Human Mammary Cancer Cell Lines Research Articles

Abstract PO1-16-04: Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression

Abstract Purpose: Low molecular weight isoforms of cyclin E (LMW-E) have been implicated in various human cancers, including triple negative breast cancer (TNBC), and are associated with a poor prognosis. However, targeted therapies for TNBC based on biomarkers are currently lacking. This study aims to investigate LMW-E as a potential therapeutic target in TNBC and evaluate the efficacy of RP-6306, a selective inhibitor of the Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1), in LMW-E-positive breast tumors. Experimental Design: Immunohistochemical (IHC) analysis was performed on pre-treatment tumor specimens from TNBC patients (n=36) to assess the correlation between LMW-E expression, CDK1 phosphorylation at Threonine 14 (pT14), and pathologic complete response to neoadjuvant chemotherapy. LMW-E inducible human mammary epithelial cells (hMEC) and breast cancer cell lines were used to investigate the regulatory effect of LMW-E on PKMYT1, the kinase responsible for CDK1 phosphorylation at T14, and the response to RP-6306, a first in-class and selective inhibitor of PKMYT1. Patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models of TNBC expressing human LMW-E (hLMW-E) were also utilized to assess LMW-E as a biomarker for predicting response to RP-6306. Results. Analysis of TNBC tumor biopsies revealed a significant positive correlation between LMW-E expression and CDK1 pT14, and both biomarkers were associated with a lack of pathological complete response to neoadjuvant chemotherapy. In vitro results using LMW-E inducible hMECs and breast cancer cell lines demonstrated that LMW-E up-regulates PKMYT1 and CDK1 pT14, acting as a PKMYT1 binding protein and enhancing PKMYT1 protein stability. High LMW-E protein levels predicted a favorable response to RP-6306, resulting in the accumulation of sub-G1 and polyploid cells, decreased tolerance to replication stress, increased DNA damage, chromosomal breakage, and apoptosis. In vivo treatment of TNBC PDX models and hLMW-E transgenic tumors with RP-6306 resulted in a significant reduction in tumor volume only in mice harboring high LMW-E tumors, while low cyclin E models showed no response. Immunohistochemical analysis confirmed increased γ-H2AX and decreased CDK1-pT14 and Ki67 levels, indicating the efficacy of RP-6306 in both PDX and transgenic models. Conclusion: This study highlights the regulatory axis from LMW-E to PKMYT1 and its predictive value for pathological complete response in TNBC patients receiving neoadjuvant chemotherapy. The selective PKMYT1 kinase inhibitor RP-6306 consistently induced DNA damage and inhibited tumor growth in in vitro and in vivo pre-clinical breast tumor models. Co-expression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306, emphasizing its therapeutic significance. Citation Format: Mi Li, Amriti Lulla, Cansu Karakas, Spiridon Tsavaschidis, Yan Wang, Tuyen Nguyun, Tuyen Bui, Gary Marshall, Kelly Hunt, Khandan Keyomarsi. Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-04.

Evaluation of in vitro immunostimulatory and cytotoxic effects of recombinant survivin protein in combination with doxorubicin and breast cancer antigen-loaded polycaprolactone nanoparticles

In this study, the immunostimulatory and anticancer activities of the doxorubicin (DOX), MCF-7 and MDA-MB-231 breast cancer antigen-loaded polycaprolactone (PCL) nanoparticles (NPs) in combination with survivin recombinant protein (RP) and an alum adjuvant are evaluated in vitro on J774 macrophage, MCF-7 and MDA-MB-231 breast cancer cell lines. A double-emulsion solvent evaporation method was used for encapsulation of DOX and antigens into PCL NPs. The physicochemical characterization of NPs included size, morphology, zeta potential, release profiles and encapsulation efficiencies, analyzed using scanning electron microscopy, a zeta-sizer and UV–vis spectrometry. The cytotoxic and inhibitory effects of NPs were determined using a methyl thiazolyl tetrazolium assay. Immunostimulatory effects of the NPs were detected by Griess reaction and ELISA tests to determine nitric oxide and cytokine levels, respectively. According to the results, DOX and antigen-loaded PCL NPs ranged between 240 nm and 290 nm. Antigen and drug-loaded NPs appear less toxic over macrophage cells in comparison with non-capsulated free agents. In addition, considerable inhibitory effects of antigen and drug-loaded NPs were observed at non-toxic concentrations, such as 25 and 50 μg ml−1, on human mammary cancer cell lines (p⩽ 0.0001). The amount of nitrite released from macrophages that were treated with antigen and DOX-encapsulated PCL NPs, in combination with alum and survivin RP, after 96 h incubation was significantly higher than the control, especially at 50 and 100 μg ml−1, and triggered macrophages to produce high quantities of IL-4 and IL-12 cytokines in contrast to the control. As a result, DOX and antigen-loaded PCL NPs in combination with survivin and alum adjuvant revealed significant immunostimulatory and inhibiting influence on macrophage and breast cancer cells, respectively. The outcomes revealed that antigen and drug-loaded PCL NPs supplemented with survivin RP and an alum adjuvant created an effective platform for the development of nanotechnology-based immunotherapeutic tools to inhibit breast cancer cells. However, these outputs should be supported by further in vivo studies.

Abstract 1339: Targeting specific extracellular matrix proteins to reactivate T cell trafficking in triple-negative breast cancer

Abstract It is now widely recognised that the tumour microenvironment (TME) is playing a crucial role in promoting tumour immune evasion by modulating the local immune infiltration. A growing body of evidence highlights the crucial role of the tumour extracellular matrix (ECM) in limiting trafficking of newly activated T cells in solid cancers, thus restricting their access to tumour and preventing the success of cancer immunotherapies. Accordingly, we are investigating whether targeting specific tumour ECM components may enhance T cell infiltration in triple-negative breast cancer (TNBC). Matrisome profiling of patient-derived fibroblasts and patient tumour tissues allowed us to identify two matrix proteins overexpressed in tumours and associated with T cell signatures, the matrix proteoglycan, versican (VCAN) and the glycoprotein, fibronectin (FN1). As there are currently no specific inhibitors of matrix proteins, we have modelled their loss-of-function using the clustered, regularly interspaced, short palindromic repeats-associated nuclease Cas9 (CRISPR-Cas9) technology with the goal of engineering cell-editable matrisomes which are enriched, reduced, or absent for specific matrix proteins. First, we constituted a panel of human and murine mammary fibroblast and TNBC cancer cell lines, which we have introduced with an inducible Cas9 protein expression. We measured VCAN and FN1 expression in the panel which showed distinct VCAN levels. Therefore, depending on VCAN basal levels, we have generated cell lines with VCAN overexpression, transient knockdown or stable knockout. Proteomic matrisome profiling of transient VCAN knockdown cells revealed that reducing VCAN may impact ECM abundance by decreasing VCAN direct binding partners. Finally, we used the edited cells to build 2D and 3D in vitro TME models to evaluate the impact of modulating VCAN expression on chimeric antigen receptor (CAR) T cell cytotoxicity and location. In parallel, we built in vivo mouse models of TNBC with controlled ECM deposition. Specifically, we have targeted mVcan and mFn1 in tumours and investigated the impact of their removal on tumour growth and immune cell infiltration. Overall, this work aims to identify immunomodulating features of the ECM which could be targeted to boost response to immunotherapies in solid cancers. Citation Format: Valentine Gauthier, Elly Tyler, Ying Liu, Tyson V. Sharp, Oliver MT Pearce. Targeting specific extracellular matrix proteins to reactivate T cell trafficking in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1339.

Herbal Honey Preparations of Curcuma Xanthorriza and Black Cumin Protect against Carcinogenesis through Antioxidant and Immunomodulatory Activities in Sprague Dawley (SD) Rats Induced with Dimethylbenz(a)anthracene

Traditionally, Curcuma xanthorriza (CX), black cumin seed (BC), and honey have been used by the Indonesian people as medicinal ingredients to treat various health symptoms. CX extracts and BC have been proven in the laboratory as chemopreventive agents, antioxidants, and immunomodulators. In this study, we developed CX extract, BC oil, and honey into herbal honey preparations (CXBCH) and hypothesized that the preparations show chemopreventive activity. The purpose of the study was to determine the CXBCH potential as chemopreventive, antioxidant, and immunomodulatory. In this experimental laboratory research, antioxidant, immunomodulatory, and cytotoxic activities were tested on human mammary cancer cell lines (T47D cells) while the chemopreventive activity of the CXBCH preparations on Sprague Dawley (SD) rats induced with dimethylbenzene(a)anthracene (DMBA). CXBCH preparations demonstrated immunomodulatory, antioxidant, and cytotoxic activities in T47D, Hela, and HTB-183 cells and in DMBA-induced SD rats, as the preparations inhibited tumor nodule formation, increased the number of CD4, CD8 and CD4CD25 cells, and glutathione-S-transferase (GST) activity, and decreased serum NO levels. CXBCH preparations display chemopreventive, antioxidant, and immunomodulatory properties.

The Use of the QbD Approach to Optimize the Co-Loading of Simvastatin and Doxorubicin in Liposomes for a Synergistic Anticancer Effect.

The present study aimed to optimize a liposomal formulation co-encapsulating simvastatin (SIM) and doxorubicin (DOX) that has future perspectives in anticancer therapy. The optimization process was performed by implementing the Quality by Design concept and by considering the results of a previous screening study. Failure Mode and Effects Analysis was used for the identification of the potential critical factors, i.e., phospholipid, SIM and DOX concentration, which were assessed in an optimization experimental design with the purpose of designing an optimal formulation. The optimal formulation, meeting the established quality profile, was additionally characterized in terms of the release profile and antiproliferative effects. During dissolution studies, a novel chronoamperometric method was used for the simultaneous quantification of SIM and DOX. The obtained data confirmed the similarity of this method with a validated HPLC method. The anticancer potential of the optimal formulation was tested against two human cancerous cell lines, namely T47D-KBluc human mammary ductal carcinoma cell line and A549 human pulmonary cancer cell line. The results highlighted that the antiproliferative effect of the optimal formulation is concentration dependent and favors a synergistic effect of the two drugs.

Establishment of a new cell line of canine inflammatory mammary cancer: IMC-118.

Canine inflammatory mammary cancer (IMC) has long been regarded as an attractive animal model for research into human inflammatory breast cancer (IBC), Although some canine mammary tumour cell lines corresponding to human mammary cancer cell lines have been established, there is still a need to supplement the canine mammary tumour cell bank. The goal of this study was to create a new type of IMC cell line. The primary tumour, IMC-118, was identified as IMC by pathology examination. Immunohistochemistry analysis revealed negative immunoreactivity to oestrogen receptor (ER), but positive immunoreactivity to progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2). Immunofluorescence (IF) analysis revealed that the IMC-118 cell line from this primary tumour was negative for ER but positive for PR and HER-2, and was also positive for epithelial and mesenchymal cell markers. This cell line was cultured stably for more than 50 passages and grew well after cryopreservation. In vivo, tumour masses and metastases in the lungs were discovered after inoculating the IMC-118 cells into the nude mice model. As a result, a novel canine IMC cell line, IMC-118, was effectively established, and could be employed as a promising model for immunotherapy and epithelial-mesenchymal transition mechanism of IMC research in both dogs and humans.

Abstract 2158: Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and mcf7 cells

Abstract Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA-sequencing and bioinformatics analyses, differentially expressed genes in significantly affected biological pathways in these cell types were validated by real-time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells. Citation Format: Heeyoun Bunch, Donguk Kim, Na Yeon Park, Jaehyeon Jeong, Keunsoo Kang, Stuart K. Calderwood, Dong-Hyung Cho, Ill Ju Bae. Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and mcf7 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2158.

Cytotoxic effect of multifruit polyphenol preparation on human breast cancer cell lines

Natural compounds are extensively used in the treatment of various diseases. Regular consumption of polyphenols plays an important role in the protection of health by reducing the risk of degenerative diseases, including cancer. The evaluation of the cytotoxic effect of the newly obtained multifruit polyphenolic preparation (composed of seven fruit) on T47D and MCF-7 breast cancer cells and MCF-12A normal cells. The PP was produced on the basis of combined ultrafiltrates obtained from chokeberry, raspberry, wild strawberry, apricot, peach, bilberry, and cranberry. The experiments were performed using human mammary gland cancer cell lines T47D (ductal cancer) and MCF-7 (adenocarcinoma) and normal breast cell line MCF-12A. Chromatographic techniques confirmed the highest contribution of cyanidin 3-O-glucoside, p-coumaroyl glucoside and chlorogenic acid in the PP. The PP exhibited dose-dependent cytotoxic effects towards MCF-7 and T47D cancer cell lines (IC50 = 1.2 μg . cm-3) and MCF-12A cells (IC50 = 0.6 μg . cm-3). The MTT cytotoxicity assay and microscopic observations confirmed the cytopathic effect of the PP on cell lines. It is supposed that berry polyphenols interfered with estrogen receptors leading to changes in the production of paracrine growth factors and therefore, PP was less cytotoxic towards the MCF-7 and T47D cell lines than against the MCF-12A cell line.

Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and MCF7 cells

Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA-sequencing and bioinformatics analyses, differentially expressed genes in significantly affected biological pathways in these cell types were validated by real-time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells.

Comparative Analysis of the Development of Acquired Radioresistance in Canine and Human Mammary Cancer Cell Lines.

Research using in vitro canine mammary cancer cell lines and naturally-occurring canine mammary tumors are not only fundamental models used to advance the understanding of cancer in veterinary patients, but are also regarded as excellent translational models of human breast cancer. Human breast cancer is commonly treated with radiotherapy; however, tumor response depends on both innate radiosensitivity and on tumor repopulation by cells that develop radioresistance. Comparative canine and human studies investigating the mechanisms of radioresistance may lead to novel cancer treatments that benefit both species. In this study, we developed a canine mammary cancer (REM-134) radioresistant (RR) cell line and investigated the cellular mechanisms related to the development of acquired radioresistance. We performed a comparative analysis of this resistant model with our previously developed human breast cancer radioresistant cell lines (MCF-7 RR, ZR-751 RR, and MDA-MB-231 RR), characterizing inherent differences through genetic, molecular, and cell biology approaches. RR cells demonstrated enhanced invasion/migration capabilities, with phenotypic evidence suggestive of epithelial-to-mesenchymal transition. Similarities were identified between the REM-134 RR, MCF-7 RR, and ZR-751 RR cell lines in relation to the pattern of expression of both epithelial and mesenchymal genes, in addition to WNT, PI3K, and MAPK pathway activation. Following the development of radioresistance, transcriptomic data indicated that parental MCF-7 and ZR-751 cell lines changed from a luminal A classification to basal/HER2-overexpressing (MCF-7 RR) and normal-like/HER2-overexpressing (ZR-751 RR). These radioresistant subtypes were similar to the REM-134 and REM-134 RR cell lines, which were classified as HER2-overexpressing. To our knowledge, our study is the first to generate a canine mammary cancer RR cell line model and provide a comparative genetic and phenotypic analysis of the mechanisms of acquired radioresistance between canine and human cancer cell lines. We demonstrate that the cellular processes that occur with the development of acquired radioresistance are similar between the human and canine cell lines; our results therefore suggest that the canine model is appropriate to study both human and canine radioresistant mammary cancers, and that treatment strategies used in human medicine may also be applicable to veterinary patients.

Synthesis and biological evaluation of 3-(2,4-dichlorophenoxymethyl)-1-phenyl-1H-pyrazole derivatives as potential antitumor agents

Series of novel 3-(2,4-dichlorophenoxymethyl)-1-phenyl-1H-pyrazole derivatives with various substituents at positions 4 and 5 were synthesized. Antitumor activity of all the synthesized compounds was evaluated by using SRB assay on four cell lines: lung carcinoma (A549), human hepatocellular carcinoma (HepG2), colon carcinoma (HCT116) and mammary gland breast cancer (MCF-7) cell lines. Some of the synthesized compounds showed promising activity in comparison with doxorubicin against lung carcinoma cell lines. Incorporating arylidene moiety at position-4 in pyrazole ring improved and displayed strong effect on the cytotoxic activity. Compound 9c incorporating 4-chlorophenyl moiety was the most potent toward all tested cell lines.

Circular RNA La-related RNA-binding protein 4 correlates with reduced tumor stage, as well as better prognosis, and promotes chemosensitivity to doxorubicin in breast cancer.

ObjectiveWe aimed to evaluate the correlation of circular RNA La‐related RNA‐binding protein 4 (circ‐LARP4) with tumor characteristics and prognosis, and its effect on chemosensitivity in breast cancer.MethodsCirc‐LARP4 from tumor and adjacent tissues of 283 female breast cancer patients underwent resection was detected by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Tumor features, disease‐free survival (DFS), and overall survival (OS) were recorded. In vitro, circ‐LARP4 in human normal mammary epithelial cells (HMEC) and breast cancer cell lines was detected by RT‐qPCR. MCF‐7 and MDA‐MB‐231 cells were transfected with circ‐LARP4 overexpression plasmid (as OE‐Circ group) and control overexpression plasmid (as OE‐Control group). Relative cell viability under different concentrations of doxorubicin was measured.ResultsCirc‐LARP4 was decreased in tumor tissues than adjacent tissues (P < .001). Tumor circ‐LARP4 negatively correlated with tumor size (P = .001), T stage (P = .009), N stage (P = .006), and TNM stage (P < .001), whereas positively correlated with DFS (P = .004) and OS (P < .001). In vitro, circ‐LARP4 was decreased MCF‐7, BT474, MDA‐MB‐231, and MDA‐MB‐468 cell lines than HMEC (all P < .001). Relatively cell viability of MCF‐7 cells (at 20 nmol/L [P < .05], 40 nmol/L [P < .01], 80 nmol/L [P < .05] of doxorubicin) and MDA‐MB‐231 cells (at 120 nmol/L [P < .05], 240 nmol/L [P < .05] of doxorubicin) was decreased in OE‐Circ group than OE‐Control group. IC50 value of doxorubicin was decreased in OE‐Circ group than OE‐Control group in MCF‐7 and MDA‐MB‐231 cell lines (both P < .01).ConclusionCirc‐LARP4 was a potential prognostic biomarker, which might improve the management of breast cancer.

SYNTHESIS AND CHARACTERIZATION OF NOVEL ORGANOBISMUTH FOR ANTIMICROBIAL AND ANTITUMOR STUDIES

Objectives: The major objective of this manuscript is to present synthesis and biomedical screening of some organic derivatives of bismuth having general formula (R3BiL2) by the method reported and characterized with the help of M.P., elemental, I.R., and NMR spectral analysis along with their antimicrobial and in vitro antitumor activity against human breast (MCF-7) and mammary cancer (EVSA-7) cell line.&#x0D; Methods: All the newly organobismuth having general formula [R3BiL2] were synthesised by the method reported especially using oxidative addition and complexation reactions.&#x0D; Results: It was found that organobismuth compounds have trigonal bipyramidal structure as per their elemental and spectral analysis and show potentiality as antimicrobial and antitumor agents.&#x0D; Conclusion: The newly synthesized organobismuth(V)substituted carboxylates were fully characterized chemically to ascertain their structure by sophisticated instrumental and spectral analysis resulted as trigonal bipyramidal structure. The compounds were also screened 1st time for antitumor and antimicrobial studies. The observations clearly indicated that organobismuth carboxylates show potent antimicrobial and antitumor activity.

ANTITUMOR AND GASTROPROTECTIVE SCREENING OF SOME NOVEL ORGANIC DERIVATIVES OF BISMUTH

Objectives: The objectives of present manuscript are to synthesize some new organic derivatives of bismuth having general formula (RBiL2; wherein R = C6F5; L = Substituted aromatic carboxylic acid), and characterized with the help of M.P., elemental, I.R., and NMR spectral analysis to ascertain the structure of novel compounds. These compounds were also characterized for their in vitro antitumor activity against human breast (MCF-7) and mammary cancer (EVSA-7) cell line along with gastroprotective (anti-ulcer) activity in rats using standard methods.&#x0D; Methods: All the newly organobismuth having general formula (RBiL2) were synthesised by earliar reported methods especially using redistribution and complexation reactions. The in-vitro antitumor and gastroprotective studies were performed by using standard protocols and methods.&#x0D; Results: It was found that the compounds were crystalline solids, air stable, and soluble in common organic solvent, have sharp melting points, and possess trigonal/pyramidal structure, exhibit higher activity than the standard ranitidine when the tests were carried out with aspirin induced and moderate activity was seen when the tests were done with ethanol induced along with potential antitumor activity against MCF-7 and EVSA-7 cell lines.&#x0D; Conclusion: The newly synthesized organobismuth complexes were characterized to ascertain their structure by sophisticated instrumental and spectral analysis resulted as trigonal/pyramidal structure. The compounds were also screened 1st time for gastroprotective and antitumor activity in vitro. The observations clearly indicate that organobismuth carboxylates as reported here show potential gastroprotective and antitumor activity in vitro.

Chromatin interactome mapping at 139 independent breast cancer risk signals

BackgroundGenome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression.ResultsWe designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers, and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals and explore the functional mechanism underlying altered risk at the 12q24 risk region.ConclusionsOur results demonstrate the power of combining genetics, computational genomics, and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.

The adverse effect of gentamicin on cell metabolism in three cultured mammary cell lines: "Are cell culture data skewed?"

Many cells are cultured in media that contains an antibiotic to prevent bacterial contamination. Mycoplasma and other bacterial contamination is a serious problem for those involved in cell culture. Antibiotics in the media helps prevent this contamination and make life easier for the investigators; as performing cell culture experiments in antibiotic free media is difficult and requires vigorous sterile technique. There are many reports of antibiotics causing mitochondrial damage. In this study, we tested the effect of gentamicin in culture media on human mammary epithelial MCF-12A and breast cancer MCF-7 and MDA-MB-231 cell lines by real time PCR, immunofluorescent microscopy, lactate assay, DNA damage assay. We found that the addition of gentamicin in media upregulated the gene expression of hypoxia inducer factor 1 alpha (HIF1a), glycolytic enzymes and glucose transporters, compared to the cells cultured in gentamicin free media. Gentamicin also increased the lactate production and inhibited mitochondrial membrane potential of the cell lines. Furthermore, the antibiotics in media induced mitochondrial reactive oxygen species causing DNA damage. We found an increase of 8-hydroxy-2’-deoxyguanosine a product of DNA oxidative damage in the media of MCF-12A, MCF-7 and MDA-MB-231 cell lines. These results showed that normal epithelial and breast cancer cells cultured in the media with gentamicin had increased HIF1a, aerobic glycolysis and DNA oxidative damage. If we use these unhealthy cells in the experiment, all data will be different, compared to cells grown in gentamicin free media. We have studied the detrimental effects of three antibiotics on mitochondrial function in the untransformed MCF-12A human mammary cell line and two human mammary cancer cell lines, MCF-7 and MB-MDA-231. The metabolic changes in all cell lines were dramatically different between those in antibiotic free media versus antibiotic containing media. There was a marked difference in gene expression of glycolytic enzymes, reactive oxygen species production and effects on membrane potential. Ironically, our first studies were done in media containing gentamicin, and repeated studies were done in gentamicin free media. The results were very different. The purpose of this report is to emphasize that metabolic cell culture data may be inaccurate because experiments were performed in cell culture media containing antibiotics. We will present evidence to support this theory.

Potentiating apoptosis and modulation of p53, Bcl2, and Bax by a novel chrysin ruthenium complex for effective chemotherapeutic efficacy against breast cancer.

Breast cancer is the most frequent cause of cancer in women. In the current study, transition metal ruthenium was complexed with flavonoid chrysin to evaluate the chemotherapeutic potential of this compound in Michigan Cancer Foundation-7 (MCF-7) human mammary cancer cell line and 7,12-dimethylbenz(α)anthracene-induced mammary cancer in female Sprague-Dawley rats. The characterizations of the complex were accomplished through UV-visible, NMR, IR, Mass spectra, and XRD techniques and antioxidant activity was assessed by DPPH, FRAP, and ABTS methods. In vitro studies included cell viability, cell cycle analysis, DNA fragmentation, and marker analysis by western blot analysis and found that complex treatment suppressed cell growth-induced cell cycle arrest and enhanced the induction of apoptosis in cancer cells. Moreover, complex treatment modulated signaling pathways including mTOR, VEGF, and p53 in the MCF-7 cells. Acute and subacute toxicity was performed in rats to determine the therapeutic doses. Breast cancer in rats was initiated by the administration of 7,12-dimethylbenz(α)anthracene (0.5 mg/100 g body weight) via single tail vein injection. The histopathological analysis after 24 weeks of carcinogenesis study depicted substantial repair of hyperplastic lesions. Immunohistochemical analysis revealed upregulation of Bax and p53 and downregulation of Bcl2 proteins and TUNEL assay showed an increase in apoptotic index in ruthenium-chrysin-treated groups as compared to the carcinogen control. Our findings from the in vitro and in vivo study support the continued investigation of ruthenium-chrysin complex possesses a potential chemotherapeutic activity against breast cancer and was efficient in reducing hyperplastic lesions in the mammary tissues of rats by inducing apoptosis.

Downregulation of Long Noncoding RNA HOTAIR and EZH2 Induces Apoptosis and Inhibits Proliferation, Invasion, and Migration of Human Breast Cancer Cells.

The long noncoding RNA HOTAIR (HOX transcript antisense intergenic RNA) has been reported to be a biomarker for various malignant tumors; however, its involvement in breast cancer is not fully understood. The aim of this study was to investigate the effects involved with long noncoding RNA HOTAIR and EZH2 (enhancer of zeste homologue 2) on the processes of proliferation, invasion, migration, and apoptosis of breast cancer cells. The expressions of HOTAIR and EZH2 in both normal human mammary epithelial cell (HBL-100) and breast cancer cell lines (MCF-7, MDA-MB-231, and SKBR-3) were detected by means of reverse transcription-quantitative polymerase chain reaction. The MCF-7 cells that exhibited the highest HOTAIR expressions were selected for further studies and divided into the control, negative control, and small interfering RNA-HOTAIR groups. The proliferation, invasion, migration, and apoptosis of breast cancer cells were evaluated by MTT assay, Scratch test, Transwell assay, and flow cytometry, respectively. The combination of HOTAIR with EZH2 and PTEN was predicted by bioinformation, with a dual-luciferase reporter gene assay providing further verification. Initially, lower expressions of HOTAIR and EZH2 in the normal human mammary epithelial cells, while higher expressions in the breast cancer cells of MCF-7, MDA-MB-231, and SKBR-3 were detected. In addition, the downregulation of HOTAIR or silencing of EZH2 was revealed to repress the proliferation, invasion, and migration, while acting to promote the apoptosis of the breast cancer cells. Furthermore, HOTAIR could bind specifically to EZH2 and PTEN, highlighting the capability of HOTAIR to inhibit the expression of PTEN by recruiting EZH2 in breast cancer, while the TCGA database demonstrated the expressions of PTEN were lower in breast cancer cells. The study suggests the higher expressions of HOTAIR and EZH2 among three breast cancer cells. Furthermore, the downregulation of HOTAIR or silencing of EZH2 was noted to inhibit the proliferation, invasion, and migration of breast cancer cells, while promoting their apoptosis.

Murine and Human Mammary Cancer Cell Lines: Functional Tests.

The biological characterization of mammary cancer cells is a prerequisite that helps the scientist understand some aspect of tumor biology. Once isolated from the tumor, cells are subjected to multiple tests that dissect their ability to growth, migrate, degrade the surrounding stroma, produce 3-dimensional structures and differentiate. Targeted inhibitors, when added to these tests, are used to unravel how specific growth factors, receptors, and intracellular translational pathways promote the ability of mammary tumor cells to achieve their biological behavior. Herein we describe a set of techniques used to put in focus the biological capacities in mammary cancer cells. When the characterization of a biological trait (e.g., proliferation) is assessable by multiple assays, we will limit the description to only one technique, possibly the easier to manage and that requires minimal laboratory equipment.

Vasculogenic Mimicry: Associated Ultrastructural Findings in Human and Canine Inflammatory Mammary Cancer Cell Lines

Vasculogenic Mimicry: Associated Ultrastructural Findings in Human and Canine Inflammatory Mammary Cancer Cell Lines