MDA-MB231 Breast Cancer Cells Research Articles

Antitumoral Potential of Lansbermin-I, a Novel Disintegrin from Porthidium lansbergii lansbergii Venom on Breast Cancer Cells.

Disintegrins from snake venoms bind with high specificity cell surface integrins, which are important pharmacological targets associated with cancer development and progression. In this study, we isolated a disintegrin from the Porthidium lansbergii lansbergii venom and evaluated its antitumoral effects on breast cancer cells. The isolation of the disintegrin was performed on RP-HPLC and the inhibition of platelet aggregation was evaluated on human platelet-rich plasma. The inhibition of cell adhesion was also evaluated in vitro on cultures of cell lines by the MTT method as well as the inhibition of breast cancer cell migration by the wound healing assay. The binding of the disintegrin to integrin subunits was verified by flow cytometry and confocal microscopy. Finally, inhibition of angiogenesis was assessed in vitro on HUVEC cells and the concentration of VEGF was measured in the cellular supernatants. The disintegrin, named Lansbermin-I, is a low molecular weight protein (< 10 kDa) that includes an RGD on its sequence identified previously. Lansbermin-I showed potent inhibition of ADP and collagen-induced platelet aggregation on human plasma and also displayed inhibitory effects on the adhesion and migration of breast cancer MCF7 and MDA-MB 231cell lines, without affecting nontumorigenic breast MCF-10A and lung BEAS cells. Additionally, Lansbermin-I prevented MCF7 cells to adhere to fibronectin and collagen, and also inhibited in vitro angiogenesis on human endothelial HUVEC cells. Our results display the first report on the antitumor and anti-metastatic effects of an RGDdisintegrin isolated from a Porthidium snake venom by possibly interfering with α2 and/or β1-containing integrins. Thus, Lansbermin-I could be an attractive model to elucidate the role of disintegrins against breast cancer development.

Targeting of prostate-specific membrane antigen for radio-ligand therapy of triple-negative breast cancer

BackgroundTriple-negative breast cancer has extremely high risk of relapse due to the lack of targeted therapies, intra- and inter-tumoral heterogeneity, and the inherent and acquired resistance to therapies. In this study, we evaluate the potential of prostate-specific membrane antigen (PSMA) as target for radio-ligand therapy (RLT).MethodsTube formation was investigated after incubation of endothelial HUVEC cells in tumor-conditioned media and monitored after staining using microscopy. A binding study with 68Ga-labeled PSMA-addressing ligand was used to indicate targeting potential of PSMA on tumor-conditioned HUVEC cells. For mimicking of the therapeutic application, tube formation potential and vitality of tumor-conditioned HUVEC cells were assessed following an incubation with radiolabeled PSMA-addressing ligand [177Lu]-PSMA-617. For in vivo experiments, NUDE mice were xenografted with triple-negative breast cancer cells MDA-MB231 or estrogen receptor expressing breast cancer cells MCF-7. Biodistribution and binding behavior of [68Ga]-PSMA-11 was investigated in both tumor models at 30 min post injection using μPET. PSMA- and CD31-specific staining was conducted to visualize PSMA expression and neovascularization in tumor tissue ex vivo.ResultsThe triple-negative breast cancer cells MDA-MB231 showed a high pro-angiogenetic potential on tube formation of endothelial HUVEC cells. The induced endothelial expression of PSMA was efficiently addressed by radiolabeled PSMA-specific ligands. 177Lu-labeled PSMA-617 strongly impaired the vitality and angiogenic potential of HUVEC cells. In vivo, as visualized by μPET, radiolabeled PSMA-ligand accumulated specifically in the triple-negative breast cancer xenograft MDA-MB231 (T/B ratio of 43.3 ± 0.9), while no [68Ga]-PSMA-11 was detected in the estrogen-sensitive MCF-7 xenograft (T/B ratio of 1.1 ± 0.1). An ex vivo immunofluorescence analysis confirmed the localization of PSMA on MDA-MB231 xenograft-associated endothelial cells and also on TNBC cells.ConclusionsHere we demonstrate PSMA as promising target for two-compartment endogenous radio-ligand therapy of triple-negative breast cancer.

Synthesis, 3D-structure and stability analyses of NRPa-308, a new promising anti-cancer agent

We report herein the synthesis of a newly described anti-cancer agent, NRPa-308. This compound antagonizes Neuropilin-1, a multi-partners transmembrane receptor overexpressed in numerous tumors, and thereby validated as promising target in oncology. The preparation of NRPa-308 proved challenging because of the orthogonality of the amide and sulphonamide bonds formation. Nevertheless, we succeeded a gram scale synthesis, according to an expeditious three steps route, without intermediate purification. This latter point is of utmost interest in reducing the ecologic impact and production costs in the perspective of further scale-up processes. The purity of NRPa-308 has been attested by means of conventional structural analyses and its crystallisation allowed a structural assessment by X-Ray diffraction. We also reported the remarkable chemical stability of this molecule in acidic, neutral and basic aqueous media. Eventually, we observed for the first time the accumulation of NRPa-308 in two types of human breast cancer cells MDA-MB231 and BT549.

Monitoring the Kinetics of the Cellular Uptake of a Metal Carbonyl Conjugated with a Lipidic Moiety in Living Cells Using Synchrotron Infrared Spectromicroscopy.

Presented here is the exploitation of synchrotron infrared spectromicroscopy to evaluate the feasibility of monitoring the cellular uptake of rhenium-tris-carbonyl-tagged (Re(CO)3) lipophilic chains in living cells. To this aim, an in-house thermostated microfluidic device was used to limit water absorption while keeping cells alive. Indeed, cells showed a high survival rate in the microfluidic device over the course of the experiment, proving the short-term biocompatibility of the device. We recorded spectra of single, living, fully hydrated breast cancer MDA-MB231 cells and could follow the penetration of the rhenium complexes for up to 2 h. Despite the strong variations observed in the uptake kinetics between individual cells, the Re(CO)3 complex was traced inside the cells at low concentration and shown to enter them on the hour time scale by active transport.

Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

Elevated proliferation rates in cancer can be visualized with positron emission tomography (PET) using 3'-deoxy-3'-l-[18F]fluorothymidine ([18F]FLT). This study investigates whether [18F]FLT transport proteins are regulated through hypoxia. Expression and function of human equilibrative nucleoside transporter (hENT)-1, hENT2, and thymidine kinase 1 (TK1) were studied under normoxic and hypoxic conditions, and assessed with [18F]FLT-PET in estrogen receptor positive (ER+)-MCF7, triple-negative MDA-MB231 breast cancer (BC) cells, and MCF10A cells (human mammary epithelial cells). Functional involvement of hENT2 [18F]FLT transport was demonstrated in all cell lines. In vitro [18F]FLT uptake was higher in MDA-MB231 than in MCF7: 242 ± 9 vs. 147 ± 18% radioactivity/mg protein after 60 min under normoxia. Hypoxia showed no significant change in radiotracer uptake. Protein analysis revealed increased hENT1 (P < 0.0963) in MDA-MB231. Hypoxia did not change expression of either hENT1, hENT2, or TK1. In vitro inhibition experiments suggested involvement of hENT1, hENT2, and human concentrative nucleoside transporters during [18F]FLT uptake into all cell lines. In vivo PET imaging revealed comparable tumor uptake in MCF7 and MDA-MB231 tumors over 60 min, reaching standardized uptake values of 0.96 ± 0.05 vs. 0.89 ± 0.08 (n = 3). Higher hENT1 expression in MDA-MB231 seems to drive nucleoside transport, whereas TK1 expression in MCF7 seems responsible for comparable [18F]FLT retention in ER+ tumors. Our study demonstrates that hypoxia does not significantly affect nucleoside transport as tested with [18F]FLT in BC.-Krys, D., Hamann, I., Wuest, M., Wuest, F. Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

Cytotoxic Potential of the Coelomic Fluid Extracted from the Sea Cucumber Holothuria tubulosa against Triple-Negative MDA-MB231 Breast Cancer Cells.

Growing evidence has demonstrated that the extracts of different holothurian species exert beneficial effects on human health. Triple negative breast cancers (TNBC) are highly malignant tumors that present a poor prognosis due to the lack of effective targeted therapies. In the attempt to identify novel compounds that might counteract TNBC cell growth, we studied the effect of the exposure of the TNBC cell line MDA-MB231 to total and filtered aqueous extracts of the coelomic fluid obtained from the sea cucumber Holoturia tubulosa, a widespread species in the Mediterranean Sea. In particular, we examined cell viability and proliferative behaviour, cell cycle distribution, apoptosis, autophagy, and mitochondrial metabolic/cell redox state. The results obtained indicate that both total and fractionated extracts are potent inhibitors of TNBC cell viability and growth, acting through both an impairment of cell cycle progression and mitochondrial transmembrane potential and a stimulation of cellular autophagy, as demonstrated by the increase of the acidic vesicular organelles and of the intracellular protein markers beclin-1, and total LC3 and LC3-II upon early exposure to the preparations. Identification of the water-soluble bioactive component(s) present in the extract merit further investigation aiming to develop novel prevention and/or treatment agents efficacious against highly metastatic breast carcinomas.

Effects of somatostatin, curcumin, and quercetin on the fatty acid profile of breast cancer cell membranes.

Breast cancer is a worldwide commonly found malignancy in women and effective treatment is regarded as a huge clinical challenge even in the presence of several treatment options. Extensive literature is available demonstrating polyphenols as phytopharmaceutical anticancer agents. Among the polyphenols, quercetin and curcumin have been reported to have a strong potential against breast cancer. However, so far, no comprehensive study has been performed to demonstrate the anticarcinogenic effects of curcumin, quercetin, and their combinations with somatostatin on the fatty acid profile of breast cancer cell membranes. We used MCF-7 and MDA-MB231 breast cancer cells incubated with curcumin and quercetin for 24 h, in the absence and presence of somatostatin, at their EC50 concentrations to evaluate membrane fatty acid based functional lipidomics together with the followup of EGFR and MAPK signaling pathways. The two cell lines gave different membrane free fatty acid reorganization. In MCF-7 cells, the following changes were observed: an increase of ω6 linoleic acid in the cells incubated with somatostatin + quercetin and quercetin and a decrease of ω3 acids in the cells incubated with somatostatin + curcumin compared to somatostatin and significant increases of monounsaturated fatty acid (MUFA), mono-trans arachidonic acid levels and docosapentaenoic acid for the cells incubated with somatostatin + quercetin compared to the control cells. In MDA-MB231 cells, incubations with curcumin, quercetin, and somatostatin + quercetin induced the most significant membrane remodeling with the increase of stearic acid, diminution of ω6 linoleic, arachidonic acids, and ω3 (docosapentaenoic and docosahexaenoic acids). Distinct signaling pathway changes were found for these cell lines. In MCF-7 cells, separate or combined incubations with somatostatin and quercetin, significantly decreased EGFR and incubation with curcumin decreased MAPK signaling. In MDA-MB231 cells, incubation with curcumin decreased AKT1 and p-AKT1 (Thr308) levels. Incubation with curcumin and quercetin decreased the EGFR levels. Our results showed that cytostatic and antioxidant treatments can be combined to induce membrane fatty acid changes, including lipid isomerization as specific free radical driven process, and to influence signaling pathways. This study aimed to contribute to the literature on these antioxidants in the treatment of breast cancer to clarify the effects and mechanisms in combination with somatostatin.

Refinements of LC-MS/MS Spectral Counting Statistics Improve Quantification of Low Abundance Proteins

Mass spectrometry-based spectral count has been a common choice of label-free proteome quantification due to the simplicity for the sample preparation and data generation. The discriminatory nature of spectral count in the MS data-dependent acquisition, however, inherently introduces the spectral count variation for low-abundance proteins in multiplicative LC-MS/MS analysis, which hampers sensitive proteome quantification. As many low-abundance proteins play important roles in cellular processes, deducing low-abundance proteins in a quantitatively reliable manner greatly expands the depth of biological insights. Here, we implemented the Moment Adjusted Imputation error model in the spectral count refinement as a post PLGEM-STN for improving sensitivity for quantitation of low-abundance proteins by reducing spectral count variability. The statistical framework, automated spectral count refinement by integrating the two statistical tools, was tested with LC-MS/MS datasets of MDA-MB468 breast cancer cells grown under normal and glucose deprivation conditions. We identified about 30% more quantifiable proteins that were found to be low-abundance proteins, which were initially filtered out by the PLGEM-STN analysis. This newly developed statistical framework provides a reliable abundance measurement of low-abundance proteins in the spectral count-based label-free proteome quantification and enabled us to detect low-abundance proteins that could be functionally important in cellular processes.

Higher Glucose Enhances Breast Cancer Cell Aggressiveness

Cancer cells overexpress several transcription factors and motor proteins, such as NFkB and kinesin, to accommodate their high energy demand as well as migratory needs via enhanced glycolysis. We hypothesize that high glucose drives cancer progression and cell aggressiveness by decreasing actin expression, increasing NFkB, and kinesin expressions, and by activating Epithelial Mesenchymal Transition (EMT). Using lowly metastatic MCF-7 and highly metastatic MDA-MB231 (MB231) breast cancer cells – highly incident cancer types – we establish how glucose metabolism regulates actin and the biochemical changes that lead to alterations of cell mechanical properties. We find that higher glucose (15 and 30 mM) increases glycolytic enzymes, glucose uptake, migration speed, kinesin, Ki-67, and NFkB expressions (biomarkers), and hybrid EMT phenotype activation (adhesion molecules/cadherins). Downregulation of actin, increased expressions of motor protein and NFkB, and decreased nuclear stiffness – induced by higher glucose – result in a significant increase in the migration speed. Moreover, glucose deprivation using the glucose analog 2-deoxyglucose decreases significantly the migration speed in both cancer cells. Thus, higher glucose promotes a more aggressive phenotype that promises to be a new target for cancer therapy and can help prevent cancer progression in diabetic patients by inhibiting glucose activated mechanisms.

Characterization of the stemness potency of mammospheres isolated from the breast cancer cell lines.

Stemness phenotype mammospheres established from cell lines and tissues taken from autopsy can be used to test and to identify the most sensitive drugs for chemotherapy. Therefore, the aim of the present study was isolation and characterization of cancer stem cells derived from MCF7, MDA-MB231, and SKBR3 breast cancer cell lines to demonstrate the stemness phenotypes of mammospheres generated for further their applications in therapeutic approaches. In this study, two luminal subtypes of cell lines, MCF7 and SKBR3 and a basal subtype cell line, MDA-MB-231, were chosen. Mammosphere culturing was implemented for breast cancer stem cells isolation and mammosphere formation efficiency. At the next step, CD44+/CD24- cell ratio, Oct4 and Nanog mRNA levels, proliferation rate, migration rate of mammospheres, and drug resistance (in third passage) were evaluated. In addition, tumorigenicity of mammospheres in the chick embryo model was evaluated and compared through the chick chorioallantoic membrane assay. Among mammospheres formed in all three cell lines, MCF7 had the highest mammosphere formation efficiency. CD24 marker (a differentiation marker for the breast cancer cells) was significantly reduced in the mammospheres generated from MCF7 and SKBR3, during three passages. Also, Oct4 and Nanog transcript levels were significantly higher in all three types of mammospheres, as compared with their cell lines. Proliferation, migration rate, and drug resistance of mammospheres generated from all three cell lines were found to be significantly higher. Tumorigenicity of MCF7 mammospheres was confirmed through tumor size measurement. Also, tumorigenicity of MCF7 and SKBR3 mammospheres was confirmed through more migration from ectoderm to mesoderm and endoderm. We succeeded to establish the technology that can be extended to tissue in the future. We have demonstrated a number of mammospheres can be generated from cell lines. Also, cells with different molecular features showed different stemness phenotypes.

A Truncated Form of the Polarity Protein SCRIB Induces Apoptosis in MDA-MB231 Cells by Regulating Components of Several Signalling Pathways

Polarity proteins are fundamental to the establishment and maintenance of breast tissue integrity, whereas alterations of their amino acid sequence and protein structure can drive breast cancer formation and progression. Scrib is a multi-functional scaffold protein whose proper localization within the cell is pivotal for its correct function. We report here that the ectopic expression of a truncated form of the polarity protein Scrib, mainly composed of the LRR domain, drives MDA-MB231 breast cancer cells to caspase 3-mediated apoptotic cell death. Furthermore, we identify the truncated Scrib as a regulator of JNK/c-Jun, Notch and Wnt/I²-catenin signalling pathways. Our results indicate that the ectopic expression of the truncated Scrib in a breast cancer cell line with a specific background of genetic mutations (KRAS, AFRGAP1, BRAF, MYCL) activates a program of intracellular molecular alterations and promotes apoptosis.

Regulation of p53 Protein Function in Response to Heat Shock

p53 protein encoded by the TP53 gene performs one of the most important functions in the process of human tumor suppression. Mutations in the DNA-binding domain of p53 change its conformation, which contributes to the formation of aberrant intracellular protein complexes including heat shock proteins (Hsp70). This can provoke an occurrence of the aggressive types of tumors including breast cancer. Thereby, the study of the regulation mechanisms of mutant p53 in these stable complexes seems extremely relevant. The aim of this work was to study the regulation of p53 protein mutant for R175H (mutp53−R175H) under heat stress in MDA–MB-231 breast cancer cells in vitro. It was found that heat shock caused a drastic decrease in the level of wtp53 (wild-type p53) and mutp53–R175H proteins. It was gradually restored after the stress ceased. We also have found that mutp53–R175H increases the intracellular level of Hsp70 in normal conditions and reduces it after heat shock. At the same time, mutp53–R175H protein changes its intracellular localization, both in normal conditions and in response to heat shock being in the composition of Hsp70 protein complexes. Thus, the behavior of wtp53 and mutp53–R175H in response to heat shock appears to differ due to different interactions with protein complexes that regulate their stability and intracellular localization.

Synthesis, characterization and anticancer activity of new Zn(II) and MoO22+ complexes of 2-amino-4,6-mercaptotriazine

The preparation of new complexes containing 2-amino-4,6-mercaptotriazine (HAT) [Zn(AT)2(H2O)2], [Zn(bpy)(AT)Cl(H2O)], [Zn(PPh3)2(AT)Cl(H2O)] and cis-[Mo2O5(AT)2] is reported. The structures of these complexes are discussed using IR, NMR, UV–Vis, and EI-mass spectra, elemental analysis, molar conductivity and thermal measurements. HAT coordinates to the central metal ions through the deprotonated cyclic-N(1) and amino-N(2) or mercapto-S(6) atoms (AT−) in mononegative bidentate manner. The stoichiometries and the formation constants of Zn(II) complexes in solution were calculated using Job's method. The anticancer activities of some of the complexes were examined against The human breast cancer MDA-MB231 cell line. The IC50 values for cell growth proliferation were ranged from 24.57 ± 0.2 to 49.89 ± 0.4 μM, while that for the standard reference drug, cisplatin, was 8.72 ± 0.5 μM.

Nuclear Receptors Are Differentially Expressed and Activated in KAIMRC1 Compared to MCF7 and MDA-MB231 Breast Cancer Cells.

We recently established a KAIMRC1 cell line that has unique features compared to the known breast cancer cell lines, MCF7 and MDA-MB231. To characterize it further, we investigated the expression profile of nuclear receptors and their respective co-factors in these cell lines. We confirm that in contrast to the triple negative cell line MDA-MB231, the MCF7 and KAIMRC1 are estrogen receptor alpha (ERa) and progesterone receptor alpha (PRa) positive, with significant lower expression of these receptors in KAIMRC1. KAIMRC1 cell is a vitamin D receptor (VDR) negative and V-ErbA-Related Protein 2 (EAR2) positive in contrast to MCF7 and MDA-MB231. Remarkably, the histone deacetylases (HDACs) are highly expressed in KAIRMC1 with HDAC6 and HDAC 7 are exclusively expressed in KAIMRC1 while thyroid hormone receptor-associated protein 80 (TRAP80), telomeric DNA binding protein 1 (TBP1) and TGF-beta receptor interacting protein (TRIP1) are absent in KAIMRC1 but present in MCF7 and MDA-MB231. In a luciferase reporter assay, the ERa coexpression is needed for estrogen receptor element (ERE)-luciferase activation by estradiol in KAIMRC1 but not in MCF7. The co-expression of exogenous Liver X receptor alpha (LXRa)/retinoid X receptor alpha (RXRa) are necessary for LXR responsive element (LXRE) activation by the GW3696 in the three cell lines. However, the activity of peroxisome proliferator-activated receptor response element (PPARE)-tk-luciferase reporter increased when peroxisome proliferator-activated receptors alpha (PPARa)/RXRa were coexpressed but the addition of PPARa agonist (GW7647) did not stimulate further the reporter. The signal of the PPARE reporter increased in a dose-dependent manner with rosiglitazone (PPARg agonist) in KAIMRC1, MCF7, and MDA-MB231 when the proliferator-activated receptors gamma (PPARg)/RXRa receptors were cotransfected. Retinoic acid-induced activation of retinoic acid receptor response element (RARE)-tk-luciferase is dependent on exogenous expression of retinoic acid receptor alpha (RARa)/RXRa heterodimer in MDA-MB 231 but not in MCF7 and KAIMRC1 cell lines. In the three cell lines, Bexarotene-induced retinoid X receptor response element (RXRE)-luciferase reporter activation was induced only if the RXRa/LXRa heterodimer were co-expressed. The vitamin D receptor response element (VDRE)-luciferase reporter activity showed another distinct feature of KAIMRC1, where only co-expression of exogenous vitamin D receptor (VDR)/RXRa heterodimer was sufficient to reach the maximum rate of activation of VDRE reporter. In the proliferation assay, nuclear receptors ligands showed a distinct effect on KAIMRC1 compared to MCF7 and MDA-MB231. Growth inhibition effects of used ligands suggest that KAIMRC1 correlate more closely to MDA-MB231 than MCF7. Vitamin D3, rosiglitazone, novel RXR compound (RXRc) and PPARa compound (GW6471) have the most profound effects. In conclusion, we showed that nuclear receptors are differentially expressed, activated and also their ligand produced distinct effects in KAIMRC1 compared to MCF7 and MDA-MB231. This finding gives us confidence that KAIMRC1 has a unique biological phenotype.

Thapsigargin, inhibitor of sarco-endoplasmic Ca2+-ATPase, effectively suppresses the expression of S100A4 in human breast cancer cell line

Thapsigargin, the SERCA ATPase inhibitor, effectively suppresses the expression of metastasis marker S100A4 in breast cancer cells MDA-MB231. It has been demonstrated that transcription of the S100A4 gene is controlled by Ca2+-signaling pathways. It has been shown that synthesis of S100A4 mRNA and protein in the MDA-MB231 cell line is effectively inhibited by thapsigargin at a concentration of 0.4-4 µM, while preserving cell survival. We assume that a change in gene transcription in response to the disruption of Ca2+ homeostasis plays a direct role in the remodeling of Ca2+-signaling pathways.

Curcuma zedoaria (christm.) roscoe inhibits proliferation of MDA-MB231 cells via caspase-cascade apoptosis

Curcuma zedoaria is a perennial herb that belongs to Zingiberaceae family, found growing lavishly in Manipur; a north eastern part of India. The traditional usages of the plant is not limited to a particular disease but extended over a large number of diseases. Anti-breast cancer activity of ethyl acetate extract of Curcuma zedoaria was conducted on MDA-MB231 breast cancer cell line with the help of MTT assay technique and the mechanistic pathway was established with the help of western blot technique, confocal microscopy, wound healing migration assay and cell cycle analysis. Cell cytotoxicity test of ethyl acetate extracts showed remarkable result on MDA-MB231 in dose dependent manner (p < 0.05). Confocal microscopy studies on MDA-MB231 cells revealed the activation of cleaved caspase 9 in treated cells. Western blot analyses confirm the regulation of cytochrome C and suppression of Bcl-2 in treated cells which suggest ethyl acetate extract of Curcuma zedoaria inhibits the cancer cells through cascase-dependent pathways. In cell cycle analysis, after the treatments of the cells; there was an increase in the percentage of SubG0/G1 cells that decreased S and G2 phase indicating the population undergoing cell death (apoptosis or necrosis) thereby, our data suggest that ethyl acetate extracts were able to induce significant apoptosis in MDA-MB-231 cells in dose dependent manner. The wound healing migration assay confirms the anti-metastatic nature of the plant extract. With the help one way ANOVA test, all the experiments were executed and it was found that ethyl acetate extract of Curcuma zedoaria inhibits MDA-MB231 cells with the down-regulation of the expression of Bcl-2 through cascase-dependent pathways therefore Curcuma zedoaria is a potent anti-cancer plant especially in breast cancer.

Thapsigargin, Inhibitor of Sarco-Endoplasmic Ca2+-ATPase, Effectively Suppresses the Expression of S100A4 Protein in Human Breast Cancer Cell Line

Thapsigargin (SERCA ATPase inhibitor) inhibited the S100A4 metastatic marker expression in MDA-MB231 breast cancer cells. We found that S100A4 gene transcription is regulated by Ca2+ signaling pathways. We found that the synthesis of S100A4 mRNA and S100A4 protein in MDA-MB231 cells was effectively suppressed by thapsigargin at a concentration of 0.4-4 μM with retaining cell viability. We assume that the change in the gene transcription in response to disturbance of Ca2+ homeostasis is directly involved in the remodeling of Ca2+ signaling pathways.

Synthesis, Ant Proliferative Activity and Docking Study of New Quercetin Derivatives against MDA-MB231 Breast Cancer Cell Lines

MDA-MB231 is a very aggressive and invasive triple negative breast cancer, which present with limited treatment options. Unlike other breast cancer types, it is characterized by absence of hormonal receptors of estrogen, progesterone as well as human epidermal growth factor receptor 2, rendering it unsuitable for hormonal therapy and a perfect candidate for chemotherapy. Quercetin is a common natural flavonoid present in many food items, which have a wide range of biological activities, likes anticancer, antiviral, antibacterial and antioxidant. This study involves the synthesis of new Quercetin derivatives and the investigation their effects against MDA-MB231 cell lines. The structures of the derivatives were established using UV, IR, 1HNMR, CHNS, EIMS and ESIMS techniques. Their antiproliferative activities were investigated in vitro using Microculture Tetrazolium (MTT) assay. The percentage cell viability following exposure to Quercetin and its derivatives (Compounds 1-5) were measured. Both compounds 1 and 4 show a significant decrease in percentage cell viability from 100% to 43.7% and 38.1% respectively. IC50 value was calculated for compound 1 and 4 and found to be 2.042 and 1.838 µM respectively, indicating that they have a potential anticancer activity against the triple negative breast cancer type. The antiproliferative activity was supported and evidenced by molecular docking study.

SAT-LB055 Using Fluidics to Model the Genomic Evolution of Metastatic Breast Cancer

Of the 200,000 new cases of breast cancer diagnosed each year in the U.S, ~80% of the cancers are estrogen receptor alpha (ER) positive. Therefore, most first line treatments are endocrine therapies that target the ER. Selective estrogen receptor modulators (SERMs) and selective estrogen receptor down-regulators (SERDs) are used in premenopausal women, and aromatase inhibitors (AIs) are often used in postmenopausal women. Many of these cancers revert to an endocrine resistant, metastatic phenotype after one or more successful first line treatment(s). Patients who present with endocrine refractory disease have no good option for therapy other than chemotherapy regimens, which ultimately fail with high rates of mortality. Interestingly, approximately 25% of patients with endocrine resistant, metastatic disease harbor a mutated ER within the cancer cells, a mutation that was not detected in the primary tumor. During metastasis, some cells from the primary tumor are shed into the bloodstream and undergo hemodynamic shear stress on route to the secondary site of metastasis. Some of these cells, known as circulating tumor cells (CTCs), can seed in secondary organs and produce metastatic lesions. Several studies have recently linked fluid shear stress (FSS) with DNA instability and tumor metastasis. To explore this possibility, we used an in vitro model to test the effect of FSS on breast cancer cells, with the ultimate aim of understanding how FSS changes genomic dynamics, possibly leading to increased mutation frequency. We have preliminary results that physiological levels of FSS induce activation of the ATR DNA damage response pathway in both MDA-MB231 and MCF-7 breast cancer cells. Whole exome sequencing results support this data by showing sequential exposure of MCF-7 cells to FSS results in both de novo somatic mutations as well as an increase in mutation frequency at several common SNP sites. These data suggest FSS may play a role in downstream genomic instability and mutagenesis. Furthermore, Western blot and migration data of sheared cells show that FSS alters the phenotype of these cells, potentially rendering them more drug resistant and/or metastatically competent. In conclusion, we have shown that FSS may play a direct role in altering genomic instability as well as the phenotypic signature in breast cancer cells, resulting in cells that may be more metastatic and aggressive. The ultimate goal of this research is to utilize this in vitro approach to identify molecular targets that can be pursued to either prevent or delay metastatic breast disease. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

A Rab4‐Regulated Endosomal Compartment Prolongs EGFR Activation in Breast Cancer Cells

Early sorting endosomes are responsible for the proper trafficking of membrane-bound receptors that can drive cancer progression. However, the morphology, cargo sorting and signaling of early endosomes remains poorly understood in breast cancer. We have identified a novel population of enlarged early endosomal vesicles in breast cancer cells and human tumor xenografts (MDAMB231 and T47D) but not in non-cancerous epithelial cells (MCF10A) using confocal and super-resolution 3D dSTORM microscopy. A detailed quantitative analysis of the whole-cell endosomal morphology, cargo sorting, maturation, and regulatory Rab-GTPases associated with this compartment in cancerous breast epithelial cell lines was performed using Airyscan microscopy and 3D rendering approaches. This analysis showed that highly aggressive triple negative MDAMB231 breast cancer cells have larger but significantly fewer EEA1-enriched early sorting endosomes compared to non-cancerous MCF10A cells. Furthermore, live-cell time-lapse imaging showed that, despite normal transferrin recycling, the epidermal growth factor (EGF) and transferrin are found together in these large endosomal vesicles following internalization in MDAMB231 but not in MCF10A cells. Most importantly, these large EEA1-positive MDAMB231 endosomes exhibited prolonged EGF-induced activation of EGFR (p1068). We investigated the role of Rab4-mediated changes on endosome structure and function and found that over-expression of wild-type Rab4 in non-cancerous MCF10A cells produced similar EEA1-positive enlarged early-endosomes with prolonged EGF induced EGFR activation (p1068). Rab4 mutants in dominant-negative or constitutively-active state did not elicit the same effects as the wild-type Rab4, indicating that the on-off cycling of Rab4 is critical for the regulation of endosomal size and number. These large endosomes contain adjacent Rab4 and Rab11 domains, are located primarily at the perinuclear region and can undergo actin-associated budding events to recycle cargo out towards the plasma membrane. Altogether, this extensive characterization of early-endosomes in breast cancer cells has identified a novel Rab4 regulated enlarged endocytic compartment leading to prolonged EGFR activation. Interestingly, our data suggests that a population of “pre-existent” large EEA1-containing endosomes is present in some breast cancer cells, challenging the current views of endosome biogenesis. This research is essential to determining receptor regulation in translational breast cancer models which can impact the optimization of anti-cancer human therapies. Alterations in endocytic regulation in cancer cells may be key to understanding the adaptability of cancer cells and lead to new therapeutic targets. Support or Funding Information This work was supported by the National Institute of Health (NIH) R01 BRG CA20772 Breast cancer cells have altered early-endocytic morphology Representative images from whole-cell Airyscan microscopy assay to quantitatively characterize early-endocytic morphology (with respect to whole-cell morphology) in non-cancerous breast epithelial cells (MCF10A)and breast cancer cell lines (MDAMB231 and T47D) with 3D rendering techniques. Example image from live-cell 4D imaging of endosomal dynamics that facilitates our comprehensive characterization of Rab4-regulated endosomes in breast cancer cells. Prolonged EGF-induced EGFR activation (p1068) in MDAMB231 compared to MCf10A This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.