Normal Breast Cancer Cell Lines Research Articles

Bunium persicum Seeds Extract in Combination with Vincristine Mediates Apoptosis in MCF-7 Cells through Regulation of Involved Genes and Proteins Expression.

Bunium persicum seeds, a member of the Apiaceae family, have historically been consumed as part of the Iranian diet. While many of this herb's biological properties have been fully investigated, there is currently no reliable information about its anticancer/cytotoxic properties. Herein, we first determined the major bioactive compounds of B. persicum seed extract (BPSE) via GC-Mass analysis. We evaluated the cytotoxicity of the extract alone as well as in combination with vincristine (VCR), a commonly used chemotherapy drug, using MTT assays on two breast cancer cell lines, MCF-7 and MDA-MB-231, as well as a normal breast cancer cell line, MCF-10A. Moreover, these compounds were evaluated in vitro for their anticancer activity using ROS assays, Real-Time PCR, Western blots, flow cytometry, and cell cycle assays. As a result of our investigation, it was determined that the extract significantly reduced the viability of cancerous cells while remaining harmless to normal cells. The combination of BPSE and VCR also resulted in synergistic effects. BPSE and/or BPSE-VCR treatment increased the intracellular ROS of MCF-7 cells by over twofold. Moreover, the IC30 of BPSE (100 μg/ml) significantly increased the BAX/BCL-2 and P53 gene expression while reducing the expression of the MYC gene. Moreover, treated cells were arrested in the G2 phase of the cell cycle. The BPSE-VCR combination synergistically reduced the NF-κB and increased the Caspase-7 proteins' expression. The percent of apoptosis in the cells treated with the extract, VCR, and their combination was 27, 11, and 50, respectively. The present study demonstrated the anticancer activity of the BPSE and its potential for application in combination therapy with VCR.

Discovery of 5-(Pyrimidin-2-ylamino)-1H-indole-2-carboxamide Derivatives as Nur77 Modulators with Selective and Potent Activity Against Triple-Negative Breast Cancer.

The orphan nuclear receptor Nur77 has been validated as a potential drug target for treating breast cancer. Therefore, focusing on the SAR study of the lead 8b (KDSPR(Nur77) = 354 nM), we found the active compound ja which exhibited improved Nur77-binding capability (KDSPR(Nur77) = 91 nM) and excellent antiproliferative activities against breast cancer cell lines. Interestingly, ja acted as a potent and selective Nur77 antagonist, displaying good potency against triple-negative breast cancer (TNBC) cell lines but did not inhibit human normal breast cancer cell line MCF-10A (SI > 20). Exceptionally, ja Nur77-dependently caused mitochondria dysfunction and induced the caspase-dependent apoptosis by mediating the TP53 phosphorylation pathway. Moreover, ja significantly suppressed MDA-MB-231 xenograft tumor growth but had no apparent side effects in mice and zebrafish. Overall, ja demonstrated to be the first Nur77 modulator mediating the TP53 phosphorylation pathway that has the potential as a novel anticancer agent for TNBC.

Arylamine N-acetyltransferase 1 deficiency inhibits drug-induced cell death in breast cancer cells: switch from cytochrome C-dependent apoptosis to necroptosis

PurposeArylamine N-acetyltransferase 1 (NAT1) deficiency has been associated with drug resistance and poor outcomes in breast cancer patients. The current study aimed to investigate drug resistance in vitro using normal breast cancer cell lines and NAT1-deficient cell lines to understand the changes induced by the lack of NAT1 that resulted in poor drug response.MethodsThe response to seven chemotherapeutic agents was quantified following NAT1 deletion using CRISPR-Cas 9 in MDA-MB-231 and T-47D cells. Apoptosis was monitored by annexin V staining and caspase 3/7 activity. Cytochrome C release and caspase 8 and 9 activities were measured by Western blots. Caspase 8 was inhibited using Z-IETD-FMK and necroptosis was inhibited using necrostatin and necrosulfonamide.ResultsCompared to parental cells, NAT1 depleted cells were resistant to drug treatment. This could be reversed following NAT1 rescue of the NAT1 deleted cells. Release of cytochrome C in response to treatment was decreased in the NAT1 depleted cells, suggesting suppression of the intrinsic apoptotic pathway. In addition, NAT1 knockout resulted in a decrease in caspase 8 activation. Treatment with necrosulfonamide showed that NAT1 deficient cells switched from intrinsic apoptosis to necroptosis when treated with the anti-cancer drug cisplatin.ConclusionsNAT1 deficiency can switch cell death from apoptosis to necroptosis resulting in decreased response to cytotoxic drugs. The absence of NAT1 in patient tumours may be a useful biomarker for selecting alternative treatments in a subset of breast cancer patients.

Ginsenoside RG1 augments doxorubicin-induced apoptotic cell death in MDA-MB-231 breast cancer cell lines.

This study determined the chemosensitizing potential of ginsenoside Rg1 in triple-negative MDA-MB-231 breast cancer cell lines. Ginsenoside Rg1 (10 µM) treated breast cancer cells were exposed to 8 nM of doxorubicin, and the chemosensitizing potential was measured by cell-based assays. Ginsenoside Rg1 (10 µM) treatment lowered the doxorubicin IC50 value to 0.01 nM. Furthermore, the ginsenoside pretreatment augments doxorubicin-mediated reactive oxygen species (ROS) generation and subsequent alterations of mitochondrial membrane potential in MDA-MB-231 cell lines. The alkaline comet assay results illustrated an increased % tail DNA during ginsenoside Rg1 plus doxorubicin treatment than doxorubicin alone treatment. In addition, the number of apoptotic cells was also increased in ginsenoside Rg1 plus doxorubicin-treated cells. Furthermore, the polymerase chain reaction array results illustrate activation of mitogen-activated protein kinase (MAPK) gene expression (AKT, ERK, and MAPK) during doxorubicin alone treatment and it has been attenuated by ginsenoside Rg1 pretreatment. Moreover, ginsenoside Rg1 treatment before doxorubicin activates the DNA damage response elements (ATM, H2AX, RAD51, and XRCC1) and subsequent apoptosis-related gene expression (p21, TP53. APAF1, Bax, CASP3, and CASP9) patterns in MDA-MB-231 cell lines. The ginsenoside Rg1 plus doxorubicin combination shows less cytotoxicity and ROS generation in MDA10A normal breast cancer cell lines. Therefore, the present results support the chemosensitizing property of ginsenoside Rg1 in triple-negative breast cancer cell lines.

Docking Protein 2 Inhibits Proliferation, Migration and Invasion of Breast Cancer Cells via the Ras/Extracellular Regulated Protein Kinases Pathway

Background: This study was designed to investigate the effects of Downstream of Tyrosine Kinase 2, Docking Protein 2 (DOK2) on breast cancer cells, and its potential mechanism in disease pathogenesis. Methods: The expression of DOK2 in human breast cancer cell lines and mammary epithelial cells were assessed by RT-qPCR and Western blot assay. CCK8 was used to evaluate cell proliferation, wound healing and transwell were used to detect cell migration and invasion. Furthermore, Western blot was used to detect the expression of migration-related proteins, MMP2, MMP9 and Ras/ERK pathway-related proteins. Results: The expression level of DOK2 was significantly lower in breast cancer MCF7 and MDA-MB-231 cells compared with the normal breast cancer cell line MCF10A. To further investigate the function of DOK2, the overexpressed plasmid of DOK2 was transfected into MCF7 and MDA-MB-231 cells, the results revealed that DOK2 markedly inhibited cell proliferation, cell migration and invasion via inhibiting the Ras/ERK pathway. Conclusions: Collectively, the data demonstrated that DOK2 could directly inhibits proliferation, migration and invasion of breast cancers via inhibiting the Ras/ERK pathway.

Abstract 1334: Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in triple negative breast cancer

Abstract Recent studies correlate Hedgehog (Hh) signaling with reduced survival rates in triple negative breast cancer (TNBC) patients. Activation of hedgehog signaling in the adjacent mesenchyme has been shown to promote tumor growth and it is a poor prognosis factor for TNBC. We developed a novel tumor-mesenchymal in vitro model of hedgehog signaling in TNBC to evaluate the role of mesenchymal cell sub-populations in the proliferative potential and stem cell markers of breast cancer cells using a custom designed multiwell array. As a source of mesenchymal cell sub-populations we evaluated myofibroblasts, mesenchymal stem cells derived from bone marrow or adipose tissue and tumor cells that undergo epithelial-mesenchymal transition (EMT). Tumor cells were culture with 1 or 2 mesenchymal cell sub-populations in adjacent compartments +/- SHH ligand for 72-96hrs. Active Hh signaling was confirmed by up-regulation of main Hh target genes (e.g. Gli1, Patch1). Paracrine Hh signaling only in bone marrow-derived mesenchymal stem cell and EMT significantly increased proliferation (10-15%) of TNBC and normal breast cancer cell lines. Addition of adipose-derived mesenchymal stem cells or pharmacological Hh inhibitors (Cyclopamine and Gant61) partially abolished tumor growth indicating that other non-canonical signals are involve in paracrine Hh-driven tumors. Cancer stem cell markers were selectively modulated in co-cultures. Our results suggest that paracrine Hh signaling-driven by bone marrow and EMT mesenchymal are potential therapeutic targets to treating TNBC. Citation Format: Karla P. Ramos, Maribella Domenech. Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in triple negative breast cancer [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 1334. doi:10.1158/1538-7445.AM2017-1334

Abstract 1230: Detection of increased O-GlcNAcylated p53 levels in MCF-7 cells after O-GlcNAcase inhibitor treatment using antibodies specific for Ser-149-O-GlcNAcylated p53 proteins

Abstract Protein O-GlcNAcylation (N-acetylglucosamine modification) plays a critical role in cell-cycle regulation, apoptosis and signal transduction. Most serine (Ser) and threonine (Thr) sites for O-GlcNAcylation are on or near to phosphorylation sites, which creates a system of mutual elimination. O-GlcNAcylation of p53, a tumor suppressor protein, at Ser-149 prevents p53 degradation and stabilizes p53 proteins expressed in MCF-7 human breast cancer cells whereas phosphorylation of the p53 at Thr-155 induces ubiquitin-dependent degradation, which decreases p53 levels. Accumulation of the p53 protein in cancer cells induces arrest of cell growth and apoptosis in response to DNA damage. Ser-149 site-specific anti-peptide antibodies have been produced using KLH conjugated with p53 peptides with and without O-GlcNAcylation at Ser-149 by immunization of rabbits for polyclonal and mice for monoclonal antibodies. Whereas polyclonal antibodies and hybridoma media for O-GlcNAcylated p53 cross-reacted with BSA conjugated with Ser-149-O-GlcNAcylated p53 peptides in ELISA or Western blot analysis, the antibodies did not cross-react with BSA conjugated with p53 peptides without O-GlcNAcylation. Anti-peptide polyclonal antibodies produced for the unmodified p53 peptides did not cross-react with BSA conjugated with Ser-149 O-GlcNAcylated p53 peptides. Using the hybridoma media for Ser-149-O-GlcNAcylated p53, Western blot analysis was carried out with nuclear lysates and cytosol-containing supernatant obtained from MCF10A (normal breast cancer cell line) and MCF-7 cells. It was found that, in MCF-10A cells, Ser-149-O-GlcNAcylated p53 proteins (∼55 kDa) were minimally expressed in the nuclear fraction but highly expressed in the cytosolic fraction whereas, in MCF-7 cells, the level of cytosolic Ser-149-O-GlcNAcylated p53 protein was low compared with the level in MCF-10A cells. MCF-7 cells were treated for 24 hr with 1% DMSO or 2 mM β-N-acetylglucosaminidase (O-GlcNAcase) inhibitor, acetazolamide, dissolved in DMSO and levels of Ser-149-O-GlcNAcylated p53 were determined by Western blot analysis using antibodies for the Ser-149-O-GlcNAcylated p53. Whereas the level of O-GlcNAcylated p53 did not increase after 1% DMSO treatment, the level dramatically increased after treatment of acetazolamide in 1% DMSO and induced cell death. Our results showed that acetazolamide treatment increased Ser-149-O-GlcNAcylated p53 level and induced cell death in MCF-7 cells, which may decrease invasiveness of the breast cancer cells. Supported by NCI SBIR Phases I and II Contracts No. N261201100073C and N261201300058C. Citation Format: Hyesook Kim, So Hee KIm, Aby Joiakim, David Kaplan, Sung Hun Bae, David Putt. Detection of increased O-GlcNAcylated p53 levels in MCF-7 cells after O-GlcNAcase inhibitor treatment using antibodies specific for Ser-149-O-GlcNAcylated p53 proteins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1230. doi:10.1158/1538-7445.AM2015-1230

Abstract 1472: The differential expression of miRNAs in breast cancer cell lines

Abstract Breast cancer is a heterogenous disease that is the second leading cause of death amongst women in the United States. Triple negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by its loss of estrogen, progesterone and HER2 receptor expression which limits its targets for effective drug therapies. Micro Ribonucleic Acids (miRNAs) are a class of small, endogenous non-coding ribonucleic acids that seem to play an essential role in gene regulation. Changes in the patterns of miRNA expression profiles are serving as potential biomarkers for tumor diagnosis, prognosis of disease-specific outcomes, and the prediction of therapeutic responses in cancer. We analyzed miRNA array (Agilent) expression profiles in triple negative breast cancer cell lines (HCC70, HCC1806, MDA-MB-157) and non-cancerous (AG11132) African American women breast cell lines. We revealed that the TNBC cells exhibited a different miRNA expression pattern when compared to the normal breast cancer cell line. We analyzed the hybridization data files using standard Microsoft Excel techniques and verified our findings using PARTEK Genomics Suite. We combined the data sets from the TNBC cell lines and compared the miRNA array expression profiles to the normal control cell line AG11132. We revealed that there were significant dysregulation of miRNAs including those of the let-7 family, miR-16, miR-31, and miR-25b-1, miR34a, and miR-95 in the triple negative cells as compared to the normal cells. These miRNAs have been described in previous scientific studies to be associated with tumorigenesis in breast cancer. These studies are initial observations that we will follow up with molecular techniques such as real-time PCR and siRNA to validate the results in order to establish more direct correlations to miRNAs and their roles in triple negative breast cancer. Citation Format: Checo J. Rorie, Brianna L. Arrington, Sherette S. Godfrey, Armeshia S. McCoy, Kashenya M. Gurley. The differential expression of miRNAs in breast cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1472. doi:10.1158/1538-7445.AM2014-1472

Toxicity evaluation of some traditional African spices on breast cancer cells and isolated rat hepatic mitochondria

BackgroundFagara leprieuri (FL), Fagara xanthoxyloïdes (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) are used in many African countries as food spices or in traditional medicine to treat several maladies. In this work, we (a) investigate whether the crude spice extracts present selective cytotoxicity for breast cancer cell lines and (b) investigate whether the same extracts affect the bioenergetics and calcium susceptibility of isolated liver mitochondrial fractions. ResultsAll extracts were cytotoxic to the cell lines studied, with the exception of MW, which was less toxic for a normal cell line. Interestingly, some of the extracts did not depolarize mitochondria in intact breast cancer MCF-7 cells, although this effect was observed in a normal breast cancer cell line (MCF-12A). All extracts increased hepatic mitochondrial state 2/4 respiration and decreased the respiratory control ratio and the transmembrane electric potential. Also, the extracts induced the mitochondrial permeability transition (MPT). ConclusionsMitochondrial toxicity may be part of the mechanism by which the spices tested cause inhibition of proliferation and death in the cell lines tested. This study also warrants caution in the excessive use of these spices for human consumption.

Abstract PD02-09: Effects of an mTOR Inhibitor RAD001 on Human Breast Cancer Stem Cells In Vitro

Abstract Background: Recent evidences have suggested that there are a rare population of cells called cancer stem cells (CSCs) in breast cancer, which have the ability of extensive self-renewal and contribute to metastasis and treatment resistance. This study evaluated the effects of an mTOR inhibitor, RAD001 (Everolimus) on breast CSCs of primary breast cancer cells and 2 breast cancer cell lines (MCF-7, MDA-MB-231) in vitro. Methods: Primarily, we isolated primary breast cancer cells from the 1cm3 axenic tissue from breast cancer patients, which was been digested in collagenase typelV in 1-3 hours. After filtration, the cells from the organoid were plated in DMEM/F12(1:1) containing 10% FBS ,100U/ml penicillin,100µg/ml streptomycin and some growth factors overnight. The next day, we sorted CD44 +, CD24-/low, ESA + cells as breast CSCs by flow cytometry from primary breast cancer cells, breast cancer cell lines MCF-7 and MDA-MB-231. And then the three CSCs were treated with different concentrations of docetaxel alone(0,20µM,40µM,80µM, 160µM), mTOR inhibitor RAD001 alone (0,10nM,100nM,1µM,10µM), or in combination with docetaxel(20µM).the Inhibition of the drugs on different Breast CSCs was quantified by methyl thiazolyl tetrazolium (MTT) assay. All employed CSCs were divided into four groups, given respectively 24h treatment of RAD001(100nM), docetaxel(20µM), combination or contrals. Apoptosis and distribution of cell cycles were examined with flow cytometry. Experiments were performed in triplicate. Result: All three kinds of breast CSCs were resistant to the standard treatment doses of docetaxel in vitro(IC50=80 µM±5µM),compared with this drug inhibited normal breast cancer cell lines(IC50=16µM±2µM). Treatment with RAD001 resulted in growth inhibition of all employed CSCs in a dose-dependent manner, which is more effective than the treatment with docetaxel alone(P<0.001), and CSCs of MDA-MB-231 proved to be most sensitive to this drug(IC20=8nM).Combination studies showed that there was an additive growth inhibitory effect of a combination treatment on three CSCs in vitro compared with treatment with RAD001 alone (P<0.001)or docetaxel alone(P<0.001). In addition, an increase in G2-M cell cycle arrest were seen in the combination treatment group when compared with controls(P<0.05), suggesting that cell cycle arrest may contribute to the increased growth inhibitory effects of combination treatment seen in this study. In primary CSCs, combined treatment induced a mount-up population of early apoptosis, the phenomenon was not taken place in the treatment of RAD001 alone, however, it's no statistical significance(P>0.05). Discussion: We conclude that RAD001 has more effective inhibitory effect than docetaxel, and enhances the cytotoxic effects of docetaxel in models of breast CSCs in vitro by inducing cell cycle arrest, indicating that combination treatment with RAD001 and docetaxel may be an efficient therapy for refractory and metastatic breast cancer. But the drug interaction of RAD001 and docetaxel in the clinical situation has to be evaluated in further studies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-09.