Phytochemical Regulation of Estrogen Responsiveness and Proliferation of Human Breast Cancer Cells PDF Download
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Author: Shyam Narayan Sundar
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Languages : en
Pages : 438
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Author: Shyam Narayan Sundar
Publisher:
ISBN:
Category :
Languages : en
Pages : 438
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Author: Kyuri Kim
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Languages : en
Pages :
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Estrogens are associated with the development and progression of breast cancer in addition to their role in normal reproductive physiology, and estrogen receptors (ER) mediate the actions of estrogen in target tissues by regulating the expression of numerous biologically important target genes. The progression of human breast cancer and the development of resistance to endocrine therapies are thought to be associated with ER phosphorylation. We generated multiple combinations of ER phospho-mutants, at residues serine 104, 106, 118, 167, 236, and 305, and examined their impact on receptor half-life, the agonist and antagonist balance of selective estrogen receptor modulators (SERMs) and selective estrogen receptor downregulators (SERDs), the regulation of ER transcriptional activity, and stimulation of cell proliferation in response to estradiol and SERMs/SERD. We showed that changes in ER affecting the phosphorylation status of the receptor greatly impact receptor function and differential SERM and SERD modulated cellular responses that could contribute to resistance to endocrine therapies in breast cancer. We also studied the regulation of microRNAs (miRNAs) by estradiol and growth factors through ER and extracellular signal-regulated kinase 2 (ERK2) in order to understand their physiological impact on breast cancer. We identified nine miRNA- encoding genes harboring overlapping ER and ERK2 binding sites close to their transcription start sites, which require ER and ERK2 for transcriptional induction as well as estradiol- mediated miRNA regulation. We then identified TP63, a target of miR-101, miR-190 and miR- 196a2, and showed that TP63 plays an important role in estradiol- or growth factor-mediated cellular response in breast cancer cells (MCF-7 and MDA-MB-231) by increasing tumor cell growth and in vitro invasion mainly controlled by miR-196a2 action. These results suggest a tumor-suppressive role of miR-196a2 in regulating TP63 expression and the aggressive behavior of breast cancers.
Author: Kevin Michael Poindexter
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Category :
Languages : en
Pages : 129
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Abstract Regulation of Anti-Proliferation in Melanoma and Breast Cancer Cells by Small Molecule Phytochemicals and Their Derivatives By Kevin Michael Poindexter Doctor of Philosophy in Endocrinology University of California, Berkeley Professor Gary L. Firestone, Chair Melanoma and breast cancer represent a significant portion of overall cancer burden, and many strategies have been developed to control their growth. Many of these current options have serious adverse effects and sometimes lead to therapy resistance which can complicate future treatment. Because of this, it is critical to investigate new classes and combinations of therapeutics that can overcome these drawbacks and retain effectiveness in multiple phenotypes. This thesis details the mechanism in which the combination of Indol-3-Carbinol (I3C) and Acetylsalicylic Acid (aspirin) act synergistically to slow the growth of human melanoma cells. In addition, this thesis demonstrates that the phytochemical Artemisinin and its derivative, Artesunate are able to inhibit the growth of luminal A estrogen receptor positive breast cancer cells. Finally, we utilize I3C derivatives to analyze the growth effects of different chemical functional groups in both melanoma and breast cancer cells. The combination of I3C and aspirin was able to synergistically control melanoma cell proliferation through an induction of G1 cell cycle arrest. Cell cycle arrest was due likely to a similar downregulation of Cyclin Dependent Kinase 2 (CDK2) levels which in turn is under the transcriptional control of the Microphthalmia-Associated Transcription Factor M (MITF-M). The combination of I3C and aspirin was able to significantly downregulate MITF-M protein and transcript expression. Luciferase analysis of the proximal region of the MITF-M promoter revealed that both compounds regulated promoter activation synergistically. The reduction in activation was mediated by two distinct pathways that met on the MITF-M promoter, I3C inhibited the activation of BRAF which caused a decrease in binding to the promoter of the transcriptional activator BRN-2 while aspirin downregulated [beta]-catenin/LEF1 binding to the promoter, again decreasing activity. Site directed mutagenesis of these sites proved that they were sufficient for the compounds' regulation of MITF-M transcriptional activity. These results uncover one mechanism in which aspirin controls melanoma growth, something that has not been explored, and the combination of aspirin and I3C could be utilized as a potential anti-cancer treatment in human melanoma. We also demonstrate that Artemisinin, a phytochemical derived from Artemisia annua and its derivative Artesunate are able to regulate the growth of human breast cancer cells through regulation of Cyclin A2, and cyclin dependent kinases 1 and 2 (CDK) critical regulators of the G1/M phase transition as well as G2 phase progression. Artesunate was able to achieve significant downregulation and induce growth effects at much smaller doses than its parent molecule indicating increased potency. Chromatin-immunoprecipitation indicated that Artemisinin was able to alter Sp1 binding to the Cyclin A promoter, potentially explaining its ability to regulate transcript levels. In the last chapter, we explore the effects on melanoma and breast cancer cell growth caused by 1-Benzyl-I3C derivatives. We find that small changes in structure alter growth inhibition by the compounds likely through changes in their ability to interact and alter function of target enzymes Human Neutrophil Elastase, and NEDD4-1. No compound was stronger than the parent molecule, 1-Benzyl-I3C, but all were able to induce significant changes in growth at concentrations lower than I3C. While this study is preliminary it provides information as to what potential derivatives might slow transformed cell growth in the future.
Author: Brian Daniel Adams
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Category :
Languages : en
Pages : 0
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Category :
Languages : en
Pages : 23
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Macrophage function can be regulated by breast cancer cells to sustain an inflammatory microenvironment that promotes cancer cell proliferation and survival. MCF-7 cells suppress IL-10 expression by co-cultured macrophages. Activated and resting THP-1 macrophages were able to induce SNAIL expression in MCF-7 cells. SNAIL has been implicated in the epithelial-mesenchymal transition which precedes metastasis. These observations suggest that tumor-associated macrophages may promote progression-related gene expression at discrete steps in the progression cascade. Both Faslodex(TradeMark) and tamoxifen were effective in up-regulating pigment epithelium-derived factor, a potent anti-angiogenic cytokine, and in down-regulating SNAIL in MCF-7 cells.
Author: Qingqing Zhang
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Category : Breast
Languages : en
Pages : 328
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Languages : en
Pages : 0
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Coactivators are nuclear proteins that interact with steroid receptors, such as estrogen receptor-alpha (ERalpha), and are required for the ability of receptors to stimulate the expression of target genes. Antiestrogen ligand are commonly utilized in the treatment of breast cancer to negatively regulate the activity of steroid receptors. However, tumors often can develop resistance to antiestrogen therapy. Therefore, as an alternative approach to inhibiting ERalpha function in breast cancer cells, we have developed antisense oligonucleotides against three of the major ERalpha coactivator proteins. These oligonucleotides decrease the expression of coactivator mRNA and protein, and in so doing, decrease the ability of ERalpha to stimulate gene expression. These oligonucleotides also decrease the proliferation of MCF-7 breast cancer cells in response to estrogen treatment. Taken together, anti sense oligonucleotide technology has the potential to regulate ERalpha action at a level that circumvents ligand control, and therefore represents a novel mechanism by which to inhibit breast cancer gene expression and proliferation, and potentially to regulate the growth of breast cancer.
Author: Virginia A. Spencer
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Category :
Languages : en
Pages : 15
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Estrogen affects both the cytoskeletal and chromatin structure of breast cancer cells. The treatment of breast cancer cells with 10 nM estradiol causes the estrogen receptor to recruit histone acetyltransferases to the promoter of the estrogen-responsive PS2 gene within 60 min, while exposure to estrogen for 72 h increases the levels of DNA-associated cytokeratins. To determine the effect of estrogen on regions downstream of estrogen- responsive promoters, quantitative PCR was performed on DNA isolated from chromatin immunoprecipitations using anti-acetylated 113 and H4 antibodies. Treatment with 10 nM estradiol induced a transient state of 113 and 114 acetylation along the PS2 promoter, exon 2, and exon 3, that was maximal after 60-120 min. After three hours, this hyperacetylation decreased to levels above those observed along the PS2 gene in untreated MCF-7 cells. To determine how estrogen influences DNA-associated cytokeratins, the levels of total, intermediate filament-assembled and DNA-associated cytokeratins were determined by Western Blotting and two dimension gel electrophoresis. The level of DNA-associated cytokeratins increased after 2 h of 10 nM estradiol treatment, while the levels of total and filament cytokeratins remained the same. This suggests that early estrogen exposure (i.e. 2 h) promotes interactions between cytokeratins and DNA.
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Languages : en
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Author: Chien-Cheng Chen
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Languages : en
Pages :
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Estrogen receptor (ER) acts as a ligand-activated transcription factor that regulates the expression of genes. The genomic mechanisms of ER action include ligand-induced dimerization of ER which binds estrogen responsive elements (EREs) in the promoters of target genes. There are also nongenomic mechanisms of ER action which are associated with membrane bound or cytosol ER-dependent activation of various protein-kinase cascades which also influence expression of target genes. Egr-1 is an immediate-early gene induced by 17B-estradiol (E2) in the rodent uterus and breast cancer cells. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region that contained serum response element (SRE3) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a MAPKK inhibitor, but not by LY294002, an inhibitor of PI3-K. These results contrasted with the hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002, suggesting that Egr-1, like c-fos, is activated through non-genomic pathways of estrogen action but through activation of different kinases. COUP-TFs are orphan nuclear receptors expressed in a variety of tissues where they regulate biological functions and organogenesis. In this study, we investigated coactivation of ERa by COUP-TF1 in cell lines transiently cotransfected with the pERE3 construct. COUP-TFI coactivated ERał-mediated transactivation, but unlike many other coactivators, COUP-TFI also enhanced transactivation of ERa when cells were cotransfected with the TAF1-ERa mutant or the 19c-ERa mutant. These data indicate that helix 12 of ERa is not required for coactivation by COUP-TFI when AF-1 of ERa is intact. However, when the AF-1 of ERa is deleted, the intact AF-2 function is required for coactivation by COUP-TFI. Analysis of multiple COUP-TFI deletion mutants showed that the DNA-binding domain and C-terminal region of COUP-TFI were important for coactivation of ERa. Point mutations of the DNA-binding domain of COUP-TFI resulted in loss of interactions with ERa, suggesting that the DNA-binding domain of COUP-TFI is important for its coactivation activity facilitating interactions with ERa. These results demonstrate that COUP-TFI coactivated ERa through a non-classical LXXLL-independent pathway.
