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Tomasz Kostrzewa,1 Izabela Nowak,2 Agnieszka Feliczak-Guzik,2 Joanna Drzeżdżon,3 Dagmara Jacewicz,3 Magdalena Górska-Ponikowska,1,4,5 Alicja Kuban-Jankowska1 1Department of Medical Chemistry, Faculty of Medicine, Medical University of Gdansk, Gdansk, 80-211, Poland; 2Department of Applied Chemistry, Faculty of Chemistry, Adam Mickiewicz University, Poznań, 61-614, Poland; 3Department of Environmental Technology, Faculty of Chemistry, University of Gdansk, Gdansk, 80-308, Poland; 4IEMEST Istituto Euro-Mediterraneo di Scienza e Tecnologia, Palermo, 90127, Italy; 5Department of Biophysics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, 70174, GermanyCorrespondence: Tomasz Kostrzewa; Alicja Kuban-Jankowska, Department of Medical Chemistry, Faculty of Medicine, Medical University of Gdansk, Gdansk, 80-211, Poland, Tel +48 58 349 14 50, Fax +48 58 349 14 56, Email tomasz.kostrzewa@gumed.edu.pl; alicja.kuban-jankowska@gumed.edu.plIntroduction: Solid lipid nanoparticles (SLN) have been considered lately as promising drug delivery system in treatment of many human diseases including cancers. We previously studied potential drug compounds that were effective inhibitors of PTP1B phosphatase – possible target for breast cancer treatment. Based on our studies, two complexes were selected for encapsulation into the SLNs, the compound 1 ([VO(dipic)(dmbipy)] · 2 H2O) and compound 2 ([VOO(dipic)](2-phepyH) · H2O). Here, we investigate the effect of encapsulation of those compounds on cell cytotoxicity against MDA-MB-231 breast cancer cell line. The study also included the stability evaluation of the obtained nanocarriers with incorporated active substances and characterization of their lipid matrix. Moreover, the cell cytotoxicity studies against the MDA-MB-231 breast cancer cell line in comparison and in combination with vincristine have been performed. Wound healing assay was carried out to observe cell migration rate.Methods: The properties of the SLNs such as particle size, zeta potential (ZP), and polydispersity index (PDI) were investigated. The morphology of SLNs was observed by scanning electron microscopy (SEM), while the crystallinity of the lipid particles was analyzed by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The cell cytotoxicity of complexes and their encapsulated forms was carried out against MDA-MB-231 breast cancer cell line using standard MTT protocols. The wound healing assay was performed using live imaging microscopy.Results: SLNs with a mean size of 160 ± 25 nm, a ZP of − 34.00 ± 0.5, and a polydispersity index of 30 ± 5% were obtained. Encapsulated forms of compounds showed significantly higher cytotoxicity also in co-incubation with vincristine. Moreover, our research shows that the best compound was complex 2 encapsulated into lipid nanoparticles.Conclusion: We observed that encapsulation of studied complexes into SLNs increases their cell cytotoxicity against MDA-MB-231 cell line and enhanced the effect of vincristine.Keywords: solid lipid nanoparticles, stability of nanoparticles, oxovanadium(IV) and dioxovanadium(V) complexes, triple-negative MDA-MB-231 breast cancer cell line, live imaging microscopy

The miRNAs have been shown to be involved in breast cancer. The aim of the present research was to evaluate the impacts of extract from Euphorbia szovitsii FischC.A. Mey on the expression level of microRNAs in triple-negative breast cancer (MDA-MB-231) cell line.The alterations in the expression level of miRNAs in MDA-MB-231 cell line exposed to the extract of E. szovitsii were determined exploiting qRT-PCR technique. The expression of MDA-MB-231 cell microRNAs including miR-15, miR-16, miR-21, miR-29, miR-34a, miR-146b, miR-151, miR-155, miR-181b, miR-221, miR-222, and Let-7 was evaluated at 24 and 48 h after treatment with the E. szovitsii extract. The treatment of MDA-MB-231 cells with E. szovitsii caused a significant elevation in the expression of miR-155, miR-146b (P0.05), miR-16, miR-21, miR-151 (P0.01), and miR-34a (P 0.001) after 24 h, and also miR-155, Let-7 (P 0.05), miR-15, miR-29, miR-151 (P 0. 01), miR-146b and miR-34a (P0.001) after 48 h.The qRT-PCR findings at 24 and 48 h after treatment revealed that the MDA-MB-231 cell line in the presence of E. szovitsii extract showed an alteration in the expression profile of miRNAs implicated in the induction of cell proliferation, apoptosis and migration. These results may be helpful in determining the anticancer activity of E. szovitsii in MDA-MB-231 cell line.

In the current study, Mesoporous silica nanoparticles (MSNs) were provided and functionalized using zinc, amine, and graphene oxide (GO) (MZNG). Then, they were applied to deliver Doxorubicin, an anticancer drug, to breast cancer cells. The characterization findings indicated that MZNG loaded with DOX had a smooth surface and a spherical shape without homogeneous distribution with a particle size of around 215 nm. The high entrapment efficiency of DOX was observed for MZNGs at pH 7.4. Cytotoxicity results indicated that free DOX had high compatibility with HFF cells compared to DOX loaded into MZNG formulations, while DOX-loaded nanoparticles significantly increased the cytotoxicity against MDA-MB-231compared to free drugs and non-loaded nanoparticles. Moreover, DOX-loaded nanoparticles displayed increased apoptotic potential in MDA-MB-231 compared to free DOX and non-loaded nanoparticles (MZNGs). Upon treatment with samples, a downregulation of MMP-9 and Bcl-2 genes and an upregulation of Bax, Caspase 3, and Mir-193 genes were found. The prepared Nano-formulation holds great promise for treating breast cancer.

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.

© 2021, Institute of Chemistry, Slovak Academy of Sciences.In this study, new sulfonyl hydrazone derivatives were synthesized. New sulfonyl hydrazones and their Pd (II) complexes were purified and characterized. Theoretical calculations of the sulfonyl hydrazone materials were done with the B3LYP method in the Gaussian program with the 6–31 (d, p) and 6–311 (d, p) basis sets. The DTA/TG studies investigated at what steps the materials were degraded. Liver tissues were collected from one male and one female healthy albino rats. The effects of the substances on these tissues were examined, and cytotoxic activity results against human breast adenocarcinoma (MDA-MB-231) and human lung epithelial (Beas-2B) were obtained. In the final stage of this study, new sulfonyl hydrazone derivatives Palladium (II) complexes were carried out using LC/MS, 1H NMR, FT-IR, 13C NMR, Magnetic Susceptibility, UV–Vis, Conductivity Measurements. HOMO–LUMO and cytotoxic activities were determined against the human breast adenocarcinoma MDA-MB-231 cell line.

Introduction: Chelidonine, a bio-active component of Chelidonium majus, has been investigated for its anti-proliferative effects on various cancer cell lines with multidrug resistance (MDR). Although the results are auspicious, its poor water solubility and low bioavailability are the main limitations for clinical applications. This study aimed to develop poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with chelidonine, in order to enhance its bioavailability for oral administration and improve the therapeutic index. Methods: Herein, we encapsulated chelidonine in PLGA nanoparticles using a single emulsion solvent evaporation method. Nanoparticles were characterized in terms of size, surface charge and morphology, encapsulation efficiency, drug loading, and in vitro drug release profile. The anti-cancer efficacy of chelidonine-loaded nanoparticles and free chelidonine was evaluated in MDA-MB-231 breast cancer cells. Results: The physicochemical characteristics showed spherical particles in nanometer size range (263 ± 19.6 nm), with negative surface charge (−20.67 ± 2.48 mv), high encapsulation efficiency (76.53 ± 3.61%), and drug loading (22.47 ± 0.09%), as well as drug release amount of 60.27±5.68% up to 10 days. Furthermore, chelidonine-loaded nanoformulations were found to improve anti-cancer potential, compared with unentrapped chelidonine. Conclusion: This in vitro study showed that the encapsulation of chelidonine, as a potent herbal drug, in a polymeric matrix enhances its bioavailability. This offers an efficient vehicle for targeted drug delivery in cancer treatment.

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.

Background Cancer of the breast is known to be among the top spreading diseases on the globe. Triple-negative breast cancer is painstaking the most destructive type of mammary tumor because it spreads faster to other parts of the body, with high chances of early relapse and mortality. This research would aim at utilizing computational methods like quantitative structure–activity relationship (QSAR), performing molecular docking studies and again to further design new effective molecules using the QSAR model parameters and to analyze the pharmacokinetics “drug-likeliness” properties of the new compounds before they could proceed to pre-clinical trials. Results The QSAR model of the derivatives was highly robust as it also conforms to the least minimum requirement for QSAR model from the statistical assessments of (R2) = 0.6715, (R2adj) = 0.61920, (Q2) = 0.5460 and (R2pred) of 0.5304, and the model parameters (AATS6i and VR1_Dze) were used in designing new derivative compounds with higher potency. The molecular docking studies between the derivative compounds and Maternal Embryonic Leucine Zipper Kinase (MELK) protein target revealed that ligand 2, 9 and 17 had the highest binding affinities of − 9.3, − 9.3 and − 8.9 kcal/mol which was found to be higher than the standard drug adriamycin with − 7.8 kcal/mol. The pharmacokinetics analysis carried out on the newly designed compounds revealed that all the compounds passed the drug-likeness test and also the Lipinski rule of five. Conclusions The results obtained from the QSAR mathematical model of parthenolide derivatives were used in designing new derivatives compounds that were more effective and potent. The molecular docking result of parthenolide derivatives showed that compounds 2, 9 and 17 had higher docking scores than the standard drug adriamycin. The compounds would serve as the most promising inhibitors (MELK). Furthermore, the pharmacokinetics analysis carried out on the newly designed compounds revealed that all the compounds passed the drug-likeness test (ADME and other physicochemical properties) and they also adhered to the Lipinski rule of five. This gives a great breakthrough in medicine in finding the cure to triple-negative breast cancer (MBA-MD-231 cell line).

Breast cancer is divided into different subtypes based on molecular characteristics, among these subtypes, Triple-negative breast cancer, has the poorest prognosis and survival with invasive. In this study, TNBC cell line was used to explore crocin anti-metastatic effect on the Wnt/β-catenin pathway. Cell proliferation assessed by MTT assay and effects of crocin on migration monitored by transwell and wound healing experiments. Expression of certain epithelial-mesenchymal transition (EMT) markers genes was evaluated by real-time PCR. β-catenin expression also examined by real-time PCR. Findings revealed crocin significantly inhibits cell proliferation and migration of tumor cells in a dose-dependent manner. Moreover, crocin decreased the expression of Vimentin, Snail, Zeb-1 and β-catenin. Also, crocin increased the expression of E-cadherin in MDA-MB-231 cell line. Results showed an association between crocin and Wnt/β-catenin signaling pathway. In conclusion, this study establishes that crocin can be a promising therapeutic for triple-negative breast cancer.

Objective(s): Breast cancer is one of the most common types of cancer. Chemotherapeutic agents used during treatment induce cytotoxic effects also on normal cells in the tissues. Anti-oxidants used in combination with chemotherapeutic agents have been shown to reduce toxicity on normal cells to a minimum, and some anti-oxidant substances have chemotherapeutic effects. Cisplatin (CDDP) is a platinum class drug that is used clinically in the treatment of many cancers. Resveratrol (RSV) is a natural polyphenol with potent anti-oxidant and anticancer properties. In this study, we aimed to investigate apoptotic effects of using cisplatin and RSV alone or in combined treatment of MDA-MB-231 cells. Materials and Methods: The cytotoxic effects of the drugs on MDA-MB-231 cells were determined by MTT method. Subsequently, the change in CDDP-induced apoptotic effect after RSV addition was examined using the AnnexinV FITC labeling, and TUNEL staining method. Activation of caspase-9, -3 in MDA-MB-231 cells was measured by flow cytometer. The mitochondrial membrane potential (MMP), the major factor on the intrinsic pathway, was measured using flowcytometry. Results: The combined dose (23 μM CDDP + 72 μM RSV) produced more cytotoxicity than the agents used alone, leading to early apoptosis (8.2%), 31% depolarization, and 23% DNA fragmentation. Caspase-9 was found to be 30.5% in this combined group and caspase-3 was 26.3%. Conclusion: RSV, an effective anti-oxidant, and CDDP as an effective drug in cancer treatment, were found to increase apoptosis when given in the MDA-MB-231 cell.

The RAC2 gene encoding GTPases involve cellular signaling of actin polymerization, cell migration, and formation of the phagocytic NADPH oxidase complex. Oncogenic mutations in the RAC2 gene have been identified in various cancers, and extensive research is in progress to delineate its signaling pathways and identify potential therapeutic targets in breast cancers. This paper explored developing a bioinformatics model system to understand the RAC2 gene expression pattern concerning estrogenic receptor status in breast cancers. We have used the MDA-MB-231 breast cancer cell line to identify RAC2 gene expression. To simplify the development of model system with one dataset, we retrieved the microarray dataset GSE27515 from the Gene Expression Omnibus (GEO) for the differential gene expression analysis. Then, network analysis, pathway enrichment analysis, volcano plot, ORA, and the up/downregulated genes were used to highlight genes involved in signaling network pathways. We observed that the RAC2 gene is upregulated in the GSM679722, GSM676923, and GSM679724 downregulated in the samples GSM676925, GSM676926, and GSM676927 from the GEO dataset. Our observation found that the RAC2 gene is upregulated in the estrogen receptor (ER) negative breast cancers and downregulated in ER-positive breast cancer, involving pathways such as focal adhesion, MAPK signaling, axon guidance, and VEGF signaling pathway.

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.

Self-assembled nanocarriers (SANs) as a novel colloidal controlled delivery for docetaxel trihydrate (DTX) were engineered by high-pressure homogenization method to overcome the several clinical problems. Drug-excipient compatibility was studied using DSC and FTIR spectroscopy. The fabricated SANs was characterized by particle size, zeta potential, and SEM. QbD based central composite design of experiment was employed for formula optimization. The cell viability of DTX-hydroalcoholic solution (DTX-HA) and DTX-loaded SANs has been determined in MDA-MB-231 cell line by MTT assay. The stability study of selected SANs formulations were carried out at various storage conditions as per ICH guidelines. The summary of results obtained shows high drug content with higher entrapment efficiency (91.23 ± 3.41% w/w) of DTX-loaded SANs. It shows diffusion controlled release of DTX over the period of 12 h which is higher than DTX-HA solution, releases the DTX within 4 h. The MTT assay expressed lower cellular viability and improved cell inhibition leads to increase cytotoxicity of formulations towards cells. The stability study reveals stability of DTX-loaded SANs formulations at various storage conditions over a period of three months. The strong experimental evidence confirms the SANs as an effective approach to formulate the controlled delivery system of antineoplastics with improved stability.

Breast cancer has been represented a challenging issue worldwide as it is one of the major leading causes of death among women. CD81 gene, a member of the tetraspanin protein family, has been associated with the development of human cancers. Genome editing technologies, particularly the CRISPR-Cas9 system, have shown rapid progress in gene function studies. In this study, we aimed to evaluate the ability of the CRISPR-Cas9 plasmid-based system to modify specific regions of the CD81 gene in the MDA-MB-231 breast cancer cell line.Using bioinformatics database search, four different single guide RNAs (sgRNAs) to target exon 3 and exon 5 of the CD81 gene were designed. The intended sgRNAs sequences were cloned into the expression plasmid pSpCas9(BB)-2A-GFP (PX458) bearing sgRNA scaffold backbone, Cas9, and EGFP coding sequences, which was confirmed by colony PCR and sequencing. Transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. Gene editing efficiency was measured qualitatively and quantitatively using the T7E1 and TIDE software, respectively.Our data show that expression constructs were successfully introduced into MDA-MB-231 cells with an acceptable transfection efficiency. Two sgRNAs that were afforded to introduce significant mutations in their target regions were detected by TIDE software (p-value0.05). To the best of our knowledge, CD81 gene editing in these cells has been investigated for the first time in this study using the CRISPR/Cas9 technique.Taken together, our data show that the CRISPR-Cas9 system can change the genomic sequence in the target area of MDA-MB-231 cells. Along with previous studies, we propose forethought when using T7E1-based quantitative indel estimates, as comparing activities of multiple gRNAs with the T7E1 assay may lead to inaccurate conclusions. Instead, estimating non-homologous end-joining events (NHEJ) by Sanger sequencing and subsequent TIDE analysis is recommended.

BackgroundThe humanSH3 domain Binding Glutamic acid Rich Like 3(SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues. However, its function is largely undetermined. The protein was found to be overexpressed in several tumors, and recent work suggested a possible relationship with EGFR family members.We aimed at further highlighting on these issues and investigated SH3BGRL3 molecular interactions and its role in cellular migration ability.ResultsWe first engineered the ErbB2-overexpressing SKBR3 cells to express exogenous SH3BGRL3, as well as wild type Myo1c or different deletion mutants. Confocal microscopy analysis indicated that SH3BGRL3 co-localized with Myo1c and ErbB2 at plasma membranes. However, co-immunoprecipitation assays and mass spectrometry demonstrated that SH3BGRL3 did not directly bind ErbB2, but specifically recognized Myo1c, on its IQ-bearing neck region. Importantly, the interaction with Myo1c was Ca2+-dependent.A role for SH3BGRL3 in cell migration was also assessed, as RNA interference of SH3BGRL3 in MDA-MB-231 cells, used as a classical migration model, remarkably impaired the migration ability of these cells. On the other side, its over-expression increased cell motility.ConclusionThe results of this study provide insights for the formulation of novel hypotheses on the putative role of SH3BGRL3 protein in the regulation of myosin-cytoskeleton dialog and in cell migration. It could be envisaged the SH3BGRL3-Myo1c interaction as a regulation mechanism for cytoskeleton dynamics. It is well known that, at low Ca2+concentrations, the IQ domains of Myo1c are bound by calmodulin. Here we found that binding of Myo1c to SH3BGRL3 requires instead the presence of Ca2+. Thus, it could be hypothesized that Myo1c conformation may be modulated by Ca2+-driven mechanisms that involve alternative binding by calmodulin or SH3BGRL3, for the regulation of cytoskeletal activity.

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.

Objective (s): Gravity could affect some system features and perform directly as an organizing field factor. Recent investigations have examined the titanium dioxide nanoparticles (TiO2 NPs) in biomedical applications, mostly in the cancer treatment field. This study aimed to evaluate the effects of simulated microgravity combined with TiO2 NPs in MDA-MB-231 cells proliferation for the first time. In other words, this study examined the utility of the microgravity environment in nano-therapy. Materials and Methods: The MDA-MB-231 human breast cancer cell line and TiO2 NPs were purchased. The 2D clinostat was applied for the simulation of the microgravity. The morphological studies, MTT cytotoxicity assay, Acridine orange/Ethidium bromide double staining studies and flow cytometry analysis were utilized.Results: The MTT assay, the morphological studies, Acridine orange/Ethidium bromide double staining studies and flow cytometry analysis confirmed the apoptosis-inducing effect of microgravity in combination with TiO2 NPs. The IC50 of simulated microgravity in the presence of TiO2 NPs was determined to be 130 µM. Furthermore, MDA-MB-231 cells exposed to microgravity adopted a different phenotype. Conclusion: Based on our observation, although the relative mechanisms need to be explored further, microgravity can strictly affect the TiO2 NPs effects on MDA-MB-231 cells. The significance of this study lied in the fact that simulating microgravity can be a powerful physical cure for cancer therapy and open new horizons for the studies in the field of biology, biophysics, and medicine.

Triple negative breast cancer is one of the most aggressive breast cancer type with abilities of early metastasis and chemoresistance. The tropical plant Citrus hystrix DC. has been reported to promote many biological activities including anticancer. However, the effect of C. hystrix against triple negative breast cancer has not yet been identified. This study aimed to evaluate the anticancer properties of C. hystrix leaf extract and its bioactive constituents citronellol and citronellal against the triple negative breast cancer MDA-MB-231 cell line. C. hystrix leaves were powdered and sequentially macerated. The in vitro anticancer effects of C. hystrix leaf extracts, and its bioactive constituents (citronellol and citronellal) were evaluated against MDA-MB-231 cell line using cytotoxic MTT assay, cell proliferation, wound scratch migration, colony formation, cell cycle, apoptosis assay, Hoechst staining, RT-qPCR, and Western blot analysis. Results showed that crude hexane extract, citronellol, and citronellal significantly reduced cell proliferation, colony formation, and cell migration by inducing cell cycle arrest, while also inducing apoptosis in MDA-MB-231 cells through inhibition of anti-apoptotic Bcl-2 expression, leading to activation of the caspase-3-dependent pathway. This study is the first report to demonstrate the effect of C. hystrix, citronellol, and citronellal against triple negative breast cancer MDA-MB-231 cells.

Background: Exosomes are membrane nanovesicles, 30 to 100 nm in diameter, secreted by most cell types. Besides playing many biological roles, especially in cell-cell communication, scientific proof indicated that the pathology of so many human cancers is closely related to many biologic elements in exosomes. They may serve as useful biomarkers for treatment, prognosis, and detection. Cancer cells produce more exosomes, inducing changes in target cells (near or distant from the tumor), such as metastasis and chemotherapy resistance. Therefore, isolation, identification, and analysis of these microvesicles seem essential. Objectives: The current study aimed at collecting and purifying microvesicles secreted from breast cancer cells and confirming the identity of the obtained exosomes using methods assessing size and morphology. Methods: In recent research, the MDA-MB-231 cell line was grown under standard conditions. Released exosomes were collected and ultra-centrifuged. Scanning (SEM) and transmission (TEM) electron microscopes, atomic force microscopy (AFM), and dynamic light scattering (DLS) were used to assess exosome size. Results: The obtained data revealed that MDA-MB-231 cells produced exosomes. The nanovesicles were isolated from the culture medium of MDA-MB-231 cells by applying different strategies, including differential centrifugation, filtration, and ultracentrifugation. The exosomes were characterized; they had a size of 30 - 100 nm and spherical shape. Conclusions: Intercellular communication can be mediated through direct cell-cell contact or transfer of secreted molecules. In the last two decades, a third mechanism for intercellular communication has emerged that involves intercellular transfer of extracellular vesicles (exosomes). Due to their many functions in the body, it is of great importance to purely isolate and recognize exosomes to understand their modes of action as the first step in the advancement of researches. However, more research is required to obtain cost-effective and efficient methods. It was found that MDA-MB-231 cells release exosomes. They are spherical and 30-100 nm in diameter. The use of a combination strategy for the first time was useful in isolating exosomes derived from MDA-MB-231 cells without disturbing their structure. Further studies are required to compile a uniform protocol for exosome isolation in medical research.

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.

Additional file 1. Co-immunoprecipitation with an anti-FLAG-coupled resin from lysates of SKBR3 cells transfected either with FLAG-SH3BGRL3 or with the empty vector. Three bands indicated with an arrow were cut and further subjected to proteomic analysis. Results are reported in Additional File 2.

Moringa oleifera Lam. (MO) is a medicinal plant distributed across the Middle East, Asia, and Africa. MO has been used in the traditional treatment of various diseases including cancer. This study aimed to perform bioassay-guided fractionation and identification of bioactive compounds from MO leaf against MDA-MB-231 breast cancer cells. MO leaf was sequentially extracted with hexane, ethyl acetate (EtOAc), and ethanol. The most effective extract was subjected to fractionation. MO extract and its derived fractions were continuously screened for anti-cancer activities. The strongest fraction was selected for re-fractionation and identification of bioactive compounds using LC-ESI-QTOF-MS/MS analysis. The best anticancer activities were related to the fraction no. 7-derived crude EtOAc extract. This fraction significantly reduced cell viability and clonogenic growth and increased cells apoptosis. Moreover, sub-fraction no. 7.7-derived fraction no. 7 was selected for the identification of bioactive compounds. There were 10 candidate compounds tentatively identified by LC-ESI-QTOF-MS. Three of identified compounds (7-octenoic acid, oleamide, and 1-phenyl-2-pentanol) showed anticancer activities by inducing cell cycle arrest and triggering apoptosis through suppressed Bcl-2 expression which subsequently promotes activation of caspase 3, indicators for the apoptosis pathway. This study identified 10 candidate compounds that may have potential in the field of anticancer substances.

Cell migration is a fundamental cellular process that plays a crucial role in many physioglogical and pathological processes such as wound healing or cancer metastasis. Many assays have been developed to examine cell migration, such as the wound healing or scratch assay, Boyden Chamber or transwell assay, and the method we will describe here, single cell migration assay. In this assay, cells are plated sparsely on a collagen coated plate and live cell imaging is performed over a period of 2 h at 1 frame per minute. After imaging is completed, cells are tracked manually using ImageJ by tracking movement of the centroid of the cell. These data points are then exported and overall distance travelled from frame to frame is determined and divided by total time imaged to determine speed of the cell. This method provides a quick way to examine effect of cellular manipulation on cell migration before proceeding to perform more complex assays.

In our effort to develop novel and powerful agents with anti-proliferative activity, two new series of 1H-benzo[f]chromene derivatives, 4a–h and 6a–h, were synthesised using heterocyclocondensation methodologies under microwave irradiation condition. The structures of the target compounds were established on the basis of their spectral data, IR, 1H NMR, 13 C NMR, 13 C NMR-DEPT/APT, and MS data. The new compounds have been examined for their anti-proliferative activity against three cancer cell lines, MCF-7, HCT-116, and HepG-2. Vinblastine and Doxorubicin have been used as positive controls in the viability assay. The obtained results confirmed that most of the tested molecules revealed strong and selective cytotoxic activity against the three cancer cell lines. Moreover, these molecules exhibited weak cytotoxicity on the HFL-1 line, which suggested that they might be ideal anticancer candidates. The SAR study of the new benzochromene compounds verified that the substituents on the phenyl ring of 1H-benzo[f]chromene nucleus, accompanied with the presence of bromine atom or methoxy group at the 8-position, increases the ability of these molecules against the different cell lines. Due to their high anti-proliferative activity, compounds 4c and 6e were selected to be examined their proficiency to inhibit the invasiveness of the highly sensitive and invasive breast cancer cell line, MDA-MB-231. The anti-invasion behaviour of these molecules against the highly sensitive, non-oestrogen, and progesterone MDA-MB-231 cell line gave rise to their decreasing metastatic effect compared to the reference drug. Furthermore, this report explores the apoptotic mechanistic pathway of the cytotoxicity of the target compounds and reveals that most of these compounds enhance the Caspase 3/7 activity that could be considered as potential anticancer agents.

Some medicinal herbs and compounds are known to target cancer cells, but the success of them as anticancer compounds depends to a large extent on their ability to activate pathways that kill cancer cells by arresting cell cycle and inducing apoptosis. The aim of the present study was to determine the anticancer effects of Euphorbia szovitsii Fisch. & C.A.Mey. on the breast cancer cells to reveal the underlying mechanism of its anti-breast cancer properties. In this experimental study, triple negative breast cancer cell line (MDA-MB-231) was cultivated in RPMI-1640 medium. Hydroalcoholic extract (70:30) of aerial parts of the plant was prepared. The cultured cells were treated with different concentrations (0–1000 μg/ml) of E. szovitsii extract for 24 and 48 h. Toxicity of the extract on MDA-MB-231 cells was examined using MTT (3-[4,5-dimethyl-2-thiazolyl]-2, 5 diphenyl tetrazolium bromide) test. The Annexin V–FITC Apoptosis Detection Kit was used to evaluate apoptosis and necrosis. Flow cytometry technique was employed to differentiate different phases of the cell cycle in the cells. Data were analyzed by GraphPad Prism and SPSS software. After 24 and 48 h, the IC50 values were respectively 76.78 (95% CI = 60.75–97.05; R = 0.8588) and 59.71 (95% CI = 46.25–77.09; R = 0.8543) μg/ml for E. szovitsii. The extract exhibited antiproliferative effects against MDA-MB-231 cells in a dose-dependent manner. Annexin V-FITC/PI assay confirmed that the extract was able to induce apoptosis in MDA-MB-231 cells. Moreover, treatment with the extract resulted in cell cycle arrest at G1 phase. Therefore, E. szovitsii could induce apoptosis and cycle arrest in the MDA-MB-231 cell line. It might be a good resource of natural products for producing anti-breast cancer drugs.

We have previously reported the anti-metastatic effects of 2-methoxy-1,4-naphthoquinone (MNQ) against MDA-MB-231 cell line.To investigate the molecular mechanism underlying the anti-metastatic effects of MNQ towards MDA-MB-231 cell line via the comparative proteomic approach.Differentially expressed proteins in MNQ-treated MDA-MB-231 cells were identified by using two-dimensional gel electrophoresis coupled with tandem mass spectrometry. Proteins and signalling pathways associated with the identified MNQ-altered proteins were studied by using Western blotting.Significant modulation of MDA-MB-231 cell proteome was observed upon treatment with MNQ in which the expressions of 19 proteins were found to be downregulated whereas another eight were upregulated (1.5 fold, p0.05). The altered proteins were mainly related to cytoskeletal functions and regulations, mRNA processing, protein modifications and oxidative stress response. Notably, two of the downregulated proteins, protein S100-A4 (S100A4) and laminin-binding protein (RPSA) are known to play key roles in driving metastasis and were verified using Western blotting. Further investigation using Western blotting also revealed that MNQ decreased the activations of pro-metastatic ERK1/2 and NF-κB signalling pathways. Moreover, MNQ was shown to stimulate the expression of the metastatic suppressor, E-cadherin.This study reports a proposed mechanism by which MNQ exerts its anti-metastatic effects against MDA-MB-231 cell line. The findings from this study offer new insights on the potential of MNQ to be developed as a novel anti-metastatic agent.

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.

Protein translocation between organelles in the cell is an important process that regulates many cellular functions. However, organelles can rarely be isolated to purity so several methods have been developed to analyse the fractions obtained by density gradient centrifugation. We present an analysis of the distribution of proteins amongst organelles in the human breast cell line, MDA-MB-231 using two approaches: an isotopic labelling and a label-free approach.

The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all-trans and 9-cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e.g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial-mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1.

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

Interaction between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) plays an important role in the progression of numerous cancer types including breast cancer by promoting tumor initiating, proliferation, invasion and metastasis. Hence, disruption of this interaction inhibits their downstream cascades and subsequently tumor growth. For this, we created two series of 8 and 10 amino acids linear peptides, derived from uPA binding region to target uPAR and studied the inhibition of proliferation in MDA-MB-231 cell line. Results revealed that all of the 10-mer peptides inhibited breast cancer cell proliferation significantly with maximum 40% inhibition of 103 peptides. Meanwhile, none of the 8-mer peptides showed significant toxicity. Current results indicate that the linear 10-mer peptides which mimic a small part of a sequence of a binding domain of uPA to uPAR could be exploited to design a novel class of anti-cancer agents.

Interaction between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) plays an important role in the progression of numerous cancer types including breast cancer by promoting tumor initiating, proliferation, invasion and metastasis. Hence, disruption of this interaction inhibits their downstream cascades and subsequently tumor growth. For this, we created two series of 8 and 10 amino acids linear peptides, derived from uPA binding region to target uPAR and studied the inhibition of proliferation in MDA-MB-231 cell line. Results revealed that all of the 10-mer peptides inhibited breast cancer cell proliferation significantly with maximum 40% inhibition of 103 peptides. Meanwhile, none of the 8-mer peptides showed significant toxicity. Current results indicate that the linear 10-mer peptides which mimic a small part of a sequence of a binding domain of uPA to uPAR could be exploited to design a novel class of anti-cancer agents.

27-O-(E)-p-coumaric acyl ursolic acid( DY-17) from Ilex latifolia is a compound of the monomer. To investigate the DY-17 inducing apoptosis in the human breast cancer cell line, the MDA-MB-231 cells were used as research object in this experiment. The proliferation activity of the MDA-MB-231 cells stimulated with the different concentrations of DY-17 (20, 40 µmol · L(-1)) was detected at different time( 12, 24, 36, 48, 60,72 h) . We surveyed the DY-17 inducing apoptosis of the MDA-MB-231 cells with the fluorescent staining technology. The rate of MDA-MB-231 cells apoptosis and necrosis was determined by flow cell cytometry (FCC). Moreover, expression of JNK, phosphorylated JNK, Bax, PARP shear and caspase-3 shear related to JNK/SAPK pathways were investigated in every group ( control group, EGF group, EGF + DY-17 40 µmol · L(1) group and EGF + SP600125 group) with Western blot. The MTT results showed that, in the presence of DY-17, the proliferation activity of MDA-MB-231 cells decreased in a dose-dependent and time-dependent manner. The apoptosis and necrosis rates of MDA-MB-231 cells with DY-17(20, 40 µmol · L(-1)) groups was respectively 31.86%, 49.91% by flow cytometry and significantly increased compared with control group under Fluores- cence microscopy. Up-regulation of the JNK phosphorylation protein expression was observed in EGF group compared with control group. In addition, markedly decreased the expression of JNK phosphorylation protein were also surveyed in EGF + DY-17 40 µmol · L(-1) group compared with EGF group. The expression of Bax, shear PARP and shear caspase-3 protein in EGF + DY-17 40 µmol · L(-1) group were significantly increased in comparison with EGF group. The results showed DY-17 induced apoptosis of human breast cancer MDA-MB-231 cell line related to down-regulating JNK/SAPK signal pathways.

Ethopharmacological relevance Uncaria tomentosa (Willd.) DC. (Rubiaceae) (Ut), also known as cat's claw, is a woody liana widely spread throughout the Amazon rainforest of Central and South America, containing many chemical constituents such as oxindole alkaloids, which are responsible for various biological activities. Since ancient times, the indigenous people of Peru have used it as a bark infusion for the treatment of a wide range of health problems gastric ulcers, arthritis and rheumatism. Recently, Ut is distributed worldwide and used as an immunomodulatory and anti-inflammatory herbal remedy. Additionally, U. tomentosa also has antitumural activity. However, little is known about the action of U. tomentosa on the purinergic system mechanisms, which is involved in tumor progression. Aim of the study Considering the pharmacological properties of U. tomentosa, we sought to evaluate the hydroalcoholic extract U tomentosa is able to influence the purinergic system in breast cancer cells, MDA-MB-231. Through the activity and expression of ectonucleotidases (NTPDase – CD39; Ecto-5′-nucleotidase – CD73) and purinergic repceptores (P2X7 and A1). Materials and methods A hydroalcoholic extract was prepared in two concentrations, 250 and 500 μg/mL. (Ut250; Ut500). The effect of these concentrations on the activity and expression of ectonucleotidases, as well as on the density of purinergic receptors were investigated in MDA-MB-231 breast cancer cells. Cells were treated with the hydroalcoholic extract of Uncaria tomentosa and/or doxorubicin (Doxo 1 μM; Ut250+Doxo; Ut500+Doxo) for 24 h. Results Although the results were not significant for the hydrolysis of the ATP, they presented an increase in the ADP hydrolysis in the Ut500+Doxo group when compared to the control group. Additionally, the activity of 5′-nucleotidase was inhibited in all groups when compared with the untreated group of cells. Inhibition of the enzyme was more evident in groups with U. tomentosa per se. The expression of CD39 was increased in the Ut250 and Ut250+Doxo groups when compared to the control group. No changes were found in the CD73 expression. Furthermore, a reduction in the density of the P2X7 receptor in all treated groups was detected. On the other hand, the density of the A1 receptor increased in all groups compared to the control group, with the exception of the Ut500+Doxo group. Conclusion Therefore, we conclude that hydroalcoholic extract of U. tomentosa may be responsible for the reduction of adenosine levels in the extracellular medium, which accelerates tumor progression. Interestingly, the dysregulation of A1 and P2X7 receptors in the MDA-MB-231 cells exacerbate the proliferation of this cells and U. tomentosa treatment may be stimulate the antitumor activity of adenosine A1 receptor and control the P2X7 effects. Our study demonstrates the significant participation of purinergic pathway in the regulation of MDA-MB-231 progression; additionally, U. tomentosa treatment alone or combined with chemotherapy may favor the action of doxorubicin.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Title, Name, Surname of candidate: Veronika Tůmová Title, Name, Surname of tutor: PharmDr. Hana Bártíková, Ph.D Title of a diploma work: The influence of selected sesquiterpenes on the effect of cytostatics for the cell line MDA-MB-231 Breast cancer is one of the most common forms of cancer. It makes up approximately one quarter of cancer cases amongst women. The rise in the incidence of this disease in western countries has, over recent years, been linked to a lifestyle that characteristically involves a sedentary way of life and a diet rich in animal fats. There are currently many therapeutic regimes available for the treatment of breast cancer which are increasingly adapted to the individual needs of the patient and their specific tumor subtype. In spite of the wide range of treatments with a variety of mechanisms of effects on cancer tissues, current treatments are limited by inadequate effectiveness, serious undesirable side effects or the occurrence of resistance. Scientists have therefore been attempting to uncover ways to prevent these undesired effects. Sesquiterpenes α humulene and trans-nerolidol are naturally-occuring substances that, through their biochemical effects, act cytotoxically...

Radix Stephaniae tetrandrae, dry roots of Stephaniae tetrandrine S. Moore (Menispermaceae), is officially and traditionally used as an analgesic and anti-hypertension drug in China. The main chemical constituents in radix Stephania tetrandrae are tetrandrine (Tet) and fangchinoline (Fan) [1]. Tet has an antiadherent effect that might result from its inhibition of Ca 2+ influx and reactive oxygen species formation, resulting in suppression of up-regulation of Mac-1 and, in turn, neutrophil adhesion to fibrinogen [2]. In the past several years, important progress had been made in the study of the mechanisms of effect of Fan on some diseases. Lin Ty et al. reported that Fan inhibited glutamate release from rat cerebral cortex nerve terminals and had been shown to possess neuroprotective properties [3]. Fan has anti- inflammatory effects [4] and antioxidant activity [5]. Most recently, Zhang YH et al. reported that Fan inhibits proliferation and cells cycle progression of aortic vascular smooth muscle cell through inhibition of ERK1/2 activation and c-fos expression [6]. However, the anti-tumor activity of Fan in breast cancer is still unknown. Objective: This study was purposed to investigate the effect of Fangchinoline (Fan) on the regulation of tumor growth and apoptosis in vivo. Methods: Immunohistochemistry (IHC) was used to determine the protein levels of p27, CD117 and ki67 in tumor tissue of xenograft. After treatment of Fan, the morphologic changes were observed by DAPI staining, Hoechst staining and TUNEL detection under fluorescent microscopy. The protein expression of Bax, Bcl-2 active Caspase-3 and cytochrome-c were measured by Western blotting.

Lactobacilli are probiotics shown to have antitumor activities. In addition, they can regulate gene expression through epigenetic mechanisms. In this study, we aimed to assess anti tumor activities of Lactobacillus acidophilus and Lactobacillus crispatus on the MDA-MB-231 breast cancer cell line. The effects of culture supernatants were determined by MTT [3-(4,5-dimethylthiazol-2-y-2,5-diphenyltetrazolium bromide] assay. Changes in expression of 5 cancer-testis antigens (CTAs), namely AKAP4, ODF4, PIWIL2, RHOXF2 and TSGA10 ,were analyzed by quantitative real time RT-PCR. The culture supernatants of the 2 lactobacilli inhibited MDA-MB-231 cell proliferation. In addition, transcriptional activity of all mentioned CTAs except AKAP4 was significantly decreased after 24 hour treatment with culture supernatants. This study shows that Lactobacillus acidophilus and Lactobacillus crispatus have antiproliferative activity against MDA-MB-231 cells. In addition, these lactobacilli could decrease transcriptional activity of 4 CTAs. Previous studies have shown that expression of CTAs is epigenetically regulated, so it is possible that lactobacilli cause this expression downregulation through epigenetic mechanisms. As expression of CTAs in cancers is usually associated with higher grades and poor prognosis, downregulation of their expression by lactobacilli may have clinical implications.

MDA-MB-231 (MDA-231) human breast cancer cells have a high proliferation rate, lack the estrogen receptor, express the intermediate filament vimentin, the hyaluronan receptor CD44, and are able to form tumors in nude mice. The MDA-231 cell line has been used in our laboratory to examine the role of the peripheral-type benzodiazepine receptor (PBR) in the progression of cancer. During these studies 2 populations of MDA-231 cells were subcloned based on the levels of PBR. The subclones proliferated at approximately the same rate, lacked the estrogen receptor, expressed vimentin and CD44, and had the same in vitro chemoinvasive and chemotactic potential. Both restriction fragment length polymorphism and comparative genomic hybridization analyses of genomic DNA from these cells indicated that both subclones are of the same genetic lineage. Only the subclone with high PBR levels, however, was able to form tumors when injected in SCID mice. These data suggest that the ability of MDA-231 cells to form tumors in vivo may depend on the amount of PBR present in the cells.

This study investigated the anticancer effect of zerumbone (ZER) and zerumbone-loaded nanostructured lipid carrier (ZER-NLC) on the human mammary gland adenocarcinoma (MDA-MB-231) cell line. The effect of ZER and ZER-NLC on MDA-MB-231 cells was determined via electron and fluorescent microscopy and flow cytometry using the Annexin V, cell cycle, and Tdt-mediated dUTP nick-end labeling assays. We demonstrated that ZER and ZER-NLC significantly suppressed the proliferation of MDA-MB-231 cells with an IC50of 5.96 ± 0.13 and 6.01 ± 0.11 μg/mL, respectively. ZER and ZER-NLC arrested MDA-MB-231 cell cycle at the G2/M phase. The induction of apoptosis by ZER and ZER-NLC was via the intrinsic pathway through the release of cytochrome c and activation of caspase-3 and caspase-9. The treatments also caused the downregulation of antiapoptotic Bcl-2, Bcl-xL proteins, and proliferating cell nuclear protein and upregulation of proapoptotic Bax protein. Therefore, loading of ZER into NLC did not compromise the anticancer effects of ZER on MDA-MB-231 cells. In conclusion, ZER-NLC, which increased the bioavailability of ZER, is an effective agent in the treatment of cancers.

The aim of this study was to establish a bioluminescent MDA-MB-231 cell line stably expressing luciferase and green fluorescent protein for the generation of a xenografted model of human triple-negative breast cancer (TNBC) in nude mice. Lentivirus vectors carrying eGFP, firefly luc2 and neo fusion genes were used to transduce the MDA-MB-231 human TNBC cells in vitro. After 8 weeks of G418 selection, eGFP and luc2 expression was determined using a fluorescence microscope and a Xenogen IVIS200 bioluminescent imaging system, respectively. The MTT, transwell invasion and wound healing assays were performed to confirm whether cellular proliferation, invasion and migration were altered by lentiviral infection. Cells were orthotopically implanted into female BALB/c nude mice to test the sensitivity and stability of reporter gene expression. Growth of the tumors was monitored with the in vivo imaging system once a week until they were large enough for experiments. The tumor tissues were resected for histology, and cancer cells were harvested for culture. The lentivirus-transduced MDA-MB-231 cells could stably express luc2 and eGFP, and the luciferase activity reached 9689 photons/sec/cell. Meanwhile, no significant difference in biological activities was observed between the lentivirus-transduced MDA-MB-231 cells and parental cells. An orthotopically implanted tumor model of human TNBCs was successfully established in BALB/c nude mice. Lentiviruses may be ideal carriers for luciferase genes due to their highly efficient infectivity and stable transgene expression. The modified MDA-MB-231 cell line stably expressing luciferase could be detected, allowing for immediate and sensitive detection of metastasis sites in nude mice. As the eGFP and luc2 combination are superior to single reporter genes in their ability to mark cells in vivo and in vitro, these cells may provide a visualizable, convenient and sensitive platform for research on the mechanisms of metastasis and the development of new antitumor drugs for human TNBC.