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Sample records for overexpressing human breast

  1. Hyaluronan synthase 2 overexpression is correlated with the tumorigenesis and metastasis of human breast cancer.

    PubMed

    Li, Peng; Xiang, Tingxiu; Li, Hongzhong; Li, Qianqian; Yang, Bing; Huang, Jing; Zhang, Xiang; Shi, Yuan; Tan, Jinxiang; Ren, Guosheng

    2015-01-01

    Extracellular matrix (ECM) is closely correlated with the malignant behavior of breast cancer cells. Hyaluronan (HA) is one of the main components of ECM, and actively regulates cell adhesion, migration and proliferation by interacting with specific cell surface receptors such as CD44 and RHAMM. HA synthase 2 (HAS2) catalyzes the sysnthesis of HA, but its role in breast tumorigenesis remains unclear. This study assessed the roles of HAS2 in malignant behavior of human breast cancer and sought to provide mechanistic insights into the biological and pivotal roles of HAS2. We observed HAS2 was overexpressed in breast cancer cell lines and invasive duct cancer tissues, compared with the nonmalignant breast cell lines and normal breast tissues. In addition, a high level of HAS2 expression was statistically correlated with lymph node metastasis. Functional assays showed that knockdown of HAS2 expression inhibited breast tumor cell proliferation in vivo and in vitro, through the induction of apoptosis or cell cycle arrest. Further studies showed that the HA were elevated in breast cancer, and HAS2 could upregulate HA expression. In conclusion, HAS2-HA system influences the biological characteristics of human breast cancer cells, and HAS2 may be a potential prognostic marker and therapeutic target in breast cancer. PMID:26722395

  2. Hyaluronan synthase 2 overexpression is correlated with the tumorigenesis and metastasis of human breast cancer

    PubMed Central

    Li, Peng; Xiang, Tingxiu; Li, Hongzhong; Li, Qianqian; Yang, Bing; Huang, Jing; Zhang, Xiang; Shi, Yuan; Tan, Jinxiang; Ren, Guosheng

    2015-01-01

    Extracellular matrix (ECM) is closely correlated with the malignant behavior of breast cancer cells. Hyaluronan (HA) is one of the main components of ECM, and actively regulates cell adhesion, migration and proliferation by interacting with specific cell surface receptors such as CD44 and RHAMM. HA synthase 2 (HAS2) catalyzes the sysnthesis of HA, but its role in breast tumorigenesis remains unclear. This study assessed the roles of HAS2 in malignant behavior of human breast cancer and sought to provide mechanistic insights into the biological and pivotal roles of HAS2. We observed HAS2 was overexpressed in breast cancer cell lines and invasive duct cancer tissues, compared with the nonmalignant breast cell lines and normal breast tissues. In addition, a high level of HAS2 expression was statistically correlated with lymph node metastasis. Functional assays showed that knockdown of HAS2 expression inhibited breast tumor cell proliferation in vivo and in vitro, through the induction of apoptosis or cell cycle arrest. Further studies showed that the HA were elevated in breast cancer, and HAS2 could upregulate HA expression. In conclusion, HAS2-HA system influences the biological characteristics of human breast cancer cells, and HAS2 may be a potential prognostic marker and therapeutic target in breast cancer. PMID:26722395

  3. DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines

    PubMed Central

    Roll, J Devon; Rivenbark, Ashley G; Jones, Wendell D; Coleman, William B

    2008-01-01

    Background DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines. Results The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A. This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers. Conclusion These observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers. PMID:18221536

  4. The gene associated with trichorhinophalangeal syndrome in humans is overexpressed in breast cancer.

    PubMed

    Radvanyi, Laszlo; Singh-Sandhu, Devender; Gallichan, Scott; Lovitt, Corey; Pedyczak, Artur; Mallo, Gustavo; Gish, Kurt; Kwok, Kevin; Hanna, Wedad; Zubovits, Judith; Armes, Jane; Venter, Deon; Hakimi, Jalil; Shortreed, Jean; Donovan, Melinda; Parrington, Mark; Dunn, Pamela; Oomen, Ray; Tartaglia, James; Berinstein, Neil L

    2005-08-01

    A comprehensive differential gene expression screen on a panel of 54 breast tumors and >200 normal tissue samples using DNA microarrays revealed 15 genes specifically overexpressed in breast cancer. One of the most prevalent genes found was trichorhinophalangeal syndrome type 1 (TRPS-1), a gene previously shown to be associated with three rare autosomal dominant genetic disorders known as the trichorhinophalangeal syndromes. A number of corroborating methodologies, including in situ hybridization, e-Northern analysis using ORF EST (ORESTES) and Unigene EST abundance analysis, immunoblot and immunofluorescence analysis of breast tumor cell lines, and immunohistochemistry, confirmed the microarray findings. Immunohistochemistry analysis found TRPS-1 protein expressed in >90% of early- and late-stage breast cancer, including ductal carcinoma in situ and invasive ductal, lobular, and papillary carcinomas. The TRPS-1 gene is also immunogenic with processed and presented peptides activating T cells found after vaccination of HLA-A2.1 transgenic mouse. Human T cell lines from HLA-A*0201+ female donors exhibiting TRPS-1-specific cytotoxic T lymphocyte activity could also be generated. PMID:16043716

  5. The gene associated with trichorhinophalangeal syndrome in humans is overexpressed in breast cancer

    PubMed Central

    Radvanyi, Laszlo; Singh-Sandhu, Devender; Gallichan, Scott; Lovitt, Corey; Pedyczak, Artur; Mallo, Gustavo; Gish, Kurt; Kwok, Kevin; Hanna, Wedad; Zubovits, Judith; Armes, Jane; Venter, Deon; Hakimi, Jalil; Shortreed, Jean; Donovan, Melinda; Parrington, Mark; Dunn, Pamela; Oomen, Ray; Tartaglia, James; Berinstein, Neil L.

    2005-01-01

    A comprehensive differential gene expression screen on a panel of 54 breast tumors and >200 normal tissue samples using DNA microarrays revealed 15 genes specifically overexpressed in breast cancer. One of the most prevalent genes found was trichorhinophalangeal syndrome type 1 (TRPS-1), a gene previously shown to be associated with three rare autosomal dominant genetic disorders known as the trichorhinophalangeal syndromes. A number of corroborating methodologies, including in situ hybridization, e-Northern analysis using ORF EST (ORESTES) and Unigene EST abundance analysis, immunoblot and immunofluorescence analysis of breast tumor cell lines, and immunohistochemistry, confirmed the microarray findings. Immunohistochemistry analysis found TRPS-1 protein expressed in >90% of early- and late-stage breast cancer, including ductal carcinoma in situ and invasive ductal, lobular, and papillary carcinomas. The TRPS-1 gene is also immunogenic with processed and presented peptides activating T cells found after vaccination of HLA-A2.1 transgenic mouse. Human T cell lines from HLA-A*0201+ female donors exhibiting TRPS-1-specific cytotoxic T lymphocyte activity could also be generated. PMID:16043716

  6. Human Epidermal Growth Factor Receptor Family-Targeted Therapies in the Treatment of HER2-Overexpressing Breast Cancer

    PubMed Central

    Eroglu, Zeynep; Tagawa, Tomoko

    2014-01-01

    Breast cancer characterized by overexpression of human epidermal growth factor receptor 2 (HER2) has been associated with more aggressive disease progression and a poorer prognosis. Although an improved understanding of breast cancer pathogenesis and the role of HER2 signaling has resulted in significant survival improvements in the past 20 years, resistance to HER2-targeted therapy remains a concern. A number of strategies to prevent or overcome resistance to HER2-targeted therapy in breast cancer are being evaluated. This article provides a comprehensive review of (a) the role of HER2 signaling in breast cancer pathogenesis, (b) potential receptor and downstream therapeutic targets in breast cancer to overcome resistance to HER2-targeted therapy, and (c) clinical trials evaluating agents targeting one or more members of the HER family and/or downstream pathways for the treatment of breast cancer, with a focus on metastatic disease. PMID:24436312

  7. EpCAM overexpression prolongs proliferative capacity of primary human breast epithelial cells and supports hyperplastic growth

    PubMed Central

    2013-01-01

    Introduction The Epithelial Cell Adhesion Molecule (EpCAM) has been shown to be strongly expressed in human breast cancer and cancer stem cells and its overexpression has been supposed to support tumor progression and metastasis. However, effects of EpCAM overexpression on normal breast epithelial cells have never been studied before. Therefore, we analyzed effects of transient adenoviral overexpression of EpCAM on proliferation, migration and differentiation of primary human mammary epithelial cells (HMECs). Methods HMECs were transfected by an adenoviral system for transient overexpression of EpCAM. Thereafter, changes in cell proliferation and migration were studied using a real time measurement system. Target gene expression was evaluated by transcriptome analysis in proliferating and polarized HMEC cultures. A Chicken Chorioallantoic Membrane (CAM) xenograft model was used to study effects on in vivo growth of HMECs. Results EpCAM overexpression in HMECs did not significantly alter gene expression profile of proliferating or growth arrested cells. Proliferating HMECs displayed predominantly glycosylated EpCAM isoforms and were inhibited in cell proliferation and migration by upregulation of p27KIP1 and p53. HMECs with overexpression of EpCAM showed a down regulation of E-cadherin. Moreover, cells were more resistant to TGF-β1 induced growth arrest and maintained longer capacities to proliferate in vitro. EpCAM overexpressing HMECs xenografts in chicken embryos showed hyperplastic growth, lack of lumen formation and increased infiltrates of the chicken leukocytes. Conclusions EpCAM revealed oncogenic features in normal human breast cells by inducing resistance to TGF-β1-mediated growth arrest and supporting a cell phenotype with longer proliferative capacities in vitro. EpCAM overexpression resulted in hyperplastic growth in vivo. Thus, we suggest that EpCAM acts as a prosurvival factor counteracting terminal differentiation processes in normal mammary glands. PMID:23758908

  8. HER-2 overexpression differentially alters transforming growth factor-? responses in luminal versus mesenchymal human breast cancer cells

    PubMed Central

    Wilson, Cindy A; Cajulis, Elaina E; Green, Jennifer L; Olsen, Taylor M; Chung, Young Ah; Damore, Michael A; Dering, Judy; Calzone, Frank J; Slamon, Dennis J

    2005-01-01

    Introduction Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-?) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-?1 on breast cancer cells in the presence or absence of overexpressed HER-2. Methods Cell proliferation assays were used to determine the effect of TGF-? on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-?1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-? signaling pathway was assessed using TGF-?1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays. Results We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-?1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-? in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-? induced pro-invasive and pro-metastatic gene signature. Conclusion HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-?. In contrast, HER-2 and TGF-? signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model. PMID:16457687

  9. Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11-q21.3 region of chromosome 17

    SciTech Connect

    Tomasetto, C.; Regnier, C.; Basset, P.

    1995-08-10

    We have performed differential screening of a human metastatic lymph node cDNA library to identify genes possibly involved during breast cancer progression. We have identified four novel genes overexpressed in malignant tissues. They were all located between q11 and q21.3, a region known to contain the c-erbB-2 oncogene and the BRCA1 breast carcinomas, and overexpression of three of them was dependent on gene amplification in breast cancer cell lines. These findings further support the concept that human chromosome 17 specifically carries genes possibly involved in breast cancer progression. 61 refs., 3 figs., 4 tabs.

  10. Notch3 overexpression causes arrest of cell cycle progression by inducing Cdh1 expression in human breast cancer cells.

    PubMed

    Chen, Chun-Fa; Dou, Xiao-Wei; Liang, Yuan-Ke; Lin, Hao-Yu; Bai, Jing-Wen; Zhang, Xi-Xun; Wei, Xiao-Long; Li, Yao-Chen; Zhang, Guo-Jun

    2016-02-01

    Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27(Kip) accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27(Kip) at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27(Kip) accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment. PMID:26694515

  11. Overexpression of p65 attenuates celecoxib-induced cell death in MDA-MB-231 human breast cancer cell line

    PubMed Central

    2013-01-01

    Background Celecoxib is a selective cyclooxygenase (COX)-2 inhibitor that has been reported to reduce the risk of breast cancer. In our previous study, celecoxib induced apoptosis and caused cell cycle arrest at the G0/G1 phase in the breast cancer cell line MDA-MB-231, and its effects were mediated by downregulation of NF-κB signaling. The NF-κB p65/RelA subunit may play a role in cell death through the activation of anti-apoptotic target genes including the inhibitor of apoptosis (IAP) and Bcl-2 families, and inhibition of protein kinase B/Akt. The aim of the present study was to investigate p65 as the potential target of celecoxib treatment and determine whether p65 overexpression can override the inhibitory effect of celecoxib on NF-κB activity and affect cell survival. Methods The effects of p65 overexpression on celecoxib-inhibited NF-κB transcriptional activity were examined by western blotting, electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assay. Cell viability and cell death were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, and the levels of cleaved poly(ADP-ribose) polymerase (PARP) and caspase. Anti-apoptotic NF-κB target genes and cell cycle regulators were examined by western blotting to screen for the expression of target genes under direct regulation by p65. Results Overexpression of p65 increased NF-κB transcriptional activity and interfered with celecoxib-mediated apoptosis as assessed by MTT assay and caspase-3, caspase-9, and PARP expressions. Exogenously overexpressed p65 upregulated NF-κB-responsive genes, including anti-apoptotic genes such as survivin and XIAP, and the cell cycle regulatory gene cyclin D1. However, p65 overexpression did not affect celecoxib-induced p-Akt inactivation, suggesting that celecoxib might have separate molecular mechanisms for regulating Akt signaling independently of its inhibition of NF-κB transcriptional activity. Conclusions p65 is a pivotal anti-apoptotic factor that can reverse celecoxib-induced growth inhibition in MDA-MB-231 cells. PMID:23402310

  12. Flaxseed oil enhances the effectiveness of trastuzumab in reducing the growth of HER2-overexpressing human breast tumors (BT-474).

    PubMed

    Mason, Julie K; Fu, Minghua; Chen, Jianmin; Thompson, Lilian U

    2015-01-01

    Flaxseed oil (FSO) reduces breast tumorigenesis and HER2 expression in animal models of luminal breast cancer. The primary treatment for HER2-overexpressing tumors is trastuzumab (TRAS). We aimed to determine the effect of 4% FSO alone and combined with TRAS on HER2-overexpressing tumor (BT-474) growth and to explore potential mechanisms with a specific focus on HER2, mitogen-activated protein kinase (MAPK) and Akt signaling and fatty acid profile. Athymic mice with established tumors were fed the basal diet (control) or 4% FSO diet, with or without TRAS (1 or 2.5 mg/kg) treatment for 4 weeks. Tumor growth, HER2 signaling biomarkers (mRNA and protein) and fatty acid profile were measured. Tumors treated with FSO alone showed no difference in tumor growth compared to control; however, compared to TRAS2.5 and other groups, FSO+TRAS2.5 caused significantly lower tumor growth and cell proliferation and higher apoptosis and the greatest lowering of signaling biomarker expressions (MAPK2, HER2 mRNA; pHER2 protein). Both TRAS and FSO had main effects of reducing the phosphorylated/total expression of Akt and MAPK protein expression. Dietary FSO altered the tumor fatty acid profile. In conclusion, 4% dietary FSO alone does not affect BT-474 tumor growth but enhances the tumor-reducing effect of TRAS (2.5 mg/kg). FSOTRAS interactive effect may be modulated by their combined reductions of HER2 signaling through the Akt and MAPK pathways leading to reduced cell proliferation and increased apoptosis. FSO alters tumor fatty acid profile that likely contributes to effects on signaling pathways. This supports FSO as a complementary treatment for HER2+ breast cancer treated with TRAS. PMID:25441844

  13. 18F-fluorodeoxy-glucose positron emission tomography marks MYC-overexpressing human basal-like breast cancers.

    PubMed

    Palaskas, Nicolaos; Larson, Steven M; Schultz, Nikolaus; Komisopoulou, Evangelia; Wong, Justin; Rohle, Dan; Campos, Carl; Yannuzzi, Nicolas; Osborne, Joseph R; Linkov, Irina; Kastenhuber, Edward R; Taschereau, Richard; Plaisier, Seema B; Tran, Chris; Heguy, Adriana; Wu, Hong; Sander, Chris; Phelps, Michael E; Brennan, Cameron; Port, Elisa; Huse, Jason T; Graeber, Thomas G; Mellinghoff, Ingo K

    2011-08-01

    In contrast to normal cells, cancer cells avidly take up glucose and metabolize it to lactate even when oxygen is abundant, a phenomenon referred to as the Warburg effect. This fundamental alteration in glucose metabolism in cancer cells enables their specific detection by positron emission tomography (PET) following i.v. injection of the glucose analogue (18)F-fluorodeoxy-glucose ((18)FDG). However, this useful imaging technique is limited by the fact that not all cancers avidly take up FDG. To identify molecular determinants of (18)FDG retention, we interrogated the transcriptomes of human-cancer cell lines and primary tumors for metabolic pathways associated with (18)FDG radiotracer uptake. From ninety-five metabolic pathways that were interrogated, the glycolysis, and several glycolysis-related pathways (pentose phosphate, carbon fixation, aminoacyl-tRNA biosynthesis, one-carbon-pool by folate) showed the greatest transcriptional enrichment. This "FDG signature" predicted FDG uptake in breast cancer cell lines and overlapped with established gene expression signatures for the "basal-like" breast cancer subtype and MYC-induced tumorigenesis in mice. Human breast cancers with nuclear MYC staining and high RNA expression of MYC target genes showed high (18)FDG-PET uptake (P < 0.005). Presence of the FDG signature was similarly associated with MYC gene copy gain, increased MYC transcript levels, and elevated expression of metabolic MYC target genes in a human breast cancer genomic dataset. Together, our findings link clinical observations of glucose uptake with a pathologic and molecular subtype of human breast cancer. Furthermore, they suggest related approaches to derive molecular determinants of radiotracer retention for other PET-imaging probes. PMID:21646475

  14. A Novel Function for the nm23-Hl Gene: Overexpression in Human Breast Carcinoma Cells Leads to the Formation of Basement Membrane and Growth Arrest

    SciTech Connect

    Howlett, Anthony R; Petersen, Ole W; Steeg, Patricia S; Bissell, Mina J

    1994-01-01

    We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia. Our purpose was to use this assay to investigate the role of the putative metastasis suppressor gene nm23-H1 in mammary development and differentiation. The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest. In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship. The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene expression and morphological-biosynthetic-growth aspects of breast differentiation in this model system. While the basement membrane microenvironment is capable of directing the differentiation of normal human breast cells, neoplastic transformation abrogates this relationship, suggesting that intrinsic cellular events are also critical to this process. The data identify nm23-H1 gene expression as one of these events, suggesting an important role in the modulation of cellular responsiveness to the microenvironment. The data also identify previously unknown growth inhibitory effects of nm23-H1 gene overexpression.

  15. Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo.

    PubMed

    Zhong, Yinan; Goltsche, Katharina; Cheng, Liang; Xie, Fang; Meng, Fenghua; Deng, Chao; Zhong, Zhiyuan; Haag, Rainer

    2016-04-01

    The therapeutic efficacy of nanoscale anticancer drug delivery systems is severely truncated by their low tumor-targetability and inefficient drug release at the target site. Here, we report the design and development of novel endosomal pH-activatable paclitaxel prodrug micelles based on hyaluronic acid-b-dendritic oligoglycerol (HA-dOG-PTX-PM) for active targeting and effective treatment of CD44-overexpressing human breast cancer xenografts in nude mice. HA-dOG-PTX-PM had a high drug content of 20.6 wt.% and an average diameter of 155 nm. The release of PTX was slow at pH 7.4 but greatly accelerated at endosomal pH. MTT assays, flow cytometry and confocal experiments showed that HA-dOG-PTX-PM possessed a high targetability and antitumor activity toward CD44 receptor overexpressing MCF-7 human breast cancer cells. The in vivo pharmacokinetics and biodistribution studies showed that HA-dOG-PTX-PM had a prolonged circulation time in the nude mice and a remarkably high accumulation in the MCF-7 tumor (6.19%ID/g at 12 h post injection). Interestingly, HA-dOG-PTX-PM could effectively treat mice bearing MCF-7 human breast tumor xenografts with little side effects, resulting in complete inhibition of tumor growth and a 100% survival rate over an experimental period of 55 days. These results indicate that hyaluronic acid-shelled acid-activatable PTX prodrug micelles have a great potential for targeted chemotherapy of CD44-positive cancers. PMID:26851390

  16. Monoclonal antibody to HER-2/neureceptor modulates repair of radiation-induced DNA damage and enhances radiosensitivity of human breast cancer cells overexpressing this oncogene.

    PubMed

    Pietras, R J; Poen, J C; Gallardo, D; Wongvipat, P N; Lee, H J; Slamon, D J

    1999-03-15

    The management of human breast cancer frequently includes radiation therapy as an important intervention, and improvement in the clinical efficacy of radiation is desirable. Overexpression of the HER-2 growth factor receptor occurs in 25-30% of human breast cancers and correlates with poor clinical outcome, including earlier local relapse following conservative surgery accompanied by radiation therapy. In breast cancer cells with overexpression of HER-2 receptor, recombinant humanized monoclonal antibodies (rhuMAbs) to HER-2 receptors (rhuMAb HER-2) decrease cell proliferation in vitro and reduce tumor formation in nude mice. Therapy with rhuMAb HER-2 enhances tumor sensitivity to radiation at doses of 1-5 Gy, exceeding remission rates obtained with radiation alone. This benefit is specific to cells with HER-2 overexpression and does not occur in cells without overexpression. Treatment of cells with radiation (2-4 Gy) alone provokes a marked increase in unscheduled DNA synthesis, a measure of DNA repair, but HER-2-overexpressing cells treated with a combination of rhuMAb HER-2 and radiation demonstrate a decrease of unscheduled DNA synthesis to 25-44% of controls. Using an alternate test of DNA repair, i.e., radiation-damaged or undamaged reporter DNA, we introduced a cytomegalovirus-driven beta3-galactosidase into HER-2-overexpressing breast cancer cells that had been treated with rhuMAb HER-2 or control. At 24 h posttransfection, the extent of repair assayed by measuring reporter DNA expression was high after exposure to radiation alone but significantly lower in cells treated with combined radiation and rhuMAb HER-2 therapy. To further characterize effects of rhuMAb HER-2 and the combination of antibody and radiation on cell growth, analyses of cell cycle phase distribution were performed. Antibody reduces the fraction of HER-2-overexpressing breast cancer cells in S phase at 24 and 48 h. Radiation treatment is also known to promote cell cycle arrest, predominantly at G1, with low S-phase fraction at 24 and 48 h. In the presence of rhuMAb HER-2, radiation elicits a similar reduction in S phase at 24 h, but a significant reversal of this arrest appears to begin 48 h postradiation exposure. The level of S-phase fraction at 48 h is significantly greater than that found at 24 h with the combined antibody-radiation therapy, suggesting that early escape from cell cycle arrest in the presence of antireceptor antibody may not allow sufficient time for completion of DNA repair in HER-2-overexpressing cells. Because it is well known that failure of adequate p21WAF1 induction after DNA damage is associated with failure of cell cycle arrest, we also assessed the activity of this critical mediator of the cellular response to DNA damage. The results show induction of p21WAF1 transcripts and protein product at 6, 12, and 24 h after radiation treatment; however, increased levels of p21WAF1 transcript and protein are not sustained in HER-2-overexpressing cells exposed to radiation in the presence of rhuMAb HER-2. Although transcript and protein levels increase at 6-12 h, they are both diminished by 24 h. Levels of p21WAF1 transcript and protein at 24 h are significantly lower than in cells treated by radiation without antibody. A reduction in the basal level of p21WAF1 transcript also occurred after 12-24 h exposure to antibody alone. The effect of HER-2 antibody may be related to tyrosine phosphorylation of p21WAF1 protein. Tyrosine phosphorylation of p21WAF1 is increased after treatment with radiation alone, but phosphorylation is blocked by combined treatment with antireceptor antibody and radiation. This dysregulation of p21WAF1 in HER-2-overexpressing breast cells after treatment with rhuMAb HER-2 and radiation appears to be independent of p53 expression levels but does correlate with reduced levels of mdm2 protein. (ABSTRACT TRUNCATED) PMID:10096569

  17. Gene amplification and overexpression of PRDM14 in breast cancers.

    PubMed

    Nishikawa, Noriko; Toyota, Minoru; Suzuki, Hiromu; Honma, Toshio; Fujikane, Tomoko; Ohmura, Tousei; Nishidate, Toshihiko; Ohe-Toyota, Mutsumi; Maruyama, Reo; Sonoda, Tomoko; Sasaki, Yasushi; Urano, Takeshi; Imai, Kohzoh; Hirata, Koichi; Tokino, Takashi

    2007-10-15

    Several genes that encode PR (PRDI-BF1 and RIZ) domain proteins (PRDM) have been linked to human cancers. To explore the role of the PR domain family genes in breast carcinogenesis, we examined the expression profiles of 16 members of the PRDM gene family in a panel of breast cancer cell lines and primary breast cancer specimens using semiquantitative real-time PCR. We found that PRDM14 mRNA is overexpressed in about two thirds of breast cancers; moreover, immunohistochemical analysis showed that expression of PRDM14 protein is also up-regulated. Analysis of the gene copy number revealed that PRDM14 is a target of gene amplification on chromosome 8q13, which is a region where gene amplification has frequently been detected in various human tumors. Introduction of PRDM14 into cancer cells enhanced cell growth and reduced their sensitivity to chemotherapeutic drugs. Conversely, knockdown of PRDM14 by siRNA induced apoptosis in breast cancer cells and increased their sensitivity to chemotherapeutic drugs, suggesting that up-regulated expression of PRDM14 may play an important role in the proliferation of breast cancer cells. That little or no expression of PRDM14 is seen in noncancerous tissues suggests that PRDM14 could be an ideal therapeutic target for the treatment of breast cancer. PMID:17942894

  18. A Novel Subset of Human Tumors That Simultaneously Overexpress Multiple E2F-responsive Genes Found in Breast, Ovarian, and Prostate Cancers

    PubMed Central

    Shackney, Stanley E; Chowdhury, Salim Akhter; Schwartz, Russell

    2014-01-01

    Reasoning that overexpression of multiple E2F-responsive genes might be a useful marker for RB1 dysfunction, we compiled a list of E2F-responsive genes from the literature and evaluated their expression in publicly available gene expression microarray data of patients with breast cancer, serous ovarian cancer, and prostate cancer. In breast cancer, a group of tumors was identified, each of which simultaneously overexpressed multiple E2F-responsive genes. Seventy percent of these genes were concerned with cell cycle progression, DNA repair, or mitosis. These E2F-responsive gene overexpressing (ERGO) tumors frequently exhibited additional evidence of Rb/E2F axis dysfunction, were mostly triple negative, and preferentially overexpressed multiple basal cytokeratins, suggesting that they overlapped substantially with the basal-like tumor subset. ERGO tumors were also identified in serous ovarian cancer and prostate cancer. In these cancer types, there was no evidence for a tumor subset comparable to the breast cancer basal-like subset. A core group of about 30 E2F-responsive genes were overexpressed in all three cancer types. Thus, it appears that disorders of the Rb/E2F axis can arise at multiple organ sites and produce tumors that simultaneously overexpress multiple E2F-responsive genes. PMID:25392696

  19. A novel human ghrelin variant (In1-ghrelin) and ghrelin-O-acyltransferase are overexpressed in breast cancer: potential pathophysiological relevance.

    PubMed

    Gahete, Manuel D; Crdoba-Chacn, Jos; Hergueta-Redondo, Marta; Martnez-Fuentes, Antonio J; Kineman, Rhonda D; Moreno-Bueno, Gema; Luque, Ral M; Castao, Justo P

    2011-01-01

    The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001), ghrelin receptor-type 1b (GHSR1b; p = 0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009), but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer. PMID:21829727

  20. A Novel Human Ghrelin Variant (In1-Ghrelin) and Ghrelin-O-Acyltransferase Are Overexpressed in Breast Cancer: Potential Pathophysiological Relevance

    PubMed Central

    Gahete, Manuel D.; Crdoba-Chacn, Jos; Hergueta-Redondo, Marta; Martnez-Fuentes, Antonio J.; Kineman, Rhonda D.; Moreno-Bueno, Gema

    2011-01-01

    The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p?=?0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p?=?0.0001), ghrelin receptor-type 1b (GHSR1b; p?=?0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p?=?0.009), but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer. PMID:21829727

  1. Correlations among p53, Her-2/neu, and ras overexpression and aneuploidy by multiparameter flow cytometry in human breast cancer: evidence for a common phenotypic evolutionary pattern in infiltrating ductal carcinomas.

    PubMed

    Smith, C A; Pollice, A A; Gu, L P; Brown, K A; Singh, S G; Janocko, L E; Johnson, R; Julian, T; Hyams, D; Wolmark, N; Sweeney, L; Silverman, J F; Shackney, S E

    2000-01-01

    Human solid tumors develop multiple genetic abnormalities that accumulate progressively in individual cells during the course of tumor evolution. We sought to determine whether there are specific sequences of occurrence of these progressive evolutionary changes in human breast cancers by performing correlated cell-by-cell measurements of cell DNA content, p53 protein, Her-2/neu protein, and ras protein by multiparameter flow cytometry in 56 primary tumor samples obtained at surgery. In addition, p53 allelic loss and Her-2/neu gene amplification were determined by fluorescence in situ hybridization in cells from the same samples. We reasoned that if there is a specific order in which genetic changes occur, the same early changes would be found consistently in the cells with the fewest abnormalities. We reasoned further that late-developing abnormalities would not occur alone in individual cells but would almost always be found together with the early changes inherited by the same cells. By these criteria, abnormalities involving p53 generally occurred early in the course of development of invasive breast cancers, whereas ras protein overexpression was found to be a late-occurring phenomenon. Within individual tumors, cellular p53 overexpression was often observed alone in individual cells, whereas ras protein overexpression was rarely observed in the absence of p53 overexpression and/or Her-2/neu overexpression in the same cells. Furthermore, the intracellular level of each abnormally expressed protein was found to increase progressively as new abnormalities were acquired. Infiltrating ductal carcinomas exhibited characteristic phenotypic patterns in which p53 allelic loss and/or p53 protein overexpression, Her-2/neu amplification and/or overexpression, aneuploidy, and ras overexpression accumulated within individual cells. However, this pattern was not a prominent feature of lobular breast cancers. All six lobular breast cancers studied were diploid. p53 allelic loss and/or early p53 overexpression, and late ras cooverexpression in the same cells were less common in lobular breast cancers than in infiltrating ductal carcinomas. Although Her-21neu overexpression was a common finding in lobular breast cancers, Her-2/neu amplification was not observed in these tumors. PMID:10656439

  2. Cyclin E2 overexpression is associated with endocrine resistance but not insensitivity to CDK2 inhibition in human breast cancer cells.

    PubMed

    Caldon, C Elizabeth; Sergio, C Marcelo; Kang, Jian; Muthukaruppan, Anita; Boersma, Marijke N; Stone, Andrew; Barraclough, Jane; Lee, Christine S; Black, Michael A; Miller, Lance D; Gee, Julia M; Nicholson, Rob I; Sutherland, Robert L; Print, Cristin G; Musgrove, Elizabeth A

    2012-07-01

    Cyclin E2, but not cyclin E1, is included in several gene signatures that predict disease progression in either tamoxifen-resistant or metastatic breast cancer. We therefore examined the role of cyclin E2 in antiestrogen resistance in vitro and its potential for therapeutic targeting through cyclin-dependent kinase (CDK) inhibition. High expression of CCNE2, but not CCNE1, was characteristic of the luminal B and HER2 subtypes of breast cancer and was strongly predictive of shorter distant metastasis-free survival following endocrine therapy. After antiestrogen treatment of MCF-7 breast cancer cells, cyclin E2 mRNA and protein were downregulated and cyclin E2-CDK2 activity decreased. However, this regulation was lost in tamoxifen-resistant (MCF-7 TAMR) cells, which overexpressed cyclin E2. Expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression contributes to the antiestrogen resistance of tamoxifen-resistant cells. Ectopic expression of cyclin E1 or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, CDK2 inhibition of E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition. Cyclin E2 overexpression is therefore a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition. CDK2 inhibitors hold promise as a component of combination therapies in endocrine-resistant disease as they effectively inhibit cyclin E1 and E2 overexpressing cells and enhance the efficacy of other therapeutics. PMID:22564725

  3. Aluminium and human breast diseases.

    PubMed

    Darbre, P D; Pugazhendhi, D; Mannello, F

    2011-11-01

    The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 28 ?g/l) compared with control healthy subjects (mean 131 10 ?g/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 ?g/l) compared with human serum (median 6 ?g/l) or human milk (median 25 ?g/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate. PMID:22099158

  4. Dual-Ligand Modified Polymer-Lipid Hybrid Nanoparticles for Docetaxel Targeting Delivery to Her2/neu Overexpressed Human Breast Cancer Cells.

    PubMed

    Yang, Zhe; Tang, Wenxin; Luo, Xingen; Zhang, Xiaofang; Zhang, Chao; Li, Hao; Gao, Di; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2015-08-01

    In this study, a dual-ligand polymer-lipid hybrid nanoparticle drug delivery vehicle comprised of an anti-HER2/neu peptide (AHNP) mimic with a modified HIV-1 Tat (mTAT) was established for the targeted treatment of Her2/neu-overexpressing cells. The resultant dual-ligand hybrid nanoparticles (NPs) consisted of a poly(lactide-co-glycolide) core, a near 90% surface coverage of the lipid monolayer, and a 5.7 nm hydrated polyethylene glycol shell. Ligand density optimization study revealed that cellular uptake efficiency of the hybrid NPs could be manipulated by controlling the surface-ligand densities. Furthermore, the cell uptake kinetics and mechanism studies showed that the dual-ligand modifications of hybrid NPs altered the cellular uptake pathway from caveolae-mediated endocytosis (CvME) to the multiple endocytic pathways, which would significantly enhance the NP internalization. Upon the systemic investigation of the cellular uptake behavior of dual-ligand hybrid NPs, docetaxel (DTX), a hydrophobic anticancer drug, was successfully encapsulated into dual-ligand hybrid NPs with high drug loading for Her2/neu-overexpressing SK-BR-3 breast cancer cell treatment. The DTX-loaded dual-ligand hybrid NPs showed a decreased burst release and a more gradual sustained drug release property. Because of the synergistic effect of dual-ligand modification, DTX-loaded dual-ligand hybrid NPs exerted substantially better therapeutic potency against SK-BR-3 cancer cells than other NP formulations and free DTX drugs. These results demonstrate that the dual-ligand hybrid NPs could be a promising vehicle for targeted drug delivery to treat breast cancer. PMID:26295141

  5. 18F-fluorodeoxy-glucose positron emission tomography (18FDG-PET) marks MYC-overexpressing human basal-like breast cancers

    PubMed Central

    Palaskas, Nicolaos; Larson, Steven M.; Schultz, Nikolaus; Komisopoulou, Evangelia; Wong, Justin; Rohle, Dan; Campos, Carl; Yannuzzi, Nicolas; Osborne, Joseph R.; Linkov, Irina; Kastenhuber, Edward R.; Taschereau, Richard; Plaisier, Seema B.; Tran, Chris; Heguy, Adriana; Wu, Hong; Sander, Chris; Phelps, Michael E.; Brennan, Cameron; Port, Elisa; Huse, Jason T.; Graeber, Thomas G.; Mellinghoff, Ingo K.

    2011-01-01

    In contrast to normal cells, cancer cells avidly take up glucose and metabolize it to lactate even when oxygen is abundant, a phenomenon referred to as the Warburg effect. This fundamental alteration in glucose metabolism in cancer cells enables their specific detection by Positron Emission Tomography (PET) following intravenous injection of the glucose analogue 18F-fluorodeoxy-glucose (18FDG). However, this useful imaging technique is limited by the fact that not all cancers avidly take up FDG. To identify molecular determinants of 18FDG-retention, we interogated the transcriptomes of human cancer cell lines and primary tumors for metabolic pathways associated with 18FDG radiotracer uptake. From 95 metabolic pathways that were interrogated, the glycolysis and several glycolysis-related pathways (pentose-phosphate, carbon fixation, aminoacyl-tRNA biosynthesis, one-carbon-pool by folate) showed the greatest transcriptional enrichment. This “FDG signature” predicted FDG-uptake in breast cancer cell lines and overlapped with established gene expression signatures for the “basal-like” breast cancer subtype and MYC-induced tumorigenesis in mice. Human breast cancers with nuclear MYC staining and high RNA expression of MYC target genes showed high 18FDG-PET uptake (p < 0.005). Presence of the FDG signature was similarly associated with MYC gene copy gain, increased MYC transcript levels, and elevated expression of metabolic MYC target genes in a human breast cancer genomic dataset. Together, our findings link clinical observations of glucose uptake with a pathologic and molecular subtype of human breast cancer. Further, they suggest related approaches to derive molecular determinants of radiotracer retention for other PET-imaging probes. PMID:21646475

  6. LETM1 overexpression is correlated with the clinical features and survival outcome of breast cancer

    PubMed Central

    Li, Nan; Zheng, Yahui; Xuan, Chouhui; Lin, Zhenhua; Piao, Longzhen; Liu, Shuangping

    2015-01-01

    Background: Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome. However, high-level expression of LETM1 has been correlated with multiple human malignancies, suggesting roles in carcinogenesis and tumor progression. This study is aimed to explore the clinicopathological characteristics and prognostic value of LETM1 overexpression in breast cancer. Methods: Immunohistochemical (IHC) staining, and immunofluorescence (IF) were performed to examine LETM1 expression in breast cancer cell line/tissues compared with adjacent normal tissues. Statistical analysis was applied to evaluate the correlation between LETM1 overexpression and the clinicopathological features of breast cancer. Survival rates were calculated using the Kaplan-Meier method, and the relationship between prognostic factors and patient survival was analyzed using the Cox proportional hazard models. Results: LETM1 protein showed cytoplasmic staining pattern in breast cancer. The strongly positive rate of LETM1 protein was 61.6% (98/159) in breast cancer, which was significantly higher than in DCIS (29.7%, 11/37), hyperplasia (16.7%, 3/18) and adjacent normal breast tissues (15.9%, 7/44). High-level expression of LETM1 protein was correlated with lymph node metastasis, poor differentiation, late clinical stage, disease-free survival (DFS) and overall survival (OS) rates in breast cancer. Moreover, multivariate analysis suggested that LETM1 emerged as a significant independent prognostic factor along with clinical stage of patients with breast cancer. Conclusions: LETM1 plays an important role in the progression of breast cancer. High level expression of LETM1 is an independent poor prognostic factor of breast cancer. PMID:26722481

  7. Overexpressed ubiquitin ligase Cullin7 in breast cancer promotes cell proliferation and invasion via down-regulating p53

    SciTech Connect

    Guo, Hongsheng; Wu, Fenping; Wang, Yan; Yan, Chong; Su, Wenmei

    2014-08-08

    Highlights: • Cullin7 is overexpressed in human breast cancer samples. • Cullin7 stimulated proliferation and invasion of breast cancer cells. • Inhibition of p53 contributes to Cullin7-induced proliferation and invasion. - Abstract: Ubiquitin ligase Cullin7 has been identified as an oncogene in some malignant diseases such as choriocarcinoma and neuroblastoma. However, the role of Cullin7 in breast cancer carcinogenesis remains unclear. In this study, we compared Cullin7 protein levels in breast cancer tissues with normal breast tissues and identified significantly higher expression of Cullin7 protein in breast cancer specimens. By overexpressing Cullin7 in breast cancer cells HCC1937, we found that Cullin7 could promote cell growth and invasion in vitro. In contrast, the cell growth and invasion was inhibited by silencing Cullin7 in breast cancer cell BT474. Moreover, we demonstrated that Cullin7 promoted breast cancer cell proliferation and invasion via down-regulating p53 expression. Thus, our study provided evidence that Cullin7 functions as a novel oncogene in breast cancer and may be a potential therapeutic target for breast cancer management.

  8. Buthionine sulphoximine-mediated sensitisation of etoposide-resistant human breast cancer MCF7 cells overexpressing the multidrug resistance-associated protein involves increased drug accumulation.

    PubMed Central

    Schneider, E.; Yamazaki, H.; Sinha, B. K.; Cowan, K. H.

    1995-01-01

    Preincubation of etoposide-resistant human MCF7 breast cancer cells (MCF7/VP) with buthionine sulphoximine (BSO) resulted in their sensitisation to etoposide and vincristine. Chemosensitisation was accompanied by elevated intracellular drug levels. In contrast, simultaneous exposure to BSO did not result in increased drug accumulation. Similar, but quantitatively smaller, effects were also observed when sensitive wild-type MCF7/WT cells were treated with BSO. In agreement with its effect on drug accumulation, BSO pretreatment also increased VP-16-stimulated cleavable complex formation between DNA topoisomerase II and cellular DNA. BSO treatment also led to a significant increase in acid-precipitable VP-16 levels in MCF7/VP, but not MCF7/WT cells. In contrast, no clear effects of BSO on drug efflux were observed and drug retention was only minimally increased after BSO treatment of both MCF7/WT and MCF7/VP cells and no difference between the two cell lines was detected. Thus, chemosensitisation by BSO appeared to be mediated through increased intracellular drug concentrations and/or protein binding. PMID:7710938

  9. SNEV overexpression extends the life span of human endothelial cells

    SciTech Connect

    Voglauer, Regina; Chang, Martina Wei-Fen; Dampier, Brigitta; Wieser, Matthias; Baumann, Kristin; Sterovsky, Thomas; Schreiber, Martin; Katinger, Hermann; Grillari, Johannes . E-mail: j.grillari@iam.boku.ac.at

    2006-04-01

    In a recent screening for genes downregulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells.

  10. HSET overexpression fuels tumor progression via centrosome clustering-independent mechanisms in breast cancer patients

    PubMed Central

    Pannu, Vaishali; Rida, Padmashree C.G.; Ogden, Angela; Turaga, Ravi Chakra; Donthamsetty, Shashikiran; Bowen, Nathan J.; Rudd, Katie; Gupta, Meenakshi V.; Reid, Michelle D.; Cantuaria, Guilherme; Walczak, Claire E.; Aneja, Ritu

    2015-01-01

    Human breast tumors harbor supernumerary centrosomes in almost 80% of tumor cells. Although amplified centrosomes compromise cell viability via multipolar spindles resulting in death-inducing aneuploidy, cancer cells tend to cluster extra centrosomes during mitosis. As a result cancer cells display bipolar spindle phenotypes to maintain a tolerable level of aneuploidy, an edge to their survival. HSET/KifC1, a kinesin-like minus-end directed microtubule motor has recently found fame as a crucial centrosome clustering molecule. Here we show that HSET promotes tumor progression via mechanisms independent of centrosome clustering. We found that HSET is overexpressed in breast carcinomas wherein nuclear HSET accumulation correlated with histological grade and predicted poor progression-free and overall survival. In addition, deregulated HSET protein expression was associated with gene amplification and/or translocation. Our data provide compelling evidence that HSET overexpression is pro-proliferative, promotes clonogenic-survival and enhances cell-cycle kinetics through G2 and M-phases. Importantly, HSET co-immunoprecipitates with survivin, and its overexpression protects survivin from proteasome-mediated degradation, resulting in its increased steady-state levels. We provide the first evidence of centrosome clustering-independent activities of HSET that fuel tumor progression and firmly establish that HSET can serve both as a potential prognostic biomarker and as a valuable cancer-selective therapeutic target. PMID:25788277

  11. The Polarity Protein Par6 Induces Cell Proliferation and Is Overexpressed in Breast Cancer

    PubMed Central

    Nolan, Marissa E.; Aranda, Victoria; Lee, Sangjun; Lakshmi, Balasubramanian; Basu, Srinjan; Allred, D. Craig; Muthuswamy, Senthil K.

    2010-01-01

    The Polarity protein complex Par6/aPKC/Cdc42 regulates polarization processes during epithelial morphogenesis, astrocyte migration and axon specification. Others and we have demonstrated that this complex is also required for disruption of apical-basal polarity during the oncogene ErbB2-induced transformation and TGF?-induced epithelial mesenchymal transition of mammary epithelial cells. Here, we report that expression of Par6 by itself in mammary epithelial cells induces epidermal growth factor independent cell proliferation and development of hyperplastic three-dimensional acini without affecting apical-basal polarity. This is dependent on the ability of Par6 to interact with aPKC and Cdc42 but not Lgl and Par3 and its ability to promote sustained activation of MEK/Erk signaling. Downregulation of Cdc42 or aPKC expression suppresses the ability of Par6 to induce proliferation, demonstrating that Par6 promotes cell proliferation by interacting with aPKC and Cdc42. We also show that Par6 is overexpressed in breast cancer derived cell lines and in both precancerous and advanced primary human breast cancers suggesting that Par6 overexpression regulates tumor initiation and progression. Thus, in addition to regulating cell polarization processes, Par6 is an inducer of cell proliferation in breast epithelial cells. PMID:18922891

  12. Steroid hormones induce HMG1 overexpression and sensitize breast cancer cells to cisplatin and carboplatin

    PubMed Central

    He, Qing; Liang, Cynthia H.; Lippard, Stephen J.

    2000-01-01

    Cisplatin is an anticancer drug that has enjoyed remarkable success against testicular tumors, but dose limiting side-effects have limited its application against a broader range of cancers. Previous studies have shown that high-mobility group (HMG) domain proteins such as HMG1 sensitize cells to cisplatin by shielding its major DNA adducts from nucleotide excision repair. Estrogen treatment increases HMG1 mRNA levels in breast cancer MCF-7 cells. Herein, we describe that treatment of human cancer cells having steroid hormone receptors with the appropriate hormone, estrogen and/or progesterone, significantly increases the potency of cisplatin and its analogue carboplatin by causing the overexpression of HMG1. These findings suggest that the proper combination of these drugs, which are already approved by the Food and Drug Administration, could have potential benefit in treating tumors such as ovarian or breast that carry the hormone receptors. PMID:10811891

  13. Rhein Induces Apoptosis in Human Breast Cancer Cells

    PubMed Central

    Chang, Ching-Yao; Chan, Hong-Lin; Lin, Hui-Yi; Way, Tzong-Der; Kao, Ming-Ching; Song, Ming-Zhang; Lin, Ying-Ju; Lin, Cheng-Wen

    2012-01-01

    Human breast cancers cells overexpressing HER2/neu are more aggressive tumors with poor prognosis, and resistance to chemotherapy. This study investigates antiproliferation effects of anthraquinone derivatives of rhubarb root on human breast cancer cells. Of 7 anthraquinone derivatives, only rhein showed antiproliferative and apoptotic effects on both HER2-overexpressing MCF-7 (MCF-7/HER2) and control vector MCF-7 (MCF-7/VEC) cells. Rhein induced dose- and time-dependent manners increase in caspase-9-mediated apoptosis correlating with activation of ROS-mediated activation of NF-?B- and p53-signaling pathways in both cell types. Therefore, this study highlighted rhein as processing anti-proliferative activity against HER2 overexpression or HER2-basal expression in breast cancer cells and playing important roles in apoptotic induction of human breast cancer cells. PMID:22007260

  14. Evaluating treatment response using DW-MRI and DCE-MRI in trastuzumab responsive and resistant HER2-overexpressing human breast cancer xenografts.

    PubMed

    Whisenant, Jennifer G; Sorace, Anna G; McIntyre, J Oliver; Kang, Hakmook; Snchez, Violeta; Loveless, Mary E; Yankeelov, Thomas E

    2014-12-01

    We report longitudinal diffusion-weighted magnetic resonance imaging (DW-MRI) and dynamic contrast enhanced (DCE)-MRI (7 T) studies designed to identify functional changes, prior to volume changes, in trastuzumab-sensitive and resistant HER2+ breast cancer xenografts. Athymic mice (N = 33) were subcutaneously implanted with trastuzumab-sensitive (BT474) or trastuzumab-resistant (HR6) breast cancer cells. Tumor-bearing animals were distributed into four groups: BT474 treated and control, HR6 treated and control. DW- and DCE-MRI were conducted at baseline, day 1, and day 4; trastuzumab (10 mg/kg) or saline was administered at baseline and day 3. Animals were sacrificed on day 4 and tumors resected for histology. Voxel-based DW- and DCE-MRI analyses were performed to generate parametric maps of ADC, K(trans), and ve. On day 1, no differences in tumor size were observed between any of the groups. On day 4, significant differences in tumor size were observed between treated vs. control BT474, treated BT474 vs. treated HR6, and treated vs. control HR6 (P < .0001). On day 1, ve was significantly higher in the BT474 treated group compared to BT474 control (P = .002) and HR6 treated (P = .004). On day 4, ve and K(trans) were significantly higher in the treated BT474 tumors compared to BT474 controls (P = .0007, P = .02, respectively). A significant decrease in Ki67 staining reinforced response in the BT474 treated group compared to BT474 controls (P = .02). This work demonstrated that quantitative MRI biomarkers have the sensitivity to differentiate treatment response in HER2+ tumors prior to changes in tumor size. PMID:25500087

  15. Utility of 18FLT-PET to Assess Treatment Response in Trastuzumab-resistant and Sensitive HER2-overexpressingHuman Breast Cancer Xenografts

    PubMed Central

    Whisenant, Jennifer G.; McIntyre, J. Oliver; Peterson, Todd E.; Kang, Hakmook; Snchez, Violeta; Manning, H. Charles; Arteaga, Carlos L.; Yankeelov, Thomas E.

    2015-01-01

    Purpose Evaluate 3-deoxy-3-[18F]-fluorothymidine (18FLT) PET as an early marker of trastuzumab response in HER2-overexpressing xenografts. Procedures Tumor-to-muscle ratios were compared between both trastuzumab-sensitive and resistant cohorts prior to and after one and two treatments. Results A significant difference (P=0.03) was observed between treated and control trastuzumab-sensitive xenografts after one treatment, which preceded between-group differences in tumor volume. Reduced Ki67 (P=0.02) and thymidine kinase 1 (TK1) (P=0.35) immunoreactivity was observed in the treated xenografts. No significant differences in volume, tumor-to-muscle ratio, or immunoreactivity were observed between treated and control trastuzumab-resistant cohorts. A significant difference (P=0.02) in tumor-to-muscle ratio was observed between trastuzumab-sensitive and resistant cohorts after two treatments; however, tumor volumes were also different (P=0.04). Ki67 (P=0.04) and TK1 (P=0.24) immunoreactivity was ~50% less in trastuzumab-sensitive xenografts.. Conclusions 18FLT-PET provided early response assessment in trastuzumab-sensitive xenografts, but only differentiated between trastuzumab-resistant and sensitive xenografts concurrent with differences in tumor size. PMID:25034624

  16. Analysis of wntless (WLS) expression in gastric, ovarian, and breast cancers reveals a strong association with HER2 overexpression.

    PubMed

    Stewart, Jonathan; James, Jacqueline; McCluggage, Glenn W; McQuaid, Stephen; Arthur, Kenneth; Boyle, David; Mullan, Paul; McArt, Darragh; Yan, Benedict; Irwin, Gareth; Harkin, D Paul; Zhengdeng, Lei; Ong, Chee-Wee; Yu, Jia; Virshup, David M; Salto-Tellez, Manuel

    2015-03-01

    The oncogenic role of WNT is well characterized. Wntless (WLS) (also known as GPR177, or Evi), a key modulator of WNT protein secretion, was recently found to be highly overexpressed in malignant astrocytomas. We hypothesized that this molecule may be aberrantly expressed in other cancers known to possess aberrant WNT signaling such as ovarian, gastric, and breast cancers. Immunohistochemical analysis using a TMA platform revealed WLS overexpression in a subset of ovarian, gastric, and breast tumors; this overexpression was associated with poorer clinical outcomes in gastric cancer (P=0.025). In addition, a strong correlation was observed between WLS expression and human epidermal growth factor receptor 2 (HER2) overexpression. Indeed, 100% of HER2-positive intestinal gastric carcinomas, 100% of HER2-positive serous ovarian carcinomas, and 64% of HER2-positive breast carcinomas coexpressed WLS protein. Although HER2 protein expression or gene amplification is an established predictive biomarker for trastuzumab response in breast and gastric cancers, a significant proportion of HER2-positive tumors display resistance to trastuzumab, which may be in part explainable by a possible mechanistic link between WLS and HER2. PMID:25258105

  17. Induction of fibronectin by HER2 overexpression triggers adhesion and invasion of breast cancer cells.

    PubMed

    Jeon, Myeongjin; Lee, Jeongmin; Nam, S J; Shin, Incheol; Lee, J E; Kim, Sangmin

    2015-04-10

    Fibronectin (FN), an extracellular matrix ligand, plays a pivotal role in cell adhesion, migration, and oncogenic transformation. Aberrant FN expression is associated with poor prognoses in various types of cancer, including breast cancer. In the current study, we investigated the relationship between FN induction and HER2 expression in breast cancer cells. Our results showed that the level of FN expression increased in response to HER family ligands, EGF and TGF-? in a time- and dose-dependent manner. On the other hand, EGF-induced FN expression decreased in response to trastuzumab, which is a HER2-targeted monoclonal antibody. However, EGF-induced FN expression was not affected by trastuzumab in JIMT-1 breast cancer cells, which are trastuzumab insensitive cells. Next, we introduced the HER2 gene into MDA-MB231 cells to verify the relationship between FN and HER2. The level of FN expression significantly increased in HER2-overexpressed MDA-MB231 cells. In contrast, the induction of FN by HER2 was significantly decreased in response to trastuzumab treatment. In addition, the induction of FN by HER2 was down-regulated by the MEK 1/2 specific inhibitor, U0126. Using conditioned culture media of vec- and HER2-overexpressed MDA-MB231 cells, we observed the cell morphology, adhesion, and invasion of MDA-MB231 cells. Interestingly, in conditioned culture media of HER2-overexpressed MDA-MB231 cells, the cell morphology was altered, and adhesion and invasion of MDA-MB231 cells significantly increased. In addition, our results showed that recombinant human FN augmented cell adhesion and invasion of MDA-MB231 cells while these inductions decreased in response to an FN inhibitor. Therefore, we demonstrated that the induction of FN by HER2 triggers cell adhesion and invasion capacities. PMID:25743092

  18. Overexpression of Kinesin Associated Protein 3 (KIFAP3) in Breast Cancer

    PubMed Central

    Telikicherla, Deepthi; Maharudraiah, Jagadeesha; Pawar, Harsh; Marimuthu, Arivusudar; Kashyap, Manoj Kumar; Ramachandra, Y. L.; Roa, Juan Carlos; Pandey, Akhilesh

    2015-01-01

    Gene expression profiling studies on breast cancer have generated catalogs of differentially expressed genes. However, many of these genes have not been investigated for their expression at the protein level. It is possible to systematically evaluate such genes in a high-throughput fashion for their overexpression at the protein level using breast cancer tissue microarrays. Our strategy involved integration of information from publicly available repositories of gene expression to prepare a list of genes upregulated at the mRNA level in breast cancer followed by curation of the published literature to identify those genes that were not tested for overexpression at the protein level. We identified Kinesin Associated Protein 3 (KIFAP3) as one such molecule for further validation at the protein level. Immunohistochemical labeling of KIFAP3 using breast cancer tissue microarrays revealed overexpression of KIFAP3 protein in 84% (240/285) of breast cancers indicating the utility of our integrated approach of combining computational analysis with experimental biology.

  19. Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications

    PubMed Central

    Iqbal, Nida; Iqbal, Naveed

    2014-01-01

    Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family having tyrosine kinase activity. Dimerization of the receptor results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways leading to cell proliferation and tumorigenesis. Amplification or overexpression of HER2 occurs in approximately 15–30% of breast cancers and 10–30% of gastric/gastroesophageal cancers and serves as a prognostic and predictive biomarker. HER2 overexpression has also been seen in other cancers like ovary, endometrium, bladder, lung, colon, and head and neck. The introduction of HER2 directed therapies has dramatically influenced the outcome of patients with HER2 positive breast and gastric/gastroesophageal cancers; however, the results have been proved disappointing in other HER2 overexpressing cancers. This review discusses the role of HER2 in various cancers and therapeutic modalities available targeting HER2. PMID:25276427

  20. Rapid Stereomicroscopic Imaging of HER2 Overexpression in Ex Vivo Breast Tissue Using Topically Applied Silica-Based Gold Nanoshells

    PubMed Central

    Bickford, Lissett R.; Langsner, Robert J.; Chang, Joseph; Kennedy, Laura C.; Agollah, Germaine D.; Drezek, Rebekah

    2012-01-01

    Tumor margin detection for patients undergoing breast conservation surgery primarily occurs postoperatively. Previously, we demonstrated that gold nanoshells rapidly enhance contrast of HER2 overexpression in ex vivo tissue sections. Our ultimate objective, however, is to discern HER2 overexpressing tissue from normal tissue in whole, nonsectioned, specimens to facilitate rapid diagnoses. Here, we use targeted nanoshells to quickly and effectively visualize HER2 receptor expression in intact ex vivo human breast tissue specimens. Punch biopsies of human breast tissue were analyzed after a brief 5-minute incubation with and without HER2-targeted silica-gold nanoshells using two-photon microscopy and stereomicroscopy. Labeling was subsequently verified using reflectance confocal microscopy, darkfield hyperspectral imaging, and immunohistochemistry to confirm levels of HER2 expression. Our results suggest that anti-HER2 nanoshells used in tandem with a near-infrared reflectance confocal microscope and a standard stereomicroscope may potentially be used to discern HER2-overexpressing cancerous tissue from normal tissue in near real time and offer a rapid supplement to current diagnostic techniques. PMID:23133450

  1. Enhanced sensitization to taxol-induced apoptosis by herceptin pretreatment in ErbB2-overexpressing breast cancer cells.

    PubMed

    Lee, Sangkyou; Yang, Wentao; Lan, Keng-Hsueh; Sellappan, Shankar; Klos, Kristine; Hortobagyi, Gabriel; Hung, Mien-Chie; Yu, Dihua

    2002-10-15

    The recombinant humanized anti-ErbB2/HER2 monoclonal antibody Herceptin (Trastuzumab) has been shown to significantly enhance the tumoricidaleffects of antitumor drugs such as paclitaxel (Taxol) in patients with ErbB2-overexpressing breast cancers. Here, we investigated the molecular mechanisms by which Herceptin enhances the antitumor effects of Taxol. Because activation of p34(Cdc2) is required for Taxol-induced apoptosis and because overexpression of ErbB2 blocks Taxol-induced apoptosis by inhibiting p34(Cdc2) activation, we studied the effect of Herceptin treatment on p34(Cdc2) kinase activation and apoptosis in Taxol-treated human breast carcinoma cell lines MDA-MB-435, SKBr3, MDA-MB-453, and 435.eB, which is an ErbB2 transfectant of MDA-MB-435. Herceptin treatment down-regulated ErbB2, reduced the inhibitory phosphorylation of Cdc2 on Tyr-15, and down-regulated the expression of p21(Cip1), a Cdc2 inhibitor. Herceptin plus Taxol treatment led to higher levels of p34(Cdc2) kinase activity and apoptosis in ErbB2-overexpressing breast cancer cells, which is likely attributable to inhibition of Cdc2-Tyr-15 phosphorylation and p21(Cip1) expression. Because significant dephosphorylation of Cdc2-Tyr-15 and down-regulation of p21(Cip1) occur at least 24 h after Herceptin treatment, we investigated whether 24 h Herceptin pretreatment will render ErbB2-overexpressing breast cancer cells more sensitive to Taxol-induced apoptosis compared with the simultaneous treatment of Herceptin plus Taxol. Indeed, Herceptin pretreatment increased Taxol-induced apoptosis and cytotoxicity in vitro and more effectively inhibited the growth of tumor xenografts with enhanced in vivo apoptosis. Thus, Herceptin treatment of ErbB2-overexpressing cells can inhibit ErbB2-mediated Cdc2-Tyr-15 phosphorylation and p21(Cip1) up-regulation, which allows effective p34(Cdc2) activation and induction of apoptosis upon Taxol treatment. Herceptin pretreatment renders ErbB2-overexpressing breast cancers more susceptible to Taxol-induced cell death, which may have important clinical therapeutic implications. PMID:12384528

  2. FOXO1A is a target for HER2 Overexpressing Breast Tumors*

    PubMed Central

    Wu, Yanyuan; Shang, Xiying; Sarkissyan, Marianna; Slamon, Dennis; Vadgama, Jaydutt V.

    2010-01-01

    Trastuzumab treatment has improved the overall survival of HER2 overexpressing breast cancer patients. However, many of these patients will eventually become resistant to treatment. The mechanisms that contribute to resistance to Trastuzumab are unknown. In this study we tested the hypothesis that targeting of the FKHR transcription factor FOXO1A in HER2 overexpressing breast tumor cells, can overcome the Trastuzumab resistance in vitro. We have demonstrated that overexpression of HER2 leads to activation of PI3K/Akt pathway and subsequent inactivation of FOXO1A in HER2 overexpressing breast cancer cells, SKBR3, BT474 and MCF7-HER2. In wildtype SKBR3 and BT474 cells, Trastuzumab downregulates active Akt and increases FOXO1A expression that leads to increase in p27kip1, and decrease in cyclin D1, and finally inhibits cell proliferation. In contrast, the effect of Trastuzumab was eliminated by the reduction of FOXO1A in HER2 overexpressing cells with constitutively active Akt1 (SKBR3/AA28 and BT474/AA9). The down-regulation of FOXO1A resulted in nuclear export of p27kip1. Blocking the constitutively active Akt by a specific Akt/protein kinase B signaling inhibitor-2 (API-2) significantly increased FOXO1A expression and rendered the cells more responsive to Trastuzumab induced growth inhibition. Re-activation of FOXO1A by stable or transient transfection also restored the growth inhibitory effects of Trastuzumab in SKBR3/AA28, BT474/AA9, and MCF7-HER2 cells. Knocking-down FOXO1A by siRNA resulted in reducing Trastuzumab induced growth inhibition. In summary, Trastuzumab can inhibit proliferation of HER2 overexpressing breast cancer cells by re-activating FOXO1A through inhibition of the PI3K/Akt pathway. FOXO1A may therefore serve as a target for HER2 overexpressing breast tumors. PMID:20551062

  3. The Effect of a DNA Repair Gene on Cellular Invasiveness: Xrcc3 Over-Expression in Breast Cancer Cells

    PubMed Central

    Martinez-Marignac, Veronica L.; Rodrigue, Amlie; Davidson, David; Couillard, Martin; Al-Moustafa, Ala-Eddin; Abramovitz, Mark; Foulkes, William D.; Masson, Jean-Yves; Aloyz, Raquel

    2011-01-01

    Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3) and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion. PMID:21283680

  4. Genetic variants in the HER2 gene: Influence on HER2 overexpression and loss of heterozygosity in breast cancer.

    PubMed

    Cresti, Nicola; Lee, Joanne; Rourke, Emma; Televantou, Despina; Jamieson, David; Verrill, Mark; Boddy, Alan V

    2016-03-01

    Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancer is an indicator of poor prognosis and is the pre-requisite for treatment with the agents targeting this member of the epidermal growth factor receptor family. In order to determine the influence of these common single-nucleotide polymorphisms (SNPs) in the HER2 gene, genomic DNA was obtained from 361 patients with breast cancer, aged between 29 and 82 years. Samples of tumour tissue were obtained from 241 (66%) patients and material for extraction of DNA is isolated from surrounding normal tissue by laser capture microdissection. Genotyping was performed using the Taqman fluorogenic 5' nuclease assay. Of the 360 patients with definitive determination of HER2 status, 49% were positive. The Ile655Val SNP had no influence on the frequency of HER2 expression. However, the proline allele of the Ala1170Pro SNP was associated with a higher frequency of HER2 overexpression (56% versus 43%, p=0.015). Where the germline genotype was homozygous, the tumour genotype was identical in every case and for both SNPs. In HER2-positive tumours, heterozygosity was maintained in only 15% and 18% of the Ile655Val and Ala1170Pro SNPs, respectively. This was lower than in the HER2-negative tumours (46% and 43%, respectively). Normal breast tissue (n=23) retained the germline genotype in all but one case. The underlying link between the Ala1170Pro SNP and HER2 positivity is not known, nor is the significance of HER2 overexpression and loss of heterozygosity in breast cancer. However, these results illustrate the complexity of HER2 genotype and overexpression in this disease. PMID:26773371

  5. Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids.

    PubMed

    Rodrguez, Cristina E; Reidel, Sara I; Bal de Kier Joff, Elisa D; Jasnis, Maria A; Fiszman, Gabriel L

    2015-01-01

    Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance. PMID:26360292

  6. Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids

    PubMed Central

    Rodrguez, Cristina E.; Reidel, Sara I.; Bal de Kier Joff, Elisa D.; Jasnis, Maria A.; Fiszman, Gabriel L.

    2015-01-01

    Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance. PMID:26360292

  7. Synthesis of folate- pegylated polyester nanoparticles encapsulating ixabepilone for targeting folate receptor overexpressing breast cancer cells.

    PubMed

    Siafaka, P; Betsiou, M; Tsolou, A; Angelou, E; Agianian, B; Koffa, M; Chaitidou, S; Karavas, E; Avgoustakis, K; Bikiaris, D

    2015-12-01

    The aim of this study was the preparation of novel polyester nanoparticles based on folic acid (FA)-functionalized poly(ethylene glycol)-poly(propylene succinate) (PEG-PPSu) copolymer and loaded with the new anticancer drug ixabepilone (IXA). These nanoparticles may serve as a more selective (targeted) treatment of breast cancer tumors overexpressing the folate receptor. The synthesized materials were characterized by (1)H-NMR, FTIR, XRD and DSC. The nanoparticles were prepared by a double emulsification and solvent evaporation method and characterized with regard to their morphology by scanning electron microscopy, drug loading with HPLC-UV and size by dynamic light scattering. An average size of 195 nm and satisfactory drug loading efficiency (3.5%) were observed. XRD data indicated that IXA was incorporated into nanoparticles in amorphous form. The nanoparticles exhibited sustained drug release properties in vitro. Based on in vitro cytotoxicity studies, the blank FA-PEG-PPSu nanoparticles were found to be non-toxic to the cells. Fluorescent nanoparticles were prepared by conjugating Rhodanine B to PEG-PPSu, and live cell, fluorescence, confocal microscopy was applied in order to demonstrate the ability of FA-PEG-PPSu nanoparticles to enter into human breast cancer cells expressing the folate receptor. PMID:26543021

  8. Neural Stem Cells Secreting Anti-HER2 Antibody Improve Survival in a Preclinical Model of HER2 Overexpressing Breast Cancer Brain Metastases.

    PubMed

    Kanojia, Deepak; Balyasnikova, Irina V; Morshed, Ramin A; Frank, Richard T; Yu, Dou; Zhang, Lingjiao; Spencer, Drew A; Kim, Julius W; Han, Yu; Yu, Dihua; Ahmed, Atique U; Aboody, Karen S; Lesniak, Maciej S

    2015-10-01

    The treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer has been revolutionized by trastuzumab. However, longer survival of these patients now predisposes them to forming HER2 positive brain metastases, as the therapeutic antibodies cannot cross the blood brain barrier. The current oncologic repertoire does not offer a rational, nontoxic targeted therapy for brain metastases. In this study, we used an established human neural stem cell line, HB1.F3 NSCs and generated a stable pool of cells secreting a high amount of functional full-length anti-HER2 antibody, equivalent to trastuzumab. Anti-HER2Ab secreted by the NSCs (HER2Ab-NSCs) specifically binds to HER2 overexpressing human breast cancer cells and inhibits PI3K-Akt signaling. This translates to HER2Ab-NSC inhibition of breast cancer cell growth in vitro. Preclinical in vivo experiments using HER2Ab overexpressing NSCs in a breast cancer brain metastases (BCBM) mouse model demonstrate that intracranial injection of HER2Ab-NSCs significantly improves survival. In effect, these NSCs provide tumor localized production of HER2Ab, minimizing any potential off-target side effects. Our results establish HER2Ab-NSCs as a novel, nontoxic, and rational therapeutic approach for the successful treatment of HER2 overexpressing BCBM, which now warrants further preclinical and clinical investigation. PMID:26260958

  9. CDK4-Mediated Phosphorylation Inhibits Smad3 Activity in Cyclin D Overexpressing Breast Cancer Cells

    PubMed Central

    Zelivianski, Stanislav; Cooley, Anne; Kall, Ron; Jeruss, Jacqueline S.

    2012-01-01

    Introduction Smad3, a component of the TGFβ signaling cascade, contributes to G1 arrest in breast cancer cells. Cyclin D1/CDK4 promotes G1/S-phase transition, and CDK phosphorylation of Smad3 has been associated with inhibition of Smad3 activity. We hypothesized that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through CDK4-mediated phosphorylation and inhibition of Smad3 and release of G1 arrest. Methods Real-time quantitative RT-PCR and immunoblotting were used to evaluate expression of study proteins in cyclin D1-overexpressing breast cancer cells. Smad3 transcriptional activity and cell cycle control were examined in cells transfected with wild type (WT) Smad3 or Smad3 with single or multiple CDK phosphorylation site mutations (M), in the presence or absence of CDK4 inhibitor or co-transfection with cdk4 siRNA. Results Transfection of the Smad3 5M construct resulted in decreased c-myc and higher p15INK4B expression. Compared with WT Smad3, overexpression of the Smad3 T8, T178, 4M, or 5M mutant constructs resulted in higher Smad3 transcriptional activity. Compared with cells transfected with WT Smad3, Smad3 transcriptional activity was higher in cells overexpressing Smad3 mutant constructs and treated with CDK4 inhibitor or transfected with cdk4 siRNA. Cells transfected with Smad3 T8 or T178 and treated with CDK4 inhibitor showed an increase in the G1 cell population. Conclusions Inhibition of CDK-mediated Smad3 phosphorylation released cyclin D1-regulated blockade of Smad3 transcriptional activity and recovered cell cycle arrest in breast cancer cells. Targeted inhibition of CDK4 activity may have a role in the treatment of cyclin D-overexpressing breast cancers. PMID:20736297

  10. Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines.

    PubMed

    Cooley, Anne; Zelivianski, Stanislav; Jeruss, Jacqueline S

    2010-12-15

    Smad3, a component of the TGFβ signaling pathway, contributes to G1 arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild type (WT) Smad3, or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1), or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers. PMID:21150326

  11. Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines

    PubMed Central

    2010-01-01

    Smad3, a component of the TGFβ signaling pathway, contributes to G1 arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild-type (WT) Smad3 or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1) or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers. PMID:21150326

  12. siRNA-Based Targeting of Cyclin E Overexpression Inhibits Breast Cancer Cell Growth and Suppresses Tumor Development in Breast Cancer Mouse Model

    PubMed Central

    Liang, Yulong; Gao, Hong; Lin, Shiaw-Yih; Goss, John A.; Brunicardi, Francis C.; Li, Kaiyi

    2010-01-01

    Cyclin E is aberrantly expressed in many types of cancer including breast cancer. High levels of the full length as well as the low molecular weight isoforms of cyclin E are associated with poor prognosis of breast cancer patients. Notably, cyclin E overexpression is also correlated with triple-negative basal-like breast cancers, which lack specific therapeutic targets. In this study, we used siRNA to target cyclin E overexpression and assessed its ability to suppress breast cancer growth in nude mice. Our results revealed that cyclin E siRNA could effectively inhibit overexpression of both full length and low molecular weight isoforms of cyclin E. We found that depletion of cyclin E promoted apoptosis of cyclin E-overexpressing cells and blocked their proliferation and transformation phenotypes. Significantly, we further demonstrated that administration of cyclin E siRNA could inhibit breast tumor growth in nude mice. In addition, we found that cyclin E siRNA synergistically enhanced the cell killing effects of doxorubicin in cell culture and this combination greatly suppressed the tumor growth in mice. In conclusion, our results indicate that cyclin E, which is overexpressed in 30% of breast cancer, may serve as a novel and effective therapeutic target. More importantly, our study clearly demonstrates a very promising therapeutic potential of cyclin E siRNA for treating the cyclin E-overexpressing breast cancers, including the very malignant triple-negative breast cancers. PMID:20877462

  13. HER-2/neu overexpression and in vitro chemosensitivity to CMF and FEC in primary breast cancer.

    PubMed

    Konecny, G; Fritz, M; Untch, M; Lebeau, A; Felber, M; Lude, S; Beryt, M; Hepp, H; Slamon, D; Pegram, M

    2001-09-01

    Available clinical and experimental data on the effect of HER-2/neu overexpression on chemosensitivity are controversial. It was the purpose of this in vitro study to define the association between HER-2/neu overexpression and the sensitivity to the chemotherapeutic drug combinations of cyclophosphamide, methotrexate and 5-fluorouracil (CMF) and 5-fluorouracil, epirubicin and cyclophosphamide (FEC) of breast cancer cells derived from 140 chemotherapy-nave patients at the time of primary surgery. Both drug combinations were tested at six different concentrations ranging from 6.25-200% peak plasma concentration (PPC). Immunohistochemical detection of HER-2/neu overexpression was performed with the HER-2/neu antibodies, CB11, TAB250 and AO485, in the same tumor specimens. Immunoreactions were determined as negative (0/1+), weakly positive (2+) and strongly positive (3+). However, the antibodies varied in their degrees of sensitivity. Breast cancer samples with strong (3+) HER-2/neu overexpression demonstrated 90% growth inhibition (IC90) at significantly lower PPC values, using the CB11 (p = 0.048), TAB250 (p = 0.007) and AO485 (p < or =0.01) antibodies, and showed 50% growth inhibition (IC50) at significantly lower PPC values, using the CB11 antibody (p = 0.01) compared to their counterparts with lower levels of HER-2/neu expression. When analyzing the group of patients with intermediate and strong HER-2/neu overexpression (2+ and 3+), an association between HER-2/neu overexpression and increased chemosensitivity was seen with the TAB250 (p = 0.044) and AO485 (p = 0.032) antibodies, but not with the CB11 antibody (p =0.8) at the IC90 level. Differences in chemosensitivity between samples with strong HER-2/neu overexpression and those with lower levels were then analyzed separately for CMF and FEC. Both regimens achieved 90% tumor growth inhibition at lower PPC values in samples with strong HER-2/neu overexpression (3+) compared to their counterparts with lower expression levels (AO485 p = 0.011 for CMF, and p = 0.09 for FEC). Cumulative concentration-response plots of tumors responding in vitro with 90% tumor cell inhibition showed a stronger dose dependence for both CMF and FEC among tumor samples with strong HER-2/neu overexpression compared to those with lower levels of expression. In conclusion, the data show that HER-2/neu overexpression was not associated with in vitro drug resistance to CMF or FEC. In contrast, tumors with strong HER-2/neu overexpression demonstrated increased dose-dependent in vitro sensitivity to both the FEC and CMF regimens. PMID:11759828

  14. Widespread p53 overexpression in human malignant tumors. An immunohistochemical study using methacarn-fixed, embedded tissue.

    PubMed Central

    Porter, P. L.; Gown, A. M.; Kramp, S. G.; Coltrera, M. D.

    1992-01-01

    p53 is a nuclear protein believed to play an important role, through mutation and overexpression, in the progression of human malignant tumors. The authors employed a monoclonal antibody, 1801, and investigated overexpression of p53 in a series of 255 malignant and benign tumors, using deparaffinized sections of methacarn-fixed tissue. Overall, immunohistochemically detected p53 overexpression was found in 39% of malignant tumors, with considerable variation within individual tumor types (34% of breast carcinomas, 92% of ovarian carcinomas, 33% of soft tissue sarcomas). Homogenous, heterogenous, and focal immunostaining patterns were noted. With rare exceptions, no immunostaining of any benign tumors was noted. No immunostaining was found in adjacent, benign tissues, or in a series of fetal tissues. This is the first demonstration of widespread p53 overexpression in alcohol-fixed, embedded tissue and confirms the major role played by p53 in human malignancies. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1731521

  15. Effect of Oncoxin Oral Solution in HER2-Overexpressing Breast Cancer.

    PubMed

    Hernández-García, Susana; González, Verena; Sanz, Eduardo; Pandiella, Atanasio

    2015-01-01

    One of the most aggressive breast cancer subtypes includes tumors with high expression of HER2. Gene expression and functional studies have shown a link between HER2 overexpression and oxidative stress. Because of this, we hypothesized that Oncoxin Oral Solution (OOS), a composite product that contains several antioxidants, could have an antitumoral effect against HER2+ tumors. Dose-response studies, biochemical and cytometric assessment of the effect of OOS on cell cycle and apoptosis, and drug combination analyses were performed on BT474 and SKBR3 cells, 2 HER2-overexpressing breast cancer cell lines. OOS reduced the proliferation of these cells, and augmented the action of lapatinib, a HER2 inhibitor used in the breast cancer clinic. Moreover, OOS decreased growth of HER2+ tumors in mice. Mechanistically, OOS provoked cell cycle blockade through upregulation of p27 expression and downregulation of cyclin D levels. OOS also caused apoptotic cell death in HER2+ breast cancer cells, as indicated by increases in PARP cleavage as well as upregulation of caspase 8 and caspase 3 activities. These results demonstrate an antitumoral action of OOS in preclinical models of HER2+ breast cancer and suggest that it can be used with anti-HER2 therapies currently adopted as standard of care in the oncology clinic. PMID:26241555

  16. LSD1 Overexpression Is Associated with Poor Prognosis in Basal-Like Breast Cancer, and Sensitivity to PARP Inhibition

    PubMed Central

    Nagasawa, Satoi; Sedukhina, Anna S.; Nakagawa, Yuko; Maeda, Ichiro; Kubota, Manabu; Ohnuma, Shigeko; Tsugawa, Koichiro; Ohta, Tomohiko; Roche-Molina, Marta; Bernal, Juan A.; Narvez, Ana J.; Jeyasekharan, Anand D.; Sato, Ko

    2015-01-01

    LSD1, a lysine-specific histone demethylase, is overexpressed in several types of cancers and linked to poor outcomes. In breast cancer, the significance of LSD1 overexpression is not clear. We have performed an in silico analysis to assess the relationship of LSD1 expression to clinical outcome. We demonstrate that LSD1 overexpression is a poor prognostic factor in breast cancer, especially in basal-like breast cancer, a subtype of breast cancer with aggressive clinical features. This link is also observed in samples of triple negative breast cancer. Interestingly, we note that overexpression of LSD1 correlates with down-regulation of BRCA1 in triple negative breast cancer. This phenomenon is also observed in in vitro models of basal-like breast cancer, and is associated with an increased sensitivity to PARP inhibitors. We propose therefore that high expression levels of the demethylase LSD1 is a potential prognostic factor of poor outcome in basal-like breast cancer, and that PARP inhibition may be a therapeutic strategy of interest in this poor prognostic subtype with overexpression of LSD1. PMID:25679396

  17. MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers

    PubMed Central

    Mohseni, Morassa; Cidado, Justin; Croessmann, Sarah; Cravero, Karen; Cimino-Mathews, Ashley; Wong, Hong Yuen; Scharpf, Rob; Zabransky, Daniel J.; Abukhdeir, Abde M.; Garay, Joseph P.; Wang, Grace M.; Beaver, Julia A.; Cochran, Rory L.; Blair, Brian G.; Rosen, D. Marc; Erlanger, Bracha; Argani, Pedram; Hurley, Paula J.; Lauring, Josh; Park, Ben Ho

    2014-01-01

    Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy. PMID:25422431

  18. Lapatinib and trastuzumab in combination with an aromatase inhibitor for the first-line treatment of metastatic hormone receptor-positive breast cancer which over-expresses human epidermal growth factor 2 (HER2): a systematic review and economic analysis.

    PubMed Central

    Fleeman, N; Bagust, A; Boland, A; Dickson, R; Dundar, Y; Moonan, M; Oyee, J; Blundell, M; Davis, H; Armstrong, A; Thorp, N

    2011-01-01

    BACKGROUND Breast cancer is the uncontrolled, abnormal growth of malignant breast tissue affecting predominantly women. Metastatic breast cancer (mBC) is an advanced stage of the disease when the disease has spread beyond the original organ. Hormone receptor status and human epidermal growth factor 2 (HER2) status are two predictive factors that are taken into consideration when estimating the prognosis of patients with breast cancer. OBJECTIVES To review the clinical effectiveness and cost-effectiveness evidence base for lapatinib (LAP) in combination with an aromatase inhibitor (AI) and trastuzumab (TRA) in combination with an AI for the first-line treatment of patients who have hormone receptor-positive (HR+)/human epidermal growth factor 2-positive (HER2+) mBC. DATA SOURCES Relevant electronic databases and websites, including MEDLINE, EMBASE and the Cochrane Library, were searched until May 2010. Further data were derived from the manufacturers' submissions for LAP + AI and TRA + AI. REVIEW METHODS A systematic review of the clinical effectiveness and cost-effectiveness of LAP + AI and TRA + AI was undertaken. As it was deemed inappropriate to compare LAP + AI with TRA + AI, two separate assessments of cost-effectiveness versus AIs alone were undertaken. RESULTS Three trials were included in the systematic review [the patient populations of the efficacy and safety of lapatinib combined with letrozole (EGF30008) trial, the efficacy and safety of trastuzumab combined with anastrozole (TAnDEM) trial and the efficacy and safety of letrozole combined with trastuzumab (eLEcTRA) trial]. As a result of differences in the exclusion criteria and because one trial was halted prematurely, comparisons across trials were believed to be inappropriate and meta-analysis was not possible. Individually, however, the findings from the trials all suggest that LAP + AI or TRA + AI results in improved progression-free survival and/or time to progression when compared with AIs alone. The trials do not show a statistically significant benefit in terms of overall survival. Two separate economic analyses were conducted based on the completed trials; neither LAP + AI nor TRA + AI was found to be cost-effective when compared with AI monotherapy. LIMITATIONS Because of differences in the EGF30008 and the TAnDEM trials, the Assessment Group believes the indirect comparisons analyses conducted by the manufacturers are inappropriate and, for the same reason, chooses not to compare LAP + AI with TRA + AI in an economic evaluation. CONCLUSIONS LAP + AI and TRA + AI appear to be clinically more effective than AI monotherapy, but neither is cost-effective compared with AIs alone. It was not possible to compare LAP + AI with TRA + AI. Future research should include research into treating mBC in the HR+/HER2+ population who are not TRA (or LAP) naive and into comparing the clinical effectiveness of AIs as monotherapy in patients with HER2+ and human epidermal growth factor 2-negative breast cancer. FUNDING The National Institute for Health Research Technology Assessment programme. PMID:22152751

  19. CHL1 is involved in human breast tumorigenesis and progression

    SciTech Connect

    He, Li-Hong; Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin ; Ma, Qin; Shi, Ye-Hui; Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin ; Ge, Jie; Zhao, Hong-Meng; Breast Surgery, Tianjin Medical University Cancer Institute and Hospital, Tianjin ; Li, Shu-Fen; Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin ; Tong, Zhong-Sheng; Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education, Tianjin Medical University Cancer Institute and Hospital, Tianjin

    2013-08-23

    Highlights: •CHL1 is down-regulation in breast cancer tissues. •Down-regulation of CHL1 is related to high grade. •Overexpression of CHL1 inhibits breast cancer cell proliferation and invasion in vitro. •CHL1 deficiency induces breast cancer cell proliferation and invasion both in vitro and in vivo. -- Abstract: Neural cell adhesion molecules (CAM) play important roles in the development and regeneration of the nervous system. The L1 family of CAMs is comprised of L1, Close Homolog of L1 (CHL1, L1CAM2), NrCAM, and Neurofascin, which are structurally related trans-membrane proteins in vertebrates. Although the L1CAM has been demonstrated play important role in carcinogenesis and progression, the function of CHL1 in human breast cancer is limited. Here, we found that CHL1 is down-regulated in human breast cancer and related to lower grade. Furthermore, overexpression of CHL1 suppresses proliferation and invasion in MDA-MB-231 cells and knockdown of CHL1 expression results in increased proliferation and invasion in MCF7 cells in vitro. Finally, CHL1 deficiency promotes tumor formation in vivo. Our results may provide a strategy for blocking breast carcinogenesis and progression.

  20. Twist overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer.

    PubMed

    Mironchik, Yelena; Winnard, Paul T; Vesuna, Farhad; Kato, Yoshinori; Wildes, Flonne; Pathak, Arvind P; Kominsky, Scott; Artemov, Dmitri; Bhujwalla, Zaver; Van Diest, Paul; Burger, Horst; Glackin, Carlotta; Raman, Venu

    2005-12-01

    Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype. PMID:16322226

  1. Cooperatively transcriptional and epigenetic regulation of sonic hedgehog overexpression drives malignant potential of breast cancer.

    PubMed

    Duan, Zhao-Heng; Wang, Hao-Chuan; Zhao, Dong-Mei; Ji, Xiao-Xin; Song, Min; Yang, Xiao-Jun; Cui, Wei

    2015-08-01

    Sonic hedgehog (Shh), a ligand of Hedgehog signaling pathway, is considered an important oncogene and an exciting potential therapeutic target in several cancers. Comprehensive understanding of the regulation mechanism of Shh in cancer cells is necessary to find an effective approach to selectively block its tumorigenic function. We and others previously demonstrated that nuclear factor-kappa B (NF-?B) activation and promoter hypomethylation contributed to the overexpression of Shh. However, the relationship between transcriptional and epigenetic regulation of Shh, and their roles in the malignant phenotype of cancer cells are still not clearly elucidated. In the present study, our data showed that the level of Shh was higher in breast cancer tissues with positive NF-?B nuclear staining and promoter hypomethylation. In addition, survival analysis revealed that Shh overexpression, but not hypomethylation and NF-?B nuclear staining, was a poor prognosis indicator for breast cancers. Moreover, invitro data demonstrated that both NF-?B activation and hypomethylation in promoter region were positively associated with the overexpression of Shh. Mechanistically, the hypomethylation in Shh promoter could facilitate NF-?B binding to its site, and subsequently cooperate to induce transcription of Shh. Furthermore, the biological function data indicated that overexpressed Shh enhanced the self-renewal capacity and migration ability of breast cancer cells, which could be augmented by promoter demethylation and NF-?B activation. Overall, our findings reveal multiple and cooperative mechanisms of Shh upregulation in cancer cells, and the roles of Shh in tumor malignant behavior, thus suggesting a new strategy for therapeutic interventions to reduce Shh in tumors and improve patients' prognosis. PMID:25990213

  2. t-Darpp overexpression in HER2-positive breast cancer confers a survival advantage in lapatinib

    PubMed Central

    Christenson, Jessica L.; Denny, Erin C.; Kane, Susan E.

    2015-01-01

    Drug resistance is a major barrier to successful cancer treatment. For patients with HER2-positive breast cancer who initially respond to therapy, the majority develop resistance within one year of treatment. Patient outcomes could improve significantly if we can find and exploit common mechanisms of acquired resistance to different targeted therapies. Overexpression of t-Darpp, a truncated form of the dual kinase/phosphatase inhibitor Darpp-32, has been linked to acquired resistance to trastuzumab, a front-line therapy for HER2-positive breast cancer. Darpp-32 reverses t-Darpp's effect on trastuzumab resistance. In this study, we examined whether t-Darpp could be involved in resistance to lapatinib, another HER2-targeted therapeutic. Lapatinib-resistant SKBR3 cells (SK/LapR) showed a marked change in the Darpp-32:t-Darpp ratio toward a predominance of t-Darpp. Overexpression of t-Darpp alone was not sufficient to confer lapatinib resistance, but cells that overexpress t-Darpp partially mimicked the molecular resistance phenotype observed in SK/LapR cells exposed to lapatinib. SK/LapR cells failed to down-regulate Survivin and failed to induce BIM accumulation in response to lapatinib; cells overexpressing t-Darpp exhibited only the failed BIM accumulation. t-Darpp knock-down reversed this phenotype. Using a fluorescence-based co-culture system, we found that cells overexpressing t-Darpp formed colonies in lapatinib within 3–4 weeks, whereas parental cells in the same co-culture did not. Overall, t-Darpp appears to mediate a survival advantage in lapatinib, possibly linked to failed lapatinib-induced BIM accumulation. t-Darpp might also be relevant to acquired resistance to other cancer drugs that rely on BIM accumulation to induce apoptosis. PMID:26430732

  3. NRBP1 is downregulated in breast cancer and NRBP1 overexpression inhibits cancer cell proliferation through Wnt/?-catenin signaling pathway.

    PubMed

    Wei, Hong; Wang, Hongbin; Ji, Qiao; Sun, Jiawei; Tao, Lin; Zhou, Xianli

    2015-01-01

    Nuclear receptor binding protein 1 (NRBP1) is a highly conserved protein that is ubiquitously expressed across cell types in humans. NRBP1 has been recently identified as an adaptor protein. It has been suggested that it plays important roles in cellular homeostasis and the pathophysiology of cancer. To determine whether NRBP1 is involved in the pathophysiology of breast cancer, we performed a correlation study between the expression level of NRBP1 and the clinicopathological features in 92 breast cancer patients. A strong correlation was detected between NRBP1 expression and advanced histopathology grades, tumor, node, and metastasis stage, tumor diameter, lymph node involvement, as well as the recurrence of breast cancer in 92 tested patients. The tumor tissues from patients also expressed lower NRBP1 than did adjacent healthy tissues. Furthermore, we overexpressed NRBP1 in MCF-7 and MDA-MB-231 breast cancer cell lines and found NRBP1 upregulation-inhibited cell proliferation by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Blocking the autocrine Wnt signaling pathway by LGK974 could remove the NRBP1-overexpression-induced inhibition in breast cancer cells. The results of this study suggest that NRBP1 plays a tumor-suppressive role in breast cancer pathophysiology, which likely acts through the Wnt/?-catenin signaling pathway. PMID:26715855

  4. NRBP1 is downregulated in breast cancer and NRBP1 overexpression inhibits cancer cell proliferation through Wnt/?-catenin signaling pathway

    PubMed Central

    Wei, Hong; Wang, Hongbin; Ji, Qiao; Sun, Jiawei; Tao, Lin; Zhou, Xianli

    2015-01-01

    Nuclear receptor binding protein 1 (NRBP1) is a highly conserved protein that is ubiquitously expressed across cell types in humans. NRBP1 has been recently identified as an adaptor protein. It has been suggested that it plays important roles in cellular homeostasis and the pathophysiology of cancer. To determine whether NRBP1 is involved in the pathophysiology of breast cancer, we performed a correlation study between the expression level of NRBP1 and the clinicopathological features in 92 breast cancer patients. A strong correlation was detected between NRBP1 expression and advanced histopathology grades, tumor, node, and metastasis stage, tumor diameter, lymph node involvement, as well as the recurrence of breast cancer in 92 tested patients. The tumor tissues from patients also expressed lower NRBP1 than did adjacent healthy tissues. Furthermore, we overexpressed NRBP1 in MCF-7 and MDA-MB-231 breast cancer cell lines and found NRBP1 upregulation-inhibited cell proliferation by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Blocking the autocrine Wnt signaling pathway by LGK974 could remove the NRBP1-overexpression-induced inhibition in breast cancer cells. The results of this study suggest that NRBP1 plays a tumor-suppressive role in breast cancer pathophysiology, which likely acts through the Wnt/?-catenin signaling pathway. PMID:26715855

  5. Involvement of B3GALNT2 overexpression in the cell growth of breast cancer.

    PubMed

    Matsuo, Taisuke; Komatsu, Masato; Yoshimaru, Tetsuro; Kiyotani, Kazuma; Miyoshi, Yasuo; Sasa, Mitsunori; Katagiri, Toyomasa

    2014-02-01

    A number of glycosyltransferases have been identified and biologically characterized in cancer cells, yet their exact pathophysiological functions are largely unknown. Here, we report the critical role of β1,3-N-acetylgalactosaminyltransferase II (B3GALNT2), which transfers N-acetylgalactosamine (GalNAc) in a β1,3 linkage to N-acetylglucosamine, in the growth of breast cancer cells. Comprehensive transcriptomics, quantitative PCR and northern blot analyses indicated this molecule to be exclusively upregulated in the majority of breast cancers. Knockdown of B3GALNT2 expression by small interfering RNA attenuated cell growth and induced apoptosis in breast cancer cells. Overexpression of B3GALNT2 in HEK293T cells prompted secretion of the gene product into the culture medium, suggesting that B3GALNT2 is potentially a secreted protein. Furthermore, we demonstrated that B3GALNT2 is N-glycosylated on both Asn-116 and Asn-174 and that this modification is necessary for its secretion in breast cancer cells. Our findings suggest that this molecule represents a promising candidate for the development of a novel therapeutic targeting drug and a potential diagnostic tumor marker for patients with breast cancer, especially TNBC. PMID:24285400

  6. Cooperation between Dmp1 Loss and Cyclin D1 Overexpression in Breast Cancer

    PubMed Central

    Zhu, Sinan; Mott, Ryan T.; Fry, Elizabeth A.; Taneja, Pankaj; Kulik, George; Sui, Guangchao; Inoue, Kazushi

    2014-01-01

    Cyclin D1 is a component of the core cell-cycle machinery and is frequently overexpressed in breast cancer. It physically interacts with the tumor suppressor Dmp1 that attenuates the oncogenic signals from Ras and HER2 by inducing Arf/p53-dependent cell-cycle arrest. Currently, the biological significance of Dmp1–cyclin D1 interplay in breast cancer has not been determined. Here, we show that cyclin D1 bound to Dmp1 to activate both Arf and Ink4a promoters and, consequently, induced apoptosis or G2/M cell-cycle delay in normal cells to protect them from neoplastic transformation. The cyclin D1–induced Ink4a/Arf gene expression was dependent on Dmp1 because the induction was not detected in Dmp1-deficient or DMP1-depleted cells. Arf/Ink4a expression was increased in pre-malignant mammary glands from Dmp1+/+;MMTV-cyclin D1 and Dmp1+/+;MMTV-D1T286A mice but significantly down-regulated in those from Dmp1-deficient mice. Selective Dmp1 deletion was found in 21% of the MMTV-D1 and D1T286A mammary carcinomas, and the Dmp1 heterozygous status significantly accelerated mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Overall, our study reveals a pivotal role of combined Dmp1 loss and cyclin D1 overexpression in breast cancer. PMID:23938323

  7. Transglutaminase 2 Overexpression in Tumor Stroma Identifies Invasive Ductal Carcinomas of Breast at High Risk of Recurrence

    PubMed Central

    Assi, Jasmeet; Srivastava, Gunjan; Matta, Ajay; Chang, Martin C.

    2013-01-01

    Introduction Molecular markers for predicting breast cancer patients at high risk of recurrence are urgently needed for more effective disease management. The impact of alterations in extracellular matrix components on tumor aggressiveness is under intense investigation. Overexpression of Transglutaminase 2 (TG2), a multifunctional enzyme, in cancer cells impacts epithelial mesenchymal transition, growth, invasion and interactions with tumor microenvironment. The objective of our study is to determine the clinical relevance of stromal TG2 overexpression and explore its potential to identify breast cancers at high risk of recurrence. Methods This retrospective study is based on immunohistochemical analysis of TG2 expression in normal breast tissues (n?=?40) and breast cancers (n?=?253) with clinical, pathological and follow-up data available for up to 12 years. TG2 expression was correlated with clinical and pathological parameters as well as disease free survival (DFS) of breast cancer patients. Results Stromal TG2 overexpression was observed in 114/253 (45.0%) breast cancer tissues as compared to breast normal tissues. Among invasive ductal carcinomas (IDC) of the breast, 97/168 (57.7%) showed strong TG2 expression in tumor stroma. Importantly, IDC patients showing stromal TG2 accumulation had significantly reduced DFS (mean DFS?=?110 months) in comparison with patients showing low expression (mean DFS?=?130 months) in Kaplan-Meier survival analysis (p<0.001). In Cox multivariate regression analysis, stromal TG2 accumulation was an independent risk factor for recurrence (p?=?0.006, Hazards ratio, H.R.?=?3.79). Notably, these breast cancer patients also showed immunostaining of N-epsilon gamma-glutamyl lysine amino residues in tumor stroma demonstrating the transamidating activity of TG2. Conclusions Accumulation of TG2 in tumor stroma is an independent risk factor for identifying breast cancer patients at high risk of recurrence. TG2 overexpression in tumor stroma may serve as a predictor of poor prognosis for IDC of the breast. PMID:24058567

  8. Downregulation of ER-?36 expression sensitizes HER2 overexpressing breast cancer cells to tamoxifen.

    PubMed

    Yin, Li; Pan, Xiaohua; Zhang, Xin-Tian; Guo, Yu-Ming; Wang, Zhao-Yi; Gong, Yaoqin; Wang, Molin

    2015-01-01

    Tamoxifen provided a successful treatment for ER-positive breast cancer for many years. However, HER2 overexpressing breast cancer cells respond poorly to tamoxifen therapy presumably by pass. The molecular mechanisms underlying development of tamoxifen resistance have not been well established. Recently, we reported that breast cancer cells with high levels of ER-?36, a variant of ER-?, were resistant to tamoxifen and knockdown of ER-?36 expression in tamoxifen resistant cells with the shRNA method restored tamoxifen sensitivity, indicating that gained ER-?36 expression is one of the underlying mechanisms of tamoxifen resistance. Here, we found that tamoxifen induced expression of ER-?36-EGFR/HER2 positive regulatory loops and tamoxifen resistant MCF7 cells (MCF7/TAM) expressed enhanced levels of the loops. Disruption of the ER-?36-EGFR/HER2 positive regulatory loops with the dual tyrosine kinase inhibitor Lapatinib or ER-?36 down-regulator Broussoflavonol B in tamoxifen resistant MCF7 cells restored tamoxifen sensitivity. In addition, we also found both Lapatinib and Broussoflavonol B increased the growth inhibitory activity of tamoxifen in tumorsphere cells derived from MCF7/TAM cells. Our results thus demonstrated that elevated expression of the ER-?36-EGFR/HER2 loops is one of the mechanisms by which ER-positive breast cancer cells escape tamoxifen therapy. Our results thus provided a rational to develop novel therapeutic approaches for tamoxifen resistant patients by targeting the ER-?36-EGFR/HER2 loops. PMID:25973295

  9. Suppression of the malignant phenotype of human glioma cells by overexpression of manganese superoxide dismutase.

    PubMed

    Zhong, W; Oberley, L W; Oberley, T D; St Clair, D K

    1997-01-30

    Manganese superoxide dismutase (MnSOD) has been previously shown to suppress the malignant phenotype of human melanoma and breast cancer cells. To test the possible role of MnSOD in glioma malignancy, MnSOD was overexpressed in wild type human glioma U118 cells and subcloned U118-9 cells by transfection of human MnSOD cDNA. The MnSOD-transfected cell lines demonstrated expression of exogenous (plasmid) MnSOD mRNA, increase in MnSOD immunoreactive protein, and a three- to eightfold increase in MnSOD enzymatic activity. The MnSOD overexpressing cell lines became less malignant as demonstrated by requiring a higher serum concentration to grow in vitro and much slower tumor growth in nude mice than the parental and neo control cell lines. These findings further support the hypothesis that MnSOD may be a tumor suppressor gene in a wide variety of human tumors. PMID:9053845

  10. R-RAS2 overexpression in tumors of the human central nervous system.

    PubMed

    Gutierrez-Erlandsson, Sylvia; Herrero-Vidal, Pedro; Fernandez-Alfara, Marcos; Hernandez-Garcia, Susana; Gonzalo-Flores, Sandra; Mudarra-Rubio, Alberto; Fresno, Manuel; Cubelos, Beatriz

    2013-01-01

    Malignant tumors of the central nervous system (CNS) are the 10th most frequent cause of cancer mortality. Despite the strong malignancy of some such tumors, oncogenic mutations are rarely found in classic members of the RAS family of small GTPases. This raises the question as to whether other RAS family members may be affected in CNS tumors, excessively activating RAS pathways. The RAS-related subfamily of GTPases is that which is most closely related to classical Ras and it currently contains 3 members: RRAS, RRAS2 and RRAS3. While R-RAS and R-RAS2 are expressed ubiquitously, R-RAS3 expression is restricted to the CNS. Significantly, both wild type and mutated RRAS2 (also known as TC21) are overexpressed in human carcinomas of the oral cavity, esophagus, stomach, skin and breast, as well as in lymphomas. Hence, we analyzed the expression of R-RAS2 mRNA and protein in a wide variety of human CNS tumors and we found the R-RAS2 protein to be overexpressed in all of the 90 CNS cancer samples studied, including glioblastomas, astrocytomas and oligodendrogliomas. However, R-Ras2 was more strongly expressed in low grade (World Health Organization grades I-II) rather than high grade (grades III-IV) tumors, suggesting that R-RAS2 is overexpressed in the early stages of malignancy. Indeed, R-RAS2 overexpression was evident in pre-malignant hyperplasias, both at the mRNA and protein levels. Nevertheless, such dramatic changes in expression were not evident for the other two subfamily members, which implies that RRAS2 is the main factor triggering neural transformation. PMID:24148564

  11. hMENA(11a) contributes to HER3-mediated resistance to PI3K inhibitors in HER2-overexpressing breast cancer cells.

    PubMed

    Trono, P; Di Modugno, F; Circo, R; Spada, S; Di Benedetto, A; Melchionna, R; Palermo, B; Matteoni, S; Soddu, S; Mottolese, M; De Maria, R; Nistic, P

    2016-02-18

    Human Mena (hMENA), an actin regulatory protein of the ENA/VASP family, cooperates with ErbB receptor family signaling in breast cancer. It is overexpressed in high-risk preneoplastic lesions and in primary breast tumors where it correlates with HER2 overexpression and an activated status of AKT and MAPK. The concomitant overexpression of hMENA and HER2 in breast cancer patients is indicative of a worse prognosis. hMENA is expressed along with alternatively expressed isoforms, hMENA(11a) and hMENA?v6 with opposite functions. A novel role for the epithelial-associated hMENA(11a) isoform in sustaining HER3 activation and pro-survival pathways in HER2-overexpressing breast cancer cells has been identified by reverse phase protein array and validated in vivo in a series of breast cancer tissues. As HER3 activation is crucial in mechanisms of cell resistance to PI3K inhibitors, we explored whether hMENA(11a) is involved in these resistance mechanisms. The specific hMENA(11a) depletion switched off the HER3-related pathway activated by PI3K inhibitors and impaired the nuclear accumulation of HER3 transcription factor FOXO3a induced by PI3K inhibitors, whereas PI3K inhibitors activated hMENA(11a) phosphorylation and affected its localization. At the functional level, we found that hMENA(11a) sustains cell proliferation and survival in response to PI3K inhibitor treatment, whereas hMENA(11a) silencing increases molecules involved in cancer cell apoptosis. As shown in three-dimensional cultures, hMENA(11a) contributes to resistance to PI3K inhibition because its depletion drastically reduced cell viability upon treatment with PI3K inhibitor BEZ235. Altogether, these results indicate that hMENA(11a) in HER2-overexpressing breast cancer cells sustains HER3/AKT axis activation and contributes to HER3-mediated resistance mechanisms to PI3K inhibitors. Thus, hMENA(11a) expression can be proposed as a marker of HER3 activation and resistance to PI3K inhibition therapies, to select patients who may benefit from these combined targeted treatments. hMENA(11a) activity could represent a new target for antiproliferative therapies in breast cancer. PMID:25961924

  12. Luminal Breast Cancer Cell Lines Overexpressing ZNF703 Are Resistant to Tamoxifen through Activation of Akt/mTOR Signaling

    PubMed Central

    Huang, Ou; Xie, Zuoquan; Jiang, Min; Geng, Meiyu; Shen, Kunwei

    2013-01-01

    Background Selective estrogen receptor modulators, such as tamoxifen, play a pivotal role in the treatment of luminal-type breast cancer. However, in clinical applications, nearly half of breast cancer patients are insensitive to tamoxifen, a small number of whom have early recurrence or disease progression when receiving tamoxifen. The underlying mechanism of this resistance has not been determined. ZNF703 is a novel oncogene in the 15% of breast cancers that harbor 8p12 amplifications. Therefore, the goal of our study was to explore the role of ZNF703 in tamoxifen resistance. Methodology/Principal Findings We used immunohistochemistry techniques to examine ZNF703 expression in stage I-III primary breast cancer specimens and found a positive expression rate of 91.3%. All patients were divided into either high or low ZNF703 expression groups. We found that high ZNF703 expression mainly occurred in ER+ and PR+ breast cancers. Furthermore, 4-hydroxytamoxifen had different modes of action in breast cancer cell lines with high or low ZNF703 expression. ZNF703 overexpression in MCF-7 breast cancer cells activated the Akt/mTOR signaling pathway, downregulated ER?, and reduced the antitumor effect of tamoxifen. Low-dose tamoxifen did not suppress, but rather, stimulated the growth of cells overexpressing ZNF703. ZNF703 knockdown in MDA-MB-134 and HCC1500 luminal B-type breast cancer cell lines by siRNA significantly decreased survival rates when cells were treated with tamoxifen. Furthermore, targeting ZNF703 with a mTOR inhibitor increased the inhibitory effects of tamoxifen in ZNF703-overexpressing cells. Conclusion/Significance Our study suggests that ZNF703 expression levels may predict tamoxifen sensitivity. Tamoxifen should be administered with caution to those patients bearing tumors with ZNF703 overexpression. However, large clinical trials and prospective clinical studies are needed to verify these results. PMID:23991038

  13. Focal adhesion kinase overexpression and its impact on human osteosarcoma

    PubMed Central

    Chen, Yong; Yang, Aizhen; Chen, Hui; Zhang, Jian; Wu, Sujia; Shi, Xin; Wang, Chen; Sun, Xiaoliang

    2015-01-01

    Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P < 0.001). There were similar differences in metastasis-free survival between groups. The Cox proportional hazards analysis showed that the FAK expression profile was an independent indicator of both overall and metastasis-free survival. siRNA-based knockdown of FAK not only dramatically reduced the migration and invasion of MG63 and 143B cells, but also had a distinct effect on osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis. PMID:26393679

  14. Activation of peroxisome proliferator-activated receptor-?/? (PPAR-?/?) inhibits human breast cancer cell line tumorigenicity.

    PubMed

    Yao, Pei-Li; Morales, Jose L; Zhu, Bokai; Kang, Boo-Hyon; Gonzalez, Frank J; Peters, Jeffrey M

    2014-04-01

    The effect of activation and overexpression of the nuclear receptor PPAR-?/? in human MDA-MB-231 (estrogen receptor-negative; ER(-)) and MCF7 (estrogen-receptor-positive; ER(+)) breast cancer cell lines was examined. Target gene induction by ligand activation of PPAR-?/? was increased by overexpression of PPAR-?/? compared with controls. Overexpression of PPAR-?/? caused a decrease in cell proliferation in MCF7 and MDA-MB-231 cells compared with controls, whereas ligand activation of PPAR-?/? further inhibited proliferation of MCF7 but not MDA-MB-231 cells. Overexpression and/or ligand activation of PPAR-?/? in MDA-MB-231 or MCF7 cells had no effect on experimental apoptosis. Decreased clonogenicity was observed in both MDA-MB-231 and MCF7 overexpressing PPAR-?/? in response to ligand activation of PPAR-?/? as compared with controls. Ectopic xenografts developed from MDA-MB-231 and MCF7 cells overexpressing PPAR-?/? were significantly smaller, and ligand activation of PPAR-?/? caused an even greater reduction in tumor volume as compared with controls. Interestingly, the decrease in MDA-MB-231 tumor size after overexpressing PPAR-?/? and ligand activation of PPAR-?/? correlated with increased necrosis. These data show that ligand activation and/or overexpression of PPAR-?/? in two human breast cancer cell lines inhibits relative breast cancer tumorigenicity and provide further support for the development of ligands for PPAR-?/? to specifically inhibit breast carcinogenesis. These new cell-based models will be invaluable tools for delineating the role of PPAR-?/? in breast cancer and evaluating the effects of PPAR-?/? agonists. PMID:24464939

  15. CD24 Overexpression Is Associated with Poor Prognosis in Luminal A and Triple-Negative Breast Cancer

    PubMed Central

    Kwon, Mi Jeong; Han, Jinil; Seo, Ji Hyun; Song, Kyoung; Jeong, Hae Min; Choi, Jong-Sun; Kim, Yu Jin; Lee, Seon-Heui; Choi, Yoon-La; Shin, Young Kee

    2015-01-01

    CD24 is associated with unfavourable prognoses in various cancers, but the prevalence of CD24 expression and its influence on clinical outcome in subtypes of breast cancers remain unclear. CD24 expression was analyzed by immunohistochemistry in 747 breast cancer tissues, and DNA methylation and histone modification status in the promoter region of CD24 were assessed using bisulfite sequencing and chromatin immunoprecipitation assay. 213 (28.5%) samples exhibited high CD24 expression in the membrane and/or cytoplasm of breast cancer cells, and CD24 overexpression was significantly correlated with the presence of lymph node metastasis and more advanced pathological stage. Patients with CD24-high tumours had significantly shorter patient survival than those with CD24-low tumours. Importantly, multivariate analysis that included tumour size, lymph node metastasis and chemotherapy demonstrated that high CD24 expression is independently associated with poorer survival in luminal A and triple-negative breast cancer (TNBC) subtypes. Furthermore, CD24 gene expression was associated with histone acetylation independent of DNA methylation, suggesting its epigenetic regulation in breast cancer. Our results suggest that CD24 overexpression is an independent unfavourable prognostic factor in breast cancer, especially for luminal A and TNBC subtypes, and CD24 may be a promising therapeutic target for specific subtypes of breast cancer. PMID:26444008

  16. Overexpression of Id1 in transgenic mice promotes mammary basal stem cell activity and breast tumorigenesis

    PubMed Central

    Won, Hee-Young; Jang, Ki-Seok; Min, Kyueng-Whan; Jang, Si-Hyong; Woo, Jong-Kyu; Oh, Seung Hyun; Kong, Gu

    2015-01-01

    Inhibitor of differentiation/DNA binding (Id)1 is a crucial regulator of mammary development and breast cancer progression. However, its effect on stemness and tumorigenesis in mammary epithelial cells remains undefined. Herein, we demonstrate that Id1 induces mammary tumorigenesis by increasing normal and malignant mammary stem cell (MaSC) activities in transgenic mice. MaSC-enriched basal cell expansion and increased self-renewal and in vivo regenerative capacity of MaSCs are observed in the mammary glands of MMTV-Id1 transgenic mice. Furthermore, MMTV-Id1 mice develop ductal hyperplasia and mammary tumors with highly expressed basal markers. Id1 also increases breast cancer stem cell (CSC) population and activity in human breast cancer lines. Moreover, the effects of Id1 on normal and malignant stem cell activities are mediated by the Wnt/c-Myc pathway. Collectively, these findings provide in vivo genetic evidence of Id1 functions as an oncogene in breast cancer and indicate that Id1 regulates mammary basal stem cells by activating the Wnt/c-Myc pathway, thereby contributing to breast tumor development. PMID:25938540

  17. Cyclin-dependent kinase 4-mediated phosphorylation inhibits Smad3 activity in cyclin D-overexpressing breast cancer cells.

    PubMed

    Zelivianski, Stanislav; Cooley, Anne; Kall, Ron; Jeruss, Jacqueline S

    2010-10-01

    Smad3, a component of the transforming growth factor β signaling cascade, contributes to G(1) arrest in breast cancer cells. Cyclin D1/cyclin-dependent kinase 4 (CDK4) promotes G(1)-S-phase transition, and CDK phosphorylation of Smad3 has been associated with inhibition of Smad3 activity. We hypothesized that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through CDK4-mediated phosphorylation and inhibition of Smad3 and release of G(1) arrest. Real-time quantitative reverse transcription-PCR and immunoblotting were used to evaluate expression of study proteins in cyclin D1-overexpressing breast cancer cells. Smad3 transcriptional activity and cell cycle control were examined in cells transfected with wild-type (WT) Smad3 or Smad3 with single or multiple CDK phosphorylation site mutations (M) in the presence or absence of the CDK4 inhibitor or cotransfection with cdk4 small interfering RNA (siRNA). Transfection of the Smad3 5M construct resulted in decreased c-myc and higher p15(INK4B) expression. Compared with WT Smad3, overexpression of the Smad3 T8, T178, 4M, or 5M mutant constructs resulted in higher Smad3 transcriptional activity. Compared with cells transfected with WT Smad3, Smad3 transcriptional activity was higher in cells overexpressing Smad3 mutant constructs and treated with the CDK4 inhibitor or transfected with cdk4 siRNA. Cells transfected with Smad3 T8 or T178 and treated with the CDK4 inhibitor showed an increase in the G(1) cell population. Inhibition of CDK-mediated Smad3 phosphorylation released cyclin D1-regulated blockade of Smad3 transcriptional activity and recovered cell cycle arrest in breast cancer cells. Targeted inhibition of CDK4 activity may have a role in the treatment of cyclin D-overexpressing breast cancers. PMID:20736297

  18. Overexpression of MMP Family Members Functions as Prognostic Biomarker for Breast Cancer Patients: A Systematic Review and Meta-Analysis

    PubMed Central

    Ren, Fanghui; Tang, Ruixue; Zhang, Xin; Madushi, Wickramaarachchi Mihiranganee; Luo, Dianzhong; Dang, Yiwu; Li, Zuyun; Wei, Kanglai; Chen, Gang

    2015-01-01

    Background Matrix metalloproteinases (MMPs) are regarded to be relevant to the prognosis of breast cancer. Numerous studies have confirmed the association between MMPs and tumor growth, invasion and metastasis in breast cancer. However, their prognostic values for survival in patients with breast cancer remain controversial. Hence, a meta-analysis was performed to clarify a more accurate estimation of the role of MMPs on prognosis of breast cancer patients. Method A systemic electronic search was conducted in PubMed, Embase and Web of science databases to identify eligible studies, which were associated with the relationship between MMPs and prognosis of breast cancer. The correlation in random-effect model was evaluated by using the hazard ratios (HRs) and 95% confidence intervals (CIs). Results A total of 28 studies covering 4944 patients were included for meta-analysis. A summary hazard ratio (HR) of all studies was calculated, as well as the sub-group HRs. The combined HRs calculated by either univariate or multivariate analysis both suggested that overexpression of MMPs had an unfavorable impact on overall survival (OS) (HR = 1.694, 95%CI: 1.3472.129, P < 0.001; HR = 1.611, 95%CI: 1.4191.830, P < 0.001, respectively). And the univariate analysis showed that patients with overexpression of MMPs had worse relapse-free survival (RFS) (HR = 1.969, 95%CI: 1.4602.655, P < 0.001) in all eligible studies. In the sub-group analyses, HRs of MMP-9 positivity with poor OS were 1.794 (95%CI: 1.3302.420, P < 0.001) and 1.709 (95%CI: 1.1572.526, P = 0.007) which were separately evaluated by univariate and multivariate analysis. A small number of articles demonstrated that MMP-2 overexpression was not related with shorter OS (HR = 1.400, 95%CI: 0.6103.029, P = 0.427). Four studies included in the OS analysis of MMPs expression in serum suggested that positive expression of serum MMPs may be an unfavorable factor (HR = 1.630, 95%CI: 1.0652.494) for breast cancer patients. No publication bias was observed in the current meta-analysis. Conclusions Our findings suggested that MMPs overexpression (especially MMP-9, MMP-2, MMPs overexpression in serum) might indicate a higher risk of poor prognosis in breast cancer. Larger prospective studies are further needed to estimate the prognostic values of MMPs overexpression. PMID:26270045

  19. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    NASA Technical Reports Server (NTRS)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  20. PAX6 overexpression is associated with the poor prognosis of invasive ductal breast cancer

    PubMed Central

    XIA, XIANGHOU; YIN, WENJUAN; ZHANG, XIPING; YU, XINGFEI; WANG, CHEN; XU, SHENHUA; FENG, WEILIANG; YANG, HONGJIAN

    2015-01-01

    Paired box 6 (PAX6) plays a significant role in the development of human neuroectodermal epithelial tissues. Previous studies have suggested that the PAX6 promoter is hypermethylated in breast cancer and that it is involved in breast cancer cell proliferation. The present study aimed to investigate the expression of PAX6 in invasive breast cancer tissues, and to evaluate its prognostic significance. Immunohistochemistry (IHC) was used to detect PAX6 expression on a breast cancer tissue microarray containing tissues from 111 patients. Associations of PAX6 expression with staging and prognosis were analyzed. PAX6 was mainly expressed in the nucleus. The PAX6 staining intensity was not associated with age, histological grade, lymph node status, tumor size, or progesterone receptor and human epidermal growth factor receptor 2 expression (all P>0.05). A high level of PAX6 staining was more frequent in estrogen receptor (ER)-negative cases compared with ER-positive cases (43.9 vs. 25.7%; P=0.049). After a median follow-up time of 110 months, the patients with low PAX6 expression exhibited an improved survival rate compared with the patients with high PAX6 expression (P<0.001). Cox analysis showed a worse survival rate in the patients with high PAX6 staining (hazard ratio, 3.458; 95% confidence interval, 1.5757.593; P=0.002). In conclusion, high tumor PAX6 staining intensity by IHC was associated with a poor prognosis in breast cancer patients. PMID:26622698

  1. Microbiota of Human Breast Tissue

    PubMed Central

    Urbaniak, Camilla; Cummins, Joanne; Brackstone, Muriel; Macklaim, Jean M.; Gloor, Gregory B.; Baban, Chwanrow K.; Scott, Leslie; O'Hanlon, Deidre M.; Burton, Jeremy P.; Francis, Kevin P.; Tangney, Mark

    2014-01-01

    In recent years, a greater appreciation for the microbes inhabiting human body sites has emerged. In the female mammary gland, milk has been shown to contain bacterial species, ostensibly reaching the ducts from the skin. We decided to investigate whether there is a microbiome within the mammary tissue. Using 16S rRNA sequencing and culture, we analyzed breast tissue from 81 women with and without cancer in Canada and Ireland. A diverse population of bacteria was detected within tissue collected from sites all around the breast in women aged 18 to 90, not all of whom had a history of lactation. The principal phylum was Proteobacteria. The most abundant taxa in the Canadian samples were Bacillus (11.4%), Acinetobacter (10.0%), Enterobacteriaceae (8.3%), Pseudomonas (6.5%), Staphylococcus (6.5%), Propionibacterium (5.8%), Comamonadaceae (5.7%), Gammaproteobacteria (5.0%), and Prevotella (5.0%). In the Irish samples the most abundant taxa were Enterobacteriaceae (30.8%), Staphylococcus (12.7%), Listeria welshimeri (12.1%), Propionibacterium (10.1%), and Pseudomonas (5.3%). None of the subjects had signs or symptoms of infection, but the presence of viable bacteria was confirmed in some samples by culture. The extent to which these organisms play a role in health or disease remains to be determined. PMID:24610844

  2. Microbiota of human breast tissue.

    PubMed

    Urbaniak, Camilla; Cummins, Joanne; Brackstone, Muriel; Macklaim, Jean M; Gloor, Gregory B; Baban, Chwanrow K; Scott, Leslie; O'Hanlon, Deidre M; Burton, Jeremy P; Francis, Kevin P; Tangney, Mark; Reid, Gregor

    2014-05-01

    In recent years, a greater appreciation for the microbes inhabiting human body sites has emerged. In the female mammary gland, milk has been shown to contain bacterial species, ostensibly reaching the ducts from the skin. We decided to investigate whether there is a microbiome within the mammary tissue. Using 16S rRNA sequencing and culture, we analyzed breast tissue from 81 women with and without cancer in Canada and Ireland. A diverse population of bacteria was detected within tissue collected from sites all around the breast in women aged 18 to 90, not all of whom had a history of lactation. The principal phylum was Proteobacteria. The most abundant taxa in the Canadian samples were Bacillus (11.4%), Acinetobacter (10.0%), Enterobacteriaceae (8.3%), Pseudomonas (6.5%), Staphylococcus (6.5%), Propionibacterium (5.8%), Comamonadaceae (5.7%), Gammaproteobacteria (5.0%), and Prevotella (5.0%). In the Irish samples the most abundant taxa were Enterobacteriaceae (30.8%), Staphylococcus (12.7%), Listeria welshimeri (12.1%), Propionibacterium (10.1%), and Pseudomonas (5.3%). None of the subjects had signs or symptoms of infection, but the presence of viable bacteria was confirmed in some samples by culture. The extent to which these organisms play a role in health or disease remains to be determined. PMID:24610844

  3. Aluminium in human breast tissue.

    PubMed

    Exley, Christopher; Charles, Lisa M; Barr, Lester; Martin, Claire; Polwart, Anthony; Darbre, Philippa D

    2007-09-01

    Aluminium is omnipresent in everyday life and increased exposure is resulting in a burgeoning body burden of this non-essential metal. Personal care products are potential contributors to the body burden of aluminium and recent evidence has linked breast cancer with aluminium-based antiperspirants. We have used graphite furnace atomic absorption spectrometry (GFAAS) to measure the aluminium content in breast biopsies obtained following mastectomies. The aluminium content of breast tissue and breast tissue fat were in the range 4-437 nmol/g dry wt. and 3-192 nmol/g oil, respectively. The aluminium content of breast tissue in the outer regions (axilla and lateral) was significantly higher (P=0.033) than the inner regions (middle and medial) of the breast. Whether differences in the regional distribution of aluminium in the breast are related to the known higher incidence of tumours in the outer upper quadrant of the breast remains to be ascertained. PMID:17629949

  4. Human cancers overexpress genes that are specific to a variety of normal human tissues

    PubMed Central

    Lotem, Joseph; Netanely, Dvir; Domany, Eytan; Sachs, Leo

    2005-01-01

    We have analyzed gene expression data from three different kinds of samples: normal human tissues, human cancer cell lines, and leukemic cells from lymphoid and myeloid leukemia pediatric patients. We have searched for genes that are overexpressed in human cancer and also show specific patterns of tissue-dependent expression in normal tissues. Using the expression data of the normal tissues, we identified 4,346 genes with a high variability of expression and clustered these genes according to their relative expression level. Of 91 stable clusters obtained, 24 clusters included genes preferentially expressed either only in hematopoietic tissues or in hematopoietic and one to two other tissues; 28 clusters included genes preferentially expressed in various nonhematopoietic tissues such as neuronal, testis, liver, kidney, muscle, lung, pancreas, and placenta. Analysis of the expression levels of these two groups of genes in the human cancer cell lines and leukemias identified genes that were highly expressed in cancer cells but not in their normal counterparts and, thus, were overexpressed in the cancers. The different cancer cell lines and leukemias varied in the number and identity of these overexpressed genes. The results indicate that many genes that are overexpressed in human cancer cells are specific to a variety of normal tissues, including normal tissues other than those from which the cancer originated. It is suggested that this general property of cancer cells plays a major role in determining the behavior of the cancers, including their metastatic potential. PMID:16339305

  5. Frequent alterations of HER2 through mutation, amplification, or overexpression in pleomorphic lobular carcinoma of the breast.

    PubMed

    Lien, Huang-Chun; Chen, Yu-Ling; Juang, Yu-Lin; Jeng, Yung-Ming

    2015-04-01

    Mutations in HER2 gene have been identified in a small subset of breast cancer cases. Identification of HER2 mutation has therapeutic implications for breast cancer, but whether a subgroup of breast cancer with a higher frequency of HER2 mutation exists, remains unknown. We analyzed HER2 mutation and pathologic factors on 73 formalin-fixed, paraffin-embedded samples, including 21 pleomorphic invasive lobular carcinoma (p-ILC) cases, 3 pleomorphic lobular carcinoma in situ (p-LCIS) cases, and 49 classic invasive lobular carcinoma (c-ILC) cases. Mutations were identified through direct sequencing. HER2 overexpression and amplification were determined through immunohistochemistry and fluorescent in situ hybridization. Six mutations were identified, including five in the 24 p-ILC or p-LCIS (p-ILC/p-LCIS) cases (20.8 %) and one in the 49 c-ILC cases (2.0 %), and the difference in frequency was significant (p = 0.013). Eight of the 24 (33.3 %) p-ILC/p-LCIS cases exhibited HER2 amplification or overexpression (amplification/overexpression), which was significantly higher than in the c-ILC cases (1/49, 2 %). Mutation and amplification/overexpression were mutually exclusive. HER2 mutations were identified more frequently in the p-ILC/p-LCIS cases with extensive apocrine change (p = 0.018). Combined HER2 alterations through mutation or amplification/overexpression were more frequently identified in p-ILC/p-LCIS cases without estrogen receptor expression. The high frequency (54.1 %, 13/24) of combined HER2 alterations in the p-ILC/p-LCIS cases suggests a crucial role of HER2 in the pathogenesis of p-ILC/p-LCIS. Because of the reported responsiveness of HER2 mutation to anti-HER2 therapy, p-ILC patients without HER2 amplification/overexpression should receive HER2 mutation analysis to identify this therapeutically relevant target. PMID:25773929

  6. Overexpression of SDF-1 activates the NF-?B pathway to induce epithelial to mesenchymal transition and cancer stem cell-like phenotypes of breast cancer cells.

    PubMed

    Kong, Lingxin; Guo, Sufen; Liu, Chunfeng; Zhao, Yiling; Feng, Chong; Liu, Yunshuang; Wang, Tao; Li, Caijuan

    2016-03-01

    The formation of EMT and EMT-induced CSC-like phenotype is crucial for the metastasis of tumor cells. The stromal cell-derived factor-1 (SDF-1) is upregulated in various human carcinomas, which is closely associated with proliferation, migration, invasion and prognosis of malignancies. However, limited attention has been directed towards the effect of SDF-1 on epithelial to mesenchymal transition (EMT) or cancer stem cell (CSC)-like phenotype formation in breast cancer cells and the related mechanism. In the present study, we screened MCF-7 cells with low SDF-1 expression level for the purpose of evaluating whether SDF-1 is involved in EMT and CSC-like phenotype formation in MCF-7 cells. The pEGFP-N1-SDF-1 plasmid was transfected into MCF-7 cells, and the stably overexpressed SDF-1 in MCF-7 cells was confirmed by real-time PCR and western blot analysis. Colony formation assay, MTT, wound healing assay and Transwell invasion assay demonstrated that overexpression of SDF-1 significantly boosted the proliferation, migration and invasion of MCF-7 cells compared with parental (P<0.05). Flow cytometry analysis revealed a notable increase of CD44+/CD24- subpopulation in SDF-1 overexpressing MCF-7 cells (P<0.001), accompanied by the apparently elevated ALDH activity and the upregulation of the stem cell markers OCT-4, Nanog, and SOX2 compared with parental (P<0.01). Besides, western blot analysis and immunofluorescence assay observed the significant decreased expression of E-cadherin and enhanced expression of slug, fibronectin and vimentin in SDF-1 overexpressed MCF-7 cells in comparison with parental (P<0.01). Further study found that overexpression of SDF-1 induced the activation of NF-?B pathway in MCF-7 cells. Conversely, suppressing or silencing p65 expression by antagonist or RNA interference could remarkably increase the expression of E-cadherin in SDF-1 overexpressed MCF-7 cells (P<0.001). Overall, the above results indicated that overexpression of SDF-1 enhanced EMT by activating the NF-?B pathway of MCF-7 cells and further induced the formation of CSC-like phenotypes, ultimately promoting the proliferation and metastasis of MCF-7 cells. Therefore, SDF-1 may further be assessed as a potential target for gene therapy of breast cancer. PMID:26782945

  7. Overexpression of HER2/neu as a Prognostic Value in Iranian Women With Early Stage Breast Cancer; A Single Institute Study

    PubMed Central

    Mirtavoos Mahyari, Hanifeh; Khosravi, Adnan; Mirtavoos Mahyari, Zeinab; Esfahani Monfared, Zahra; Khosravi, Negin

    2014-01-01

    Background: Patients with early stage breast cancer with same treatment strategy can have markedly different outcomes. Human epidermal growth factor receptor 2 (HER2/nue) gene amplification or the subsequent overexpression of protein has been proved to be associated with patient's outcome and response to anthracyclins-based regimens. Objectives: This study assessed prognostic value of HER2/nue marker in patients with early stage breast cancer who received adjuvant chemotherapy with anthracyclins-based regimens. Materials and Methods: Fifty tissue samples from patients with primary breast cancer of moderate risk receiving sequential adjuvant chemotherapy with anthracyclins-based regimens were assessed to evaluate HER2/nue gene status (quantified by Immunohistochemistry and fluorescence in situ hybridization) retrospectively. Besides, correlation of HER2/neu with patients' characteristics and outcome was studied. Results: HER2/neu amplification was identified in 19 (38%) of 50 patients. No significant difference regarding HER2/neu status was seen in clinic pathological characteristics of patients. Although Progression Free Survival (PFS) was shorter in HER2 overexpressed group, but uni/multivariate analysis adjusted for HER2 overexpression, nodal involvement, hormone receptor status, age and tumor size revealed no significant predictive and/or prognostic value for HER2 regarding PFS. Conclusions: This study on a limited number of patients treated with adjutant anthracyclins-based regimens, revealed that HER2/neu is not a unique strong predictor for outcome, thus according to combination of HER2/neu status and other clinical factors, it is necessary to distinguish patients at high risk of recurrence. PMID:25763209

  8. Claudin-20 promotes an aggressive phenotype in human breast cancer cells

    PubMed Central

    Martin, Tracey A; Lane, Jane; Ozupek, Hulya; Jiang, Wen G

    2013-01-01

    Claudin-20 is a member of the Claudin family of transmembrane proteins located in the tight junction (TJ) of cells of epithelial origin. Due to the increasing evidence supporting the role of TJ proteins in preventing tumor cell metastatic behavior, this study sought to evaluate the distribution of Claudin-20 in human breast cancer and the effect of Claudin-20 overexpression in human breast cancer cells. Q-PCR data from breast cancer primary tumors (n = 114) and matched background tissue (n = 30) showed that high claudin-20 expression was correlated with poor survival of patients with breast cancer (p = 0.022). Following transformation of the breast cancer cell lines MDA-MB-231 and MCF7 with a Claudin-20 expression construct functional assays were performed to ascertain changes in cell behavior. Claudin-20 transformed cells showed significantly increased invasion (p < 0.005) and were significantly less adhesive than wild type cells (p < 0.05). There was no effect on growth (either in vitro or in vivo) for either cell line. Overexpression of Claudin-20 resulted in reduced transepithelial resistance (induced by the motogen HGF at 25 ng/ml, p = 0.0007). Interestingly, this was not mirrored by paracellular permeability, as overexpression of Claudin-20 caused a decrease in permeability. The introduction of Claudin-20 into human breast cancer cells resulted in breast cancer cells with an aggressive phenotype and reduced trans-epithelial resistance. There was no corresponding decrease in paracellular permeability, indicating that this Claudin has a differential function in epithelial TJ. This provides further insight into the importance of correctly functioning TJ in preventing the progression of human breast cancer. PMID:24665404

  9. Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+ breast cancer patients

    PubMed Central

    Scaltriti, Maurizio; Eichhorn, Pieter J.; Corts, Javier; Prudkin, Ludmila; Aura, Claudia; Jimnez, Jos; Chandarlapaty, Sarat; Serra, Violeta; Prat, Aleix; Ibrahim, Yasir H.; Guzmn, Marta; Gili, Magui; Rodrguez, Olga; Rodrguez, Sonia; Prez, Jos; Green, Simon R.; Mai, Sabine; Rosen, Neal; Hudis, Clifford; Baselga, Jos

    2011-01-01

    Clinical benefits from trastuzumab and other anti-HER2 therapies in patients with HER2 amplified breast cancer remain limited by primary or acquired resistance. To identify potential mechanisms of resistance, we established trastuzumab-resistant HER2 amplified breast cancer cells by chronic exposure to trastuzumab treatment. Genomewide copy-number variation analyses of the resistant cells compared with parental cells revealed a focal amplification of genomic DNA containing the cyclin E gene. In a cohort of 34 HER2+ patients treated with trastuzumab-based therapy, we found that cyclin E amplification/overexpression was associated with a worse clinical benefit (33.3% compared with 87.5%, P < 0.02) and a lower progression-free survival (6 mo vs. 14 mo, P < 0.002) compared with nonoverexpressing cyclin E tumors. To dissect the potential role of cyclin E in trastuzumab resistance, we studied the effects of cyclin E overexpression and cyclin E suppression. Cyclin E overexpression resulted in resistance to trastuzumab both in vitro and in vivo. Inhibition of cyclin E activity in cyclin E-amplified trastuzumab resistant clones, either by knockdown of cyclin E expression or treatment with cyclin-dependent kinase 2 (CDK2) inhibitors, led to a dramatic decrease in proliferation and enhanced apoptosis. In vivo, CDK2 inhibition significantly reduced tumor growth of trastuzumab-resistant xenografts. Our findings point to a causative role for cyclin E overexpression and the consequent increase in CDK2 activity in trastuzumab resistance and suggest that treatment with CDK2 inhibitors may be a valid strategy in patients with breast tumors with HER2 and cyclin E coamplification/overexpression. PMID:21321214

  10. Biologic role of activated leukocyte cell adhesion molecule overexpression in breast cancer cell lines and clinical tumor tissue.

    PubMed

    Hein, Sibyll; Mller, Volkmar; Khler, Nadine; Wikman, Harriet; Krenkel, Sylke; Streichert, Thomas; Schweizer, Michaela; Riethdorf, Sabine; Assmann, Volker; Ihnen, Maike; Beck, Katrin; Issa, Rana; Jnicke, Fritz; Pantel, Klaus; Milde-Langosch, Karin

    2011-09-01

    The activated leukocyte cell adhesion molecule (ALCAM) is overexpressed in many mammary tumors, but controversial results about its role and prognostic impact in breast cancer have been reported. Therefore, we evaluated the biologic effects of ALCAM expression in two breast cancer cell lines and a larger cohort of mammary carcinomas. By stable transfections, MCF7 cells with ALCAM overexpression and MDA-MB231 cells with reduced ALCAM levels were generated and analyzed in functional assays and cDNA microarrays. In addition, an immunohistochemical study on 347 patients with breast cancer with long-term follow-up and analysis of disseminated tumor cells (DTCs) was performed. In both cell lines, high ALCAM expression was associated with reduced cell motility. In addition, ALCAM silencing in MDA-MB231 cells resulted in lower invasive potential, whereas high ALCAM expression was associated with increased apoptosis in both cell lines. Among genes which were differentially expressed in clones with altered ALCAM expression, there was an overlap of 15 genes between both cell lines, among them cathepsin D, keratin 7, gelsolin, and ets2 whose deregulation was validated by western blot analysis. In MDA-MB231 cells, we observed a correlation with VEGF expression which was validated by enzyme-linked immuno sorbent assay (ELISA). Our IHC results on primary breast carcinomas showed that ALCAM expression was associated with an estrogen receptor-positive phenotype. In addition, strong ALCAM immunostaining correlated with nodal involvement and the presence of tumor cells in bone marrow. By Kaplan-Meier analysis, strong ALCAM expression in ductal carcinomas correlated with shorter recurrence-free intervals (P=0.048) and overall survival (OAS, P=0.003). Our results indicate that the biologic role of ALCAM in breast cancer is complex, but overexpression might be relevant for outcome in ductal carcinomas. PMID:20972617

  11. PAQR3 expression is downregulated in human breast cancers and correlated with HER2 expression

    PubMed Central

    Li, Zhenghu; Ling, Zhi-Qiang; Guo, Weiwei; Lu, Xiao-Xiao; Pan, Yi; Wang, Zhenzhen; Chen, Yan

    2015-01-01

    PAQR3 is a newly discovered tumor suppressor and its functional role in breast cancer has not been well characterized. We report here that PAQR3 is associated with the progression and survival of human patients with breast cancer, as well as cell proliferation and migration of human breast cancer cells. PAQR3 mRNA level was robustly downregulated in human breast cancer samples compared with their corresponding para-cancerous histological normal tissues (n = 82, P < 0.0001). The mRNA level of PAQR3 was negatively correlated with HER2 expression (P < 0.0001) and disease-free survival of the patients (P < 0.0001). PAQR3 overexpression inhibited cell proliferation, colony formation and migration of breast cancer cells including MCF7, SKBR3, MDA-MD-231, MDA-MD-468 and MDA-MD-453 cells. Knockdown of PAQR3 in MDA-MD-231 cells elevated cell proliferation and migration. Inhibition of HER2 by trastuzumab increased PAQR3 expression in SKBR3 cells. In conclusion, PAQR3 expression is frequently downregulated in human breast cancers inversely correlated with HER2 expression. PAQR3 is able to modulate the proliferation and migration of breast cancer cells. Our data indicate that PAQR3 functions as a tumor suppressor in the development of human breast cancers. PMID:25900239

  12. Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines

    SciTech Connect

    Lee, Won Jae; Roberts-Thomson, Sarah J.; Monteith, Gregory R. . E-mail: G.Monteith@pharmacy.uq.edu.au

    2005-11-25

    There is evidence to suggest that plasma membrane Ca{sup 2+}-ATPase (PMCA) isoforms are important mediators sssof mammary gland physiology. PMCA2 in particular is upregulated extensively during lactation. Expression of other isoforms such as PMCA4 may influence mammary gland epithelial cell proliferation and aberrant regulation of PMCA isoform expression may lead or contribute to mammary gland pathophysiology in the form of breast cancers. To explore whether PMCA2 and PMCA4 expression may be deregulated in breast cancer, we compared mRNA expression of these PMCA isoforms in tumorigenic and non-tumorigenic human breast epithelial cell lines using real time RT-PCR. PMCA2 mRNA has a higher level of expression in some breast cancer cell lines and is overexpressed more than 100-fold in ZR-75-1 cells, compared to non-tumorigenic 184B5 cells. Although differences in PMCA4 mRNA levels were observed between breast cell lines, they were not of the magnitude observed for PMCA2. We conclude that PMCA2 mRNA can be highly overexpressed in some breast cancer cells. The significance of PMCA2 overexpression on tumorigenicity and its possible correlation with other properties such as invasiveness requires further study.

  13. SET and MYND domain-containing protein 3 is overexpressed in human glioma and contributes to tumorigenicity.

    PubMed

    Dai, Bin; Wan, Weiqing; Zhang, Peng; Zhang, Yisong; Pan, Changcun; Meng, Guolu; Xiao, Xinru; Wu, Zhen; Jia, Wang; Zhang, Junting; Zhang, Liwei

    2015-11-01

    SET and MYND domain-containing protein3 (SMYD3) is a histone H3 lysine4 (H3K4) di- and tri-methyltransferase that forms a transcriptional complex with RNA polymeraseII and plays an important role in early embryonic lineage commitment through the activation of lineage-specific genes. SMYD3 activates the transcription of oncogenes and cell cycle genes in gastric and breast cancer cells. However, the contribution of SMYD3 in glioma tumorigenesis remains unknown. Here, we determined the expression of SMYD3 and assessed its clinical significance in human glioma. We found that SMYD3 was overexpressed in human glioma but not in normal brain tissue. The level of SMYD3 protein expression in human glioma tissues was directly correlated with the glioma grade. The level of SMYD3 protein expression in human glioma tissues was inversely correlated with patient survival. Enforced SMYD3 expression promoted glioma LN-18 cell proliferation. Inhibition of SMYD3 expression in glioma T98G cells suppressed their anchorage?independent growth invitro and tumorigenicity invivo. Furthermore, we found that SMYD3 regulated the expression of p53 protein, which is essential in SMYD3?induced cell growth in glioma cells. These results showed that SMYD3 is overexpressed in human glioma and contributes to glioma tumorigenicity through p53. Therefore, SMYD3 may be a new potential therapeutic target for human malignant glioma. PMID:26328527

  14. ERβ overexpression results in endocrine therapy resistance and poor prognosis in postmenopausal ERα-positive breast cancer patients

    PubMed Central

    GUO, LIYING; ZHANG, YU; YILAMU, DILIMINA; LIU, SHA; GUO, CHENMING

    2016-01-01

    The aim of the present study was to investigate the role of estrogen receptor (ER) β in the prognosis of ERα-positive breast cancer in postmenopausal women, and its effect on the efficacy of endocrine therapy. Tissue specimens from 195 patients with postmenopausal breast cancer were analyzed. ERβ expression levels were detected using immunohistochemical staining. Kaplan-Meier analysis was performed to assess patient survival, and the difference in survival was analyzed using the log-rank test. Cox regression was utilized to evaluate prognostic factors. The results revealed that the disease-free survival rate decreased dramatically as ERβ expression levels increased in all postmenopausal ERα-positive breast cancer patients, and ERβ expression was identified to be an indicator of poor prognosis in cases of this disease. Similarly, in postmenopausal ERα-positive breast cancer patients undergoing endocrine therapy, high ERβ expression levels reduced the disease-free survival rate and were correlated with poor patient prognosis. However, in such patients who were not treated with endocrine therapy, disease-free survival rate and prognosis were not significantly affected by ERβ expression. In conclusion, ERβ overexpression led to endocrine therapy resistance and poor prognosis in postmenopausal ERα-positive breast cancer patients, suggesting that ERβ may affect breast cancer prognosis via an increase in endocrine therapy resistance. PMID:26893775

  15. WNT-1 inducible signaling pathway protein-1 enhances growth and tumorigenesis in human breast cancer

    PubMed Central

    Chiang, Kun-Chun; Yeh, Chun-Nan; Chung, Li-Chuan; Feng, Tsui-Hsia; Sun, Chi-Chin; Chen, Miin-Fu; Jan, Yi-Yin; Yeh, Ta-Sen; Chen, Shin-Cheh; Juang, Horng-Heng

    2015-01-01

    WNT1 inducible signaling pathway protein 1 (WISP1) plays a key role in many cellular functions in a highly tissue-specific manner; however the role of WISP1 in breast cancer is still poorly understood. Here, we demonstrate that WISP1 acts as an oncogene in human breast cancer. We demonstrated that human breast cancer tissues had higher WISP1 mRNA expression than normal breast tissues and that treatment of recombinant WISP1 enhanced breast cancer cell proliferation. Further, ectopic expression of WISP1 increased the growth of breast cancer cells in vitro and in vivo. WISP1 transfection also induced epithelial-mesenchymal-transition (EMT) in MCF-7 cells, leading to higher migration and invasion. During this EMT-inducing process, E-cadherin was repressed and N-cadherin, snail, and β-catenin were upregulated. Filamentous actin (F-actin) remodeling and polarization were also observed after WISP1 transfection into MCF-7 cells. Moreover, forced overexpression of WISP1 blocked the expression of NDRG1, a breast cancer tumor suppressor gene. Our study provides novel evidence that WISP1-modulated NDRG1 gene expression is dependent on a DNA fragment (−128 to +46) located within the human NDRG1 promoter. Thus, we concluded that WISP1 is a human breast cancer oncogene and is a potential therapeutic target. PMID:25732125

  16. HOXB7-S3 inhibits the proliferation and invasion of MCF-7 human breast cancer cells

    PubMed Central

    MA, RUI; ZHANG, DAN; HU, PENG-CHAO; LI, QUN; LIN, CONG-YAO

    2015-01-01

    Homeobox B7 (HOXB7) has been found to be overexpressed in numerous types of human cancer. However, the role of HOXB7 in breast cancer remains to be elucidated. The aim of the present study was to investigate the effects of HOXB7 on the proliferation and invasion of breast cancer cells. Initially, reverse transcription quantitative polymerase chain reaction and western blotting were respectively employed to detect the mRNA and protein expression levels of the HOXB7 gene in the MDA-MB-231 and MCF-7 human breast cancer cell lines. Subsequently, small interfering RNAs designed to interfere with the expression of HOXB7 were used to knockdown the expression of HOXB7 in the MCF-7 cell line, the effects of which on cell proliferation, the apoptotic rate and invasion capacity were measured using a Cell Counting kit-8 assay, flow cytometry and transwell chambers, respectively. The results demonstrated that HOXB7 mRNA and protein were all overexpressed in MDA-MB-231 and MCF-7 breast cancer cell lines. Furthermore, HOXB7-S3 effectively inhibited the proliferation and invasion of MCF-7 breast cancer cells. In conclusion, these results demonstrated that HOXB7 may be a potential therapeutic target in human breast cancer. PMID:26135503

  17. HOXC10 is overexpressed in breast cancer and transcriptionally regulated by estrogen via involvement of histone methylases MLL3 and MLL4.

    PubMed

    Ansari, Khairul I; Hussain, Imran; Kasiri, Sahba; Mandal, Subhrangsu S

    2012-02-01

    HOXC10 is a critical player in the development of spinal cord, formation of neurons, and associated with human leukemia. We found that HOXC10 is overexpressed in breast cancer and transcriptionally regulated by estrogen (17?-estradiol, E(2)). The HOXC10 promoter contains several estrogen response elements (ERE1-7, half-sites). A luciferase-based reporter assay showed that ERE1 and ERE6 of HOXC10 promoter are E(2) responsive. ER? and ER? play critical roles in E(2)-mediated activation of HOXC10. Knockdown of ER? and ER? downregulated E(2)-induced HOXC10 expression. ER? and ER? bind to ERE1 and ERE6 regions in an E(2)-dependent manner. Additionally, knockdown of histone methylases MLL3 and MLL4 (but not MLL1 and MLL2) diminished E(2)-induced expression of HOXC10. MLL3 and MLL4 were bound to the ERE1 and ERE6 regions of HOXC10 promoter in an E(2)-dependent manner. Overall, we demonstrated that HOXC10 is overexpressed in breast cancer, and it is an E(2)-responsive gene. Histone methylases MLL3 and MLL4, along with ERs, regulate HOXC10 gene expression in the presence of E(2). PMID:22143955

  18. PI3K inhibition results in enhanced HER signaling and acquired ERK dependency in HER2-overexpressing breast cancer

    PubMed Central

    Serra, V; Scaltriti, M; Prudkin, L; Eichhorn, P J A; Ibrahim, Y H; Chandarlapaty, S; Markman, B; Rodriguez, O; Guzman, M; Rodriguez, S; Gili, M; Russillo, M; Parra, J L; Singh, S; Arribas, J; Rosen, N; Baselga, J

    2011-01-01

    There is a strong rationale to therapeutically target the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in breast cancer since it is highly deregulated in this disease and it also mediates resistance to anti-HER2 therapies. However, initial studies with rapalogs, allosteric inhibitors of mTORC1, have resulted in limited clinical efficacy probably due to the release of a negative regulatory feedback loop that triggers AKT and ERK signaling. Since activation of AKT occurs via PI3K, we decided to explore whether PI3K inhibitors prevent the activation of these compensatory pathways. Using HER2-overexpressing breast cancer cells as a model, we observed that PI3K inhibitors abolished AKT activation. However, PI3K inhibition resulted in a compensatory activation of the ERK signaling pathway. This enhanced ERK signaling occurred as a result of activation of HER family receptors as evidenced by induction of HER receptors dimerization and phosphorylation, increased expression of HER3 and binding of adaptor molecules to HER2 and HER3. The activation of ERK was prevented with either MEK inhibitors or anti-HER2 monoclonal antibodies and tyrosine kinase inhibitors. Combined administration of PI3K inhibitors with either HER2 or MEK inhibitors resulted in decreased proliferation, enhanced cell death and superior anti-tumor activity compared with single agent PI3K inhibitors. Our findings indicate that PI3K inhibition in HER2-overexpressing breast cancer activates a new compensatory pathway that results in ERK dependency. Combined anti-MEK or anti-HER2 therapy with PI3K inhibitors may be required in order to achieve optimal efficacy in HER2-overexpressing breast cancer. This approach warrants clinical evaluation. PMID:21278786

  19. Overexpression of 14-3-3θ promotes tumor metastasis and indicates poor prognosis in breast carcinoma

    PubMed Central

    Fan, Jing; Tong, Chao; Yang, Jixin; Wei, Hongliang; Yi, Jun; Ling, Rui

    2014-01-01

    An isoform of the 14-3-3 protein family, 14-3-3θ has been linked with tumor cell proliferation and apoptosis. However, the role of 14-3-3θ in the progression of breast cancer remains unknown. Here, we report that 14-3-3θ plays a critical role in breast cancer metastasis and prognosis. The expression of 14-3-3θ was markedly higher in breast cancer tissues compared to adjacent normal tissues. A hospital-based study cohort of 216 breast cancer patients was evaluated in this study. The level of 14-3-3θ expression was determined and correlated based upon tumor clinicopathological features, disease-free survival, and overall survival. We found that overexpression of 14-3-3θ was correlated with advanced TNM stage (P < 0.05), lymph node metastasis (P < 0.05), and ER negative status (P < 0.05). Breast cancer patients with high 14-3-3θ expression had a shorter overall survival and a higher rate of recurrence than those with low 14-3-3θ expression. Additionally, knockdown of 14-3-3θ expression in breast cancer cells inhibited metastasis in vitro. Similarly, an in vivo assay showed that 14-3-3θ knockdown dramatically suppressed the growth of breast cancer xenografts and inhibited tumor cell metastasis in a lung metastasis model. Thus, this study provided the first evidence that 14-3-3θ is a novel tumor suppressor and may serve as a candidate prognostic biomarker and target for new therapies in metastatic breast cancer. PMID:24371149

  20. Epigenetic effects of human breast milk.

    PubMed

    Verduci, Elvira; Banderali, Giuseppe; Barberi, Salvatore; Radaelli, Giovanni; Lops, Alessandra; Betti, Federica; Riva, Enrica; Giovannini, Marcello

    2014-04-01

    A current aim of nutrigenetics is to personalize nutritional practices according to genetic variations that influence the way of digestion and metabolism of nutrients introduced with the diet. Nutritional epigenetics concerns knowledge about the effects of nutrients on gene expression. Nutrition in early life or in critical periods of development, may have a role in modulating gene expression, and, therefore, have later effects on health. Human breast milk is well-known for its ability in preventing several acute and chronic diseases. Indeed, breastfed children may have lower risk of neonatal necrotizing enterocolitis, infectious diseases, and also of non-communicable diseases, such as obesity and related-disorders. Beneficial effects of human breast milk on health may be associated in part with its peculiar components, possible also via epigenetic processes. This paper discusses about presumed epigenetic effects of human breast milk and components. While evidence suggests that a direct relationship may exist of some components of human breast milk with epigenetic changes, the mechanisms involved are still unclear. Studies have to be conducted to clarify the actual role of human breast milk on genetic expression, in particular when linked to the risk of non-communicable diseases, to potentially benefit the infant's health and his later life. PMID:24763114

  1. Epigenetic Effects of Human Breast Milk

    PubMed Central

    Verduci, Elvira; Banderali, Giuseppe; Barberi, Salvatore; Radaelli, Giovanni; Lops, Alessandra; Betti, Federica; Riva, Enrica; Giovannini, Marcello

    2014-01-01

    A current aim of nutrigenetics is to personalize nutritional practices according to genetic variations that influence the way of digestion and metabolism of nutrients introduced with the diet. Nutritional epigenetics concerns knowledge about the effects of nutrients on gene expression. Nutrition in early life or in critical periods of development, may have a role in modulating gene expression, and, therefore, have later effects on health. Human breast milk is well-known for its ability in preventing several acute and chronic diseases. Indeed, breastfed children may have lower risk of neonatal necrotizing enterocolitis, infectious diseases, and also of non-communicable diseases, such as obesity and related-disorders. Beneficial effects of human breast milk on health may be associated in part with its peculiar components, possible also via epigenetic processes. This paper discusses about presumed epigenetic effects of human breast milk and components. While evidence suggests that a direct relationship may exist of some components of human breast milk with epigenetic changes, the mechanisms involved are still unclear. Studies have to be conducted to clarify the actual role of human breast milk on genetic expression, in particular when linked to the risk of non-communicable diseases, to potentially benefit the infant’s health and his later life. PMID:24763114

  2. Diosgenin, a naturally occurring steroid, suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt, mTOR and JNK phosphorylation.

    PubMed

    Chiang, Chun-Te; Way, Tzong-Der; Tsai, Shang-Jie; Lin, Jen-Kun

    2007-12-22

    Fatty acid synthase (FAS) expression is markedly elevated in HER2-overexpressing breast cancer cells. In this study, diosgenin, a plant-derived steroid, was found to be effective in suppressing FAS expression in HER2-overexpressing breast cancer cells. Diosgenin preferentially inhibited proliferation and induced apoptosis in HER2-overexpressing cancer cells. Furthermore, diosgenin inhibited the phosphorylation of Akt and mTOR, and enhanced phosphorylation of JNK. The use of pharmacological inhibitors revealed that the modulation of Akt, mTOR and JNK phosphorylation was required for diosgenin-induced FAS suppression. Finally, we showed that diosgenin could enhance paclitaxel-induced cytotoxicity in HER2-overexpressing cancer cells. These results suggested that diosgenin has the potential to advance as chemopreventive or chemotherapeutic agent for cancers that overexpress HER2. PMID:18022396

  3. Trastuzumab treatment improves brain metastasis outcomes through control and durable prolongation of systemic extracranial disease in HER2-overexpressing breast cancer patients

    PubMed Central

    Park, Y H; Park, M J; Ji, S H; Yi, S Y; Lim, D H; Nam, D H; Lee, J-I; Park, W; Choi, D H; Huh, S J; Ahn, J S; Kang, W K; Park, K; Im, Y-H

    2009-01-01

    In patients with human epidermal growth factor receptor-2 (HER2)-overexpressing breast cancer, treatment with trastuzumab has been shown to markedly improve the outcome. We investigated the role of trastuzumab on brain metastasis (BM) in HER2-positive breast cancer patients. From 1999 to 2006, 251 patients were treated with palliative chemotherapy for HER2-positive metastatic breast cancer at Samsung Medical Center. The medical records of these patients were analysed to study the effects of trastuzumab on BM prevalence and outcomes. Patients were grouped according to trastuzumab therapy: pre-T (no trastuzumab therapy) vs post-T (trastuzumab therapy). The development of BM between the two treatment groups was significantly different (37.8% for post-T vs 25.0% for pre-T, P=0.028). Patients who had received trastuzumab had longer times to BM compared with patients who were not treated with trastuzumab (median 15 months for post-T group vs 10 months for pre-T group, P=0.035). Time to death (TTD) from BM was significantly longer in the post-T group than in the pre-T group (median 14.9 vs 4.0 months, P=0.0005). Extracranial disease control at the time of BM, 12 months or more of progression-free survival of extracranial disease and treatment with lapatinib were independent prognostic factors for TTD from BM. PMID:19240719

  4. A phase II study of lapatinib and bevacizumab as treatment for HER2-overexpressing metastatic breast cancer.

    PubMed

    Rugo, Hope S; Chien, A Jo; Franco, Sandra X; Stopeck, Alison T; Glencer, Alexa; Lahiri, Soumi; Arbushites, Michael C; Scott, Janet; Park, John W; Hudis, Clifford; Nulsen, Ben; Dickler, Maura N

    2012-07-01

    Preclinical data have demonstrated that the combination of antihuman epidermal growth factor receptor-2 (anti-HER2) and antivascular endothelial growth factor (anti-VEGF)--targeted agents has antitumor activity; these data indicate certain patients with HER2-overexpressing breast cancer may derive clinical benefit from this combination. The purpose of this single-arm phase II study was to determine the efficacy and safety of the dual-targeting combination of lapatinib and bevacizumab. Women with HER2-overexpressing advanced breast cancer received 1,500 mg oral lapatinib daily plus 10 mg/kg IV bevacizumab every 2 weeks. The primary endpoint was progression-free survival (PFS) at week 12; secondary endpoints included overall tumor response rate (ORR), clinical benefit rate (CBR), duration of response, time-to-response, PFS, and safety. Circulating tumor cells (CTC) and circulating endothelial cells (CEC) were measured at baseline and during study treatment as potential response markers. Fifty-two patients with stage IV disease were enrolled. The 12-week investigator-assessed PFS rate was 69.2% (95% confidence interval [CI]: 54.9, 81.3). Median PFS was 24.7 weeks (95% CI: 20.4, 35.1), and the CBR was 30.8% (95% CI: 18.7, 45.1). Of 45 patients with measurable disease, 6 were determined to have a partial response per Response Evaluation Criteria in Solid Tumors (ORR: 13.3%; 95% CI: 5.1, 26.8). The most common adverse events (AEs) included diarrhea, rash, and fatigue; most of these were either grade 1 or 2. Clinical responses were correlated with decreases in CTC and CEC. Lapatinib plus bevacizumab was active in patients with HER2-overexpressing breast cancer. The AE profile of the combination was consistent with the known profiles for these agents. PMID:22198412

  5. Human mammary microenvironment better regulates the biology of human breast cancer in humanized mouse model.

    PubMed

    Zheng, Ming-Jie; Wang, Jue; Xu, Lu; Zha, Xiao-Ming; Zhao, Yi; Ling, Li-Jun; Wang, Shui

    2015-02-01

    During the past decades, many efforts have been made in mimicking the clinical progress of human cancer in mouse models. Previously, we developed a human breast tissue-derived (HB) mouse model. Theoretically, it may mimic the interactions between "species-specific" mammary microenvironment of human origin and human breast cancer cells. However, detailed evidences are absent. The present study (in vivo, cellular, and molecular experiments) was designed to explore the regulatory role of human mammary microenvironment in the progress of human breast cancer cells. Subcutaneous (SUB), mammary fat pad (MFP), and HB mouse models were developed for in vivo comparisons. Then, the orthotopic tumor masses from three different mouse models were collected for primary culture. Finally, the biology of primary cultured human breast cancer cells was compared by cellular and molecular experiments. Results of in vivo mouse models indicated that human breast cancer cells grew better in human mammary microenvironment. Cellular and molecular experiments confirmed that primary cultured human breast cancer cells from HB mouse model showed a better proliferative and anti-apoptotic biology than those from SUB to MFP mouse models. Meanwhile, primary cultured human breast cancer cells from HB mouse model also obtained the migratory and invasive biology for "species-specific" tissue metastasis to human tissues. Comprehensive analyses suggest that "species-specific" mammary microenvironment of human origin better regulates the biology of human breast cancer cells in our humanized mouse model of breast cancer, which is more consistent with the clinical progress of human breast cancer. PMID:25572806

  6. Human Cripto-1 overexpression in the mouse mammary gland results in the development of hyperplasia and adenocarcinoma.

    PubMed

    Wechselberger, Christian; Strizzi, Luigi; Kenney, Nicholas; Hirota, Morihisa; Sun, Youping; Ebert, Andreas; Orozco, Olivia; Bianco, Caterina; Khan, Nadia I; Wallace-Jones, Brenda; Normanno, Nicola; Adkins, Heather; Sanicola, Michele; Salomon, David S

    2005-06-01

    Human Cripto-1 (CR-1) is overexpressed in approximately 80% of human breast, colon and lung carcinomas. Mouse Cr-1 upregulation is also observed in a number of transgenic (Tg) mouse mammary tumors. To determine whether CR-1 can alter mammary gland development and/or may contribute to tumorigenesis in vivo, we have generated Tg mouse lines that express human CR-1 under the transcriptional control of the mouse mammary tumor virus (MMTV). Stable Tg MMTV/CR-1 FVB/N lines expressing different levels of CR-1 were analysed. Virgin female MMTV/CR-1 Tg mice exhibited enhanced ductal branching, dilated ducts, intraductal hyperplasia, hyperplastic alveolar nodules and condensation of the mammary stroma. Virgin aged MMTV/CR-1 Tg mice also possessed persistent end buds. In aged multiparous MMTV/CR-1 mice, the hyperplastic phenotype was most pronounced with multifocal hyperplasias. In the highest CR-1-expressing subline, G4, 38% (12/31) of the multiparous animals aged 12-20 months developed hyperplasias and approximately 33% (11/31) developed papillary adenocarcinomas. The long latency period suggests that additional genetic alterations are required to facilitate mammary tumor formation in conjunction with CR-1. This is the first in vivo study that shows hyperplasia and tumor growth in CR-1-overexpressing animals. PMID:15897912

  7. The septin-binding protein anillin is overexpressed in diverse human tumors.

    PubMed

    Hall, Peter A; Todd, Christopher B; Hyland, Paula L; McDade, Simon S; Grabsch, Heike; Dattani, Mit; Hillan, Kenneth J; Russell, S E Hilary

    2005-10-01

    Anillin is an actin-binding protein that can bind septins and is a component of the cytokinetic ring. We assessed the anillin expression in 7,579 human tissue samples and cell lines by DNA microarray analysis. Anillin is expressed ubiquitously but with variable levels of expression, being highest in the central nervous system. The median level of anillin mRNA expression was higher in tumors than normal tissues (median fold increase 2.58; 95% confidence intervals, 2.19-5.68, P < 0.0001) except in the central nervous system where anillin mRNA levels were lower in tumors. We developed a sensitive reverse transcription-PCR strategy to show that anillin mRNA is expressed in cell lines and in cDNA panels derived from fetal and adult tissues, thus validating the microarray data. We compared anillin with Ki67 mRNA expression and found a significant linear relationship between anillin and Ki67 mRNA expression (Spearmann r approximately 0.6, P < 0.0001). Anillin mRNA expression was analyzed during tumor progression in breast, ovarian, kidney, colorectal, hepatic, lung, endometrial, and pancreatic tumors and in all tissues there was progressive increase in anillin mRNA expression from normal to benign to malignant to metastatic disease. Finally, we used anti-anillin sera and found nuclear anillin immunoreactivity to be widespread in normal tissues, often not correlating with proliferative compartments. These data provide insight into the existence of nonproliferation-associated activities of anillin and roles in interphase nuclei. Thus, anillin is overexpressed in diverse common human tumors, but not simply as a consequence of being a proliferation marker. Anillin may have potential as a novel biomarker. PMID:16203764

  8. Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice

    PubMed Central

    Wege, Anja K; Schmidt, Marcus; Ueberham, Elke; Ponnath, Marvin; Ortmann, Olaf; Brockhoff, Gero; Lehmann, Jörg

    2014-01-01

    Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγnull (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4+ T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy. PMID:24870377

  9. Olfactory deficits in mice overexpressing human wildtype α-synuclein

    PubMed Central

    Fleming, Sheila M.; Tetreault, Nicole A.; Mulligan, Caitlin K.; Hutson, Ché B.; Masliah, Eliezer; Chesselet, Marie-Françoise

    2011-01-01

    Accumulation of α-synuclein in neurons of the central and peripheral nervous system is a hallmark of sporadic Parkinson’s disease (PD) and mutations that increase α-synuclein levels cause familial PD. Transgenic mice overexpressing α-synuclein under the Thy1 promoter (Thy1-aSyn) have high levels of α-synuclein expression throughout the brain but no loss of nigrostriatal dopamine neurons up to 8 months, suggesting that they may be useful to model pre-clinical stages of PD. Olfactory dysfunction often precedes the onset of the cardinal motor symptoms of PD by several years and includes deficits in odor detection, discrimination and identification. In the present study, we measured olfactory function in 3- and 9-month-old male Thy1-aSyn mice with a buried pellet test based on latency to find an exposed or hidden odorant, a block test based on exposure to self and non-self odors, and a habituation/dishabituation test based on exposure to non-social odors. In a separate group of mice, α-synuclein immunoreactivity was assessed in the olfactory bulb. Compared with wildtype littermates, Thy1-aSyn mice could still detect and habituate to odors but showed olfactory impairments in aspects of all three testing paradigms. Thy1-aSyn mice also displayed proteinase K-resistant α-synuclein inclusions throughout the olfactory bulb. These data indicate that overexpression of α-synuclein is sufficient to cause olfactory deficits in mice similar to that observed in patients with PD. Furthermore, the buried pellet and block tests provided sufficient power for the detection of a 50% drug effect, indicating their usefulness for testing novel neuroprotective therapies. PMID:18702696

  10. Comprehensive analysis of long non-coding RNAs in human breast cancer clinical subtypes.

    PubMed

    Su, Xiaoping; Malouf, Gabriel G; Chen, Yunxin; Zhang, Jianping; Yao, Hui; Valero, Vicente; Weinstein, John N; Spano, Jean-Philippe; Meric-Bernstam, Funda; Khayat, David; Esteva, Francisco J

    2014-10-30

    Accumulating evidence highlights the potential role of long non-coding RNAs (lncRNAs) as biomarkers and therapeutic targets in solid tumors. However, the role of lncRNA expression in human breast cancer biology, prognosis and molecular classification remains unknown. Herein, we established the lncRNA profile of 658 infiltrating ductal carcinomas of the breast from The Cancer Genome Atlas project. We found lncRNA expression to correlate with the gene expression and chromatin landscape of human mammary epithelial cells (non-transformed) and the breast cancer cell line MCF-7. Unsupervised consensus clustering of lncRNA revealed four subgroups that displayed different prognoses. Gene set enrichment analysis for cis- and trans-acting lncRNAs showed enrichment for breast cancer signatures driven by master regulators of breast carcinogenesis. Interestingly, the lncRNA HOTAIR was significantly overexpressed in the HER2-enriched subgroup, while the lncRNA HOTAIRM1 was significantly overexpressed in the basal-like subgroup. Estrogen receptor (ESR1) expression was associated with distinct lncRNA networks in lncRNA clusters III and IV. Importantly, almost two thirds of the lncRNAs were marked by enhancer chromatin modifications (i.e., H3K27ac), suggesting that expressed lncRNA in breast cancer drives carcinogenesis through increased activity of neighboring genes. In summary, our study depicts the first lncRNA subtype classification in breast cancer and provides the framework for future studies to assess the interplay between lncRNAs and the breast cancer epigenome. PMID:25296969

  11. Localization of decorin gene expression in normal human breast tissue and in benign and malignant tumors of the human breast.

    PubMed

    Bostrm, Pia; Sainio, Annele; Kakko, Tanja; Savontaus, Mikko; Sderstrm, Mirva; Jrvelinen, Hannu

    2013-01-01

    The small extracellular matrix proteoglycan decorin which possesses a potent antitumor activity has been shown to be present in various amounts in the stroma of several tumors including those of the breast. Regarding decorin in breast malignancies the published data are conflicting, i.e., whether breast cancer cells express it or not. Here, we first compared decorin gene expression levels between healthy human breast tissue and selected types of human breast cancer using GeneSapiens databank. Next, we localized decorin mRNA in tissue specimen of normal human breast, intraductal breast papillomas and various histologic types of human breast cancer using in situ hybridization (ISH) with digoxigenin-labeled RNA probes for decorin. We also examined the effect of decorin transduction on the behavior of cultured human breast cancer MCF7 cells. Analysis of GeneSapiens databank revealed that in various human breast cancers decorin expression is significant. However, ISH results clearly demonstrated that human breast cancer cells independently of the type of the cancer do not express decorin mRNA. This was also true for papilloma-forming cells of the human breast. Indeed, decorin gene expression in healthy human breast tissue as well as in benign and malignant tumors of human breast was shown to take place solely in cells of the original stroma. Decorin transduction using decorin adenoviral vector in decorin-negative MCF7 cells resulted in a significant decrease in the proliferation of these cells and changed cell cohesion. Decorin-transduced MCF7 cells also exhibited increased apoptosis. In conclusion, our study shows that in human breast tissue only cells of the original stroma are capable of decorin gene expression. Our study also shows that transduction of decorin in decorin-negative human breast cancer cells markedly modulates the growth pattern of these cells. PMID:23007289

  12. Epigenetic Mechanisms Leading to Overexpression of HMGA Proteins in Human Pituitary Adenomas

    PubMed Central

    D’Angelo, Daniela; Esposito, Francesco; Fusco, Alfredo

    2015-01-01

    Overexpression of the high-mobility group A (HMGA)1 and HMGA2 proteins is a feature of all human pituitary adenoma (PAs) subtypes. However, amplification and/or rearrangement of the HMGA2 have been described in human prolactinomas, but rarely in other pituitary subtypes, and no genomic amplification of HMGA1 was detected in PAs. Here, we summarize the functional role of HMGA proteins in pituitary tumorigenesis and the epigenetic mechanisms contributing to HMGA overexpression in these tumors focusing on recent studies indicating a critical role of non-coding RNAs in modulating HMGA protein levels. PMID:26137461

  13. Effect of CH-35, a novel anti-tumor colchicine analogue, on breast cancer cells overexpressing the ?III isotype of tubulin.

    PubMed

    Yeh, Lee-Chuan C; Banerjee, Asok; Prasad, Veena; Tuszynski, Jack A; Weis, Alexander L; Bakos, Tamas; Yeh, I-Tien; Luduea, Richard F; Lee, John C

    2016-02-01

    The subunit protein of microtubules is tubulin, which has been the target for some of the most successful and widely used anti-tumor drugs. Most of the drugs that target tubulin bind to the ? subunit. There are many isotypes of ?-tubulin and their distributions differ among different tissues. The ?III isotype is over-expressed in many tumors, particularly those that are aggressive, metastatic, and drug resistant. We have previously reported the design and synthesis of a series of compounds to fit the colchicine site on ?III but not on the other isotypes. In the current study, we tested the toxicity and the anti-tumor activity of one of these compounds, CH-35, on the human breast tumor MDA-MB-231 over-expressing ?III in a xenogeneic mouse model. We found that CH-35 was as toxic as Taxol in vivo. Although the ?III-over-expressing cells developed into very fast-growing tumors, CH-35 was more effective against this tumor than was Taxol. Our results suggest that CH-35 is a promising candidate for future drug development. PMID:26686345

  14. A phase II trial of trabectedin in triple-negative and HER2-overexpressing metastatic breast cancer.

    PubMed

    Blum, Joanne L; Gonalves, Anthony; Efrat, Noa; Debled, Marc; Conte, Pierfranco; Richards, Paul D; Richards, Donald; Lardelli, Pilar; Nieto, Antonio; Cullell-Young, Martin; Delaloge, Suzette

    2016-01-01

    Trabectedin is an alkylating agent that binds to the minor groove of DNA. Early studies with trabectedin suggested efficacy in triple-negative and HER2-overexpressing metastatic breast cancer (MBC). The efficacy and safety of trabectedin in pretreated patients with these tumors were evaluated in this parallel-cohort phase II trial. Patients received a 3-h infusion of trabectedin 1.3mg/m(2) intravenously every 3weeks until progression or unmanageable/unacceptable toxicity. The primary objective was to evaluate the efficacy using the objective response rate (ORR) as per Response Evaluation Criteria In Solid Tumors (RECIST). Secondary objectives comprised time-to-event endpoints and safety assessed with the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v.3.0. Patients with heavily pretreated triple-negative (n=50) or HER2-overexpressing (n=37) MBC were enrolled. No confirmed responses were found in triple-negative MBC patients, with median progression-free survival (PFS) of 2.2months (95% CI 1.3-2.7months). Confirmed partial responses occurred in 4 of 34 evaluable HER2-overexpressing MBC patients (ORR=12%; 95% CI 3-27%) and lasted a median of 12.5months (95% CI, 6.2-14.7months); median PFS was 3.8months (95% CI, 1.8-5.5months). Most trabectedin-related adverse events were mild or moderate, and the most frequent were fatigue, nausea, vomiting, constipation, and anorexia. Severe neutropenia and transaminase increases were non-cumulative and transient and were mostly managed by infusion delays or dose reductions. Single-agent trabectedin is well tolerated in aggressive MBC and has moderate activity in HER2-overexpressing tumors. Further studies are warranted to evaluate trabectedin combined with HER2-targeted treatments in this subtype. PMID:26749361

  15. Late ROS accumulation and radiosensitivity in SOD1-overexpressing human glioma cells.

    PubMed

    Gao, Zhen; Sarsour, Ehab H; Kalen, Amanda L; Li, Ling; Kumar, Maneesh G; Goswami, Prabhat C

    2008-12-01

    This study investigates the hypothesis that CuZn superoxide dismutase (SOD1) overexpression confers radioresistance to human glioma cells by regulating the late accumulation of reactive oxygen species (ROS) and the G(2)/M-checkpoint pathway. U118-9 human glioma cells (wild type, neo vector control, and stably overexpressing SOD1) were irradiated (0-10 Gy) and assayed for cell survival, cellular ROS levels, cell-cycle-phase distributions, and cyclin B1 expression. SOD1-overexpressing cells were radioresistant compared to wild-type (wt) and neo vector control (neo) cells. Irradiated wt and neo cells showed a significant increase (approximately twofold) in DHE fluorescence beginning at 2 days postirradiation, which remained elevated at 8 days postirradiation. Interestingly, the late accumulation of ROS was suppressed in irradiated SOD1-overexpressing cells. The increase in ROS levels was followed by a decrease in cell growth and viability and an increase in the percentage of cells with sub-G(1) DNA content. SOD1 overexpression enhanced radiation-induced G(2) accumulation within 24 h postirradiation, which was accompanied by a decrease in cyclin B1 mRNA and protein levels. These results support the hypothesis that long after radiation exposure a "metabolic redox response" regulates radiosensitivity of human glioma cells. PMID:18790046

  16. Late ROS-accumulation and Radiosensitivity in CuZnSOD Overexpressing Human Glioma Cells

    PubMed Central

    Gao, Zhen; Sarsour, Ehab H.; Kalen, Amanda L.; Li, Ling; Kumar, Maneesh G.; Goswami, Prabhat C.

    2008-01-01

    This study investigates the hypothesis that CuZn-superoxide dismutase (SOD1) overexpression confers radioresistance to human glioma cells by regulating the late accumulation of reactive oxygen species (ROS) and G2/M checkpoint pathway. U118-9 human glioma cells (wild type, neo vector control, and stably overexpressing SOD1) were irradiated (0-10 Gy) and assayed for cell survival, cellular ROS levels, cell cycle phase distributions, and cyclin B1 expression. SOD1 overexpressing cells were radioresistant compared to wild type (wt) and neo vector control (neo) cells. Irradiated wt and neo cells showed a significant increase (~2-fold) in DHE-fluorescence beginning at 2 d post-irradiation, which remained elevated at 8 d post-irradiation. Interestingly, the late accumulation of ROS was suppressed in irradiated SOD1-overexpressing cells. The increase in ROS levels was followed by a decrease in cell growth and viability, and an increase in the percentage of cells with sub G1 DNA content. SOD1 overexpression enhanced radiation-induced G2-accumulation within 24 h post-irradiation, which was accompanied with a decrease in cyclin B1 mRNA and protein levels. These results support the hypothesis that long after the radiation exposure a “metabolic redox-response” regulates radiosensitivity of human glioma cells. PMID:18790046

  17. Drug Efflux Transporters Are Overexpressed in Short-Term Tamoxifen-Induced MCF7 Breast Cancer Cells

    PubMed Central

    Krisnamurti, Desak Gede Budi; Louisa, Melva; Anggraeni, Erlia; Wanandi, Septelia Inawati

    2016-01-01

    Tamoxifen is the first line drug used in the treatment of estrogen receptor-positive (ER+) breast cancer. The development of multidrug resistance (MDR) to tamoxifen remains a major challenge in the treatment of cancer. One of the mechanisms related to MDR is decrease of drug influx via overexpression of drug efflux transporters such as P-glycoprotein (P-gp/MDR1), multidrug resistance associated protein (MRP), or BCRP (breast cancer resistance protein). We aimed to investigate whether the sensitivity of tamoxifen to the cells is maintained through the short period and whether the expressions of several drug efflux transporters have been upregulated. We exposed MCF7 breast cancer cells with tamoxifen 1 μM for 10 passages (MCF7 (T)). The result showed that MCF7 began to lose their sensitivity to tamoxifen from the second passage. MCF7 (T) also showed a significant increase in all transporters examined compared with MCF7 parent cells. The result also showed a significant increase of CC50 in MCF7 (T) compared to that in MCF7 (97.54 μM and 3.04 μM, resp.). In conclusion, we suggest that the expression of several drug efflux transporters such as P-glycoprotein, MRP2, and BCRP might be used and further studied as a marker in the development of tamoxifen resistance. PMID:26981116

  18. Excretion of drugs in human breast milk

    SciTech Connect

    Welch, R.M.; Findlay, J.W.

    1981-01-01

    The present report briefly discusses some of the morphological, physiological, and compositional aspects of animal and human breast milk and how these characteristics might be important for the accumulation of drugs and foreign compounds. In addition, a study is described confirming the presence of caffeine, codeine, morphine, phenacetin, acetaminophen, and salicylic acid in the breast milk of a lactating mother following oral administration of a combination analgesic containing aspirin, phenacetin, caffeine, and codeine. Although the study is limited to one subject, it has provided critically needed data on the rates of appearance in, and elimination of these drugs from, breast milk. A similar amount of information is presented on phenacetin, also a component of the analgesic mixture, which has not been previously reported to enter human milk. The distribution of these drugs between the slightly more acidic breast milk and the relatively neutral plasma is consistent with their weakly basic, acidic, or relatively neutral properties. In general, the study shows that codeine and morphine milk concentrations are higher than, salicylic acid milk levels are much lower than, and phenacetin, caffeine, and acetaminophen milk concentrations are relatively similar to their respective plasma levels. It is projected, from estimated steady-state milk concentrations of the drugs and their metabolites studied, that very low percentages of the therapeutic dosages (less than 0.7%) would be excreted in mother's milk, too low an amount to be clinically significant to the infant.

  19. Chemical Biomarkers of Human Breast Milk Pollution

    PubMed Central

    Massart, Francesco; Gherarducci, Giulia; Marchi, Benedetta; Saggese, Giuseppe

    2008-01-01

    Human milk is, without question, the best source of nutrition for infants containing the optimal balance of fats, carbohydrates and proteins for developing babies. Breastfeeding provides a range of benefits for growth, immunity and development building a powerful bond between mother and her child. Recognition of the manifold benefits of breast milk has led to the adoption of breast-feeding policies by numerous health and professional organizations such as the World Health Organization and American Academy of Pediatrics. In industrially developed as well as in developing nations, human milk contamination by toxic chemicals such as heavy metals, dioxins and organohalogen compounds, however, is widespread and is the consequence of decades of inadequately controlled pollution. Through breastfeeding, the mother may transfer to the suckling infant potentially toxic chemicals to which the mother has previously been exposed. In the present review, environmental exposure, acquisition and current levels of old and emerging classes of breast milk pollutants are systematically presented. Although scientific evidences indicated that the advantages of breast-feeding outweigh any risks from contaminants, it is important to identify contaminant trends, to locate disproportionately exposed populations, and to take public health measures to improve chemical BM pollution as possible. PMID:19578503

  20. LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

    PubMed Central

    Saha, Tapas

    2012-01-01

    Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer. PMID:22874552

  1. Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone.

    PubMed

    Liu, Sijin; Goldstein, Robert H; Scepansky, Ellen M; Rosenblatt, Michael

    2009-11-15

    Rho-associated kinase (ROCK) signaling plays a fundamental role in regulating cell morphology, adhesion, and motility. Aberrant expression of ROCK is related to tumor metastases and poor clinical outcome. Here, we show that ROCK expression is increased in metastatic human mammary tumors and breast cancer cell lines compared with nonmetastatic tumors and cell lines. Overexpression of ROCK confers a metastatic phenotype on the nonmetastatic MCF-7 cell line. Inhibition of ROCK activity, by either a specific ROCK inhibitor (Y27632) or ROCK-targeted small interfering RNAs, reduces cell migration and proliferation in vitro and metastasis to bone in vivo using a novel "human breast cancer metastasis to human bone" mouse model. Expression of the c-Myc-regulated miR-17-92 cluster is shown to be elevated in metastatic breast cancer cells compared with nonmetastatic cells and diminished by Y27632 treatment. Furthermore, blockade of miR-17 is shown to decrease breast cancer cell invasion/migration in vitro and metastasis in vivo. Together, these findings suggest that augmented ROCK signaling contributes to breast cancer metastasis. The effects of ROCK on tumor cell invasion/motility and growth may derive from regulating cytoskeletal actin-myosin contraction and modulating the c-Myc pathway, including c-Myc-dependent microRNAs. Inhibition of ROCK or the pathway it stimulates, therefore, may represent a novel approach for treatment of breast cancer metastases. PMID:19887617

  2. Overexpressing Human Membrane Proteins in Stably Transfected and Clonal Human Embryonic Kidney 293S Cells

    PubMed Central

    Chaudhary, Sarika; Pak, John E.; Gruswitz, Franz; Sharma, Vinay

    2013-01-01

    X-ray crystal structures of human membrane proteins, while potentially being of extremely high impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we utilize to overexpress human membrane proteins from clonal HEK293S GnTI- cells, and was recently used in our 2.1 X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled-up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of ~0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost-effective, can be used to express proteins that would otherwise be toxic to mammalian cells, and can be completed in 810 weeks. PMID:22322218

  3. Co-Overexpression of GEP100 and AMAP1 Proteins Correlates with Rapid Local Recurrence after Breast Conservative Therapy

    PubMed Central

    Kinoshita, Rumiko; Nam, Jin-Min; Ito, Yoichi M.; Hatanaka, Kanako C.; Hashimoto, Ari; Handa, Haruka; Otsuka, Yutaro; Hashimoto, Shigeru; Onodera, Yasuhito; Hosoda, Mitsuchika; Onodera, Shunsuke; Shimizu, Shinichi; Tanaka, Shinya; Shirato, Hiroki; Tanino, Mishie; Sabe, Hisataka

    2013-01-01

    A major problem of current cancer research and therapy is prediction of tumor recurrence after initial treatment, rather than the simple biological characterization of the malignancy and proliferative properties of tumors. Breast conservation therapy (BCT) is a well-approved, standard treatment for patients with early stages of breast cancer, which consists of lumpectomy and whole-breast irradiation. In spite of extensive studies, only 'age' and 'Ki-67 positivity' have been identified to be well correlated with local recurrence after BCT. An Arf6 pathway, activated by GEP100 under receptor tyrosine kinases (RTKs) and employs AMAP1 as its effector, is crucial for invasion and metastasis of some breast cancer cells. This pathway activates ?1 integrins and perturbs E-cadherin-based adhesions, hence appears to be integral for epithelial-mesenchymal transdifferentiation (EMT). We here show that expression of the Arf6 pathway components statistically correlates with rapid local recurrence after BCT. We retrospectively analyzed four hundred seventy-nine patients who received BCT in Hokkaido University Hospital, and found 20 patients had local recurrence. We then analyzed pathological samples of patients who experienced local recurrence by use of Kaplan-Meier analysis, Stepwise regression analysis and the t-test, coupled with immunostaining, and found that co-overexpression of GEP100 and AMAP1 correlates with rapidity of the local recurrence. Their margin-status, node-positivity, and estrogen receptor (ER)- or progesterone receptor (PgR)-positivity did not correlated with the rapidity. This study is the first to show that expression of a certain set of proteins correlates with the rapidity of local recurrence. Our results are useful not only for prediction, but highlight the possibility of developing novel strategies to block local recurrence. We also discuss why mRNAs encoding these proteins have not been identified to correlate with local recurrence by previous conventional gene expression profiling analyses. PMID:24116160

  4. Paralemmin-1 is over-expressed in estrogen-receptor positive breast cancers

    PubMed Central

    2012-01-01

    Background Paralemmin-1 is a phosphoprotein lipid-anchored to the cytoplasmic face of membranes where it functions in membrane dynamics, maintenance of cell shape, and process formation. Expression of paralemmin-1 and its major splice variant (? exon 8) as well as the extent of posttranslational modifications are tissue- and development-specific. Paralemmin-1 expression in normal breast and breast cancer tissue has not been described previously. Results Paralemmin-1 mRNA and protein expression was evaluated in ten breast cell lines, 26 primary tumors, and 10 reduction mammoplasty (RM) tissues using real time RT-PCR. Paralemmin-1 splice variants were assessed in tumor and RM tissues using a series of primers and RT-PCR. Paralemmin-1 protein expression was examined in cell lines using Western Blots and in 31 ductal carcinomas in situ, 65 infiltrating ductal carcinomas, and 40 RM tissues using immunohistochemistry. Paralemmin-1 mRNA levels were higher in breast cancers than in RM tissue and estrogen receptor (ER)-positive tumors had higher transcript levels than ER-negative tumors. The ? exon 8 splice variant was detected more frequently in tumor than in RM tissues. Protein expression was consistent with mRNA results showing higher paralemmin-1 expression in ER-positive tumors. Conclusions The differential expression of paralemmin-1 in a subset of breast cancers suggests the existence of variation in membrane dynamics that may be exploited to improve diagnosis or provide a therapeutic target. PMID:22574838

  5. The significance of Brf1 overexpression in human hepatocellular carcinoma.

    PubMed

    Zhong, Qian; Xi, Shaoyan; Liang, Jianzhong; Shi, Ganggang; Huang, Yi; Zhang, Yanmei; Levy, Daniel; Zhong, Shuping

    2016-02-01

    Brf1 (TFIIB-related factor 1) plays a crucial role in cell transformation and tumorigenesis. However, the significance of Brf1 expression in human HCC (hepatocellular carcinoma) cases remains to be addressed. In this study, biopsies of human HCC, liver tumor samples of mice and cell lines of normal and tumor liver were utilized to determine the alteration of Brf1 expression using cytological and molecular biological approaches. Brf1 expression is increased in human HCC cases, which is correlated with shorter survival times. Levels of Brf1 and Pol III (RNA polymerase III-dependent) gene transcription in HCC patients with alcohol consumption are higher than the cases of non-HCC with or without alcohol intake. Induction of Brf1 and Pol III genes by ethanol in hepatoma cells is higher than in non-tumor cells. Ethanol increases the rate of cell transformation. Repression of Brf1 inhibits alcohol-promoted cell transformation. Alcohol consumption enhances Brf1 expression to promote cell transformation. These studies demonstrate that Brf1 is a new biomarker of HCC. PMID:26701855

  6. Significance of twist and iNOS expression in human breast carcinoma.

    PubMed

    Ranganathan, Santhalakshmi; Krishnan, Arunkumar; Sivasithambaram, Niranjali Devaraj

    2016-01-01

    Twist is a basic helix-loop-helix transcription factor family normally expressed during embryonic development and apparently activated in variety of tumours. Overexpression of twist is correlated with uncontrolled cell proliferation, differentiation, invasion and metastasis. Twist expression is associated with oestrogen receptor (ER); however, the molecular mechanism behind involvement of twist in progression of breast cancer is still unclear. Nitric oxide synthases (NOSs) which cause damage to the cellular DNA are also shown to be involved in cancer progression. The present study involves total number of n=85 breast biopsies, which include 19 non-cancer and 66 cancerous lesions. We analysed twist, iNOS and ER expression pattern in human breast carcinomas by RT-PCR and also analysed twist cellular localisation by immunohistochemical analysis. iNOS expression pattern was correlated with different stages of breast carcinoma. Twist expression was significantly increased in cancer lesions when compared to the non-cancer. The breast cancer lesions positive to ER showed positivity to twist (72%) as well. The higher stages of cancer lesions showed a significant expression of twist localised in cytoplasm of the cancer cells. Collectively these data indicate that up-regulation of twist is correlated with the ER presenting breast cancer, and iNOS expression was positively correlated with tumour-node metastasis (TNM) staging of breast cancer. These findings suggest that expression of twist and iNOS may have a functional role in cancer progression. PMID:26590086

  7. Tamoxifen stimulates the growth of cyclin D1-overexpressing breast cancer cells by promoting the activation of signal transducer and activator of transcription 3.

    PubMed

    Ishii, Yuki; Waxman, Samuel; Germain, Doris

    2008-02-01

    De novo or acquired resistance to tamoxifen is a major clinical challenge for the management of estrogen receptor (ER)-positive breast cancers. Although cyclin D1 overexpression is associated with a better outcome for breast cancer patients, its overexpression is also linked to tamoxifen resistance. We previously reported that the beneficial effect of cyclin D1 correlates with its ability to repress the antiapoptotic transcription factor signal transducer and activator of transcription 3 (STAT3). In contrast, molecular pathways linking overexpression of cyclin D1 to tamoxifen resistance have not been established. In the current study, the effect of tamoxifen on the growth of genetically matched high or low cyclin D1-expressing breast cancer cells was characterized and the interactions between cyclin D1, ER, and STAT3 in response to tamoxifen treatment were determined. We show that repression of STAT3 by cyclin D1 inhibits cell growth on Matrigel and in tumors in vivo; however, treatment with tamoxifen abolishes cyclin D1-mediated repression of STAT3 and growth suppression. We show that tamoxifen induces a redistribution of cyclin D1 from STAT3 to the ER, which results in the activation of both STAT3 and the ER. These results offer a molecular mechanism for the dual effect of cyclin D1 overexpression in breast cancer and support the notion that the level of cyclin D1 expression and activated STAT3 are important markers to predict response to tamoxifen treatment. PMID:18245487

  8. Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

  9. Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

    SciTech Connect

    Lu, Li; Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin; Song, Wen-Hui; Yan, Ba-Yi; Yang, Gui-Jiao; Li, Ang; Yang, Wu-Lin

    2014-01-24

    Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

  10. Overexpression of mutant p53 and c-erbB-2 proteins and breast tumour take in mice.

    PubMed Central

    Mehta, R. R.; Graves, J. M.; Warso, M. A.; Das Gupta, T. K.

    1995-01-01

    We established a panel of 17 xenografts from primary human breast carcinomas. We examined which characteristics of the original tumours and the xenografts facilitate growth in animals. Tumours expressing medium or strong immunoreactivity for p53 protein had significantly (P < 0.05) higher incidence (92%) of in vivo tumour take than those showing weak or negative immunoreactivity (9.1%). No such association was observed between either c-erbB-2 or epidermal growth factor receptor (EGFR) expression in the original tumours and their in vivo tumour take. Following subcutaneous (s.c.) transplantation of original breast tumours or established xenografts, 7/17 tumours showed metastatic disease spread to distant sites (mainly lungs). This study suggests that selective growth of highly aggressive tumours occurs during in vivo propagation of malignant tumours, and these tumours will be of particular interest in evaluating various chemotherapeutic agents for breast cancer management. Images Figure 1 PMID:7577462

  11. A canine model of Alzheimer's disease generated by overexpressing a mutated human amyloid precursor protein.

    PubMed

    Lee, Geun-Shik; Jeong, Yeon Woo; Kim, Joung Joo; Park, Sun Woo; Ko, Kyeong Hee; Kang, Mina; Kim, Yu Kyung; Jung, Eui-Man; Moon, Changjong; Hyun, Sang Hwan; Hwang, Kyu-Chan; Kim, Nam-Hyung; Shin, Taeyoung; Jeung, Eui-Bae; Hwang, Woo Suk

    2014-04-01

    Canines are considered the most authentic model for studying multifactorial human diseases, as these animals typically share a common environment with man. Somatic cell nuclear transfer (SCNT) technology along with genetic engineering of nuclear donor cells provides a unique opportunity for examining human diseases using transgenic canines. In the present study, we generated transgenic canines that overexpressed the human amyloid precursor protein (APP) gene containing well-characterized familial Alzheimer's disease (AD) mutations. We successfully obtained five out of six live puppies by SCNT. This was confirmed by observing the expression of green fluorescence protein in the body as a visual transgenic marker and the overexpression of the mutated APP gene in the brain. The transgenic canines developed AD-like symptoms, such as enlarged ventricles, an atrophied hippocampus, and ?-amyloid plaques in the brain. Thus, the transgenic canines we created can serve as a novel animal model for studying human AD. PMID:24481173

  12. Overexpression of sonic hedgehog in the triple negative breast cancer: clinicopathological characteristics of high burden breast cancer patients from Bangladesh

    PubMed Central

    Noman, A. S.; Uddin, M.; Rahman, M. Z.; Nayeem, M. J.; Alam, S. S.; Khatun, Z.; Wahiduzzaman, M.; Sultana, A.; Rahman, M. L.; Ali, M. Y.; Barua, D.; Ahmed, I.; Islam, M. S.; Aboussekhra, A.; Yeger, H.; Farhat, W. A.; Islam, S. S.

    2016-01-01

    Dysregulation of Hedgehog (Hh) signaling pathway has been documented in mammary gland development and breast cancer (BC) progression. Despite the remarkable progress in therapeutic interventions, BC related mortality in Bangladesh increased in the last decade. Triple negative breast cancer (TNBC) still presents a critical therapeutic challenge. Thus effective targeted therapy is urgently needed. In this study, we report the clinicopathological characteristics and prognosis of BC patients from Bangladesh. Routine immunohistochemical analysis and high throughput RNA-Seq data from the TCGA library were used to analyze the expression pattern and association of high and low level of Shh expression in a collection of BC patients with a long-term follow-up. High levels of Shh were observed in a subset of BC tumors with poor prognostic pathological features. Higher level of Shh expression correlated with a significantly poorer overall survival of patients compared with patients whose tumors expressed a low level of Shh. These data support the contention that Shh could be a novel biomarker for breast cancer that is involved in mediating the aggressive phenotype of BC. We propose that BC patients exhibiting a higher level of Shh expression, representing a subset of BC patients, would be amenable to Shh targeted therapy. PMID:26727947

  13. Overexpression of sonic hedgehog in the triple negative breast cancer: clinicopathological characteristics of high burden breast cancer patients from Bangladesh.

    PubMed

    Noman, A S; Uddin, M; Rahman, M Z; Nayeem, M J; Alam, S S; Khatun, Z; Wahiduzzaman, M; Sultana, A; Rahman, M L; Ali, M Y; Barua, D; Ahmed, I; Islam, M S; Aboussekhra, A; Yeger, H; Farhat, W A; Islam, S S

    2016-01-01

    Dysregulation of Hedgehog (Hh) signaling pathway has been documented in mammary gland development and breast cancer (BC) progression. Despite the remarkable progress in therapeutic interventions, BC related mortality in Bangladesh increased in the last decade. Triple negative breast cancer (TNBC) still presents a critical therapeutic challenge. Thus effective targeted therapy is urgently needed. In this study, we report the clinicopathological characteristics and prognosis of BC patients from Bangladesh. Routine immunohistochemical analysis and high throughput RNA-Seq data from the TCGA library were used to analyze the expression pattern and association of high and low level of Shh expression in a collection of BC patients with a long-term follow-up. High levels of Shh were observed in a subset of BC tumors with poor prognostic pathological features. Higher level of Shh expression correlated with a significantly poorer overall survival of patients compared with patients whose tumors expressed a low level of Shh. These data support the contention that Shh could be a novel biomarker for breast cancer that is involved in mediating the aggressive phenotype of BC. We propose that BC patients exhibiting a higher level of Shh expression, representing a subset of BC patients, would be amenable to Shh targeted therapy. PMID:26727947

  14. ODAM Expression Inhibits Human Breast Cancer Tumorigenesis

    PubMed Central

    Kestler, Daniel P.; Foster, James S.; Bruker, Charles T.; Prenshaw, John W.; Kennel, Stephen J.; Wall, Jonathan S.; Weiss, Deborah T.; Solomon, Alan

    2011-01-01

    We have posited that Odontogenic Ameloblast Associated Protein (ODAM) serves as a novel prognostic biomarker in breast cancer and now have investigated its potential role in regulating tumor growth and metastasis. Human breast cancer MDA-MB-231 cells were transfected with a recombinant ODAM plasmid construct (or, as a control, the plasmid vector alone). ODAM expression increased adhesion and apoptosis of the transfected MDA-MB-231 cells and suppressed their growth rate, migratory activity, and capability to invade extracellular matrix-coated membranes. Implantation of such cells into mouse mammary fat pads resulted in significantly smaller tumors than occurred in animals that received control cells; furthermore, ODAM-expressing cells, when injected intravenously into mice, failed to metastasize, whereas the control-transfected counterparts produced extensive lung lesions. Our finding that induction of ODAM expression in human breast cancer cells markedly inhibited their neoplastic properties provides further evidence for the regulatory role of this molecule in tumorigenesis and, consequently, is of potential clinical import. PMID:21603257

  15. Bone metastasis in a novel breast cancer mouse model containing human breast and human bone.

    PubMed

    Xia, Tian-Song; Wang, Guo-Zhu; Ding, Qiang; Liu, Xiao-An; Zhou, Wen-Bin; Zhang, Yi-Fen; Zha, Xiao-Ming; Du, Qing; Ni, Xiao-Jian; Wang, Jue; Miao, Su-Yu; Wang, Shui

    2012-04-01

    In practice, investigations for bone metastasis of breast cancer rely heavily on models in vivo. Lacking of such ideal model makes it difficult to study the whole process or accurate mechanism of each step of this metastatic disease. Development of xenograft mouse models has made great contributions in this area. Currently, the best animal model of breast cancer metastasizing to bone is NOD/SCID-hu models containing human bone, which makes it possible to let the breast cancer cells and the bone target of osteotropic metastasis be both of human origin. We have developed a novel mouse model containing both human bone and breast, and proved it functional and reliable. In this study, a set of human breast cancer cell line including MDA-MB-231, MDA-MB-231BO, MCF-7, ZR-75-1 and SUM1315 were characterized their osteotropism in this model. A specific cell line SUM1315 made species-specific bone metastasis, certifying the osteotropism-identification utility of the novel mouse model. Furthermore, gene expression and microRNA expression profiling analysis were done to the two SUM1315 derived sub lines isolated and purified from the orthotopic and metastatic xenograft. In addition, to demonstrate the disparity between the "spontaneous" and "forced" bone metastasis in mouse model, MDA-MB-231 cells were inoculated into both the human implants in this model simultaneously, and then primary cultured and profiling analyzed. Supported by overall results of profiling analyses, this study suggested the novel model was a useful tool for understanding, preventing and treating bone metastasis of breast cancer, meanwhile it had provided significant information for further investigations. PMID:21638054

  16. Survivin family proteins as novel molecular determinants of doxorubicin resistance in organotypic human breast tumors

    PubMed Central

    2014-01-01

    Introduction The molecular determinants of breast cancer resistance to first-line anthracycline-containing chemotherapy are unknown. Methods We examined the response to doxorubicin of organotypic cultures of primary human breast tumors ex vivo with respect to cell proliferation, DNA damage and modulation of apoptosis. Samples were analyzed for genome-wide modulation of cell death pathways, differential activation of p53, and the role of survivin family molecules in drug resistance. Rational drug combination regimens were explored by high-throughput screening, and validated in model breast cancer cell types. Results Doxorubicin treatment segregated organotypic human breast tumors into distinct Responder or Non Responder groups, characterized by differential proliferative index, stabilization of p53, and induction of apoptosis. Conversely, tumor histotype, hormone receptor or human epidermal growth factor receptor-2 (HER2) status did not influence chemotherapy sensitivity. Global analysis of cell death pathways identified survivin and its alternatively spliced form, survivin-?Ex3 as uniquely overexpressed in Non Responder breast tumors. Forced expression of survivin-?Ex3 preserved cell viability and prevented doxorubicin-induced apoptosis in breast cancer cell types. High-throughput pharmacologic targeting of survivin family proteins with a small-molecule survivin suppressant currently in the clinic (YM155) selectively potentiated the effect of doxorubicin, but not other chemotherapeutics in breast cancer cell types, and induced tumor cell apoptosis. Conclusions Survivin family proteins are novel effectors of doxorubicin resistance in chemotherapy-naive breast cancer. The incorporation of survivin antagonist(s) in anthracycline-containing regimens may have improved clinical activity in these patients. PMID:24886669

  17. Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

    SciTech Connect

    Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria . E-mail: m.barile@biologia.uniba.it

    2006-06-09

    FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.

  18. MUC1-C ONCOPROTEIN INDUCES TAMOXIFEN RESISTANCE IN HUMAN BREAST CANCER CELLS

    PubMed Central

    Kharbanda, Akriti; Rajabi, Hasan; Jin, Caining; Raina, Deepak; Kufe, Donald

    2013-01-01

    Resistance of estrogen receptor positive (ER+) breast cancer cells to tamoxifen has been linked in part to activation of (i) certain receptor tyrosine kinases, such as HER2, and (ii) the PI3K?AKT pathway. Mucin 1 (MUC1) is aberrantly overexpressed in about 90% of human breast cancers and the oncogenic MUC1-C subunit associates with ER?. The present studies using HER2 overexpressing BT-474 breast cancer cells, which are constitutively resistant to tamoxifen, demonstrate that silencing MUC1-C is associated with (i) downregulation of p-HER2 levels, and (ii) sensitivity to tamoxifen-induced growth inhibition and loss of clonogenic survival. The results also demonstate that overexpression of MUC1-C in tamoxifen-sensitive MCF-7 breast cancer cells results in upregulation of p-AKT and tamoxifen resistance. We show that MUC1-C forms complexes with ER? on the estrogen-responsive promoter of the Rab31 gene and that MUC1-C blocks tamoxifen-induced decreases in ER? occupancy. MUC1-C also attenuated tamoxifen-induced decreases in (i) recruitment of the coactivator CREB binding protein, (ii) Rab31 promoter activation, and (ii) Rab31 mRNA and protein levels. The importance of MUC1-C is further supported by the demonstration that targeting MUC1-C with the cell-penetrating peptide inhibitor, GO-203, sensitizes tamoxifen-resistant cells to tamoxifen treatment. Moreover, we show that targeting MUC1-C in combination with tamoxifen is highly synergistic in the treatment of tamoxifen-resistant breast cancer cells. These findings indicate that MUC1-C contributes to tamoxifen resistance and provide support for the investigation of MUC1-C inhibitors in the setting of tamoxifen refractory disease. PMID:23538857

  19. The Stromal Overexpression of CD10 in Invasive Breast Cancer and its Association with Clincophathologic Factors

    PubMed Central

    Taghizadeh-Kermani, Ali; Jafarian, Amir Hossein; Ashabyamin, Reza; Seilanian-Toosi, Mehdi; Pourali, Leila; Asadi, Mehdi; Mashhadi, Leila

    2014-01-01

    Background Breast carcinoma is the most common non-skin malignancy in women. More recently, it has been suggested that extracellular proteinase has also regulated growth factors and cytokines that might contribute to tumor progression. CD10 is a 90-110kd cell surface zinc-dependent metalloproteinase. Since CD10 is structurally similar to matrix metalloproteinase and stromelysin, it might facilitate cancer cell invasion and/or metastasis. The aim of this study was investigation the rate of CD10 expression in the stromal cells of invasive ductal breast carcinomas, Immunohistochemical aspects, then any other aspects to be able to clarify its correlation with other clinicopathological factors of this disease. Methods One hundred patients with histopathologic diagnosis of invasive ductal carcinoma and 50 patients with fibroadenoma of breast (as the control group) have selected, then 150 paraffin blocks have obtained. The stained slides by immunohistochemistry method for CD10 marker have examined separately by two pathologists, and discrepancies have reviewed in common session to get the final result. Results Stromal CD10 has detected in 28% of the IDC. No kind of immunoreactivity has identified in the stromal cells of normal breast. Stromal CD10 expression in IDC has significantly correlated with increasing tumor size (p<0.001), increasing histologic grade (p<0.001), the presence of nodal metastases (p<0.001) and estrogen receptor negative status (p=0.003). Conclusion Stromal CD10 expression in IDC has closely correlated with invasion and metastasis and it might play an important role in the pathogenesis of IDC. PMID:25250143

  20. Human Breast Progenitor Cell Numbers Are Regulated by WNT and TBX3

    PubMed Central

    Arendt, Lisa M.; St. Laurent, Jessica; Wronski, Ania; Caballero, Silvia; Lyle, Stephen R.; Naber, Stephen P.; Kuperwasser, Charlotte

    2014-01-01

    Background Although human breast development is mediated by hormonal and non-hormonal means, the mechanisms that regulate breast progenitor cell activity remain to be clarified. This limited understanding of breast progenitor cells has been due in part to the lack of appropriate model systems to detect and characterize their properties. Methods To examine the effects of WNT signaling and TBX3 expression on progenitor activity in the breast, primary human mammary epithelial cells (MEC) were isolated from reduction mammoplasty tissues and transduced with lentivirus to overexpress WNT1 or TBX3 or reduce expression of their cognate receptors using shRNA. Changes in progenitor activity were quantified using characterized assays. We identified WNT family members expressed by cell populations within the epithelium and assessed alterations in expression of WNT family ligands by MECs in response to TBX3 overexpression and treatment with estrogen and progesterone. Results Growth of MECs on collagen gels resulted in the formation of distinct luminal acinar and basal ductal colonies. Overexpression of TBX3 in MECs resulted in increased ductal colonies, while shTBX3 expression diminished both colony types. Increased WNT1 expression led to enhanced acinar colony formation, shLRP6 decreased both types of colonies. Estrogen stimulated the formation of acinar colonies in control MEC, but not shLRP6 MEC. Formation of ductal colonies was enhanced in response to progesterone. However, while shLRP6 decreased MEC responsiveness to progesterone, shTBX3 expression did not alter this response. Conclusions We identified two phenotypically distinguishable lineage-committed progenitor cells that contribute to different structural elements and are regulated via hormonal and non-hormonal mechanisms. WNT signaling regulates both types of progenitor activity. Progesterone favors the expansion of ductal progenitor cells, while estrogen stimulates the expansion of acinar progenitor cells. Paracrine WNT signaling is stimulated by estrogen and progesterone, while autocrine WNT signaling is induced by the embryonic T-box transcription factor TBX3. PMID:25350852

  1. Expression and regulation of Cyr61 in human breast cancer cell lines.

    PubMed

    Tsai, Miaw-Sheue; Bogart, Daphne F; Li, Patricia; Mehmi, Inderjit; Lupu, Ruth

    2002-01-31

    We have shown that Cyr61, an angiogenic regulator, is overexpressed in invasive and metastatic human breast cancer cells and tumor biopsies. We have further demonstrated that Cyr61 promotes acquisition of estrogen-independence and anti-estrogen resistance in vivo in breast cancer cells. Moreover, we have demonstrated that Cyr61 induces tumor formation and tumor vascularization in vivo, events mediated through the activation of the MAPK and the Akt signaling pathways. Here we investigate how Cyr61 expression is regulated in both estrogen receptor (ER)-positive and ER-negative breast cancer cells. We demonstrate that Cyr61 mRNA and protein expression is inducible by estrogen and anti-estrogens in ER-positive breast cancer cells. We show that a labile protein as well as a negative regulator might be involved in Cyr61 expression in estrogen-dependent breast cancer cells. Other important regulators of Cyr61 expression in breast cancer cells that we found are the phorbol ester TPA, vitamin D, and retinoic acid. TPA causes positive regulation of Cyr61 expression in ER-positive MCF-7 cells. Vitamin D induces a transient stimulatory effect on Cyr61 gene expression. Lastly, retinoic acid has a negative effect on Cyr61 expression and downregulates its expression in MCF-7 cells. Interestingly, most of these effects are not seen in aggressive breast cancer cells that do not express ER and express high levels of Cyr61, such as the MDA-MB-231 cells. Our results are in agreement with our knowledge that Cyr61 promotes tumor growth, and that tumor-promoting agents have a positive impact on cells that express low levels of Cyr61, such as the ER-positive breast cancer cells; however, these agents have no significant effect on cells that express high levels of Cyr61. Our findings suggest an association between increased Cyr61 expression and an aggressive phenotype of breast cancer cells. PMID:11840342

  2. Increased level of phosphorylated akt measured by chemiluminescence-linked immunosorbent assay is a predictor of poor prognosis in primary breast cancer overexpressing ErbB-2

    PubMed Central

    Cicenas, Jonas; Urban, Patrick; Vuaroqueaux, Vincent; Labuhn, Martin; Kng, Willy; Wight, Edward; Mayhew, Mark; Eppenberger, Urs; Eppenberger-Castori, Serenella

    2005-01-01

    Introduction Akt1, Akt2 and Akt3 kinases are downstream components of phosphoinositol 3-kinase derived signals from receptor tyrosine kinases, which influence cell growth, proliferation and survival. Akt2 overexpression and amplification have been described in breast, ovarian and pancreatic cancers. The present study was designed to investigate the prognostic significance of activated Akt in primary breast cancer and its association with other tumour biomarkers. Methods Using a two-site chemiluminescence-linked immunosorbent assay, we measured the quantitative expression levels of total phosphorylated (P-S473) Akt (Akt1/Akt2/Akt3) on cytosol fractions obtained from fresh frozen tissue samples of 156 primary breast cancer patients. Results Akt phosphorylation was not associated with nodal status or ErbB-2 protein expression levels. High levels of phosphorylated Akt correlated (P < 0.01) with poor prognosis, and the significance of this correlation increased (P < 0.001) in the subset of patients with ErbB-2 overexpressing tumours. In addition, phosphorylated Akt was found to be associated with mRNA expression levels of several proliferation markers (e.g. thymidylate synthase), measured using quantitative real-time RT-PCR. Conclusion Our findings demonstrate that, in breast cancer patients, Akt activation is associated with tumour proliferation and poor prognosis, particularly in the subset of patients with ErbB2-overexpressing tumours. PMID:15987444

  3. Anti-HER2 immunoliposomes for selective delivery of electron paramagnetic resonance imaging probes to HER2-overexpressing breast tumor cells

    PubMed Central

    Burks, Scott R.; Macedo, Luciana F.; Barth, Eugene D.; Tkaczuk, Katherine H.; Martin, Stuart S.; Rosen, Gerald M.; Halpern, Howard J.; Brodie, Angela M.

    2014-01-01

    Electron paramagnetic resonance (EPR) imaging is an emerging modality that can detect and localize paramagnetic molecular probes (so-called spin probes) in vivo. We previously demonstrated that nitroxide spin probes can be encapsulated in liposomes at concentrations exceeding 100 mM, at which nitroxides exhibit a concentration-dependent quenching of their EPR signal that is analogous to the self-quenching of fluorescent molecules. Therefore, intact liposomes encapsulating high concentrations of nitroxides exhibit greatly attenuated EPR spectral signals, and endocytosis of such liposomes represents a cell-activated contrast-generating mechanism. After endocytosis, the encapsulated nitroxide is liberated and becomes greatly diluted in the intracellular milieu. This dequenches the nitroxides to generate a robust intracellular EPR signal. It is therefore possible to deliver a high concentration of nitroxides to cells while minimizing background signal from unendocytosed liposomes. We report here that intracellular EPR signal can be selectively generated in a specific cell type by exploiting its expression of Human Epidermal Growth Factor Receptor 2 (HER2). When targeted by anti-HER2 immunoliposomes encapsulating quenched nitroxides, Hc7 cells, which are novel HER2-overexpressing cells derived from the MCF7 breast tumor cell line, endocytose the liposomes copiously, in contrast to the parent MCF7 cells or control CV1 cells, which do not express HER2. HER2-dependent liposomal delivery enables Hc7 cells to accumulate 750 ?M nitroxide intracellularly. Through the use of phantom models, we verify that this concentration of nitroxides is more than sufficient for EPR imaging, thus laying the foundation for using EPR imaging to visualize HER2-overexpressing Hc7 tumors in animals. PMID:20066490

  4. Anti-HER2 immunoliposomes for selective delivery of electron paramagnetic resonance imaging probes to HER2-overexpressing breast tumor cells.

    PubMed

    Burks, Scott R; Macedo, Luciana F; Barth, Eugene D; Tkaczuk, Katherine H; Martin, Stuart S; Rosen, Gerald M; Halpern, Howard J; Brodie, Angela M; Kao, Joseph P Y

    2010-11-01

    Electron paramagnetic resonance (EPR) imaging is an emerging modality that can detect and localize paramagnetic molecular probes (so-called spin probes) in vivo. We previously demonstrated that nitroxide spin probes can be encapsulated in liposomes at concentrations exceeding 100 mM, at which nitroxides exhibit a concentration-dependent quenching of their EPR signal that is analogous to the self-quenching of fluorescent molecules. Therefore, intact liposomes encapsulating high concentrations of nitroxides exhibit greatly attenuated EPR spectral signals, and endocytosis of such liposomes represents a cell-activated contrast-generating mechanism. After endocytosis, the encapsulated nitroxide is liberated and becomes greatly diluted in the intracellular milieu. This dequenches the nitroxides to generate a robust intracellular EPR signal. It is therefore possible to deliver a high concentration of nitroxides to cells while minimizing background signal from unendocytosed liposomes. We report here that intracellular EPR signal can be selectively generated in a specific cell type by exploiting its expression of Human Epidermal Growth Factor Receptor 2 (HER2). When targeted by anti-HER2 immunoliposomes encapsulating quenched nitroxides, Hc7 cells, which are novel HER2-overexpressing cells derived from the MCF7 breast tumor cell line, endocytose the liposomes copiously, in contrast to the parent MCF7 cells or control CV1 cells, which do not express HER2. HER2-dependent liposomal delivery enables Hc7 cells to accumulate 750 ?M nitroxide intracellularly. Through the use of phantom models, we verify that this concentration of nitroxides is more than sufficient for EPR imaging, thus laying the foundation for using EPR imaging to visualize HER2-overexpressing Hc7 tumors in animals. PMID:20066490

  5. Clinicopathologic significance of the basal-like subtype of breast cancer: a comparison with hormone receptor and Her2/neu-overexpressing phenotypes.

    PubMed

    Kim, Mi-Jung; Ro, Jae Y; Ahn, Sei-Hyun; Kim, Hak Hee; Kim, Sung-Bae; Gong, Gyungyub

    2006-09-01

    DNA microarray profiling studies have led to the classification of invasive breast carcinoma into luminal/estrogen receptor-positive, normal breast-like, Her2/neu-overexpressing, and basal-like types. Among these groups, the basal-like subtype is associated with the poorest clinical outcome in Western countries. To date, the clinicopathologic characteristics of the basal-like carcinomas, compared with other subtypes, have not been described in the Korean population. In this study, we used tissue microarray to examine the expression of basal cytokeratins (CK) (CK5 and CK14) and luminal CK (CK8/18), epidermal growth factor receptor, c-kit, hormone receptors (HRs), p53, and Her2/neu in 776 consecutive patients diagnosed with invasive breast carcinoma from January 1993 to December 1998 and categorized these cases into 5 subgroups (basal-like, HR-expressing, Her2/neu-overexpressing, HR and Her2/neu-expressing, and null subtypes negative for all markers), based on the immunohistochemical data. We identified cases of 114 (14.7%) basal-like, 345 (44.5%) HR-expressing, 133 (17.1%) Her2/neu-overexpressing, 61 (7.8%) HR and Her2/neu-expressing, and 123 (15.9%) null subtypes. Histologically, most basal-like breast cancers were invasive ductal carcinoma, not otherwise specified (98 cases, 86.0%), with high nuclear and/or histologic grades, and most metaplastic carcinomas (6 [75.0%] of 8 cases) were the basal-like subtype. Both basal-like and Her2/neu-overexpressing subtypes were associated with larger tumor sizes (mean, 3.6 and 3.3 cm, respectively) than the HR-expressing group (mean, 2.8 cm) (P = .001 and P = .036, respectively). Nodal stage of Her2/neu-overexpressing subtype was higher than that of basal-like subtype; however, overall stage was not different between the 2 groups (P = .010 and .123, respectively). Distant metastasis was most frequently observed in the Her2/neu-overexpressing subtype (33.8%), which was prognostically the worst subgroup of breast cancers. In contrast to previous findings from Western countries, our analyses reveal that the Her2/neu status is the most important prognostic factor of breast cancers. PMID:16938528

  6. Proteomic Identification of Mitochondrial Targets of Arginase in Human Breast Cancer

    PubMed Central

    Singh, Rajan; Avliyakulov, Nuraly K.; Braga, Melissa; Haykinson, Michael J.; Martinez, Luis; Singh, Vikash; Parveen, Meher; Chaudhuri, Gautam; Pervin, Shehla

    2013-01-01

    We have previously reported arginase expression in human breast cancer cells and demonstrated that the inhibition of arginase by N? hydroxy L-arginine (NOHA) in MDA-MB-468 cells induces apoptosis. However, arginase expression and its possible molecular targets in human breast tumor samples and potential clinical implications have not been fully elucidated. Here, we demonstrate arginase expression in human breast tumor samples, and several established breast cancer cell lines, in which NOHA treatment selectively inhibits cell proliferation. The over-expression of Bcl2 in MDA-MB-468 cells abolished NOHA-induced apoptosis, suggesting that the mitochondria may be the main site of NOHAs action. We, therefore, undertook a proteomics approach to identify key mitochondrial targets of arginase in MDA-MB-468 cells. We identified 54 non-mitochondrial and 13 mitochondrial proteins that were differentially expressed in control and NOHA treated groups. Mitochondrial serine hydroxymethyltransferase (mSHMT) was identified as one of the most promising targets of arginase. Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression. MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression. Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression. As mSHMT is a key player in folate metabolism, our data provides a novel link between arginine and folate metabolism in human breast cancer, both of which are critical for tumor cell proliferation. PMID:24223914

  7. The Polycomb group protein RING1B is overexpressed in ductal breast carcinoma and is required to sustain FAK steady state levels in breast cancer epithelial cells.

    PubMed

    Bosch, Almudena; Panoutsopoulou, Konstantina; Corominas, Josep Maria; Gimeno, Ramn; Moreno-Bueno, Gema; Martn-Caballero, Juan; Morales, Saleta; Lobato, Tania; Martnez-Romero, Carles; Farias, Eduardo F; Mayol, Xavier; Cano, Amparo; Hernndez-Muoz, Inmaculada

    2014-04-30

    In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgf?-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy. PMID:24742605

  8. Constitutive overexpression of cyclin D1 but not cyclin E confers acute resistance to antiestrogens in T-47D breast cancer cells.

    PubMed

    Hui, Rina; Finney, Georgina L; Carroll, Jason S; Lee, Christine S L; Musgrove, Elizabeth A; Sutherland, Robert L

    2002-12-01

    Cyclin D1 and cyclin E are overexpressed in approximately 45% and 30% of breast cancers, respectively, and adverse associations with patient outcome have been reported. The potential roles of cyclin D1 and cyclin E expression as markers of therapeutic responsiveness to the pure steroidal antiestrogen ICI 182780 were investigated using T-47D breast cancer cell lines constitutively overexpressing cyclin D1 or cyclin E. Measurement of S phase fraction, phosphorylation states of the retinoblastoma protein, and cyclin E-cyclin-dependent kinase (Cdk) 2 activity demonstrated that overexpression of cyclin D1 decreased sensitivity to antiestrogen inhibition at 24 and 48 h. Overexpression of cyclin E produced a less pronounced early cell cycle effect indicating only partial resistance to antiestrogen inhibition in the short-term. In ICI 182780-treated cyclin D1-overexpressing cells, sufficient Cdk activity was retained to allow retinoblastoma protein phosphorylation and cell proliferation, despite an increase in the association of p21 and p27 with cyclin D1-Cdk4/6 and cyclin E-Cdk2 complexes. After longer-term (>7 days) treatment, antiestrogens inhibited colony growth in cyclin D1- or cyclin E-overexpressing breast cancer cells, but with an approximately 2-2.5-fold decrease in dose sensitivity. This was associated with a fall in cyclin D1 levels, a reduction in the half-life of cyclin D1 protein and a decline in cyclin E-Cdk2 activity in cyclin D1-overexpressing cells, and the maintenance of cyclin E-p27 association in the cyclin E-overexpressing cells. These data confirm that cyclin D1 expression and cyclin E-p27 association play important roles in antiestrogen action, and suggest that cyclin D1 or cyclin E overexpression has subtle effects on antiestrogen sensitivity. Additional studies to elucidate the contribution of alterations in cyclin D1 stability to antiestrogen action and to assess the relationship between antiestrogen sensitivity and expression of cyclin D1, cyclin E, or p27 in a clinical setting are required. PMID:12460907

  9. Apigenin induces caspase-dependent apoptosis by inhibiting signal transducer and activator of transcription 3 signaling in HER2-overexpressing SKBR3 breast cancer cells.

    PubMed

    Seo, Hye-Sook; Ku, Jin Mo; Choi, Han-Seok; Woo, Jong-Kyu; Jang, Bo-Hyoung; Go, Hoyeon; Shin, Yong Cheol; Ko, Seong-Gyu

    2015-08-01

    Phytoestrogens have been demonstrated to inhibit tumor induction; however, their molecular mechanisms of action have remained elusive. The present study aimed to investigate the effects of a phytoestrogen, apigenin, on proliferation and apoptosis of the human epidermal growth factor receptor 2 (HER2)-expressing breast cancer cell line SKBR3. Proliferation assay, MTT assay, fluorescence-activated cell sorting analysis, western blot analysis, immunocytochemistry, reverse transcription-polymerase chain reaction and ELISA assay were used in the present study. The results of the present study indicated that apigenin inhibited the proliferation of SKBR3 cells in a dose-and time-dependent manner. This inhibition of growth was accompanied by an increase in the sub-G0/G1 apoptotic population. Furthermore, apigenin enhanced the expression levels of cleaved caspase-8 and -3, and induced the cleavage of poly(adenosine diphosphate ribose) polymerase in SKBR3 cells, confirming that apigenin promotes apoptosis via a caspase-dependent pathway. Apigenin additionally reduced the expression of phosphorylated (p)-janus kinase 2 and p-signal transducer and activator of transcription 3 (STAT3), inhibited CoCl2-induced vascular endothelial growth factor (VEGF) secretion and decreased the nuclear localization of STAT3. The STAT3 inhibitor S31-201 decreased the cellular proliferation rate and reduced the expression of p-STAT3 and VEGF. Therefore, these results suggested that apigenin induced apoptosis via the inhibition of STAT3 signaling in SKBR3 cells. In conclusion, the results of the present study indicated that apigenin may be a potentially useful compound for the prevention or treatment of HER2-overexpressing breast cancer. PMID:25936427

  10. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    SciTech Connect

    Yu, Wei; Chai, Hongyan; Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue; Yang, Guifang; Cai, Xiaojun; Falck, John R.; Yang, Jing; Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071

    2012-10-01

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have been studied in vitro and in vivo. ► CYP4Z1 regulates expression and production of VEGF-A and TIMP-2. ► CYP4Z1-induced angiogenesis is associated with PI3K and ERK1/2 activation. ► CYP4Z1 may be an attractive target for anti-cancer therapy.

  11. Dystonia, facial dysmorphism, intellectual disability and breast cancer associated with a chromosome 13q34 duplication and overexpression of TFDP1: case report

    PubMed Central

    2013-01-01

    Background Dystonia is a movement disorder characterized by involuntary sustained muscle contractions causing twisting and repetitive movements or abnormal postures. Some cases of primary and neurodegenerative dystonia have been associated with mutations in individual genes critical to the G1-S checkpoint pathway (THAP1, ATM, CIZ1 and TAF1). Secondary dystonia is also a relatively common clinical sign in many neurogenetic disorders. However, the contribution of structural variation in the genome to the etiopathogenesis of dystonia remains largely unexplored. Case presentation Cytogenetic analyses with the Affymetrix Genome-Wide Human SNP Array 6.0 identified a chromosome 13q34 duplication in a 36year-old female with global developmental delay, facial dysmorphism, tall stature, breast cancer and dystonia, and her neurologically-normal father. Dystonia improved with bilateral globus pallidus interna (GPi) deep brain stimulation (DBS). Genomic breakpoint analysis, quantitative PCR (qPCR) and leukocyte gene expression were used to characterize the structural variant. The 218,345bp duplication was found to include ADPRHL1, DCUN1D2, and TMCO3, and a 69bp fragment from a long terminal repeat (LTR) located within Intron 3 of TFDP1. The 3' breakpoint was located within Exon 1 of a TFDP1 long non-coding RNA (NR_026580.1). In the affected subject and her father, gene expression was higher for all three genes located within the duplication. However, in comparison to her father, mother and neurologically-normal controls, the affected subject also showed marked overexpression (2) of the transcription factor TFDP1 (NM_007111.4). Whole-exome sequencing identified an SGCE variant (c.1295G?>?A, p.Ser432His) that could possibly have contributed to the development of dystonia in the proband. No pathogenic mutations were identified in BRCA1 or BRCA2. Conclusion Overexpression of TFDP1 has been associated with breast cancer and may also be linked to the tall stature, dysmorphism and dystonia seen in our patient. PMID:23849371

  12. Overexpressed human heme Oxygenase-1 decreases adipogenesis in pigs and porcine adipose-derived stem cells.

    PubMed

    Park, Eun Jung; Koo, Ok Jae; Lee, Byeong Chun

    2015-11-27

    Adipose-derived mesenchymal stem cells (ADSC) are multipotent, which means they are able to differentiate into several lineages invivo and invitro under proper conditions. This indicates it is possible to determine the direction of differentiation of ADSC by controlling the microenvironment. Heme oxygenase 1 (HO-1), a type of antioxidant enzyme, attenuates adipogenicity and obesity. We produced transgenic pigs overexpressing human HO-1 (hHO-1-Tg), and found that these animals have little fatty tissue when autopsied. To determine whether overexpressed human HO-1 suppresses adipogenesis in pigs, we analyzed body weight increases of hHO-1-Tg pigs and wild type (WT) pigs of the same strain, and induced adipogenic differentiation of ADSC derived from WT and hHO-1-Tg pigs. The hHO-1-Tg pigs had lower body weights than WT pigs from 16 weeks of age until they died. In addition, hHO-1-Tg ADSC showed reduced adipogenic differentiation and expression of adipogenic molecular markers such as PPAR? and C/EBP? compared to WT ADSC. These results suggest that HO-1 overexpression reduces adipogenesis both invivo and invitro, which could support identification of therapeutic targets of obesity and related metabolic diseases. PMID:26471299

  13. Overexpression of transcription factor FOXC2 in cultured human podocytes upregulates injury markers and increases motility.

    PubMed

    Datta, Neeta; Lindfors, Sonja; Miura, Naoyuki; Saleem, Moin A; Lehtonen, Sanna

    2016-01-01

    Obesity and diabetes-related kidney diseases associate with renal failure and cardiovascular morbidity, and represent a major health issue worldwide. However, the molecular mechanisms leading to their development remain poorly understood. We observed increased expression of transcription factor FoxC2 in the podocytes of obese Zucker rats that are insulin resistant and albuminuric. We also found that depletion of adiponectin, an adipocyte-derived hormone whose secretion is decreased in obesity, upregulated FOXC2 in differentiated human podocytes in vitro. Overexpression of FOXC2 in cultured human podocytes led to increased nuclear expression of FOXC2 associated with a change of cellular morphology. This was accompanied by upregulation of vimentin, a key mesenchymal marker, and active beta-catenin, associated with podocyte injury. We also observed re-organization of the actin cytoskeleton, disrupted localization of the tight junction protein ZO-1, and increased motility of podocytes overexpressing FOXC2. These data indicate that the expression of FOXC2 in podocytes needs to be tightly regulated, and that its overexpression induces a chain of cellular events leading to podocyte dysfunction. These changes may lead to podocyte detachment and depletion ultimately contributing to albuminuria. We also suggest a novel molecular mechanism linking obesity-induced decrease in adiponectin to podocyte dysfunction via upregulation of FOXC2. PMID:26524507

  14. Delta9-tetrahydrocannabinol inhibits cell cycle progression in human breast cancer cells through Cdc2 regulation.

    PubMed

    Caffarel, Mara M; Sarri, David; Palacios, Jos; Guzmn, Manuel; Snchez, Cristina

    2006-07-01

    It has been proposed that cannabinoids are involved in the control of cell fate. Thus, these compounds can modulate proliferation, differentiation, and survival in different manners depending on the cell type and its physiopathologic context. However, little is known about the effect of cannabinoids on the cell cycle, the main process controlling cell fate. Here, we show that Delta(9)-tetrahydrocannabinol (THC), through activation of CB(2) cannabinoid receptors, reduces human breast cancer cell proliferation by blocking the progression of the cell cycle and by inducing apoptosis. In particular, THC arrests cells in G(2)-M via down-regulation of Cdc2, as suggested by the decreased sensitivity to THC acquired by Cdc2-overexpressing cells. Of interest, the proliferation pattern of normal human mammary epithelial cells was much less affected by THC. We also analyzed by real-time quantitative PCR the expression of CB(1) and CB(2) cannabinoid receptors in a series of human breast tumor and nontumor samples. We found a correlation between CB(2) expression and histologic grade of the tumors. There was also an association between CB(2) expression and other markers of prognostic and predictive value, such as estrogen receptor, progesterone receptor, and ERBB2/HER-2 oncogene. Importantly, no significant CB(2) expression was detected in nontumor breast tissue. Taken together, these data might set the bases for a cannabinoid therapy for the management of breast cancer. PMID:16818634

  15. Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells

    PubMed Central

    Ravacci, Graziela Rosa; Brentani, Maria Mitzi; Tortelli, Tharcisio Citrângulo; Torrinhas, Raquel Suzana M. M.; Santos, Jéssica Reis; Logullo, Angela Flávia; Waitzberg, Dan Linetzky

    2015-01-01

    In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36), transport (FABP4), and storage (DGAT) of exogenous fatty acids (FA), as well as increased activation of “de novo” FA synthesis (FASN). We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPARγ pathway. Inhibition of the mTORC1 pathway markers, p70S6 K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR) was detected in HB4aC5.2. Based on these results, a PPARγ selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4) in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPARγ activity. This reprogramming was inhibited by DHA. PMID:26640797

  16. Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells.

    PubMed

    Ravacci, Graziela Rosa; Brentani, Maria Mitzi; Tortelli, Tharcisio Citrngulo; Torrinhas, Raquel Suzana M M; Santos, Jssica Reis; Logullo, Angela Flvia; Waitzberg, Dan Linetzky

    2015-01-01

    In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36), transport (FABP4), and storage (DGAT) of exogenous fatty acids (FA), as well as increased activation of "de novo" FA synthesis (FASN). We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPAR? pathway. Inhibition of the mTORC1 pathway markers, p70S6?K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR) was detected in HB4aC5.2. Based on these results, a PPAR? selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4) in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPAR? activity. This reprogramming was inhibited by DHA. PMID:26640797

  17. Over-expression of miR-675 in formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients

    PubMed Central

    Zhai, Ling-Ling; Wang, Peng; Zhou, Ling-Yu; Yin, Jia-Yu; Tang, Qin; Zhang, Tin-Juan; Wang, Yu-Xin; Yang, Dong-Qin; Lin, Jiang; Deng, Zhao-Qun

    2015-01-01

    Background: Dysregulation of miR-675 has been found in a variety of solid tumors. MiR-675 has been suggested as having both oncogenic and tumor suppression properties in cancer. However, there is no evidence whether miR-675 is involved in breast cancer. The objective of this study was to evaluate the expression status of miR-675 and its clinical relevance in breast cancer patients. Methods: The expression level of miR-675 was detected in 100 breast cancer patients and 38 cancer-free controls using real-time quantitative PCR. The clinicopathological characteristics of miR-675 in breast cancer were also investigated. All statistical analyses were performed using SPSS 20.0. Results: The study showed that miR-675 was significantly up-regulated in breast cancer patients compared with controls (P < 0.01). There was no significant difference in age, lymph nodes stage, ER status and PR status between patients with and without miR-675 over-expression (P > 0.05). The frequency of miR-675 over-expression was higher in the patients of histological grade I-II than in others (50% versus 9%, P = 0.011). The expression level of miR-675 had a high correlation with miR-24/93/98/378 in breast cancer patients. Conclusions: Taken together, our study demonstrated that miR-675 in formalin-fixed paraffin-embedded (FFPE) tissues might serve as a good source for biomarker discovery and breast cancer validation. PMID:26379923

  18. Screening of HER2 Overexpressed Breast Cancer Subtype In Vivo by the Validation of High-Performance, Long-Term, and Noninvasive Fluorescence Tracer.

    PubMed

    Ding, Jie; Zhou, Ying; Li, Jingjing; Jiang, Liping; He, Zhiwei; Zhu, Jun-Jie

    2015-12-15

    The high-performance and noninvasive screening of heterogeneous tumor subtypes in vivo is particularly desirable for the diagnosis and symptomatic treatment of cancer. Therefore, we report a near-infrared (NIR) fluorescence tracer "smartly identified HER2" (SI-HER2) for rapid, accurate, and highly specific screening of HER2 overexpressed breast cancer. An antibody against HER2 protein receptor, EP1045Y, was conjugated with NIR emitting CdSeTe/CdS/ZnS QDs via polyhistidine-driven self-assembly approach. The further adsorption of black hole quencher 3 on antibody enabled a "turn on" fluorescence response of the fluorescence tracer to HER2 protein receptor. Aside from the capability of differentiating the HER2 overexpressed MCF-7 cells from its counterparts, the fluorescence tracer can also accurately and rapidly identify the HER2 overexpressed breast tumor subtype in two tumors-bearing mouse model, providing a platform for the investigation of advanced pathways to distinguish the different breast cancer subtypes. PMID:26598802

  19. Mutations in p53 as potential molecular markers for human breast cancer

    SciTech Connect

    Runnebaum, I.B.; Nagarajan, M.; Bowman, M.; Soto, D.; Sukumar, S. )

    1991-12-01

    Based on the high incidence of loss of heterozygosity for loci on chromosome 17p in the vicinity of the p53 locus in human breast tumors. The authors investigated the frequency and effects of mutations in the p53 tumor suppressor gene in mammary neoplasia. They examined the p53 gene in 20 breast cancer cell lines and 59 primary breast tumors. Northern blot analysis, immunoprecipitation, and nucleotide sequencing analysis revealed aberrant mRNA expression, over-expression of protein, and point mutations in the p53 gene in 50% of the cell line tested. A multiplex PCR assay was developed to search for deletions in the p53 genomic locus. Multiplex PCR of genomic DNA showed that up to 36% of primary tumors contained aberrations in the p53 locus. Mutations in exons 5-9 of the p53 gene were found in 10 out of 59 (17%) of the primary tumors studied by single-stranded conformation polymorphism analysis. They conclude that, compared to amplification of HER2/NEU, MYC, or INT2 oncogene loci, p53 gene mutations and deletions are the most frequently observed genetic change in breast cancer related to a single gene. Correlated to disease status, p53 gene mutations could prove to be a valuable marker for diagnosis and/or prognosis of breast neoplasia.

  20. Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

    SciTech Connect

    Xu, Hanwen; Pirisi, Lucia; Creek, Kim E.

    2015-01-01

    Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling.

  1. Integrin activation controls metastasis in human breast cancer

    NASA Astrophysics Data System (ADS)

    Felding-Habermann, Brunhilde; O'Toole, Timothy E.; Smith, Jeffrey W.; Fransvea, Emilia; Ruggeri, Zaverio M.; Ginsberg, Mark H.; Hughes, Paul E.; Pampori, Nisar; Shattil, Sanford J.; Saven, Alan; Mueller, Barbara M.

    2001-02-01

    Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin v3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin v3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated αvβ3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant αvβ3D723R, but not αvβ3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin αvβ3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

  2. A Comprehensive Outline of Trastuzumab Resistance Biomarkers in HER2 Overexpressing Breast Cancer.

    PubMed

    Menyhrt, Otlia; Santarpia, Libero; Gy?rffy, Balzs

    2015-01-01

    The introduction of trastuzumab for anti-HER2 therapy dramatically changed the clinical outcome for HER2 (ERBB2, neu) positive breast cancer patients. Today, patients eligible for trastuzumab are selected using HER2 expression/amplification status of the primary tumor. However, acquired and inherent resistance to anti-HER2 therapy in these patients poses a significant challenge, and better patient stratification will be needed to improve clinical response. Here, we provide a wide-ranging overview of potential biomarkers capable of stratifying patients regarding their response to trastuzumab. These include HER2 amplification, impaired access to the binding site (p95HER2, ?16HER-2, MUC4), augmented signaling through other ERBB family receptors (HER1, HER3, HER4) and their ligands, activation of HER2 targets by alternate heterodimers (EphA2, IGF-1R, GDF15, MUC1*), signaling triggered by downstream members (PIK3CA, PTEN, SRC, mTOR), altered expression of cell cycle and apoptotic regulators (CDKs, p27(kip1), Bcl-2), hormone receptor status, resistance to antibody-dependent cellular cytotoxicity (Fc?R), and altered miRNA expression signatures. Multigenic molecular profile analyses have revealed further genes not directly associated with classical oncogenic pathways. Although numerous biomarkers have shown promise in pre-clinical studies, many have delivered controversial results when evaluated in clinical trials. One of the keys for targeting ERBB2 will be to consider the entire ERBB family and downstream associated pathways responsible for the malignant transformation. The heterogeneity of the disease is likely to represent a significant obstacle to accurately predicting the course of resistance. The future most probably involves the incorporation of multiple biomarkers into a unified predictor enabling selection of patients for superior targeted drug administration. PMID:26452383

  3. Over-expression of muscle glycogen synthase in human diabetic nephropathy.

    PubMed

    Gatica, Rodrigo; Bertinat, Romina; Silva, Pamela; Kairath, Pamela; Slebe, Felipe; Pardo, Fabián; Ramírez, María J; Slebe, Juan C; Campistol, José M; Nualart, Francisco; Caelles, Carme; Yáñez, Alejandro J

    2015-03-01

    Diabetic nephropathy (DN) is a major complication of diabetic patients and the leading cause of end-stage renal disease. Glomerular dysfunction plays a critical role in DN, but deterioration of renal function also correlates with tubular alterations. Human DN is characterized by glycogen accumulation in tubules. Although this pathological feature has long been recognized, little information exists about the triggering mechanism. In this study, we detected over-expression of muscle glycogen synthase (MGS) in diabetic human kidney. This enhanced expression suggests the participation of MGS in renal metabolic changes associated with diabetes. HK2 human renal cell line exhibited an intrinsic ability to synthesize glycogen, which was enhanced after over-expression of protein targeting to glycogen. A correlation between increased glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogen-induced damage during long-term diabetes. PMID:25371328

  4. Overexpression of p53 protein in basal cell carcinomas of human skin.

    PubMed Central

    Shea, C. R.; McNutt, N. S.; Volkenandt, M.; Lugo, J.; Prioleau, P. G.; Albino, A. P.

    1992-01-01

    Basal cell carcinoma (BCC) of the skin is the most common human cancer, but its molecular-genetic pathogenesis is unclear. In many other types of cancer, mutations of the tumor-suppressor gene p53 occur frequently and may lead to overexpression of a long-lived mutant form of p53 protein. In this study, overexpression of p53 protein was detected immunohistochemically in 30 (83%) of 36 specimens of BCC of the head and neck. The same regions of tumor typically were reactive both with a monoclonal antibody (PAb240) specific for the mutant protein and with one (PAb1801) directed against an epitope common to both wild-type and mutant p53 protein. Keratinocytes of chronically sun-exposed epidermis adjacent to BCCs also focally overexpressed p53 protein in the majority of cases, whereas those of sun-protected buttock skin did not. Mutation of p53 may form an important part of the pathogenetic sequence in a majority of cases of BCC. Images Figure 1 Figure 2 Figure 3 PMID:1632467

  5. Overexpression of protein disulfide isomerases enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe.

    PubMed

    Mukaiyama, Hiroyuki; Tohda, Hideki; Takegawa, Kaoru

    2010-04-01

    Although the fission yeast Schizosaccharomyces pombe has been used for high-level heterologous protein production, the productivity of secreted human serum transferrin (hTF) has been low, presumably, because the protein harbors twenty disulfide bonds and two N-glycosylation sites. In the present study, we found that overexpression of endogenous putative protein disulfide isomerase (PDI) improved productivity. Whole genome sequence analysis of S. pombe revealed five putative PDI genes and overexpression of two of them, SPAC17H9.14c and SPBC3D6.13c (SpPdi2p or SpPdi3p, respectively), significantly improved the productivity of secreted hTF. GFP-fused SpPdi2p and SpPdi3p were found to localize to the endoplasmic reticulum. Co-overexpression of SpPdi2p or SpPdi3p with hTF coupled with modifications to the growth medium reported in our previous study were able to increase the level of secreted hTF approximately 30-fold relative to conventional conditions. PMID:20013338

  6. Overexpression of human ?-defensin 2 promotes growth and invasion during esophageal carcinogenesis

    PubMed Central

    Shi, Ni; Jin, Feng; Zhang, Xiaoli; Clinton, Steven K.; Pan, Zui; Chen, Tong

    2014-01-01

    Human ?-defensin 2 (HBD-2) is an antimicrobial peptide produced by mucosal surfaces in response to microbial exposure or inflammatory cytokines. Although HBD-2 is expressed in the esophagus in response to stress and infectious agents, little is known regarding its expression and functional role in esophageal carcinogenesis. In the current investigation, normal esophagus and N-nitrosomethylbenzylamine (NMBA)-induced precancerous and papillomatous lesions of the rat esophagus were characterized for HBD-2 encoding gene Defb4 and protein. HBD-2 was found to be overexpressed in esophagi of rats treated with NMBA compared to animals in control group. Results of Real-time PCR, Western blot and immunohistochemistry demonstrated a positive correlation between the overexpression of HBD-2 and the progression of rat squamous cell carcinogenesis (SCC) in the esophagus. We also observed that HBD-2 is overexpressed in tumor tissues removed from patients with esophageal SCC. Moreover, Defb4 silencing in vitro suppresses the tumor cell proliferation, mobility and invasion in esophageal SCC cell line KYSE-150. The results from this study provide experimental evidence that HBD-2 may play an oncogenic role in the initiation and progression of esophageal SCC and thus serves as a target for chemopreventive and therapeutic interventions. PMID:25226614

  7. COOPERATIVITY OF THE MUC1 ONCOPROTEIN AND STAT1 PATHWAY IN POOR PROGNOSIS HUMAN BREAST CANCER

    PubMed Central

    Khodarev, Nikolai; Ahmad, Rehan; Rajabi, Hasan; Pitroda, Sean; Kufe, Turner; McClary, Cain; Joshi, Maya Datt; MacDermed, Dhara; Weichselbaum, Ralph; Kufe, Donald

    2009-01-01

    Signal transducer and activator of transcription 1 (STAT1) is activated in the inflammatory response to interferons. The MUC1 oncoprotein is overexpressed in human breast cancers. Analysis of genes differentially expressed in MUC1-transformed cells has identified a network linking MUC1 and STAT1 that is associated with cellular growth and inflammation. The results further demonstrate that the MUC1-C subunit associates with STAT1 in cells and that the MUC1-C cytoplasmic domain binds directly to the STAT1 DNA binding domain. The interaction between MUC1-C and STAT1 is inducible by IFN? in non-malignant epithelial cells and constitutive in breast cancer cells. Moreover, the MUC1-STAT1 interaction contributes to the activation of STAT1 target genes, including MUC1 itself. Analysis of two independent databases demonstrated that MUC1 and STAT1 are coexpressed in about 15% of primary human breast tumors. Coexpression of MUC1 and the STAT1 pathway was found to be significantly associated with decreased recurrence-free and overall survival. These findings indicate that (i) MUC1 and STAT1 function in an auto-inductive loop, and (ii) activation of both MUC1 and the STAT1 pathway in breast tumors confers a poor prognosis for patients. PMID:19915608

  8. Cooperativity of the MUC1 oncoprotein and STAT1 pathway in poor prognosis human breast cancer.

    PubMed

    Khodarev, N; Ahmad, R; Rajabi, H; Pitroda, S; Kufe, T; McClary, C; Joshi, M D; MacDermed, D; Weichselbaum, R; Kufe, D

    2010-02-11

    Signal transducer and activator of transcription 1 (STAT1) is activated in the inflammatory response to interferons. The MUC1 oncoprotein is overexpressed in human breast cancers. Analysis of genes differentially expressed in MUC1-transformed cells has identified a network linking MUC1 and STAT1 that is associated with cellular growth and inflammation. The results further show that the MUC1-C subunit associates with STAT1 in cells and the MUC1-C cytoplasmic domain binds directly to the STAT1 DNA-binding domain. The interaction between MUC1-C and STAT1 is inducible by IFNgamma in non-malignant epithelial cells and constitutive in breast cancer cells. Moreover, the MUC1-STAT1 interaction contributes to the activation of STAT1 target genes, including MUC1 itself. Analysis of two independent databases showed that MUC1 and STAT1 are coexpressed in about 15% of primary human breast tumors. Coexpression of MUC1 and the STAT1 pathway was found to be significantly associated with decreased recurrence-free and overall survival. These findings indicate that (i) MUC1 and STAT1 function in an auto-inductive loop, and (ii) activation of both MUC1 and the STAT1 pathway in breast tumors confers a poor prognosis for patients. PMID:19915608

  9. Targeting uPAR with Antagonistic Recombinant Human Antibodies in Aggressive Breast Cancer

    PubMed Central

    LeBeau, Aaron M.; Duriseti, Sai; Murphy, Stephanie T.; Pepin, Francois; Hann, Byron; Gray, Joe W.; VanBrocklin, Henry F.; Craik, Charles S.

    2013-01-01

    Components of the plasminogen activation system (PAS) which are overexpressed in aggressive breast cancer subtypes offer appealing targets for development of new diagnostics and therapeutics. By comparing gene expression data in patient populations and cultured cell lines, we identified elevated levels of the urokinase plasminogen activation receptor (uPAR, PLAUR) in highly aggressive breast cancer subtypes and cell lines. Recombinant human anti-uPAR antagonistic antibodies exhibited potent binding in vitro to the surface of cancer cells expressing uPAR. In vivo these antibodies detected uPAR expression in triple negative breast cancer (TNBC) tumor xenografts using near infrared (NIR) imaging and 111In single-photon emission computed tomography (SPECT). Antibody-based uPAR imaging probes accurately detected small disseminated lesions in a tumor metastasis model, complementing the current clinical imaging standard 18F-fluorodeoxyglucose (FDG) at detecting non-glucose-avid metastatic lesions. A monotherapy study using the antagonistic antibodies resulted in a significant decrease in tumor growth in a TNBC xenograft model. Additionally, a radioimmunotherapy (RIT) study, using the anti-uPAR antibodies conjugated to the therapeutic radioisotope 177Lu, found that they were effective at reducing tumor burden in vivo. Taken together, our results offer a preclinical proof of concept for uPAR targeting as a strategy for breast cancer diagnosis and therapy using this novel human antibody technology. PMID:23400595

  10. Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

    PubMed

    Tsai, Steven C; Chang, David F; Hong, Chang-Mu; Xia, Ping; Senadheera, Dinithi; Trump, Lisa; Mishra, Suparna; Lutzko, Carolyn

    2014-06-15

    Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC. PMID:24627557

  11. c-Myc dependent expression of pro-apoptotic Bim renders HER2-overexpressing breast cancer cells dependent on anti-apoptotic Mcl-1

    PubMed Central

    2011-01-01

    Background Anti-apoptotic signals induced downstream of HER2 are known to contribute to the resistance to current treatments of breast cancer cells that overexpress this member of the EGFR family. Whether or not some of these signals are also involved in tumor maintenance by counteracting constitutive death signals is much less understood. To address this, we investigated what role anti- and pro-apoptotic Bcl-2 family members, key regulators of cancer cell survival, might play in the viability of HER2 overexpressing breast cancer cells. Methods We used cell lines as an in vitro model of HER2-overexpressing cells in order to evaluate how anti-apoptotic Bcl-2, Bcl-xL and Mcl-1, and pro-apoptotic Puma and Bim impact on their survival, and to investigate how the constitutive expression of these proteins is regulated. Expression of the proteins of interest was confirmed using lysates from HER2-overexpressing tumors and through analysis of publicly available RNA expression data. Results We show that the depletion of Mcl-1 is sufficient to induce apoptosis in HER2-overexpressing breast cancer cells. This Mcl-1 dependence is due to Bim expression and it directly results from oncogenic signaling, as depletion of the oncoprotein c-Myc, which occupies regions of the Bim promoter as evaluated in ChIP assays, decreases Bim levels and mitigates Mcl-1 dependence. Consistently, a reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion. Western blot analysis confirms that naïve HER2-overexpressing tumors constitutively express detectable levels of Mcl-1 and Bim, while expression data hint on enrichment for Mcl-1 transcripts in these tumors. Conclusions This work establishes that, in HER2-overexpressing tumors, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of cancer cell death. PMID:21899728

  12. In vitro comparative models for canine and human breast cancers

    PubMed Central

    VISAN, SIMONA; BALACESCU, OVIDIU; BERINDAN-NEAGOE, IOANA; CATOI, CORNEL

    2016-01-01

    During the past four decades, an increased number of similarities between canine mammary tumors and human breast cancer have been reported: molecular, histological, morphological, clinical and epidemiological, which lead to comparative oncological studies. One of the most important goals in human and veterinary oncology is to discover potential molecular biomarkers that could detect breast cancer in an early stage and to develop new effective therapies. Recently, cancer cell lines have successfully been used as an in vitro model to study the biology of cancer, to investigate molecular pathways and to test the efficiency of anticancer drugs. Moreover, establishment of an experimental animal model for the study of human breast cancer will improve testing potential anti-cancer therapies and the discovery of effective therapeutic schemes suitable for human clinical trials. In this review, we collected data from previous studies that strengthen the value of canine mammary cancer cell lines as an in vitro model for the study of human breast cancer. PMID:27004024

  13. Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

    SciTech Connect

    Ghezali, Lamia; Leger, David Yannick; Limami, Youness; Cook-Moreau, Jeanne; Beneytout, Jean-Louis; Liagre, Bertrand

    2013-04-15

    Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression.

  14. Atypical Scrapie Prions from Sheep and Lack of Disease in Transgenic Mice Overexpressing Human Prion Protein

    PubMed Central

    Joiner, Susan; Linehan, Jacqueline M.; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M.; Griffiths, Peter C.; Groschup, Martin H.; Hope, James; Brandner, Sebastian; Asante, Emmanuel A.; Collinge, John

    2013-01-01

    Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants. PMID:24188521

  15. Overexpression of copper zinc superoxide dismutase suppresses human glioma cell growth.

    PubMed

    Zhang, Ying; Zhao, Weiling; Zhang, Hannah J; Domann, Frederick E; Oberley, Larry W

    2002-02-15

    Copper zinc superoxide dismutase (CuZnSOD) is an essential primary antioxidant enzyme that converts superoxide radical to hydrogen peroxide and molecular oxygen in the cytoplasm. Cytosolic glutathione peroxidase (GPx) converts hydrogen peroxide into water. The overall goal of the present study was to explore the possible role of the antioxidant enzyme CuZnSOD in expression of the malignant phenotype. We hypothesized that overexpression of CuZnSOD would lead to the suppression of at least part of the human malignant phenotype. To test this hypothesis, human CuZnSOD cDNA was transfected into U118-9 human malignant glioma cells. CuZnSOD activity levels increased 1.5-, 2.0-, 2.6-, and 3.5-fold, respectively, in four table transfected cell lines compared with wild type and vector controls. Overexpression of CuZnSOD altered cellular antioxidant enzyme profiles, including those of manganese superoxide dismutase, catalase, and GPx. The transfected clone with the highest CuZnSOD:GPx ratio (3.5) showed a 42% inhibition of tumor cell growth in vitro. The decreased rate of tumor cell growth in vitro was strongly correlated with the enzyme activity ratio of CuZnSOD:GPx. Glioma cells that stably overexpressed CuZnSOD demonstrated additional suppressive effects on the malignant phenotype when compared with the parental cells and vector controls. These cells showed decreased plating efficiency, elongated cell population doubling time, lower clonogenic fraction in soft agar, and, more significantly, inhibition of tumor formation in nude mice. This work suggested that CuZnSOD is a new tumor suppressor gene. Increased intracellular ROS levels were found in cells with high activity ratios of CuZnSOD:GPx. Change in the cellular redox status, especially change attributable to the accumulation of hydrogen peroxide or other hydroperoxides, is a possible reason to explain the suppression of tumor growth observed in CuZnSOD-overexpressing cells. PMID:11861405

  16. Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

    PubMed Central

    Ziegler, Yvonne S.; Moresco, James J.; Tu, Patricia G.; Yates, John R.; Nardulli, Ann M.

    2014-01-01

    The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease. PMID:25029196

  17. Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

    SciTech Connect

    Tsukahara, Tamotsu; Haniu, Hisao

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer We examined the correlation between PPAR{gamma} expression and cell proliferation. Black-Right-Pointing-Pointer PPAR{gamma} overexpression reduces cell viability. Black-Right-Pointing-Pointer We show the synergistic effect of cell growth inhibition by a PPAR{gamma} agonist. -- Abstract: Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPAR{gamma} is expressed at considerable levels in human colon cancer cells. This suggests that PPAR{gamma} expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPAR{gamma} expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPAR{gamma} mRNA and protein in these cells were in the order HT-29 > LOVO > Caco-2 > DLD-1. We also found that PPAR{gamma} overexpression promoted cell growth inhibition in PPAR{gamma} lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPAR{gamma} expression and the cells' sensitivity for proliferation.

  18. Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase.

    PubMed

    Jeong, Yeon Woo; Lee, Geun-Shik; Kim, Joung Joo; Park, Sun Woo; Ko, Kyeong Hee; Kang, Mina; Kim, Yu Kyung; Jung, Eui-Man; Hyun, Sang Hwan; Shin, Taeyoung; Jeung, Eui-Bae; Hwang, Woo Suk

    2012-08-01

    Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus. PMID:22580743

  19. Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase.

    PubMed Central

    Gmez-Fabre, P M; Aledo, J C; Del Castillo-Olivares, A; Alonso, F J; Nez De Castro, I; Campos, J A; Mrquez, J

    2000-01-01

    Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of lambdagt10 cDNA library screening and the rapid amplification of cDNA ends ('RACE') technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver. PMID:10620514

  20. Overexpression of Human and Fly Frataxins in Drosophila Provokes Deleterious Effects at Biochemical, Physiological and Developmental Levels

    PubMed Central

    Soriano, Sirena; Botella, Jos A.; Schneuwly, Stephan; Martnez-Sebastin, Mara J.; Molt, Mara D.

    2011-01-01

    Background Friedreich's ataxia (FA), the most frequent form of inherited ataxias in the Caucasian population, is caused by a reduced expression of frataxin, a highly conserved protein. Model organisms have contributed greatly in the efforts to decipher the function of frataxin; however, the precise function of this protein remains elusive. Overexpression studies are a useful approach to investigate the mechanistic actions of frataxin; however, the existing literature reports contradictory results. To further investigate the effect of frataxin overexpression, we analyzed the consequences of overexpressing human (FXN) and fly (FH) frataxins in Drosophila. Methodology/Principal Findings We obtained transgenic flies that overexpressed human or fly frataxins in a general pattern and in different tissues using the UAS-GAL4 system. For both frataxins, we observed deleterious effects at the biochemical, histological and behavioral levels. Oxidative stress is a relevant factor in the frataxin overexpression phenotypes. Systemic frataxin overexpression reduces Drosophila viability and impairs the normal embryonic development of muscle and the peripheral nervous system. A reduction in the level of aconitase activity and a decrease in the level of NDUF3 were also observed in the transgenic flies that overexpressed frataxin. Frataxin overexpression in the nervous system reduces life span, impairs locomotor ability and causes brain degeneration. Frataxin aggregation and a misfolding of this protein have been shown not to be the mechanism that is responsible for the phenotypes that have been observed. Nevertheless, the expression of human frataxin rescues the aconitase activity in the fh knockdown mutant. Conclusion/Significance Our results provide in vivo evidence of a functional equivalence for human and fly frataxins and indicate that the control of frataxin expression is important for treatments that aim to increase frataxin levels. PMID:21779322

  1. A novel potent tumour promoter aberrantly overexpressed in most human cancers

    PubMed Central

    Takahashi, Atsushi; Tokita, Hisashi; Takahashi, Kenzo; Takeoka, Tomoharu; Murayama, Kosho; Tomotsune, Daihachiro; Ohira, Miki; Iwamatsu, Akihiro; Ohara, Kazuaki; Yazaki, Kazufumi; Koda, Tadayuki; Nakagawara, Akira; Tani, Kenzaburo

    2011-01-01

    The complexity and heterogeneity of tumours have hindered efforts to identify commonalities among different cancers. Furthermore, because we have limited information on the prevalence and nature of ubiquitous molecular events that occur in neoplasms, it is unfeasible to implement molecular-targeted cancer screening and prevention. Here, we found that the FEAT protein is overexpressed in most human cancers, but weakly expressed in normal tissues including the testis, brain, and liver. Transgenic mice that ectopically expressed FEAT in the thymus, spleen, liver, and lung spontaneously developed invasive malignant lymphoma (48%, 19/40) and lung-metastasizing liver cancer (hepatocellular carcinoma) (35%, 14/40) that models human hepatocarcinogenesis, indicating the FEAT protein potently drives tumorigenesis in vivo. Gene expression profiling suggested that FEAT drives receptor tyrosine kinase and hedgehog signalling pathways. These findings demonstrate that integrated efforts to identify FEAT-like ubiquitous oncoproteins are useful and may provide promising approaches for cost-effective cancer screening and prevention. PMID:22355534

  2. Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

    PubMed Central

    GAO, SUJIE; CHEN, XUEBO; JIN, HONGYONG; REN, SHENGNAN; LIU, ZHUO; FANG, XUEDONG; ZHANG, GUIZHEN

    2016-01-01

    ErbB2 is known to upregulate glycolysis in breast cancer, however, the precise mechanisms remain unclear. In the present study, ErbB2 upregulated Hexokinase II (HK II) activity by increasing the binding of HK II to the mitochondrial outer membrane. Dysregulated glucose metabolism in high ErbB2-expressing breast cancer cells induces susceptibility to glucose starvation and glycolysis inhibition. Additionally, HK II has a tendency to dissociate from the mitochondria outer membrane in ErbB2-overexpressing cells following treatment with the HK II inhibitor, 3-BrPA. Furthermore, 3-BrPA treatment results in decreased mitochondria membrane potential and release of cytochrome c into cytoplasm in ErbB2-overexpressing cells, leading to activation of the mitochondrial apoptotic signaling pathway. In summary, the results demonstrate a novel mechanism for ErbB2-activated glycolysis and reveal that 3-BrPA is effective in reducing ErbB2-positive breast cancer cell viability by targeting HK II in vitro and in vivo. PMID:26893781

  3. Human Umbilical Cord Matrix Mesenchymal Stem Cells Suppress the Growth of Breast Cancer by Expression of Tumor Suppressor Genes

    PubMed Central

    Ohta, Naomi; Ishiguro, Susumu; Kawabata, Atsushi; Uppalapati, Deepthi; Pyle, Marla; Troyer, Deryl; De, Supriyo; Zhang, Yongqing; Becker, Kevin G.; Tamura, Masaaki

    2015-01-01

    Human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analyses of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Their different tumoricidal abilities were clarified by analyzing gene expression profiles in the two types of UCMSC. Microarray analysis revealed differential gene expression between untreated nave UCMSC and those co-cultured with species-matched breast carcinoma cells. The analyses screened 17 differentially expressed genes that are commonly detected in both human and rat UCMSC. The comparison between the two sets of gene expression profiles identified two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), that were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species breast carcinoma cells. Over-expression of FST, but not ADRP, in human UCMSC enhanced their ability to suppress the growth of MDA-231 cells. The growth of MDA-231 cells was also significantly lower when they were cultured in medium conditioned with FST, but not ADRP over-expressing human UCMSC. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-over-expressing human UCMSC significantly attenuated the tumor burden. These results suggest that FST may play an important role in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and also implies that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression. PMID:25942583

  4. ERBB4 is over-expressed in human colon cancer and enhances cellular transformation.

    PubMed

    Williams, Christopher S; Bernard, Jessica K; Demory Beckler, Michelle; Almohazey, Dana; Washington, Mary Kay; Smith, Jesse J; Frey, Mark R

    2015-07-01

    The ERBB4 receptor tyrosine kinase promotes colonocyte survival. Herein, we tested whether ERBB4's antiapoptotic signaling promotes transformation and colorectal tumorigenesis. ERBB4 alterations in a The Cancer Genome Atlas colorectal cancer (CRC) data set stratified survival, and in a combined Moffitt Cancer Center and Vanderbilt Medical Center CRC expression data set, ERBB4 message levels were increased at all tumor stages. Similarly, western blot and immunohistochemistry on additional CRC tissue banks showed elevated ERBB4 protein in tumors. ERBB4 was highly expressed in aggressive, dedifferentiated CRC cell lines, and its knockdown in LIM2405 cells reduced anchorage-independent colony formation. In nude mouse xenograft studies, ERBB4 alone was insufficient to induce tumor establishment of non-transformed mouse colonocytes, but its over-expression in cells harboring Apc(min) and v-Ha-Ras caused a doubling of tumor size. ERBB4-expressing xenografts displayed increased activation of survival pathways, including epidermal growth factor receptor and Akt phosphorylation and COX-2 expression, and decreased apoptotic signals. Finally, ERBB4 deletion from mouse intestinal epithelium impaired stem cell replication and in vitro enteroid establishment. In summary, we report that ERBB4 is over-expressed in human CRC, and in experimental systems enhances the survival and growth of cells driven by Ras and/or WNT signaling. Chronic ERBB4 over-expression in the context of, for example, inflammation may contribute to colorectal carcinogenesis. Tumors with high receptor levels are likely to have enhanced cell survival signaling through epidermal growth factor receptor, PI3K and COX-2. These results suggest ERBB4 as a novel therapeutic target in a subset of CRC. PMID:25916654

  5. Bovine Leukemia Virus DNA in Human Breast Tissue

    PubMed Central

    Shen, Hua Min; Jensen, Hanne M.; Choi, K. Yeon; Sun, Dejun; Nuovo, Gerard

    2014-01-01

    Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease. PMID:24750974

  6. Expression and prognostic role of SKIP in human breast carcinoma.

    PubMed

    Liu, Xiaobing; Ni, Qichao; Xu, Junfei; Sheng, Chenyi; Wang, Qingqing; Chen, Jinpeng; Yang, Shuyun; Wang, Hua

    2014-04-01

    Ski-interacting protein (SKIP) is a nuclear hormone receptor-interacting cofactor, interactions with the proto-oncogene Ski, appears to modulate a number of signalling pathways involved in control of cell proliferation and differentiation, and may play a critical role in oncogenesis. In the present study, to investigate the potential roles of SKIP in breast cancer, expression patterns, interaction and the correlation with clinical/prognostic factors of SKIP and Ki-67 were examined among patients with breast cancer. Immunohistochemistry and Western blot analysis were performed for SKIP in 85 breast carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance. We found that SKIP was over expressed in breast carcinoma as compared with the adjacent normal tissues. High expression of SKIP was positively associated with histological grade (P = 0.01) and Ki-67 (P = 0.004). Univariate analysis showed that SKIP expression was associated with a poor prognosis (P = 0.006). While in vitro, following release of breast cancer cell lines from serum starvation, the expression of SKIP was up-regulated, whereas p27 was down-regulated. In addition, we employed small interfering RNA (siRNA) technique to knock down SKIP expression and observed it effects on MDA-MB-231 cells growth. SKIP depletion by siRNA inhibited cell proliferation, blocked S phase and decreased cyclin A and cyclin B levels. On the basis of these results, we suggested that SKIP overexpression was involved in the pathogenesis of breast cancer, which might serve as a future target for breast cancer. PMID:24150787

  7. Clinical impact of human breast milk metabolomics.

    PubMed

    Cesare Marincola, Flaminia; Dessì, Angelica; Corbu, Sara; Reali, Alessandra; Fanos, Vassilios

    2015-12-01

    Metabolomics is a research field concerned with the analysis of metabolome, the complete set of metabolites in a given cell, tissue, or biological sample. Being able to provide a molecular snapshot of biological systems, metabolomics has emerged as a functional methodology in a wide range of research areas such as toxicology, pharmacology, food technology, nutrition, microbial biotechnology, systems biology, and plant biotechnology. In this review, we emphasize the applications of metabolomics in investigating the human breast milk (HBM) metabolome. HBM is the recommended source of nutrition for infants since it contains the optimal balance of nutrients for developing babies, and it provides a range of benefits for growth, immunity, and development. The molecular mechanisms beyond the inter- and intra-variability of HBM that make its composition unique are yet to be well-characterized. Although still in its infancy, the study of HBM metabolome has already proven itself to be of great value in providing insights into this biochemical variability in relation to mother phenotype, diet, disease, and lifestyle. The results of these investigations lay the foundation for further developments useful to identify normal and aberrant biochemical changes as well as to develop strategies to promote healthy infant feeding practices. PMID:25689794

  8. Cysteine-rich 61-connective tissue growth factor-nephroblastoma-overexpressed 5 (CCN5)/Wnt-1-induced signaling protein-2 (WISP-2) regulates microRNA-10b via hypoxia-inducible factor-1?-TWIST signaling networks in human breast cancer cells.

    PubMed

    Haque, Inamul; Banerjee, Snigdha; Mehta, Smita; De, Archana; Majumder, Monami; Mayo, Matthew S; Kambhampati, Suman; Campbell, Donald R; Banerjee, Sushanta K

    2011-12-16

    MicroRNAs (miRNAs) are naturally occurring single-stranded RNA molecules that post-transcriptionally regulate the expression of target mRNA transcripts. Many of these target mRNA transcripts are involved in regulating processes commonly altered during tumorigenesis and metastatic growth. These include cell proliferation, differentiation, apoptosis, migration, and invasion. Among the several miRNAs, miRNA-10b (miR-10b) expression is increased in metastatic breast cancer cells and positively regulates cell migration and invasion through the suppression of the homeobox D10 (HOXD10) tumor suppressor signaling pathway. In breast metastatic cells, miR-10b expression is enhanced by a transcription factor TWIST1. We find that miR-10b expression in breast cancer cells can be suppressed by CCN5, and this CCN5 effect is mediated through the inhibition of TWIST1 expression. Moreover, CCN5-induced inhibition of TWIST1 expression is mediated through the translational inhibition/modification of hypoxia-inducible factor-1? via impeding JNK signaling pathway. Collectively, these studies suggest a novel regulatory pathway exists through which CCN5 exerts its anti-invasive function. On the basis of these findings, it is plausible that reactivation of CCN5 in miR-10b-positive invasive/metastatic breast cancers alone or in combination with current therapeutic regimens could provide a unique, alternative strategy to existing breast cancer therapy. PMID:22020939

  9. CIP2A is overexpressed in human ovarian cancer and regulates cell proliferation and apoptosis.

    PubMed

    Fang, Yuanyuan; Li, Zhengtao; Wang, Xiuxia; Zhang, Shulan

    2012-12-01

    CIP2A is a recently characterized oncoprotein which involves in the progression of several human malignancies. This study aimed to investigate its clinical significance and biological function in ovarian cancer. CIP2A expression was analyzed in 152 archived ovarian cancer specimens using immunohistochemistry. One hundred cases (65.79 %) showed CIP2A overexpression, including 63 of 92 serous carcinomas (68.48 %), 21 of 33 endometrioid carcinomas (63.64 %), 12 of 23 mucinous carcinomas (52.17 %), and 4 of 4 clear cell carcinomas (100 %). There is no significant difference of CIP2A expression between serous tumors and all other morphologies combined. CIP2A overexpression positively correlated with advanced FIGO stage (p = 0.0336) and tumor grade (p = 0.0213). siRNA knockdown was performed in A2780 and SKOV3 cell lines. MTT, colony formation assay, and flow cytometry were carried out to assess the role of CIP2A in proliferation, cell cycle, and apoptosis. CIP2A depletion in ovarian cancer cell lines inhibited proliferation, blocked cell cycle progression, and increased paclitaxel-induced apoptosis. Furthermore, CIP2A depletion downregulated cyclin D1, c-myc, phospho-Rb, Bcl-2, and phospho-AKT expression. These results validate the role of CIP2A as a clinically relevant oncoprotein and establish CIP2A as a promising therapeutic target of ovarian cancer. PMID:22923389

  10. Genetically modified human keratinocytes overexpressing PDGF-A enhance the performance of a composite skin graft.

    PubMed

    Eming, S A; Medalie, D A; Tompkins, R G; Yarmush, M L; Morgan, J R

    1998-03-01

    Skin loss due to burns and ulcers is a major medical problem. Bioengineered skin substitutes that use cultured keratinocytes as an epidermal layer with or without analogues of the dermis are one strategy for skin repair. However, none can achieve definitive wound closure, function, or cosmesis comparable to split-thickness autografts. Moreover, autograft donor sites, which require time to heal, may be limited or have attendant problems such as infection or functional/cosmetic deficiencies. To determine if the performance of composite skin grafts of keratinocytes on a dermal analogue could be enhanced, human keratinocytes were genetically modified to overexpress platelet-derived growth factor A chain (PDGF-A). Composite grafts of modified keratinocytes seeded onto acellular dermis, prepared from cryopreserved cadaver skin, secreted PDGF-AA protein in vitro [90 ng/graft (1.5 x 1.5 cm)/24 hr]. To test their performance in a wound healing model, composite grafts were transplanted to full-thickness excisional wounds on the back of athymic mice. PDGF-A grafts formed a stratified differentiated epidermis similar to control grafts. The acellular dermis was repopulated with host fibrovascular cells and by day 7, the PDGF-A grafts had significantly more cells in the dermis and increased staining for murine collagen types I and IV. At this early time point, wound contraction was also significantly inhibited in PDGF-A grafts versus control grafts. Thus, PDGF-A overexpression improves graft performance during the first critical week after transplantation. PMID:9525314

  11. Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents.

    PubMed Central

    Kaina, B; Lohrer, H; Karin, M; Herrlich, P

    1990-01-01

    Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions. Images PMID:2320583

  12. Overexpression of Wip1 Is Associated with Biologic Behavior in Human Clear Cell Renal Cell Carcinoma

    PubMed Central

    Liu, Sulai; Qi, Lin; Han, Weqing; Wan, Xinxing; Jiang, Shusuan; Li, Yuan; Xie, Yu; Liu, Longfei; Zeng, Fuhua; Liu, Zhizhong; Zu, Xiongbing

    2014-01-01

    Wild-type p53-induced phosphatase (Wip1 or PPM1D) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of Wip1 in RCC remains unclear. The present study investigated its abnormal expression and dysfunctions in clear cell renal cell carcinoma (ccRCC) in vitro. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of Wip1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of Wip1 was significantly associated with depth of invasion (P<0.001), Distant metastasis (P?=?0.001), lymph node status (P<0.001) and Fuhrman grade (P<0.001). Wip1 knockdown inhibited the proliferation, migration and invasion of 786-O and RLC-310 cells, whereas Wip1 overexpression promoted the growth and aggressive phenotype of 786-O and RLC-310 cells in vitro. The uni- and multivariate analyses indicated that expression of Wip1 was an independent predictor for survival of ccRCC patients (P?=?0.003, P?=?0.027 respectively). Wip1- negative patients had a higher tumor-free/overall survival rate than patients with high Wip1 expression (P?=?0.001, P?=?0.002 respectively). Overexpression of Wip1 is useful in the prediction of survival in ccRCC patients.

  13. Overexpression of Recombinant Human Beta Interferon (rhINF-β) in Periplasmic Space of Escherichia coli

    PubMed Central

    Morowvat, Mohammad Hossein; Babaeipour, Valiollah; Rajabi-Memari, Hamid; Vahidi, Hossein; Maghsoudi, Nader

    2014-01-01

    Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon (rhINF-β) was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 (DE3). The sugar, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rhINF-β production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-β protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD600nm = 3.42 was found to be 35 % of the total protein content of the strain which translates to 0.32 g L-1. The constructed vector could efficiently overexpress the rhINF-β into the periplasmic space of E. coli. The obtained yield of the produced rhINF-β was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins. PMID:24711841

  14. Fragmentation of the Golgi apparatus induced by the overexpression of wild-type and mutant human tau forms in neurons.

    PubMed

    Liazoghli, Dalinda; Perreault, Sebastien; Micheva, Kristina D; Desjardins, Mylne; Leclerc, Nicole

    2005-05-01

    Tau is a microtubule-associated protein enriched in the axonal compartment. In several neurodegenerative diseases including Alzheimer's disease, hyperphosphorylated tau accumulates in the somatodendritic compartment, self-aggregates, and forms neurofibrillary tangles. A fragmentation of the neuronal Golgi apparatus (GA) was also observed in Alzheimer's disease. In the present study, we examined the effect of overexpressing human tau on the organization of the neuronal GA in rat hippocampal cultures and in JNPL3 mice expressing tau mutant P301L. GA fragmentation was noted in a significantly higher percentage of hippocampal neurons overexpressing wild-type human tau than in control neurons over-expressing green fluorescent protein (GFP) alone. Most importantly, in neurons overexpressing mutant forms of human tau (P301L, V337M, or R406W), the percentage of neurons with a fragmented GA was 10% higher than that of neurons overexpressing wild-type human tau. In JNPL3 mice, a significantly higher percentage of motor neurons presented a fragmented GA compared to control mice. Interestingly, fragmentation of the GA was more frequent in neurons containing an accumulation and aggregation of hyperphosphorylated tau in the cell body than in neurons without these features. In both primary hippocampal neurons and JNPL3 mice, the tau-induced GA fragmentation was not caused by apoptosis. The pre-sent results implicate tau in GA fragmentation and show that this event occurs before the formation of neurofibrillary tangles. PMID:15855649

  15. Fragmentation of the Golgi Apparatus Induced by the Overexpression of Wild-Type and Mutant Human Tau Forms in Neurons

    PubMed Central

    Liazoghli, Dalinda; Perreault, Sebastien; Micheva, Kristina D.; Desjardins, Mylène; Leclerc, Nicole

    2005-01-01

    Tau is a microtubule-associated protein enriched in the axonal compartment. In several neurodegene-rative diseases including Alzheimer’s disease, hyperphosphorylated tau accumulates in the somatodendritic compartment, self-aggregates, and forms neurofibrillary tangles. A fragmentation of the neuronal Golgi apparatus (GA) was also observed in Alzheimer’s disease. In the present study, we examined the effect of overexpressing human tau on the organization of the neuronal GA in rat hippocampal cultures and in JNPL3 mice expressing tau mutant P301L. GA fragmentation was noted in a significantly higher percentage of hippocampal neurons overexpressing wild-type human tau than in control neurons over-expressing green fluorescent protein (GFP) alone. Most importantly, in neurons overexpressing mutant forms of human tau (P301L, V337M, or R406W), the percentage of neurons with a fragmented GA was 10% higher than that of neurons overexpressing wild-type human tau. In JNPL3 mice, a significantly higher percentage of motor neurons presented a fragmented GA compared to control mice. Interestingly, fragmentation of the GA was more frequent in neurons containing an accumulation and aggregation of hyperphosphorylated tau in the cell body than in neurons without these features. In both primary hippocampal neurons and JNPL3 mice, the tau-induced GA fragmentation was not caused by apoptosis. The pre-sent results implicate tau in GA fragmentation and show that this event occurs before the formation of neurofibrillary tangles. PMID:15855649

  16. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

    SciTech Connect

    Watanabe, H.; Grubb, J.H.; Sly, W.S. )

    1990-10-01

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.

  17. Correlation of CD44 expression with proliferative activity of normal human breast epithelial cells in culture.

    PubMed

    Cooper, N L; Bardy, P; Bacani, J; Kuusk, U; Dougherty, G J; Eaves, C J; Emerman, J T

    1998-07-01

    A number of studies have shown that certain variant isoforms of CD44 are overexpressed in human breast cancer, suggesting their use as indicators of the presence of malignant cells. We now show that CD44 isoform mRNA and protein expression is upregulated in normal human breast epithelial cells (HBEC) when these cells are stimulated to proliferate in culture. Reverse transcription-PCR analysis of cultured normal HBEC revealed complex patterns of CD44 mRNA expression that were indistinguishable from patterns previously shown to be characteristic of tissue samples containing malignant HBEC. CD44v6-expressing cells were identified in cultures generated from FACS-purified populations of either normal luminal (CALLA-MUC-1+) or myoepithelial (CALLA+MUC-1-) cells, even though immunohistochemical analysis of normal breast tissue sections confirmed CD44v6 expression to be limited to the myoepithelium in vivo. Increased expression of both CD44v mRNA and protein in cultured populations of normal HBEC was shown to correlate positively with the proportion of cells that were proliferating (Ki-67+) independent of cell density. These results indicate that activation of CD44 variant isoform expression in HBEC occurs as a normal response to factors that stimulate their proliferation and suggests caution in the use of this marker to identify malignant cells. PMID:9822219

  18. Overexpression of Long Non-Coding RNA HOTAIR Promotes Tumor Growth and Metastasis in Human Osteosarcoma

    PubMed Central

    Wang, Bo; Su, Yun; Yang, Qun; Lv, Decheng; Zhang, Weiguo; Tang, Kai; Wang, Hong; Zhang, Rui; Liu, Yang

    2015-01-01

    Human osteosarcoma usually presented a high tendency to metastatic spread and caused poor outcomes, however, the underlying mechanism was still largely unknown. In the present study, using a series of in vitro experiments and an animal model, we investigated the roles of HOX antisense intergenic RNA (HOTAIR) during the proliferation and invasion of osteosarcoma. According with our results, HOTAIR was commonly overexpressed in osteosarcoma, which significantly correlated with advanced tumor stage, highly histological grade and poor prognosis. In vitro and in vivo experiments demonstrated that knockdown of HOTAIR could notably suppress cellular proliferation, inhibit invasion and decrease the secretion of MMP2 and MMP9 in osteosarcoma. Collectively, our results suggested that HOTAIR might be a potent therapeutic target for osteosarcoma. PMID:25728753

  19. Rad: A member of the Ras family overexpressed in muscle of type II diabetic humans

    SciTech Connect

    Reynet, C.; Kahn, C.R. )

    1993-11-26

    To identify the gene or genes associated with insulin resistance in Type II (non-insulin-dependent) diabetes mellitus, subtraction libraries were prepared from skeletal muscle of normal and diabetic humans and screened with subtracted probes. Only one clone out of 4000 was selectively overexpressed in Type II diabetic muscle as compared to muscle of non-diabetic or Type I diabetic individuals. This clone encoded a new 290 kilodalton member of the Ras-guanosine triphosphatase superfamily and was termed Rad (Ras associated with diabetes). Messenger ribonucleic acid of Rad was expressed primarily in skeletal and cardiac muscle and was increased an average of 8.6-fold in the muscle of Type II diabetics as compared to normal individuals.

  20. Diosgenin, a plant steroid, induces apoptosis in human rheumatoid arthritis synoviocytes with cyclooxygenase-2 overexpression

    PubMed Central

    Liagre, Bertrand; Vergne-Salle, Pascale; Corbiere, Cecile; Charissoux, Jean L; Beneytout, Jean L

    2004-01-01

    In the present study, we have shown for the first time that a plant steroid, diosgenin, causes an inhibition of the growth of fibroblast-like synoviocytes from human rheumatoid arthritis, with apoptosis induction associated with cyclooxygenase-2 (COX-2) up-regulation. Celecoxib, a selective COX-2 inhibitor, provoked a large decrease in diosgenin-induced apoptosis even in the presence of exogenous prostaglandin E2, whereas interleukin-1?, a COX-2 inducer, strongly increased diosgenin-induced apoptosis of these synoviocytes. These findings suggest that the proapoptotic effect of diosgenin is associated with overexpression of COX-2 correlated with overproduction of endogenous prostaglandin E2. We also observed a loss of mitochondrial membrane potential, caspase-3 activation, and DNA fragmentation after diosgenin treatment. PMID:15225373

  1. High-mobility group A1 proteins are overexpressed in human leukaemias.

    PubMed Central

    Pierantoni, Giovanna Maria; Agosti, Valter; Fedele, Monica; Bond, Heather; Caliendo, Irene; Chiappetta, Gennaro; Lo Coco, Francesco; Pane, Fabrizio; Turco, Maria Caterina; Morrone, Giovanni; Venuta, Salvatore; Fusco, Alfredo

    2003-01-01

    High-mobility group A (HMGA) proteins are non-histone nuclear proteins that bind DNA and several transcription factors. They are involved in the regulation of chromatin structure and function. HMGA protein expression is low in normal adult tissues, but abundant during embryonic development and in several human tumours. Rearrangements of the HMGA genes have been frequently detected in human benign tumours of mesenchymal origin, e.g. lipomas, lung hamartomas and uterine leiomiomas. HMGA proteins have been implicated in the control of cell growth and differentiation of the pre-adipocytic cell line 3T3-L1. In an attempt to better understand the role of HMGA1 proteins in haematological neoplasias and in the differentiation of haematopietic cells, we have investigated their expression in human leukaemias and in leukaemic cell lines induced to terminal differentiation. Here we report HMGA1 overexpression in most fresh human leukaemias of different origin and in several leukaemic cell lines. Moreover, differentiation of three cell lines towards the megakaryocytic phenotype was associated with HMGA1 protein induction, whereas induction of erythroid and monocytic differentiation generally resulted in reduced HMGA1 expression. PMID:12573034

  2. The potential utility of acetyltanshinone IIA in the treatment of HER2-overexpressed breast cancer: Induction of cancer cell death by targeting apoptotic and metabolic signaling pathways

    PubMed Central

    Guerram, Mounia; Jiang, Zhen-Zhou; Yousef, Bashir Alsiddig; Hamdi, Aida Mejda; Hassan, Hozeifa Mohamed; Yuan, Zi-Qiao; Luo, Hou-Wei; Zhu, Xiong; Zhang, Lu-Yong

    2015-01-01

    Increased lipogenesis and protein synthesis is a hallmark of cancer cell proliferation, survival, and metastatic progression and is under intense investigation as a potential antineoplastic target. Acetyltanshinone IIA (ATA) is a compound that was obtained from chemical modifications of tanshinone IIA (TIIA), a potent anticancer agent extracted from the dried roots of the Chinese herbal medicine Salvia miltiorrhiza Bunge. A previous investigation indicated that ATA is more effective in inhibiting the growth of breast cancer especially cells with HER2 overexpression. However, the molecular mechanism(s) mediating this cytotoxic effect on HER2-positive breast cancer remained undefined. Studies described here report that ATA induced G1/S phase arrest and apoptosis in the HER2-positive MDA-MB-453, SK-BR-3, and BT-474 breast cancer cell lines. Mechanistic investigations revealed that the ATA-induced apoptosis effect is associated with remarkably down-regulation of receptor tyrosine kinases (RTKs) EGFR/HER2 and inhibition of their downstream pro-survival signaling pathways. Interestingly, ATA was found to trigger oxidative and endoplasmic reticulum (ER) stresses and to activate AMP activated protein kinase (AMPK) leading to inactivation of key enzymes involved in lipid and protein biogenesis. Intraperitoneal administration of ATA significantly inhibited the growth of MDA-MB-453 xenografts in athymic mice without causing weight loss and any other side effects. Additionally, transwell migration, invasion, and wound healing assays revealed that ATA could suppress tumor angiogenesis in vitro. Taken together, our data suggest that ATA may have broad utility in the treatment of HER2-overexpressed breast cancers. PMID:26068969

  3. Inhibition of Cell Growth and Induction of Apoptosis by Antrodia camphorata in HER-2/neu-Overexpressing Breast Cancer Cells through the Induction of ROS, Depletion of HER-2/neu, and Disruption of the PI3K/Akt Signaling Pathway.

    PubMed

    Lee, Chuan-Chen; Yang, Hsin-Ling; Way, Tzong-Der; Kumar, K J Senthil; Juan, Ying-Chen; Cho, Hsin-Ju; Lin, Kai-Yuan; Hsu, Li-Sung; Chen, Ssu-Ching; Hseu, You-Cheng

    2012-01-01

    Previously, we demonstrated that a submerged fermentation culture of Antrodia camphorata (AC) promotes cell-cycle arrest and apoptosis in human estrogen receptor-positive/negative breast cancer cells. However, whether AC is effective against HER-2/neu-overexpressing breast cancers has not been thoroughly elucidated. In the present study, we showed that AC exhibited a significant cytotoxic effect against HER-2/neu-overexpressing MDA-MB-453 and BT-474 cells. Immunoblot analysis demonstrated that HER-2/neu and their tyrosine phosphorylation were inhibited by AC in a dose-dependent manner. An increase in intracellular reactive oxygen species (ROS) was observed in AC-treated cells, whereas antioxidant N-acetylcysteine (NAC) significantly prevented AC induced HER-2/neu depletion and cell death, which directly indicates that AC-induced HER-2/neu depletion and cell death was mediated by ROS generation. Also, AC significantly downregulated the expression of cyclin D1, cyclin E, and CDK4 followed by the suppression of PI3K/Akt, and their downstream effectors GSK-3β and β-catenin. Notably, AC-treatment induced apoptotic cell death, which was associated with sub-G1 accumulation, DNA fragmentation, mitochondrial dysfunction, cytochrome c release, caspase-3/-9 activation, PARP degradation, and Bcl-2/Bax dysregulation. Assays for colony formation also confirmed the growth-inhibitory effects of AC. This is the first report confirming the anticancer activity of this potentially beneficial mushroom against human HER-2/neu-overexpressing breast cancers. PMID:22701509

  4. Inhibition of Cell Growth and Induction of Apoptosis by Antrodia camphorata in HER-2/neu-Overexpressing Breast Cancer Cells through the Induction of ROS, Depletion of HER-2/neu, and Disruption of the PI3K/Akt Signaling Pathway

    PubMed Central

    Lee, Chuan-Chen; Yang, Hsin-Ling; Way, Tzong-Der; Kumar, K. J. Senthil; Juan, Ying-Chen; Cho, Hsin-Ju; Lin, Kai-Yuan; Hsu, Li-Sung; Chen, Ssu-Ching; Hseu, You-Cheng

    2012-01-01

    Previously, we demonstrated that a submerged fermentation culture of Antrodia camphorata (AC) promotes cell-cycle arrest and apoptosis in human estrogen receptor-positive/negative breast cancer cells. However, whether AC is effective against HER-2/neu-overexpressing breast cancers has not been thoroughly elucidated. In the present study, we showed that AC exhibited a significant cytotoxic effect against HER-2/neu-overexpressing MDA-MB-453 and BT-474 cells. Immunoblot analysis demonstrated that HER-2/neu and their tyrosine phosphorylation were inhibited by AC in a dose-dependent manner. An increase in intracellular reactive oxygen species (ROS) was observed in AC-treated cells, whereas antioxidant N-acetylcysteine (NAC) significantly prevented AC induced HER-2/neu depletion and cell death, which directly indicates that AC-induced HER-2/neu depletion and cell death was mediated by ROS generation. Also, AC significantly downregulated the expression of cyclin D1, cyclin E, and CDK4 followed by the suppression of PI3K/Akt, and their downstream effectors GSK-3β and β-catenin. Notably, AC-treatment induced apoptotic cell death, which was associated with sub-G1 accumulation, DNA fragmentation, mitochondrial dysfunction, cytochrome c release, caspase-3/-9 activation, PARP degradation, and Bcl-2/Bax dysregulation. Assays for colony formation also confirmed the growth-inhibitory effects of AC. This is the first report confirming the anticancer activity of this potentially beneficial mushroom against human HER-2/neu-overexpressing breast cancers. PMID:22701509

  5. Involvement of pp60c-src with two major signaling pathways in human breast cancer.

    PubMed Central

    Luttrell, D K; Lee, A; Lansing, T J; Crosby, R M; Jung, K D; Willard, D; Luther, M; Rodriguez, M; Berman, J; Gilmer, T M

    1994-01-01

    The phosphotyrosine residues of receptor tyrosine kinases serve as unique binding sites for proteins involved in intracellular signaling, which contain SRC homology 2 (SH2) domains. Since overexpression or activation of the pp60c-src kinase has been reported in a number of human tumors, including primary human breast carcinomas, we examined the interactions of the SH2 and SH3 domains of human SRC with target proteins in human carcinoma cell lines. Glutathione S-transferase fusion proteins containing either the SH2, SH3, or the entire SH3/SH2 region of human SRC were used to affinity purify tyrosine-phosphorylated proteins from human breast carcinoma cell lines. We show here that in human breast carcinoma cell lines, the SRC SH2 domain binds to activated epidermal growth factor receptor (EGFR) and p185HER2/neu. SRC SH2 binding to EGFR was also observed in a nontumorigenic cell line after hormone stimulation. Endogenous pp60c-src was found to tightly associate with tyrosine-phosphorylated EGFR. Association of the SRC SH2 with the EGFR was blocked by tyrosyl phosphopeptides containing the sequences surrounding tyrosine-530, the regulatory site in the SRC C terminus, or sequences surrounding the major sites of autophosphorylation in the EGFR. These results raise the possibility that association of pp60c-src with these receptor tyrosine kinases is an integral part of the signaling events mediated by these receptors and may contribute to malignant transformation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7506422

  6. Function of RasGRP3 in the formation and progression of human breast cancer

    PubMed Central

    2014-01-01

    Introduction Ras guanine nucleotide exchange factors (RasGEFs) mediate the activation of the Ras signaling pathway that is over activated in many human cancers. The RasGRP3, an activator of H-Ras and R-Ras protein exerts oncogenic effects and the overexpression of the protein is observed in numerous malignant cancer types. Here, we investigated the putative alteration of expression and potential function of RasGRP3 in the formation and progression of human breast cancer. Methods The RasGRP3 and phosphoRasGRP3 expressions were examined in human invasive ductal adenocarcinoma derived samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) both in mRNA (Q-PCR) and protein (Western blot; immunohistochemistry) levels. To explore the biological function of the protein, RasGRP3 knockdown cultures were established. To assess the role of RasGRP3 in the viability of cells, annexin-V/PI staining and MitoProbe™ DilC1 (5) assay were performed. To clarify the function of the protein in cell proliferation and in the development of chemotherapeutic resistance, CyQuant assay was performed. To observe the RasGRP3 function in tumor formation, the Severe combined immunodeficiency (SCID) mouse model was used. To investigate the role of the protein in Ras-related signaling Q-PCR and Western blot experiments were performed. Results RasGRP3 expression was elevated in human breast tumor tissue samples as well as in multiple human breast cancer cell lines. Down-regulation of RasGRP3 expression in breast cancer cells decreased cell proliferation, induced apoptosis in MCF7 cells, and sensitized T-47D cells to the action of drugs Tamoxifen and trastuzumab (Herceptin). Gene silencing of RasGRP3 reduced tumor formation in mouse xenografts as well. Inhibition of RasGRP3 expression also reduced Akt, ERK1/2 and estrogen receptor alpha phosphorylation downstream from IGF-I insulin like growth factor-I (IGF-I) or epidermal growth factor (EGF) stimulation confirming the functional role of RasGRP3 in the altered behavior of these cells. Conclusions Taken together, our results suggest that the Ras activator RasGRP3 may have a role in the pathological behavior of breast cancer cells and may constitute a therapeutic target for human breast cancer. PMID:24779681

  7. E2F7 overexpression leads to tamoxifen resistance in breast cancer cells by competing with E2F1 at miR-15a/16 promoter

    PubMed Central

    Chu, Junjun; Zhu, Yinghua; Liu, Yujie; Sun, Lijuan; Lv, Xiaobin; Wu, Yanqin; Hu, Pengnan; Su, Fengxi; Gong, Chang; Song, Erwei; Liu, Bodu; Liu, Qiang

    2015-01-01

    About 50–70% of breast cancers are estrogen receptor α (ERα) positive and most of them are sensitive to endocrine therapy including tamoxifen. However, one third of these patients will eventually develop resistance and relapse. We found that the expression of miR-15a and miR-16 were significantly decreased in tamoxifen resistant ER positive breast cancer cell lines. Exogenous expression of miR-15a/16 mimics re-sensitized resistant cells to tamoxifen by inhibiting Cyclin E1 and B cell lymphoma-2 (Bcl-2) to induce cell growth arrest and apoptosis respectively. Further, we identified that a repressive member of E2F family, E2F7, was responsible for the suppression of miR-15a/16 cluster by competing with E2F1 for E2F binding site at the promoter of their host gene DLEU2. Moreover, high expression of E2F7 is correlated with high risk of relapse and poor prognosis in breast cancer patients receiving tamoxifen treatment. Together, our results suggest that overexpression of E2F7 represses miR-15a/16 and then increases Cyclin E1 and Bcl-2 that result in tamoxifen resistance. E2F7 may be a valuable prognostic marker and a therapeutic target of tamoxifen resistance in breast cancer. PMID:26397135

  8. Comprehensive molecular portraits of human breast tumors

    PubMed Central

    2012-01-01

    Summary We analyzed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse phase protein arrays. Our ability to integrate information across platforms provided key insights into previously-defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at > 10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the Luminal A subtype. We identified two novel protein expression-defined subgroups, possibly contributed by stromal/microenvironmental elements, and integrated analyses identified specific signaling pathways dominant in each molecular subtype including a HER2/p-HER2/HER1/p-HER1 signature within the HER2-Enriched expression subtype. Comparison of Basal-like breast tumors with high-grade Serous Ovarian tumors showed many molecular commonalities, suggesting a related etiology and similar therapeutic opportunities. The biologic finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biologic subtypes of breast cancer. PMID:23000897

  9. Assessing sequential oncogene amplification in human breast cancer

    SciTech Connect

    Janocko, L.E.; Lucke, J.F.; Groft, D.W.; Brown, K.A.

    1995-09-01

    Studies of amplification and/or overexpression of c-myc, HER-2/neu, and H-ras in breast cancer have shown that each is associated with a poor prognosis. The purpose of this study was to explore the possibility that there is a preferred sequence of amplification of these oncogenes in breast cancer. The frequencies of amplification and patterns of co-amplification of c-myc, HER-2/neu, and H-ras were studied in a group of 84 breast cancers. The data suggested a preferred sequence of amplification that consisted of c-myc amplification-HER-2/neu amplification-H-ras amplification. This model was supported by loglinear analysis. In addition, the levels of amplification of JC-A, a DNA fragment newly isolated from a patient with advanced breast cancer, were studied in 61 of these cases. The data suggested that JC-A amplification occurred early. Loglinear analysis supported a model in which JC-A amplification occurred either before or after c-myc amplification but was unrelated to Her-2/neu or ras amplification. 35 refs., 1 tab.

  10. Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer

    PubMed Central

    2013-01-01

    Introduction Elevated expression of erbB3 rendered erbB2-overexpressing breast cancer cells resistant to paclitaxel via PI-3K/Akt-dependent upregulation of Survivin. It is unclear whether an erbB3-targeted therapy may abrogate erbB2-mediated paclitaxel resistance in breast cancer. Here, we study the antitumor activity of an anti-erbB3 antibody MM-121/SAR256212 in combination with paclitaxel against erbB2-overexpressing breast cancer. Methods Cell growth assays were used to determine cell viability. Cells undergoing apoptosis were quantified by a specific apoptotic ELISA. Western blot analyses were performed to assess the protein expression and activation. Lentiviral vector containing shRNA was used to specifically knockdown Survivin. Tumor xenografts were established by inoculation of BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with paclitaxel and/or MM-121/SAR256212 to determine whether the antibody (Ab) enhances paclitaxels antitumor activity. Immunohistochemistry was carried out to study the combinatorial effects on tumor cell proliferation and induction of apoptosis in vivo. Results MM-121 significantly facilitated paclitaxel-mediated anti-proliferative/anti-survival effects on SKBR3 cells transfected with a control vector or erbB3 cDNA. It specifically downregulated Survivin associated with inactivation of erbB2, erbB3, and Akt. MM-121 enhances paclitaxel-induced poly(ADP-ribose) polymerase (PARP) cleavage, activation of caspase-8 and -3, and apoptosis in both paclitaxel-sensitive and -resistant cells. Specific knockdown of Survivin in the trastuzumab-resistant BT474-HR20 cells dramatically enhanced paclitaxel-induced apoptosis, suggesting that increased Survivin caused a cross-resistance to paclitaxel. Furthermore, the studies using a tumor xenograft model-established from BT474-HR20 cells revealed that either MM-121 (10mg/kg) or low-dose (7.5mg/kg) paclitaxel had no effect on tumor growth, their combinations significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed that the combinations of MM-121 and paclitaxel significantly reduced the cells with positive staining for Ki-67 and Survivin, and increased the cells with cleaved caspase-3. Conclusions The combinations of MM-121 and paclitaxel not only inhibit tumor cell proliferation, but also promote erbB2-overexpressing breast cancer cells to undergo apoptosis via downregulation of Survivin in vitro and in vivo, suggesting that inactivation of erbB3 with MM-121 enhances paclitaxel-mediated antitumor activity against erbB2-overexpressing breast cancers. Our data supports further exploration of the combinatorial regimens consisting of MM-121 and paclitaxel in breast cancer patients with erbB2-overexpressing tumors, particularly those resistant to paclitaxel. PMID:24168763

  11. Cdx2 Polymorphism Affects the Activities of Vitamin D Receptor in Human Breast Cancer Cell Lines and Human Breast Carcinomas

    PubMed Central

    Di Benedetto, Anna; Korita, Etleva; Goeman, Frauke; Sacconi, Andrea; Biagioni, Francesca; Blandino, Giovanni; Strano, Sabrina; Muti, Paola; Mottolese, Marcella; Falvo, Elisabetta

    2015-01-01

    Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression. PMID:25849303

  12. Development of realistic physical breast phantoms matched to virtual breast phantoms based on human subject data

    SciTech Connect

    Kiarashi, Nooshin; Nolte, Adam C.; Sturgeon, Gregory M.; Ghate, Sujata V.; Segars, William P.; Nolte, Loren W.; Samei, Ehsan; and others

    2015-07-15

    Purpose: Physical phantoms are essential for the development, optimization, and evaluation of x-ray breast imaging systems. Recognizing the major effect of anatomy on image quality and clinical performance, such phantoms should ideally reflect the three-dimensional structure of the human breast. Currently, there is no commercially available three-dimensional physical breast phantom that is anthropomorphic. The authors present the development of a new suite of physical breast phantoms based on human data. Methods: The phantoms were designed to match the extended cardiac-torso virtual breast phantoms that were based on dedicated breast computed tomography images of human subjects. The phantoms were fabricated by high-resolution multimaterial additive manufacturing (3D printing) technology. The glandular equivalency of the photopolymer materials was measured relative to breast tissue-equivalent plastic materials. Based on the current state-of-the-art in the technology and available materials, two variations were fabricated. The first was a dual-material phantom, the Doublet. Fibroglandular tissue and skin were represented by the most radiographically dense material available; adipose tissue was represented by the least radiographically dense material. The second variation, the Singlet, was fabricated with a single material to represent fibroglandular tissue and skin. It was subsequently filled with adipose-equivalent materials including oil, beeswax, and permanent urethane-based polymer. Simulated microcalcification clusters were further included in the phantoms via crushed eggshells. The phantoms were imaged and characterized visually and quantitatively. Results: The mammographic projections and tomosynthesis reconstructed images of the fabricated phantoms yielded realistic breast background. The mammograms of the phantoms demonstrated close correlation with simulated mammographic projection images of the corresponding virtual phantoms. Furthermore, power-law descriptions of the phantom images were in general agreement with real human images. The Singlet approach offered more realistic contrast as compared to the Doublet approach, but at the expense of air bubbles and air pockets that formed during the filling process. Conclusions: The presented physical breast phantoms and their matching virtual breast phantoms offer realistic breast anatomy, patient variability, and ease of use, making them a potential candidate for performing both system quality control testing and virtual clinical trials.

  13. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    PubMed Central

    Yu, Wei; Chai, Hongyan; Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue; Yang, Guifang; Cai, Xiaojun; Falck, John R.; Yang, Jing

    2012-01-01

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. PMID:22841774

  14. Hydroxytyrosol protects against oxidative DNA damage in human breast cells.

    PubMed

    Warleta, Fernando; Quesada, Cristina Snchez; Campos, Mara; Allouche, Yosra; Beltrn, Gabriel; Gaforio, Jos J

    2011-10-01

    Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol's effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells. PMID:22254082

  15. G protein-coupled receptors GPR4 and TDAG8 are oncogenic and overexpressed in human cancers.

    PubMed

    Sin, Wun Chey; Zhang, Yaoping; Zhong, Wendy; Adhikarakunnathu, Sree; Powers, Scott; Hoey, Tim; An, Songzhu; Yang, Jianxin

    2004-08-19

    The GPR4 subfamily consists of four G protein-coupled receptors that share significant sequence homology. In addition to GPR4, this subfamily includes OGR1, TDAG8 and G2A. G2A has previously been shown to be a potent transforming oncogene for murine 3T3 cells. Here we show that GPR4 also malignantly transforms NIH3T3 cells and that TDAG8 malignantly transforms the normal mammary epithelial cell line NMuMG. Overexpression of GPR4 or TDAG8 in HEK293 cells led to transcriptional activation from SRE- and CRE-driven promoters, independent of exogenously added ligand. TDAG8 and GPR4 are also overexpressed in a range of human cancer tissues. Our results suggest that GPR4 and TDAG8 overexpression in human tumors plays a role in driving or maintaining tumor formation. PMID:15221007

  16. Immunotherapy of human tumour xenografts overexpressing the EGF receptor with rat antibodies that block growth factor-receptor interaction.

    PubMed Central

    Modjtahedi, H.; Eccles, S.; Box, G.; Styles, J.; Dean, C.

    1993-01-01

    Athymic mice bearing xenografts of human tumours that overexpress the receptor (EGFR) for EGF and TGF alpha have been used to evaluate the therapeutic potential of three new rat monoclonal antibodies (mAbs) directed against two distinct epitopes on the extracellular domain of the human EGFR. The antibodies, ICR16 (IgG2a), ICR62 (IgG2b) and ICR64 (IgG1), have been shown (Modjtahedi et al., 1993) to be potent inhibitors of the growth in vitro of a number of human squamous cell carcinomas because they block receptor-ligand interaction. When given i.p. at 200 micrograms dose, the three antibodies were found to induce complete regression of xenografts of the HN5 tumour if treatment with antibody commenced at the time of tumour implantation (total doses: ICR16, 3.0 mg; ICR62, 1.2 mg; ICR64, 2.2 mg). More importantly when treatment was delayed until the tumours were established (mean diam. 0.5 cm) both ICR16 and ICR62 induced complete or almost complete regression of the tumours. Furthermore, treatment with a total dose of only 0.44 mg of ICR62 was found to induce complete remission of xenografts of the breast carcinoma MDA-MB 468, but ICR16 was less effective at this dose of antibody and only 4/8 tumours regressed completely. ICR16 and ICR62 were poor inhibitors of the growth in vitro of the vulval carcinoma A431, but both induced a substantial delay in the growth of xenografts of this tumour and 4/8 tumours regressed completely in the mice treated with ICR62 (total dose 2.2 mg). Although ICR16 and ICR64 were more effective than ICR62 as growth inhibitors in vitro, ICR62 was found to be substantially better at inducing regression of the tumour xenografts due perhaps to additional activation of host immune effector functions by the IgG2b antibody. We conclude that these antibodies may be useful therapeutic agents that can be used alone without conjugation to other cytotoxic moieties. PMID:7679281

  17. Role of WISP-2/CCN5 in the Maintenance of a Differentiated and Noninvasive Phenotype in Human Breast Cancer Cells?

    PubMed Central

    Fritah, Asma; Saucier, Ccile; De Wever, Olivier; Bracke, Marc; Biche, Ivan; Lidereau, Rosette; Gespach, Christian; Drouot, Sylvain; Redeuilh, Grard; Sabbah, Michle

    2008-01-01

    WISP-2/CCN5 is an estrogen-regulated member of the connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family of the cell growth and differentiation regulators. The WISP-2/CCN5 mRNA transcript is undetectable in normal human mammary cells, as well as in highly aggressive breast cancer cell lines, in contrast with its higher level in the breast cancer cell lines characterized by a more differentiated phenotype. We report here that knockdown of WISP-2/CCN5 by RNA interference in estrogen receptor alpha (ER?)-positive MCF-7 breast cancer cells induced an estradiol-independent growth linked to a loss of ER? expression and promoted epithelial-to-mesenchymal transdifferentiation. In contrast, forced expression of WISP-2/CCN5 directed MCF-7 cells toward a more differentiated phenotype. When introduced into the poorly differentiated, estrogen-independent, and invasive MDA-MB-231 breast cancer cells, WISP-2/CCN5 was able to reduce their proliferative and invasive phenotypes. In a series of ER?-positive tumor biopsies, we found a positive correlation between the expression of WISP-2/CCN5 and ID2, a transcriptional regulator of differentiation in normal and transformed breast cells. We propose that WISP-2/CCN5 is an important regulator involved in the maintenance of a differentiated phenotype in breast tumor epithelial cells and may play a role in tumor cell invasion and metastasis. PMID:18070926

  18. Expression of K+ channels in normal and cancerous human breast.

    PubMed

    Brevet, Marie; Ahidouch, Ahmed; Sevestre, Henri; Merviel, Philippe; El Hiani, Yassine; Robbe, Micheline; Ouadid-Ahidouch, Halima

    2008-08-01

    Potassium (K+) channels contribute to the regulation of cell proliferation and apoptosis and are also involved in tumor generation and malignant growth. Using immunohistochemical analysis, we investigated the expression of four K+ channels GIRK1 (G-Protein Inwardly Rectifying Potassium Channel 1), Ca2+-activated K channel (K Ca 1.1), voltage activated K+ channels (KV 1.1 and KV 1.3) and of the anti-apoptotic protein Bcl2 in normal and cancerous breast tissues and compared their expression with clinicopathological data. GIRK1 was overexpressed in carcinomatous tissues. In contrast, K V 1.1 and K V 1.3 were less expressed in cancerous tissue. The expression of Bcl-2 was similar in both tissues. As to the clinicopathological data, a correlation between K Ca 1.1 channel and estrogen receptor (ER) expression was observed. GIRK1 was overexpressed in breast carcinoma suggesting its involvement in proliferation and oncogenesis and its possible use as a putative pharmaceutical target. The correlation between K Ca 1.1 channel and ER suggests the involvement of this channel in proliferation. The loss of expression of the two channels K V 1.1 and K V 1.3 may correspond to their role in apoptosis. PMID:18498071

  19. The overexpression membrane type 1 matrix metalloproteinase is associated with the progression and prognosis in breast cancer

    PubMed Central

    Li, Yongping; Cai, Guohong; Yuan, Shifang; Jun, Yi; Li, Nanlin; Wang, Ling; Chen, Feng; Ling, Rui; Yun, Jun

    2015-01-01

    Membrane type 1 matrix metalloproteinase (MT1-MMP) has been demonstrated to play an important role in tumor progression. The aim of the present study was to analyze the expression of MT1-MMP in breast cancer and its correlation with clinicopathologic characteristics, including the survival of breast cancer patients. In our results, MT-MMP1 was up-expressed in breast cancer tissues compared with ductal hyperplasia tissues in microarray data (GSE2429). MT1-MMP mRNA and protein expression was markedly higher in breast cancer tissues than in normal breast tissues (P=0.005 and P=0.037, respectively). Using immunohistochemistry, high levels of MT1-MMP protein were positively correlated with the status of clinical stage (I-II vs. III-IV; P=0.043), lymph node metastasis (absence vs. presence; P=0.024), and distant metastasis (No vs. Yes; P=0.017) of breast cancer patients. Patients with higher MT1-MMP expression had a significantly shorter overall survival time than did patients with low MT1-MMP expression. Multivariate analysis indicated that the level of MT1-MMP expression was an independent prognostic indicator (P<0.001) for the survival of patients with breast cancer. In conclusions, MT1-MMP plays an important role on breast cancer aggressiveness and prognosis and may act as a promising target for prognostic prediction. PMID:25755834

  20. Mistic's membrane association and its assistance in overexpression of a human GPCR are independent processes.

    PubMed

    Marino, Jacopo; Bordag, Natalie; Keller, Sandro; Zerbe, Oliver

    2015-01-01

    The interaction of the Bacillus subtilis protein Mistic with the bacterial membrane and its role in promoting the overexpression of other membrane proteins are still matters of debate. In this study, we aimed to determine whether individual helical fragments of Mistic are sufficient for its interaction with membranes in vivo and in vitro. To this end, fragments encompassing each of Mistic's helical segments and combinations of them were produced as GFP-fusions, and their cellular localization was studied in Escherichia coli. Furthermore, peptides corresponding to the four helical fragments were synthesized by solid-phase peptide synthesis, and their ability to acquire secondary structure in a variety of lipids and detergents was studied by circular dichroism spectroscopy. Both types of experiments demonstrate that the third helical fragment of Mistic interacts only with LDAO micelles but does not partition into lipid bilayers. Interestingly, the other three helices interact with membranes in vivo and in vitro. Nevertheless, all of these short sequences can replace full-length Mistic as N-terminal fusions to achieve overexpression of a human G-protein-coupled receptor in E. coli, although with different effects on quantity and quality of the protein produced. A bioinformatic analysis of the Mistic family expanded the number of homologs from 4 to 20, including proteins outside the genus Bacillus. This information allowed us to discover a highly conserved Shine-Dalgarno sequence in the operon mstX-yugO that is important for downstream translation of the potassium ion channel yugO. PMID:25297828

  1. Chaperones Ameliorate Beta Cell Dysfunction Associated with Human Islet Amyloid Polypeptide Overexpression

    PubMed Central

    Alcarraz-Vizán, Gema; Visa, Montse; Vidal-Fàbrega, Laia; Servitja, Joan-Marc; Novials, Anna

    2014-01-01

    In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP). The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR), perturbing endoplasmic reticulum (ER) homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP) or protein disulfite isomerase (PDI), and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA) or 4-phenylbutyrate (PBA), alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes. PMID:25010593

  2. Polymorphic nucleotides within the human IL-4 promoter that mediate overexpression of the gene.

    PubMed

    Song, Z; Casolaro, V; Chen, R; Georas, S N; Monos, D; Ono, S J

    1996-01-15

    Atopy, which predisposes individuals to develop asthma, severe systemic anaphylaxis, and atopic dermatitis, is usually associated with dramatically elevated total serum IgE levels and is thought to be controlled by a major susceptibility gene and multiple minor susceptibility genes. A recent sib-pair analysis revealed a tight linkage between markers on 5q31.1 and a major susceptibility gene controlling total serum IgE levels. Due to its location within this cluster and its biologic role in Ig class switching and Th2 cell differentiation, the IL-4 gene has emerged as one major candidate for the atopy gene. In one model, polymorphisms within IL-4 regulatory elements might result in overexpression of the gene, amplifying Th2 cell differentiation and class switching to IgE. In support of this model, we report that the human IL-4 promoter exists in multiple allelic forms that exhibit distinct transcriptional activities in IL-4-positive T cells. A particular allele has an unusually high transcriptional activity. A nucleotide substitution within a recently described OAP40 element located just upstream of an NF-AT site (P sequence) appears to be largely responsible for the increased promotor strength of this particular allelic form of the IL-4 promoter. In EMSAs, this substitution results in a markedly enhanced affinity for sequence-specific complexes exhibiting an AP-1 specificity. The identification of allelic nucleotides, which results in overexpression of the IL-4 gene, provides specific targets for a comprehensive screening of atopic and nonatopic individuals and may provide a clue for genetic predisposition for atopy. PMID:8543789

  3. Mistic's membrane association and its assistance in overexpression of a human GPCR are independent processes

    PubMed Central

    Marino, Jacopo; Bordag, Natalie; Keller, Sandro; Zerbe, Oliver

    2015-01-01

    The interaction of the Bacillus subtilis protein Mistic with the bacterial membrane and its role in promoting the overexpression of other membrane proteins are still matters of debate. In this study, we aimed to determine whether individual helical fragments of Mistic are sufficient for its interaction with membranes in vivo and in vitro. To this end, fragments encompassing each of Mistic's helical segments and combinations of them were produced as GFP-fusions, and their cellular localization was studied in Escherichia coli. Furthermore, peptides corresponding to the four helical fragments were synthesized by solid-phase peptide synthesis, and their ability to acquire secondary structure in a variety of lipids and detergents was studied by circular dichroism spectroscopy. Both types of experiments demonstrate that the third helical fragment of Mistic interacts only with LDAO micelles but does not partition into lipid bilayers. Interestingly, the other three helices interact with membranes in vivo and in vitro. Nevertheless, all of these short sequences can replace full-length Mistic as N-terminal fusions to achieve overexpression of a human G-protein-coupled receptor in E. coli, although with different effects on quantity and quality of the protein produced. A bioinformatic analysis of the Mistic family expanded the number of homologs from 4 to 20, including proteins outside the genus Bacillus. This information allowed us to discover a highly conserved Shine-Dalgarno sequence in the operon mstX-yugO that is important for downstream translation of the potassium ion channel yugO. PMID:25297828

  4. Profilin-1 overexpression in MDA-MB-231 breast cancer cells is associated with alterations in proteomics biomarkers of cell proliferation, survival, and motility as revealed by global proteomics analyses.

    PubMed

    Coumans, Joëlle V F; Gau, David; Poljak, Anne; Wasinger, Valerie; Roy, Partha; Moens, Pierre D J

    2014-12-01

    Despite early screening programs and new therapeutic strategies, metastatic breast cancer is still the leading cause of cancer death in women in industrialized countries and regions. There is a need for novel biomarkers of susceptibility, progression, and therapeutic response. Global analyses or systems science approaches with omics technologies offer concrete ways forward in biomarker discovery for breast cancer. Previous studies have shown that expression of profilin-1 (PFN1), a ubiquitously expressed actin-binding protein, is downregulated in invasive and metastatic breast cancer. It has also been reported that PFN1 overexpression can suppress tumorigenic ability and motility/invasiveness of breast cancer cells. To obtain insights into the underlying molecular mechanisms of how elevating PFN1 level induces these phenotypic changes in breast cancer cells, we investigated the alteration in global protein expression profiles of breast cancer cells upon stable overexpression of PFN1 by a combination of three different proteome analysis methods (2-DE, iTRAQ, label-free). Using MDA-MB-231 as a model breast cancer cell line, we provide evidence that PFN1 overexpression is associated with alterations in the expression of proteins that have been functionally linked to cell proliferation (FKPB1A, HDGF, MIF, PRDX1, TXNRD1, LGALS1, STMN1, LASP1, S100A11, S100A6), survival (HSPE1, HSPB1, HSPD1, HSPA5 and PPIA, YWHAZ, CFL1, NME1) and motility (CFL1, CORO1B, PFN2, PLS3, FLNA, FLNB, NME2, ARHGDIB). In view of the pleotropic effects of PFN1 overexpression in breast cancer cells as suggested by these new findings, we propose that PFN1-induced phenotypic changes in cancer cells involve multiple mechanisms. Our data reported here might also offer innovative strategies for identification and validation of novel therapeutic targets and companion diagnostics for persons with, or susceptibility to, breast cancer. PMID:25454514

  5. microRNA, cell cycle, and human breast cancer.

    PubMed

    Yu, Zuoren; Baserga, Renato; Chen, Lide; Wang, Chenguang; Lisanti, Michael P; Pestell, Richard G

    2010-03-01

    The discovery of microRNAs as a novel class of gene expression regulators has led to a new strategy for disease diagnostics and therapeutics. Cell cycle, cell proliferation, and tumorigenesis are all regulated by microRNAs. Several general principles linking microRNAs and cancer have been recently reviewed; therefore, the current review focuses specifically on the perspective of microRNAs in control of cell cycle, stem cells, and heterotypic signaling, as well as the role of these processes in breast cancer. Altered abundance of cell cycle regulation proteins and aberrant expression of microRNAs frequently coexist in human breast cancers. Altered microRNA expression in breast cancer cell lines is associated with altered cell cycle progression and cell proliferation. Indeed, recent studies have demonstrated a causal role for microRNA in governing breast tumor suppression or collaborative oncogenesis. This review summarizes the current understanding of the role for microRNA in regulating the cell cycle and summarizes the evidence for aberrant microRNA expression in breast cancer. The new evidence for microRNA regulation by annotated genes and the involvement of microRNA in breast cancer metastasis are discussed, as is the potential for microRNA to improve breast cancer diagnosis and therapy. PMID:20075198

  6. Early Human Papilloma Virus (HPV) Oncogenic Influences in Breast Cancer

    PubMed Central

    Ngan, Christopher; Lawson, James S.; Clay, Rosemary; Delprado, Warick; Whitaker, Noel J.; Glenn, Wendy K.

    2015-01-01

    BACKGROUND Human papilloma viruses (HPVs) may act early in breast oncogenesis (“hit-and-run” phenomena). METHODS The authors used immunohistochemistry for the identification of HPV E7 oncogenic protein expression in 32 sets of benign and subsequent breast cancer specimens from the same Australian patients. RESULTS HPV E7 oncoprotein was clearly expressed in the nuclei of 23 (72%) of the 32 benign specimens and 20 (62.5%) of the subsequent 32 breast cancer specimens in the same patients. There was no HPV E7 protein expression in seven (30%) of the 23 breast cancer specimens that had prior HPV E7 protein-positive benign breast biopsies in the same patients. CONCLUSIONS This observation suggests that HPV oncogenic influences occur early in some breast cancers. This finding confirms the previous observations. This early influence of HPVs may be the reason why there is no increase in the prevalence of HPV-associated breast cancer in immunocompromised patients as compared to HPV-associated cervical cancer. PMID:26691275

  7. The Oncogenic Potential of Human Cytomegalovirus and Breast Cancer

    PubMed Central

    Herbein, Georges; Kumar, Amit

    2014-01-01

    Breast cancer is the leading causes of cancer-related death among women. The vast majority of breast cancers are carcinomas that originate from cells lining the milk-forming ducts of the mammary gland. Numerous articles indicate that breast tumors exhibit diverse phenotypes depending on their distinct physiopathological signatures, clinical courses, and therapeutic possibilities. The human cytomegalovirus (HCMV) is a multifaceted highly host specific betaherpesvirus that is regarded as asymptomatic or mildly pathogenic virus in immunocompetent host. HCMV may cause serious in utero infections as well as acute and chronic complications in immunocompromised individual. The involvement of HCMV in late inflammatory complications underscores its possible role in inflammatory diseases and cancer. HCMV targets a variety of cell types in vivo, including macrophages, epithelial cells, endothelial cells, fibroblasts, stromal cells, neuronal cells, smooth muscle cells, and hepatocytes. HCMV can be detected in the milk after delivery and thereby HCMV could spread to adjacent mammary epithelial cells. HCMV also infects macrophages and induces an atypical M1/M2 phenotype, close to the tumor-associated macrophage phenotype, which is associated with the release of cytokines involved in cancer initiation or promotion and breast cancer of poor prognosis. HCMV antigens and DNA have been detected in tissue biopsies of breast cancers and elevation in serum HCMV IgG antibody levels has been reported to precede the development of breast cancer in some women. In this review, we will discuss the potential role of HCMV in the initiation and progression of breast cancer. PMID:25202681

  8. Six1 Overexpression at Early Stages of HPV16-mediated Transformation of Human Keratinocytes Promotes Differentiation Resistance and EMT

    PubMed Central

    Xu, Hanwen; Pirisi, Lucia; Creek, Kim E.

    2014-01-01

    Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial-mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. PMID:25463612

  9. Herceptin functionalized microfluidic polydimethylsiloxane devices for the capture of human epidermal growth factor receptor 2 positive circulating breast cancer cells

    PubMed Central

    Thierry, Benjamin; Kurkuri, Mahaveer; Shi, Jun Yan; Lwin, Lwin Ei Mon Phyo; Palms, Dennis

    2010-01-01

    Building on recent breakthroughs in the field of microfluidic-based capture of rare cancer cells circulating in the blood, the present article reports on the use of Herceptin functionalized PDMS devices designed to efficiently capture from blood cancer cells, overexpressing the tyrosine kinase human epidermal growth factor receptor (HER2). The identification of patients overexpressing HER2 is critical as it typically associates with an aggressive disease course in breast cancer and poor prognosis. Importantly, HER2 positive patients have been found to significantly benefit from Herceptin (Trastuzumab), a humanized monoclonal antibody (MAb) against HER2. Disposable PDMS devices prepared using standard soft lithography were functionalized by the plasma polymerization of an epoxy-containing monomer. The epoxy-rich thin film (AGEpp) thus created could be conjugated with Herceptin either directly or through a polyethylene glycol interlayer. The properties and reactivity toward the monoclonal antibody conjugation of these coatings were determined using x-ray photoelectron spectroscopy; direct conjugation provided a good compromise in reactivity and resistance to biologically nonspecific fouling and was selected. Using the breast cancer cell line SK-BR-3 as a model for cells overexpressing HER2, the immunocapture efficacy of the Herceptin functionalized PDMS was demonstrated in model studies. Validation studies confirmed the ability of the device to efficiently capture (?80% capture yield) HER2 positive cells from full blood. PMID:21045921

  10. Human neural stem cells over-expressing choline acetyltransferase restore cognition in rat model of cognitive dysfunction.

    PubMed

    Park, Dongsun; Lee, Hong Jun; Joo, Seong Soo; Bae, Dae-Kwon; Yang, Goeun; Yang, Yun-Hui; Lim, Inja; Matsuo, Akinori; Tooyama, Ikuo; Kim, Yun-Bae; Kim, Seung U

    2012-04-01

    A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model. PMID:22245157

  11. MicroRNA Regulation of Human Breast Cancer Stem Cells.

    PubMed

    Shimono, Yohei; Mukohyama, Junko; Nakamura, Shun-Ichi; Minami, Hironobu

    2015-01-01

    MicroRNAs (miRNAs) are involved in virtually all biological processes, including stem cell maintenance, differentiation, and development. The dysregulation of miRNAs is associated with many human diseases including cancer. We have identified a set of miRNAs differentially expressed between human breast cancer stem cells (CSCs) and non-tumorigenic cancer cells. In addition, these miRNAs are similarly upregulated or downregulated in normal mammary stem/progenitor cells. In this review, we mainly describe the miRNAs that are dysregulated in human breast CSCs directly isolated from clinical specimens. The miRNAs and their clusters, such as the miR-200 clusters, miR-183 cluster, miR-221-222 cluster, let-7, miR-142 and miR-214, target the genes and pathways important for stem cell maintenance, such as the self-renewal gene BMI1, apoptosis, Wnt signaling, Notch signaling, and epithelial-to-mesenchymal transition. In addition, the current evidence shows that metastatic breast CSCs acquire a phenotype that is different from the CSCs in a primary site. Thus, clarifying the miRNA regulation of the metastatic breast CSCs will further advance our understanding of the roles of human breast CSCs in tumor progression. PMID:26712794

  12. MicroRNA Regulation of Human Breast Cancer Stem Cells

    PubMed Central

    Shimono, Yohei; Mukohyama, Junko; Nakamura, Shun-ichi; Minami, Hironobu

    2015-01-01

    MicroRNAs (miRNAs) are involved in virtually all biological processes, including stem cell maintenance, differentiation, and development. The dysregulation of miRNAs is associated with many human diseases including cancer. We have identified a set of miRNAs differentially expressed between human breast cancer stem cells (CSCs) and non-tumorigenic cancer cells. In addition, these miRNAs are similarly upregulated or downregulated in normal mammary stem/progenitor cells. In this review, we mainly describe the miRNAs that are dysregulated in human breast CSCs directly isolated from clinical specimens. The miRNAs and their clusters, such as the miR-200 clusters, miR-183 cluster, miR-221-222 cluster, let-7, miR-142 and miR-214, target the genes and pathways important for stem cell maintenance, such as the self-renewal gene BMI1, apoptosis, Wnt signaling, Notch signaling, and epithelial-to-mesenchymal transition. In addition, the current evidence shows that metastatic breast CSCs acquire a phenotype that is different from the CSCs in a primary site. Thus, clarifying the miRNA regulation of the metastatic breast CSCs will further advance our understanding of the roles of human breast CSCs in tumor progression. PMID:26712794

  13. Development of a new metastatic human breast carcinoma xenograft line.

    PubMed Central

    Mehta, R. R.; Graves, J. M.; Shilkaitis, A.; Das Gupta, T. K.

    1998-01-01

    Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research. However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15%. Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel. Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously. In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c. in athymic mice. We have studied in detail the characteristics of the xenograft and the patient's tumour from which the xenograft line originated. Both the xenograft and the patient's tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR. In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen. This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages. This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research. Images p596-a Figure 1 Figure 2 Figure 3 Figure 5 Figure 4 Figure 9 PMID:9484817

  14. Overexpressed genes associated with hormones in terminal ductal lobular units identified by global transcriptome analysis: An insight into the anatomic origin of breast cancer.

    PubMed

    Yang, Jianmin; Yu, Haijing; Zhang, Liang; Deng, Hua; Wang, Qi; Li, Wenping; Zhang, Anqin; Gao, Hongyi; Yin, Aihua

    2016-03-01

    Although human breast ducts and terminal ductal lobular units (TDLUs) share the same cell types, ample evidence shows that TDLUs are the predominant site for the origin of breast cancer. Yet, there is still limited information concerning the molecular mechanisms. Analysis of transcriptomic profiles in TDLUs may provide insight into early breast tumorigenesis. We compared genome-wide expression profiles of 8 matched sets of breast main duct and TDLU samples, using significance analysis of microarray (SAM) software to screen differentially expressed genes (DEGs) with fold-change >2.0 and q-value <0.05. Moreover, we used Gene Ontology for functional enrichment analysis. We identified 472 DEGs between the two tissue types, and confirmed 17 randomly chosen DEGs by quantitative reverse transcription-PCR (qRT-PCR). Notably, hormone-related pathways were highly enriched in the TDLU samples, including various hormone-related DEGs that are associated with breast carcinogenesis and tumor progression. Oncogenic upregulation in TDLUs indicates a potential inappropriate or excessive response to successive hormone stimulus during the proliferation, differentiation and lactation cycles of the human mammary gland. Imbalanced hormone reactions may finally result in the early onset of neoplastic transformation that occurs mostly in breast TDLUs. PMID:26707748

  15. Epigenetic and transcriptional determinants of the human breast

    PubMed Central

    Gascard, Philippe; Bilenky, Misha; Sigaroudinia, Mahvash; Zhao, Jianxin; Li, Luolan; Carles, Annaick; Delaney, Allen; Tam, Angela; Kamoh, Baljit; Cho, Stephanie; Griffith, Malachi; Chu, Andy; Robertson, Gordon; Cheung, Dorothy; Li, Irene; Heravi-Moussavi, Alireza; Moksa, Michelle; Mingay, Matthew; Hussainkhel, Angela; Davis, Brad; Nagarajan, Raman P.; Hong, Chibo; Echipare, Lorigail; OGeen, Henriette; Hangauer, Matthew J.; Cheng, Jeffrey B.; Neel, Dana; Hu, Donglei; McManus, Michael T.; Moore, Richard; Mungall, Andrew; Ma, Yussanne; Plettner, Patrick; Ziv, Elad; Wang, Ting; Farnham, Peggy J.; Jones, Steven J.M.; Marra, Marco A.; Tlsty, Thea D.; Costello, Joseph F.; Hirst, Martin

    2015-01-01

    While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells. PMID:25690954

  16. PTEN and NEDD4 in Human Breast Carcinoma.

    PubMed

    Chen, Yilun; van de Vijver, Marc J; Hibshoosh, Hanina; Parsons, Ramon; Saal, Lao H

    2016-01-01

    PTEN is an important tumor suppressor gene that antagonizes the oncogenic PI3K/AKT signaling pathway and has functions in the nucleus for maintaining genome integrity. Although PTEN inactivation by mutation is infrequent in breast cancer, transcript and protein levels are deficient in >25 % of cases. The E3 ubiquitin ligase NEDD4 (also known as NEDD4-1) has been reported to negatively regulate PTEN protein levels through poly-ubiquitination and proteolysis in carcinomas of the prostate, lung, and bladder, but its effect on PTEN in the breast has not been studied extensively. To investigate whether NEDD4 contributes to low PTEN levels in human breast cancer, we analyzed the expression of these proteins by immunohistochemistry across a large Swedish cohort of breast tumor specimens, and their transcript expression levels by microarrays. For both NEDD4 and PTEN, their transcript expression was significantly correlated to their protein expression. However, comparing NEDD4 expression to PTEN expression, either no association or a positive correlation was observed at the protein and transcript levels. This unexpected observation was further corroborated in two independent breast cancer cohorts from The Netherlands Cancer Institute and The Cancer Genome Atlas. Our results suggest that NEDD4 is not responsible for the frequent down-regulation of the PTEN protein in human breast carcinoma. PMID:26276352

  17. Prodigiosin down-regulates survivin to facilitate paclitaxel sensitization in human breast carcinoma cell lines

    SciTech Connect

    Ho, T.-F.; Peng, Y.-T.; Chuang, S.-M.; Lin, S.-C.; Feng, B.-L.; Lu, C.-H.; Yu, W.-J.; Chang, J.-S. Chang, C.-C.

    2009-03-01

    Prodigiosin is a bacterial metabolite with potent anticancer activity, which is attributed to its proapoptotic effect selectively active in malignant cells. Still, the molecular mechanisms whereby prodigiosin induces apoptosis remain largely unknown. In particular, the role of survivin, a vital inhibitor of apoptosis, in prodigiosin-induced apoptosis has never been addressed before and hence was the primary goal of this study. Our results showed that prodigiosin dose-dependently induced down-regulation of survivin in multiple breast carcinoma cell lines, including MCF-7, T-47D and MDA-MB-231. This down-regulation is mainly regulated at the level of transcription, as prodigiosin reduced the levels of both survivin mRNA and survivin promoter activity but failed to rescue survivin expression when proteasome-mediated degradation is abolished. Importantly, overexpression of survivin rendered cells more resistant to prodigiosin, indicating an essential role of survivin down-regulation in prodigiosin-induced apoptosis. In addition, we found that prodigiosin synergistically enhanced cell death induced by paclitaxel, a chemotherapy drug known to up-regulate survivin that in turn confers its own resistance. This paclitaxel sensitization effect of prodigiosin is ascribed to the lowering of survivin expression, because prodigiosin was shown to counteract survivin induction by paclitaxel and, notably, the sensitization effect was severely abrogated in cells that overexpress survivin. Taken together, our results argue that down-regulation of survivin is an integral component mediating prodigiosin-induced apoptosis in human breast cancer cells, and further suggest the potential of prodigiosin to sensitize anticancer drugs, including paclitaxel, in the treatment of breast cancer.

  18. Chronic wasting disease prions are not transmissible to transgenic mice overexpressing human prion protein

    PubMed Central

    Sandberg, Malin K.; Al-Doujaily, Huda; Sigurdson, Christina J.; Glatzel, Markus; O'Malley, Catherine; Powell, Caroline; Asante, Emmanuel A.; Linehan, Jacqueline M.; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Collinge, John

    2010-01-01

    Chronic wasting disease (CWD) is a prion disease that affects free-ranging and captive cervids, including mule deer, white-tailed deer, Rocky Mountain elk and moose. CWD-infected cervids have been reported in 14 USA states, two Canadian provinces and in South Korea. The possibility of a zoonotic transmission of CWD prions via diet is of particular concern in North America where hunting of cervids is a popular sport. To investigate the potential public health risks posed by CWD prions, we have investigated whether intracerebral inoculation of brain and spinal cord from CWD-infected mule deer transmits prion infection to transgenic mice overexpressing human prion protein with methionine or valine at polymorphic residue 129. These transgenic mice have been utilized in extensive transmission studies of human and animal prion disease and are susceptible to BSE and vCJD prions, allowing comparison with CWD. Here, we show that these mice proved entirely resistant to infection with mule deer CWD prions arguing that the transmission barrier associated with this prion strain/host combination is greater than that observed with classical BSE prions. However, it is possible that CWD may be caused by multiple prion strains. Further studies will be required to evaluate the transmission properties of distinct cervid prion strains as they are characterized. PMID:20610667

  19. Overexpression of ROCK1 and ROCK2 inhibits human laryngeal squamous cell carcinoma.

    PubMed

    Zhang, Junbo; He, Xue; Ma, Yueying; Liu, Yanli; Shi, Huaiyin; Guo, Weiwei; Liu, Liangfa

    2015-01-01

    Rho-associated coiled-coil containing protein kinase (ROCK) over-expression has been implicated in the progression of many tumor types. The aim of this study was to explore the roles of ROCK1 and ROCK2 in human laryngeal squamous cell carcinoma (LSCC). ROCK1 and ROCK2 expression levels were examined in 50 cases of human LSCC samples by immunohistochemistry. Effects of ROCK1 and ROCK2 on LSCC cell proliferation and motility were investigated in the presence of the ROCK inhibitor Y-27632. The results showed that ROCK1 expression was positively correlated with tumor size and lymph node metastasis (P < 0.05); ROCK2 positively correlated with tumor size (P < 0.05). Inhibition of ROCK1 and ROCK2 by Y-27632 significantly inhibits proliferation, migration, and invasion of LSCC cells. Our data indicate that expression of ROCK1 and ROCK2 are closely associated with tumor growth and lymph node metastasis of LSCC. Thus, these two ROCK isoforms may be useful as molecular makers for LSCC diagnosis and may be useful therapeutic targets as well. PMID:25755711

  20. Glypican-1 is frequently overexpressed in human gliomas and enhances FGF-2 signaling in glioma cells.

    PubMed

    Su, Gui; Meyer, Kristy; Nandini, Chilkunda D; Qiao, Dianhua; Salamat, Shahriar; Friedl, Andreas

    2006-06-01

    Signaling by fibroblast growth factor 2 (FGF-2), an autocrine stimulator of glioma growth, is regulated by heparan sulfate proteoglycans (HSPGs) via a ternary complex with FGF-2 and the FGF receptor (FGFR). To characterize glioma growth signaling, we examined whether altered HSPGs contribute to loss of growth control in gliomas. In a screen of five human glioma cell lines, U118 and U251 cell HSPGs activated FGF-2 signaling via FGFR1c. The direct comparison of U251 glioma cells with normal astrocyte HSPGs demonstrated that the glioma HSPGs had a significantly elevated ability to promote FGF-2-dependent mitogenic signaling via FGFR1c. This enhanced activity correlated with a higher level of overall sulfation, specifically the abundance of 2S- and 6S-containing disaccharides. Glioma cell expression of the cell-surface HSPG glypican-1 closely mirrored the FGF-2 coactivator activity. Furthermore, forced expression of glypican-1 in (glypican-1-deficient) U87 glioma cells enhanced their FGF-2 response. Immunohistochemical analysis revealed a highly significant overexpression of glypican-1 in human astrocytoma and oligodendroglioma samples compared with nonneoplastic gliosis. In summary, these observations suggest that altered HSPGs contribute to enhanced signaling of FGF-2 via FGFR1c in gliomas with glypican-1 playing a significant role in this mitogenic pathway. PMID:16723715

  1. Assessment of the Selenoprotein M (SELM) Over-Expression on Human Hepatocellular Carcinoma Tissues by Immunohistochemistry

    PubMed Central

    Guerriero, E.; Accardo, M.; Capone, F.; Colonna, G.; Castello, G.; Costantini, S.

    2014-01-01

    Selenium is an essential trace mineral of fundamental importance to human healthy and exerts its biological function through selenoproteins. In particular, Selenoprotein M (SELM) is located in the endoplasmic reticulum and contains the common redox motif of cysteine-X-X-selenocysteine type. It attracts great attention due to its high expression in brain and its potential roles as antioxidant, neuroprotective, and cytosolic calcium regulator. Recently, our group found SELM over-expression in human hepatocellular carcinoma (HCC) cell lines. In this report some paraffin-embedded tissues from liver biopsy of patients with hepatitis C virus (HCV)-related cirrhosis and HCC were immunohistochemically stained and SELM expression scoring was evaluated. Our results evidence for the first time an increase of SELM expression in HCC liver tissues, and its gradual expression raise associated with an increased malignancy grade. Therefore, we propose to use i) SELM as putative marker for HCC as well as ii) simple immunohistochemistry technique to distinguish between the different grades of malignancy. PMID:25578973

  2. Molecular Signaling Involved in Oxysterol-Induced ?1-Integrin Over-Expression in Human Macrophages

    PubMed Central

    Gargiulo, Simona; Gamba, Paola; Testa, Gabriella; Sottero, Barbara; Maina, Marco; Guina, Tina; Biasi, Fiorella; Poli, Giuseppe; Leonarduzzi, Gabriella

    2012-01-01

    The hypercholesterolemia-atherosclerosis association is now established; hypercholesterolemia may induce vascular-cell activation, subsequently increasing expression of adhesion molecules, cytokines, chemokines, growth factors, and other key inflammatory molecules. Among inflammatory molecules expressed by vascular cells, integrins play a critical role in regulating macrophage activation and migration to the site of inflammation, by mediating cell-cell and cell-extracellular matrix interactions. The main lipid oxidation products present in oxidized LDL that may be responsible for inflammatory processes in atherogenesis, are cholesterol oxidation products, known as oxysterols. This study demonstrates the effect of an oxysterol mixture, compatible with that detectable in human hypercholesterolemic plasma, on the expression and synthesis of ?1-integrin in cells of the macrophage lineage. The molecular signaling whereby oxysterols induce ?1-integrin up-regulation is also comprehensively investigated. Over-expression of ?1-integrin depends on activation of classic and novel members of protein kinase C and extracellular signal-regulated kinases 1 and 2, as well as of the up-stream G-protein (Gq and G13), c-Src, and phospholipase C. In addition, the localization of ?1-integrin in advanced human carotid plaques is highlighted, marking its importance in atherosclerotic plaque progression. PMID:23203064

  3. Overexpression of FRAT1 is associated with malignant phenotype and poor prognosis in human gliomas.

    PubMed

    Guo, Geng; Zhong, Cheng-liang; Liu, Yang; Mao, Xing-gang; Zhang, Zheng; Jin, Ji; Liu, Jing; Yang, Liu; Mao, Jin-ming; Guo, Yu-hong; Zhao, Yuan-li

    2015-01-01

    Glioma is the most common malignancy of the central nervous system. Approximately 40 percent of intracranial tumors are diagnosed as gliomas. Difficulties in treatment are associated closely with the malignant phenotype, which is characterized by excessive proliferation, relentless invasion, and angiogenesis. Although the comprehensive treatment level of brain glioma is continuously progressing, the outcome of this malignancy has not been improved drastically. Therefore, the identification of new biomarkers for diagnosis and therapy of this malignancy is of significant scientific and clinical value. FRAT1 is a positive regulator of the Wnt/?-catenin signaling pathway and is overexpressed in many human tumors. In the present study, we investigated the expression status of FRAT1 in 68 patients with human gliomas and its correlation with the pathologic grade, proliferation, invasion, angiogenesis, and prognostic significance. These findings suggest that FRAT1 may be an important factor in the tumorigenesis and progression of glioma and could be explored as a potential biomarker for pathological diagnosis, an indicator for prognosis, and a target for biological therapy of malignancy. PMID:25922553

  4. CAPER, a novel regulator of human breast cancer progression

    PubMed Central

    Mercier, Isabelle; Gonzales, Donna M; Quann, Kevin; Pestell, Timothy G; Molchansky, Alexander; Sotgia, Federica; Hulit, James; Gandara, Ricardo; Wang, Chenguang; Pestell, Richard G; Lisanti, Michael P; Jasmin, Jean-Franois

    2014-01-01

    CAPER is an estrogen receptor (ER) co-activator that was recently shown to be involved in human breast cancer pathogenesis. Indeed, we reported increased expression of CAPER in human breast cancer specimens. We demonstrated that CAPER was undetectable or expressed at relatively low levels in normal breast tissue and assumed a cytoplasmic distribution. In contrast, CAPER was expressed at higher levels in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) specimens, where it assumed a predominantly nuclear distribution. However, the functional role of CAPER in human breast cancer initiation and progression remained unknown. Here, we used a lentiviral-mediated gene silencing approach to reduce the expression of CAPER in the ER-positive human breast cancer cell line MCF-7. The proliferation and tumorigenicity of MCF-7 cells stably expressing control or human CAPER shRNAs was then determined via both in vitro and in vivo experiments. Knockdown of CAPER expression significantly reduced the proliferation of MCF-7 cells in vitro. Importantly, nude mice injected with MCF-7 cells harboring CAPER shRNAs developed smaller tumors than mice injected with MCF-7 cells harboring control shRNAs. Mechanistically, tumors derived from mice injected with MCF-7 cells harboring CAPER shRNAs displayed reduced expression of the cell cycle regulators PCNA, MCM7, and cyclin D1, and the protein synthesis marker 4EBP1. In conclusion, knockdown of CAPER expression markedly reduced human breast cancer cell proliferation in both in vitro and in vivo settings. Mechanistically, knockdown of CAPER abrogated the activity of proliferative and protein synthesis pathways. PMID:24621503

  5. CAPER, a novel regulator of human breast cancer progression.

    PubMed

    Mercier, Isabelle; Gonzales, Donna M; Quann, Kevin; Pestell, Timothy G; Molchansky, Alexander; Sotgia, Federica; Hulit, James; Gandara, Ricardo; Wang, Chenguang; Pestell, Richard G; Lisanti, Michael P; Jasmin, Jean-Franois

    2014-01-01

    CAPER is an estrogen receptor (ER) co-activator that was recently shown to be involved in human breast cancer pathogenesis. Indeed, we reported increased expression of CAPER in human breast cancer specimens. We demonstrated that CAPER was undetectable or expressed at relatively low levels in normal breast tissue and assumed a cytoplasmic distribution. In contrast, CAPER was expressed at higher levels in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) specimens, where it assumed a predominantly nuclear distribution. However, the functional role of CAPER in human breast cancer initiation and progression remained unknown. Here, we used a lentiviral-mediated gene silencing approach to reduce the expression of CAPER in the ER-positive human breast cancer cell line MCF-7. The proliferation and tumorigenicity of MCF-7 cells stably expressing control or human CAPER shRNAs was then determined via both in vitro and in vivo experiments. Knockdown of CAPER expression significantly reduced the proliferation of MCF-7 cells in vitro. Importantly, nude mice injected with MCF-7 cells harboring CAPER shRNAs developed smaller tumors than mice injected with MCF-7 cells harboring control shRNAs. Mechanistically, tumors derived from mice injected with MCF-7 cells harboring CAPER shRNAs displayed reduced expression of the cell cycle regulators PCNA, MCM7, and cyclin D1, and the protein synthesis marker 4EBP1. In conclusion, knockdown of CAPER expression markedly reduced human breast cancer cell proliferation in both in vitro and in vivo settings. Mechanistically, knockdown of CAPER abrogated the activity of proliferative and protein synthesis pathways. PMID:24621503

  6. Human neural stem cell tropism to metastatic breast cancer.

    PubMed

    Zhao, Donghong; Najbauer, Joseph; Annala, Alexander J; Garcia, Elizabeth; Metz, Marianne Z; Gutova, Margarita; Polewski, Monika D; Gilchrist, Megan; Glackin, Carlotta A; Kim, Seung U; Aboody, Karen S

    2012-02-01

    Metastasis to multiple organs is the primary cause of mortality in breast cancer patients. The poor prognosis for patients with metastatic breast cancer and toxic side effects of currently available treatments necessitate the development of effective tumor-selective therapies. Neural stem cells (NSCs) possess inherent tumor tropic properties that enable them to overcome many obstacles of drug delivery that limit effective chemotherapy strategies for breast cancer. We report that increased NSC tropism to breast tumor cell lines is strongly correlated with the invasiveness of cancer cells. Interleukin 6 (IL-6) was identified as a major cytokine mediating NSC tropism to invasive breast cancer cells. We show for the first time in a preclinical mouse model of metastatic human breast cancer that NSCs preferentially target tumor metastases in multiple organs, including liver, lung, lymph nodes, and femur, versus the primary intramammary fat pad tumor. For proof-of-concept of stem cell-mediated breast cancer therapy, NSCs were genetically modified to secrete rabbit carboxylesterase (rCE), an enzyme that activates the CPT-11 prodrug to SN-38, a potent topoisomerase I inhibitor, to effect tumor-localized chemotherapy. In vitro data demonstrate that exposure of breast cancer cells to conditioned media from rCE-secreting NSCs (NSC.rCE) increased their sensitivity to CPT-11 by 200-fold. In vivo, treatment of tumor-bearing mice with NSC.rCE cells in combination with CPT-11 resulted in reduction of metastatic tumor burden in lung and lymph nodes. These data suggest that NSC-mediated enzyme/prodrug therapy may be more effective and less toxic than currently available chemotherapy strategies for breast cancer metastases. PMID:22084033

  7. Aberrant hypomethylation-mediated CD147 overexpression promotes aggressive tumor progression in human prostate cancer.

    PubMed

    Liang, Yu-Xiang; Mo, Ru-Jun; He, Hui-Chan; Chen, Jia-Hong; Zou, Jun; Han, Zhao-Dong; Lu, Jian-Ming; Cai, Chao; Zeng, Yan-Ru; Zhong, Wei-De; Wu, Chin-Lee

    2015-05-01

    Our previous study revealed the potential role of CD147 in human prostate cancer (PCa). Here, we investigated the CD147 promoter methylation status and the correlation with tumorigenicity in human PCa. CD147 mRNA and protein expression levels were both significantly higher in the 4 PCa cell lines, than in the 2 non-tumorigenic benign human prostatic epithelial cell lines (all P<0.01). We showed hypomethylation of promoter regions of CD147 in PCa cell lines with significant CD147 expression as compared to non-tumorigenic benign human prostatic epithelial cell lines slowly expressing CD147. Additionally, the treatment of methylated cell lines with 5-aza-2'-deoxycytidine increased CD147 expression significantly in low-expressing cell lines and also activated the expression of matrix metalloproteinase (MMP)-2, which may be one of the most important downstream targets of CD147. Furthermore, PCa tissues displayed decreased DNA methylation in the promoter region of CD147 compared to the corresponding non-cancerous prostate tissues, and methylation intensity correlated inversely with the CD147 mRNA levels. There was a significant negative correlation between CD147 mRNA levels and the number of methylated sites in PCa tissues (r=-0.467, P<0.01). In conclusion, our data offer convincing evidence for the first time that the DNA promoter hypomethylation of CD147 may be one of the regulatory mechanisms involved in the cancer-related overexpression of CD147 and may play a crucial role in the tumorigenesis of PCa. PMID:25813864

  8. Cyclo-oxygenase 2 expression is associated with angiogenesis and lymph node metastasis in human breast cancer

    PubMed Central

    Costa, C; Soares, R; Reis-Filho, J S; Leito, D; Amendoeira, I; Schmitt, F C

    2002-01-01

    Aims: Cyclo-oxygenases 1 and 2 (COX-1 and COX-2) are key enzymes in prostaglandin biosynthesis. COX-2 is induced by a wide variety of stimuli, and present during inflammation. COX-2 overexpression has been observed in colon, head and neck, lung, prostate, stomach, and breast cancer. In colon and gastric cancer, COX-2 expression was associated with angiogenesis. The aim of this study was to determine the relation between COX-2 expression and angiogenesis in breast cancer, and to correlate the expression of this enzyme with classic clinicopathological parameters. Methods: COX-2 expression was investigated by immunohistochemistry and western blotting analysis. The expression of COX-2 was then related to age, histological grade, nodal status, oestrogen receptor status, p53 expression,c-erb-B2 overexpression, mitotic counts, MIB-1 labelling index, apoptotic index, sialyl-Tn expression, transforming growth factor ? expression, microvessel density, and disease free survival in 46 patients with invasive ductal breast carcinoma. Results: By means of immunohistochemistry, COX-2 expression was detected in eight of the 46 carcinomas studied. Western blotting showed COX-2 protein expression in the same breast tumours, but not in normal adjacent tissues. The density of microvessels immunostained with anti-F-VIII related antigen was significantly higher in patients with COX-2 expression than in those without expression (p = 0.03). In addition, COX-2 was significantly associated with the presence of sialyl-Tn expression (p = 0.02), lymph node metastasis (p = 0.03), a high apoptotic index (p = 0.03), and a short disease free survival (p = 0.03) in univariate analyses. Conclusions: These data suggest that COX-2 expression is associated with angiogenesis, lymph node metastasis, and apoptosis in human breast cancer. Moreover, these results warrant further studies with larger series of patients to confirm the association with short disease free survival in patients with breast cancer. PMID:12037025

  9. Overexpression of Rsf-1 correlates with pathological type, p53 status and survival in primary breast cancer

    PubMed Central

    Ren, Jie; Chen, Qiu-Chen; Jin, Feng; Wu, Hui-Zhe; He, Miao; Zhao, Lin; Yu, Zhao-Jin; Yao, Wei-Fan; Mi, Xiao-Yi; Wang, En-Hua; Wei, Min-Jie

    2014-01-01

    Aim: The incidence of breast cancer in developing countries still increasing, to identify novel molecular markers associated with carcinogenesis and prognosis of breast cancer still being implemented. The largest subunit of Remodeling and spacing factor (RSF), Rsf-1, mediates ATPase-dependent chromatin remodeling. Its oncogenic properties have been demonstrated in certain carcinomas. The aim of this study was to examine the prognostic value of Rsf-1 in patients with primary breast carcinoma. Methods: A total of 537 patients with primary breast cancer, and 54 with benign breast hyperplasia, were performed resection surgery in the same period were enrolled. Rsf-1 immunoexpression was retrospectively assessed by immunohistochemistry (IHC). As well as, it relationship with clinicopathological factors and patient survival (LRFS, DFS and OS) was investigated. Results: Compared with benign breast hyperplasia tissues, higher percentage of Rsf-1 positive expression was detected in malignant breast carcinomas. Based on IHC staining extent × intensity scores and ROC analysis, 278 of 526 cancers (52.9%) had high-expression (cut-off values 2.5) of Rsf-1, which correlated significantly to pathologic subtypes of breast cancer (DCIS vs. IDC, P < 0.001; ILC vs. IDC, P = 0.036), bigger tumor size (P = 0.030), higher TNM stage (P = 0.044), and p53-positive expression. In addition, there was a trend that high-expression of Rsf-1 associated with younger age (P = 0.053). We further prove that combined positive-expression of Rsf-1 and p53 (Rsf-1 (+)/p53 (+)) was correlated with the bigger tumor size (P = 0.018), and higher TNM stage (P = 0.024). Kaplan-Meier survival analysis showed that Rsf-1 high-expression and combined positive-expression of Rsf-1 and p53 (Rsf-1 (+)/p53 (+)) exhibited a significant correlation with poor overall survival of patients with primary breast cancer, and no association has been identified in relation to LRFS or DFS. Especially, Univariate and multivariate survival analysis demonstrated Rsf-1 expression is an independent prognostic parameter for the overall survival of patients with breast cancer. Conclusions: High-expression of Rsf-1 is associated with pathologic subtypes of breast cancer, aggressive phenotype, p53 positive and poor clinical outcome, which confers tumor aggressiveness through chromatin remodeling, and targeting Rsf-1 gene and the pathway it related may provide new therapeutic avenues for treating breast cancer. PMID:25337201

  10. Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

    SciTech Connect

    Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun; Choi, Yun Jin; Kim, Yong Sang; Ko, Kinarm; Koh, Yong-Gon

    2014-08-08

    Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.

  11. T Cell Coinhibition and Immunotherapy in Human Breast Cancer

    PubMed Central

    Janakiram, Murali; Abadi, Yael M.; Sparano, Joseph A.; Zang, Xingxing

    2014-01-01

    Costimulation and coinhibition generated by the B7 family and their receptor CD28 family have key roles in regulating T lymphocyte activation and tolerance. These pathways are very attractive therapeutic targets for human cancers including breast cancer. Gene polymorphisms of B7x (B7-H4/B7S1), PD-1 (CD279), and CTLA-4 (CD152) are associated with increased risk of developing breast cancer although the underlying mechanisms are unclear. In human breast cancer microenvironment, up-regulation of coinhibitory B7/CD28 members B7x, B7-H3 (CD276), and PD-L1 (B7-H1/CD274) on tumor cells as well as PD-1 and PD-L1 on tumor-infiltrating immune cells are emerging as immune evasion pathways. Chemotherapy can affect the expression of these molecules, and therefore may dampen the immune response against breast cancer. Immunotherapy targeting T cell coinhibition as monotherapy or combined with standard therapies are in early stages of clinical development, but hold great promise for treatment of human breast cancer. PMID:23114578

  12. Cation-selective transporters are critical to the AMPK-mediated antiproliferative effects of metformin in human breast cancer cells.

    PubMed

    Cai, Hao; Zhang, Yunhui; Han, Tianxiang Kevin; Everett, Ruth S; Thakker, Dhiren R

    2016-05-01

    The antidiabetic drug metformin exerts antineoplastic effects against breast cancer and other cancers. One mechanism by which metformin is believed to exert its anticancer effect involves activation of its intracellular target, adenosine monophosphate-activated protein kinase (AMPK), which is also implicated in the antidiabetic effect of metformin. It is proposed that in cancer cells, AMPK activation leads to inhibition of the mammalian target of rapamycin (mTOR) and the downstream pS6K that regulates cell proliferation. Due to its hydrophilic and cationic nature, metformin requires cation-selective transporters to enter cells and activate AMPK. This study demonstrates that expression levels of cation-selective transporters correlate with the antiproliferative and antitumor efficacy of metformin in breast cancer. Metformin uptake and antiproliferative activity were compared between a cation-selective transporter-deficient human breast cancer cell line, BT-20, and a BT-20 cell line that was engineered to overexpress organic cation transporter 3 (OCT3), a representative of cation-selective transporters and a predominant transporter in human breast tumors. Metformin uptake was minimal in BT-20 cells, but increased by >13-fold in OCT3-BT20 cells, and its antiproliferative potency was >4-fold in OCT3-BT20 versus BT-20 cells. This increase in antiproliferative activity was associated with greater AMPK phosphorylation and decreased pS6K phosphorylation in OCT3-BT20 cells. In vitro data were corroborated by in vivo observations of significantly greater antitumor efficacy of metformin in xenograft mice bearing OCT3-overexpressing tumors versus low transporter-expressing wildtype tumors. Collectively, these findings establish a clear relationship between cation-selective transporter expression, the AMPK-mTOR-pS6K signaling cascade, and the antiproliferative activity of metformin in breast cancer. PMID:26669511

  13. The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells.

    PubMed

    Honda, Makoto; Minami, Itsunari; Tooi, Norie; Morone, Nobuhiro; Nishioka, Hisae; Uemura, Kengo; Kinoshita, Ayae; Heuser, John E; Nakatsuji, Norio; Aiba, Kazuhiro

    2016-01-15

    Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-? 42 (A?42)/A?40 and A?43/A?40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery. PMID:26687948

  14. Over-expression of human endosulfatase-1 exacerbates cadmium-induced injury to transformed human lung cells in vitro

    SciTech Connect

    Zhang, Huiying; Department of Environmental and Molecular Toxicology, College of Agriculture and Life Sciences, NC State University, Raleigh, NC 27695 ; Newman, Donna R.; Bonner, James C.; Sannes, Philip L.

    2012-11-15

    Environmental exposure to cadmium is known to cause damage to alveolar epithelial cells of the lung, impair their capacity to repair, and result in permanent structural alterations. Cell surface heparan sulfate proteoglycans (HSPGs) can modulate cell responses to injury through their interactions with soluble effector molecules. These interactions are often sulfate specific, and the removal of sulfate groups from HS side chains could be expected to influence cellular injury, such as that caused by exposure to cadmium. The goal of this study was to define the role 6-O-sulfate plays in cellular responses to cadmium exposure in two pulmonary epithelial cancer cell lines (H292 and A549) and in normal human primary alveolar type II (hAT2) cells. Sulfate levels were modified by transduced transient over-expression of 6-O-endosulfatase (HSulf-1), a membrane-bound enzyme which specifically removes 6-O-sulfate groups from HSPG side chains. Results showed that cadmium decreased cell viability and activated apoptosis pathways at low concentrations in hAT2 cells but not in the cancer cells. HSulf-1 over-expression, on the contrary, decreased cell viability and activated apoptosis pathways in H292 and A549 cells but not in hAT2 cells. When combined with cadmium, HSulf-1 over-expression further decreased cell viability and exacerbated the activation of apoptosis pathways in the transformed cells but did not add to the toxicity in hAT2 cells. The finding that HSulf-1 sensitizes these cancer cells and intensifies the injury induced by cadmium suggests that 6-O-sulfate groups on HSPGs may play important roles in protection against certain environmental toxicants, such as heavy metals. -- Highlights: ► Primary human lung alveolar type 2 (hAT2) cells and H292 and A549 cells were used. ► Cadmium induced apoptosis in hAT2 cells but not in H292 or A549 cells. ► HSulf-1exacerbates apoptosis induced by cadmium in H292 and A549 but not hAT2 cells.

  15. Human breast milk: A review on its composition and bioactivity.

    PubMed

    Andreas, Nicholas J; Kampmann, Beate; Mehring Le-Doare, Kirsty

    2015-11-01

    Breast milk is the perfect nutrition for infants, a result of millions of years of evolution, finely attuning it to the requirements of the infant. Breast milk contains many complex proteins, lipids and carbohydrates, the concentrations of which alter dramatically over a single feed, as well as over lactation, to reflect the infant's needs. In addition to providing a source of nutrition for infants, breast milk contains a myriad of biologically active components. These molecules possess diverse roles, both guiding the development of the infants immune system and intestinal microbiota. Orchestrating the development of the microbiota are the human milk oligosaccharides, the synthesis of which are determined by the maternal genotype. In this review, we discuss the composition of breast milk and the factors that affect it during the course of breast feeding. Understanding the components of breast milk and their functions will allow for the improvement of clinical practices, infant feeding and our understanding of immune responses to infection and vaccination in infants. PMID:26375355

  16. Imaging the Redox States of Human Breast Cancer Core Biopsies

    PubMed Central

    Xu, H. N.; Tchou, J.; Chance, B.; Li, L. Z.

    2016-01-01

    Currently, the gold standard to establish benign vs. malignant breast tissue diagnosis requires an invasive biopsy followed by tissue fixation for subsequent histopathological examination. This process takes at least 24 h resulting in tissues that are less suitable for molecular, functional, or metabolic analysis. We have recently conducted redox scanning (cryogenic NADH/flavoprotein fluorescence imaging) on snap-frozen breast tissue biopsy samples obtained from human breast cancer patients at the time of their breast cancer surgery. The redox state was readily determined by the redox scanner at liquid nitrogen temperature with extraordinary sensitivity, giving oxidized flavoproteins (Fp) an up to tenfold discrimination of cancer to non-cancer of breast in our preliminary data. Our finding suggests that the identified metabolic parameters could discriminate between cancer and non-cancer breast tissues without subjecting tissues to fixatives. The remainder of the frozen tissue is available for additional analysis such as molecular analysis and conventional histopathology. We propose that this novel redox scanning procedure may assist in tissue diagnosis in ex vivo tissues. PMID:22879054

  17. A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone

    PubMed Central

    Thibaudeau, Laure; Taubenberger, Anna V.; Holzapfel, Boris M.; Quent, Verena M.; Fuehrmann, Tobias; Hesami, Parisa; Brown, Toby D.; Dalton, Paul D.; Power, Carl A.; Hollier, Brett G.; Hutmacher, Dietmar W.

    2014-01-01

    ABSTRACT The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact ‘organ’ bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo. PMID:24713276

  18. A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone.

    PubMed

    Thibaudeau, Laure; Taubenberger, Anna V; Holzapfel, Boris M; Quent, Verena M; Fuehrmann, Tobias; Hesami, Parisa; Brown, Toby D; Dalton, Paul D; Power, Carl A; Hollier, Brett G; Hutmacher, Dietmar W

    2014-02-01

    The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact 'organ' bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo. PMID:24713276

  19. In vivo over-expression of KGF mimic human middle ear cholesteatoma.

    PubMed

    Yamamoto-Fukuda, Tomomi; Akiyama, Naotaro; Shibata, Yasuaki; Takahashi, Haruo; Ikeda, Tohru; Koji, Takehiko

    2015-10-01

    We reported previously that keratinocyte growth factor (KGF), a mesenchymal cell-derived paracrine growth factor, plays an important role in middle ear cholesteatoma formation, which is characterized by marked proliferation of epithelial cells. Here, we investigated whether KGF, the main factor that induces cholesteatoma, overexpression in vivo results in the formation of cholesteatoma. Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal (EAC) of rats once (short-term model) or five times on every fourth day (long-term model). Ears transfected with empty vector were used as controls. Successful transfection of plasmids into epithelial and stromal cells was confirmed by Flag immunohistochemistry. In the short-term model, the intensity of KGF protein was the strongest in hKGF transfected ear at day 4. KGF expression induced epithelial cell proliferation, reaching a peak level at day 4 and then decreased later, while in the long-term model, KGF expression in the EAC led to middle ear cholesteatoma formation. In conclusion, we described here a new experimental model of human middle ear cholesteatoma, and demonstrated that KGF and KGF receptor paracrine action play an essential role in middle ear cholesteatoma formation in an in vivo model. PMID:25138153

  20. Fluorogold induces persistent neurological deficits and circling behavior in mice over-expressing human mutant tau.

    PubMed

    He, Zhen

    2009-02-01

    An increasing number of applications use nanospecie-fluorescent labeling technology; however, no established guidelines are available to warrant their safety for potential clinical use. Here, rTg4510 transgenic mice and their littermate controls were injected with fluorogold, a nanospecie tracer, or phosphate buffered saline (PBS) targeted to the right amygdala. No significant abnormal behavior was detected in any mice injected with PBS. After fluorogold injection, however, rTg4510 mice displayed persistent left-sided neurological deficits and left circling behavior for up to 14 days post-injection, while control mice demonstrated a transient syndrome. Mortality occurred only in rTg4510 mice and statistically significant differences appeared independent of age. An immunofluorescent study revealed TUNEL positive cells that were heavily and extensively distributed in the periamygdalar region that overlapped with the fluorogold deposit region in rTg4510 mice, whereas control mice showed only sporadic distribution of TUNEL-positive cells. Co-localization of TUNEL and caspase-3 active peptide immunoreactivity was identified in a subset of the cells, indicating an involvement of caspase-dependent apoptotic mechanisms. In conclusion, fluorogold induces damage in the central nervous system most noticeably in mice over-expressing human mutant tau. PMID:19355926

  1. Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

    SciTech Connect

    Diermeier, Simone; Horvath, Gabor; Knuechel-Clarke, Ruth; Hofstaedter, Ferdinand; Szoellosi, Janos; Brockhoff, Gero . E-mail: Gero.Brockhoff@klinik.uni-regensburg.de

    2005-04-01

    Background: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. Methods: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. Results: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. Conclusion: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and heterointeractions, as well as the presence or absence of growth factors. C-erbB2 overexpression alone is insufficient to predict the impact of growth factors and antibodies on cell proliferation. The optimization and specification of therapeutic approaches based on erbB-receptor targeting requires to account for EGFR coexpression as well as the potential presence of erbB-receptor relevant growth factors.

  2. Insulin like growth factor binding protein-7 reduces growth of human breast cancer cells and xenografted tumors.

    PubMed

    Amemiya, Y; Yang, W; Benatar, T; Nofech-Mozes, S; Yee, A; Kahn, H; Holloway, C; Seth, Arun

    2011-04-01

    Previously, we have shown that insulin-like growth factor binding protein-7 (IGFBP-7) expression is inversely correlated with disease progression in breast cancer and is associated with poor outcome. To further investigate the role of IGFBP-7 in the growth and metastatic behavior of breast cancer, primary breast tumors and metastatic tumors derived from the same patients were analyzed for IGFBP-7 expression. Immunohistochemical analysis revealed that IGFBP-7 is downregulated in half of the human metastatic breast tumors tested. IGFBP-7 has been linked to suppression of oncogenic pathways and can directly restore cellular senescence in melanomas, leading to their regression. It is possible that breast tumors with metastatic potential have escaped from IGFBP-7-induced suppression by its down-regulation. Twenty-two human primary breast tumor specimens were transplanted into human-bone NOD/SCID mice. One of the two triple negative primary breast tumors was serially xenotransplanted more than five times. Each serial transplant resulted in increased tumor take and rate of growth. Expression of IGFBP-7 was downregulated upon each serial implantation. To investigate the role of IGFBP-7 in breast tumor suppression, IGFBP-7 was overexpressed in the triple negative MDA-MB-468 human breast cancer line by stable transfection of a pSec-tag2-IGFBP-7 vector. The parental MDA-MB-468 breast cancer cells expressed extremely low levels of endogenous IGFBP-7. The production of IGFBP-7 protein by the MDA-MB-468 cells stably transfected with IGFBP-7 was confirmed by immunoblotting with anti-IGFBP-7 antibody. Ectopic overexpression of IGFBP-7 significantly reduced the growth of the IGFBP-7 transfected MDA-MB-468 cells compared to the parental MDA-MB-468 cells. We also assessed the role of IGFBP-7 on cell migration, a key determinant of malignant progression and metastasis. When parental MDA-MB-468 cells were treated with various amounts of conditioned medium derived from the IGFBP-7 overexpressing cell line, a significant difference in cell migration rate was observed between untreated and treated cells. IGFBP-7 strongly suppressed the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK-1/2, suggesting that IGFBP-7 mediates its anti-proliferative effects through negative feedback signaling. Levels of phospho-ERK-1/2 were higher in the parental MDA-MB-468 than in IGFBP-7-expressing cells derived from it. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468 transfected cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 controls. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein. PMID:20464481

  3. Globular adiponectin enhances invasion in human breast cancer cells

    PubMed Central

    FALK LIBBY, EMILY; LIU, JIANZHONG; LI, YI; LEWIS, MONICA J.; DEMARK-WAHNEFRIED, WENDY; HURST, DOUGLAS R.

    2016-01-01

    Every year, a large number of women succumb to metastatic breast cancer due to a lack of curative approaches for this disease. Adiponectin (AdipoQ) is the most abundant of the adipocyte-secreted adipokines. In recent years, there has been an interest in the use of AdipoQ and AdipoQ receptor agonists as therapeutic agents for the treatment of breast cancer. However, while multiple epidemiological studies have previously indicated that low levels of circulating plasma AdipoQ portend poor prognosis in patients with breast cancer, recent studies have reported that elevated expression levels of AdipoQ in breast tissue are correlated with advanced stages of the disease. Thus, the aim of the present study was to clarify the mechanism by which AdipoQ in breast tissue acts directly on tumor cells to regulate the early steps of breast cancer metastasis. In the present study, the effects of different AdipoQ isoforms on the metastatic potential of human breast cancer cells were investigated. The results revealed that globular adiponectin (gAd) promoted invasive cell morphology and significantly increased the migration and invasion abilities of breast cancer cells, whereas full-length adiponectin (fAd) had no effect on these cells. Additionally, gAd, but not fAd, increased the expression levels of microtubule-associated protein 1 light chain 3 beta (LC3B)-II and intracellular LC3B puncta, which are indicators of autophagosome formation, thus suggesting autophagic induction by gAd. Furthermore, the inhibition of autophagic function by autophagy-related protein 7 knockdown attenuated the gAd-induced increase in invasiveness in breast cancer cells. Therefore, the results of the present study suggested that a specific AdipoQ isoform may enhance breast cancer invasion, possibly via autophagic induction. Understanding the roles of the different AdipoQ isoforms as microenvironmental regulatory molecules may aid the development of effective AdipoQ-based treatments for breast cancer. PMID:26870258

  4. Transgenic mice overexpressing human G972R IRS-1 show impaired insulin action and insulin secretion.

    PubMed Central

    Hribal, Marta L; Tornei, F; Pujol, A; Menghini, R; Barcaroli, D; Lauro, D; Amoruso, R; Lauro, R; Bosch, F; Sesti, G; Federici, M

    2008-01-01

    Molecular scanning of human insulin receptor substrate (Irs) genes revealed a single lrs1 prevalent variant, a glycine to arginine change at codon 972 (G972R); previous in vitro studies had demonstrated that the presence of this variant results in an impaired activation of the insulin signalling pathway, while human studies gave controversial results regarding its role in the pathogenesis of insulin resistance and related diseases. To address in vivo impact of this IRS-1 variant on whole body glucose homeostasis and insulin signalling, we have generated transgenic mice overexpressing it (Tg972) and evaluated insulin action in the liver, skeletal muscle and adipose tissue and assessed glucose homeostasis both under a normal diet and a high-fat diet. We found that Tg972 mice developed age-related glucose and insulin intolerance and hyperglycaemia, with insulin levels comparatively low. Glucose utilization and insulin signalling were impaired in all key insulin target tissues in Tg972 mice. There were no differences in pancreatic morphology between Tg972 and wild-type mice, however when insulin secretion was evaluated in isolated islets, it was significantly reduced in Tg972 mice islets at any glucose concentration tested. Under a high-fat diet, Tg972 mice had increased body and adipose tissue weight, and were more prone to develop diet-induced glucose and insulin intolerance. So, we believe that Tg972 mice may represent a useful model to elucidate the interaction between genetic and environmental factors in insulin resistance pathogenesis. Furthermore, they may become an important tool to test novel tailored therapies. PMID:18208559

  5. Bag1 proteins regulate growth and survival of ZR-75-1 human breast cancer cells.

    PubMed

    Kudoh, Masafumi; Knee, Deborah A; Takayama, Shinichi; Reed, John C

    2002-03-15

    Bag1 proteins bind heat shock protein M(r) 70,000 (Hsp 70) family molecular chaperones and regulate diverse pathways involved in cell proliferation, apoptosis, and stress responses. Four isoforms of Bag1 can be produced from a single gene in humans, including a nuclear-targeted long version (Bag1L)and a shorter cytosolic isoform (Bag1). Because overexpression of Bag1and Bag1L has been reported in breast cancers, we explored the effects of Bag1 and Bag1L on the growth of ZR-75-1 human breast cancer cells cultured in vitro and in tumor xenograft models using immunocompromised mice. Cells stably transfected with expression plasmids encoding either Bag1 or Bag1L displayed comparable rates of growth in cultures containing 10% serum, compared with control-transfected ZR-75-1 cells. In contrast, ZR-75-1 cells stably expressing mutants of Bag1 or Bag1L, which lack the COOH-terminal domain (DeltaC) required for heat shock protein M(r) 70,000 binding, displayed retarded growth rates. When cultured without serum, the viability of control-transfected, as well as Bag1DeltaC- and Bag1LDeltaC-expressing, cells declined with time, whereas Bag1- and Bag1L-overexpressing ZR-75-1 cells survived for over a week in culture. Caspase protease activation induced by serum deprivation was also prevented by stable expression of either Bag1 or Bag1L in ZR-75-1 cells. In addition, sensitivity to anchorage dependence was restored partially in ZR-75-1 cells expressing dominant-negative Bag1DeltaC and Bag1LDeltaC. In tumor xenograft studies involving injection of ZR-75-1 cells into mammary fat pads of female nu/nu mice, ZR-75-1 cells expressing Bag1 or Bag1L formed 1.4-1.6-fold larger tumors compared with control-transfected cells, whereas tumors formed by Bag1DeltaC- and Bag1LDeltaC-expressing cells grew very slowly and reached sizes < one-third of tumors generated by Neo-control ZR-75-1 cells. Altogether, these findings demonstrate that Bag1 and Bag1L provoke similar changes in breast cancer cell growth and survival and suggest that interference with Bag1 or Bag1L function might be a useful strategy for opposing breast cancer. PMID:11912172

  6. SALL4 is essential for cancer cell proliferation and is overexpressed at early clinical stages in breast cancer.

    PubMed

    Kobayashi, Daisuke; Kuribayshi, Kageaki; Tanaka, Maki; Watanabe, Naoki

    2011-04-01

    Few target molecules have been identified that enable the diagnosis of breast cancer with a high sensitivity and specificity, especially in the early clinical stages of cancer. Here, we present the first evidence for diagnostic performance of gene expression for SALL4, a transcription factor that plays an essential role in the embryonic development and self-renewal of embryonic stem (ES) cells, in breast cancer. The sensitivity and specificity of SALL4 was 80.4 and 80.0%, respectively, as estimated using the cut-off value obtained from the analysis of the receiver operating characteristic curve. Furthermore, comparison of paired cancer and non-cancer tissues from the same breast cancer patient revealed elevated SALL4 mRNA levels in 86.1% (31/36) of the specimens. No obvious correlations were detected between clinicopathological factors and SALL4 mRNA expression; however, SALL4 mRNA was expressed at a high level even in the early clinical stages of the cancer. An siRNA experiment to determine the significance of SALL4 expression showed complete inhibition of proliferation in breast cancer MCF7 cells. This inhibitory effect of siRNA was induced by cell cycle arrest mainly at the G1 phase, leading to increased cell volume. These results suggest that SALL4 mRNA may be a new tool to support the diagnosis of breast cancer, and it may also represent a novel therapeutic target. PMID:21274508

  7. Overexpression of miR-100 inhibits cell proliferation, migration, and chemosensitivity in human glioblastoma through FGFR3

    PubMed Central

    Luan, Yongxin; Zhang, Shuyan; Zuo, Ling; Zhou, Lixiang

    2015-01-01

    Background Glioblastoma multiforme is one of the most deadly forms of brain cancer. We investigated the regulatory effects of microRNA-100 (miR-100) on cell proliferation, migration, and chemosensitivity in human glioblastoma. Methods miR-100 expression was assessed by quantitative real-time polymerase chain reaction in both glioblastoma cells and human tumors. Lentiviruses of miR-100 mimics and inhibitors were transfected into U251 and T98G cells. The regulatory effects of either overexpressing or downregulating miR-100 on glioblastoma were evaluated by a viability assay, growth assay, migration assay, chemosensitivity assay, and an in vivo tumor transplantation assay. Expression of fibroblast growth factor receptor 3 (FGFR3), the bioinformatically predicted target of miR-100, was examined by Western blot in glioblastoma. FGFR3 was then ectopically overexpressed in U251 and T98G cells, and its effects on miR-100-mediated cancer regulation were evaluated by growth, migration, and chemosensitivity assays. Results MiR-100 was markedly downregulated in both glioblastoma cell lines and human tumors. Overexpressing miR-100 through lentiviral transfection in U251 and T98G cells significantly inhibited cancer growth (both in vitro and in vivo) and migration and increased chemosensitivity to cisplatin and 1, 3-bis (2-chloroethyl)-l-nitrosourea, whereas downregulation of miR-100 had no effects on development of cancer. FGFR3 was directly regulated by miR-100 in glioblastoma. Ectopically overexpressing FGFR3 was able to ameliorate the anticancer effects of upregulation of miR-100 on glioblastoma growth, migration, and chemosensitivity. Conclusion MiR-100 was generally downregulated in glioblastoma. Overexpressing miR-100 had anticancer effects on glioblastoma, likely through regulation of FGFR3. The MiR-100/FGFR3 signaling pathway might be a biochemical target for treatment in patients with glioblastoma. PMID:26604796

  8. Aging of stromal-derived human breast fibroblasts might contribute to breast cancer progression.

    PubMed

    Martens, John W M; Sieuwerts, Anieta M; Bolt-deVries, Joan; Bosma, Peter T; Swiggers, Susan J J; Klijn, Jan G M; Foekens, John A

    2003-02-01

    Age is an important factor in the development and spread of breast cancer. Stromal cells also contribute to breast cancer growth and metastasis through the production of extracellular matrix (ECM) modifiers such as urokinase type plasminogen activator (uPA), its receptor (uPAR), its inhibitors (PAI-1 and PAI-2), matrix metalloproteinases (MMPs), and growth factors, including the fibroblast and insulin-like growth factors (FGF's and IGF's). In the present study we have investigated whether breast fibroblasts aged in vitro through passage in culture display altered levels of the plasminogen activator system and growth factors that are known to modulate that system. With real-time RT-PCR we found that during passage human breast fibroblasts, whether derived from the tumour burden or from matched adjacent normal breast tissue, exhibited a consistent increase in PAI-1 and FGF-1 and a decrease in MMP-2 mRNA expression. In addition, in 5 out of 7 fibroblast strains we observed an induction of uPA expression in combination with a reduced IGF-1 expression. Interestingly, while during aging MMP-2 protein increased in all tumour-derived fibroblast strains, these protein levels were reduced in all normal tissue- derived fibroblasts. No other clear-cut age-dependent alterations were found in the all-together 25 factors investigated. We furthermore demonstrate in one tumour-derived fibroblast strain that the increases in uPA and PAI-1 mRNA and MMP-2 protein production are inversely related to the telomere length. Artificially increasing telomere length in this fibroblast strain by expressing human telomerase reverse transcriptase (hTERT) prevented senescence and resulted in late passage cultures with early passage uPA, PAI-1 and MMP-2 levels. Our results show that aging accompanied by telomere loss induces PAI-1 and FGF-1 mRNA expression in all breast fibroblast strains, increases uPA and decreases IGF-1 mRNA expression in a subset, and increases MMP-2 protein expression only in tumour-derived breast fibroblasts. These age-induced levels of PAI-1, FGF-1, uPA and MMP-2 in stromal breast fibroblast could contribute to breast cancer progression. PMID:12574821

  9. Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors

    PubMed Central

    Rycaj, Kiera; Plummer, Joshua B.; Li, Ming; Yin, Bingnan; Frerich, Katherine; Garza, Jeremy G.; Shen, Jianjun; Lin, Kevin; Yan, Peisha; Glynn, Sharon A.; Dorsey, Tiffany H.; Hunt, Kelly K.; Ambs, Stefan; Johanning, Gary L.

    2012-01-01

    Background The envelope (env) protein of the human endogenous retrovirus type K (HERV-K) family is commonly expressed on the surface of breast cancer cells. We assessed whether HERV-K env is a potential target for antibody-based immunotherapy of breast cancer. Methods We examined the expression of HERV-K env protein in various malignant (MDA-MB-231, MCF-7, SKBR3, MDA-MB-453, T47D, and ZR-75-1) and nonmalignant (MCF-10A and MCF-10AT) human breast cell lines by immunoblot, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry. Anti-HERV-K env monoclonal antibodies (mAbs; 6H5, 4D1, 4E11, 6E11, and 4E6) were used to target expression of HERV-K, and antitumor effects were assessed by quantifying growth and apoptosis of breast cancer cells in vitro, and tumor growth in vivo in mice (n = 5 per group) bearing xenograft tumors. The mechanisms responsible for 6H5 mAbmediated effects were investigated by microarray assays, flow cytometry, immunoblot, and immunofluorescence staining. The expression of HERV-K env protein was assessed in primary breast tumors (n = 223) by immunohistochemistry. All statistical tests were two-sided. Results The expression of HERV-K env protein in malignant breast cancer cell lines was substantially higher than nonmalignant breast cells. AntiHERV-K-specific mAbs inhibited growth and induced apoptosis of breast cancer cells in vitro. Mice treated with 6H5 mAb showed statistically significantly reduced growth of xenograft tumors compared with mice treated with control immunoglobulin (control [mIgG] vs 6H5 mAb, for tumors originating from MDA-MB-231 cells, mean size = 1448.33 vs 475.44 mm3; difference = 972.89 mm3, 95% CI = 470.17 to 1475.61 mm3; P < .001). Several proteins involved in the apoptotic signaling pathways were overexpressed in vitro in 6H5 mAbtreated malignant breast cells compared with mIgG-treated control. HERV-K expression was detected in 148 (66%) of 223 primary breast tumors, and a higher rate of lymph node metastasis was associated with HERV-K-positive compared with HERV-K-negative tumors (43% vs 23%, P = .003). Conclusion Monoclonal antibodies against HERV-K env protein show potential as novel immunotherapeutic agents for breast cancer therapy. PMID:22247020

  10. Clinicopathological correlation and prognostic significance of sonic hedgehog protein overexpression in human gastric cancer

    PubMed Central

    Niu, Yanyang; Li, Fang; Tang, Bo; Shi, Yan; Hao, Yingxue; Yu, Peiwu

    2014-01-01

    Objectives: This study investigated the expression of Sonic Hedgehog (Shh) protein in gastric cancer, and correlated it with clinicopathological parameters. The prognostic significance of Shh protein was analyzed. Methods: Shh protein expression was evaluated in 113 cases of gastric cancer and 60 cases of normal gastric mucosa. The immunoreactivity was scored semi quantitatively as: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong. All cases were further classified into two groups, namely non-overexpression group with score 0 or 1, and overexpression group with score 2 or 3. The overexpression of Shh protein was correlated with clinicopathological parameters. Survival analysis was then performed to determine the Shh protein prognostic significance in gastric cancer. Results: In immunohistochemistry study, nineteen (31.7%) normal gastric mucosa revealed Shh protein overexpression, while eighty-one (71.7%) gastric cancer revealed overexpression. The expression of Shh protein were significantly higher in gastric cancer tissues than in normal gastric mucosa (P < 0.001), which was statistically correlated with age (P = 0.006), tumor differentiation (P < 0.001), depth of invasion (P = 0.042), pathologic staging (P = 0.017), and nodal metastasis (P = 0.019). We found no significant difference in both overall and disease free survival rates between Shh overexpression and non-expression groups P = 0.168 and 0.071). However, Shh overexpression emerged as a significant independent prognostic factor in multivariate Cox regression analysis (hazard ratio 1.187, P = 0.041). Conclusions: Shh protein expression is upregulated and is statistically correlated with age, tumor differentiation, depth of invasion, pathologic staging, and nodal metastasis. The Shh protein overexpression is a significant independent prognostic factor in multivariate Cox regression analysis in gastric cancer. PMID:25197388

  11. Can estrogen receptor overexpression in normal tissues due to previous estrogen deprivation explain the fulvestrant efficacy in breast cancer therapy?

    PubMed

    Kurbel, Sven

    2012-12-01

    Fulvestrant is a down-regulator of estrogen receptors (ERs) with still evolving optimal dosage for ER-positive breast cancer patients. The CONFIRM phase III trial in women with advanced breast cancer proved fulvestrant 500-mg to be associated with a longer time till progression (TTP) than the 250-mg schedule. Detailed results suggest that the fulvestrant in both schedules depended on the previous endocrine therapy. All complete responses and the only significant TTP difference between the two schedules was found among women previously treated with tamoxifen (TAM) and not in women after aromatase inhibitors (AIs). Noting that TAM competes with estrogen binding to ERs is important, so the optimal TAM dosage produces drug concentrations comparable to concentrations of available ER ligands. All AIs diminish production of the main ER ligand, so the optimal AI dosage depends on the overall pool of aromatase molecules in the body. Both treatments are not directly related to the pool of available ERs in the body. Here proposed interpretation is that estrogen deprivation due to years of endocrine breast cancer therapy increases ER expression in breast cancer cells and in other healthy estrogen target tissues. The breast cancer exposure to fulvestrant depends on the presence of all ERs in the body. Only when this overall pool is sufficiently saturated with fulvestrant, we can expect to achieve some breast cancer response due to down-regulation of ER in cancer tissue. The CONFIRM data suggest that among patients switching from TAM to fulvestrant, only the 500-mg schedule could down-regulate the moderately enlarged total body ER pool and thus induce breast cancer regression. In patients switching from previous AI treatments, both 250 and 500-mg schedules were unable to prolong the TTP, suggesting that in both doses, fulvestrant showed no efficacy since the overall ER pool was more enlarged after AIs. Fulvestrant might be more effective before TAM and AIs, in the first line endocrine therapy of metastatic breast cancer, since an unaltered ER pool in normal tissues is expected in this setting. PMID:23062772

  12. Ocular input for human melatonin regulation: relevance to breast cancer

    NASA Technical Reports Server (NTRS)

    Glickman, Gena; Levin, Robert; Brainard, George C.

    2002-01-01

    The impact of breast cancer on women across the world has been extensive and severe. As prevalence of breast cancer is greatest in industrialized regions, exposure to light at night has been proposed as a potential risk factor. This theory is supported by the epidemiological observations of decreased breast cancer in blind women and increased breast cancer in women who do shift-work. In addition, human, animal and in vitro studies which have investigated the melatonin-cancer dynamic indicate an apparent relationship between light, melatonin and cancer, albeit complex. Recent developments in understanding melatonin regulation by light in humans are examined, with particular attention to factors that contribute to the sensitivity of the light-induced melatonin suppression response. Specifically, the role of spectral characteristics of light is addressed, and recent relevant action spectrum studies in humans and other mammalian species are discussed. Across five action spectra for circadian and other non-visual responses, a peak sensitivity between 446-484 nm was identified. Under highly controlled exposure circumstances, less than 1 lux of monochromatic light elicited a significant suppression of nocturnal melatonin. In view of the possible link between light exposure, melatonin suppression and cancer risk, it is important to continue to identify the basic related ocular physiology. Visual performance, rather than circadian function, has been the primary focus of architectural lighting systems. It is now necessary to reevaluate lighting strategies, with consideration of circadian influences, in an effort to maximize physiological homeostasis and health.

  13. The use of ?-conotoxin ImI to actualize the targeted delivery of paclitaxel micelles to ?7 nAChR-overexpressing breast cancer.

    PubMed

    Mei, Dong; Lin, Zhiqiang; Fu, Jijun; He, Bing; Gao, Wei; Ma, Ling; Dai, Wenbing; Zhang, Hua; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Lu, Wanliang; Zhou, Demin; Zhang, Qiang

    2015-02-01

    Alpha7 nicotinic acetylcholine receptor (?7 nAChR), a ligand-gated ion channel, is increasingly emerging as a new tumor target owing to its expression specificity and significancy for cancer. In an attempt to increase the targeted drug delivery to the ?7 nAChR-overexpressing tumors, herein, ?-conotoxin ImI, a disulfide-rich toxin with highly affinity for ?7 nAChR, was modified on the PEG-DSPE micelles (ImI-PMs) for the first time. The DLS, TEM and HPLC detections showed the spherical nanoparticle morphology about 20 nm with negative charge and high drug encapsulation. The ligand modification did not induce significant differences. The immunofluorescence assay confirmed the expression level of ?7 nAChR in MCF-7 cells. In vitro and in vivo experiments demonstrated that the ?7 nAChR-targeted nanomedicines could deliver more specifically and faster into ?7 nAChR-overexpressing MCF-7 cells. Furthermore, fluo-3/AM fluorescence imaging technique indicated that the increased specificity was attributed to the ligand-receptor interaction, and the inducitivity for intracellular Ca(2+) transient by ImI was still remained after modification. Moreover, paclitaxel, a clinical frequently-used anti-tumor drug for breast cancer, was loaded in ImI-modified nanomedicines to evaluate the targeting efficacy. Besides of exhibiting greater cytotoxicity and inducing more cell apoptosis in vitro, paclitaxel-loaded ImI-PMs displayed stronger anti-tumor efficacy in MCF-7 tumor-bearing nu/nu mice. Finally, the active targeting system showed low systemic toxicity and myelosuppression evidenced by less changes in body weight, white blood cells, neutrophilic granulocyte and platelet counts. In conclusion, ?7 nAChR is also a promising target for anti-tumor drug delivery and in this case, ?-conotoxin ImI-modified nanocarrier is a potential delivery system for targeting ?7 nAChR-overexpressing tumors. PMID:25542793

  14. Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2+ breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry

    PubMed Central

    Caldern-Gonzlez, Karla Grisel; Valero Rustarazo, Ma Luz; Labra-Barrios, Maria Luisa; Bazn-Mndez, Csar Isaac; Tavera-Tapia, Alejandra; Herrera-Aguirre, Mar;aEsther; Snchez del Pino, Manuel M.; Gallegos-Prez, Jos Luis; Gonzlez-Mrquez, Humberto; Hernndez-Hernndez, Jose Manuel; Len-vila, Gloria; Rodrguez-Cuevas, Sergio; Guisa-Hohenstein, Fernando; Luna-Arias, Juan Pedro

    2015-01-01

    Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry (Caldern-Gonzlez et al. [1] in press). PMID:26217805

  15. Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2(+) breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry.

    PubMed

    Caldern-Gonzlez, Karla Grisel; Valero Rustarazo, Ma Luz; Labra-Barrios, Maria Luisa; Bazn-Mndez, Csar Isaac; Tavera-Tapia, Alejandra; Herrera-Aguirre, Mar aEsther; Snchez Del Pino, Manuel M; Gallegos-Prez, Jos Luis; Gonzlez-Mrquez, Humberto; Hernndez-Hernndez, Jose Manuel; Len-vila, Gloria; Rodrguez-Cuevas, Sergio; Guisa-Hohenstein, Fernando; Luna-Arias, Juan Pedro

    2015-09-01

    Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article "Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry" (Caldern-Gonzlez et al. [1] in press). PMID:26217805

  16. Transgenic rats overexpressing the human MrgX3 gene show cataracts and an abnormal skin phenotype

    SciTech Connect

    Kaisho, Yoshihiko . E-mail: Kaisho_Yoshihiko@takeda.co.jp; Watanabe, Takuya; Nakata, Mitsugu; Yano, Takashi; Yasuhara, Yoshitaka; Shimakawa, Kozo; Mori, Ikuo; Sakura, Yasufumi; Terao, Yasuko; Matsui, Hideki; Taketomi, Shigehisa

    2005-05-13

    The human MrgX3 gene, belonging to the mrgs/SNSRs (mass related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.

  17. Simultaneous topographic and recognition imaging of epidermal growth factor receptor (EGFR) on single human breast cancer cells.

    PubMed

    Xiao, Lifu; Chen, Qian; Wu, Yangzhe; Qi, Xiaojun; Zhou, Anhong

    2015-10-01

    Epidermal growth factor receptor (EGFR) plays an important role in signaling pathway of the development of breast cancer cells. Since EGFR overexpresses in most breast cancer cells, it is regarded as a biomarker molecule of breast cancer cells. Here we demonstrated a new AFM technique-topography and recognition (TREC) imaging-to simultaneously obtain highly sensitive and specific molecular recognition images and high-resolution topographic images of EGFR on single breast cancer cells. PMID:26002322

  18. Emotional memory impairments induced by AAV-mediated overexpression of human ?-synuclein in dopaminergic neurons of the ventral tegmental area.

    PubMed

    Alvarsson, A; Caudal, D; Bjrklund, A; Svenningsson, P

    2016-01-01

    Parkinson's disease (PD) is associated with extensive degeneration of dopaminergic neurons originating in the substantia nigra pars compacta, but neuronal loss is also found in the ventral tegmental area (VTA). The VTA projects to areas involved in cognitive and emotional processes, including hippocampus, amygdala, nucleus accumbens and prefrontal cortex, and has thus been proposed to play a role in emotional memory impairments in PD. Since the formation of ?-synuclein inclusions throughout the central nervous system is a pathological hallmark of PD, we studied the progressive effects of ?-synuclein overexpression in the VTA on motor functions, emotional behaviour and emotional memory. Adeno-associated viral (AAV) vectors encoding either human ?-synuclein or green fluorescent protein (GFP) were injected stereotactically into the VTA, and behaviour was monitored 3 and 8 weeks following AAV injection. At week 8, there was a 22% reduction of TH+ neurons in the VTA. We demonstrate that ?-synuclein overexpression in dopaminergic neurons of the VTA induced mild motor deficits that appeared 3 weeks following AAV-?-synuclein injection and were aggravated at week 8. No depressive- or anxiety-like behaviours were found. To address emotional memory, we used the passive avoidance test, a one-trial associative learning paradigm based on contextual conditioning which requires minimal training. Interestingly, emotional memory impairments were found in ?-synuclein overexpressing animals at week 8. These findings indicate that ?-synuclein overexpression induces progressive memory impairments likely caused by a loss of function of mesolimbic dopaminergic projections. PMID:26341317

  19. Excretion of mefloquine in human breast milk.

    PubMed

    Edstein, M D; Veenendaal, J R; Hyslop, R

    1988-01-01

    Concentrations of mefloquine in plasma and breast milk were measured in 2 women following the administration of one Lariam tablet (250 mg mefloquine). The milk-to-plasma ratio of mefloquine based on the area under the plasma and milk concentration curves was 0.13 and 0.16. After a maternal mefloquine dose of 3.73 mg/kg and assuming a daily milk ingestion of 1 litre over a week, the maximum amount of drug ingested by an infant would be 0.14 mg/kg. During lactation the plasma clearance and apparent volume of distribution of mefloquine were about 50% less than the same parameters calculated after lactation had ceased. PMID:3262044

  20. Bioconjugation of Calcium Phosphate Nanoparticles for Selective Targeting of Human Breast and Pancreatic Cancers In Vivo

    PubMed Central

    Sharma, Rahul; Barth, Brian M.; Altino?lu, Erhan ?.; Morgan, Thomas T.; Shanmugavelandy, Sriram S.; Kaiser, James M.; McGovern, Christopher; Matters, Gail L.; Smith, Jill P.; Kester, Mark; Adair, James H.

    2010-01-01

    The early diagnosis of cancer is the critical element in successful treatment and long term favorable patient prognoses. The high rate of mortality is mainly attributed to the tendency for late diagnoses as symptoms may not occur until the disease has metastasized, as well as the lack of effective systemic therapies. Late diagnosis is often associated with the lack of timely sensitive imaging modalities. The promise of nanotechnology is presently limited by the inability to simultaneously seek, treat and image cancerous lesions. This study describes the design and synthesis of fluorescent calcium phosphosilicate nanocomposite particles (CPNPs) that can be systemically targeted to breast and pancreatic cancer lesions. The CPNPs are a ~20nm diameter composite composed of an amorphous calcium phosphate matrix doped with silicate in which a near infra-red imaging agent indocyanine green (ICG) is embedded. In the present studies, we describe and validate CPNP bioconjugation of human holotransferrin, anti-CD71 antibody, and short gastrin peptides via an avidin-biotin- or a novel PEG-maleimide-coupling strategy. The conjugation of biotinylated human holotransferrin (diferric transferrin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to avidin conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors, which are highly expressed on breast cancer cells. Similarly, the conjugation of biotinylated pentagastrin to Avidin-CPNPs and decagastrin (gastrin-10) to PEG-CPNPs via PEG-maleimide coupling permits targeting of gastrin receptors, which are over-expressed in pancreatic cancer lesions. These bioconjugated CPNPs have the potential to perform as a theranostic modality, simultaneously enhancing drug delivery, targeting and imaging of breast and pancreatic cancer tumors. PMID:20180585

  1. Generation and characterization of a breast carcinoma model by PyMT overexpression in mammary epithelial cells of tree shrew, an animal close to primates in evolution.

    PubMed

    Ge, Guang-Zhe; Xia, Hou-Jun; He, Bao-Li; Zhang, Hai-Lin; Liu, Wen-Jing; Shao, Ming; Wang, Chun-Yan; Xiao, Ji; Ge, Fei; Li, Fu-Bing; Li, Yi; Chen, Ceshi

    2016-02-01

    The tree shrew is becoming an attractive experimental animal model for human breast cancer owing to a closer relationship to primates/humans than rodents. Tree shrews are superior to classical primates because tree shrew are easier to manipulate, maintain and propagate. It is required to establish a high-efficiency tree shrew breast cancer model for etiological research and drug assessment. Our previous studies suggest that 7,12-dimethylbenz(a)anthracene (DMBA) and medroxyprogesterone acetate (MPA) induce breast tumors in tree shrews with a low frequency (<50%) and long latency (∼7-month), making these methods less than ideal. We induced mammary tumors in tree shrew (Tupaia belangeri chinensis) by injection of lentivirus expressing the PyMT oncogene into mammary ducts of 22 animals. Most tree shrews developed mammary tumors with a latency of about three weeks, and by 7 weeks all injected tree shrews had developed mammary tumors. Among these, papillary carcinoma is the predominant tumor type. One case showed lymph node and lung metastasis. Interestingly, the expression levels of phosphorylated AKT, ERK and STAT3 were elevated in 41-68% of PyMT-induced mammary tumors, but not all tumors. Finally, we observed that the growth of PyMT-induced tree shrew mammary tumors was significantly inhibited by Cisplatin and Epidoxorubicin. PyMT-induced tree shrew mammary tumor model may be suitable for further breast cancer research and drug development, due to its high efficiency and short latency. PMID:26296387

  2. Characterization of human breast cancer by scanning acoustic microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Di; Malyarenko, Eugene; Seviaryn, Fedar; Yuan, Ye; Sherman, Mark; Bandyopadhyay, Sudeshna; Gierach, Gretchen; Greenway, Christopher W.; Maeva, Elena; Strumban, Emil; Duric, Neb; Maev, Roman

    2013-03-01

    Objectives: The purpose of this study was to characterize human breast cancer tissues by the measurement of microacoustic properties. Methods: We investigated eight breast cancer patients using acoustic microscopy. For each patient, seven blocks of tumor tissue were collected from seven different positions around a tumor mass. Frozen sections (10 micrometer, ?m) of human breast cancer tissues without staining and fixation were examined in a scanning acoustic microscope with focused transducers at 80 and 200 MHz. Hematoxylin and Eosin (H and E) stained sections from the same frozen breast cancer tissues were imaged by optical microscopy for comparison. Results: The results of acoustic imaging showed that acoustic attenuation and sound speed in cancer cell-rich tissue regions were significantly decreased compared with the surrounding tissue regions, where most components are normal cells/tissues, such as fibroblasts, connective tissue and lymphocytes. Our observation also showed that the ultrasonic properties were influenced by arrangements of cells and tissue patterns. Conclusions: Our data demonstrate that attenuation and sound speed imaging can provide biomechanical information of the tumor and normal tissues. The results also demonstrate the potential of acoustic microscopy as an auxiliary method for operative detection and localization of cancer affected regions.

  3. Combating HER2-overexpressing breast cancer through induction of calreticulin exposure by Tras-Permut CrossMab

    PubMed Central

    Zhang, Fan; Zhang, Jie; Liu, Moyan; Zhao, Lichao; LingHu, RuiXia; Feng, Fan; Gao, Xudong; Jiao, Shunchang; Zhao, Lei; Hu, Yi; Yang, Junlan

    2015-01-01

    Although trastuzumab has succeeded in breast cancer treatment, acquired resistance is one of the prime obstacles for breast cancer therapies. There is an urgent need to develop novel HER2 antibodies against trastuzumab resistance. Here, we first rational designed avidity-imporved trastuzumab and pertuzumab variants, and explored the correlation between the binding avidity improvement and their antitumor activities. After characterization of a pertuzumab variant L56TY with potent antitumor activities, a bispecific immunoglobulin G-like CrossMab (Tras-Permut CrossMab) was generated from trastuzumab and binding avidity-improved pertuzumab variant L56TY. Although, the antitumor efficacy of trastuzumab was not enhanced by improving its binding avidity, binding avidity improvement could significantly increase the anti-proliferative and antibody-dependent cellular cytotoxicity (ADCC) activities of pertuzumab. Further studies showed that Tras-Permut CrossMab exhibited exceptional high efficiency to inhibit the progression of trastuzumab-resistant breast cancer. Notably, we found that calreticulin (CRT) exposure induced by Tras-Permut CrossMab was essential for induction of tumor-specific T cell immunity against tumor recurrence. These data indicated that simultaneous blockade of HER2 protein by Tras-Permut CrossMab could trigger CRT exposure and subsequently induce potent tumor-specific T cell immunity, suggesting it could be a promising therapeutic strategy against trastuzumab resistance. PMID:25949918

  4. Overexpression of myocardin induces partial transdifferentiation of human?induced pluripotent stem cell?derived mesenchymal stem cells into cardiomyocytes

    PubMed Central

    Zhang, Jiao; Ho, Jenny Chung?Yee; Chan, Yau?Chi; Lian, Qizhou; Siu, Chung?Wah; Tse, Hung?Fat

    2014-01-01

    Abstract Mesenchymal stem cells (MSCs) derived from human?induced pluripotent stem cells (iPSCs) show superior proliferative capacity and therapeutic potential than those derived from bone marrow (BM). Ectopic expression of myocardin further improved the therapeutic potential of BM?MSCs in a mouse model of myocardial infarction. The aim was of this study was to assess whether forced myocardin expression in iPSC?MSCs could further enhance their transdifferentiation to cardiomyocytes and improve their electrophysiological properties for cardiac regeneration. Myocardin was overexpressed in iPSC?MSCs using viral vectors (adenovirus or lentivirus). The expression of smooth muscle cell and cardiomyocyte markers, and ion channel genes was examined by reverse transcription?polymerase chain reaction (RT?PCR), immunofluorescence staining and patch clamp. The conduction velocity of the neonatal rat ventricular cardiomyocytes cocultured with iPSC?MSC monolayer was measured by multielectrode arrays recording plate. Myocardin induced the expression of ??MHC, GATA4, ??actinin, cardiac MHC, MYH11, calponin, and SM ??actin, but not cTnT, ??MHC, and MLC2v in iPSC?MSCs. Overexpression of myocardin in iPSC?MSC enhanced the expression of SCN9A and CACNA1C, but reduced that of KCa3.1 and Kir2.2 in iPSC?MSCs. Moreover, BKCa, IKir, ICl, Ito and INa.TTX were detected in iPSC?MSC with myocardin overexpression; while only BKCa, IKir, ICl, IKDR, and IKCa were noted in iPSC?MSC transfected with green florescence protein. Furthermore, the conduction velocity of iPSC?MSC was significantly increased after myocardin overexpression. Overexpression of myocardin in iPSC?MSCs resulted in partial transdifferentiation into cardiomyocytes phenotype and improved the electrical conduction during integration with mature cardiomyocytes. PMID:24744906

  5. Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer.

    PubMed Central

    Moscow, J A; Townsend, A J; Goldsmith, M E; Whang-Peng, J; Vickers, P J; Poisson, R; Legault-Poisson, S; Myers, C E; Cowan, K H

    1988-01-01

    The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs). We have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (AdrR MCF7), the anionic isozyme of glutathione S-transferase (GST pi). Hybridization with this GST pi cDNA, GST pi-1, demonstrated that increased GST pi activity in AdrR MCF7 cells is associated with overexpression but not with amplification of the gene. We mapped the GST pi gene to human chromosome 11q13 by in situ hybridization. Since multidrug resistance and GST pi overexpression are associated with the loss of ERs in AdrR MCF7 cells, we examined several other breast cancer cell lines that were not selected for drug resistance. In each of these cell lines we found an inverse association between GST pi expression and ER content. We also examined RNA from 21 primary breast cancers and found a similar association between GST pi expression and ER content in vivo. GST pi mRNA content in 11 ER-positive tumors (less than or equal to 10 fmol/mg of protein) was significantly different from the GST pi content of 10 ER-negative tumors (P = 0.002; Mann-Whitney Wilcoxon test for two independent samples). The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GST pi than ER-positive tumors. Images PMID:2842775

  6. Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer

    SciTech Connect

    Moscow, J.A.; Townsend, A.J.; Goldsmith, M.E.; Whang-Peng, J.; Vickers, P.J.; Poisson, R.; Legault-Poisson, S.; Myers, C.E.; Cowan, K.H.

    1988-09-01

    The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs). The authors have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (Adr/sup R/ MCF7), the anionic isozyme of glutathione S-transferase (GST/pi/). Hybridization with this GST/pi/ cDNA, GST/pi/-1, demonstrated that increased GST/pi/ activity in Adr/sup R/ MCF7 cells is associated with overexpression but not with amplification of the gene. They mapped the GST/pi/ gene to human chromosome 11q13 by in situ hybridization. Since multidrug resistance and GST/pi/ overexpression are associated with the loss of ERs in Adr/sup R/ MCF7 cells, they examined several other breast cancer cell lines that were not selected for drug resistance. In each of these cell lines they found an inverse association between GST/pi/ expression and ER content. They also examined RNA from 21 primary breast cancers and found a similar association between GST/pi/ expression and ER content in vivo. The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GST/pi/ than ER-positive tumors.

  7. Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction

    SciTech Connect

    Fang, Xian-Ying; Chen, Wei; Fan, Jun-Ting; Song, Ran; Wang, Lu; Gu, Yan-Hong; Zeng, Guang-Zhi; Shen, Yan; Wu, Xue-Feng; Tan, Ning-Hua; Xu, Qiang; Sun, Yang

    2013-02-15

    In the present paper, we examined the effects of a natural cyclopeptide RA-V on human breast cancer cells and the underlying mechanisms. RA-V significantly inhibited the growth of human breast cancer MCF-7, MDA-MB-231 cells and murine breast cancer 4T1 cells. In addition, RA-V triggered mitochondrial apoptotic pathway which was indicated by the loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase cascade. Further study showed that RA-V dramatically inhibited phosphorylation of AKT and 3-phosphoinositide dependent protein kinase 1 (PDK1) in MCF-7 cells. Moreover, RA-V disrupted the interaction between PDK1 and AKT in MCF-7 cells. Furthermore, RA-V-induced apoptosis could be enhanced by phosphatidylinositol 3-kinase inhibitor or attenuated by over-expression of AKT in all the three kinds of breast cancer cells. Taken together, this study shows that RA-V, which can induce mitochondria-mediated apoptosis, exerts strong anti-tumor activity against human breast cancer. The underlying anti-cancer mechanism of RA-V is related to the blockage of the interaction between PDK1 and AKT. - Highlights: ► Plant cyclopeptide RA-V kills human breast cancer cells. ► RA-V triggered mitochondrial apoptotic pathway in human breast cancer cells. ► RA-V inhibited phosphorylation of AKT and PDK1 in breast cancer MCF-7 cells. ► Its mechanism is related to the blockage of the interaction between PDK1 and AKT.

  8. Ubiquitin-specific protease 28 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity by regulating MYC expression.

    PubMed

    Wang, Zengwu; Song, Qimin; Xue, Jian; Zhao, Yumei; Qin, Shiqiang

    2016-02-01

    The transcription factor MYC, which is dysregulated in the majority of gliomas, is difficult to target directly. Deubiquitinase ubiquitin-specific protease 28 (USP28) stabilizes oncogenic factors, including MYC. However, the contribution of USP28 in tumorigenesis, particularly in glioma, is unknown. Here, we determined the expression of USP28 and assessed its clinical significance in human glioma. We found that USP28 is overexpressed in human glioma but not in normal brain tissue. The level of USP28 protein expression in human glioma tissues was directly correlated with glioma grade. Meanwhile, the level of USP28 protein expression in human glioblastoma tissues was inversely correlated with patient survival. Enforced USP28 expression promotes SW1783 glioma cell proliferation. Moreover, gliomas that arose from USP28-transfected SW1783 cells displayed tumorigenicity in nude mouse model systems. Inhibition of USP28 expression in glioblastoma U373 cells suppressed anchorage-independent growth invitro and tumorigenicity invivo. Furthermore, USP28 regulates the expression of MYC protein, which is essential in USP28-induced cell growth in glioma cells. These results showed that USP28 is overexpressed in human glioblastomas and it contributes to glioma tumorigenicity. Therefore, USP28 could be a new target of therapy for human malignant glioma. PMID:26209720

  9. Development and Evaluation of a Cetuximab-based Humanized Single Chain Antibody Against EGFR-overexpressing Tumors.

    PubMed

    Veisi, K; Farajnia, S; Zarghami, N; Khorshid, H R K; Samadi, N; Safdari, Y; Ahmadzadeh, V

    2015-12-01

    Production of humanized single chain antibodies (hscFv) can potentially be a powerful solution to limitations imposed by large size and murine nature of cetuximab. The present study describes generation of a cetuximab-based hscFv using CDR-grafting method. Cetuximab CDRs were grafted on frameworks selected from human germline antibody sequence repertoire. The strategy employed in selecting human sequences was the highest sequence similarity of variable domains between human and parental antibodies as well as similarity in the CDRs canonical structures. To maintain the binding affinity, the parental vernier zone residues were retained murine in hscFv. Recombinant hscFv was expressed in E. coli and affinity purified by Ni-NTA column. The potency of hscFv in targeting EGFR was evaluated using A431, a cell line over-expressing EGFR. Dot blot and ELISA tests were used to assess the reactivity and MTT assay to evaluate the growth inhibition of hscFv on A431 cell line. The humanization of cetuximab variable regions resulted in 22.2% increase in humanness of hscFv. Reactivity analyses of hscFv on A431 cells showed better binding affinity and higher growth inhibition effect (2.6 times) comparing to murine counterpart. In conclusion, hscFv produced in this study displayed reduced potential immunogenicity as well as enhanced cytotoxic effect on EGFR- overexpressing tumor cells. PMID:25333654

  10. Pluripotency genes overexpressed in primate embryonic stem cells are localized on homologues of human chromosomes 16, 17, 19, and X.

    PubMed

    Ben-Yehudah, Ahmi; Navara, Christopher S; Redinger, Carrie J; Mich-Basso, Jocelyn D; Castro, Carlos A; Oliver, Stacie; Chensny, Lara J; Richards, Thomas J; Kaminski, Naftali; Schatten, Gerald

    2010-01-01

    While human embryonic stem cells (hESCs) are predisposed toward chromosomal aneploidities on 12, 17, 20, and X, rendering them susceptible to transformation, the specific genes expressed are not yet known. Here, by identifying the genes overexpressed in pluripotent rhesus ESCs (nhpESCs) and comparing them both to their genetically identical differentiated progeny (teratoma fibroblasts) and to genetically related differentiated parental cells (parental skin fibroblasts from whom gametes were used for ESC derivation), we find that some of those overexpressed genes in nhpESCs cluster preferentially on rhesus chromosomes 16, 19, 20, and X, homologues of human chromosomes 17, 19, 16, and X, respectively. Differentiated parental skin fibroblasts display gene expression profiles closer to nhpESC profiles than to teratoma cells, which are genetically identical to the pluripotent nhpESCs. Twenty over- and underexpressed pluripotency modulators, some implicated in neurogenesis, have been identified. The overexpression of some of these genes discovered using pedigreed nhpESCs derived from prime embryos generated by fertile primates, which is impossible to perform with the anonymously donated clinically discarded embryos from which hESCs are derived, independently confirms the importance of chromosome 17 and X regions in pluripotency and suggests specific candidates for targeting differentiation and transformation decisions. PMID:19854689

  11. GPER mediates estrogen-induced signaling and proliferations in human breast epithelial cells, and normal and malignant breast

    PubMed Central

    Scaling, Allison L.

    2014-01-01

    17?-estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor ?. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized non-tumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant human tissue, revealing a role for GPER in estrogen-induced breast physiology and pathology. PMID:24718936

  12. Urinary isothiocyanate levels, brassica, and human breast cancer.

    PubMed

    Fowke, Jay H; Chung, Fung-Lung; Jin, Fan; Qi, Dai; Cai, Qiuyin; Conaway, Cliff; Cheng, Jia-Rong; Shu, Xiao-Ou; Gao, Yu-Tang; Zheng, Wei

    2003-07-15

    Brassica vegetable consumption (e.g., Chinese cabbage) provides isothiocyanates (ITC) and other glucosinolate derivatives capable of inducing Phase II enzymes [e.g., glutathione S-transferases (GSTM1, GSTT1, and GSTP1) and NADPH quinine oxidoreductase] and apoptosis, altering steroid hormone metabolism, regulating estrogen receptor response, and stabilizing cellular proliferation. Asian populations consuming large amounts of Brassica have a lower breast cancer incidence compared with Western populations; however, the association between Brassica consumption and breast cancer risk is uncertain. It is difficult to estimate glucosinolate exposure and degradation in humans, possibly limiting epidemiological investigations of Brassica and cancer associations. We conducted a case control investigation of breast cancer in Shanghai, China, using urinary ITC levels as a biological measure of glucosinolate intake and degradation in populations with habitual Brassica intake. A representative subgroup of 337 cases providing presurgery, fasting, and first-morning urine specimens was one-to-one matched (age, menopausal status, date of urine collection, and day of laboratory assay) to population controls. Urinary ITC levels were inversely associated with breast cancer [odds ratio (OR) (Quartile 1) = 1 (ref); OR(Q2) = 0.9, 95% confidence interval (0.6, 1.4); OR(Q3) = 0.7, (0.5, 1.1); OR(Q4) = 0.5, (0.3, 0.8), adjusted for age, menopausal status, soy protein, fibroadenoma history, family breast cancer, physical activity, waist-to-hip ratio, body mass index, age at menarche, and parity in conditional logistic model]. This protective association persisted within post and premenopausal women. In contrast, total Brassica intake estimated from a food frequency questionnaire was not associated with breast cancer. Trends in the association between urinary ITC and breast cancer were more consistent with homozygous deletion of GSTM1 or GSTT1, the AAgenotype of GSTP1 (A313G), or with the C allele of NADPH quinine oxidoreductase (C609T), although interactions were not statistically significant. In conclusion, greater Brassica vegetable consumption, as measured by the urinary ITC biomarker, was associated with significantly reduced breast cancer risk among Chinese women. PMID:12873994

  13. FT-Raman spectroscopy study of human breast tissue

    NASA Astrophysics Data System (ADS)

    Bitar Carter, Renata A.; Martin, Airton A.; Netto, Mario M.; Soares, Fernando A.

    2004-07-01

    Optical spectroscopy has been extensively studied as a potential in vivo diagnostic tool to provide information about the chemical and morphologic structure of tissue. Raman Spectroscpy is an inelastic scattering process that can provide a wealth of spectral features that can be related to the specific molecular structure of the sample. This article reports results of an in vitro study of the FT-Raman human breast tissue spectra. An Nd:YAG laser at 1064nm was used as the excitation source in the FT-Raman Spectrometer. The neoplastic human breast samples, both Fibroadenoma and ICD, were obtained during therapeutical routine medical procedures required by the primary disease, and the non-diseased human tissue was obtained in plastic surgery. No sample preparation was needed for the FT-Raman spectra collection. The FT-Raman spectra were recorded from normal, benign (Fibroadenomas) and malignant (IDC-Intraductal Carcinoma) samples, adding up 51 different areas. The main spectral differences of a typical FT-Raman spectra of a Normal (Non-diseased), Fibroadenoma, and Infiltrating Ductal Carcinoma (IDC) breast tissue at the interval of 600 to 1800cm-1, which may differentiate diagnostically the sample, were found in the bands of 1230 to 1295cm-1, 1440 to 1460 cm-1 and 1650 to 1680 cm-1, assigned to the vibrational bands of the carbohydrate-amide III, proteins and lipids, and carbohydrate-amide I, respectively.

  14. Analyzing the regulation of metabolic pathways in human breast cancer

    PubMed Central

    2010-01-01

    Background Tumor therapy mainly attacks the metabolism to interfere the tumor's anabolism and signaling of proliferative second messengers. However, the metabolic demands of different cancers are very heterogeneous and depend on their origin of tissue, age, gender and other clinical parameters. We investigated tumor specific regulation in the metabolism of breast cancer. Methods For this, we mapped gene expression data from microarrays onto the corresponding enzymes and their metabolic reaction network. We used Haar Wavelet transforms on optimally arranged grid representations of metabolic pathways as a pattern recognition method to detect orchestrated regulation of neighboring enzymes in the network. Significant combined expression patterns were used to select metabolic pathways showing shifted regulation of the aggressive tumors. Results Besides up-regulation for energy production and nucleotide anabolism, we found an interesting cellular switch in the interplay of biosynthesis of steroids and bile acids. The biosynthesis of steroids was up-regulated for estrogen synthesis which is needed for proliferative signaling in breast cancer. In turn, the decomposition of steroid precursors was blocked by down-regulation of the bile acid pathway. Conclusion We applied an intelligent pattern recognition method for analyzing the regulation of metabolism and elucidated substantial regulation of human breast cancer at the interplay of cholesterol biosynthesis and bile acid metabolism pointing to specific breast cancer treatment. PMID:20831783

  15. Overexpression of MicroRNA-200c Predicts Poor Outcome in Patients with PR-Negative Breast Cancer

    PubMed Central

    Tuomarila, Marie; Luostari, Kaisa; Soini, Ylermi; Kataja, Vesa

    2014-01-01

    Micro-RNAs are small, noncoding RNAs that act as tumor suppressors or oncogenes. MiR-200c is a member of the miR-200 family; it is known to be dysregulated in invasive breast carcinoma. MiR-200c maintains the epithelial-mesenchymal transition and inhibits cell migration and invasion. Recent studies showed that miR-200c regulated steroid hormone receptors, estrogen receptors (ER), and progesterone receptors (PR). The present study aimed to detect miR-200c in 172 invasive breast carcinoma cases selected from a prospective cohort enrolled in Kuopio, Eastern Finland, between 1990 and 1995. MiR-200c expression was determined with relative q-PCR, and results were compared to clinicopathological variables and patient outcome. We found that PR status combined with miR-200c expression was a significant marker of outcome. High miR-200c expression was associated with reduced survival in PR-negative cases (n?=?68); low miR-200c expression indicated reduced survival in PR-positive cases (n?=?86) (Cox regression: P?=?0.002, OR?=?3.433; and P?=?0.004, OR?=?4.176, respectively). In PR-negative cases, high miR-200c expression was associated with shortened relapse-free survival (Cox regression: P?=?0.001, OR?=?3.613); increased local/distant recurrence (Logistic regression: P?=?0.006, OR?=?3.965); and more frequent distant metastasis (Logistic regression: P?=?0.015, OR?=?3.390). We also found that high grade and low stage tumors were positively correlated with high miR-200c expression (Logistic regression for high grade tumors: P?=?0.002, OR?=?2.791 and for high stage tumors: P?=?0.035, OR?=?0.285). Our results indicated that miR-200c may play a role in invasive breast carcinoma. Furthermore, miR-200c combined with PR status provided a refined predictor of outcome. In future, a larger study is required to confirm our results. This data may provide a basis for new research targetprogesterone receptorregulated microRNAs in breast cancer. PMID:25329395

  16. Frequent copy number gains at 1q21 and 1q32 are associated with overexpression of the ETS transcription factors ETV3 and ELF3 in breast cancer irrespective of molecular subtypes.

    PubMed

    Mesquita, Bárbara; Lopes, Paula; Rodrigues, Ana; Pereira, Deolinda; Afonso, Mariana; Leal, Conceição; Henrique, Rui; Lind, Guro E; Jerónimo, Carmen; Lothe, Ragnhild A; Teixeira, Manuel R

    2013-02-01

    Several ETS transcription factors are involved in the pathogenesis of human cancers by different mechanisms. As gene copy number gain/amplification is an alternative mechanism of oncogenic activation and 1q gain is the most common copy number change in breast carcinoma, we investigated how that genomic change impacts in the expression of the three 1q ETS family members ETV3, ELK4, and ELF3. We have first evaluated 141 breast carcinomas for genome-wide copy number changes by chromosomal CGH and showed that 1q21 and 1q32 were the two chromosome bands with most frequent genomic copy number gains. Second, we confirmed by FISH with locus-specific BAC clones that cases showing 1q gain/amplification by CGH showed copy number increase of the ETS genes ETV3 (located in 1q21~23), ELF3, and ELK4 (both in 1q32). Third, gene expression levels of the three 1q ETS genes, as well as their potential targets MYC and CRISP3, were evaluated by quantitative real-time PCR. We here show for the first time that the most common genomic copy number gains in breast cancer, 1q21 and 1q32, are associated with overexpression of the ETS transcription factors ETV3 and ELF3 (but not ELK4) at these loci irrespective of molecular subtypes. Among the three 1q ETS genes, ELF3 has a relevant role in breast carcinogenesis and is also the most likely target of the 1q copy number increase. The basal-like molecular subtype presented the worst prognosis regarding disease-specific survival, but no additional prognostic value was found for 1q copy number status or ELF3 expression. In addition, we show that there is a correlation between the expression of the oncogene MYC, irrespectively of copy number gain at its loci in 8q24, and the expression of both the transcriptional repressor ETV3 and the androgen respondent ELK4. PMID:23329352

  17. Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer

    PubMed Central

    Wolff, Antonio C.; Hammond, M. Elizabeth H.; Hicks, David G.; Dowsett, Mitch; McShane, Lisa M.; Allison, Kimberly H.; Allred, Donald C.; Bartlett, John M.S.; Bilous, Michael; Fitzgibbons, Patrick; Hanna, Wedad; Jenkins, Robert B.; Mangu, Pamela B.; Paik, Soonmyung; Perez, Edith A.; Press, Michael F.; Spears, Patricia A.; Vance, Gail H.; Viale, Giuseppe; Hayes, Daniel F.

    2014-01-01

    Purpose To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. Methods ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. Results The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. Recommendations The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to >10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing. PMID:24099077

  18. Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line.

    PubMed

    Laud, K; Gourdou, I; Pessemesse, L; Peyrat, J P; Djiane, J

    2002-02-25

    To evaluate whether leptin plays a putative role in breast tumorigenesis, we studied the expression of its long and short receptor isoforms in various tumoral breast tissues. We applied semiquantitative RT-PCR method to RNA extracted from 20 breast cancer biopsies and two human breast cancer cell lines (T47-D and MCF-7). Our results showed the expression of both leptin receptor transcripts in all tumoral tissues examined. By in situ hybridization experiments, we localized leptin receptors in proliferating epithelial cells. Study of leptin effects on human breast cancer cells growth was performed by [3H]-thymidine incorporation method and colorimetric MTT assay. We demonstrated that leptin (50-100 ng/ml) significantly stimulates proliferation of the human breast cancer cell line T47-D (P<0.05). Western blot analysis indicated that leptin induces a time-dependent activation of mitogen-activated protein kinases (MAPKinase) 1 and 2 in T47-D cell line. Moreover, the specific MAPK-inhibitor PD 98059 blocked cell proliferation induced by leptin. In conclusion, we demonstrate that leptin receptors are expressed in breast cancer and that leptin induces proliferation in the T47-D cell line via activation of the MAPKinases pathway. These data suggest that leptin and its receptors may be implicated in mammary cell proliferation and in breast cancer pathogenesis. PMID:11911959

  19. [Effects and costs of adjuvant chemotherapy for operable lymph node positive breast cancer with HER2/neu overexpression].

    PubMed

    Vos, E J; Linn, S C; Rodenhuis, S

    2006-04-01

    Newer forms ofadjuvant chemotherapy can considerably improve the prognosis for breast cancer. The benefits that can be achieved are particularly high for young women (< 50 years) with an unfavourable risk profile (tumour-positive axilliary nodes). The recent application of taxans and trastuzumab has sharply increased the costs of an adjuvant treatment for high-risk mammary breast carcinoma. The cost increase can especially be attributed to trastuzumab. The additional costs of cytostatics (10,079 Euro per life-year gained) appear to be justified if the following is taken into account: women under the age of 50 years still have a life expectancy of approximately 33 years, many have socially relevant positions, and that cure also prevents such things as absence through illness and inability to work as well as expensive palliative care. The pharmaceutical industry spends approximately the same amount on research and innovation as it does on advertising. By reducing marketing costs, there will be more room to lessen the costs of new and socially relevant medications. Ultimately, the pressing question remains on why the Dutch government does not fully compensate hospitals in the Netherlands for the introduction of new, potentially life-saving medications. At present, a substantial percentage of the costs has to be paid by the hospitals themselves out of the regular hospital budget, which is not meant for this. This is happening at the expense of other care to an increasing extent. PMID:16649394

  20. Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia

    SciTech Connect

    Wang, Suna Zhou, Yifu; Andreyev, Oleg; Hoyt, Robert F.; Singh, Avneesh; Hunt, Timothy; Horvath, Keith A.

    2014-04-15

    Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative {sup RT}PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions. - Highlights: • FABP3 expression pattern was studied in 12 human hypoxic-MSCs. • FABP3 mRNA and proteins are upregulated in the MSCs under hypoxic conditions. • Overexpression of FABP3 inhibits cell growth but advanced the MSC survival under hypoxia. • Overexpression of FABP3 down-regulate the cell cycle and stem cell signaling pathways.

  1. A human breast cell model of preinvasive to invasive transition.

    PubMed

    Rizki, Aylin; Weaver, Valerie M; Lee, Sun-Young; Rozenberg, Gabriela I; Chin, Koei; Myers, Connie A; Bascom, Jamie L; Mott, Joni D; Semeiks, Jeremy R; Grate, Leslie R; Mian, I Saira; Borowsky, Alexander D; Jensen, Roy A; Idowu, Michael O; Chen, Fanqing; Chen, David J; Petersen, Ole W; Gray, Joe W; Bissell, Mina J

    2008-03-01

    A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from preinvasive to invasive phenotype as it may occur "spontaneously" in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted basement membrane cultures. These cells remained noninvasive; however, unlike their nonmalignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher-grade tumors. To find functionally significant changes in transition from preinvasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between preinvasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP9, MMP13, MMP15, and MMP17 was up-regulated in the invasive cells. Using small interfering RNA-based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which preinvasive breast cells could acquire invasiveness in a metaplastic context. PMID:18316601

  2. PHOSPHOLIPASE D (PLD) DRIVES CELL INVASION, TUMOR GROWTH AND METASTASIS IN A HUMAN BREAST CANCER XENOGRAPH MODEL

    PubMed Central

    Henkels, Karen M.; Boivin, Gregory P.; Dudley, Emily S.; Berberich, Steven J.; Gomez-Cambronero, Julian

    2014-01-01

    Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based shRNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, p<0.05) and their onset delayed when compared to control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (FIPI and NOPT). These inhibitors led to significant (>70%, p<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid (PA), WASp, Grb2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows that PLD has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target. PMID:23752189

  3. Detection of Human Papillomavirus DNA in Patients with Breast Tumor in China

    PubMed Central

    Li, Jie; Ding, Jie; Zhai, Kan

    2015-01-01

    The presence of HPV in breast tissue and the potential causal association between human papillomavirus (HPV) and breast cancer (BC) remains controversial. The aim of the present study was to compare the HPV prevalence in BC tissues, adjacent normal breast tissues and breast benign disease tissues and to investigate the possible association between HPV and breast tumor development in Chinese women. Paraffin-embedded specimens from 187 pairs of BCs including tumor and normal breast tissue adjacent to tumors and 92 breast benign lesions between June 2009 and July 2014 were investigated by nested polymerase chain reaction (PCR) and type-specific PCR, respectively. With strictly quality control, HPV positive infection was detected in three BC tissues. No HPV positive infection was detected in all normal breast tissue adjacent to tumors and benign breast tissues. Through our detailed analysis, rare HPV infection in this study suggests that HPV might not be associated with BC progression. PMID:26295705

  4. Overexpression of ?2,3sialyl T-antigen in breast cancer determined by miniaturized glycosyltransferase assays and confirmed using tissue microarray immunohistochemical analysis

    PubMed Central

    Patil, Shilpa A.; Bshara, Wiam; Morrison, Carl; Chandrasekaran, E. V.; Matta, Khushi L.; Neelamegham, Sriram

    2014-01-01

    Glycan structure alterations during cancer regulate disease progression and represent clinical biomarkers. The study determined the degree to which changes in glycosyl transferase activities during cancer can be related to aberrant cell-surface tumor associated carbohydrate structures (TACA). To this end, changes in sialyltransferase (sialylT), fucosyltransferase (fucT) and galactosyltransferase (galT) activity were measured in normal and tumor tissue using a miniaturized enzyme activity assay and synthetic glycoconjugates bearing terminal LacNAc Type-I (Gal?1,3GlcNAc), LacNAc Type-II (Gal?1,4GlcNAc), and mucin core-1/Type-III (Gal?1,3GalNAc) structures. These data were related to TACA using tissue microarrays containing 115 breast and 26 colon cancer specimen. The results show that primary human breast and colon tumors, but not adjacent normal tissue, express elevated ?1,3 galT and ?2,3sialylT activity that can form ?2,3sialylated Type-III glycans (Sia?2,3Gal?1,3GalNAc). Prostate tumors did not exhibit such elevated enzymatic activities. ?1,3/4fucT activity was higher in breast, but not colon tissue. The enzymology based prediction of enhanced ?2,3sialylated Type-III structures in breast tumors was verified using histochemical analysis of tissue sections and tissue microarrays. Here, the binding of two markers that recognize Gal?1,3GalNAc (peanut lectin and mAb A78-G/A7) was elevated in breast tumor, but not normal control, only upon sialidase treatment. These antigens were also upregulated in colon tumors though to a lesser extent. ?2,3sialylated Type-III expression correlated inversely with patient HER2 expression and breast metastatic potential. Overall, enzymology measurements of glycoT activity predict glycan structure changes during cancer. High expression of the ?2,3sialylated T-antigen O-glycans occur in breast tumors. A transformation from linear core-1 glycan to other epitopes may accompany metastasis. PMID:25142811

  5. Enhanced tethered-flight duration and locomotor activity by overexpression of the human gene SOD1 in Drosophila motorneurons

    PubMed Central

    Petrosyan, Agavni; Hsieh, I-Hui; Phillips, John P.; Saberi, Kourosh

    2015-01-01

    Mutation of the human gene superoxide dismutase (hSOD1) is associated with the fatal neurodegenerative disease familial amyotrophic lateral sclerosis (Lou Gehrigs disease). Selective overexpression of hSOD1 in Drosophila motorneurons increases lifespan to 140% of normal. The current study was designed to determine resistance to lifespan decline and failure of sensorimotor functions by overexpressing hSOD1 in Drosophilas motorneurons. First, we measured the ability to maintain continuous flight and wingbeat frequency (WBF) as a function of age (5 to 50 days). Flies overexpressing hSOD1 under the D42-GAL4 activator were able to sustain flight significantly longer than controls, with the largest effect observed in the middle stages of life. The hSOD1-expressed line also had, on average, slower wingbeat frequencies in late, but not early life relative to age-matched controls. Second, we examined locomotor (exploratory walking) behavior in late life when flies had lost the ability to fly (age ? 60 d). hSOD1-expressed flies showed significantly more robust walking activity relative to controls. Findings show patterns of functional decline dissimilar to those reported for other life-extended lines, and suggest that the hSOD1 gene not only delays death but enhances sensorimotor abilities critical to survival even in late life. PMID:25983632

  6. Enhanced tethered-flight duration and locomotor activity by overexpression of the human gene SOD1 in Drosophila motorneurons.

    PubMed

    Petrosyan, Agavni; Hsieh, I-Hui; Phillips, John P; Saberi, Kourosh

    2015-03-01

    Mutation of the human gene superoxide dismutase (hSOD1) is associated with the fatal neurodegenerative disease familial amyotrophic lateral sclerosis (Lou Gehrig's disease). Selective overexpression of hSOD1 in Drosophila motorneurons increases lifespan to 140% of normal. The current study was designed to determine resistance to lifespan decline and failure of sensorimotor functions by overexpressing hSOD1 in Drosophila's motorneurons. First, we measured the ability to maintain continuous flight and wingbeat frequency (WBF) as a function of age (5 to 50 days). Flies overexpressing hSOD1 under the D42-GAL4 activator were able to sustain flight significantly longer than controls, with the largest effect observed in the middle stages of life. The hSOD1-expressed line also had, on average, slower wingbeat frequencies in late, but not early life relative to age-matched controls. Second, we examined locomotor (exploratory walking) behavior in late life when flies had lost the ability to fly (age ? 60 d). hSOD1-expressed flies showed significantly more robust walking activity relative to controls. Findings show patterns of functional decline dissimilar to those reported for other life-extended lines, and suggest that the hSOD1 gene not only delays death but enhances sensorimotor abilities critical to survival even in late life. PMID:25983632

  7. Overexpression of uncoupling protein 2 inhibits the high glucose-induced apoptosis of human umbilical vein endothelial cells

    PubMed Central

    HE, YING; LUAN, ZHOU; FU, XUNAN; XU, XUN

    2016-01-01

    Ectopic apoptosis of vascular cells plays a critical role in the early stage development of diabetic retinopathy (DR). Uncoupling protein 2 (UCP2) is a mitochondrial modulator which protects against endothelial dysfunction. However, the role which UCP2 plays in endothelial apoptosis and its association with DR was unclear. In the present study, we investigated whether UCP2 functioned as an inhibitor of DR in endothelial cells. Firstly, we noted that in UCP2-knockout mice retinal cell death and damage in vivo was similar to that of db/db diabetic mice. Additionally, UCP2 knockdown induced caspase-3 activation and exaggerated high glucose (HG)-induced apoptosis of human umbilical vein endothelial cells (HUVECs). Conversely, adenovirus-mediated UCP2 overexpression inhibited the apoptosis of HUVECs and HG-induced caspase-3 activation. Furthermore, HG treatment resulted in the opening of the permeability transition pore (PTP) and liberation of cytochrome c from mitochondria to the cytosol in HUVECs. Notably, UCP2 overexpression inhibited these processes. Furthermore, adenovirus-mediated UCP2 overexpression led to a significant increase in intracellular nitric oxide (NO) levels and a decrease in reactive oxygen species (ROS) generation in HUVECs. Collectively, these data suggest that UCP2 plays an anti-apoptotic role in endothelial cells. Thus, we suggest that approaches which augment UCP2 expression in vascular endothelial cells aid in preventing the early stage development and progression of DR. PMID:26846204

  8. Galectin-1 is overexpressed in CD133+ human lung adenocarcinoma cells and promotes their growth and invasiveness

    PubMed Central

    Zhou, Xuefeng; Li, Dan; Wang, Xianguo; Zhang, Bo; Zhu, Hua; Zhao, Jinping

    2015-01-01

    Previous studies demonstrated that a subpopulation of cancer cells, which are CD133 positive (CD133+) feature higher invasive and metastatic abilities, are called cancer stem cells (CSCs). By using tumor cells derived from patients with lung adenocarcinoma, we found that galectin-1 is highly overexpressed in the CD133+ cancer cells as compared to the normal cancer cells (CD133−) from the same patients. We overexpressed galectin-1 in CD133− cancer cells and downregulated it in CSCs. We found that overexpression of galectin-1 promoted invasiveness of CD133− cells, while knockdown of galectin-1 suppressed proliferation, colony formation and invasiveness of CSCs. Furthermore, tumor growth was significantly inhibited in CSCs xenografts with knockdown of galectin-1 as compared to CSCs treated with scramble siRNAs. Biochemical studies revealed that galectin-1 knockdown led to the suppression of COX-2/PGE2 and AKT/mTOR pathways, indicating galectin-1 might control the phenotypes of CSCs by regulating these signaling pathways. Finally, a retrospective study revealed that galectin-1 levels in blood circulation negatively correlates with overall survival and positively correlates with lymph node metastasis of the patients. Taken together, these findings suggested that galectin-1 plays a major role on the tumorigenesis and invasiveness of CD133+ cancer cells and might serve as a potential therapeutic target for treatment of human patients with lung adenocarcinoma. PMID:25605013

  9. PTEN overexpression improves cisplatin-resistance of human ovarian cancer cells through upregulating KRT10 expression

    SciTech Connect

    Wu, Huijuan; Wang, Ke; Liu, Wenxin; Hao, Quan

    2014-02-07

    Highlights: • Overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin. • KRT10 is a downstream molecule of PTEN involved in the resistance-reversing effect. • Overexpression of KRT10 enhanced the chemosensitivity of C13K cells to cisplatin. - Abstract: Multi-drug resistance (MDR) is a common cause of the failure of chemotherapy in ovarian cancer. PTEN, a tumor suppressor gene, has been demonstrated to be able to reverse cisplatin-resistance in ovarian cancer cell line C13K. However, the downstream molecules of PTEN involved in the resistance-reversing effect have not been completely clarified. Therefore, we screened the downstream molecules of PTEN and studied their interactions in C13K ovarian cancer cells using a 3D culture model. Firstly, we constructed an ovarian cancer cell line stably expressing PTEN, C13K/PTEN. MTT assay showed that overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin, but not to paclitaxel. Then we examined the differently expressed proteins that interacted with PTEN in C13K/PTEN cells with or without cisplatin treatment by co-immunoprecipitation. KRT10 was identified as a differently expressed protein in cisplatin-treated C13K/PTEN cells. Further study confirmed that cisplatin could induce upregulation of KRT10 mRNA and protein in C13K/PTEN cells and there was a directly interaction between KRT10 and PTEN. Forced expression of KRT10 in C13K cells also enhanced cisplatin-induced proliferation inhibition and apoptosis of C13K cells. In addition, KRT10 siRNA blocked cisplatin-induced proliferation inhibition of C13K/PTEN cells. In conclusion, our data demonstrate that KRT10 is a downstream molecule of PTEN which improves cisplatin-resistance of ovarian cancer and forced KRT10 overexpression may also act as a therapeutic method for overcoming MDR in ovarian cancer.

  10. Detection of c-erbB-2 mRNAs Using Dig-Labelled Oligonucleotide Probe with in situ Hybridisation in Human Breast Carcinoma: Comparison with Immunohistochemical Results

    PubMed Central

    ztrk, Melek; Bolkent, Sema; Yilmazer, Selma; Kaner, Gltekin; nal, Hilal

    1998-01-01

    Amplification and overexpression of the c-erbB-2 oncogene are of prognostic significance in human breast cancer. Overexpression of c-erbB-2 is the result of gene amplification. However, increased transcript levels of c-erbB-2 are also detected in the absence of gene amplification. In this study for the detection of the overexpression mRNA in situ hybridisation (ISH) and immunohistochemistry (IHC) were used. Our aim was to develop the suitable mRNA ISH protocol for formalin-fixed paraffin-embedded material and to compare the localisation of transcripts and protein products in 20 primary breast carcinomas. Sections were immunostained with monoclonal c-erbB-2 antibody. In ISH method digoxigenin-labelled oligoprobe was used for the detection of c-erbB-2 mRNAs. We determined optimal condition for the ISH procedure (e.g., probe concentration, digestion, post washing). c-erbB-2 protein overproduction was detected in 11/20 cases with IHC. The mRNA signals were observed in malignant cell cytoplasm in 6/20 cases by ISH. ISH positive signals were found in only one case without detected overexpression of the protein. There were cell to cell variations in the hybridisation signals even within individual tumours. The ISH and IHC positive signals for c-erbB-2 was observed mostly in infiltrating ductal carcinomas that belong to aggressive lesions. PMID:9762367

  11. Overexpression and lack of copy number variation in the BMI-1 gene in human glioma

    PubMed Central

    MADATHAN KANDY, SIBIN; ISHWARA BHAT, DHANANJAYA; CHOPPAVARAPU, LAVANYA; SUVATHA, ARATI; GHATI KASTURIRANGAN, CHETAN

    2015-01-01

    Malignant gliomas are neoplasms of the brain that are associated with a poor prognosis. The B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) gene is one of the major cancer stem cell factors responsible for treatment failure in glioma. In the present study, the DNA-RNA-protein alterations in the BMI-1 gene were assessed in 50 glioma samples. Copy number variations in the BMI-1 gene were analyzed using SYBR Green quantitative polymerase chain reaction. Gene expression analysis was performed using a Taqman assay and protein quantitation was performed using western blotting. A comparative Ct analysis showed the absence of copy number variations in all glioma samples. BMI-1 mRNA expression was found to be overexpressed in 36 out of 50 samples (72.0%), and 37 out of 50 samples showed overexpression (74.0%) of BMI-1 protein; this was statistically significant when compared with non-glioma tissues. It was observed that the protein and RNA expression in glioma were concordant. In this study on the BMI-1 gene, transcription and translation in glioma were observed and BMI-1 overexpression was found to be a common phenomenon. PMID:26722333

  12. S-phase delay in human hepatocellular carcinoma cells induced by overexpression of integrin β1

    PubMed Central

    Liang, Yu-Long; Lei, Ting-Wen; Wu, Heng; Su, Jian-Min; Wang, Li-Ying; Lei, Qun-Ying; Zha, Xi-Liang

    2003-01-01

    AIM: To clarify the mechanisms of integrin overexpression in negatively regulating the cell cycle control of hepatocellular carcinoma cells SMMC-7721. METHODS: The cell cycle pattern was determined by flow cytometry. The mRNA and protein expression levels were assayed by RT-PCR and Western blot, respectively. Stable transfection was performed by Lipofectamine 2000 reagent, and cells were screened by G418. RESULTS: Overexpression of α5β1 or β1 integrin induced S-phase delay in SMMC-7721 cells, and this delay was possibly due to the accumulation of cyclin-dependent kinase inhibitors (CKIs) p21cip1 and p27kip1. The decrease of protein kinase B (PKB) phosphorylation was present in this signaling pathway, but focal adhesion kinase (FAK) was not involved. When phosphorylation of PKB was solely blocked by wortmannin, p27kip1 protein level was increased. Moreover, S-phase delay was recurred when attachment of the parental SMMC-7721 cells was inhibited by the preparation of poly-HEME, and this cell cycle pattern was similar to that of β1-7721 or α5β1-7721 cells. CONCLUSION: S-phase delay induced by overexpression of integrin β1 subunit is attributed to the decrease of PKB phosphorylation and subsequent increases of p21cip1 and p27kip1 proteins, and may be involved in the unoccupied α5β1 because of lack of its ligands. PMID:12918102

  13. Elements related to heterogeneity of antibody-dependent cell cytotoxicity in patients under trastuzumab therapy for primary operable breast cancer overexpressing Her2.

    PubMed

    Varchetta, Stefania; Gibelli, Nadia; Oliviero, Barbara; Nardini, Elena; Gennari, Roberto; Gatti, Giovanna; Silva, Luzemira Santos; Villani, Laura; Tagliabue, Elda; Mnard, Sylvie; Costa, Alberto; Fagnoni, Francesco F

    2007-12-15

    Preliminary results from a pilot trial on trastuzumab's mechanism of action against operable breast tumors overexpressing Her2 suggested a role for antibody-dependent cell cytotoxicity (ADCC). To examine factors affecting ADCC intensity and variability, we extended this study to the phenotypic and functional analysis of circulating mononuclear cells in 18 patients. ADCC was induced by trastuzumab therapy in 15 of 18 patients (83%). Inability to develop ADCC in three patients did not depend on inadequate levels of trastuzumab because further increase in its concentration in vitro was ineffective. Rather, susceptibility to develop ADCC was fairly predicted by test with trastuzumab before therapy and was correlated to the number of lymphocytes coexpressing CD16 and CD56. Phenotypic analysis at the end of ADCC evaluating down-regulation of CD16, and up-regulation of CD69 and CD107a, confirmed that natural killer (NK) cells and CD56(+) T cells were involved in productive engagement of trastuzumab. Also, the killing efficiency of CD16(+) lymphocytes was influenced by 158 V/F polymorphism of Fc gamma RIII (CD16), whereas variations of CD247 on NK cells were consistent with trends between ADCC before and after therapy. Complete pathologic response was observed in one patient showing ADCC of outstanding intensity, whereas four cases of partial response showed intermediate ADCC; none of the three patients unable to mount ADCC had significant tumor regression. These data indicate that quantity and lytic efficiency of CD16(+) lymphocytes are major factors for ADCC induction by trastuzumab, and confirm that breast cancer responses to short-term trastuzumab monotherapy may depend on involvement of the ADCC mechanism. PMID:18089830

  14. A Genome-Wide Over-Expression Screen Identifies Genes Involved in Phagocytosis in the Human Protozoan Parasite, Entamoeba histolytica

    PubMed Central

    King, Ada V.; Welter, Brenda H.; Koushik, Amrita B.; Gordon, Lindsay N.; Temesvari, Lesly A.

    2012-01-01

    Functional genomics and forward genetics seek to assign function to all known genes in a genome. Entamoeba histolytica is a protozoan parasite for which forward genetics approaches have not been extensively applied. It is the causative agent of amoebic dysentery and liver abscess, and infection is prevalent in developing countries that cannot prevent its fecal-oral spread. It is responsible for considerable global morbidity and mortality. Given that the E. histolytica genome has been sequenced, it should be possible to apply genomic approaches to discover gene function. We used a genome-wide over-expression screen to uncover genes regulating an important virulence function of E. histolytica, namely phagocytosis. We developed an episomal E. histolytica cDNA over-expression library, transfected the collection of plasmids into trophozoites, and applied a high-throughput screen to identify phagocytosis mutants in the population of over-expressing cells. The screen was based on the phagocytic uptake of human red blood cells loaded with the metabolic toxin, tubercidin. Expression plasmids were isolated from trophozoites that survived exposure to tubercidin-charged erythrocytes (phagocytosis mutants), and the cDNAs were sequenced. We isolated the gene encoding profilin, a well-characterized cytoskeleton-regulating protein with a known role in phagocytosis. This supports the validity of our approach. Furthermore, we assigned a phagocytic role to several genes not previously known to function in this manner. To our knowledge, this is the first genome-wide forward genetics screen to be applied to this pathogen. The study demonstrates the power of forward genetics in revealing genes regulating virulence in E. histolytica. In addition, the study validates an E. histolytica cDNA over-expression library as a valuable tool for functional genomics. PMID:22905196

  15. Depressive-like phenotype induced by AAV-mediated overexpression of human α-synuclein in midbrain dopaminergic neurons.

    PubMed

    Caudal, D; Alvarsson, A; Björklund, A; Svenningsson, P

    2015-11-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague-Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8 weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8 weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3 weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic-pituitary-adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to alterations in proteins involved in synaptic plasticity. PMID:26363495

  16. Biological determinants of radiation-induced human breast cancer

    SciTech Connect

    Feig, S.A.

    1980-01-01

    This is the second in a three part series on the hypothetical risk from x-ray mammography. It will review those aspects of breast anatomy, histology, physiology, and pathology which are pertinent to radiation carcinogenesis. Radiation-induced breast cancers are histologically identical to the naturally occurring type in that they arise from the ductal epithelium and consist of a similar proportion of infiltrating and intraductal lesions. Possible explanations for the increased resistance to radiation effect in women over 30 years of age at time of exposure include regression of the glandular target tissue, hormonal changes, and parity. Examples of age-related sensitivity and hormonal dependence in other radiation-induced human and animal tumors will be discussed.

  17. MicroRNA expression profile of MCF-7 human breast cancer cells and the effect of green tea polyphenon-60.

    PubMed

    Fix, Lindsey N; Shah, Maitri; Efferth, Thomas; Farwell, Mary A; Zhang, Baohong

    2010-01-01

    This study reports for the first time the microRNA expression profile of human breast cancer MCF-7 cells and the effect of green tea. Although hundreds of miRNAs have been identified in humans, only a small proportion (25.6%) of miRNAs are expressed in MCF-7 cells. Low concentration treatment with Polyphenon-60 significantly alters the miRNA expression profile in MCF-7 cells. Twenty three miRNAs have been identified with differential expression after a 48 h treatment with 10 ?g/ml Polyphenon-60 (green tea extract). These miRNAs include miR-21 and miR-27 that were found to be down-regulated following treatment with green tea. These two miRNAs have previously been identified as being overexpressed in MCF-7 breast cancer cells, with miR-21 specifically implicated in down-regulating the tumor suppressor gene, tropomyosin-1. This data supports the hypothesis that Polyphenon-60-induced modification of the breast cancer miRNA expression profile contributes to the efficacy of green tea treatment. The resulting decrease in carcinogenesis is further supported by the altered miRNA regulation of potential oncogenes and tumor-suppressor genes. PMID:20952761

  18. Interleukin-4 receptor alpha overexpression in human bladder cancer correlates with the pathological grade and stage of the disease

    PubMed Central

    Joshi, Bharat H; Leland, Pamela; Lababidi, Samir; Varrichio, Frederick; Puri, Raj K

    2014-01-01

    Previously, we have demonstrated that interleukin-4 receptor ? (IL-4R?) is overexpressed on a variety of human cancers and can serve as target for IL-4 immunotoxin comprised of IL-4 and a mutated Pseudomonas exotoxin. However, its expression and association with grade and clinical stage of bladder cancer has not been studied. IL-4R? expression was examined in human bladder cancer cell lines, mouse xenografts, and biopsy specimens at mRNA and protein levels by real-time RT-PCR and IHC/ISH techniques. We also examined the effect of IL-4 on proliferation and invasion of bladder carcinoma cell lines. For tissue microarray (TMA) results, we analyzed the precision data using exact binomial proportion with exact two-sided P-values. We used CochranArmitage Statistics with exact two-sided P-values to examine the trend analysis of IL-4R? over grade or stage of the bladder cancer specimens. The influence of age and gender covariates was also analyzed using multiple logistic regression models. IL-4R? is overexpressed in five bladder cancer cell lines, while normal bladder and human umbilical vein cell lines (HUVEC) expressed at low levels. Two other chains of IL-4 receptor complex, IL-2R?C and IL-13R?1, were absent or weakly expressed. IL-4 modestly inhibited the cell proliferation, but enhanced cell invasion of bladder cancer cell lines in a concentration-dependent manner. Bladder cancer xenografts in immunodeficient mice also maintained IL-4R? overexpression in vivo. Analysis of tumor biopsy specimens in TMAs revealed significantly higher IL-4R? immunostaining (?2+) in Grade 2 (85%) and Grade 3 (97%) compared to Grade 1 tumors (0%) (P ? 0.0001). Similarly, 9% stage I tumors were positive for IL-4R? (?2+) compared to 84% stage II (P ? 0.0001) and 100% stages IIIIV tumors (P ? 0.0001). IL-13R?1 was also expressed in tumor tissues but at low levels and it did not show any correlation with the grade and stage of disease. However, the IL-2R?C was not expressed. Ten normal bladder specimens demonstrated ?1+ staining for IL-4R? and IL-13R?1 and no staining for IL-2R?C. These results demonstrate that IL-4R? is overexpressed in human bladder cancer, which correlates with advanced grade and stage of the disease. Thus, IL-4R? may be a bladder tumor-associated protein and a prognostic biomarker. PMID:25208941

  19. Interleukin-4 receptor alpha overexpression in human bladder cancer correlates with the pathological grade and stage of the disease.

    PubMed

    Joshi, Bharat H; Leland, Pamela; Lababidi, Samir; Varrichio, Frederick; Puri, Raj K

    2014-12-01

    Previously, we have demonstrated that interleukin-4 receptor ? (IL-4R?) is overexpressed on a variety of human cancers and can serve as target for IL-4 immunotoxin comprised of IL-4 and a mutated Pseudomonas exotoxin. However, its expression and association with grade and clinical stage of bladder cancer has not been studied. IL-4R? expression was examined in human bladder cancer cell lines, mouse xenografts, and biopsy specimens at mRNA and protein levels by real-time RT-PCR and IHC/ISH techniques. We also examined the effect of IL-4 on proliferation and invasion of bladder carcinoma cell lines. For tissue microarray (TMA) results, we analyzed the precision data using exact binomial proportion with exact two-sided P-values. We used Cochran-Armitage Statistics with exact two-sided P-values to examine the trend analysis of IL-4R? over grade or stage of the bladder cancer specimens. The influence of age and gender covariates was also analyzed using multiple logistic regression models. IL-4R? is overexpressed in five bladder cancer cell lines, while normal bladder and human umbilical vein cell lines (HUVEC) expressed at low levels. Two other chains of IL-4 receptor complex, IL-2R?C and IL-13R?1, were absent or weakly expressed. IL-4 modestly inhibited the cell proliferation, but enhanced cell invasion of bladder cancer cell lines in a concentration-dependent manner. Bladder cancer xenografts in immunodeficient mice also maintained IL-4R? overexpression in vivo. Analysis of tumor biopsy specimens in TMAs revealed significantly higher IL-4R? immunostaining (? 2+) in Grade 2 (85%) and Grade 3 (97%) compared to Grade 1 tumors (0%) (P ? 0.0001). Similarly, 9% stage I tumors were positive for IL-4R? (? 2+) compared to 84% stage II (P ? 0.0001) and 100% stages III-IV tumors (P ? 0.0001). IL-13R?1 was also expressed in tumor tissues but at low levels and it did not show any correlation with the grade and stage of disease. However, the IL-2R?C was not expressed. Ten normal bladder specimens demonstrated ? 1+ staining for IL-4R? and IL-13R?1 and no staining for IL-2R?C. These results demonstrate that IL-4R? is overexpressed in human bladder cancer, which correlates with advanced grade and stage of the disease. Thus, IL-4R? may be a bladder tumor-associated protein and a prognostic biomarker. PMID:25208941

  20. The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1.

    PubMed

    Drakulic, Danijela; Vicentic, Jelena Marjanovic; Schwirtlich, Marija; Tosic, Jelena; Krstic, Aleksandar; Klajn, Andrijana; Stevanovic, Milena

    2015-03-01

    The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis. PMID:25761220

  1. Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma

    PubMed Central

    2010-01-01

    Background Since hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, it is still important to understand hepatocarcinogenesis mechanisms and identify effective markers for tumor progression to improve prognosis. Amplification and overexpression of Tropomyosin3 (TPM3) are frequently observed in HCC, but its biological meanings have not been properly defined. In this study, we aimed to elucidate the roles of TPM3 and related molecular mechanisms. Methods TPM3-siRNA was transfected into 2 HCC cell lines, HepG2 and SNU-475, which had shown overexpression of TPM3. Knockdown of TPM3 was verified by real-time qRT-PCR and western blotting targeting TPM3. Migration and invasion potentials were examined using transwell membrane assays. Cell growth capacity was examined by colony formation and soft agar assays. Results Silencing TPM3 resulted in significant suppression of migration and invasion capacities in both HCC cell lines. To elucidate the mechanisms behind suppressed migration and invasiveness, we examined expression levels of Snail and E-cadherin known to be related to epithelial-mesenchymal transition (EMT) after TPM3 knockdown. In the TPM3 knockdown cells, E-cadherin expression was significantly upregulated and Snail downregulated compared with negative control. TPM3 knockdown also inhibited colony formation and anchorage independent growth of HCC cells. Conclusions Based on our findings, we formulate a hypothesis that overexpression of TPM3 activates Snail mediated EMT, which will repress E-cadherin expression and that it confers migration or invasion potentials to HCC cells during hepatocarcinogenesis. To our knowledge, this is the first evidence that TPM3 gets involved in migration and invasion of HCCs by modifying EMT pathway. PMID:20356415

  2. SOX2 overexpression affects neural differentiation of human pluripotent NT2/D1 cells.

    PubMed

    Klajn, A; Drakulic, D; Tosic, M; Pavkovic, Z; Schwirtlich, M; Stevanovic, M

    2014-11-01

    SOX2 is one of the key transcription factors involved in maintenance of neural progenitor identity. However, its function during the process of neural differentiation, including phases of lineage-specification and terminal differentiation, is still poorly understood. Considering growing evidence indicating that SOX2 expression level must be tightly controlled for proper neural development, the aim of this research was to analyze the effects of constitutive SOX2 overexpression on outcome of retinoic acid-induced neural differentiation of pluripotent NT2/D1 cells. We demonstrated that in spite of constitutive SOX2 overexpression, NT2/D1 cells were able to reach final phases of neural differentiation yielding both neuronal and glial cells. However, SOX2 overexpression reduced the number of mature MAP2-positive neurons while no difference in the number of GFAP-positive astrocytes was detected. In-depth analysis at single-cell level showed that SOX2 downregulation was in correlation with both neuronal and glial phenotype acquisitions. Interestingly, while in mature neurons SOX2 was completely downregulated, astrocytes with low level of SOX2 expression were detected. Nevertheless, cells with high level of SOX2 expression were incapable of entering in either of two differentiation pathways, neurogenesis or gliogenesis. Accordingly, our results indicate that fine balance between undifferentiated state and neural differentiation depends on SOX2 expression level. Unlike neurons, astrocytes could maintain low level of SOX2 expression after they acquired glial fate. Further studies are needed to determine whether differences in the level of SOX2 expression in GFAP-positive astrocytes are in correlation with their self-renewal capacity, differentiation status, and/or their phenotypic characteristics. PMID:25540002

  3. MicroRNA-101 inhibits cell progression and increases paclitaxel sensitivity by suppressing MCL-1 expression in human triple-negative breast cancer

    PubMed Central

    Chen, Jianping; Song, Cailu; Yang, Lu; Liu, Peng; Wang, Neng; Xie, Xinhua; Lin, Xiaoti; Xie, Xiaoming

    2015-01-01

    Triple-negative breast cancer is the most aggressive breast cancer subtype. The aim of our study was to investigate the functional role of both miR-101 and MCL-1 in the sensitivity of human triple-negative breast cancer (TNBC) to paclitaxel. We found that the expression of miR-101 was strongly decreased in triple-negative breast cancer tissues and cell lines. The expression of miR-101 was not associated with clinical stage or lymph node infiltration in TNBC. Ectopic overexpression of miR-101 inhibit growth and induced apoptosis in vitro and suppressed tumorigenicity in vivo. MCL-1 was significantly overexpressed in most of the TNBC tissues and cell lines. Luciferase assay results confirmed MCL-1 as a direct target gene of miR-101. MiR-101 inhibited MCL-1 expression in TNBC cells and transplanted tumors. There was a negative correlation between the level of expression of miR-101 and MCL-1 in TNBC tissues. Suppression of MCL-1 enhanced the sensitivity of MDA-MB-435 cells to paclitaxel. Furthermore, miR-101 increased paclitaxel sensitivity by inhibiting MCL-1 expression. Our findings provide significant insight into the molecular mechanisms of TNBC carcinogenesis and may have clinical relevance for the development of novel, targeted therapies for TNBC. PMID:26036638

  4. Genomic complexity profiling reveals that HORMAD1 overexpression contributes to homologous recombination deficiency in triple-negative breast cancers

    PubMed Central

    Watkins, Johnathan; Weekes, Daniel; Shah, Vandna; Gazinska, Patrycja; Joshi, Shalaka; Sidhu, Bhavna; Gillett, Cheryl; Pinder, Sarah; Vanoli, Fabio; Jasin, Maria; Mayrhofer, Markus; Isaksson, Anders; Cheang, Maggie C.U.; Mirza, Hasan; Frankum, Jessica; Lord, Christopher J.; Ashworth, Alan; Vinayak, Shaveta; Ford, James M.; Telli, Melinda L.; Grigoriadis, Anita; Tutt, Andrew N.J.

    2015-01-01

    Triple-negative breast cancers (TNBCs) are characterised by a wide spectrum of genomic alterations, some of which might be caused by defects in DNA repair processes such as homologous recombination (HR). Despite this understanding, associating particular patterns of genomic instability with response to therapy has been challenging. Here, we show that Allelic-imbalanced Copy Number Aberrations (AiCNA) are more prevalent in TNBCs that respond to platinum-based chemotherapy, thus providing a candidate predictive biomarker for this disease. Furthermore, we show that a high level of AiCNA is linked with elevated expression of a meiosis-associated gene HORMAD1. Elevated HORMAD1 expression suppresses RAD51-dependent HR and drives the use of alternative forms of DNA repair, the generation of AiCNAs as well as sensitising cancer cells to HR targeting therapies. Our data therefore provides a mechanistic association between HORMAD1 expression, a specific pattern of genomic instability and an association with response to platinum-based chemotherapy in TNBC. PMID:25770156

  5. Marker evaluation of human breast and bladder cancers

    SciTech Connect

    Mayall, B.H.; Carroll, P.R.; Chen, Ling-Chun; Cohen, M.B.; Goodson, W.H. III; Smith, H.S.; Waldman, F.M. )

    1990-11-02

    We are investigating multiple markers in human breast and bladder cancers. Our aim is to identify markers that are clinically relevant and that contribute to our understanding of the disease process in individual patients. Good markers accurately assess the malignant potential of a cancer in an individual patient. Thus, they help identify those cancers that will recur, and they may be used to predict more accurately time to recurrence, response to treatment, and overall prognosis. Therapy and patient management may then be optimized to the individual patient. Relevant markers reflect the underlying pathobiology of individual tumors. As a tissue undergoes transformation from benign to malignant, the cells lose their differentiated phenotype. As a generalization, the more the cellular phenotype, cellular proliferation and cellular genotype depart from normal, the more advanced is the tumor in its biological evolution and the more likely it is that the patient has a poor prognosis. We use three studies to illustrate our investigation of potential tumor markers. Breast cancers are labeled in vivo with 5-bromodeoxyuridine (BrdUrd) to give a direct measure of the tumor labeling index. Bladder cancers are analyzed immunocytochemically using an antibody against proliferation. Finally, the techniques of molecular genetics are used to detect allelic loss in breast cancers. 6 refs., 3 figs.

  6. Mathematical analysis of mammary ducts in lactating human breast.

    PubMed

    Mortazavi, S Negin; Geddes, Donna; Hassiotou, Foteini; Hassanipour, Fatemeh

    2014-01-01

    This work studies a simple model for milk transport through lactating human breast ducts, and describes mathematically the mass transfer from alveolar sacs through the mammary ducts to the nipple. In this model both the phenomena of diffusion in the sacs and conventional flow in ducts have been considered. The ensuing analysis reveals that there is an optimal range of bifurcation numbers leading to the easiest milk flow based on the minimum flow resistance. This model formulates certain difficult-to-measure values like diameter of the alveolar sacs, and the total length of the milk path as a function of easy-to-measure properties such as milk fluid properties and macroscopic measurements of the breast. Alveolar dimensions from breast tissues of six lactating women are measured and reported in this paper. The theoretically calculated alveoli diameters for optimum milk flow (as a function of bifurcation numbers) show excellent match with our biological data on alveolar dimensions. Also, the mathematical model indicates that for minimum milk flow resistance the glandular tissue must be within a short distance from the base of the nipple, an observation that matches well with the latest anatomical and physiological research. PMID:25571286

  7. The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation.

    PubMed

    De Petrocellis, L; Melck, D; Palmisano, A; Bisogno, T; Laezza, C; Bifulco, M; Di Marzo, V

    1998-07-01

    Anandamide was the first brain metabolite shown to act as a ligand of "central" CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 microM and 83-92% maximal inhibition at 5-10 microM. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 microM anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55, 940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1-0.5 microM) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor. PMID:9653194

  8. The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation

    PubMed Central

    De Petrocellis, Luciano; Melck, Dominique; Palmisano, Antonella; Bisogno, Tiziana; Laezza, Chiara; Bifulco, Maurizio; Di Marzo, Vincenzo

    1998-01-01

    Anandamide was the first brain metabolite shown to act as a ligand of central CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 ?M and 8392% maximal inhibition at 510 ?M. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 ?M anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55,940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.10.5 ?M) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor. PMID:9653194

  9. KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis

    PubMed Central

    Narla, Goutham; DiFeo, Analisa; Fernandez, Yolanda; Dhanasekaran, Saravana; Huang, Fei; Sangodkar, Jaya; Hod, Eldad; Leake, Devin; Friedman, Scott L.; Hall, Simon J.; Chinnaiyan, Arul M.; Gerald, William L.; Rubin, Mark A.; Martignetti, John A.

    2008-01-01

    Metastatic prostate cancer (PCa) is one of the leading causes of death from cancer in men. The molecular mechanisms underlying the transition from localized tumor to hormone-refractory metastatic PCa remain largely unknown, and their identification is key for predicting prognosis and targeted therapy. Here we demonstrated that increased expression of a splice variant of the Kruppel-like factor 6 (KLF6) tumor suppressor gene, known as KLF6-SV1, in tumors from men after prostatectomy predicted markedly poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that KLF6-SV1 levels were specifically upregulated in hormone-refractory metastatic PCa. In 2 complementary mouse models of metastatic PCa, KLF6-SV1overexpressing PCa cells were shown by in vivo and ex vivo bioluminescent imaging to metastasize more rapidly and to disseminate to lymph nodes, bone, and brain more often. Interestingly, while KLF6-SV1 overexpression increased metastasis, it did not affect localized tumor growth. KLF6-SV1 inhibition using RNAi induced spontaneous apoptosis in cultured PCa cell lines and suppressed tumor growth in mice. Together, these findings demonstrate that KLF6-SV1 expression levels in PCa tumors at the time of diagnosis can predict the metastatic behavior of the tumor; thus, KLF-SV1 may represent a novel therapeutic target. PMID:18596922

  10. Predicting the Important Enzymes in Human Breast Milk Digestion

    PubMed Central

    2015-01-01

    Human milk is known to contain several proteases, but little is known about whether these enzymes are active, which proteins they cleave, and their relative contribution to milk protein digestion in vivo. This study analyzed the mass spectrometry-identified protein fragments found in pooled human milk by comparing their cleavage sites with the enzyme specificity patterns of an array of enzymes. The results indicate that several enzymes are actively taking part in the digestion of human milk proteins within the mammary gland, including plasmin and/or trypsin, elastase, cathepsin D, pepsin, chymotrypsin, a glutamyl endopeptidase-like enzyme, and proline endopeptidase. Two proteins were most affected by enzyme hydrolysis: β-casein and polymeric immunoglobulin receptor. In contrast, other highly abundant milk proteins such as α-lactalbumin and lactoferrin appear to have undergone no proteolytic cleavage. A peptide sequence containing a known antimicrobial peptide is released in breast milk by elastase and cathepsin D. PMID:24620897

  11. Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands.

    PubMed

    Itoh, Kohji; Kobayashi, Isao; Nishioka, So-Ichiro; Sezutsu, Hideki; Machii, Hiroaki; Tamura, Toshiki

    2016-01-01

    Since 2000, transgenic silkworms have been developed to produce recombinant proteins with therapeutic potential for future clinical use, including antibody preparations. Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by mutations of lysosomal enzymes associated with excessive accumulation of natural substrates and neurovisceral symptoms. Over the past few years, enzyme replacement therapy (ERT) with human lysosomal enzymes produced by genetically engineered mammalian cell lines has been used clinically to treat several patients with an LSD involving multi-organ symptoms. ERT is based on the incorporation of recombinant glycoenzymes by their binding to glycan receptors on the surface of target cells and their subsequent delivery to lysosomes. However, ERT has several disadvantages, including difficulty mass producing human enzymes, dangers of pathogen contamination, and high costs. Recently, the current authors have succeeded in producing transgenic silkworms overexpressing human lysosomal enzymes in the silk glands and the authors have purified catalytically active enzymes from the middle silk glands. Silk gland-derived human enzymes carrying high-mannose and pauci-mannose N-glycans were endocytosed by monocytes via the mannose receptor pathway and were then delivered to lysosomes. Conjugates with cell-penetrating peptides were also taken up by cultured fibroblasts derived from patients with enzyme deficiencies to restore intracellular catalytic activity and reduce the excessive accumulation of substrates in patient fibroblasts. Transgenic silkworms overexpressing human lysosomal enzymes in the silk glands could serve as future bioresources that provide safe therapeutic enzymes for the treatment of LSDs. Combining recent developments in transglycosylation technology with microbial endoglycosidases will promote the development of therapeutic glycoproteins as bio-medicines. PMID:26971553

  12. Overexpression of mimecan in human aortic smooth muscle cells inhibits cell proliferation and enhances apoptosis and migration

    PubMed Central

    ZHANG, HUI-JIE; WANG, JING; LIU, HUI-FANG; ZHANG, XIAO-NA; ZHAN, MING; CHEN, FENG-LING

    2015-01-01

    The pathogenesis of atherosclerosis is multifactorial. The proliferation and migration of vascular smooth muscle cells (VSMCs) are significant in the genesis and development of atherosclerosis plaques, and the degradation of VSMCs plays a crucial role in the process. Mimecan is a member of the Keratan sulfate family of proteoglycans, which are leucine-rich proteoglycans. It has been demonstrated that mimecan is associated with arteriogenesis and atherosclerosis. In the present study, the effect of mimecan on the characteristics of cultured human aortic smooth muscle cells (HASMCs) was investigated. In vitro, human mimecan was stably overexpressed in HASMCs using a lentiviral system. It was observed that the proliferation rate of HASMCs transduced with mimecan was lower compared with that of control cells; overexpression of mimecan induced HASMC apoptosis. To determine the effect of mimecan on HASMC migration, a Transwell cell culture chamber and sterile cloning cylinder assays were used, and it was noted that mimecan enhanced the migration of HASMCs horizontally and vertically. These data indicated that mimecan may be involved in the pathogenesis of atherosclerosis by regulating the proliferation, apoptosis and migration of VSMCs. PMID:26170933

  13. Mapping MRI/MRS Parameters with Genetic Over-expression Profiles In Human Prostate Cancer: Demonstrating the Potential

    PubMed Central

    Lenkinski, Robert E.; Bloch, B. Nicholas; Liu, Fangbing; Frangioni, John V.; Perner, Sven; Rubin, Mark A.; Genega, Elizabeth; Rofsky, Neil M.; Gaston, Sandra M.

    2009-01-01

    Magnetic resonance imaging (MRI) and MR spectroscopy can probe a variety of physiological (e.g. blood vessel permeability) and metabolic characteristics of prostate cancer. However, little is known about the changes in gene expression that underlie the spectral and imaging features observed in prostate cancer. Tumor induced changes in vascular permeability and angiogenesis are thought to contribute to patterns of dynamic contrast enhanced (DCE) MRI images of prostate cancer even though the genetic basis of tumor vasculogenesis is complex and the specific mechanisms underlying these DCEMRI features have not yet been determined. In order to identify the changes in gene expression that correspond to MRS and DCEMRI patterns in human prostate cancers, we have utilized tissue print micropeel techniques to generate whole mount molecular maps of radical prostatectomy specimens that correspond to pre-surgical MRI/MRS studies. These molecular maps include RNA expression profiles from both Affymetrix GeneChip microarrays and quantitative reverse transcriptase PCR (qrt-PCR) analysis, as well as immunohistochemical studies. Using these methods on patients with prostate cancer, we found robust over-expression of choline kinase a in the majority of primary tumors. We also observed overexpression of neuropeptide Y (NPY), a newly identified angiogenic factor, in a subset of DCEMRI positive prostate cancers. These studies set the stage for establishing MRI/MRS parameters as validated biomarkers for human prostate cancer. PMID:18752015

  14. Androgen receptor is overexpressed in boys with severe hypospadias, and ZEB1 regulates androgen receptor expression in human foreskin cells

    PubMed Central

    Qiao, Liang; Tasian, Gregory E.; Zhang, Haiyang; Cao, Mei; Ferretti, Max; Cunha, Gerald R.; Baskin, Laurence S.

    2012-01-01

    INTRODUCTION ZEB1 is overexpressed in patients with severe hypospadias. We examined the interaction between ZeB1 and the androgen receptor (AR) in vitro and the expression of AR in boys with hypospadias. RESULTS ZEB1 and AR colocalize to the nucleus. Estrogen upregulated ZEB1 and AR expression. Chromatin immunoprecipitation (ChIP) demonstrated that ZEB1 binds to an E-box sequence in the AR gene promoter. AR expression is higher in subjects with severe hypospadias than those with mild hypospadias and control subjects (P < 0.05). ZEB1 physically interacts with AR in human foreskin cells. DISCUSSION AR is overexpressed in patients with severe hypospadias. Environmental estrogenic compounds may increase the risk of hypospadias by facilitating the interaction between ZEB1 and AR. METHODS Hs68 cells, a fibroblast cell line derived from neonatal human foreskin, were exposed to 0, 10, and 100 nmol/l of estrogen, after which the cellular localization of ZEB1 and AR was assessed using immunocytochemistry. To determine if ZEB1 interacted with the AR gene, ChIP was performed using ZEB1 antibody and polymerase chain reaction (PCR) for AR. Second, AR expression was quantified using real-time PcR and western blot in normal subjects (n = 32), and subjects with mild (n = 16) and severe hypospadia (n = 16). PMID:22391641

  15. Polymeric micelles as a diagnostic tool for image-guided drug delivery and radiotherapy of HER2 overexpressing breast cancer

    NASA Astrophysics Data System (ADS)

    Hoang, Nu Bryan

    Block copolymer micelles have emerged as a viable formulation strategy with several drugs relying on this technology in clinical evaluation. To date, information on the tumor penetration and intratumoral distribution of block copolymer micelles (BCM) has been quite limited. Thus, there is impetus to develop a radiolabeled formulation that can be used to gain invaluable insight into the intratumoral distribution of the BCMs. This information could then be used to direct formulation strategies as a means to optimize treatment outcomes. This thesis describes the synthesis and characterization of a targeted block copolymer micelle system based on poly(ethylene glycol)-block -poly(epsilon-caprolactone) labeled with the radionuclide Indium-111 (111In). The incorporation of the imageable component, 111In permits pursuit of image-guided drug delivery for real-time monitoring of tumor localization and intratumoral distribution. Intracellular trafficking of drugs and therapies such as Auger electron emitting radionuclides to perinuclear and nuclear regions of cells is critical to realizing their full therapeutic potential. HER2 specific antibodies (trastuzumab fab fragments) and nuclear localization signal peptides were conjugated to the surface of the BCMs to direct uptake in HER2 expressing cells and subsequent localization in the cell nucleus. Cell uptake was HER2 density dependent, confirming receptor-mediated internalization of the BCMs. Importantly, conjugation of NLS resulted in a significant increase in nuclear uptake of the radionuclide 111In. Successful nuclear targeting was shown to improve the antiproliferative effect of the Auger electrons. In addition, a significant radiation enhancement effect was observed by concurrent delivery of low-dose MTX and 111In in all breast cancer cell lines evaluated. Imaging enabled the accurate quantification of the specific tumor uptake of the micelles and visualization of their degree of tumor penetration in relation to microvessel density. Ultimately, the 111In-micelles could be used for such diverse applications as detection of malignancies, molecular characterization of tumors, improved therapy guidance and targeted anti-cancer treatment.

  16. Critical roles of DMP1 in human epidermal growth factor receptor 2/neu-Arf-p53 signaling and breast cancer development.

    PubMed

    Taneja, Pankaj; Maglic, Dejan; Kai, Fumitake; Sugiyama, Takayuki; Kendig, Robert D; Frazier, Donna P; Willingham, Mark C; Inoue, Kazushi

    2010-11-15

    Human epidermal growth factor receptor 2 (HER2) overexpression stimulates cell growth in p53-mutated cells while it inhibits cell proliferation in those with wild-type p53, but the molecular mechanism is unknown. The Dmp1 promoter was activated by HER2/neu through the phosphatidylinositol-3'-kinase-Akt-NF-κB pathway, which in turn stimulated Arf transcription. Binding of p65 and p52 subunits of NF-κB was shown to the Dmp1 promoter and that of Dmp1 to the Arf promoter on HER2/neu overexpression. Both Dmp1 and p53 were induced in premalignant lesions from mouse mammary tumor virus-neu mice, and mammary tumorigenesis was significantly accelerated in both Dmp1+/- and Dmp1-/- mice. Selective deletion of Dmp1 and/or overexpression of Tbx2/Pokemon was found in >50% of wild-type HER2/neu carcinomas, although the involvement of Arf, Mdm2, or p53 was rare. Tumors from Dmp1+/-, Dmp1-/-, and wild-type neu mice with hemizygous Dmp1 deletion showed significant downregulation of Arf and p21Cip1/WAF1, showing p53 inactivity and more aggressive phenotypes than tumors without Dmp1 deletion. Notably, endogenous hDMP1 mRNA decreased when HER2 was depleted in human breast cancer cells. Our study shows the pivotal roles of Dmp1 in HER2/neu-p53 signaling and breast carcinogenesis. PMID:21062982

  17. Elevation of Soluble Guanylate Cyclase Suppresses Proliferation and Survival of Human Breast Cancer Cells

    PubMed Central

    Chen, Chen-Yu; Shiah, Shine-Gwo; Kung, Hsing-Jien; King, Kuang-Liang; Su, Liang-Chen; Chang, Shi-Chuan; Chang, Chung-Ho

    2015-01-01

    Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of ?1 and ?1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC ?1 and sGC?1 mRNAs. However, levels of sGC?1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-dC) increased mRNA levels of both sGC?1 and sGC?1 in MDA-MB-231 cells but only sGC?1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGC?1 in MDA-MB-231 cells and promoter of sGC?1 in MCF-7 cells were methylated. Promoter hypermethylation of sGC?1 and sGC?1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells. PMID:25928539

  18. Diallyl trisulfide inhibits estrogen receptor-? activity in human breast cancer cells.

    PubMed

    Hahm, Eun-Ryeong; Singh, Shivendra V

    2014-02-01

    Organosulfur compounds from garlic effectively inhibit growth of transplanted as well as spontaneous cancers in preclinical animal models without any adverse side effects. However, the mechanisms underlying anticancer effect of this class of compounds are not fully understood. This study reports, for the first time, that garlic organosulfide diallyl trisulfide (DATS) inhibits estrogen receptor-? (ER-?) activity in human breast cancer cells. Exposure of MCF-7 and T47D cells to DATS resulted in downregulation of ER-? protein, which peaked between 12- and 24-h post-treatment. DATS was relatively more effective in suppressing ER-? protein expression compared with its mono and disulfide analogs. The 17?-estradiol (E2)-induced expression of pS2 and cyclin D1, ER-? target gene products, was also decreased in the presence of DATS. Downregulation of ER-? protein expression resulting from DATS treatment was accompanied by a decrease in nuclear levels of ER-? protein, ER-? mRNA suppression, and inhibition of ERE2e1b-luciferase reporter activity. DATS-mediated inhibition of cell viability and apoptosis induction were not affected in the presence of E2. In agreement with these results, ectopic expression of ER-? in MDA-MB-231 cell line failed to confer any protection against cell proliferation inhibition or apoptosis induction resulting from DATS exposure. DATS treatment caused a decrease in protein levels of peptidyl-prolyl cis-trans isomerase (Pin1), and overexpression of Pin1 partially attenuated ER-? downregulation by DATS. DATS-induced apoptosis was modestly but significantly augmented by overexpression of Pin1. In conclusion, this study identifies ER-? as a novel target of DATS in mammary cancer cells. PMID:24487688

  19. Estrogen induces Vav1 expression in human breast cancer cells.

    PubMed

    Du, Ming-juan; Chen, Xiang-dong; Zhou, Xiao-li; Wan, Ya-juan; Lan, Bei; Zhang, Cui-zhu; Cao, Youjia

    2014-01-01

    Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17?-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be ? form, not ?. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ER? might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells. PMID:24905577

  20. Estrogen Induces Vav1 Expression in Human Breast Cancer Cells

    PubMed Central

    Du, Ming-juan; Chen, Xiang-dong; Zhou, Xiao-li; Wan, Ya-juan; Lan, Bei; Zhang, Cui-zhu; Cao, Youjia

    2014-01-01

    Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17?-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be ? form, not ?. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ER? might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells. PMID:24905577

  1. PTEN overexpression improves cisplatin-resistance of human ovarian cancer cells through upregulating KRT10 expression.

    PubMed

    Wu, Huijuan; Wang, Ke; Liu, Wenxin; Hao, Quan

    2014-02-01

    Multi-drug resistance (MDR) is a common cause of the failure of chemotherapy in ovarian cancer. PTEN, a tumor suppressor gene, has been demonstrated to be able to reverse cisplatin-resistance in ovarian cancer cell line C13K. However, the downstream molecules of PTEN involved in the resistance-reversing effect have not been completely clarified. Therefore, we screened the downstream molecules of PTEN and studied their interactions in C13K ovarian cancer cells using a 3D culture model. Firstly, we constructed an ovarian cancer cell line stably expressing PTEN, C13K/PTEN. MTT assay showed that overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin, but not to paclitaxel. Then we examined the differently expressed proteins that interacted with PTEN in C13K/PTEN cells with or without cisplatin treatment by co-immunoprecipitation. KRT10 was identified as a differently expressed protein in cisplatin-treated C13K/PTEN cells. Further study confirmed that cisplatin could induce upregulation of KRT10 mRNA and protein in C13K/PTEN cells and there was a directly interaction between KRT10 and PTEN. Forced expression of KRT10 in C13K cells also enhanced cisplatin-induced proliferation inhibition and apoptosis of C13K cells. In addition, KRT10 siRNA blocked cisplatin-induced proliferation inhibition of C13K/PTEN cells. In conclusion, our data demonstrate that KRT10 is a downstream molecule of PTEN which improves cisplatin-resistance of ovarian cancer and forced KRT10 overexpression may also act as a therapeutic method for overcoming MDR in ovarian cancer. PMID:24434152

  2. Expression of Bcl-2 by human bone marrow mast cells and its overexpression in mast cell leukemia.

    PubMed

    Cerveró, C; Escribano, L; San Miguel, J F; Díaz-Agustín, B; Bravo, P; Villarrubia, J; García-Sanz, R; Velasco, J L; Herrera, P; Vargas, M; González, M; Navarro, J L; Orfao, A

    1999-03-01

    Bcl-2 protein plays a major role in the prevention of programmed cell death of differentiating cells. In the present study, the expression of cytoplasmic bcl-2 by human Bone Marrow Mast Cells (BMMC) from both normal and pathological bone marrow samples was examined. A total of 35 subjects corresponding to 9 healthy volunteers, 8 cases of adult indolent systemic mast cell disease (SMCD), 4 cases of pediatric mastocytosis (PM), 11 cases of hematological malignancies (HM), 2 cases of reactive bone marrow, and 1 case of mast cell leukemia (MCL) were analyzed. The expression of bcl-2 was studied using quantitative three-color flow cytometry. We also studied the molecular configuration of the bcl-2 gene and other relatives by Southern blot and polymerase chain reaction (PCR) in the MCL case. Bcl-2 expression was detected in BMMC from all samples analyzed. No significant differences on the expression of bcl-2 were detected between BMMC from healthy subjects and patients with SMCD, PM, HM, and reactive bone marrow. By contrast, bcl-2 protein was overexpressed in BMMC from MCL patient without gene rearrangement. Our results show that bcl-2 protein was constitutively expressed by BMMC. BMMC from MCL display overexpression of bcl-2, which could not be related to molecular rearrangements involving the bcl-2 gene. The expression of this protein by mature MC may play a role in the prevention of MC apoptosis and thus help to explain the long survival of these cells. The overexpression of bcl-2 by BMMC in MCL may help to explain their resistance to chemotherapy-induced apoptosis. PMID:10072109

  3. Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia.

    PubMed Central

    Lee, P J; Alam, J; Wiegand, G W; Choi, A M

    1996-01-01

    Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to biliverdin, which is reduced by biliverdin reductase to bilirubin. Heme oxygenase-1 (HO-1) is inducible not only by its heme substrate, but also by a variety of agents causing oxidative stress. Although much is known about the regulation of HO-1 expression, the functional significance of HO-1 induction after oxidant insult is still poorly understood. We hypothesize and provide evidence that HO-1 induction serves to protect cells against oxidant stress. Human pulmonary epithelial cells (A549 cells) stably transfected with the rat HO-1 cDNA exhibit marked increases of HO-1 mRNA levels which were correlated with increased HO enzyme activity. Cells that overexpress HO-1 (A549-A4) exhibited a marked decrease in cell growth compared with wild-type A549 (A549-WT) cells or A549 cells transfected with control DNA (A549-neo). This slowing of cell growth was associated with an increased number of cells in G0/G1 phase during the exponential growth phase and decreased entry into the S phase, as determined by flow cytometric analysis of propidium iodide-stained cells and pulse experiments with bromodeoxyuridine. Furthermore, the A549-A4 cells accumulated at the G2/M phase and failed to progress through the cell cycle when stimulated with serum, whereas the A549-neo control cells exhibited normal cell cycle progression. Interestingly, the A549-A4 cells also exhibited marked resistance to hyperoxic oxidant insult. Tin protoporphyrin, a selective inhibitor of HO, reversed the growth arrest and ablated the increased survival against hyperoxia observed in the A549-A4 cells overexpressing HO-1. Taken together, our data suggest that overexpression of HO-1 results in cell growth arrest, which may facilitate cellular protection against non-heme-mediated oxidant insult such as hyperoxia. Images Fig. 1 Fig. 4 PMID:8816811

  4. Role of human neutrophil gelatinase associated lipocalin (NGAL) and Matrix Metalloproteinase-9 (MMP-9) overexpression in neoplastic colon polyps

    PubMed Central

    Odabasi, Mehmet; Yesil, Atakan; Ozkara, Selvinaz; Paker, Nurcan; Ozkan, Sevil; Eris, Cengiz; Yildiz, Mehmet Kamil; Abuoglu, Hac? Hasan; Gunay, Emre; Teke?in, Kemal

    2014-01-01

    To explore the role of Human neutrophil gelatinase associated lipocalin (NGAL) and Matrix Metalloproteinase-9 (MMP-9) overexpression in neoplastic polyps and might used as a marker to separate those from non-noeplastic polyps. The study was performed on total 65 cases, 40% (n = 26) of them females and 60% (n = 39) of them males, in Haydarpasa Numune Education and Research Hospital between March 2012 and June 2012. The assessment of immunostained sections was performed by a random principle by one experinced pathologists to the clinico-pathological data. NGAL expression was based on the presence of cytoplasmic and membranous staining. The NGAL intensities of the cases show highly statistically significantly difference according to the pathological results (p < 0.01). The NGAL prevalences of the cases show highly statistically significantly difference according to the pathological results (p < 0.01). The NGAL ID scores of the cases show highly statistically significantly difference according to the pathological results (p < 0.01). We could hypothesize that NGAL and MMP-9 overexpression in neoplastic polyps might be used as a marker to separate those from non-noeplastic polyps. However, in this study, we determined that NGAL overexpression could not distinguish dysplasia from adenocancer. Finally, we suggest NGAL and MMP-9 as an immunohistochemical marker for colonic dysplasia. To determine dysplasia in early steps of colorectal adenoma-carcinoma sequence, it could help to determine new targets in preventive cancer therapy for colorectal cancer. We suggest development of standards for study method, introduction to routine practice by investigating in future studies including many patients. PMID:25356142

  5. Role of human neutrophil gelatinase associated lipocalin (NGAL) and Matrix Metalloproteinase-9 (MMP-9) overexpression in neoplastic colon polyps.

    PubMed

    Odabasi, Mehmet; Yesil, Atakan; Ozkara, Selvinaz; Paker, Nurcan; Ozkan, Sevil; Eris, Cengiz; Yildiz, Mehmet Kamil; Abuoglu, Hac? Hasan; Gunay, Emre; Teke?in, Kemal

    2014-01-01

    To explore the role of Human neutrophil gelatinase associated lipocalin (NGAL) and Matrix Metalloproteinase-9 (MMP-9) overexpression in neoplastic polyps and might used as a marker to separate those from non-noeplastic polyps. The study was performed on total 65 cases, 40% (n = 26) of them females and 60% (n = 39) of them males, in Haydarpasa Numune Education and Research Hospital between March 2012 and June 2012. The assessment of immunostained sections was performed by a random principle by one experinced pathologists to the clinico-pathological data. NGAL expression was based on the presence of cytoplasmic and membranous staining. The NGAL intensities of the cases show highly statistically significantly difference according to the pathological results (p < 0.01). The NGAL prevalences of the cases show highly statistically significantly difference according to the pathological results (p < 0.01). The NGAL ID scores of the cases show highly statistically significantly difference according to the pathological results (p < 0.01). We could hypothesize that NGAL and MMP-9 overexpression in neoplastic polyps might be used as a marker to separate those from non-noeplastic polyps. However, in this study, we determined that NGAL overexpression could not distinguish dysplasia from adenocancer. Finally, we suggest NGAL and MMP-9 as an immunohistochemical marker for colonic dysplasia. To determine dysplasia in early steps of colorectal adenoma-carcinoma sequence, it could help to determine new targets in preventive cancer therapy for colorectal cancer. We suggest development of standards for study method, introduction to routine practice by investigating in future studies including many patients. PMID:25356142

  6. Insulin-like growth factor 1 receptor expression in breast cancer tissue and mammographic density

    PubMed Central

    SUN, WOO-YOUNG; YUN, HYO-YOUNG; SONG, YOUNG-JIN; KIM, HEON; LEE, OK-JUN; NAM, SEOK-JIN; KOO, JA-SEUNG

    2015-01-01

    The aim of this study was to evaluate the association between insulin-like growth factor 1 receptor (IGF-1R) expression in breast cancer tissue and mammographic density and the clinical significance of IGF-1R overexpression. A total of 167 patients with primary invasive breast cancer were analyzed. Mammographic breast density and IGF-1R overexpression were correlated with clinicopathological parameters and analyzed by overall survival (OS) and disease-free survival (DFS). Increased breast tissue density was significantly associated with age, body mass index, menopausal status, histological grade and IGF-1R overexpression in the univariate analysis and with age (P=0.001), histological grade (P=0.045) and IGF-1R overexpression (P=0.021) in the multivariate analysis. IGF-1R overexpression was significantly associated with dense breast tissue in patients aged >40 years (P=0.002). IGF-1R overexpression in breast cancer in premenopausal women was associated with human epidermal growth factor receptor 2 (HER-2) positivity (P=0.016) and worse DFS (P=0.0414). There was no significant difference in OS and DFS between dense and non-dense breast tissue. IGF-1R expression in breast cancer tissue was significantly associated with mammographic breast tissue density in patients aged >40 years. It appears that IGF-1R expression in breast cancer tissue plays an important role in breast cancer in patients with dense breast tissue. In premenopausal women, IGF-1R overexpression in breast cancer tissue was significantly associated with HER-2 positivity and poor DFS. PMID:26137269

  7. Glyphosate induces human breast cancer cells growth via estrogen receptors.

    PubMed

    Thongprakaisang, Siriporn; Thiantanawat, Apinya; Rangkadilok, Nuchanart; Suriyo, Tawit; Satayavivad, Jutamaad

    2013-09-01

    Glyphosate is an active ingredient of the most widely used herbicide and it is believed to be less toxic than other pesticides. However, several recent studies showed its potential adverse health effects to humans as it may be an endocrine disruptor. This study focuses on the effects of pure glyphosate on estrogen receptors (ERs) mediated transcriptional activity and their expressions. Glyphosate exerted proliferative effects only in human hormone-dependent breast cancer, T47D cells, but not in hormone-independent breast cancer, MDA-MB231 cells, at 10? to 10??M in estrogen withdrawal condition. The proliferative concentrations of glyphosate that induced the activation of estrogen response element (ERE) transcription activity were 5-13 fold of control in T47D-KBluc cells and this activation was inhibited by an estrogen antagonist, ICI 182780, indicating that the estrogenic activity of glyphosate was mediated via ERs. Furthermore, glyphosate also altered both ER? and ? expression. These results indicated that low and environmentally relevant concentrations of glyphosate possessed estrogenic activity. Glyphosate-based herbicides are widely used for soybean cultivation, and our results also found that there was an additive estrogenic effect between glyphosate and genistein, a phytoestrogen in soybeans. However, these additive effects of glyphosate contamination in soybeans need further animal study. PMID:23756170

  8. Broccoli and watercress suppress matrix metalloproteinase-9 activity and invasiveness of human MDA-MB-231 breast cancer cells

    SciTech Connect

    Rose, Peter . E-mail: bchpcr@nus.edu.sg; Huang, Qing; Ong, Choon Nam; Whiteman, Matt

    2005-12-01

    A high dietary intake of cruciferous vegetables has been associated with a reduction in numerous human pathologies particularly cancer. In the current study, we examined the inhibitory effects of broccoli (Brassica oleracea var. italica) and watercress (Rorripa nasturtium aquaticum) extracts on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cancer cell invasion and matrix metalloproteinase-9 activity using human MDA-MB-231 breast cancer cells. Aberrant overexpression of matrix metalloproteinases, including metalloproteinase-9, is associated with increased invasive potential in cancer cell lines. Our results demonstrate that extracts of broccoli and Rorripa suppressed TPA-induced MMP-9 activity and invasiveness in a concentration dependant manner as determined by zymographic analysis. Furthermore, fractionation of individual extracts followed by liquid chromatography mass spectroscopy analysis (LC-MS) revealed that the inhibitory effects of each vegetable were associated with the presence of 4-methysulfinylbutyl (sulforaphane) and 7-methylsulphinylheptyl isothiocyanates. Taken together, our data indicate that isothiocyanates derived form broccoli and Rorripa inhibit metalloproteinase 9 activities and also suppress the invasive potential of human MDA-MB-231 breast cancer cells in vitro. The inhibitory effects observed in the current study may contribute to the suppression of carcinogenesis by diets high in cruciferous vegetables.

  9. Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells

    PubMed Central

    Acua-Macas, Isabel; Vera, Eunice; Vzquez-Snchez, Alma Yolanda; Mendoza-Garrido, Mara Eugenia; Camacho, Javier

    2015-01-01

    Oncogenic ether -go-go-1 (Eag1) potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF) regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively) were obtained from the American Type Culture Collection and cultured following the manufacturers recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development. PMID:26527881

  10. Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells.

    PubMed

    Acua-Macas, Isabel; Vera, Eunice; Vzquez-Snchez, Alma Yolanda; Mendoza-Garrido, Mara Eugenia; Camacho, Javier

    2015-01-01

    Oncogenic ether -go-go-1 (Eag1) potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF) regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively) were obtained from the American Type Culture Collection and cultured following the manufacturer's recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development. PMID:26527881

  11. Differential effects of estrogen-dependent transactivation vs. transrepression by the estrogen receptor on invasiveness of HER2 overexpressing breast cancer cells.

    PubMed

    Patki, Mugdha; Salazar, Marcela d'alincourt; Trumbly, Robert; Ratnam, Manohar

    2015-02-13

    Estrogen (E2) supports breast cancer cell growth but suppresses invasiveness and both actions are antagonized by anti-estrogens. As a consequence, anti-estrogen treatment may increase the invasive potential of estrogen receptor (ER)+ tumor cell sub-populations that are endocrine resistant due to HER2 amplification. Either transactivation or transrepression by E2/ER could lead to both up- and down-regulation of many genes. Inhibition of the transactivation function of ER is adequate to inhibit E2-dependent growth. However, the impact of inhibiting E2-dependent transactivation vs. transrepression by ER on regulation of invasiveness by E2 is less clear. Here we dissect the roles of ER-mediated transactivation and transrepression in the regulation of invasiveness of ER+/HER2+ breast cancer cells by E2. Knocking down the general ER co-activators CBP and p300 prevented activation by E2 of its classical target genes but did not interfere with the ability of E2 to repress its direct target genes known to support invasiveness and tumor progression; there was also no effect on invasiveness or the ability of E2 to regulate invasiveness. On the other hand, overexpression of a co-repressor binding site mutant of ER (L372R) prevented E2-dependent transrepression but not transactivation. The mutant ER abrogated the ability of E2 to suppress invasiveness. E2 can partially down-regulate HER2 but knocking down HER2 below E2-regulated levels did not affect invasiveness or the ability of E2 to regulate invasiveness, although it did inhibit growth. Therefore, in ER+/HER2+ cells, the E2-dependent transrepression by ER rather than its transactivation function is critical for regulation of invasiveness and this is independent of HER2 regulation by E2. The findings suggest that selective inhibitors of transactivation by ER may be more beneficial in reducing tumor progression than conventional anti-estrogens that also antagonize E2-dependent transrepression. PMID:25582774

  12. Optimisation of Over-Expression in E. coli and Biophysical Characterisation of Human Membrane Protein Synaptogyrin 1

    PubMed Central

    Löw, Christian; Jegerschöld, Caroline; Kovermann, Michael; Moberg, Per; Nordlund, Pär

    2012-01-01

    Progress in functional and structural studies of integral membrane proteins (IMPs) is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP) synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3) strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3) strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs. PMID:22675529

  13. Overexpression of orphan G-protein-coupled receptor, Gpr49, in human hepatocellular carcinomas with beta-catenin mutations.

    PubMed

    Yamamoto, Yoshiya; Sakamoto, Michiie; Fujii, Gen; Tsuiji, Hitomi; Kenetaka, Kengo; Asaka, Masahiro; Hirohashi, Setsuo

    2003-03-01

    To identify the genes responsible for carcinogenesis and progression of hepatocellular carcinoma (HCC), we screened differentially expressed genes in several human HCC cell lines. Among these genes, Gpr49 was up-regulated in PLC/PRF/5 and HepG2. Gpr49 is a member of the glycoprotein hormone receptor subfamily, which includes the thyroid-stimulating hormone receptor (TSHR). However, Gpr49 remains to be an orphan G-protein-coupled receptor. By real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, overexpression (>3-fold increase compared with the corresponding noncancerous liver tissue) of Gpr49 mRNA was observed in 18 of 38 (47%) HCCs compared with corresponding noncancerous livers. Clinicopathologically, overexpression of Gpr49 was frequently observed in HCC with mutation in beta-catenin exon 3 (14 of 16 cases, 87.5%). Moreover, introduction of mutant beta-catenin into mouse hepatocytes in culture caused up-regulation of the Gpr49 mouse homologue. Therefore, Gpr49 is likely to be a target gene activated by Wnt-signaling in HCC. In conclusion, although much is still unknown, Gpr49 may be critically involved in the development of HCCs with beta-catenin mutations and has the potential to be a new therapeutic target in the treatment of HCC. PMID:12601349

  14. Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

    SciTech Connect

    Huang Shurong; Risques, Rosa Ana; Martin, George M.; Rabinovitch, Peter S.; Oshima, Junko

    2008-01-01

    LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.

  15. Evaluation of gene delivery strategies to efficiently overexpress functional HLA-G on human bone marrow stromal cells

    PubMed Central

    Boura, Joana S; Vance, Melisa; Yin, Weihong; Madeira, Catarina; Lobato da Silva, Cludia; Porada, Christopher D; Almeida-Porada, Graa

    2014-01-01

    Mesenchymal stromal cells (MSC) constitutively express low levels of human leukocyte antigen-G (HLA-G), which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immunomodulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1) and a viral system (Lv-HLA-G1) using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK) cellmediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR)9 expression is the mechanism responsible for the abrogation of HLA-G1s immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSCs immunomodulatory properties. PMID:25279386

  16. Evaluation of gene delivery strategies to efficiently overexpress functional HLA-G on human bone marrow stromal cells.

    PubMed

    Boura, Joana S; Vance, Melisa; Yin, Weihong; Madeira, Catarina; Lobato da Silva, Cludia; Porada, Christopher D; Almeida-Porada, Graa

    2014-09-01

    Mesenchymal stromal cells (MSC) constitutively express low levels of human leukocyte antigen-G (HLA-G), which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immuno-modulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1) and a viral system (Lv-HLA-G1) using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK) cell-mediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR)9 expression is the mechanism responsible for the abrogation of HLA-G1's immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSC's immunomodulatory properties. PMID:25279386

  17. Human epidermal growth factor receptor 2 overexpression and amplification in endoscopic biopsies and resection specimens in esophageal and junctional adenocarcinoma.

    PubMed

    van Hagen, P; Biermann, K; Boers, J E; Stoss, O; Sleddens, H F; van Lanschot, J J B; Dinjens, W N M; Rueschoff, J; Wijnhoven, B P L

    2015-01-01

    Human epidermal growth factor receptor 2 (HER2) is overexpressed in a subset of esophageal adenocarcinomas. Frequently, biopsy material is used for evaluation of HER2 status. The aim of the study was to determine if HER2 expression in preoperative endoscopic biopsies is representative for the entire tumor. Preoperative endoscopic biopsies and matched resection specimens were collected from 75 patients who underwent esophagectomy for esophageal adenocarcinoma. Immunohistochemical staining (IHC) on HER2 and dual-color in situ hybridization (ISH) were performed. HER2 status was determined by following a clinical algorithm, first determining HER2 overexpression on immunohistochemistry and, when equivocal (2+), determining HER2 amplification on ISH. Seventy-one of 75 (95%) biopsies and 69/75 (92%) resection specimens could be analyzed due to technical failure. HER2 positivity was seen in 18/71 (25%) biopsies and in 15/69 (22%) resection specimens. Overall, HER2 status in the biopsy was concordant with HER2 status in the resection specimen in 94% of cases. Interobserver agreement on IHC scoring for all three observers was 83% in biopsies and 85% in resection specimens. HER2 positivity was detected in 22% of esophageal adenocarcinomas. Although interobserver agreement was moderate, HER2 status of a primary tumor can be reliably determined based on the endoscopically obtained pretreatment biopsy. PMID:24611982

  18. DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro

    PubMed Central

    Tan, Yan; Shi, Yu-sheng; Wu, Xi-dong; Liang, Hui-yu; Gao, Yu-bo; Li, Shu-ji; Zhang, Xing-mei; Wang, Fang; Gao, Tian-ming

    2013-01-01

    Aim: Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule in cells, thus may act as effective vehicles for drug or siRNA delivery. In this study we investigated the DNA aptamers that target human glioblastoma multiforme (GBM) cells overexpressing epidermal growth factor receptor variant III (EGFRvIII), which was linked to radiation and chemotherapeutic resistance of this most aggressive brain tumor. Methods: A 73-mer ssDNA library containing molecules with 30 nt of random sequence flanked by two primer hybridization sites was chosen as the initial library. Cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) method was used to select the DNA aptamers that target EGFRvIII. The binding affinity of the aptamers was measured using a cell-based biotin-avidin ELISA. Results: After 14 rounds of selection, four DNA aptamers (32, 41, 43, and 47) that specifically bound to the EGFRvIII-overexpressing human glioma U87? cells with Kd values of less than 100 nmol/L were discovered. These aptamers were able to distinguish the U87? cells from the negative control human glioma U87MG cells and HEK293 cells. Aptamer 32 specifically bound to the EGFRvIII protein with an affinity similar to the EGFR antibody (Kd values of aptamer 32 and the EGFR antibody were 0.620.04 and 0.320.01 nmol/L, respectively), and this aptamer was localized in the cell nucleus. Conclusion: The DNA aptamers are promising molecular probes for the diagnosis and treatment of GBM. PMID:24304919

  19. A non-randomized dose-escalation Phase I trial of a protein-based immunotherapeutic for the treatment of breast cancer patients with HER2-overexpressing tumors.

    PubMed

    Limentani, Steven A; Campone, Mario; Dorval, Thierry; Curigliano, Giuseppe; de Boer, Richard; Vogel, Charles; White, Shane; Bachelot, Thomas; Canon, Jean-Luc; Disis, Mary; Awada, Ahmad; Berlière, Martine; Amant, Frédéric; Levine, Ellis; Burny, Wivine; Callegaro, Andrea; de Sousa Alves, Pedro Miguel; Louahed, Jamila; Brichard, Vincent; Lehmann, Frédéric F

    2016-04-01

    This Phase I dose-escalation study (NCT00058526) assessed the safety and immunogenicity of an anti-cancer immunotherapeutic (recombinant HER2 protein (dHER2) combined with the immunostimulant AS15) in patients with early-stage HER2-overexpressing breast cancer (BC). Sixty-one trastuzumab-naive patients with stage II-III HER2-positive BC received the dHER2 immunotherapeutic after surgical resection and adjuvant therapy. They were allocated into four cohorts receiving different doses of dHER2 (20, 100, 500 µg) combined with a fixed AS15 dose. Safety and immunogenicity (dHER2-specific antibody responses) were assessed. After completing the immunization schedule (three or six doses over 14 weeks) and a six-month follow-up, the patients were followed for 5 years for late toxicity, long-term immunogenicity, and clinical status. The immunizations were well tolerated, and increasing doses of dHER2 had no impact on the frequency or severity of adverse events. Few late toxicities were reported, and after 5 years 45/54 patients (83.3 %) were still alive, while 28/45 (62 %) with known disease status were disease free. Regarding the immunogenicity of the compound, a positive association was found between the dHER2 dose, the immunization schedule, and the prevalence of dHER2-specific humoral responses. Among the patients receiving the most intense immunization schedule with the highest dHER2 dose, 6/8 maintained their dHER2-specific antibody response 5 years after immunization. The dHER2 immunotherapeutic had an acceptable safety profile in early HER2-positive BC patients. dHER2-specific antibody responses were induced, with the rate of responders increasing with the dHER2 dose and the number and frequency of immunizations. PMID:26993131

  20. On-chip immunoelectrophoresis of extracellular vesicles released from human breast cancer cells.

    PubMed

    Akagi, Takanori; Kato, Kei; Kobayashi, Masashi; Kosaka, Nobuyoshi; Ochiya, Takahiro; Ichiki, Takanori

    2015-01-01

    Extracellular vesicles (EVs) including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker) antibody showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG) isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker) antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer. PMID:25928805

  1. On-Chip Immunoelectrophoresis of Extracellular Vesicles Released from Human Breast Cancer Cells

    PubMed Central

    Akagi, Takanori; Kato, Kei; Kobayashi, Masashi; Kosaka, Nobuyoshi; Ochiya, Takahiro; Ichiki, Takanori

    2015-01-01

    Extracellular vesicles (EVs) including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker) antibody showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG) isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker) antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer. PMID:25928805

  2. Breast Cancer classification using extracted parameters from a terahertz dielectric model of human breast tissue.

    PubMed

    Truong, Bao C Q; Tuan, H D; Fitzgerald, Anthony J; Wallace, Vincent P; Tuan Nghia Nguyen; Nguyen, H T

    2015-08-01

    Our previous study proposed a dielectric model for human breast tissue and provided initial analysis of classification potential of the eight model parameters and their multiparameter combinations with the support vector machine (SVM). A combination of three model parameters could achieve a leave-one-out cross validation accuracy of 93.2%. However, the SVM approach fails to exploit the combinations of more than three model parameters for classification improvement. Thus, the Bayesian neural network (BNN) method is employed to overcome this problem based on its advantages of handling our small data and high complexity of the multiparamter combinations. The BNN successfully classifies the data using the combinations of four model parameters with an accuracy, estimated by leave-one-out cross validation, of 97.3%. Overall performance assessed by leaveone-out and repeated random-subsampling cross validations for all examined combinations is also remarkably improved by BNN. The results indicate the advance of BNN as compared to SVM in utilising the model parameters for detecting tumour from normal breast tissue. PMID:26736874

  3. Estrogen deprivation causes estradiol hypersensitivity in human breast cancer cells.

    PubMed

    Masamura, S; Santner, S J; Heitjan, D F; Santen, R J

    1995-10-01

    Genetic and environmental factors can modulate the level of sensitivity to various hormones, including estrogens. Enhanced sensitivity to estradiol (E2) has been demonstrated in several biological conditions, such as in sheep during the nonbreeding season, in untreated patients with Turner's syndrome, and in the prepubertal state in normal girls. We postulated that secondary responses to hormonal therapy in patients with breast cancer could also result from enhanced E2 sensitivity, developing as an adaptive mechanism to E2 deprivation. The present study used the MCF-7 human breast cancer cell line as a model system to test the concept that enhanced sensitivity to E2 may occur as a result of adaptation to low E2 levels. After depriving MCF-7 cells of estrogens in tissue culture medium for periods of 1-6 months, we established conditions under which replication could be stimulated maximally by 10(-14)-10(-15) mol/L E2. In contrast, wild-type cells not exposed to estrogen deprivation required 10(-10) mol/L E2 to grow at the same rate. Further, the concentration of the antiestrogen, ICI 164384, needed to inhibit growth by 50% in estrogen-deprived cells was much lower than that required in wild-type cells (i.e. 10(-15) vs. 10(-9) mol/L). Nude mice implanted with these estrogen-deprived cells demonstrated an earlier appearance of palpable tumors in response to E2 than animals bearing wild-type cells. Reexposure to 10(-10)-10(-9) mol/L E2, either in vivo or in vitro, returned these cells to the level of estrogen sensitivity observed in wild-type cells. Taken together, these observations suggest that breast cancer cells can adapt to low levels of estrogens by enhancing their sensitivity to E2. PMID:7559875

  4. Effects of Recombinant Human Prolactin on Breast Milk Composition

    PubMed Central

    Powe, Camille E.; Puopolo, Karen M.; Newburg, David S.; Lnnerdal, Bo; Chen, Ceng; Allen, Maureen; Merewood, Anne; Worden, Susan

    2011-01-01

    OBJECTIVE: The objective of this study was to determine the impact of recombinant human prolactin (r-hPRL) on the nutritional and immunologic composition of breast milk. METHODS: We conducted 2 trials of r-hPRL treatment. In the first study, mothers with documented prolactin deficiency were given r-hPRL every 12 hours in a 28-day, open-label trial. In the second study, mothers with lactation insufficiency that developed while they were pumping breast milk for their preterm infants were given r-hPRL daily in a 7-day, double-blind, placebo-controlled trial. Breast milk characteristics were compared before and during 7 days of treatment. RESULTS: Among subjects treated with r-hPRL (N = 11), milk volumes (73 36 to 146 54 mL/day; P < .001) and milk lactose levels (155 15 to 184 8 mmol/L; P = .01) increased, whereas milk sodium levels decreased (12.1 2.0 to 8.3 0.5 mmol/L; P = .02). Milk calcium levels increased in subjects treated with r-hPRL twice daily (2.8 0.6 to 5.0 0.9 mmol/L; P = .03). Total neutral (1.5 0.3 to 2.5 0.4 g/L; P = .04) and acidic (33 4 to 60 6 mg/L; P = .02) oligosaccharide levels increased in r-hPRL-treated subjects, whereas total daily milk immunoglobulin A secretionwas unchanged. CONCLUSIONS: r-hPRL treatment increased milk volume and induced changes in milk composition similar to those that occur during normal lactogenesis. r-hPRL also increased antimicrobially active oligosaccharide concentrations. These effects were achieved for women with both prolactin deficiency and lactation insufficiency. PMID:21262884

  5. First trimester human placental factors induce breast cancer cell autophagy.

    PubMed

    Epstein Shochet, G; Drucker, L; Pasmanik-Chor, M; Pomeranz, M; Fishman, A; Tartakover Matalon, S; Lishner, M

    2015-02-01

    Placental factors, progesterone included, facilitate breast cancer cell line (BCCL) motility and thus may contribute to the advanced breast cancer found during pregnancy. Cancer and placental implantations are similar; the last is accompanied by extravillous trophoblast cell invasion and autophagy which are interlinked. We aimed to analyze the effect of first trimester human placenta on BCCL autophagy. BCCLs (MCF-7/T47D) were cultured with placental explants (60 h) or placental supernatants (24 h). Following cultures, BCCLs were sorted out for RNA/protein extraction. RNA served for microarray/qPCR (BNIP3) and protein for Western blot (HIF1?, LC3BII) analyses. Inhibitors were added to the placenta-MCF-7 coculture or placental supernatants (autophagy inhibitor-3MA, progesterone receptor (PR) inhibitor-RU486, and HIF1? inhibitor-Vitexin) in order to evaluate their effects on BCCL motility and LC3BII/HIF1? expression. LC3BII (an autophagy marker) expression was elevated in BCCLs following placental explant coculture and exposure to placental supernatants. The autophagy inhibitor (3MA) repressed the placenta-induced MCF-7/T47D migration, establishing a connection between BCCL autophagy and migration. Microarray analysis of MCF-7 following placenta-MCF-7 coculture showed that "HIF1? pathway," a known autophagy facilitator, was significantly manipulated. Indeed, placental factors elevated HIF1? and its target BNIP3 in the BCCLs, verifying array results. Lastly, PR inhibitor reduced HIF1? expression and both PR and HIF1? inhibitors reduced MCF-7 LC3BII expression and motility, suggesting involvement of the PR-HIF1? axis in the autophagy process. Placental factors induced BCCL autophagy that is interlinked to their motility. This suggests that autophagy-related molecules may serve as targets for therapy in pregnancy-associated breast cancer. PMID:25656679

  6. Association of hsa-miR‑145 overexpression in human testicular cells with male infertility.

    PubMed

    Zhang, Liyuan; Ding, Xianping; Nie, Shuangshuang; Li-Ling, Jesse; Zhang, Yuping; Zhang, Hui; Chen, Lin; Li, Lingxiao; Ding, Min

    2015-06-01

    MicroRNAs (miRs) have crucial functions in spermatogenesis and implications for male infertility. In the present study, Homo sapiens (hsa)‑miR‑145 was designed and cloned into the eukaryotic expression plasmid pGenesil‑1. The recombinant plasmids were transfected into Hs 1.tes normal testicular cells and NTERA‑2 testicular cancer cells. Quantitative polymerase chain reaction of hsa‑miR‑145 indicated that pGenesil‑1‑miR‑145 effectively upregulated the expression of hsa‑miR‑145 in vitro. hsa‑miR‑145 overexpression inhibited the mRNA and protein expression of sex-determining region Y Box 9 in Hs 1.tes cells. The proliferation rates of NTERA‑2 cells transfected with pGenesil‑1‑miR‑145 were significantly decreased. High expression levels of miR‑145 promoted cell apoptosis in NTERA‑2 cells. The results revealed that altered hsa‑miR‑145 expression in testicular cells affects the regulation of target genes associated with male infertility. PMID:25633044

  7. GPX4 and GPX7 Over-Expression in Human Hepatocellular Carcinoma Tissues

    PubMed Central

    Guerriero, E.; Capone, F.; Accardo, M.; Sorice, A.; Costantini, M.; Colonna, G.; Castello, G.

    2015-01-01

    Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is still one of the most fatal cancers. Hence, it needs to identify always new putative markers to improve its diagnosis and prognosis. The selenium is an essential trace mineral implicated as a key factor in the early stage of cancer and exerts its biological function through the selenoproteins. In the last years our group has been studying the involvement of some selenoproteins in HCC. However, no many data are reported in literature about the correlation between HCC and the glutathione peroxidases (GPXs), both selenium and non selenium-containing GPXs. In this paper we have evaluated the GPX4 and GPX7 expression in some paraffin-embedded tissues from liver biopsy of patients with hepatitis C virus (HCV)-related cirrhosis and HCC by immunohistochemistry and RT-qPCR analysis. Our results evidenced that i) GPX4 and GPX7 had a statistically significant over-expression in HCC tissues compared to cirrhotic counterparts used as non tumor tissues, and ii) their expression was higher in grade III HCC tissues with respect to grade I-II samples. Therefore, we propose to use GPX4 and GPX7 as possible markers for improving HCC diagnosis/prognosis. PMID:26708178

  8. The Expression of the Nectin Complex in Human Breast Cancer and the Role of Nectin-3 in the Control of Tight Junctions during Metastasis

    PubMed Central

    Martin, Tracey A.; Lane, Jane; Harrison, Gregory M.; Jiang, Wen G.

    2013-01-01

    Introduction Nectins are a family of integral protein molecules involved in the formation of functioning Adherens and Tight Junctions (TJ). Aberrant expression is associated with cancer progression but little is known how this effects changes in cell behaviour. This study aimed to ascertain the distribution of Nectins-1 to -4 in human breast cancer and the effect on junctional integrity of Nectin-3 modulation in human endothelial and breast cancer cells. Methods A human breast tissue cohort was processed for Q-PCR and immunohistochemistry for analysis of Nectin-1/-2/-3/-4. Nectin-3 over-expression was induced in the human breast cancer cell line MDA-MB-231 and the human endothelial cell line HECV. Functional testing was carried out to ascertain changes in cell behaviour. Results Q-PCR revealed a distinct reduction in node positive tumours and in patients with poor outcome. There was increased expression of Nectin-1/-2 in patients with metastatic disease, Nectin-3/-4 was reduced. IHC revealed that Nectin-3 expression showed clear changes in distribution between normal and cancerous cells. Nectin-3 over-expression in MDA-MB-231 cells showed reduced invasion and migration even when treated with HGF. Changes in barrier function resulted in MDAN3 cells showing less change in resistance after 2h treatment with HGF (p<0.001). Nectin-3 transformed endothelial cells were significantly more adhesive, irrespective of treatment with HGF (p<0.05) and had reduced growth. Barrier function revealed that transformed HECV cells had significantly tighter junctions that wildtype cells when treated with HGF (p<0.0001). HGF-induced changes in permeability were also reduced. Overexpression of Nectin-3 produced endothelial cells with significantly reduced ability to form tubules (p<0.0001). Immunoprecipitation studies discovered hitherto novel associations for Nectin-3. Moreover, HGF appeared to exert an effect on Nectin-3 via tyrosine and threonine phosphorylation. Conclusions Nectin-3 may be a key component in the formation of cell junctions and be a putative suppressor molecule to the invasion of breast cancer cells. PMID:24386110

  9. Met induces diverse mammary carcinomas in mice and is associated with human basal breast cancer.

    PubMed

    Graveel, Carrie R; DeGroot, Jack D; Su, Yanli; Koeman, Julie; Dykema, Karl; Leung, Samuel; Snider, Jacqueline; Davies, Sherri R; Swiatek, Pamela J; Cottingham, Sandra; Watson, Mark A; Ellis, Matthew J; Sigler, Robert E; Furge, Kyle A; Vande Woude, George F

    2009-08-01

    Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. The oncogene MET is associated with decreased survival in breast cancer, yet the role that MET plays in the various breast cancer subtypes is unclear. We describe a knockin mouse with mutationally activated Met (Met(mut)) that develops a high incidence of diverse mammary tumors with basal characteristics, including metaplasia, absence of progesterone receptor and ERBB2 expression, and expression of cytokeratin 5. With gene expression and tissue microarray analysis, we show that high MET expression in human breast cancers significantly correlated with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers. Few treatment options exist for breast cancers of the basal or trastuzumab-resistant ERBB2 subtypes. We conclude from these studies that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target. PMID:19567831

  10. Effect of soy isoflavones on the growth of human breast tumors: findings from preclinical studies

    PubMed Central

    Kwon, Youngjoo

    2014-01-01

    Breast cancer is the most common cancer among women worldwide, and many women with breast cancer live more than 5years after their diagnosis. Breast cancer patients and survivors have a greater interest in taking soy foods and isoflavone supplements. However, the effect of isoflavones on breast cancer remains controversial. Thus, it is critical to determine if and when isoflavones are beneficial or detrimental to breast cancer patients. According to the available preclinical data, high concentrations of isoflavones inhibit the proliferation of breast cancer cells, regardless of their estrogen receptor (ER) status. In comparison, genistein, a major isoflavone, has stimulated tumor growth at low concentrations and mitigated tamoxifen efficacy in ER-positive breast cancer. Studies have indicated that the relative levels of genistein and estrogen at the target site are important to determine the genistein effect on the ER-positive tumor growth. However, studies using ovariectomized mice and subcutaneous xenograft models might not truly reflect estrogen concentrations in human breast tumors. Moreover, it may be an oversimplification that isoflavones stimulate hormone-dependent tumor growth due to their potential estrogenic effect since studies also suggest nonestrogenic anticancer effects of isoflavones and ER-independent anticancer activity of tamoxifen. Therefore, the concentrations of isoflavones and estrogen in human breast tumors should be considered better in future preclinical studies and the parameters that can estimate those levels in breast tumors are required in human clinical/epidemiological investigation. In addition, it will be important to identify the molecular mechanisms that either inhibit or promote the growth of breast cancer cells by soy isoflavones, and use those molecules to evaluate the relevance of the preclinical findings to the human disease and to predict the health effects of isoflavones in human breast tumors. PMID:25493176

  11. Human dCTP pyrophosphatase 1 promotes breast cancer cell growth and stemness through the modulation on 5-methyl-dCTP metabolism and global hypomethylation

    PubMed Central

    Song, F-f; Xia, L-l; Ji, P; Tang, Y-b; Huang, Z-m; Zhu, L; Zhang, J; Wang, J-q; Zhao, G-p; Ge, H-l; Zhang, Y; Wang, Y

    2015-01-01

    Human DCTPP1 (dCTP pyrophosphatase 1), also known as XTP3-transactivated protein A, belongs to MazG-like nucleoside triphosphate pyrophosphatase (NTP-PPase) superfamily. Being a newly identified pyrophosphatase, its relevance to tumorigenesis and the mechanisms are not well investigated. In the present study, we have confirmed our previous study that DCTPP1 was significantly hyperexpressed in breast cancer and further demonstrated its strong association with tumor progression and poor prognosis in breast cancer. Knockdown of DCTPP1 in breast cancer cell line MCF-7 cells remarkably retarded proliferation and colony formation in vitro. The capacity of mammosphere formation of MCF-7 was suppressed with the silence of DCTPP1, which was consistent with the enhanced mammosphere-forming ability in DCTPP1-overexpressed MDA-MB-231 cells. To further dissect the mechanisms of DCTPP1 in promoting tumor cell growth and stemness maintenance, its biochemical properties and biological functions were investigated. DCTPP1 displayed bioactive form with tetrameric structure similar to other MazG domain-containing pyrophosphatases based on structure simulation. A substrate preference for dCTP and its methylated or halogen-modified derivatives over the other canonical (deoxy-) NTPs was demonstrated from enzymatic assay. This substrate preference was also proved in breast cancer cells that the intracellular 5-methyl-dCTP level increased in DCTPP1-deficient MCF-7 cells but decreased in DCTPP1-overexpressed MDA-MB-231 cells. Moreover, global methylation level was elevated in DCTPP1-knockdown MCF-7 cells or mammosphere-forming MCF-7 cells but decreased significantly in DCTPP1-overexpressed MDA-MB-231 cells and its mammospheres. Our results thus indicated that human DCTPP1 was capable of modulating the concentration of intracellular 5-methyl-dCTP. This in turn affected global methylation, contributing to a known phenomenon of hypomethylation related to the cancer cell growth and stemness maintenance. Our current investigations point to the pathological functions of DCTPP1 overexpression in breast cancer cells with aberrant dCTP metabolism and epigenetic modification. PMID:26075750

  12. Prostaglandin E2 production and metabolism in human breast cancer cells and breast fibroblasts. Regulation by inflammatory mediators.

    PubMed Central

    Schrey, M. P.; Patel, K. V.

    1995-01-01

    Malignant human breast tumours contain high levels of prostaglandin E2 (PGE2). However, the mechanisms controlling PGE2 production in breast cancer are unknown. This in vitro study investigates the capacity for PGE2 synthesis and metabolism in several human breast cancer cell lines and early passage human breast fibroblasts and seeks to identify potential regulatory factors which may control these pathways. Basal PGE2 production rose up to 30-fold in breast fibroblast lines on addition of exogenous arachidonic acid (10 microM), whereas no such changes were observed in six out of seven cancer cell lines, with the exception of modest increases in MDA-MB-231 cells. Interleukin 1 beta (IL-1 beta) also induced PGE2 production in breast fibroblasts in the presence of excess substrate, consistent with cyclo-oxygenase induction by the cytokine. Under these conditions only Hs578T cells and MDA-MB-231 cells demonstrated large increases in PGE2 in response to IL-1 beta or phorbol ester; no such responses were seen in MCF-7, T47-D, ZR-75-1, BT-20 or CLF-90-1 cells. In the absence of added arachidonate, bradykinin (BK) and endothelin-1 (ET-1), potentiated PGE2 production in IL-1 beta-treated fibroblasts, possibly by mobilising endogenous substrate. PGE2 also stimulated ET-1 production by breast cancer cells. In co-cultures with T47-D cells both basal and stimulated PGE2 production by breast fibroblasts was greatly reduced. This appeared to be due to metabolic inactivation by the cancer cell since T47-D cells readily converted PGE2 to 15-keto-PGE2. This apparent 15-hydroxy-PG dehydrogenase activity was stimulated by TPA and inhibited by cycloheximide. In conclusion, breast fibroblasts, particularly under the influence of inflammatory mediators, provide a potentially rich source for PGE2 production in breast tumours, whereas significant contributions from the epithelial tumour component may be restricted to cancer cells exhibiting an invasive phenotype. Metabolic inactivation by the cancer cells may also play an important role in the regulation of breast tumour PGE2 levels. PMID:8519653

  13. Overexpression of TRIB2 in human lung cancers contributes to tumorigenesis through downregulation of C/EBP?

    PubMed Central

    Grandinetti, KB; Stevens, TA; Ha, S; Salamone, RJ; Walker, JR; Zhang, J; Agarwalla, S; Tenen, DG; Peters, EC; Reddy, VA

    2012-01-01

    Lung cancer is the most common cause of cancer-related mortality worldwide. Here, we report elevated expression of tribbles homolog 2 (TRIB2) in primary human lung tumors and in non-small cell lung cancer cells that express low levels of differentiation-inducing transcription factor CCAAT/enhancer-binding protein alpha (C/EBP?). In approximately 1020% of cases, elevated TRIB2 expression resulted from gene amplification. TRIB2 knockdown was found to inhibit cell proliferation and in vivo tumor growth. In addition, TRIB2 knockdown led to morphological changes similar to C/EBP? overexpression and correlated with increased expression and activity of C/EBP?. TRIB2-mediated regulation of C/EBP? was found to occur through the association of TRIB2 with the E3 ligase TRIM21. Together, these data identify TRIB2 as a potential driver of lung tumorigenesis through a mechanism that involves downregulation of C/EBP?. PMID:21399661

  14. Overexpression of TRIB2 in human lung cancers contributes to tumorigenesis through downregulation of C/EBP?.

    PubMed

    Grandinetti, K B; Stevens, T A; Ha, S; Salamone, R J; Walker, J R; Zhang, J; Agarwalla, S; Tenen, D G; Peters, E C; Reddy, V A

    2011-07-28

    Lung cancer is the most common cause of cancer-related mortality worldwide. Here, we report elevated expression of tribbles homolog 2 (TRIB2) in primary human lung tumors and in non-small cell lung cancer cells that express low levels of differentiation-inducing transcription factor CCAAT/enhancer-binding protein alpha (C/EBP?). In approximately 10-20% of cases, elevated TRIB2 expression resulted from gene amplification. TRIB2 knockdown was found to inhibit cell proliferation and in vivo tumor growth. In addition, TRIB2 knockdown led to morphological changes similar to C/EBP? overexpression and correlated with increased expression and activity of C/EBP?. TRIB2-mediated regulation of C/EBP? was found to occur through the association of TRIB2 with the E3 ligase TRIM21. Together, these data identify TRIB2 as a potential driver of lung tumorigenesis through a mechanism that involves downregulation of C/EBP?. PMID:21399661

  15. Directing Cardiomyogenic Differentiation of Human Pluripotent Stem Cells by Plasmid-Based Transient Overexpression of Cardiac Transcription Factors

    PubMed Central

    Hartung, Susann; Schwanke, Kristin; Haase, Alexandra; David, Robert; Franz, Wolfgang-Michael

    2013-01-01

    Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) possess a high potential for regenerative medicine. Previous publications suggested that viral transduction of a defined set of transcription factors (TFs) known to play pivotal roles in heart development also increases cardiomyogenesis in vitro upon overexpression in mouse or human ES cells. To circumvent issues associated with viral approaches such as insertional mutagenesis, we have established a transient transfection system for straightforward testing of TF combinations. Applying this method, the transfection efficiency and the temporal pattern of transgene expression were extensively assessed in hPSCs by quantitative real time-polymerase chain reaction (qRT-PCR), TF-specific immunofluorescence analysis, and flow cytometry. Testing TF combinations in our approach revealed that BAF60C, GATA4, and MESP1 (BGM) were most effective for cardiac forward programming in human induced pluripotent stem cell lines and human ES cells as well. Removal of BAF60C slightly diminished formation of CM-like cells, whereas depletion of GATA4 or MESP1 abolished cardiomyogenesis. Each of these TFs alone had no inductive effect. In addition, we have noted sensitivity of CM formation to cell density effects, which highlights the necessity for cautious analysis when interpreting TF-directed lineage induction. In summary, this is the first report on TF-induced cardiomyogenesis of hPSCs applying a transient, nonintegrating method of cell transfection. PMID:23157212

  16. Nicotine-induced human breast cancer cell proliferation attenuated by garcinol through down-regulation of the nicotinic receptor and cyclin D3 proteins.

    PubMed

    Chen, Ching-Shyang; Lee, Chia-Hwa; Hsieh, Chang-Da; Ho, Chi-Tang; Pan, Min-Hsiung; Huang, Ching-Shui; Tu, Shih-Hsin; Wang, Ying-Jan; Chen, Li-Ching; Chang, Yu-Jia; Wei, Po-Li; Yang, Yi-Yuan; Wu, Chih-Hsiung; Ho, Yuan-Soon

    2011-01-01

    Previous studies have demonstrated that the persistent exposure of human bronchial epithelial cells to nicotine (Nic) through nicotinic acetylcholine receptors increases cyclin D1 promoter activity and protein expression. The main purpose of this study is to elucidate the carcinogenic role of cyclin D3, which is involved in breast tumorigenesis when induced by Nic. Real-time PCR analysis revealed that cyclin D3 is highly expressed at the mRNA level in surgically dissected breast tumor tissue, compared to the surrounding normal tissue (tumor/normal fold ratio = 17.93, n = 74). To test whether Nic/nicotinic acetylcholine receptor (nAChR) binding could affect cyclin D3 expression in human breast cancer cells, the transformed cell line MCF-10A-Nic (DOX) was generated from normal breast epithelial cells (MCF-10A) with inducible α9-nAChR gene expression, using the adenovirus tetracycline-regulated Tet-off system. Tet-regulated overexpression of α9-nAChR in MCF-10A-Nic (DOX) xenografted BALB/c-nu/nu mice resulted in a significant induction of cyclin D3. In contrast, cyclin D3 expression was down-regulated in α9-nAChR knock-down (siRNA) MDA-MB-231-xenografted tumors in NOD.CB17-PRKDC(SCID)/J(NOD-SCID) mice. Furthermore, we found that Nic-induced human breast cancer (MDA-MB-231) cell proliferation was inhibited by 1 μM of garcinol (Gar), isolated from the edible fruit Garcinia indica, through down-regulation of α9-nAChR and cyclin D3 expression. These results suggest that α9-nAChR-mediated cyclin D3 overexpression is important for nicotine-induced transformation of normal human breast epithelial cells. The homeostatic regulation of cyclin D3 has the potential to be a molecular target for antitumor chemotherapeutic or chemopreventive purposes in clinical breast cancer patients. PMID:20229177

  17. Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells

    SciTech Connect

    Wang, Jing; Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province ; Yang, Qifeng; Haffty, Bruce G.; Li, Xiaoyan; Moran, Meena S.

    2013-02-08

    Highlights: ► Fulvestrant radiosensitizes MCF-7 cells. ► Fulvestrant increases G1 arrest and decreases S phase in MCF-7 cells. ► Fulvestrant down-regulates DNA-PKcs and RAD51 in MCF-7 cells. -- Abstract: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT increases breast cancer cell radiosensitivity compared with radiation alone. These findings have salient implications for designing clinical trials using fulvestrant and radiation therapy.

  18. Defective renal water handling in transgenic mice over-expressing human CD39/NTPDase1.

    PubMed

    Zhang, Yue; Morris, Kaiya L; Sparrow, Shannon K; Dwyer, Karen M; Enjyoji, Keiichi; Robson, Simon C; Kishore, Bellamkonda K

    2012-08-01

    Ectonucleoside triphosphate diphosphohydrolase-1 hydrolyzes extracellular ATP and ADP to AMP. Previously, we showed that CD39 is expressed at several sites within the kidney and thus may impact the availability of type 2 purinergic receptor (P2-R) ligands. Because P2-Rs appear to regulate urinary concentrating ability, we have evaluated renal water handling in transgenic mice (TG) globally overexpressing hCD39. Under basal conditions, TG mice exhibited significantly impaired urinary concentration and decreased protein abundance of AQP2 in the kidney compared with wild-type (WT) mice. Urinary excretion of total nitrates/nitrites was significantly higher in TG mice, but the excretion of AVP or PGE(2) was equivalent to control WT mice. There were no significant differences in electrolyte-free water clearance or fractional excretion of sodium. Under stable hydrated conditions (gelled diet feeding), the differences between the WT and TG mice were negated, but the decrease in urine osmolality persisted. When water deprived, TG mice failed to adequately concentrate urine and exhibited impaired AVP responses. However, the increases in urinary osmolalities in response to subacute dDAVP or chronic AVP treatment were similar in TG and WT mice. These observations suggest that TG mice have impaired urinary concentrating ability despite normal AVP levels. We also note impaired AVP release in response to water deprivation but that TG kidneys are responsive to exogenous dDAVP or AVP. We infer that heightened nucleotide scavenging by increased levels of CD39 altered the release of endogenous AVP in response to dehydration. We propose that ectonucleotidases and modulated purinergic signaling impact urinary concentration and indicate potential utility of targeted therapy for the treatment of water balance disorders. PMID:22622462

  19. First Mnks degrading agents block phosphorylation of eIF4E, induce apoptosis, inhibit cell growth, migration and invasion in triple negative and Her2-overexpressing breast cancer cell lines

    PubMed Central

    Ramalingam, Senthilmurugan; Gediya, Lalji; Kwegyir-Afful, Andrew K.; Ramamurthy, Vidya P.; Purushottamachar, Puranik; Mbatia, Hannah; Njar, Vincent C. O.

    2014-01-01

    Some retinoic acid metabolism blocking agents (RAMBAs) are known to exhibit a wide range of anticancer activities by mechanisms that are still not completely resolved. This study investigated the anticancer efficacy and mechanism(s) of novel RAMBA retinamides (RRs) in triple negative and Her-2 overexpressing breast cancer cells. Specifically, we examined the possibility that RRs affect the translational machinery in these breast cancer (BC) cells. Recent findings suggest that overexpression of eukaryotic translation initiation factor 4E (eIF4E) in breast cancers critically augments CAP-dependent mRNA translation and synthesis of proteins involved in cell growth, cell proliferation, invasion and apoptosis evasion. The oncogenic potential of eIF4E is strictly dependent on serine209 phosphorylation by upstream MAPK-interacting kinases (Mnks). Targeting Mnk/eIF4E pathway for blocking Mnk function and eIF4E phosphorylation is therefore a novel approach for treating BCs, particularly for Her2-positive and triple negative breast cancers that have no indications for endocrine therapy or effective treatment regimes. We report for the first time that the degradation of Mnk1 by RRs in BC cells blocks eIF4E phosphorylation and subsequently inhibits cell growth, colonization, invasion, and migration and induce apoptosis. Most importantly, the anticancer efficacy of RRs was mediated via degrading Mnk rather than inhibiting its kinase activity like Mnk inhibitors (cercosporamide and CGP57380). Furthermore, RRs potencies on peIF4E down-regulation and growth inhibition were superior to those of two clinically relevant retinoids and the Mnk inhibitors. Together our findings provide the first preclinical proof-of-concept of novel Mnk degrading agents for Mnk/eIF4E based therapeutic treatment of breast cancers. PMID:24504069

  20. Mode of action of the retrogene product SNAI1P, a SNAIL homolog, in human breast cancer cells.

    PubMed

    Mittal, Mukul K; Myers, Jeremy N; Bailey, Charvann K; Misra, Smita; Chaudhuri, Gautam

    2010-03-01

    SNAI1P, a protein coded by a retrogene, is a member of the SNAI family of E2-box binding transcriptional repressors. To evaluate whether the mode of action of SNAI1P is similar to those of the other predominant members of the SNAI family, we studied its action on human claudin 7 (CLDN7) gene promoter which has seven E2-boxes. We over-expressed FLAG-tagged SNAI1P in MCF7 and MDA-MB-468 cells. SNAI1P inhibited the expression of CLDN7 in these recombinant cells. SNAI1P also inhibited cloned CLDN7 gene promoter activity in human breast cancer cells. ChIP assays revealed that SNAI1P is recruited on the CLDN7 gene promoter along with the co-repressor CtBP1 and the effector HDAC1. Treatment of the cells with trichostatin A, an inhibitor of HDAC1, abrogated the repressor activity of SNAI1P. These data suggest that SNAI1P inhibits CLDN7 gene promoter epigenetically in breast cancer cells through chromatin remodeling. PMID:19277896

  1. Constitutive overexpression of a growth-regulated gene in transformed Chinese hamster and human cells

    SciTech Connect

    Anisowicz, A.; Bardwell, L.; Sager, R.

    1987-10-01

    Comparison by subtractive hybridization of mRNAs revealed a moderately abundant message in highly tumorigenic CHEF/16 cells present at very low levels in closely related nontumorigenic CHEF/18 cells. After cloning and sequencing the corresponding cDNA, computer comparison showed closest homology with the human connective tissue-activating peptide III (CTAP III). The human tumor cell cDNA hybridizing with the Chinese hamster clone was isolated, sequenced, and found to have closer similarity to the Chinese hamster gene than to CTAP III. Thus, the cloned cDNAs from Chinese hamster and human cells represent a different gene, named gro. Studies of its transcriptional regulation have shown that expression is tightly regulated by growth status in normal Chinese hamster and human cells and relaxed in the tumorigenic cells so far examined.

  2. Overexpression of E2F3 promotes proliferation of functional human ? cells without induction of apoptosis

    PubMed Central

    Rady, Brian; Chen, Yanmei; Vaca, Pilar; Wang, Qian; Wang, Yong; Salmon, Patrick; Oberholzer, Jos

    2013-01-01

    The mechanisms that control proliferation, or lack thereof, in adult human ? cells are poorly understood. Controlled induction of proliferation could dramatically expand the clinical application of islet cell transplantation and represents an important component of regenerative approaches to a functional cure of diabetes. Adult human ? cells are particularly resistant to common proliferative targets and often dedifferentiate during proliferation. Here we show that expression of the transcription factor E2F3 has a role in regulating ?-cell quiescence and proliferation. We found human islets have virtually no expression of the pro-proliferative G1/S transcription factors E2F13, but an abundance of inhibitory E2Fs 46. In proliferative human insulinomas, inhibitory E2Fs were absent, while E2F3 is expressed. Using this pattern as a roadmap for proliferation, we demonstrated that ectopic expression of nuclear E2F3 induced significant expansion of insulin-positive cells in both rat and human islets. These cells did not undergo apoptosis and retained their glucose-responsive insulin secretion, showing the ability to reverse diabetes in mice. Our results suggest that E2F46 may help maintain quiescence in human ? cells and identify E2F3 as a novel target to induce proliferation of functional ? cells. Refinement of this approach may increase the islets available for cell-based therapies and research and could provide important cues for understanding in vivo proliferation of ? cells. PMID:23907129

  3. Antitumor effects of crocin on human breast cancer cells

    PubMed Central

    Lu, Pengwei; Lin, Huan; Gu, Yuanting; Li, Lin; Guo, Hong; Wang, Fang; Qiu, Xinguang

    2015-01-01

    Crocin is a chemical extracted from saffron and it is the most important kind of pigment of saffron. It has been proposed as a promising candidate for cancer prevention. In this study, we investigate the growth inhibition and the apoptosis of MCF-7 cells induced by Crocin, and explore the underlying molecular mechanism. We found that Crocin can significantly inhibit the proliferation of MCF-7 cells, and induce their apoptosis through mitochondrial signaling pathways including the activation of Caspase-8, upregulation of Bax, the disruption of mitochondrial membrane potential (MMP), and the release of cytochrome c. The studies showed that Crocin induced apoptosis of MCF-7 cells partially through caspase-8 mediated mitochondrial pathway. Therefore, we postulate that Crocin might have cancer-preventive and cancer-therapeutic benefit for human breast cancer. PMID:26884946

  4. Multiplexed ion beam imaging (MIBI) of human breast tumors

    PubMed Central

    Angelo, Michael; Bendall, Sean C.; Finck, Rachel; Hale, Matthew B.; Hitzman, Chuck; Borowsky, Alexander D.; Levenson, Richard M.; Lowe, John B.; Liu, Scot D.; Zhao, Shuchun; Natkunam, Yasodha; Nolan, Garry P.

    2014-01-01

    Immunohistochemistry (IHC) is a tool for visualizing protein expression employed as part of the diagnostic work-up for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded (FFPE) human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI will provide new insights by integrating tissue microarchitecture with highly multiplexed protein expression patterns, and will be valuable for basic research, drug discovery and clinical diagnostics. PMID:24584119

  5. Multiplexed ion beam imaging of human breast tumors.

    PubMed

    Angelo, Michael; Bendall, Sean C; Finck, Rachel; Hale, Matthew B; Hitzman, Chuck; Borowsky, Alexander D; Levenson, Richard M; Lowe, John B; Liu, Scot D; Zhao, Shuchun; Natkunam, Yasodha; Nolan, Garry P

    2014-04-01

    Immunohistochemistry (IHC) is a tool for visualizing protein expression that is employed as part of the diagnostic workup for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI can provide new insights into disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics. PMID:24584119

  6. The emerging importance of α-L-fucose in human breast cancer: a review

    PubMed Central

    Listinsky, Jay J; Siegal, Gene P; Listinsky, Catherine M

    2011-01-01

    Breast cancer cells incorporate the simple sugar alpha-L-fucose (fucose) into glycoproteins and glycolipids which, in turn, are expressed as part of the malignant phenotype. We have noted that fucose is not simply a bystander molecule, but, in fact, contributes to many of the fundamental oncologic properties of breast cancer cells. Here, we summarize the evidence from us and others that fucose is necessary for key functions of neoplastic progression including hematogenous metastasis, tumor invasion through extracellular matrices including basement membranes and up-regulation of the Notch signaling system, with implications for epithelial-to-mesenchymal transition and activation of breast cancer stem cells. Additionally, certain breast cancer biomarkers are fucose-rich while a well-known marker of breast cancer progression, soluble E-selectin, is a known counter-receptor of fucosylated selectin ligands. We provide illustrative examples and supportive evidence drawn from work with human breast cancer cell lines in vitro as well as clinical studies with human pathologic material. And finally, we discuss evidence that fucose (or its absence) is central to the mechanisms of action of several experimental targeted therapies which may prove useful in breast cancer treatment. We propose that alpha-L-fucose is essential in order to construct first, the malignant and then the metastatic phenotype of many human breast cancers. This knowledge may inform the search for novel treatment approaches in breast cancer. PMID:21904652

  7. Immune profiling in human breast cancer using high-sensitivity detection and analysis techniques

    PubMed Central

    Weightman, Michael J; Bradley, John; Skinner, John M

    2015-01-01

    Objectives Evaluation of immune profiles in human breast cancer using high-sensitivity detection and analysis methods. Design Cohort comparative analysis studies of breast tissue. Setting Human hospital and laboratory healthcare facilities. Participants Women over 18 years. Main outcome measures Evaluation of the comparative immunophenotype of human breast carcinoma and normal breast tissues. Results Leukocyte density and specific subgroups of lymphocytes and macrophages were generally higher in breast cancers compared to normal breast tissues. CD3, CD4, CD45RO, CD45RA(2H4), CD45 and HLA Class II (on TIL) were significantly expressed on breast tumour tissues compared with normal tissues (p?Breast carcinoma showed predominance of CD4 T-cells of mainly memory phenotype. Normal breast tissues showed low leukocyte infiltration. Further correlation of these findings with clinical outcome, including survival, is proceeding with encouraging results. PMID:26464809

  8. Pharmacokinetic interactions of breast cancer chemotherapeutics with human doxorubicin reductases.

    PubMed

    Hofman, Jakub; Skarka, Adam; Havrankova, Jana; Wsol, Vladimir

    2015-08-01

    Paclitaxel (PTX), docetaxel (DTX), 5-fluorouracil (5-FU), cyclophosphamide (CYC) or tamoxifen (TMX) are combined with doxorubicin (DOX) in first-line chemotherapy regimens that are indicated for breast cancer patients. Although the efficacies of these drugs in combination treatments have been demonstrated in clinical practice, their possible interference with DOX metabolism has not been described in detail to date. In the present study, we investigated the possible interactions of human carbonyl reducing enzymes with 5-FU, PTX, DTX, CYC and TMX. First, the reducing activities of carbonyl reducing enzymes toward DOX were tested using incubations with purified recombinant enzymes. In the subsequent studies, we investigated the possible effects of the tested anticancer agents on the DOX-reducing activities of the most potent enzymes (AKR1C3, CBR1 and AKR1A1) and on the DOX metabolism driven by MCF7, HepG2 and human liver cytosols. In both of these assays, we observed that CYC and its active metabolites inhibited DOX metabolism. In the final study, we tracked the changes in AKR1C3, CBR1 and AKR1A1 expression levels following exposure to the tested cytostatics in MCF7 and HepG2 cells. Consequently, no significant changes in the expression levels of tested enzymes were detected in either cell line. Based on these findings, it is feasible to presume that inhibition rather than induction plays a role in the interactions of the tested anticancer agents with DOX-reducing enzymes. In conclusion, our results describe important molecular events that occur during combination breast cancer therapies and might modulate pharmacokinetic DOX resistance and/or behaviour. PMID:25986883

  9. Gene Expression Analysis in Human Breast Cancer Associated Blood Vessels

    PubMed Central

    Jones, Dylan T.; Lechertier, Tanguy; Mitter, Richard; Herbert, John M. J.; Bicknell, Roy; Jones, J. Louise; Li, Ji-Liang; Buffa, Francesca; Harris, Adrian L.; Hodivala-Dilke, Kairbaan

    2012-01-01

    Angiogenesis is essential for solid tumour growth, whilst the molecular profiles of tumour blood vessels have been reported to be different between cancer types. Although presently available anti-angiogenic strategies are providing some promise for the treatment of some cancers it is perhaps not surprisingly that, none of the anti-angiogenic agents available work on all tumours. Thus, the discovery of novel anti-angiogenic targets, relevant to individual cancer types, is required. Using Affymetrix microarray analysis of laser-captured, CD31-positive blood vessels we have identified 63 genes that are upregulated significantly (572 fold) in angiogenic blood vessels associated with human invasive ductal carcinoma (IDC) of the breast as compared with blood vessels in normal human breast. We tested the angiogenic capacity of a subset of these genes. Genes were selected based on either their known cellular functions, their enriched expression in endothelial cells and/or their sensitivity to anti-VEGF treatment; all features implicating their involvement in angiogenesis. For example, RRM2, a ribonucleotide reductase involved in DNA synthesis, was upregulated 32-fold in IDC-associated blood vessels; ATF1, a nuclear activating transcription factor involved in cellular growth and survival was upregulated 23-fold in IDC-associated blood vessels and HEX-B, a hexosaminidase involved in the breakdown of GM2 gangliosides, was upregulated 8-fold in IDC-associated blood vessels. Furthermore, in silico analysis confirmed that AFT1 and HEX-B also were enriched in endothelial cells when compared with non-endothelial cells. None of these genes have been reported previously to be involved in neovascularisation. However, our data establish that siRNA depletion of Rrm2, Atf1 or Hex-B had significant anti-angiogenic effects in VEGF-stimulated ex vivo mouse aortic ring assays. Overall, our results provide proof-of-principle that our approach can identify a cohort of potentially novel anti-angiogenic targets that are likley to be, but not exclusivley, relevant to breast cancer. PMID:23056178

  10. CREB-1 and AP-1 transcription factors JunD and Fra-2 regulate bone sialoprotein gene expression in human breast cancer cells.

    PubMed

    Detry, C; Lamour, V; Castronovo, V; Bellahcne, A

    2008-02-01

    Bone sialoprotein (BSP) expression is detected in a variety of human osteotropic cancers. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. In this study, we examined the transcriptional regulation of BSP gene expression in MDA-MB-231 and MCF-7 human breast cancer cells compared with Saos-2 human osteoblast-like cells. BSP human promoter deletion analyses delineated a -56/-84 region, which comprises a cAMP response element (CRE) that was sufficient for maximal promoter activity in breast cancer cell lines. We found that the basic fibroblast growth factor response element (FRE) also located in the proximal promoter was a crucial regulator of human BSP promoter activity in Saos-2 but not in breast cancer cells. Promoter activity experiments in combination with DNA mobility shift assays demonstrated that BSP promoter activity is under the control of the CRE element, through CREB-1, JunD and Fra-2 binding, in MDA-MB-231, MCF-7 and in Saos-2 cells. Forskolin, a protein kinase A pathway activator, failed to enhance BSP transcriptional activity suggesting that CRE site behaves as a constitutive rather than an inducible element in these cell lines. Over-expression of JunD and Fra-2 increased BSP promoter activity and upregulated endogenous BSP protein expression in MCF-7 and Saos-2 cells while siRNA-mediated inhibition of both factors expression significantly reduced BSP protein level in MDA-MB-231. Collectively, these data provide with new transcriptional mechanisms, implicating CREB and AP-1 factors, that control BSP gene expression in breast cancer cells. PMID:18088579

  11. Development of Anatomically Realistic Numerical Breast Phantoms with Accurate Dielectric Properties for Modeling Microwave Interactions with the Human Breast

    PubMed Central

    Zastrow, Earl; Davis, Shakti K.; Lazebnik, Mariya; Kelcz, Frederick; Van Veen, Barry D.; Hagness, Susan C.

    2008-01-01

    Computational electromagnetics models of microwave interactions with the human breast serve as an invaluable tool for exploring the feasibility of new technologies and improving design concepts related to microwave breast cancer detection and treatment. In this paper we report the development of a collection of anatomically realistic 3D numerical breast phantoms of varying shape, size, and radiographic density which can be readily used in FDTD computational electromagnetics models. The phantoms are derived from T1-weighted magnetic resonance images (MRIs) of prone patients. Each MRI is transformed into a uniform grid of dielectric properties using several steps. First, the structure of each phantom is identified by applying image processing techniques to the MRI. Next, the voxel intensities of the MRI are converted to frequency-dependent and tissue-dependent dielectric properties of normal breast tissues via a piecewise-linear map. The dielectric properties of normal breast tissue are taken from the recently completed large-scale experimental study of normal breast tissue dielectric properties conducted by the Universities of Wisconsin and Calgary. The comprehensive collection of numerical phantoms is made available to the scientific community through an online repository. PMID:19126460

  12. Progesterone and estrogen receptors segregate into different cell subpopulations in the normal human breast.

    PubMed

    Hilton, H N; Graham, J D; Kantimm, S; Santucci, N; Cloosterman, D; Huschtscha, L I; Mote, P A; Clarke, C L

    2012-09-25

    Progesterone is critical in normal breast development and its synthetic derivatives are emerging as major drivers of breast cancer risk. The recent demonstration that progesterone regulates the stem cell compartment in the murine mammary gland, despite the absence of progesterone receptor (PR) in mammary stem cells, highlights the fact that PR distribution in progenitor cell subsets in the human breast remains to be conclusively shown. By utilising two independent cell sorting strategies to fractionate cells into distinct subpopulations enriched for different cell lineage characteristics, we have demonstrated a consistent enrichment of PR transcripts, relative to estrogen receptor transcripts, in the bipotent progenitor subfraction in the normal human breast. We have also shown co-expression of both steroid hormone receptors with basal markers in a subset of human breast cells, and finally we have demonstrated that PR+ bipotent progenitor cells are estrogen-insensitive, and that estrogen regulates PR in mature luminal cells only. PMID:22580007

  13. Breast Cancer

    MedlinePLUS

    ... Sledding, Skiing, Snowboarding, Skating Crushes What's a Booger? Breast Cancer KidsHealth > For Kids > Breast Cancer Print A ... for it when they are older. What Is Breast Cancer? The human body is made of tiny ...

  14. ALDH1A1-overexpressing cells are differentiated cells but not cancer stem or progenitor cells in human hepatocellular carcinoma

    PubMed Central

    Tanaka, Kaori; Tomita, Hiroyuki; Hisamatsu, Kenji; Nakashima, Takayuki; Hatano, Yuichiro; Sasaki, Yoshiyuki; Osada, Shinji; Tanaka, Takuji; Miyazaki, Tatsuhiko; Yoshida, Kazuhiro; Hara, Akira

    2015-01-01

    Aldehyde dehydrogenase 1A1 (ALDH1A1) is considered to be a cancer stem cell marker in several human malignancies. However, the role of ALDH1A1 in hepatocellular carcinoma (HCC) has not been well elucidated. In this study, we investigated the relationship between ALDH1A1 and clinicopathological findings and examined whether ALDH1A1 deserves to be a cancer stem cell marker in HCC. Sixty HCC samples obtained from surgical resection were collected for immunohistochemical (IHC) staining. Of these 60 samples, 47 samples of HCC tumorous and non-tumorous tissues were evaluated with qRT-PCR. There was no significant difference in the ALDH1A1-mRNA level between tumorous and non-tumorous tissues. Tumorous ALDH1A1-mRNA level had no relationship with the clinicopathological features. Immunoreactivity of ALDH1A1 was classified into two groups based on the percentage of ALDH1A1-overexpressing cells. The ALDH1A1-high group was significantly associated with low serum levels of α-fetoprotein, small tumor diameter, very little lymphovascular invasion, more differentiated pathology and good stage. The ALDH1A1-high group showed more favorable prognosis for recurrence-free survival. In double-staining IHC, ALDH1A1 was not co-expressed with BMI1, EpCAM, CD13, CD24, CD90 and CD133, which reported as cancer stem cell markers in HCC. In conclusion, ALDH1A1-overexpressing cells could appear to be differentiated cells rather than cancer stem cells in HCC. PMID:26160842

  15. Soluble overexpression and purification of bioactive human CCL2 in E. coli by maltose-binding protein.

    PubMed

    Vu, Thu Trang Thi; Koo, Bon-Kyung; Song, Jung-A; Chong, Seon-Ha; Park, Cho Rong; Nguyen, Minh Tan; Jeong, Boram; Ryu, Han-Bong; Seong, Jae Young; Jang, Yeon Jin; Robinson, Robert Charles; Choe, Han

    2015-03-01

    Human chemokine (C-C motif) ligand 2 (hCCL2) is a small cytokine in the CC chemokine family that attracts monocytes, memory T lymphocytes, and natural killer cells to the site of tissue injury- or infection-induced inflammation. hCCL2 has been implicated in the pathogeneses of diseases characterized by monocytic infiltrates, including psoriasis, rheumatoid arthritis, atherosclerosis, multiple sclerosis, and insulin-resistant diabetes. The prokaryotic overexpression of hCCL2 has been investigated previously in an attempt to develop biomedical applications for this factor, but this has been hampered by protein misfolding and aggregation into inclusion bodies. In our present study, we screened 7 protein tags-Trx, GST, MBP, NusA, His8, PDI, and PDIb'a'-for their ability to allow the soluble overexpression of hCCL2. Three tags-MBP, His8, and PDI-solubilized more than half of the expressed hCCL2 fusion proteins. Lowering the expression temperature to 18C significantly further improved the solubility of all fusion proteins. MBP was chosen for further study based on its solubility, expression level, ease of purification, and tag size. MBP-CCL2 was purified using conventional chromatography and cleaved using TEV or Factor Xa proteases. Biological activity was assessed using luciferase and cell migration assays. Factor Xa-cleaved hCCL2 was found to be active and TEV-cleaved hCCL2 showed relatively less activity. This is probably because the additional glycine residues present at the N-terminus of hCCL2 following TEV digestion interfere with the binding of hCCL2 to its receptor. PMID:25391768

  16. Surface plasmon resonance immunosensor for ErbB2 breast cancer biomarker determination in human serum and raw cancer cell lysates.

    PubMed

    Eletxigerra, Unai; Martinez-Perdiguero, Josu; Barderas, Rodrigo; Pingarrn, Jos M; Campuzano, Susana; Merino, Santos

    2016-01-28

    A highly sensitive surface plasmon resonance (SPR) immunosensor for the important ErbB2 breast cancer biomarker has been developed. Optimization of the assay has been carried out, including signal enhancement employing gold nanoparticles (GNPs). The effect of the signal amplification of the GNPs has been also studied. The assay has been tested with clinically relevant matrices. Results in 50% human serum yielded a LOD of 180pgmL(-1) which is a concentration 83 times lower than the clinical cut-off. Raw lysates from model breast cancer cell lines (SK-BR-3, MCF-7 and MDA-MB-436) have been also assayed and higher quantities of the ErbB2 protein were clearly observed in the ErbB2 over-expressing case (SK-BR-3). The results confirmed that the simple and highly sensitive SPR immunosensor represents a feasible tool for ErbB2 detection. PMID:26755150

  17. Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

    SciTech Connect

    Perez-Pinera, Pablo; Chang, Y.; Astudillo, A.; Mortimer, J.; Deuel, T.F. . E-mail: tfdeuel@scripps.edu

    2007-06-29

    Pleiotrophin (PTN, Ptn) is an 18 kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway in PTN-stimulated cells, not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTP{beta}/{zeta} signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the 'dotted' pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTP{beta}/{zeta} signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.

  18. Increased Infectivity of Anchorless Mouse Scrapie Prions in Transgenic Mice Overexpressing Human Prion Protein

    PubMed Central

    Phillips, Katie; Meade-White, Kimberly; Striebel, James; Chesebro, Bruce

    2015-01-01

    ABSTRACT Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. IMPORTANCE Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection. PMID:25810548

  19. Overexpression of Telomerase Protects Human and Murine Lung Epithelial Cells from Fas- and Bleomycin-Induced Apoptosis via FLIP Upregulation

    PubMed Central

    Arish, Nissim; Cohen, Pazit Y.; Golan-Gerstl, Regina; Fridlender, Zvi; Dayan, Mark Richter; Zisman, Philip; Breuer, Raphael; Wallach-Dayan, Shulamit B.

    2015-01-01

    High doses of bleomycin administered to patients with lymphomas and other tumors lead to significant lung toxicity in general, and to apoptosis of epithelial cells, in particular. Apoptosis of alveolar epithelium is an important step in the pathogenesis of bleomycin-induced pulmonary fibrosis. The Fas-FasL pathway is one of the main apoptotic pathways involved. Telomerase is a ribonucleoprotein RNA-dependent DNA polymerase complex consisting of an RNA template and a catalytic protein, telomerase reverse transcriptase (TERT). Telomerase also possess extra-telomeric roles, including modulation of transcription of anti-apoptotic genes, differentiation signals, and more. We hypothesized that telomerase overexpression affects Fas-induced epithelial cell apoptosis by an extra-telomeric role such as regulation of anti-apoptotic genes, specifically FLICE-like inhibitory protein (FLIP). Telomerase in mouse (MLE) and human (A549) lung epithelial cell lines was upregulated by transient transfection using cDNA hTERT expression vector. Telomerase activity was detected using a real-time PCR-based system. Bleomycin, and bleomycin-induced Fas-mediated apoptosis following treatment with anti-Fas activating mAb or control IgG, were assessed by Annexin V staining, FACS analysis, and confocal microscopy; caspase cleavage by Western blot; FLIP or Fas molecule detection by Western blot and flow cytometry. hTERT transfection of lung epithelial cells resulted in a 100% increase in their telomerase activity. Fas-induced lung epithelial cell apoptosis was significantly reduced in hTERT-transfected cells compared to controls in all experiments. Lung epithelial cells with increased telomerase activity had higher levels of FLIP expression but membrane Fas expression was unchanged. Upregulation of hTERT+ in human lung epithelial cells and subsequent downregulation of FLIP by shFLIP-RNA annulled hTERT-mediated resistance to apoptosis. Telomerase-mediated FLIP overexpression may be a novel mechanism to confer protection from apoptosis in bleomycin-exposed human lung epithelial cells. PMID:25951185

  20. MiR-492 contributes to cell proliferation and cell cycle of human breast cancer cells by suppressing SOX7 expression.

    PubMed

    Shen, Fei; Cai, Wen-Song; Feng, Zhe; Li, Jiang-Lin; Chen, Ji-Wei; Cao, Jie; Xu, Bo

    2015-03-01

    MicroRNAs (miRNAs) have emerged as important regulators that potentially play critical roles in cancer cell biological processes. Previous studies have shown that miR-492 plays an important role in cell tumorigenesis in multiple kinds of human cancer cells. However, the underlying mechanisms of this microRNA in breast cancer remain largely unknown. In the present study, we investigated miR-492's role in cell proliferation of breast cancer. MiR-492 expression was markedly upregulated in breast cancer tissues and breast cancer cells. Overexpression of miR-492 promoted the proliferation and anchorage-independent growth of breast cancer cells. Bioinformatics analysis further revealed sex-determining region Y-box 7 (SOX7), a putative tumor suppressor, as a potential target of miR-492. Data from luciferase reporter assays showed that miR-492 directly binds to the 3'-untranslated region (3'-UTR) of SOX7 messenger RNA (mRNA) and repressed expression at both transcriptional and translational levels. Ectopic expression of miR-492 led to downregulation of SOX7 protein, which resulted in the upregulation of cyclin D1 and c-Myc. In functional assays, SOX7 silenced in miR-492-in-transfected ZR-75-30 cells has positive effect to promote cell proliferation, suggesting that direct SOX7 downregulation is required for miR-492-induced cell proliferation and cell cycle of breast cancer. In sum, these results suggest that miR-492 represents a potential onco-miR and participates in breast cancer carcinogenesis by suppressing SOX7 expression. PMID:25407488

  1. Long-term efficacy and downstream mechanism of anti-annexinA2 monoclonal antibody (anti-ANX A2 mAb) in a pre-clinical model of aggressive human breast cancer.

    PubMed

    Sharma, Mahesh C; Tuszynski, George P; Blackman, Marc R; Sharma, Meena

    2016-04-01

    There is considerable direct evidence that calcium binding protein ANX A2 is a potential target for treating aggressive breast cancer. The most compelling data are based on the finding of ANX A2 overexpression in aggressive triple negative human breast cancer (TNBC) cell lines and in human breast cancer tissues. Previously, we and others reported a unique role of ANX A2 in cancer invasion, including breast cancer. Moreover, we demonstrated that anti-ANX A2 mAb-mediated immunoneutralization of ANX A2 inhibited invasive human breast cancer growth in a xenograft model. We further evaluated the long-term effects of multiple treatments with anti-ANX A2 mAb and its mechanism of inhibition on human breast tumor growth. We now demonstrate that three treatments with anti-ANX A2 mAb led to significant inhibition of breast tumor growth in immunodeficient mice, and that the anti-tumor response was demonstrable from day 94. After treatment, we followed tumor growth for 172 days and demonstrated 67% inhibition of tumor growth without detectable adverse effects. Biochemical analysis demonstrated that anti-ANX A2 mAb treatment caused significant inhibition of conversion of tissue plasminogen activator (tPA) in the tumor microenvironment. This led to disruption of plasmin generation that consequently inhibited activation of MMP-9 and MMP-2. These results suggest that ANX A2 plays an important role in aggressive breast tumor growth by regulating proteolytic pathways in the tumor microenvironment. ANX A2 may represent a new target for the development of therapeutics for treatment of aggressive breast cancer. PMID:26797420

  2. Generation of bi-transgenic pigs overexpressing human lactoferrin and lysozyme in milk.

    PubMed

    Cui, Dan; Li, Jia; Zhang, Linlin; Liu, Shen; Wen, Xiao; Li, Qiuyan; Zhao, Yaofeng; Hu, Xiaoxiang; Zhang, Ran; Li, Ning

    2015-04-01

    Intensive swine production industry uses antibiotics to treat diseases and improve pig growth. This can not only cause antibiotic resistance, but can also pollute the environment or eventually affect human public health. To date, human lactoferrin (hLF) and human lysozyme (hLZ) have been known as non-adaptive but interactive antimicrobial members and could act in concert against bacteria, which contribute to host defense. Therefore, their expression in pigs might be an alternative strategy for replacing antibiotics in the pig production industry. In our study, we produced hLF and hLZ bi-transgenic pigs and assessed the milk's antibacterial ability. Integration of both transgenes was confirmed by PCR and southern blot. Both the hLF and hLZ were expressed in the mammary gland of bi-transgenic pigs, as detected by western blotting. The expression amounts were 6.5 g/L for hLF and 1.1 mg/L for hLZ using ELISA. Interestingly, pig milk containing hLF and hLZ had synergistic antimicrobial activity. Our results suggest an alternative approach for avoiding the use of antibiotics in the pig industry, which would be of great benefit to the commercial swine production. PMID:25236863

  3. Downregulation of COX-2 and CYP 4A signaling by isoliquiritigenin inhibits human breast cancer metastasis through preventing anoikis resistance, migration and invasion.

    PubMed

    Zheng, Hao; Li, Ying; Wang, Yuzhong; Zhao, Haixia; Zhang, Jing; Chai, Hongyan; Tang, Tian; Yue, Jiang; Guo, Austin M; Yang, Jing

    2014-10-01

    Flavonoids exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Anoikis resistance occurs at multiple key stages of the metastatic cascade. Here, we demonstrate that isoliquiritigenin (ISL), a flavonoid from Glycyrrhiza glabra, inhibits human breast cancer metastasis by preventing anoikis resistance, migration and invasion through downregulating cyclooxygenase (COX)-2 and cytochrome P450 (CYP) 4A signaling. ISL induced anoikis in MDA-MB-231 and BT-549 human breast cancer cells as evidenced by flow cytometry and the detection of caspase cleavage. Moreover, ISL inhibited the mRNA expression of phospholipase A2, COX-2 and CYP 4A and decreased the secretion of prostaglandin E2 (PGE2) and 20-hydroxyeicosatetraenoic acid (20-HETE) in detached MDA-MB-231 cells. In addition, it decreased the levels of phospho-PI3K (Tyr(458)), phospho-PDK (Ser(241)) and phospho-Akt (Thr(308)). Conversely, the exogenous addition of PGE2, WIT003 (a 20-HETE analog) and an EP4 agonist (CAY10580) or overexpression of constitutively active Akt reversed ISL-induced anoikis. ISL exerted the in vitro anti-migratory and anti-invasive activities, whereas the addition of PGE2, WIT003 and CAY10580 or overexpression of constitutively active Akt reversed the in vitro anti-migratory and anti-invasive activities of ISL in MDA-MB-231 cells. Notably, ISL inhibited the in vivo lung metastasis of MDA-MB-231 cells, together with decreased intratumoral levels of PGE2, 20-HETE and phospho-Akt (Thr(308)). In conclusion, ISL inhibits breast cancer metastasis by preventing anoikis resistance, migration and invasion via downregulating COX-2 and CYP 4A signaling. It suggests that ISL could be a promising multi-target agent for preventing breast cancer metastasis, and anoikis could represent a novel mechanism through which flavonoids may exert the anti-metastatic activities. PMID:25094029

  4. Overexpression of Pterin-4a-carbinolamine Dehydratase/Dimerization Cofactor of Hepatocyte Nuclear Factor 1 in Human Colon Cancer

    PubMed Central

    Eskinazi, Rally; Thny, Beat; Svoboda, Michal; Robberecht, Patrick; Dassesse, Donald; Heizmann, Claus W.; Van Laethem, Jean-Luc; Resibois, Anne

    1999-01-01

    Pterin-4a-carbinolamine dehydratase (PCD) is a bifunctional protein also known as DCoH (dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)). PCD/DCoH modulates the DNA binding specificity of HNF1, thus acting on its transcriptional activity. In addition, it participates in the recycling of tetrahydrobiopterin (BH4), an essential cofactor of several metabolic reactions. We investigated colorectal tumors and colorectal tumor cell lines as compared to normal colon samples in search of a potential differential expression of PCD/DCoH. Immunohistochemistry was conducted on 20 human colorectal tumors and 20 normal samples using a specific polyclonal antibody. Immunoblotting and RT-PCR analysis for PCD/DCoH and HNF1 were also performed on both human tissues and CACO-2 and HT-29 cell lines. All of the 20 tumors and both colon cancer cell lines presented a strong and widespread immunoreactivity for PCD/DCoH, contrasting with the absence of expression in the normal epithelia. We thus report the massive overexpression of PCD/DCoH in colon tumors, which is in striking contrast with the absence of staining in normal counterparts. The sharp contrast in the expression of a modulator of transcriptional activity between tumoral and normal cells may have a physiopathological role. PCD/DCoH could potentially be a new marker of malignant colon cells in vivo. PMID:10514393

  5. Bioconjugation of calcium phosphosilicate composite nanoparticles for selective targeting of human breast and pancreatic cancers in vivo.

    PubMed

    Barth, Brian M; Sharma, Rahul; Altino?lu, Erhan I; Morgan, Thomas T; Shanmugavelandy, Sriram S; Kaiser, James M; McGovern, Christopher; Matters, Gail L; Smith, Jill P; Kester, Mark; Adair, James H

    2010-03-23

    The early diagnosis of cancer is the critical element in successful treatment and long-term favorable patient prognoses. The high rate of mortality is mainly attributed to the tendency for late diagnoses as symptoms may not occur until the disease has metastasized, as well as the lack of effective systemic therapies. Late diagnosis is often associated with the lack of timely sensitive imaging modalities. The promise of nanotechnology is presently limited by the inability to simultaneously seek, treat, and image cancerous lesions. This study describes the design and synthesis of fluorescent calcium phosphosilicate nanocomposite particles (CPNPs) that can be systemically targeted to breast and pancreatic cancer lesions. The CPNPs are a approximately 20 nm diameter composite composed of an amorphous calcium phosphate matrix doped with silicate in which a near-infrared imaging agent, indocyanine green (ICG), is embedded. In the present studies, we describe and validate CPNP bioconjugation of human holotransferrin, anti-CD71 antibody, and short gastrin peptides via an avidin-biotin or a novel PEG-maleimide coupling strategy. The conjugation of biotinylated human holotransferrin (diferric transferrin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to avidin-conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors, which are highly expressed on breast cancer cells. Similarly, the conjugation of biotinylated pentagastrin to Avidin-CPNPs and decagastrin (gastrin-10) to PEG-CPNPs via PEG-maleimide coupling permits targeting of gastrin receptors, which are overexpressed in pancreatic cancer lesions. These bioconjugated CPNPs have the potential to perform as a theranostic modality, simultaneously enhancing drug delivery, targeting, and imaging of breast and pancreatic cancer tumors. PMID:20180585

  6. Overexpression of p53 activated by small activating RNA suppresses the growth of human prostate cancer cells.

    PubMed

    Ge, Qiangqiang; Wang, Chenghe; Ruan, Yajun; Chen, Zhong; Liu, Jihong; Ye, Zhangqun

    2016-01-01

    Previous research has reported that a particular double-stranded RNA, named dsP53-285, has the capacity to induce expression of the tumor suppressor gene TP53 in chimpanzee cells by targeting its promoter. Usually, it is the wild-type p53 protein, rather than mutants, which exhibits potent cancer-inhibiting effects. In addition, nonhuman primates, such as chimpanzees, share almost identical genome sequences with humans. This prompted us to speculate whether dsP53-285 can trigger wild-type p53 protein expression in human prostate cancer (PCa) cells and consequently suppress cell growth. The human PCa cell lines LNCaP and DU145 were transfected with dsP53-285 for 72 hours. Compared with the dsControl and mock transfection groups, expression of both p53 messenger RNA and p53 protein was significantly enhanced after dsP53-285 transfection, and this enhancement was followed by upregulation of p21, which indirectly indicated that dsP53-285 induced wild-type p53 expression. Moreover, overexpression of wild-type p53 mediated by dsP53-285 downregulated the expression of Cyclin D1 and cyclin-dependent kinase 4/6, thereby inducing PCa cell cycle arrest in G0/G1 phase and then inhibiting cell proliferation and clonogenicity. More importantly, dsP53-285 suppressed PCa cells mainly by modulating wild-type p53 expression. In conclusion, our study provides evidence that dsP53-285 can significantly stimulate wild-type p53 expression in the human PCa cell lines LNCaP and DU145 and can exert potent antitumor effects. PMID:26811691

  7. Overexpression of p53 activated by small activating RNA suppresses the growth of human prostate cancer cells

    PubMed Central

    Ge, Qiangqiang; Wang, Chenghe; Ruan, Yajun; Chen, Zhong; Liu, Jihong; Ye, Zhangqun

    2016-01-01

    Previous research has reported that a particular double-stranded RNA, named dsP53-285, has the capacity to induce expression of the tumor suppressor gene TP53 in chimpanzee cells by targeting its promoter. Usually, it is the wild-type p53 protein, rather than mutants, which exhibits potent cancer-inhibiting effects. In addition, nonhuman primates, such as chimpanzees, share almost identical genome sequences with humans. This prompted us to speculate whether dsP53-285 can trigger wild-type p53 protein expression in human prostate cancer (PCa) cells and consequently suppress cell growth. The human PCa cell lines LNCaP and DU145 were transfected with dsP53-285 for 72 hours. Compared with the dsControl and mock transfection groups, expression of both p53 messenger RNA and p53 protein was significantly enhanced after dsP53-285 transfection, and this enhancement was followed by upregulation of p21, which indirectly indicated that dsP53-285 induced wild-type p53 expression. Moreover, overexpression of wild-type p53 mediated by dsP53-285 downregulated the expression of Cyclin D1 and cyclin-dependent kinase 4/6, thereby inducing PCa cell cycle arrest in G0/G1 phase and then inhibiting cell proliferation and clonogenicity. More importantly, dsP53-285 suppressed PCa cells mainly by modulating wild-type p53 expression. In conclusion, our study provides evidence that dsP53-285 can significantly stimulate wild-type p53 expression in the human PCa cell lines LNCaP and DU145 and can exert potent antitumor effects. PMID:26811691

  8. Differential contextual responses of normal human breast epithelium to ionizing radiation in a mouse xenograft model.

    PubMed

    Coates, Philip J; Appleyard, M Virginia C L; Murray, Karen; Ackland, Caroline; Gardner, June; Brown, Douglas C; Adamson, Dougal J A; Jordan, Lee B; Purdie, Colin A; Munro, Alastair J; Wright, Eric G; Dewar, John A; Thompson, Alastair M

    2010-12-01

    Radiotherapy is a key treatment option for breast cancer, yet the molecular responses of normal human breast epithelial cells to ionizing radiation are unclear. A murine subcutaneous xenograft model was developed in which nonneoplastic human breast tissue was maintained with the preservation of normal tissue architecture, allowing us to study for the first time the radiation response of normal human breast tissue in situ. Ionizing radiation induced dose-dependent p53 stabilization and p53 phosphorylation, together with the induction of p21(CDKN1A) and apoptosis of normal breast epithelium. Although p53 was stabilized in both luminal and basal cells, induction of Ser392-phosphorylated p53 and p21 was higher in basal cells and varied along the length of the ductal system. Basal breast epithelial cells expressed ?Np63, which was unchanged on irradiation. Although stromal responses themselves were minimal, the response of normal breast epithelium to ionizing radiation differed according to the stromal setting. We also demonstrated a dose-dependent induction of ?-H2AX foci in epithelial cells that was similarly dependent on the stromal environment and differed between basal and luminal epithelial cells. The intrinsic differences between human mammary cell types in response to in vivo irradiation are consistent with clinical observation that therapeutic ionizing radiation is associated with the development of basal-type breast carcinomas. Furthermore, there may be clinically important stromal-epithelial interactions that influence DNA damage responses in the normal breast. These findings demonstrate highly complex responses of normal human breast epithelium following ionizing radiation exposure and emphasize the importance of studying whole-tissue effects rather than single-cell systems. PMID:21084272

  9. Automated quantification of aligned collagen for human breast carcinoma prognosis

    PubMed Central

    Bredfeldt, Jeremy S.; Liu, Yuming; Conklin, Matthew W.; Keely, Patricia J.; Mackie, Thomas R.; Eliceiri, Kevin W.

    2014-01-01

    Background: Mortality in cancer patients is directly attributable to the ability of cancer cells to metastasize to distant sites from the primary tumor. This migration of tumor cells begins with a remodeling of the local tumor microenvironment, including changes to the extracellular matrix and the recruitment of stromal cells, both of which facilitate invasion of tumor cells into the bloodstream. In breast cancer, it has been proposed that the alignment of collagen fibers surrounding tumor epithelial cells can serve as a quantitative image-based biomarker for survival of invasive ductal carcinoma patients. Specific types of collagen alignment have been identified for their prognostic value and now these tumor associated collagen signatures (TACS) are central to several clinical specimen imaging trials. Here, we implement the semi-automated acquisition and analysis of this TACS candidate biomarker and demonstrate a protocol that will allow consistent scoring to be performed throughout large patient cohorts. Methods: Using large field of view high resolution microscopy techniques, image processing and supervised learning methods, we are able to quantify and score features of collagen fiber alignment with respect to adjacent tumor-stromal boundaries. Results: Our semi-automated technique produced scores that have statistically significant correlation with scores generated by a panel of three human observers. In addition, our system generated classification scores that accurately predicted survival in a cohort of 196 breast cancer patients. Feature rank analysis reveals that TACS positive fibers are more well-aligned with each other, are of generally lower density, and terminate within or near groups of epithelial cells at larger angles of interaction. Conclusion: These results demonstrate the utility of a supervised learning protocol for streamlining the analysis of collagen alignment with respect to tumor stromal boundaries. PMID:25250186

  10. Salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells

    SciTech Connect

    Hu, Xiaolan; Zhang, Xianqi; Qiu, Shuifeng; Yu, Daihua; Lin, Shuxin

    2010-07-16

    Research highlights: {yields} Salidroside inhibits the growth of human breast cancer cells. {yields} Salidroside induces cell-cycle arrest of human breast cancer cells. {yields} Salidroside induces apoptosis of human breast cancer cell lines. -- Abstract: Recently, salidroside (p-hydroxyphenethyl-{beta}-D-glucoside) has been identified as one of the most potent compounds isolated from plants of the Rhodiola genus used widely in traditional Chinese medicine, but pharmacokinetic data on the compound are unavailable. We were the first to report the cytotoxic effects of salidroside on cancer cell lines derived from different tissues, and we found that human breast cancer MDA-MB-231 cells (estrogen receptor negative) were sensitive to the inhibitory action of low-concentration salidroside. To further investigate the cytotoxic effects of salidroside on breast cancer cells and reveal possible ER-related differences in response to salidroside, we used MDA-MB-231 cells and MCF-7 cells (estrogen receptor-positive) as models to study possible molecular mechanisms; we evaluated the effects of salidroside on cell growth characteristics, such as proliferation, cell cycle duration, and apoptosis, and on the expression of apoptosis-related molecules. Our results demonstrated for the first time that salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells and may be a promising candidate for breast cancer treatment.

  11. Analyses of Resected Human Brain Metastases of Breast Cancer Reveal the Association between Up-regulation of Hexokinase 2 and Poor Prognosis

    PubMed Central

    Palmieri, Diane; Fitzgerald, Daniel; Shreeve, S. Martin; Hua, Emily; Bronder, Julie L.; Weil, Robert J.; Davis, Sean; Stark, Andreas M.; Merino, Maria J.; Kurek, Raffael; Mehdorn, H. Maximilian; Davis, Gary; Steinberg, Seth M.; Meltzer, Paul S.; Aldape, Kenneth; Steeg, Patricia S.

    2009-01-01

    Brain metastases of breast cancer appear to be increasing in incidence as systemic therapy improves. Metastatic disease in the brain is associated with high morbidity and mortality. We present the first gene expression analysis of laser captured epithelial cells from resected human brain metastases of breast cancer compared to unlinked primary breast tumors. The tumors were matched for histology, TNM stage and hormone receptor status. Most differentially expressed genes were down-regulated in the brain metastases which included, surprisingly, many genes associated with metastasis. Q-PCR analysis confirmed statistically significant differences or strong trends in the expression of six genes: BMP1, PEDF, LAM?3, SIAH, STHMN3 and TSPD2. Hexokinase 2 (HK2) was also of interest because of its increased expression in brain metastases. HK2 is important in glucose metabolism and apoptosis. In agreement with our micro