MCF-7 Mammary Carcinoma Research Articles

Exopolysaccharides from the Green Microalga Strain Coelastrella sp. BGV-Isolation, Characterization, and Assessment of Anticancer Potential.

Algal metabolites have been extensively studied as potential anticancer therapeutics. Among them, polysaccharides have attracted much attention because of their beneficial biological effects and safety. In the present research, the chemical characteristics, antitumor, and proapoptotic activities of extracellular polysaccharides (EPS) isolated from a new Bulgarian strain of the green microalga Coelastrella sp. BGV were investigated. A fast and convenient method of precipitation with cold ethanol was used to isolate EPS from the culture medium. The chemical characteristics of the isolated EPS were examined by colorimetric and spectrophotometric analyses, HPSEC-RID and HPLC-UV chromatography, and FT-IR spectroscopy. The results showed that the isolated EPS sample consists of three carbohydrate fractions with different molecular weights (11.5 × 104 Da, 30.7 × 104 Da, and 72.4 × 104 Da, respectively) and contains 7.14 (w/w%) protein. HPLC-UV analysis revealed the presence of galactose and fucose. The total uronic acid content in the sample was 4.5 (w/w%). The IR-FT spectrum of EPS revealed the presence of various functional groups typical of a polysaccharide (or proteoglycan) composed primarily of neutral sugars. The anticancer potential of the obtained EPS was assessed using cell lines with cancerous and non-cancerous origins as in vitro experimental models. The results of the performed MTT assay showed that EPS reduced the viability of the cervical and mammary carcinoma cell lines HeLa and MCF-7, while the control non-cancer cell lines BALB/3T3 and HaCaT were less affected. The HeLa cell line showed the highest sensitivity to the effects of EPS and was therefore used for further studies of its anticancer potential. The ability of EPS to inhibit cancer cell migration was demonstrated by wound-healing (scratch) assay. The cell cycle FACS analysis indicated that the EPS treatment induced significant increases in the sub G1 cell population and decreases of the percentages of cells in the G1, S, and G2-M phases, compared to the control. The fluorescent microscopy studies performed using three different staining methods in combination with Annexin V-FITC flow cytometric analysis clearly demonstrate the ability of EPS to induce cancer cell death via the apoptosis pathway. Moreover, an altered pattern and intensity of the immunocytochemical staining for the apoptosis- and proliferation-related proteins p53, bcl2, and Ki67 was detected in EPS-treated HeLa cancer cells as compared to the untreated controls. The obtained results characterize the new local strain of green microalgae Coelastrella sp. BGV as a producer of EPS with selective antitumor activity and provide an opportunity for further studies of its pharmacological and biotechnological potential.

Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells

The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as well as therapeutic side effects such as fibrosis. We screened an in-house library of 178 substances derived from marine sponges, endophytic fungi, and higher plants, and determined their senolytic activities towards DNA damage-induced senescent HCT116 colon carcinoma cells. The Pan-PI3K-inhibitor wortmannin and its clinical derivative, PX-866, were identified to act as senolytics. PX-866 potently induced apoptotic cell death in senescent HCT116, MCF-7 mammary carcinoma, and A549 lung carcinoma cells, independently of whether senescence was induced by ionizing radiation or by chemotherapeutics, but not in proliferating cells. Other Pan-PI3K inhibitors, such as the FDA-approved drug BAY80-6946 (Copanlisib, Aliqopa®), also efficiently and specifically eliminated senescent cells. Interestingly, only the simultaneous inhibition of both PI3K class I alpha (with BYL-719 (Alpelisib, Piqray®)) and delta (with CAL-101 (Idelalisib, Zydelig®)) isoforms was sufficient to induce senolysis, whereas single application of these inhibitors had no effect. On the molecular level, inhibition of PI3Ks resulted in an increased proteasomal degradation of the CDK inhibitor p21WAF1/CIP1 in all tumor cell lines analyzed. This led to a timely induction of apoptosis in senescent tumor cells. Taken together, the senolytic properties of PI3K-inhibitors reveal a novel dimension of these promising compounds, which holds particular potential when employed alongside DNA damaging agents in combination tumor therapies.

Automated Layer Analysis (ALAn): An Image Analysis Tool for the Unbiased Characterization of Mammalian Epithelial Architecture in Culture.

Cultured mammalian cells are a common model system for the study of epithelial biology and mechanics. Epithelia are often considered as pseudo-two dimensional and thus imaged and analyzed with respect to the apical tissue surface. We found that the three-dimensional architecture of epithelial monolayers can vary widely even within small culture wells, and that layers that appear organized in the plane of the tissue can show gross disorganization in the apical-basal plane. Epithelial cell shapes should be analyzed in 3D to understand the architecture and maturity of the cultured tissue to accurately compare between experiments. Here, we present a detailed protocol for the use of our image analysis pipeline, Automated Layer Analysis (ALAn), developed to quantitatively characterize the architecture of cultured epithelial layers. ALAn is based on a set of rules that are applied to the spatial distributions of DNA and actin signals in the apical-basal (depth) dimension of cultured layers obtained from imaging cultured cell layers using a confocal microscope. ALAn facilitates reproducibility across experiments, investigations, and labs, providing users with quantitative, unbiased characterization of epithelial architecture and maturity. Key features • This protocol was developed to spatially analyze epithelial monolayers in an automated and unbiased fashion. • ALAn requires two inputs: the spatial distributions of nuclei and actin in cultured cells obtained using confocal fluorescence microscopy. • ALAn code is written in Python3 using the Jupyter Notebook interactive format. • Optimized for use in Marbin-Darby Canine Kidney (MDCK) cells and successfully applied to characterize human MCF-7 mammary gland-derived and Caco-2 colon carcinoma cells. • This protocol utilizes Imaris software to segment nuclei but may be adapted for an alternative method. ALAn requires the centroid coordinates and volume of nuclei.

Synthesis and Cytotoxicity Evaluation of Tyrosine-5 Fluorinated Analogues of RA-VII, An Antitumor Bicyclic Hexapeptide

AbstractRA-VII analogues fluorinated at each ε-position of Tyr-5 were designed. The synthesis of these peptide analogues commenced with the preparation of atropisomeric fluorocycloisodityrosines from protected 3-fluoro-L-tyrosyl-3-boronyl-L-tyrosine mediated by copper(II) acetate and 4-(dimethylamino)pyridine. After N-methylation, the tetrapeptide segment was coupled with the N-terminus of each fluorocycloisodityrosine to afford a hexapeptide. After removal of the C- and N-terminal protecting groups, each peptide was subjected to macrocyclization to produce an analogue. The analogue with a β-oriented fluorine atom was equipotent to RA-VII with regard to cytotoxicity toward human mammary carcinoma MCF-7 cells, and showed 2.1-fold and 1.4-fold lower cytotoxicities than RA-VII toward human promyelocytic leukemia HL-60 and human colorectal cancer HCT-116 cells, respectively, whereas the analogue with an α-oriented fluorine atom showed 7.7-fold, 6.0-fold, and 14-fold lower cytotoxicities than RA-VII toward those cells, respectively.

The Aryl Hydrocarbon Receptor Ligand 6-Formylindolo(3,2-b)carbazole Promotes Estrogen Receptor Alpha and c-Fos Protein Degradation and Inhibits MCF-7 Cell Proliferation and Migration

Introduction: Worldwide, breast cancer is the most common cancer in women and is the main cause of death among all neoplasia in this group. Luminal A breast cancer represents approximately 70% of all breast cancers and is treated with hormone therapies targeting estrogen receptor alpha (ERα). Unfortunately, patients develop drug resistance leading to recurrence of neoplasia due to estrogen-independent ERα reactivation. Therefore, it is crucial to identify new molecular targets downstream ERα signaling pathway that allows the implementation of better treatments to improve the outcome of breast cancer patients. Overexpression of c-Fos, an ERα gene target, has been associated with increased cell motility, malignancy, metastasis, and invasion while its neutralization results in decreased breast cancer tumorigenesis. The aryl hydrocarbon receptor (AHR) ligands halogenated and polycyclic aromatic hydrocarbons, highly toxic compounds, down regulate c-Fos and ERα levels. The present study aimed to evaluate whether 6-formylindolo(3,2-b)carbazole (FICZ), a no toxic AHR agonist, modifies c-Fos levels in MCF-7 mammary carcinoma cells as well as to determine its effects on cell proliferation and migration. In addition, the possible mechanism through which FICZ mediates c-Fos levels in MCF-7 cells was investigated. Methods: Initially, the effect of FICZ on c-Fos mRNA and protein levels in MCF-7 cells, untreated or treated with estradiol, was evaluated by qPCR and Western blot. 2,3,7,8-Tetrachloro-dibenzo-p-dioxin, an AHR prototype agonist, was used as a positive control. Next, we examined the effect of FICZ on MCF-7 cell proliferation and migration by cell counting, MTT, 3H-thymidine incorporation, and scratch-wound assays. Finally, the involvement of proteasome 26S on ERα and c-Fos protein degradation was investigated by the use of MG132 and Western blot. Results: The data show that FICZ treatment downregulates c-Fos mRNA and protein levels, most likely by promoting ERα proteasome degradation, blocking MCF-7 cell proliferation and migration. The results also demonstrate that liganded ERα was required for FICZ-mediated ERα degradation. Conclusions: Activation of AHR results in a decreased MCF-7 cell proliferation and migration by ERα and c-Fos down regulation. Targeting AHR might be a promising therapy for breast cancer treatment, particularly when estrogen-independent ERα reactivation presents.

Senescence Marker Protein 30 (SMP30): A Novel Pan-Species Diagnostic Marker for the Histopathological Diagnosis of Breast Cancer in Humans and Animals.

Senescence marker protein 30 (SMP30) is a cell survival factor playing an important role in vitamin C synthesis and antiapoptosis. Moreover, its cytoprotective role suggests a possibility to be related to cancer cell survival. Mammary carcinoma is a common cancer in both humans and animals. Because of its histopathological diversity, especially in the early stage, histopathological diagnosis may be complicated; therefore, a diagnostic marker is helpful for confirmation. The present study analyzed the expression pattern of SMP30 in mammary carcinoma in humans, dogs, and cats. Immunohistochemistry, immunofluorescence, and western blot analysis were used to investigate SMP30 expression patterns. The expression was specifically observed in neoplastic glandular epithelial cells. The expression increased with the malignancy of glandular epithelial cells with a highly proliferative status. However, SMP30 expression was low in normal mammary gland tissues or well-differentiated adenoma tissues. The patterns were consistently reproduced in canine primary mammary carcinoma cells and MCF-7 and MDA-MB-231 human carcinoma cell lines. This study provides useful information to understand SMP30 expression in various stages of mammary carcinoma and to suggest its utility as a pan-species diagnostic marker, thereby helping to establish strategies for diagnosing mammary carcinoma in several species.

Reactivity and Anticancer Assessment of 4-Hydroxyquinoline Derivatives

Ethyl 4-hydroxy-7-(trifluoromethyl)quinoline-3-carboxylate (1) exhibits a moderate cytotoxic activity against the MCF-7 mammary gland cancer cell line and HePG2 hepatocellular carcinoma cell line and a weak activity against the HCT-116 human colorectal carcinoma cell line. In order to enhance the cytotoxic activity of this compound, it was modified by changing the side-chain substituent and/or forming a new heterocyclic ring fused to the pyridine ring. Heating compound 1 with chloroacetyl chloride gave a mixture of two isomeric O-acylation products ethyl 4-(2-chloroacetoxy)-7-(trifluoromethyl)-quinoline-3-carboxylate and 3-chloro-8-(trifluoromethyl)-2H-pyrano[3,2-c]quinoline-2,4(3H)-dione, whereas the reaction with acetyl chloride in NaOH formed an N-acylation product 1-acetyl-4-oxo-7-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid. The reactions of compound 1 with urea, thiourea, hydrazine hydrate, hydroxylamine, o-phenylenediamine, phenyl isothiocyanate, and ethyl acetoacetate yielded the corresponding condensation products 1-[4-oxo-7-(trifluoromethyl)-3,4-dihydroquinoline-3-carbonyl)]urea, 1-[4-oxo-7-(trifluoromethyl)-3,4-dihydroquinoline-3-carbonyl)]thiourea, 7-(trifluoromethyl)-1,2-dihydropyrazolo[4,3-c]quinolin-3-one, 7-(trifluoromethyl)isoxazolo[4,5-c]quinolin-3(2H)-one, 3-(trifluoromethyl)-13H-benzo[2,3][1,4]diazepino[6,5-c]quinolin-7-ol, 3-phenyl-2-thioxo-8-(trifluoromethyl)-2,3-dihydro-4H-[1,3]oxazino[5,6-c]quinolin-4-one and ethyl 8-(trifluoromethyl)-2-methyl-4-oxo-4H-pyrano[3,2-c]quinolone 3-carboxylate, respectively. The structures of the synthesized compounds were confirmed by elemental analysis and spectral data.

Distinct cellular responses to replication stress leading to apoptosis or senescence.

Replication stress (RS) is a major driver of genomic instability and tumorigenesis. Here, we investigated whether RS induced by the nucleotide analog fludarabine and specific kinase inhibitors [e.g. targeting checkpoint kinase 1 (Chk1) or ataxia telangiectasia and Rad3‐related (ATR)] led to apoptosis or senescence in four cancer cell lines differing in TP53 mutation status and expression of lamin A/C (LA/C). RS resulted in uneven chromatin condensation in all cell types, as evidenced by the presence of metaphasic chromosomes with unrepaired DNA damage, as well as detection of less condensed chromatin in the same nucleus, frequent ultrafine anaphase bridges, and micronuclei. We observed that responses to these chromatin changes may be distinct in individual cell types, suggesting that expression of lamin A/C and lamin B1 (LB1) may play an important role in the transition of damaged cells to senescence. MCF7 mammary carcinoma cells harboring wild‐type p53 (WT‐p53) and LA/C responded to RS by transition to senescence with a significant reduction of lamin B receptor and LB1 proteins. In contrast, a lymphoid cancer cell line WSU‐NHL (WT‐p53) lacking LA/C and expressing low levels of LB1 died after several hours, while lines MEC‐1 and SU‐DHL‐4, both with mutated p53, and SU‐DHL‐4 with mutations in LA/C, died at different rates by apoptosis. Our results show that, in addition to being influenced by p53 mutation status, the response to RS (apoptosis or senescence) may also be influenced by lamin A/C and LB1 status.

Antibacterial and cytotoxic activities of the Syzygium polyanthum leaf extract from Malaysia.

Background and Aim:The increasing prevalence of drug resistance eventually leads scientist to discover new drugs that could solve the problem. Since ancient immemorial times, medicinal plants generally known as herbs were widely used in every culture throughout the world. In fact, currently up to 70,000 plant species have been screened for biological activities and about 70% ends up for commercialization. Therefore, this study was aimed to evaluate the potential cytotoxic and antibacterial effect of Syzygium polyanthum leaves which are local Malaysia plants, against 4T1 and MCF-7 mammary carcinoma cells, respectively, and also against bacteria causing mastitis in cows.Materials and Methods:The cytotoxic effect of hydromethanolic extract of S. polyanthum against 4T1 and MCF-7 mammary carcinoma cells was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cells were treated with the concentration of extracts ranging from 15.63 µg/mL to 1000 µg/ml for 72 h, and the percentage of cell survivability was determined based on minimum concentration that was able to allow at least 50% growth of cancer cells (IC50) after 72 h. The antibacterial activity was tested against common bacteria causing mastitis in cow. The bacteria were isolated from milk samples. The antibacterial activity of the extract was determined by disk diffusion method and susceptibility test based on minimum inhibitory concentration (MIC).Results:Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus intermedius were isolated from the milk samples that positive for mastitis. The MIC values range from 7.12 mm to 13.5 mm. The extract exhibits the widest zone of inhibition (13.5±0.20 mm) at 1000 mg/ml of concentrations. The extract relatively has low cytotoxicity effect against 4T1 and MCF-7 cells with IC50 values ranging from 672.57±59.42 and 126.05±50.89 µg/ml, respectively.Conclusion:S. polyanthum exerts weak antibacterial activity and cytotoxic effect to mammary carcinoma cells. The extract does not toxic to cells. However, further study is recommended, especially, this plant should be tested for in vivo.

Abstract LB-398: DNA-PKcs protects irradiated cells from persistent DNA damage signaling and accelerated senescence

Abstract The DNA-dependent protein kinase catalytic subunit DNA-PKcs has long been considered as a promising target in cancer, based on its roles in DNA damage response and the potential for inhibitors to enhance sensitivity to genotoxic therapy. Multiple investigational DNA-PK inhibitors remain under evaluation, though several have failed in development and none have reached the clinic. DNA-PKcs has been assigned diverse roles in DNA damage response that overlap with ATM including direct phosphorylation of H2AX to form γH2AX and promoting double-strand break (DSB) repair by conventional non-homologous end-joining (NHEJ). Nonetheless, a recent report implicated DNA-PKcs as a negative regulator of ATM. This may resolve the paradox that while ATM inhibitors block H2AX phosphorylation, prevent ionizing radiation induced foci (IRIF) formation and suppress terminal arrest and onset of cellular senescence, DNA-PK inhibitors induce persistent γH2AX, delay IRIF resolution and promote senescence. Toward better defining DNA-PKcs roles in radiation response, we examined MCF7 mammary carcinoma cells for effects of DNA-PKcs shRNA knockdown or treatment with specific inhibitors on DNA damage response and senescence after irradiation. We confirmed a requirement for DNA-PK in resolution of γH2AX and IRIF by 24 h after irradiation. Rather than return to proliferation, surviving cells with DNA-PK defects developed characteristic features of senescence within five days in culture. To examine if this effect of DNA-PKcs inhibition was mediated by blocking DSB repair, we irradiated MCF7 cell lines expressing shRNAs targeting NHEJ factors Ku70, Ku80, XLF, XRCC4, PAXX, and LIG4 as well as scrambled control in the presence or absence of DNA-PKcs inhibitor NU7441. Strikingly, IRIF resolution at 24 h was similar to control in cell lines lacking NHEJ factors while NU7441 increased IRIF persistence and enhanced senescence in all cell lines. These results suggest that a critical role for DNA-PKcs in radiation response is to attenuate DNA damage signaling and prevent cellular senescence. Treating irradiated DNA-PKcs knockdown cells with the ATM inhibitor KU55933 at 24 h resolved persistent γH2AX and IRIF and blocked senescence. Taken together, our data demonstrate that DNA-PKcs regulates IRIF resolution and cell senescence by targeting ATM functions rather than directly affecting DSB repair. Citation Format: Yue Liu, Elena V. Efimova, Aishwarya Ramamurthy, Stephen J. Kron. DNA-PKcs protects irradiated cells from persistent DNA damage signaling and accelerated senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-398.

Two new alkylresorcinol derivatives from the leaves of Scyphocephalium ochocoa

Abstract Two new alkylresorcinol derivatives, namely ochocinone A and ochocinone B (1, 2), as well as a known alkylresorcinol oleiferinone (3) were isolated from the methanol extract of the leaves of Scyphocephalium ochocoa. The structures of these compounds were established by detailed spectroscopic analysis and by comparison with the spectral data of related known compounds. Compounds 1–3 showed significant immunomodulatory activity and cytotoxic effect on the mammary carcinoma MCF-7 cell line.

Self-Assembling Benzothiazole-Based Gelators: A Mechanistic Understanding of in Vitro Bioactivation and Gelation.

Low molecular weight gelators (LMWGs) of chemotherapeutic drugs represent a valid alternative to the existing polymer-based formulations used for targeted delivery of anticancer drugs. Herein we report the design and development of novel self-assembling gelators of the antitumor benzothiazole 5F 203 (1). Two different types of derivatives of 1 were synthesized, formed by an amide (2) and a carbamate (3a-3d) linker, respectively, which showed potent in vitro antitumor activity against MCF-7 mammary and IGROV-1 ovarian carcinoma cells. In contrast, MRC-5 fibroblasts were inherently resistant to the above derivatives (GI50 > 10 μM), thus revealing stark selectivity against the malignant cell lines over the nontransformed fibroblasts. Western blots assays demonstrated induction of CYP1A1 by 1 and its derivatives only in sensitive malignant cells (MCF-7), corroborating conservation of a CYP1A1-mediated mechanism of action. The ability to form stable gels under relatively high strains was supported by rheological tests; in addition, their inner morphology was characterized as possessing a crossed-linked nanostructure, with the formation of thick aggregates with variable widths between 1100 and 400 nm and lengths from 8 to 32 μm. Finally, in vitro dissolution studies proved the ability of hydrogel 2 to release 48% of 2 within 80 h, therefore demonstrating its ability to act as a platform for localized delivery.

Development of novel inorganic strontium nanoparticles for delivery of pDNA and siRNA to breast cancer cell

Event Abstract Back to Event Development of novel inorganic strontium nanoparticles for delivery of pDNA and siRNA to breast cancer cell ATHIRAH BAKHTIAR1*, Md Ezharul H. Chowdhury2, Iekhsan Othman3 and Anuar Zaini3 1 Faculty of Pharmacy, Mahsa University, Malaysia 2 Monash University Malaysia, Malaysia 3 Monash University, Australia Background Recent studies have been focused on developing smart nanoparticles for efficient delivery of transgenes and siRNAs into cancerous cells of an animal model through active and passive targeting. Precipitation reaction is one of the facile and convenient ways to generate inorganic strontium nanoparticles, in which an insoluble salt is formed after mixing two water-soluble salts. Advantages in comparison with other methods include requiring only simple equipment, ability to prepare and control particle size and composition in near ambient temperature and pressure. The approach would pave a new way for facile, low toxicity and large-scale synthesis of inorganic nanoparticles with well-controlled dimensions and properties. Methods The study involved the formation of 5 different types of inorganic strontium nanocrystals via precipitation reactions, with various observational studies involving pH, temperature, salt concentration and time of incubation. Strontium particle sizes were determined using zetasizer and scanning electron microscope prior to binding studies. The nanoparticles were then subjected to various cellular studies including cellular uptake and cytotoxicity studies through incorporation of p53 and MAPK siRNA, forming gene-nanoparticle complexes onto human and mice mammary carcinoma cells, MCF-7 and 4T1 cells, respectively. Unloaded strontium nanoparticles formed were also observed for toxicity effect against both cells. Results Studies showed that strontium fluoride and strontium carbonate were smaller in size in comparison to other strontium salt particles, averaging in 100-300nm in diameter. Additionally, cellular studies revealed that gene-loaded strontium sulfite and strontium fluoride were responsible for greater cytotoxicity effect, in comparison naked gene treatment. Low toxicity effect was seen with unloaded strontium nanoparticles. Studies were compared against established inorganic carbonate apatite nanoparticles. Conclusion Nano-sized strontium particles showed great potential to be applied for in vivo studies, ideally seen with strontium sulfite and strontium fluoride, via the gene delivery system in the cellular level. Keywords: breast cancer, pDNA delivery, siRNA, Strontium, Nanoparticles Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Poster Presentation Topic: Cancer Citation: BAKHTIAR A, Chowdhury MH, Othman I and Zaini A (2019). Development of novel inorganic strontium nanoparticles for delivery of pDNA and siRNA to breast cancer cell. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00137 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. ATHIRAH BAKHTIAR, Faculty of Pharmacy, Mahsa University, Bandar Saujana Putra, Selangor, 42610, Malaysia, athirahbakhtiar@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers ATHIRAH BAKHTIAR Md Ezharul H Chowdhury Iekhsan Othman Anuar Zaini Google ATHIRAH BAKHTIAR Md Ezharul H Chowdhury Iekhsan Othman Anuar Zaini Google Scholar ATHIRAH BAKHTIAR Md Ezharul H Chowdhury Iekhsan Othman Anuar Zaini PubMed ATHIRAH BAKHTIAR Md Ezharul H Chowdhury Iekhsan Othman Anuar Zaini Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract 4419: HER2 expression and 17β-estradiol induce ACSL4 expression in estrogen receptor positive mammary carcinoma cells

Abstract Breast cancer is a multifactorial disease involving several molecular changes and a high proliferation rate often under the activation of hormonal receptors. Therefore, cancer cells require the metabolic machinery for membrane synthesis and to promote signaling pathways. Amongst biomolecules required for efficient cell growth are polyunsaturated fatty acids, like arachidonic acid, that are essential nutrients whose cellular uptake is partly governed by long chain acyl-CoA synthetases (ACSL). In this study, we investigated the impact of 17β-estradiol and Human Epidermal growth factor Receptor 2 (HER2) on cellular uptake of polyunsaturated fatty acids and on the expression of ACSLs. The administration of 17β-estradiol in steroid-starved MCF-7 and T47D mammary carcinoma cells induced a significant increase in cellular uptake of arachidonic acid (AA) and eicosapentaenoic acid (EPA), and in ACSL4 protein content (p<0.05). There was no change in the expression of the ACSL1, ACSL3 and ACSL6 isoforms. Increased ACSL4 protein expression was not accompanied by changes in ACSL4 mRNA expression, but was associated with a significant increase in the protein half-life (T1/2=26±2h) compared to untreated cells (T1/2=8±0.5h). Silencing of ERα reversed the impact of 17β-estradiol on ACSL4 protein expression and half-life. Silencing of ACSL4 eliminated the 17β-estradiol-induced increase in cellular polyunsaturated fatty acid uptake, cell migration and invasion capacities. ACSL4 silencing also prevented the 17β-estradiol-induced increases in p-Akt and p-GSK3β, as well as the 17β-estradiol-induced decrease in E-cadherin expression, important events in epithelial to mesenchymal transition. In addition, the forced expression of HER2 in MCF7 cells induced an increase in cellular uptake of AA and in the expression of ACSL4 measured by both qPCR and western blots. This suggests that HER2-induces ACSL4 expression by a mechanism that differs from that of 17β-estradiol. Further investigations are planned to study the implication of ACSL4 on cellular functions induced by HER2. Overall, these results demonstrate that an induction of ACSL4 protein expression in ERα positive MCF-7 and T47D cells is implicated in 17β-estradiol-induced cellular migration and invasion capacities. Citation Format: Maroua Mbarik, Anissa Belkaid, Marc E. Surette. HER2 expression and 17β-estradiol induce ACSL4 expression in estrogen receptor positive mammary carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4419. doi:10.1158/1538-7445.AM2017-4419

17β-estradiol-induced ACSL4 protein expression promotes an invasive phenotype in estrogen receptor positive mammary carcinoma cells.

Long chain acyl-CoA synthase-4 (ACSL4) expression has been associated with an aggressive phenotype in breast carcinoma cells, whereas its role in ERα-positive breast cancer has not been studied. ACSL4 prefers 20-carbon polyunsaturated fatty acid (PUFA) substrates, and along with other ACSLs has been associated with cellular uptake of exogenous fatty acids. 17β-estradiol induces proliferation and invasive capacities in ERα+ve breast carcinoma that is associated with modifications of cellular lipid metabolism. In this study, treatment of steroid-starved ERα-positive MCF-7 and T47D mammary carcinoma cells with 17β-estradiol resulted in increased cellular uptake of the PUFA arachidonic acid (AA) and eicosapentaenoic acid (EPA), important building blocks for cellular membranes, and increased ACSL4 protein levels. There was no change in the expression of the ACSL1, ACSL3 and ACSL6 protein isotypes. Increased ACSL4 protein expression was not accompanied by changes in ACSL4 mRNA expression, but was associated with a significant increase in the protein half-life compared to untreated cells. ERα silencing reversed the impact of 17β-estradiol on ACSL4 protein levels and half-life. Silencing of ACSL4 eliminated the 17β-estradiol-induced increase in AA and EPA uptake, as well as the 17β-estradiol-induced cell migration, proliferation and invasion capacities. ASCL4 silencing also prevented the 17β-estradiol induced increases in p-Akt and p-GSK3β, and decrease in E-cadherin expression, important events in epithelial to mesenchymal transition. Taken together, these results demonstrate that ACSL4 is a target of 17β-estradiol-stimulated ERα and is required for the cellular uptake of exogenous PUFA and the manifestation of a more malignant phenotype in ERα+ve breast carcinoma cells.

Development and characterization of MCF7 mammary carcinoma xenografts in a non-immunocompromised rat model

Purpose: To investigate the development of mammary tumours in female Sprague-dawley rats through a simple subcutaneous injection of human adenocarcinoma breast cells (MCF7) in combination with basement membrane matrix (BME).Methods: Nine Sprague-Dawley rats were divided into three groups. Group A received no injection, group B was injected with MCF7 cells at a cell density of 7.2 x 106/ml, and group C was co-injected with MCF7 and BME at 7.2 x 106/ml and 3.158 mg/ml, respectively. Tumour growth was observed over a duration of 70 days. Hematological analysis was performed using differential blood cell counts. Histological evaluation was carried out using standard LM techniques and H&E staining.Results: At day 35, RBC concentration across all groups was 8.10 x 106/mm3, whereas by day 70 the range decreased to (7.64 – 7.87) x 106/mm3. White blood cells (WBCs) were within normal range up to day 35, but monocytes and lymphocytes displayed an increase in concentration for group C. Mammary tissues from the thoracic region showed evidence of MCF7 cellular proliferation in both groups B and C.Conclusion: This study reveals that BME enhances tumour growth. Further studies are required to investigate optimization strategies for the development of mammary tumours in alternative recipient animal.Keywords: Tumour induction, MCF7, Histopathology, Thoracic mammary gland, Mammary tumour, Basement membrane matrix

Abstract 4124: Sub-cytotoxic concentrations of a novel polyphenol fatty acid ester derivative arrest breast cancer cell proliferation and metastasis

Abstract In this study, the antiproliferative and antimetastatic properties of phloridzin docosahexaenoate (PZ-DHA), a novel polyphenol fatty acid ester derivative, were explored using in vitro and in vivo models of mammary carcinoma. PZ-DHA combines phloridzin (PZ, a dihydrochalcone found in apple peels) with docosahexaenoic acid (DHA, an omega-3 fatty acid). The regioselective acylation reaction was catalyzed by lipase B enzyme from Candida antarctica. PZ-DHA-induced selective cytotoxicity towards breast cancer cells (triple-negative mammary carcinoma cells, MDA-MB-231, MDA-MB-468, and 4T1; estrogen receptor-positive mammary carcinoma cells, MCF-7 and T-47D) was observed compared to human mammary epithelial cells (HMECs and MCF 10A) using the MTS assay. Significantly less (p<0.05) cytotoxicity of PZ-DHA for normal cells was also confirmed using flow cytometric analysis of annexin-V-FLUOS/propidium iodide-stained MCF 10A and human dermal fibroblasts compared to MDA-MB-231 cells. Flow cytometric analysis of Oregon Green 488-stained MDA-MB-231 cells showed an antiproliferative effect of PZ-DHA at sub-cytotoxic concentrations (10 to 30 μM). Cell cycle analysis showed that MDA-MB-231 replication was arrested at the G2/M phase of the cell cycle following treatment with PZ-DHA at sub-cytotoxic concentrations. PZ-DHA suppressed the migration of MDA-MB-231 and 4T1 cells in vitro in wound healing and cell migration assays. Reduced expression of proteins (β-catenin, slug, Zinc finger E-box-binding homeobox 1, vimentin and matrix metalloproteinase-2) involved in the epithelial-to-mesenchymal transition was demonstrated by western blot analysis of PZ-DHA-treated MDA-MB-231 cell lysates. Finally, 4T1 tumor bearing-female BALB/c mice that received intra-peritoneal injections of PZ-DHA showed a significant reduction (p<0.05, n = 9) in primary tumor volume at the mammary fat pad and fewer metastatic lesions in the lungs compared to the saline-treated control mice, suggesting an in vivo antimetastatic effect of PZ-DHA. In conclusion, findings of this study reveal that PZ-DHA suppresses mammary carcinoma cell proliferation and metastasis, suggesting a potential clinical application to prevent breast cancer progression in patients. Citation Format: Wasundara Fernando, Melanie R. Power Coombs, David W. Hoskin, H. P. Vasantha Rupasinghe. Sub-cytotoxic concentrations of a novel polyphenol fatty acid ester derivative arrest breast cancer cell proliferation and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4124.

Gamma-Retrovirus Integration Marks Cell Type-Specific Cancer Genes: A Novel Profiling Tool in Cancer Genomics.

Retroviruses have been foundational in cancer research since early studies identified proto-oncogenes as targets for insertional mutagenesis. Integration of murine gamma-retroviruses into the host genome favours promoters and enhancers and entails interaction of viral integrase with host BET/bromodomain factors. We report that this integration pattern is conserved in feline leukaemia virus (FeLV), a gamma-retrovirus that infects many human cell types. Analysis of FeLV insertion sites in the MCF-7 mammary carcinoma cell line revealed strong bias towards active chromatin marks with no evidence of significant post-integration growth selection. The most prominent FeLV integration targets had little overlap with the most abundantly expressed transcripts, but were strongly enriched for annotated cancer genes. A meta-analysis based on several gamma-retrovirus integration profiling (GRIP) studies in human cells (CD34+, K562, HepG2) revealed a similar cancer gene bias but also remarkable cell-type specificity, with prominent exceptions including a universal integration hotspot at the long non-coding RNA MALAT1. Comparison of GRIP targets with databases of super-enhancers from the same cell lines showed that these have only limited overlap and that GRIP provides unique insights into the upstream drivers of cell growth. These observations elucidate the oncogenic potency of the gamma-retroviruses and support the wider application of GRIP to identify the genes and growth regulatory circuits that drive distinct cancer types.

Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent.

IntroductionCannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems. The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands.MethodsThe CB2-selective agonist JWH-015 was used to investigate mechanisms downstream of CB2 activation in mouse and human breast cancer cell lines in vitro and in a murine mammary tumor model.ResultsJWH-015 treatment significantly reduced primary tumor burden and metastasis of luciferase-tagged murine mammary carcinoma 4T1 cells in immunocompetent mice in vivo. Furthermore, JWH-015 reduced the viability of murine 4T1 and human MCF7 mammary carcinoma cells in vitro by inducing apoptosis. JWH-015-mediated reduction of breast cancer cell viability was not dependent on Gαi signaling in vitro or modified by classical pharmacological blockade of CB1, GPR55, TRPV1, or TRPA1 receptors. JWH-015 effects were calcium dependent and induced changes in MAPK/ERK signaling.ConclusionThe results of this work characterize the actions of a CB2-selective agonist on breast cancer cells in a syngeneic murine model representing how a clinical presentation of cancer progression and metastasis may be significantly modulated by a G-protein-coupled receptor.

EBP1 protein modulates the expression of human MHC class II molecules in non-hematopoietic cancer cells.

Many solid tumours including melanoma, glioblastoma, and breast carcinomas express MHC class II molecules (MHC II). The surface expression of these molecules confers to non-hematopoietic tumour cells the role of non-professional antigen presenting cells and the ability to potentially stimulate tumour-specific CD4+ T cell response. We studied EBP1, an ErbB3 binding protein, and the effects of p48 and p42 isoforms on the MHC II expression in U87 glioblastoma, M14 melanoma and MCF7 mammary carcinoma cell lines. We found that overexpression of p48 increases MHC II transcription in U87 and M14, through upregulation of CIITA transactivator and STAT1 phosphorylation. In addition, p48 protein influences MHC II expression by increasing mRNA stability. In melanoma and glioblastoma cell lines, p48 isoform functions as oncogene promoting tumour growth, while p42 isoform, that does not affect MHC II expression, acts as a tumour suppressor by blocking cell growth and inducing apoptosis. In contrast, p48 seems to act as tumour suppressor in breast carcinoma inhibiting proliferation, favouring apoptosis, and inducing a slight increase of MHC II expression similar to p42. Our data highlight the tissue specificity function of EBP1 isoforms and demonstrate that only the oncogene p48 activates MHC II expression in human solid tumours, via STAT1 phosphorylation, in order to affect tumour progression by triggering specific immune response.