagen X demonstrated a higher rate of invasion suggesting that collagen X may play a role in enhancing the metastatic potential of breast cancer cells. Understanding the role collagen X plays in breast cancer metastasis may provide a mechanism for developing diagnostic and prognostic strategies for identifying patients whose breast cancer is more prone to metastasize. Citation Format: McKiernan K, Jimenez H, McEachern M, Hubbard J, O'Connell M. Role of collagen X in enhancing the metastatic potential of breast cancer cells using a MDA-MB-231 cell line model [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-16.

AIM: Despite current advances in therapies and the gradual decline in breast cancer-related mortality, metastasis remains a major therapeutic challenge for treatment. Energy reprogramming is now recognized to be an important part of tumorigenic processes, but its relevance in metastatic dissemination has yet to be elucidated. METHODS: Using the MDA-MB-231HM.LNm5 cell line, a novel, highly metastatic variant line derived from TN human breast adenocarcinoma MDA-MB-231 line, alteration in growth and energy metabolisms associated with enhanced metastatic potential were described. Glycolysis and oxidative phosphorylation (OXPHOS) was characterized using the seahorse XF analyzer. Whole transcriptome sequencing (RNA-seq) and quantitative real-time PCR (RT-qPCR) was used to ascertain expression differences in metabolic genes. RESULTS: We observed reduced proliferation, and an elevation of both glycolytic and OXPHOS metabolism in the highly metastatic daughter line. The elevated metabolic rate is only partially reflected by transcript levels of relevant metabolic regulators. Heightened mitochondrial respiration is potentially underpinned by increased expression mitochondrial electron transport chain (ETC) components. However, increased glycolysis was not underpinned by up-regulation of metabolic genes encoding enzymes participating in glycolysis. CONCLUSION: Our results indicate breast tumour cells with elevated metastatic propensity are more metabolic active. We also identified differentially expressed metabolic genes, such as IDH2, that may play a part in the metastatic process beyond energy reprogramming.

A new combination of sulforaphane (a natural compound obtained from Brassicaceae vegetables) and the cytostatic drug doxorubicin was entrapped in nanometer-sized liposomes. In vitro experiments were performed to investigate the cytotoxicity of these structures on the human breast cancer cell line MDA-MB-231. Confocal microscopy studies revealed enhanced cellular endocytotic internalization, followed by the release of the examined combination from the lysosomes. The in vitro interaction analysis using the Chou-Talalay approach showed high synergistic activity of the examined combination. This synergistic activity enables a considerable reduction in cytostatic dosage and an increase in cancer treatment efficiency.

Triple-negative breast cancers (TNBCs) are aggressive cancers that do not benefit from hormonal therapy or therapies that target HER2 receptors. Insulin-like growth factor 1 receptor (IGF-1R), which has been shown to be overexpressed in breast cancer, activates numerous downstream kinases that associate with cell proliferation and survival. This study compared the effects caused by dual treatments targeting IGF-1R, PI3K, mTORC, or MEK with those by single treatments in a TNBC cell line, MDA-MB-231. We used small-molecule kinase inhibitors, namely, NVP-AEW541, NVP-BKM120, KU0063794, and PD0325901 to target IGF-1R, PI3K, mTORC, and MEK, respectively. Combination treatments of PD0325901 with NVP-AEW541, NVP-BKM120 or KU0063794 and NVP-AEW541 with KU0063794 demonstrated a significant synergistic growth inhibition. These dual treatments increased apoptosis and/or cell cycle arrest at G0/G1 phase and enhanced the inhibition of phosphorylation of Akt or downstream molecules of mTORC1, as compared to the single treatments. Our study suggests that targeting multiple kinases in IGF-1R signaling may be a promising therapeutic approach.

Background: Breast cancer is the most diagnosed and the major cause of cancer death in women worldwide. Metastasis is the main cause of these deaths. The metastatic cascade involves multiple steps and it has been described that adrenergic receptors can modulate this process at multiple levels. However, β-adrenergic action in breast cancer is controversial. We have previously shown that β-adrenergic agonists inhibit cell proliferation and tumor growth of numerous breast cancer models. Objective: The purpose of the present investigation was to evaluate adrenergic effect in parameters related to tumor progression (migration, invasion and metastases) in two human breast cancer cell lines. Method: Migration was assessed in IBH-6 and MDA-MB-231 cells by time-lapse videomicroscopy and modified Boyden chambers. Invasion was evaluated by Transwells coated with Matrigel and expression of pro-metastatic genes was determined by RT-qPCR. Experimental metastases studies were performed by injection of the cells in the tail vein of NSG immuno-deficient mice. Results: In both cell lines, salbutamol (β2-agonist) and propranolol (β-blocker) significantly diminished cell migration while epinephrine exerted opposite effects. Moreover, salbutamol inhibited invasion of both breast cancer cell lines and enhanced adhesion to extracellular matrix. Salbutamol treatment was also able to decrease the expression of pro-metastatic genes in MDA-MB-231 cells. Finally, this compound decreased the number and size of MDA-MB-231 lung experimental metastases in NSG immuno- deficient mice. No effect on the establishment of IBH-6 metastases was observed. Conclusion: Our results suggest that salbutamol could be an effective adjuvant drug for the treatment of metastatic breast cancer. Fil: Rivero, Ezequiel Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Perez, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Gargiulo, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Entschladen, Frank. Witten/Herdecke University; Alemania Fil: Zänker, Kurt. Witten/herdecke University; Alemania Fil: Bruzzone, Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Luthy, Isabel Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

Eukaryotic elongation factor 2 kinase (eEF-2K) is an unusual alpha kinase involved in protein synthesis through phosphorylation of elongation factor 2 (EF2). eEF-2K is highly overexpressed in breast cancer, and its activity is associated with significantly shortened patient survival and proven to be a potential molecular target in breast cancer. The crystal structure of eEF-2K remains unknown, and there is no potent, safe, and effective inhibitor available for clinical applications. We designed and synthesized several generations of potential inhibitors. The effect of the inhibitors at the binding pocket of eEF-2K was analyzed after developing a 3D target model by using a domain of another ?-kinase called myosin heavy-chain kinase A (MHCKA) that closely resembles eEF-2K. In silico studies showed that compounds with a coumarin-chalcone core have high predicted binding affinities for eEF-2K. Using in vitro studies in highly aggressive and invasive (MDA-MB-436, MDA-MB-231, and BT20) and noninvazive (MCF-7) breast cancer cells, we identified a lead compound that was highly effective in inhibiting eEF-2K activity at submicromolar concentrations and at inhibiting cell proliferation by induction of apoptosis with no toxicity in normal breast epithelial cells. In vivo systemic administration of the lead compound encapsulated in single lipid-based liposomal nanoparticles twice a week significantly suppressed growth of MDA-MB-231 tumors in orthotopic breast cancer models in nude mice with no observed toxicity. In conclusion, our study provides a highly potent and in vivo effective novel small-molecule eEF-2K inhibitor that may be used as a molecularly targeted therapy breast cancer or other eEF-2K-dependent tumors. © 2021 American Chemical Society. 1R01CA244344; University of Texas MD Anderson Cancer Center; Türkiye Bilimsel ve Teknolojik Araştirma Kurumu, TÜBITAK: 215S008, TUBITAK-BIDEB 2214A This study was funded by The Scientific and Technological Research Council of Turkey (TUBITAK) (grant number 215S008 and TUBITAK-BIDEB 2214A program, F.C.O.) and The University of Texas-MD Anderson Cancer Center Bridge fund (B.O. and N.K.) and NIH-NCI 1R01CA244344 grants (B.O. and N.K.).

A hallmark of cancer cells is their ability to invade surrounding tissues and form metastases. Cell-extracellular matrix (ECM)-adhesion proteins are crucial in metastasis, connecting tumor ECM with actin cytoskeleton thus enabling cells to respond to mechanical cues. Vasodilator-stimulated phosphoprotein (VASP) is an actin-polymerization regulator which interacts with cell-ECM adhesion protein Migfilin, and regulates cell migration. We compared VASP expression in MCF-7 and MDA-MB-231 breast cancer (BC) cells and found that more invasive MDA-MB-231 cells overexpress VASP. We then utilized a 3-dimensional (3D) approach to study metastasis in MDA-MB-231 cells using a system that considers mechanical forces exerted by the ECM. We prepared 3D collagen I gels of increasing concentration, imaged them by atomic force microscopy, and used them to either embed cells or tumor spheroids, in the presence or absence of VASP. We show, for the first time, that VASP silencing downregulated Migfilin, β-catenin and urokinase plasminogen activator both in 2D and 3D, suggesting a matrix-independent mechanism. Tumor spheroids lacking VASP demonstrated impaired invasion, indicating VASP's involvement in metastasis, which was corroborated by Kaplan-Meier plotter showing high VASP expression to be associated with poor remission-free survival in lymph node-positive BC patients. Hence, VASP may be a novel BC metastasis biomarker. © 2017 Elsevier Inc. 352 281 292 281-292

Great strides have been made in triple negative breast cancer (TNBC) treatment, which represents 20% of total predicted annual US breast cancer (BC) cases. Despite the development of several therapeutics, TNBC patients have poor overall survival rate, compared to other BC patients, justifying the urgent need to discover new entities for use to control TNBC. Chalcones are important natural products with diverse bioactivities including anticancer effects. This study aimed to design, synthesize and validate novel chalcone leads as potential therapies for TNBC. Fourteen novel chalcone analogs were designed and synthesized comprising alicyclic amines (pyrrolidine, morpholine and piperidine) or nitrogen mustard (Bis-(2-chloroethyl) amine) substituents. Among them, compound 14 ((E)-3-(4-(Bis(2-chloroethyl) amino) phenyl)-1-(3-methoxyphenyl) prop-2-en-1-one) was identified as the most effective against TNBC and other BC phenotypes, with anti-proliferative IC50 values ranging between 3.94 and 9.22 μM against the TNBC cell lines MDA-MB-231 and MDA-MB-468, as well as against the estrogen positive MCF-7 cell line. Chalcone 14 effectively suppressed the colony formation capacity of MDA-MB-231, MDA-MB-468, and MCF-7 cell lines at 5 and 10 μM treatment concentrations. Furthermore, compound 14 has significantly inhibited cell invasion and migration of MDA-MB-231 and MCF-7 BC cell lines. Additionally, compound 14 had significantly promoted apoptosis by upregulating BAX and downregulating Bcl-2 proteins. Compound 14 induced significant cell cycle arrest of TNBC cells at the G2/M phase. It also induced a reversal of Epithelial Mesenchymal Transition (EMT) by upregulating the epithelial markers E-cadherin and Pan-cadherin and downregulating FAK. Furthermore, it had dramatically diminished new vessel formation (vasculogenesis) in chick chorioallantoic membrane (CAM) model by 60.20 ± 8.47%. Chalcone 14 inhibited 46.41 ± 0.71% of the TNBC MAD-MB-231 cells growth in a nude mouse orthotopic xenograft model in comparison with vehicle control treated animals. Collectively, this study results propose chalcone 14 as a promising lead molecule for the control of TNBC as well as other breast cancer phenotypes. This research work was supported by Grants# GCC-2017-2 and QUST-1-CPH-2018-18 from Qatar University ; and GSRA award# GSRA4-2-0418-17024 from Qatar National Research Fund (a member of Qatar Foundation). Scopus

Effective control of breast cancer has been primarily hampered by a lack of tumor specificity in treatments. One potential way to improve targeting specificity is to develop novel vectors that specifically bind to and are internalized by tumor cells. Through a phage display library, an 11-L-amino acid peptide, PI (sequence, CASPSGALRSC), was selected. PI was labeled with fluorescein isothiocyanate (FITC) and named PI-FITC. Subsequently, the specific affinity of PI-FITC to MDA-MB-231 human breast cancer cells and other cancer cell lines was observed by confocal microscopy. Our previous study established that PI-FITC also shows affinity to Calu-1 human lung carcinoma cells and major histocompatibility complex class I antigen molecules; therefore, the cytomembrane proteins of the cell lines were analyzed to determine those that were common to the two cell lines and may be associated with transmembrane transduction. To further test the delivery ability of PI to MDA-MB-231 cells, PI-glutathione-S-transferase (GST) was constructed and the internalization of this fusion protein was visualized by immunofluorescence microscopy. The results revealed that PI exhibited specific affinity to MDA-MB-231 cells. Use of membrane transport inhibitors indicated that macropinocytosis and caveolin-mediated endocytosis may be involved in the endocytosis of PI. In addition, 11 membrane proteins common to MDA-MB-231 and Calu-1 may be associated with transmembrane transduction. In summary, PI was able to deliver PI-GST into MDA-MB-231 cells. Thus, PI could be modified to be a potential vector, and may contribute to the development of targeted therapeutic strategies for breast cancer.

The main aim of this study is to assess the safety and antitumor efficacy of a palladium(II) (Pd)-saccharinate complex with terpyridine. To characterize the Pd(II) complex in vitro, its cytotoxicity was evaluated using a water-soluble tetrazolium salt cell viability assay and the mechanism of cell death was assessed by DNA fragmentation/condensation and live cell imaging analyses. The antitumor efficacy and safety of the Pd(II) complex in-vivo were examined by analyzing reduction in tumor size, changes in body and organ weight, histopathological analysis of liver, kidney, and tumor sections, and biochemical analysis of serum in C57BL/6 mice. Our results showed that the Pd(II) complex was more cytotoxic to cancer cells than noncancer cell lines and caused cell death through apoptotic pathways. The treatment of the Pd(II) complex in tumor-bearing mice effectively reduced the tumor size at half the dose used for cisplatin. The Pd(II) complex appeared to exert less liver damage than the cisplatin-based complex on changes in the hepatic enzymes levels in the serum. Hence, the complex appears to be a potential chemotherapeutic drug with high antitumor efficacy and fewer hepatotoxic complications, providing an avenue for further studies. Anti-Cancer Drugs 28: 898-910 Copyright (C) 2017 Wolters Kluwer Health, Inc. All rights reserved. Scientific and Technological Research Institute of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) This work was supported by the internal funding of The Scientific and Technological Research Institute of Turkey (TUBITAK). WOS:000408162400009 28657910 Q3

// Vasiliki Gkretsi 1 , Andreas Stylianou 1 , Maria Louca 1 , Triantafyllos Stylianopoulos 1 1 Cancer Biophysics Laboratory, Department of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia, Cyprus Correspondence to: Triantafyllos Stylianopoulos, email: tstylian@ucy.ac.cy Keywords: Ras suppressor-1, extracellular matrix stiffness, invasion, tumor spheroids, atomic force microscopy Received: July 16, 2016 Accepted: February 20, 2017 Published: March 09, 2017 ABSTRACT Breast cancer (BC) is the most common malignant disease in women, with most patients dying from metastasis to distant organs, making discovery of novel metastasis biomarkers and therapeutic targets imperative. Extracellular matrix (ECM)-related adhesion proteins as well as tumor matrix stiffness are important determinants for metastasis. As traditional two-dimensional culture does not take into account ECM stiffness, we employed 3-dimensional collagen I gels of increasing concentration and stiffness to embed BC cells of different invasiveness (MCF-7, MDA-MB-231 and MDA-MB-231-LM2) or tumor spheroids. We tested the expression of cell-ECM adhesion proteins and found that Ras Suppressor-1 (RSU-1) is significantly upregulated in increased stiffness conditions. Interestingly, RSU-1 siRNA-mediated silencing inhibited Urokinase Plasminogen Activator, and metalloproteinase-13, whereas tumor spheroids formed from RSU-1-depleted cells lost their invasive capacity in all cell lines and stiffness conditions. Kaplan-Meier survival plot analysis corroborated our findings showing that high RSU-1 expression is associated with poor prognosis for distant metastasis-free and remission-free survival in BC patients. Taken together, our results indicate the important role of RSU-1 in BC metastasis and set the foundations for its validation as potential BC metastasis marker.

Background: Due to the aggressive clinical behavior, poor outcome, and lack of effective specific targeted therapies, triple-negative breast cancer (TNBC) has currently been recognized as one of the most malignant types of tumors. In the present study, we investigated the cytotoxic effect of ziyuglycoside I, one of the major components extracted from Chinese anti-tumor herbal Radix Sanguisorbae, on the TNBC cell line MDA-MB-231. Methods: The underlying molecular mechanism of the cytotoxic effect ziyuglycoside I on MDA-MB-231 cells was investigated with cell viability assay, flow cytometric analysis and Western blot. Results: Compared to normal mammary gland Hs 578Bst cells, treatment of ziyuglycoside I resulted in a significant growth inhibitory effect on MDA-MB-231 cells. Ziyuglycoside I induced the G2/M phase arrest and apoptosis of MDA-MB-231 cells in a dose-dependent manner. These effects were found to be partially mediated through the up-regulation of p53 and p21WAF1, elevated Bax/Bcl-2 ratio, and the activation of both intrinsic (mitochondrial-initiated) and extrinsic (Fas/FasL-initiated) apoptotic pathways. Furthermore, the p53 specific siRNA attenuated these effects. Conclusion: Our study suggested that ziyuglycoside I-triggered MDA-MB-231 cell cycle arrest and apoptosis were probably mediated by p53. This suggests that ziyuglycoside I might be a potential drug candidate for treating TNBC.

Background: Triple Negative Breast Cancer (TNBC) commonly displays Epidermal growth factor receptor (EGFR). Effective EGFR degradation results in the suppression of tumor in various models. Studies have addressed the relevance of this strategy in the treatment of TNBC. In the present study, we examined the effect of 17 β- estradiol on EGFR expression in MDA-MB-231 (TNBC) cell line and assessed whether 17 β-estradiol degrades EGFR by ubiquitination pathway. Objectives: The objectives of this study are to treat MDA-MB-231 cell lines with Cycloheximide with or without 17β-estrdiol to observe whether 17β-estradiol leads to EGFR degradation and to treat with MG-132 to assess whether degradation occurs through ubiquitination pathway. Methods: MDA-MB-231 cells were treated with 17β-estradiol (E2) and EGFR expression was studied by western blotting at different intervals by using Cycloheximide chase. To assess ubiquitination pathway of degradation of EGFR in MDA-MB-231 cell line, MG-132 was used. Results: EGFR expression was reduced with β-estradiol treatment in MDA-MB-231 cell line with Cycloheximide chase. Upon Treatment with MG-132 and E2, EGFR expression did not reduce, suggesting that Estrogen degrades EGFR by ubiquitination pathway. Conclusion: Estrogen degrades EGFR in MDA-MB-231 cells and this degradation occurs by ubiquitination.

Purpose: Breast cancer is one of the most commonly diagnosed types of cancer worldwide. This cancer is treated with various methods like mastectomy, chemotherapy, hormone replacement therapy, and radiotherapy. Among them, targeted therapy, which specifically targets cancerous cells by using strategies like microRNA replacement therapy, is considered a new approach in treating breast cancer. Methods: Data analysis from TCGA datasets were used to confirm the decreased expression of miR-146a in breast cancer. MTT assay was used to evaluate the viability of MDA-MB-231 cells after miR-146a restoration. A wound-healing assay was used to observe migration in the MDA-MB-231 cell line and the effect of miR-146a on the migration. Finally, qRT-PCR was used as a method to determine the effect of miR-146a on the expression of CXCR4, β-catenin, MMP2, MMP9, and Vimentin genes are known to be involved in invasion and migration in MDA-MB-231 cell lines in breast cancer. Results: The bioinformatic study showed miR-146a expression decreased in breast tumors compared to adjusted normally. Our results indicated that miR-146a is not involved in apoptosis in the MDA-MB-231 cell line, while it is highly effective in migration inhibition. MMP9, MMP2, CXCR4, and Vimentin expressions were suppressed by miR-146a induction; however, it induced the expression of β-catenin which all result in inhibition of migration. Conclusion: Regulatory molecules, such as miR-146a, are effective in breast cancer targeted therapy. As cancer is a complicated disorder, therefore the combination of therapies, based on these targets might lead to novel therapeutic strategies.

Triple-negative breast cancer (TNBC) subtype is one of the most aggressive tumors with no definite receptor, hence has a limited number of effective chemotherapeutics. Doxorubicin (Dox) is the first-line drug for the TNBC treatment, acting as a DNA intercalator and topoisomerase II (Topo II) inhibitor; however, it has been observed to exhibit strong cardiotoxicity. Tanshinone IIA (Tan IIA) has a previously confirmed antitumor activity against breast cancer in addition to its well-known cardioprotective effect. In our study, molecular docking reveals the potential activity of Tan IIA as a DNA intercalator and Topo II inhibitor as a recommended possible mechanism of action compared to Dox as a reference drug. Moreover, we wanted to assess a new use for Tan IIA in the TNBC treatment in combination with Dox to broaden its therapeutic window and compensate for its cardiotoxic effects by the cardioprotective effects of Tan IIA. On performing the in vitro MTT assay to evaluate the cytotoxic effects of Dox, Tan IIA and their combinations at different concentrations of the MDA-MB-231 cell line revealed the potential activity of Tan IIA compared to Dox, in addition to the confirmed synergistic effects of Dox/Tan IIA combinations on TNBC cell lines. Furthermore, the analysis of the gene expression profiles of PI3K, mTOR, and AKT as anti-apoptotic genes were examined in the MDA-MB-231 cell line after the treatment with the IC50 of Dox, Tan IIA, and their combination, and compared to the control using PCR. These results confirmed the synergistic effect of the Dox/Tan IIA combination that exceeded that of Dox alone.

Cancer is a major health problem which is considered as one of the most reasons of death worldwide. Breast cancer is a second cause of women death and estimated to have 2.1 million new cases. Traditional treatment of cancer based on different clinical approaches is included surgery, chemotherapy, radio therapy, hormone therapy and combination therapy which affect both cancer and normal cells. The present study investigated the anticancer potential of Cerastes vipera venom in comparison with Cisplatin drug on MDA-MB-231 breast cancer cell line. Cytotoxicity activity of tested material was evaluated using MTT assay. Cell cycle arrest was performed using propidium iodine DNA staining. Apoptotic detection was determined using annexin V-FTIC and for the detection of the genes Caspase 3, Caspase 8 & Caspase 9 weredetermined by ELISA. The C. vipera venom, Cisplatin and PLA2 had IC50 as 3.4, 10.3, 35.1 μg/mL with MDA-MB-231 cell line respectively. Cell cycle has arrest of cells in G2/M of C. vipera venom and PLA2. Tested venoms have apoptotic effect against MDA-MB-231 cell line through elevation of Caspase 3, Caspase 8 & Caspase 9 genes.

Triple-negative breast cancer (TNBC) is the most outrageous subtype of breast cancer. Emphasizing the urge of new approach in cancer therapy, combinational drug therapy may be proven as an effective strategy. In our previous study, we reported that coralyne (COR) with paclitaxel (PTX) efficiently decreases the proliferation of MDA-MB-231 compared with MCF-7 cell line. Thus, we studied the effect of COR and PTX in combination on apoptosis of MDA-MB-231 cell line. In silico results demonstrated that COR intercalates DNA at a minor groove. In vitro approaches revealed that in combination (COR and PTX) increases the efficacy of apoptosis in MDA-MB-231 cell line by a significant increase in G1/S phase arrest, DNA fragmentation, and change in mitochondria membrane potential. The expression of ATM and ATR a serine/threonine-protein kinase, ataxia telangiectasia and Rad3-related protein were depleted with an increase in time from 24 to 48 hours in concurrent with increased levels of γH2AX indicating that DNA damage routes cells to enter apoptosis. This was confirmed by high levels of caspase-3 and cytochrome c. Also, the decrease in the expression levels of matrix metalloproteinase-9 confirmed the antimetastatic efficacy of COR + PTX. The present study indicates that the synergistic effect of COR and PTX can enhance apoptosis in MDA-MB-231 cell line and may be proven as a potential anticancer therapy against TNBC.

Background: Autophagy has an essential role in cellular energetic balance, cell cycle, and cell death, so the change in autophagy level is crucial in many human diseases such as cancer. Herbal medicine has been widely used to treat cancer. Bowman–Birk protease inhibitor (BBI), a protease inhibitor extracted from soybean, has antitumorigenic, anti-inflammatory, and anti-angiogenic activities. In this study, we evaluated the effect of BBI on the growth of breast cancer cell line and transcript level of autophagy and apoptosis-related genes. Materials and Methods: BBI was purified from soybean by ion-exchange chromatography method. The viability of MDA-MB-231 cells that were treated with BBI was measured by MTT assay, and the transcript level of genes involved in autophagy and apoptosis was measured by real-time–polymerase chain reaction (PCR) technique. Results: The results of BBI purification showed that 100 g of the ethanolic fraction yielded 300-mg BBI with more than 95% purity. MTT results revealed that BBI inhibited the cell growth of MDA-MB-231 cell line in a dose-dependent manner, with an IC50 of 200 μg/mL. The results of real-time reverse transcription-PCR exhibited that BBI altered the expression of Atg5, Beclin1, light chain 3-II, and sequestosome1 and increased the Bax/Bcl2 ratio in MDA-MB-231 cell line. Conclusion: According to our results, BBI could inhibit autophagy and induce apoptosis in MDA-MB-231 cell line. Thus, BBI may be used as a therapeutic drug in the treatment of breast cancer whether alone or with chemotherapeutic drugs.

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 IV6Neu5Ac-nLc4Cer and GalNAc-GM1b (IV3Neu5Ac-Gg5Cer) not reported previously, were identified in both CSCs and non-CSCs. IV6Neu5Ac-nLc4Cer had increased expression in both CSCs and non-CSCs of both cell lines after the treatment with 1, while GM3 (II3Neu5Ac-LacCer) had increased expression only on both cell subpopulations in MDA-MB-231 cell line. GalNAc-GM1b, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) and GM2 (II3Neu5Ac-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.

Background: RX-5902 (Supinoxin) is a novel anti-cancer compound that targets phosphorylated p68 RNA helicase, a member of the DEAD box family of helicases, affecting upstream and downstream molecules in the Wnt canonical pathway. As a single agent, RX-5902 exhibits strong growth inhibition in both in vitro and in vivo settings. Specifically, RX-5902 enhances survival and tumor growth inhibition in numerous xenograft models, including ovarian, renal and breast. We have previously shown RX-5902 inhibits cell growth in a dose-dependent fashion in the triple-negative breast cancer (TNBC) xenograft MDA-MB231. In the current study, we have expanded our investigation of the therapeutic potential of RX-5902 against TNBC using both in vitro and in vivo preclinical models. Methods: RX-5902 was provided by Rexahn, Inc. (Rockville, MD). Cell proliferation was measured using the Cell-Titer Glo luminescent cell viability assay (Promega). Apoptosis was assessed using Incucyte Caspase 3/7 Green apoptosis assay (Essenbioscience). Immunoblots of MDA-MB-231 cell line were probed for ß-catenin (Cell Signaling). Syngeneic 4T1 murine TNBC mice were obtained from Sippr-BK Laboratory Animal Co (Shanghai, China) and tumor volumes were measured twice a week. When the mean tumor volumes reached ˜90 mm3, mice were randomized and treated with vehicle or RX-5902 PO daily alone or in combination with anti-CTLA4 or anti-PD-1 BIW for 3 weeks. Tumor growth inhibition (TGI) was calculated at Day 25. Results: A panel of 18 TNBC cell lines were treated with RX-5902 and effects on cell proliferation were measured by the Cell Titer-Glo assay. Using 100nM as a cutoff, 14 sensitive lines and 4 resistant lines were identified, with an average IC50 of 56 nM in the sensitive lines. Of these, we chose 2 sensitive lines (MDA-MB-231, HCC1806) and 2 resistant lines (MDA-MB-436 and CAL-120) and assessed induction of apoptosis by the Incucyte caspase activity assay. Robust induction of apoptosis was observed in both sensitive lines (N=3). These lines were then subjected to cell cycle analysis by flow cytometry, which revealed a pronounced G2/M cell cycle arrest and aneuploidy following exposure to RX-5902. Western blot analysis of the MDA-MB-231 cell line showed decreases in the Wnt pathway-related protein nuclear ß-catenin in doses ranging from 20 nM to 200 nM. Finally, the therapeutic efficacy of RX-5902 was assessed as a single agent and in combination with two immune-oncology agents in the treatment of the TNBC 4T1 animal model. RX-5902 as a single agent showed dose dependency in the 4T1 model, and when given in combination with either anti-CTLA4 or anti-PD1 showed an additive effect (p Conclusions: RX-5902 showed efficacy against several in vitro and in vivo preclinical models of TNBC. RX-5902 resulted in G2/M arrest and induced apoptosis in sensitive TNBC cell lines and decreases in nuclear beta-catenin. In vivo, RX-5902 demonstrated additive anti-tumor effects when combined with either anti-CTLA4 or anti-PD1 immunotherapies. Together, these finding indicate that RX-5902 may have important clinical implications for the treatment of TNBC. A phase 2a clinical study in metastatic TNBC is ongoing..training_cert Citation Format: Tentler JJ, Frank JG, Kim DJ, George C, Lee YB, Ely B, Tan AC, Kim J, Pitts TM, Capasso A, Dailey KL, Eckhardt G, Diamond JR. Preclinical studies of RX-5902, a beta-catenin modulator in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-16.

Tumor cells modulate immune responses by secreting exosomes. Tumor exosomes can affect the metabolism of immune cells and increase immune inhibitory molecules such as programmed cell death protein 1 (PD-1). PD-1 inhibits the glycolysis pathway in immune cells. We investigated the role of tumor exosomes in how metabolic changes occur through the PD1-GLUT1-HK2 metabolic axisin peripheral blood mononuclear cells (PBMCs). The MDA-MB-231 cell line was cultured, serum samples from breast cancer patients were collected, and exosomes purified from serum samples and the MDA-MB-231 cell line. PBMCs were treated with purified exosomes for 72 h and, the expression of PD1-GLUT1-HK2 genes was measured by real-time PCR. Our study results showed relative expression of the HK2 gene in both groups treated with MDA-MB-231 cell line exosomes and serum exosomes of breast cancer patients was significantly increased compared to the control group (p < 0.0001). Also, the relative expression of the PD1 gene and GLUT1 gene showed a significant increase compared to the control group only in the group treated with MDA-MB-231 cell line exosomes (p < 0.0001). Therefore, Breast cancer exosomes increased the expression of key genes in the glycolysis pathway, increasing the glycolysis pathway in PBMCs. Increased expression of PD-1 could not prevent the expression of critical genes in the glycolysis pathway as in previous studies.

Background Propolis and its major constituent – caffeic acid phenethyl ester (CAPE) have good abilities on antitumor and anti-inflammation. However, little is known about the actions of propolis and CAPE on tumor in inflammatory microenvironment, and inflammatory responses play decisive roles at different stages of tumor development. To understand the effects and mechanisms of ethanol-extracted Chinese propolis (EECP) and its major constituent - CAPE in inflammation-stimulated tumor, we investigated their effects on Toll-like receptor 4 (TLR4) signaling pathway which plays a crucial role in breast cancer MDA-MB-231 cell line. Methods 80% confluent breast cancer MDA-MB-231 cells were stimulated with 1 μg/mL lipopolysaccaride (LPS). Then the cells were divided for treatment by CAPE (25 μg/mL) and EECP (25, 50 and 100 μg/mL), respectively. Cell viability, nitric oxide (NO) production and cell migration were measured by sulforhodamine B assay, chemical method and scratch assay. The levels of TLR4, MyD88, IRAK4, TRIF, caspase 3, PARP, LC3B and p62 were investigated through western blotting. The expression of TLR4, LC3B and nuclear factor-κB p65 (NF-κB p65) were tested by immunofluorescence microscopy assay. Results Treatment of different concentrations of EECP (25, 50 and 100 μg/mL) and CAPE (25 μg/mL) significantly inhibited LPS-stimulated MDA-MB-231 cell line proliferation, migration and NO production. Furthermore, EECP and CAPE activated caspase3 and PARP to induce cell apoptosis, and also upregulated LC3-II and decreased p62 level to induce autophagy during the process. TLR4 signaling pathway molecules such as TLR4, MyD88, IRAK4, TRIF and NF-κB p65 were all down-regulated after EECP and CAPE treatment in LPS-stimulated MDA-MB-231 cells. Conclusions These findings indicated that EECP and its major constituent - CAPE inhibited breast cancer MDA-MB-231 cells proliferation in inflammatory microenvironment through activating apoptosis, autophagy and inhibiting TLR4 signaling pathway. EECP and CAPE may hold promising prospects in treating inflammation-induced tumor.

Lapatinib, one of the tyrosine kinase inhibitors (TKIs), is used to reduce epidermal growth factor family proteins overexpression. This study aims to assess the cytotoxic and genotoxic effects of lapatinib on the triple negative breast cancer cell line “MDA-MB-231”. We investigated the cytotoxicity of lapatinib by MTT assay, mode of cell death using apoptosis-necrosis assay, DNA damage using micronucleus test, EGFR protein expression by immunocytochemistry, and assessed its effect on EGFR (7p11.2 locus) and TP53 (17p13 locus) genes using interphase-FISH technique. Lapatinib induced cytotoxicity on MDA-MB-231 cell line by elevating the concentration and its IC 50 value was 32.5 μM after 24 h. Lapatinib increased apoptotic cells and micronuclei in binucleated cells gradually by increasing the concentration for 24 h. The EGFR protein expression was reduced by double fold that expressed in non-treated cells. Lapatinib enhanced deletion of EGFR gene signals highly significantly from the lowest concentration. Alternatively, lapatinib amplified signals of TP53 gene effectively by raising the concentration. In conclusion, lapatinib induced cytotoxic and genotoxic effects on MDA-MB-231 cell line. However, laptinib reduced the EGFR protein expression and EGFR signals, it raised the apoptotic cells and TP53 gene signals, which triggered extensive DNA damage. Therefore, lapatinib is an effective TKI in triple negative breast cancer cells as elucidated by its mode of cell death.

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like ( WBP2NL) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.

Annona muricataL., known as graviola, is an evergreen plant of the tropical regions and is a rich source of natural products. Graviola has various biological activities, and it is best known for its anticancer activity. This study aimed to investigate the effects of crude graviola extractin vitroon breast cancer cells; in particular, we aimed to identify an agent against triple negative breast cancer (TNBC). We used the TNBC MDA-MB-231 cell line as the experimental model and the ER(+) non-TNBC MCF-7 breast cancer cell line as the control. We identified annonaceous acetogenins, including annonacin isomers, characteristic to this plant by using liquid chromatography tandem mass spectrometry (LC/MS/MS). We observed a significant decrease in the cell viability in both cell lines within 48 h, whereas impaired cell motility and invasiveness were observed only in the MDA-MB-231 cell line. While the MCF-7 cells showed an ER-dependent mechanism of apoptosis, the apoptosis of MDA-MB-231 cells was governed by an intrinsic apoptotic pathway triggered by graviola leaf extract (GLE).

Breast cancer suppressor candidate-1 (BCSC-1; also termed von Willebrand factor A domain containing 5A and LOH11CR2A) is a newly identified candidate tumor suppressor gene that has been implicated in several types of cancer in previous studies. However, there have been few reports about the association between BCSC-1 and human breast cancer in recent years. In the present study, the expression of BCSC-1 in breast cancer was determined by immunohistochemistry (IHC) staining of tissue microarrays and clinical tissue specimens. Subsequently, BCSC-1 gene expression was evaluated in different breast cancer cell lines by quantitative polymerase chain reaction and the MDA-MB-231 cell line was selected for further use in subsequent experiments, due to its low BCSC-1 expression. An MDA-MB-231 cell line with stable overexpression of BCSC-1 was established through transfection with plasmid containing the BCSC-1 gene, and then screening for G418 resistance. Wound-healing, migration and invasion assays were conducted to detect the effect of BCSC-1 on MDA-MB-231 cells. Furthermore, changes in matrix metalloproteinases (MMPs), osteopontin (OPN) and the nuclear factor-κB (NF-κB) pathway were detected in the current study. Additionally, stable silencing of BCSC-1 expression in MCF-7 cells was performed using a lentivirus. The results of IHC indicated that BCSC-1 is expressed at low levels in breast cancer tissues compared with in normal breast tissue. Results of the wound healing, migration and invasion assays demonstrated that BCSC-1 overexpression reduced the metastasis ability of MDA-MB-231 cells in vitro. Further research confirmed that the BCSC-1 overexpression reduced the expression levels of MMP7, MMP9 and OPN, and the phosphorylation of NF-κB p65. Furthermore, inhibition of BCSC-1 via lentivirus-mediated RNA interference revealed that the downregulation of BCSC-1 increased the invasive ability of MCF-7 cells. In summary, the results demonstrated that BCSC-1 is expressed at low levels in breast cancer tissues, and that it can suppress human breast cancer cell migration and invasion, potentially altering the expression of MMP7, MMP9, OPN, and the activity of the NF-κB pathway. Therefore, BCSC-1 may be useful as a biomarker for the treatment of breast cancer in the future.

Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer.

Hypoxic microenvironment in breast tumors has been associated with tumor aggressiveness, therapy resistance, and worse prognosis. HIF-1α (hypoxia inducible factor-1α) plays important roles in hypoxia by regulating a set of genes, such as VEGFA (vascular endothelial growth factor), which is related to angiogenesis. Studies have shown that DNA methylation acts on the gene control of HIF1A in hypoxia. However, little is known about the HIF1A transcriptional regulation in aggressive breast cancer MDA-MB-231 cell line. Therefore, the present study analyzed the effect of a demethylating agent on the transcription levels of HIF1A, and its target gene VEGFA, in the MDA-MB-231 cell line, in hypoxia. Herein, we demonstrated that 5′-aza-2′-deoxycitidine treatment in hypoxia reverted and raised HIF1A mRNA levels, but decreased VEGFA mRNA levels. Additionally, in both normoxia and hypoxia, HIF-1α protein levels increased in response to the demethylating treatment. The results of the current study indicated that HIF1A could be regulated by DNA methylation in normoxia and hypoxia. Furthermore, our results indicated a distinct regulation of VEGFA transcripts, independent of HIF-1α, in 5′-aza-dC-treated MDA-MB-231 cells in hypoxia.

Background The malignant potential of triple negative breast cancer (TNBC) is also dependent on a sub-population of cells with a stem-like phenotype. Among the cancer stem cell markers, CD133 and EpCAM strongly correlate with breast tumor aggressiveness, suggesting that simultaneous targeting of the two surface antigens may be beneficial in treatment of TNBC. Since in TNBC-derived cells we demonstrated that PLC-β2 induces the conversion of CD133high to CD133low cells, here we explored its possible role in down-modulating the expression of both CD133 and EpCAM and, ultimately, in reducing the number of TNBC cells with a stem-like phenotype. Methods A magnetic step-by-step cell isolation with antibodies directed against CD133 and/or EpCAM was performed on the TNBC-derived MDA-MB-231 cell line. In the same cell model, PLC-β2 was over-expressed or down-modulated and cell proliferation and invasion capability were evaluated by Real-time cell assays. The surface expression of CD133, EpCAM and CD44 in the different experimental conditions were measured by multi-color flow cytometry immunophenotyping. Results A CD133+/EpCAM+ sub-population with high proliferation rate and invasion capability is present in the MDA-MB-231 cell line. Over-expression of PLC-β2 in CD133+/EpCAM+ cells reduced the surface expression of both CD133 and EpCAM, as well as proliferation and invasion capability of this cellular subset. On the other hand, the up-modulation of PLC-β2 in the whole MDA-MB-231 cell population reduced the number of cells with a CD44+/CD133+/EpCAM+ stem-like phenotype. Conclusions Since selective targeting of the cells with the highest aggressive potential may have a great clinical importance for TNBC, the up-modulation of PLC-β2, reducing the number of cells with a stem-like phenotype, may be a promising goal for novel therapies aimed to prevent the progression of aggressive breast tumors. Electronic supplementary material The online version of this article (10.1186/s12885-017-3592-y) contains supplementary material, which is available to authorized users.

© 2017 The Royal Society of Chemistry. Tricyclic alkaloid-like compounds were synthesised in a few steps, via the bridging Ritter reaction. The compounds were evaluated for their antiproliferative activity against the MCF-7 and the aggressive MDA-MB-231 breast cancer cells. The anti-cancer activities of 2c were found to be selective towards the aggressive and more challenging to treat triple negative (MDA-MB-231) cell line while exhibiting no antiproliferative activities towards the MCF-7 cells at the highest concentration tested (50 μM). The IC50 of compound 2c was determined to be 7.9 μM for the MDA-MB-231 cell line. Furthermore, 2c arrested cell cycle at the G2/M phase and induced apoptosis in a dose-dependent manner. Besides in-house anti-cancer screening, compound 3 was selected for anti-cancer screening by the National Cancer Institute and was found to have broad anti-cancer activity with selectivity against particular leukaemia, colon, melanoma, and breast cancer cell lines. Cytotoxicities of compounds 2c and 3 were also tested against noncancerous mammalian cells (VERO cell line), and found to be selective towards cancerous cells. The facile synthetic route, unique chemical structures and the biological data make these alkaloid-like compounds worthwhile lead compounds for further anti-cancer drug development.

Highlights • Unguinol and Aspergillusidone D inhibit cell viability at low μM concentrations. • These depsidones increase the amount of apoptotic cells in the MDA-MB-231 cell line. • Unguinol causes cell cycle arrest of MDA-MB-231 cells in the G2/M-phase. • Apoptosis and cell cycle arrest were seen at clinically high concentrations (>60 μM)., There is an ongoing search for new compounds to lower the mortality and recurrence of breast cancer, especially triple-negative breast cancer. Naturally occurring depsidones, extracted from the fungus Aspergillus, are known for their wide range of biological activities such as cytotoxicity, aromatase inhibition, radical scavenging, and antioxidant properties. Research showed the potential of depsidones as a treatment option for hormone receptor-positive breast cancer treatment, yet its effects on hormone receptor-negative breast cancer are still unkown. This study, therefore, investigated the potential of two depsidones (Unguinol and Aspergillusidone D) to induce apoptosis, cell cycle arrest and cytotoxicity, and reduce cell proliferation in the triple-negative MDA-MB-231 breast cancer cell line. Results were compared with the effects of the cytostatic drug doxorubicin, antimitotic agent colchicine and endogenous hormones 17β-estradiol, testosterone and dihydrotestosterone. The cytostatic drugs and hormones affected the MDA-MB-231 cell line comparable to other studies, showing the usefulness of this model to study the effects of depsidones on a triple-negative breast cancer cell line. At sub μM levels, Unguinol and Aspergillusidone D did not influence cell proliferation, while cell viability was reduced at concentrations higher than 50 μM. Both depsidones induced apoptosis, albeit not statistically significantly. In addition, Unguinol induced cell cycle arrest in MDA-MB-231 cells at 100 μM. Our research shows the potential of two depsidones to reduce triple-negative breast cancer cell survival. Therefore, this group of compounds may be promising in the search for new cancer treatments, especially when looking at similar depsidones.

Cancer stem cells (CSC) contribute to the high incidence of metastatic recurrence in triple-negative breast cancer (TNBC) and elucidating the mechanisms controlling CSC properties should provide novel targets for therapeutic development that improves patient outcomes. The transcription factor BCL11A is highly differentially expressed in TNBC versus all other subtypes and controls breast CSC phenotypes such as marker expression and tumor initiation. However, the genes that BCL11A targets to control CSC biology and whether BCL11A promotes metastatic progression are unknown. To address these questions, we assessed the impact of transiently silencing the expression of BCL11A in TNBC cells using siRNA transfections. Reducing BCL11A expression did not impact TNBC cell viability or in vitro migratory capacity. However, silencing BCL11A significantly decreased invasion of TNBC cell lines, in vitro. Furthermore, stable shRNA-mediated silencing of BCL11A in the highly metastatic MDA-MB-231 cell line reduced metastatic outgrowth when xenografted into immunocompromised mice, suggesting that BCL11A is critical for metastatic progression of TNBC. To identify the BCL11A-regulated transcriptome in TNBC, we performed RNA-seq analysis of cells transiently transfected with non-targeting or BCL11A-targeted siRNAs in the MDA-MB-231 cell line. Consistent with the impact of BCL11A on invasion, numerous genes involved in adhesion and extracellular matrix were reduced with BCL11A silencing. The gene encoding the matrix metalloproteinase, MMP1, was the most significantly differentially expressed gene, with a 10-fold decrease in RNA expression following BCL11A suppression. We confirmed this down-regulation by both qRT-PCR and western blots of independent samples, as well as in the HCC1143 TNBC cell line. Current studies are assessing the functional impact of MMP1 regulation by BCL11A on invasion and metastatic progression of TNBC. Citation Format: Darcie D. Seachrist, Natasha N. Ingles, Molly M. Hannigan, Donny D. Licatalosi, Ruth A. Keri. BCL11A is necessary for the expression of extracellular matrix genes and metastatic progression of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 32.

Background and Objective: Beauvericin (BEA), a cyclic hexadepsipeptide mycotoxin, is a potent inhibitor of the acyl-CoA: cholesterol acyltransferase enzyme 1 (ACAT1), involved in multiple tumor-correlated pathways. However, the binding mechanisms between BEA and ACAT1 were not elucidated. Methods: BEA was purified from a mangrove entophytic Fusarium sp. KL11. Single-crystal X-ray diffraction was used to determine the structure of BEA. Wound healing assays of BEA against KB cell line and MDA-MB-231 cell line were evaluated. Inhibitory potency of BEA against ACAT1 was determined by ELISA assays. Molecular docking was carried out to illuminate the bonding mechanism between BEA and ACAT1. Results: The structure of BEA was confirmed by X-ray diffraction, indicating a monoclinic crystal system with P21 space group (α = 90°, β = 92.2216(9)°, γ= 90°). BEA displayed migration-inhibitory activities against KB cells and MDA-MB-231 cells in vitro. ELISA assays revealed that the protein expression level of ACAT1 in KB cells was significantly decreased after BEA treatment (P ＜0.05). Molecular docking demonstrated that BEA formed hydrogen bond with His425 and pi-pi staking with Tyr429 in ACAT1. Conclusions: BEA sufficiently inhibited the proliferation and migration of KB cells and MDA-MB-231 cells by downregulating ACAT1 expression. In addition, BEA potentially possessed a strong binding affinity with ACAT1. BEA may serve as a potential lead compound for the development of a new ACAT1-targeted anticancer drug.

Immune checkpoint inhibitors (ICI) targeting PD-1/PD-L1 have been approved for the treatment of a variety of cancers. However, the efficacy of antibody-based ICIs could be further improved by mitigating anti-drug antibodies, proteolytic cleavage, and on-target off-tumor toxicity. One strategy for accomplishing this is through the use of extracellular vesicles (EVs), cell derived submicron vesicles with many unique properties. We constructed an engineered MDA-MB-231 cell line for harvesting EVs. This was accomplished by overexpressing a high-affinity variant human PD-1 protein (havPD-1), while simultaneously knocking out intrinsic PD-L1 and beta-2 microglobulin. The engineered havPD-1 EVs reduced PD-L1 overexpressing cancer cell proliferation and induced cellular apoptosis. Moreover, the EVs were shown to efficiently block PD-L1 mediated T cell suppression. Meanwhile antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity were not observed. The havPD-1 EVs treatment resulted in robust anti-tumor activity in both preventative co-implantation and therapeutic xenograft tumor models reconstituted with human T cells. The efficacy of the havPD-1 EVs was shown to be comparable to clinical anti-PD1 monoclonal antibodies. Additionally, loading the havPD-1 EVs with a potent PARP inhibitor was shown to further augment treatment efficacy. In brief, the engineered universal EVs harboring havPD-1 proteins can be used for cancer concurrent immunotherapy and chemotherapy., Graphical abstract Universal extracellular vesicles as immune checkpoint inhibitors and chemotherapeutics nanocarriers for cancer combination therapy.Image 1, Highlights • It is the first attempt to develop extracellular vesicles (EV) as direct agents for immune checkpoint therapy. • The HLA-I knock-out EVs could be off-the-self universal donors for EV-based therapy. • The EV-based concurrent immunotherapy and chemotherapy can significantly improve treatment efficacy.

Breast cancers exhibit different response to drug treatment. In this work, we analyze and compare the effect of two anticancer drugs differing in their primary action, miltefosine and cisplatin (cis-Pt), on two different breast cancer (the low—(MCF-7) and high—(MDA-MB-231) metastatic) cell lines, and one normal epithelial (MCF-10A) breast cell lines. The effect of cip-Pt and miltefosine on the thermodynamic behavior of the cancer cell lines was analyzed by differential scanning calorimetry, the cell morphology and viability were determined by optical microscopy and MTT test. We revealed distinct effects of miltefosine and cis-Pt on the thermodynamic behavior and viability of the cancer and normal cells. Importantly, the normal MCF-10A cells were drastically affected by miltefosine, while not by cis-Pt. MDA-MB-231 cell line, on the other hand, is more susceptible to cis-Pt than MCF-7 cells, while both cancer cell lines are equally affected by miltefosine. The drug-associated alteration of the thermal unfolding of the cells constituents correlated with the changes in the cell viability. The altered thermodynamic behavior of the cancer cells upon the drug treatment strongly indicates altered conformations of the proteins in cancer cell membrane and cellular matrix, and the DNA-containing structures.

Indoleamine 2, 3-dioxygenase (IDO) catabolizes tryptophan, mediates immunomodulatory functions, and is released by stromal cells such as mesenchymal stem cells. The aims of this study were to investigate the effects of IDO silencing on immunosuppressive function of adipose-derived mesenchymal stem cells (ASCs), T cells phenotype, and the proliferation/migration of tumor cells. ASCs isolated from adipose tissues of healthy women were transfected with IDO-siRNA. Galectin-3, transforming growth factor-β1, hepatocyte growth factor, and interleukin-10 as immunomodulators were measured in ASCs using qRT-PCR. T cells phenotype, interferon-γ, and interleukin-17 expression were evaluated in peripheral blood lymphocytes (PBLs) cocultured with IDO silenced-ASCs by flow cytometry and qRT-PCR, respectively. Scratch assay was applied to assess the proliferation/migration of MDA-MB-231 cell line. Galectin-3 was upregulated (p ˂ 0.05) while hepatocyte growth factor was downregulated (p ˂ 0.05) in IDO-silenced ASCs compared to control groups. Regulatory T cells were inhibited in PBLs cocultured with IDO-silenced ASCs; also T helper2 was decreased in PBLs cocultured with IDO-silenced ASCs relative to the scramble group. IDO-silenced ASCs caused interferon-γ overexpression but interleukin-17 downregulation in PBLs. The proliferation/migration of MDA-MB-231 was suppressed after exposing to condition media of IDO-silenced ASCs compared with condition media of untransfected (p < 0.01) and scramble-transfected ASCs (p < 0.05). The results exhibited the weakened capacity of IDO-silenced ASCs for suppressing the immune cells and promoting the tumor cells' proliferation/migration. IDO suppression may be utilized as a strategy for cancer treatment. Simultaneous blocking of immunomodulators along with IDO inhibitors may show more effects on boosting the efficiency of immune-based cancer therapies.

Sex-determining region Y-box 3 (SOX3) protein, a SOX transcriptions factors group, has been identified as a key regulator in several diseases, including cancer. Downregulation of transcriptions factors in invasive ductal carcinoma (IDC) can interfere in neoplasia development, increasing its aggressiveness. We investigated SOX3 protein expression and its correlation with apoptosis in the MDA-MB-231 cell line, as SOX3 and Pro-Caspase-3 immunoexpression in paraffin-embedded invasive ductal carcinoma tissue samples from patients (n = 27). Breast cancer cell line MDA-MD-231 transfected with pEF1-SOX3 + and pEF1-Empty vector followed by cytotoxicity assay (MTT), Annexin-V FITC PI for apoptosis percentage assessment by flow cytometry, qPCR for apoptotic-related gene expression, immunofluorescence, and immunohistochemistry to SOX3 immunolocalization in culture cells, and paraffin-embedded invasive ductal carcinoma tissue samples.Apoptotic rate was higher in cells transfected with pEF1-SOX3 + (56%) than controls (10%). MDA-MB-231 transfected with pEF1-SOX3 + presented upregulation of pro-apoptotic mRNA from CASP3, CASP8, CASP9, and BAX genes, contrasting with downregulation antiapoptotic mRNA from BCL2, compared to non-transfected cells and cells transfected with pEF1-empty vector (p 0.005). SOX3 protein nuclear expression was detected in 14% (4/27 cases) of ductal carcinoma cases, and pro-Caspase-3 expression was positive in 50% of the cases.Data suggest that SOX3 transcription factor upregulates apoptosis in breast cancer cell line MDA-MB-231, and has a down nuclear expression in ductal carcinoma cases, and need to be investigated as a tumor suppressor protein, and its loss of expression and non-nuclear action turn the cells resistant to apoptosis. Further studies are necessary to understand how SOX3 protein regulates the promoter regions of genes involved in apoptosis.

DNAJC25 gene is a member of the HSP40 (DNAJ) family, and it was suggested as a tumor suppressor gene in hepatocellular carcinoma. The aim of this study was to analyze the expression, genetic/epigenetic alterations, and prognostic value of the DNAJC25 gene in breast cancer. DNAJC25 transcript levels are upregulated in BT-20 and ZR-75-1 cell lines and downregulated in MDA-MB-231 cell line compared to the non-tumorigenic mammary epithelial cell line (MCF 10A) (P< 0.05). According to UALCAN in-silico tool, clinical breast cancer samples show significantly reduced levels of DNAJC25 mRNA levels relative to the normal samples (P=1.47e-02). The Kaplan–Meier plotter tool shows that high DNAJC25 expression is favorable for post-progression survival in breast cancer (P=0.0035). Point mutations or copy number variations of DNAJC25 are uncommon in clinical breast cancer samples. Combined bisulfite restriction analysis (COBRA) results showed that DNAJC25 promoter is not methylated in breast cell lines. Promoter hypomethylation was also observed in normal and tumor clinical samples (Beta-value < 0.25). In conclusion, DNAJC25 is suggested as a tumor suppressor candidate having limited biomarker potential in breast cancer. Functional studies are essential to reveal its role in breast carcinogenesis.