Browsing by Subject "breast cancer"
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Item Anti-inflammatory and Cytotoxic Activities of Mango (Mangifera indica L. var Keitt) Polyphenols in Cancer and Non-cancer Breast Fibroblasts in Vitro(2013-08-12) Arbizu Berrocal, ShirleyBreast cancer is the leading cause of cancer death among women worldwide and polyphenols are under investigation as an alternative to conventional treatment approaches of breast cancer. The anti-inflammatory and anti-proliferative activities of polyphenols have been demonstrated in many studies, yet cellular targets and the underlying cellular mechanisms remain unclear. The overall goal of this study was to investigate the anti-inflammatory and cytotoxic properties of polyphenol compounds extracted from the mango variety Keitt in MCF-12A breast non-cancer and MDA-MB231 breast cancer cells by assessing the modulation of signaling pathways involved in inflammation and carcinogenesis. Mango polyphenols were identified by HPLC-MS analysis. The generation of reactive oxygen species was performed using fluorescence intensity in the DCFH-DA assay. Gene expression was analyzed by qRT-PCR, and protein expression was conducted by Western Blotting and Multiplex Bead assay analysis. Bioactive compounds identified in the mango pulp by HPLC-MS included a great variety of polyphenols such as gallic acid, galloyl glucosides with different degree of polymerization and other polyphenols. The anti-inflammatory activities of mango polyphenols were evaluated in MCF-12A non cancer breast fibroblasts. An inflammatory microenvironment for MCF-12A breast cells was induced with tumor necrosis factor alpha (TNF-?). The generation of reactive oxygen species was suppressed significantly compared to cells induced with TNF-?, where there was no significant difference between the concentrations of mango polyphenol extract. Results showed a significant down-regulation of mRNA and protein expression of inflammatory genes involved in the PI3K/AKT pathway and related downstream targets such as NF-?B and mTOR involved in biological processes including cell growth, proliferation and survival. Moreover, mango polyphenols had a significant impact on the miRNA-126-PI3K/AKT axis which plays an important role in inflammation and carcinogenesis, suggesting a potential anti-inflammatory underlying mechanism. The cytotoxic effects of mango polyphenols were investigated in MDA-MB231 breast cancer cells. Mango polyphenols decreased the production of reactive oxygen species; however no significant differences were found between the tested concentrations of mango polyphenols. The gene expression of proapoptotic factors involved in the intrinsic mitochondrial pathway such as cytochrome C and caspase-3 were significantly regulated after mango polyphenol treatment. In addition, the suppression of the PI3K/AKT/mTOR pathway and downstream effectors such as HIF-1? and VEGF as well as the disruption of the miRNA-21-PTEN/AKT axis were identified as potential underlying mechanism of the cytotoxic properties of mango polyphenols. Overall, findings from this study show that mango polyphenols counteract inflammatory and cancerous cell signaling processes; therefore the potential of mango polyphenols in the prevention of breast-cancer focusing on the PI3K/AKT/mTOR-axis should be further investigated.Item Combination of Manumycin A and Mebendazole in Human Breast Cancer Cell Lines(2011-12-12) Haddadin, Rania; Chow, Diana; Yeung, Jim; Bond, Richard; Liang, Dong; Giovanella, BappinoWe evaluated the combination of MA and Mbz in wild-type and HER2 transfected MDA-MB-231 and MCF-7 human breast cancer cell-lines in vitro and in xenografted mouse model. Methods: XTT colorimetric and SRB assays were used to determine cell viability in culture after single and combination treatment. Flow cytometry and western blotting were used to test the role of cell cycle arrest and apoptosis in cytotoxicity of single and combination treatment. We used PI for cell cycle and Annexin-V-FITC for apoptosis. We probed for Cyclins E and B and cleaved PARP. In vivo MDA-MB-231cell pair was used for dorsal subcutaneous xenogratfs in nu/nu Swiss mice. MA and Mbz were administered ip in single and combination treatments. The change in tumor volume was used to assess effectiveness. Results: MA and Mbz were cytotoxic in all four cell-lines at micro-molar levels. Mbz is more effective in MDA-MB-231 cells. MA 1st and Mbz1st showed additional benefit in MDA-MB-231/ErbB2 and MCF-7/Her18 cells, respectively. MA arrested MCF-7cells at G1/S and MDA-MB-231 cells at G2/M phase. No cleaved PARP was detected at 89kDa in all four cell-lines. In vivo, concurrent treatment showed additional benefit in MDA-MB-23/ErbB2. Mbz1st treatment showed additional benefit in male but not female mice with MDA-MB-231 xenografts. Liver histopathology showed necrosic, apoptosic and microangiopathic changes with combination treatment.Discussion: MA and Mbz were cytotoxic in all four cell-lines at micro-molar levels, with Mbz being more effective in MDA-MB-231 cells. Combination therapy showed additional benefit over single agent treatment in HER2 transfected but not wild-type cells. Apoptotic cell death did not play a major role in cytotoxicty. Sequence of drug administration, drug concentration, ratio of MA to Mbz, and targeted cells affect final outcome of combination treatment in vitro. Sequence of drug administration, type of cancer cells, and gender affect treatment outcome in vivo. Liver toxicity was observed with combination treatment. Conclusion: We were able to identify factors affecting MA and Mbz combination outcome. This combination is antagonistic with some exceptions. We are the first to show anticancer activity of Mbz in breast cancer xenografts using a microemulsion formulation.Item Comparative activation of estrogen receptor alpha (er alpha) by endocrine disruptors(2009-05-15) Wu, FeiEstrogen receptor ? (ER?) is a ligand activated transcription factor. Many widely used synthetic compounds and natural chemicals can activate ER?. The compounds investigated in this study include 17?-estradiol (E2), diethylstilbestrol (DES), antiestrogens ICI 182,780, 4-hydroxytamoxifen, the phytoestrogen resveratrol, and the xenoestrogens bisphenol A (BPA), nonylphenol (NP), octylphenol (OP), endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1- trichloroethane (HPTE) and 2,3,4,5-tetrachlorobiphenylol-4-ol (HO-PCB-Cl4). With the exception of the antiestrogens, all the compounds induced transactivation in MCF-7 or MDA-MB-231 cells transfected with wild-type ER? and a construct (pERE3) containing three tandem estrogen responsive elements (EREs) linked to a luciferase gene. However, these compounds differentially activated C-terminal deletion mutants of ER?. For example, neither E2 nor DES induced transactivation in MCF-7 transfected with ER?(1-537) due to partial deletion of helix 12 of ER?; however, OP, NP, resveratrol, kepone and HPTE induced this ER? mutant, demonstrating that the estrogenic activity of these synthetic compounds do not require activation function 2 (AF-2) of ER?. This study also investigated the effects of xenoestrogens on activation of reporter gene activity in MCF-7 and MDA-MB-231 cells transfected with a construct (pSp13) containing three tandem GC-rich Sp binding sites linked to the luciferase gene. In MCF-7 cells, antiestrogen-induced activation of ER?/Sp1 required the zinc finger motifs of ER?, whereas activation by estrogen and some xenoestrogens activation, such as endosulfan, NP and OP required the H12 of ER?. In contrast, xenoestrogens, such as HPTE, BPA, kepone and HO-PCBCl4, significantly induced transactivation of all four ER? deletion mutants tested in this study. Moreover, RNA interference assays demonstrated structuredependent differences in activation of ER?/Sp1, ER?/Sp3 and ER?/Sp4. The in vivo activities of E2, ICI 182,780, BPA and NP were further investigated in a transgenic mouse model containing pSp13 transgene. All the compounds induced luciferase activity in the mouse uterus; however activities observed in the penis and testis of male and stomach of both male and female mice were structure-dependent,. These results demonstrate that various ER ligands differentially activate ER? in breast cancer cells and transgenic mice, and their activities are dependent on ER? variants, promoter-, cell-context and selective use of different Sp proteins, suggesting these structurally diverse compounds are selective ER modulators (SERMs).Item Gene silencing in cancer cells using siRNA : genetic and functional studies(Texas A&M University, 2004-09-30) Abdel Rahim, Ma'en AhmadSequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17?-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 ? S phase progression. Surprisingly, TCDD alone induced G0/G1 ? S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 ? S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 ? S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.Item Lgr4 in Breast Cancer Stem Cells(2014-12-11) Zeng, LiBreast cancer is the most commonly diagnosed cancer among American women. G-protein coupled receptors (GPCR) comprise a huge family protein with almost 800 members. GPCRs sense molecules or other stimuli outside the cell, and activate intracellular signals. Consequently, a large proportion of modern drugs target these receptors. Lgr4 is a GPCR implicated in the development of multiple organs; in the mammary gland, it is expressed in the basal epithelial subpopulation and controls organ development by regulating stem cell activity through the wnt/?-catenin pathway. High breast tumor expression of Lgr4 correlated with a high risk of tumor relapse after chemokine therapy and an elevated risk of bone metastasis. We crossed mice bearing a gene trap cassette in the Lgr4 locus with several breast cancer mouse models such as MMTV-Wnt1 and MMTV-PyMT to study the consequences of Lgr4 expression ablation in breast cancer progression. We found that the absence of Lgr4 significantly delayed tumor progression in both MMTV-Wnt1 and MMTV-PyMT mouse models. Meanwhile, Lgr4 ablation led to diminished lung metastases in MMTV-PyMT tumors and several breast cancer cell lines. Further studies revealed that the repression of tumor progression and metastasis formation was due to a decreased cancer stem cell number in tumors with Lgr4 down-regulation, as well as blocking of epithelial-mesenchymal transition. Mechanistic studies suggested that Lgr4 is a master regulator which modulates multiple pathways (Wnt, EGFR, MMP) in breast cancer. Our findings clarify the role of Lgr4 in tumor progression and metastasis formation, and provide a potential therapeutic target in breast cancer treatment.Item Mechanisms of growth inhibition induced by methylene-substituted and ring-substituted dims in breast cancer cells(2009-05-15) Vanderlaag, Kathryn ElisabethOne in 8 women will be diagnosed with breast cancer in the United States and estrogen receptor (ER) status largely influences the type and subsequent success of treatment employed. Although ER-positive breast cancer can be treated with endocrine therapy, the more invasive ER-negative breast cancer is non-responsive to this therapy and cytotoxic agents are often utilized which are associated with many adverse side effects. Consequently, there is a genuine need to develop more effective, less toxic treatments for invasive breast cancer. Indole-3-carbinol is a phytochemical found in cruciferous vegetables and one of its major metabolites, 3,3?-diindolylmethane (DIM), exhibits a broad range of anticancer and antitumorigenic activities. ER-negative MDA-MB-231 and MDA-MB-453 breast cancer cell growth was inhibited after treatment with a novel series of methylenesubstituted DIMs (C-DIMs), namely 1,1-bis(3?-indolyl)-1-(p-substitutedphenyl) methanes containing trifluoromethyl (DIM-C-pPhCF3), t-butyl (DIM-C-pPhtBu) and phenyl (DIM-C-pPhC6H5) groups. In addition, DIM-C-pPhC6H5 (40 mg/kg/d) inhibited tumor growth in nude mice bearing MDA-MB-231 cells as xenografts. Treatment of breast cancer cells with C-DIMs lead to downregulation of cyclin D1 and induction of non-steroidal anti-inflammatory drug-activated gene 1. Detection of necrosis, caspasedependent or caspase-independent apoptosis were not observed in breast cancer cells treated with C-DIMs, however autophagic cell death was induced by C-DIMs. DIM and ring-substituted DIMs have exhibited antitumorigenic activity in tumor murine mammary models. An investigation into the mechanism of cell death induced by DIM and 5,5?-dibromoDIM (5,5?-diBrDIM) in both ER-positive (MCF-7) and ERnegative (MDA-MB-231) breast cancer cells revealed modulation of several key signaling pathways involved in growth control. Both DIM and 5,5?-diBrDIM downregulated cyclin D1, although only 5,5?-diBrDIM induced a depolarization of the mitochondrial membrane. In addition, apoptosis was observed in MCF-7 cells treated with 5,5?-diBrDIM but not MDA-MB-231 cells. In summary, C-DIMs may represent new mechanism-based agents for treatment of breast cancer through induction of autophagic cell death. The ring-substituted DIMs correspond to a novel class of uncharged mitochondrial poisons that are also highly effective in inhibiting breast cancer cell growth. Results of this research provide evidence for the potential role of two new series of DIM analogs for the treatment of highly aggressive breast cancer.Item MicroRNA expression in canine mammary cancer(Texas A&M University, 2008-10-10) Boggs, Rene' MichelleMicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan? Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.Item Photoacoustic computed tomography in biological tissues: algorithms and breast imaging(Texas A&M University, 2004-11-15) Xu, MinghuaPhotoacoustic computed tomography (PAT) has great potential for application in the biomedical field. It best combines the high contrast of electromagnetic absorption and the high resolution of ultrasonic waves in biological tissues. In Chapter II, we present time-domain reconstruction algorithms for PAT. First, a formal reconstruction formula for arbitrary measurement geometry is presented. Then, we derive a universal and exact back-projection formula for three commonly used measurement geometries, including spherical, planar and cylindrical surfaces. We also find this back-projection formula can be extended to arbitrary measurement surfaces under certain conditions. A method to implement the back-projection algorithm is also given. Finally, numerical simulations are performed to demonstrate the performance of the back-projection formula. In Chapter III, we present a theoretical analysis of the spatial resolution of PAT for the first time. The three common geometries as well as other general cases are investigated. The point-spread functions (PSF's) related to the bandwidth and the sensing aperture of the detector are derived. Both the full-width-at-half-maximum of the PSF and the Rayleigh criterion are used to define the spatial resolution. In Chapter IV, we first present a theoretical analysis of spatial sampling in the PA measurement for three common geometries. Then, based on the sampling theorem, we propose an optimal sampling strategy for the PA measurement. Optimal spatial sampling periods for different geometries are derived. The aliasing effects on the PAT images are also discussed. Finally, we conduct numerical simulations to test the proposed optimal sampling strategy and also to demonstrate how the aliasing related to spatially discrete sampling affects the PAT image. In Chapter V, we first describe a prototype of the RF-induced PAT imaging system that we have built. Then, we present experiments of phantom samples as well as a preliminary study of breast imaging for cancer detection.Item Role of estrogen receptor alpha (ER alpha) insulin-like growth factor (IGF)-I-induced responses in MCF-7 breast cancer cells(2009-05-15) Zhang, ShuInsulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that induces proliferation and activation of kinase pathways in MCF-7 breast cancer cells. The role of estrogen receptor ? (ER?) in mediating responses induced by IGF-I was investigated in cells transfected with small inhibitory RNA for ER? (iER?) or cotreated with the pure antiestrogen ICI 182780. The results showed that IGF-I-dependent phosphorylation of Akt and MAPK, induction of G1?S-phase progression and enhanced expression of cyclin D1 and cyclin E were dependent on ER?. Moreover, these IGF-I-induced responses were also inhibited by the antiestrogen ICI 182780, suggesting that the effects of ICI 182780 as an inhibitor of IGF-I induced responses in breast cancer cells are primarily related to downregulation of ER?. Chemoprotective phytochemicals exhibit multiple activities and interact with several cellular receptors, including the aryl hydrocarbon receptor (AhR). We investigated the AhR agonist/antagonist activities of the following flavonoids: chrysin, phloretin, kaempferol, galangin, naringenin, genistein, quercetin, myricetin, luteolin, baicalein, daidzein, apigenin, and diosmin, in MCF-7 breast cancer cells, HepG2 human liver cells and mouse Hepa-1 cells. The dietary phytochemicals exhibited substantial cell context?dependent AhR agonist as well as antagonist activities, and these are factors that must be considered in risk assessment of overall exposures to AhR agonists. Halogenated aromatic hydrocarbons (HAHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8- pentachlorodibenzo-p-dioxin (PeCDD), 3,3?,4,4?,5-pentachlorobiphenyl (PCBP), 2,3,7,8- tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) bind and activate the aryl hydrocarbon receptor (AhR). It has been assumed that these compounds only differ in their potencies. The SAhRM-like activity of the 5 HAHs was investigated by determining ligand structure dependent differences in their induction of CYP1A1 and interactions of the AhR with a series of coactivators in a mammalian two-hybrid assay in three AhR-responsive cell lines, including mouse Hepa-1, Human HEK293 and human Panc1 cells. There were multiple structure-dependent differences in activation of luciferase activity in these cell lines transfected with VP-AhR and six different GAL4-coactivator chimeras and a GAL4-response element-luciferase promoter construct. The results show that HAHs selectively interact with coactivators and these interactions are dependent on cell-context, and even among HAHs, these compounds exhibit selective receptor modulator activity.Item The Role of single minded 2 short in mammary gland development and breast cancer(2009-05-15) Kwak, Hyeong-ilSingle minded 2 (Sim2) is a member of the basic helix-loop-helix Per-ARNT-Sim (Period-Arylhydrocarbon Nuclear Translocator-Single minded) family. Human SIM2 is involved in the etiology of the Down?s phenotype. In addition to the physical and mental deficiencies associated with DS, it has become apparent that women with DS are 10-25 times less likely to develop breast cancer in comparison to age-matched normal populations. Such significant effects on breast cancer susceptibility are thought to result from gene dosage effects of one or more tumor suppressor genes on chromosome 21. Here we report the identification and transcriptional characterization of mouse Sim2s, a splice variant of Sim2, which is missing the carboxyl Pro/Ala-rich repressive domain. Similar to full-length Sim2, Sim2s interacts with ARNT and to a lesser extent, ARNT2. The effects of Sim2s on transcriptional regulation through hypoxia-, dioxin- and central midline response elements are different than that of full length Sim2. Specifically, Sim2s exerts a less repressive effect on hypoxia-induced gene expression than full length Sim2, but is just as effective as Sim2 at repressing TCDD-induced gene expression from a dioxin response element. Interestingly, Sim2s binds to and activates expression from a central midline response element-controlled reporter through an ARNT transactivation domain-dependent mechanism. Forced expression of SIM2s in MDA-MB-435 breast cancer cells significantly inhibited proliferation, reduced anchorage-independent growth, and decreased invasive potential. SIM2s directly decreased expression of matrix metalloprotease-3, a known mediator of breast cancer metastasis. In addition, loss of Sim2 in the mouse mammary gland increased ductal branching, accelerated lobuloalveolar-like precocious hyperplasia, and decreased cell apoptosis, suggesting that SIM2s is a mammary tumor suppressor. Sim2-/- mammary glands lose E-cadherin expression, suggesting that Sim2s plays a role in regulating E-cadherin/beta-catenin signaling. Loss of Sim2 in the mammary glands also resulted in dramatically increased MMP3 expression. The mechanism of SIM2smediated repression of MMP3 was found to be due to its ability to inhibit AP-1 binding to the MMP3 promoter. These results suggest that SIM2s contributes to the breast cancer protective effects observed in DS individuals.