Mitochondria are the main sites for adenosine triphosphate (ATP) generation within most cells. Structural and functional alterations of mitochondria due to genetic abnormalities of mitochondria can cause respiratory chain dysfunction. In this study, the important role of mitochondria in energy metabolism was determined by comparing the effect of mitochondrial DNA (mtDNA) mutations on growth patterns and oxidative phosphorylation (OXPHOS) enzyme activities of six isolated clones (B5, B12, D4, D9, E1 and E8); as well as the effect of ATP supplement to culture using the slowest growing clone. The isolated clones had shown distinct growth pattern and morphology. The difference in proliferation rates among the clones was ascertained by the doubling times (B5=26.4h. B12=43.2h. D4=25.7h. D9=33.6h. E1=26.9h and E8=28.8h). The clone's slow growth rate was likely the result of mitochondrial mutations in the 16S rRNA gene, ND1, ND4, ND6 and COX III. Five heteroplasmic mutations were found in clone B12 (G2480T, C2513G, A2520T, C9527T and C14263G), one heteroplasmic mutation in clone D9 (A4137G) and one homoplasmic mutation in clone D4 (C11496). The mutations in clone B12 appeared to be deleterious to the cell by disrupting mitochondrial OXPHOS activities and reducing energy output. Additionally, extracellular ATP supplement to OXPHOS deficient clone B12 facilitated cell growth and enhances the gene expression. Increased expression of mtDNA-encoded respiratory chain complexes observed in clone B12 compared to clone D4 may reflect mitochondrial genomic adaptation to perturbations in cellular energy requirements. The stimulation of mitochondrial biogenesis may be a cellular response in compensation for defects in OXPHOS associated with mtDNA mutations. My data support the hypothesis that the variability in functional manifestations of mtDNA is attributed to the nature of the mutation, number of mutation and the gene specifically affected. These results will help to further our understanding of the relationship between mitochondrial mutation and cellular function.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-07-25T18:53:17Z No. of bitstreams: 1 PRISCYLLA_ANDRADE_VOLKART_DIS.pdf: 1703523 bytes, checksum: 625ed4371e92d211496ba44a5d264d21 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-26T14:20:44Z (GMT) No. of bitstreams: 1 PRISCYLLA_ANDRADE_VOLKART_DIS.pdf: 1703523 bytes, checksum: 625ed4371e92d211496ba44a5d264d21 (MD5) / Made available in DSpace on 2017-07-26T14:27:43Z (GMT). No. of bitstreams: 1 PRISCYLLA_ANDRADE_VOLKART_DIS.pdf: 1703523 bytes, checksum: 625ed4371e92d211496ba44a5d264d21 (MD5) Previous issue date: 2017-03-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The redox-active chemical properties of trace element copper make it an essential cofactor for several cellular mechanisms, as for ROS production. Polyphenols have been used in the pro-oxidant mechanism of action of interaction with endogenous Cu (II) ions for the production of reactive oxygen species in excess as a treatment for malignancies, leading to apoptosis. This work studied Cu (II) ions in the complex formation mechanism with Resveratrol in MCF-7 cells model. We analyzed the selectivity of Resveratrol in relation to the cupric metal ion using HPLC (High-Performance Liquid Chromatography) and the formation of the compound Resveratrol-Copper through UV-VIS (Ultra-Violet Visible Spectrophotometry). We analyzed the cellular morphology and location of the metal ion by MET-EDS (Electronic Transmission microscopy with Dispersive X-ray Spectroscopy). Cell death by apoptosis and quantification of ROS in said cell line with copper enrichment and treated with Resveratrol was made by flow cytometry. The results show the selectivity of the polyphenol compound by copper ion as well as the formation of the complex Resveratrol-Copper in extracellular conditions, however even with verification of endogenous copper accumulation in physiological conditions in vitro, the formation of that complex did not occur because there was no production of ROS and therefore, no cell death. In short, our research reveals that for in vitro conditions for the MCF-7 line, there's no Resveratrol-Copper complex formation as observed in sub-lethal quantities of chemically enriched cells with CuSO4 and treated with Resveratrol. / As propriedades qu?micas redox-ativas do oligoelemento cobre o tornam cofator essencial para diversos mecanismos celulares como o de produ??o de ROS. Polifen?is v?m sendo utilizados no mecanismo de a??o pr?-oxidante de intera??o com ?ons Cu (II) end?geno para a produ??o dessas esp?cies oxig?nio reativas em excesso como tratamento de malignidades, levando a apoptose. O presente trabalho estudou os ?ons Cu (II) no mecanismo de forma??o do complexo com Resveratrol em modelo de c?lulas MCF-7. Analisamos a seletividade do Resveratrol frente ao ?on met?lico c?prico utilizando CLAE (Cromatografia L?quida de Alta Efici?ncia) e verificou-se a forma??o do complexo Resveratrol-Cobre por meio de UV-VIS (Espectrofotometria de Ultra-Violeta Vis?vel). Analisou-se a morfologia celular e localiza??o do ?on met?lico por MET-EDS (Microscopia de Transmiss?o Eletr?nica com Espectroscopia Dispersiva de Raios-X). Morte celular por apoptose e quantifica??o de ROS na linhagem enriquecida com cobre e tratadas com Resveratrol foi feita por Citometria de Fluxo. Os resultados mostram a seletividade do composto polifen?lico pelo ?on cobre bem como a forma??o do complexo Resveratrol-Cobre em condi??es extracelulares, por?m mesmo com a verifica??o de ac?mulo de cobre end?geno, em condi??es fisiol?gicas in vitro n?o foi constatada a forma??o do referido, pois n?o houve forma??o de ROS e morte celular conseguinte. Em suma, Nossa pesquisa revela que em condi??es in vitro para a linhagem MCF-7, n?o h? forma??o de complexo Resveratrol-Cobre como observado quimicamente em quantidades sub-letais de c?lulas enriquecidas com CuSO4 e tratadas com Resveratrol.

After menopause, a woman's production of 17-estradiol, the predominant female sex hormone, declines. This change is associated with increased risk of osteoporosis/osteopenia and atraumatic bone fracture, cardiovascular disease, and breast and ovarian cancers. Phytoestrogens are non-steroidal compounds isolated from plants that have antagonistic, weak agonistic, or super-agonistic estrogenic effects in mammalian tissues; they have emerged as a potential therapeutic to alleviate post-menopausal symptoms. While some epidemiological evidence indicates that dietary consumption of phytoestrogens can alleviate post-menopausal health risks, other research suggests that phytoestrogens may not be completely safe. The research presented in this thesis indicates that a high concentration and sustained dose of phytoestrogens may be necessary to achieve antiestrogenic effects. MCF-7 cells, an estrogen-sensitive breast adenocarcinoma cell line, were used as a model system, and proliferating cell nuclear antigen (PCNA) was used as a marker of cell proliferation. Immunoblotting shows that genistein, a commercially purified phytoestrogen, promotes cell proliferation when administered for 24 hours, but may reduce proliferation when cells were treated for 48 hours. Genistein and estrogen have an additive effect on cells that were treated simultaneously with both hormones for 24 hours. In contrast, Promensilâ„¢, an over-the-counter phytoestrogen dietary supplement, was able to abolish expression of PCNA after 48 hours, and at high concentrations prevented estrogen-induced upregulation of PCNA after 48 hours. The clinical significance of these findings is that phytoestrogens may reduce the risk of breast cancer, but only after sustained high doses, which may be difficult if patient non-compliance is at issue. Additionally, because cell proliferation and not cell survival was investigated, we cannot say whether phytoestrogens are cytotoxic to breast cancer cells, only that they reduce proliferation.

Thesis (M.Sc. (Biochemistry)) --University of Limpopo, 2017 / Cancer is a broad group of various diseases characterised by unregulated cell proliferation which leads to the formation of tumours (Vickers, 2004). Some tumours remain confined to their site of origin while some gain the ability to spread to other parts of the body, a process known as metastasis (Weiss, 1990). The burden of cancer continues to rise, due to inefficient prevention strategies and serious side effects, as well as the cost of cancer regimens (Sondhi et al., 2010). Medicinal plants represent a reservoir of bioactive compounds that can be useful in the management of cancer with less or no side effects (Wong et al., 2012). The aim of this study was to investigate the anti-cancer effects of M. balsamina leaf extract in breast MCF-7 cancer cells. In this study, M. balsamina leaves powder was extracted using acetone. The biological effect of the extract was assessed on the viability of MCF-7 cells using the MTT assay. The extract’s ability to induce apoptosis was assessed using the Hoechst/propidium iodide dual staining method. Its anti-metastatic potential was investigated by determining its effect on MCF-7 cell migration, attachment and invasion using wound healing, adhesion, invasion assay, respectively. The human apoptosis antibody and human angiogenesis antibody array kits were used to determine the effect of the extract on the expression levels of proteins involved in apoptosis and metastasis, respectively. Treatment of MCF-7 cells with different concentrations of the extract showed a significant decrease in cell viability after 48 h incubation at 10 - 20 µg/ml. The decrease in cell viability was associated with the induction of apoptosis as seen by nuclear condensation and loss of membrane permeability in cells treated with the extract. Inhibition of migration, adhesion and invasiveness of the MCF-7 cells was seen in the treated cells. The extract also modulated proteins implicated in cell apoptosis, adhesion, migration and invasion such as Bcl-2 family of proteins, IGFBP, uPA, MMPs. In conclusion, based on the results, the extract show pro-apoptotic and anti-metastasis potential. Thus M. balsamina can be considered as a potential source of compounds with anti-cancer activity

Steroid receptor coactivators modulate the final outcome of hormone induced gene transcription by steroid receptors. E6-associated protein (E6-AP), an E3 ubiquitin ligase, acts as a coactivator of steroid receptors, including estrogen receptor (ER). In this study, we elucidated the contribution of E6-AP to ER-dependent gene transcription in breast cancer cells. siRNA-mediated knockdown of E6-AP abrogates transcription of classic ER target genes, GREB1 and pS2, suggesting that E6-AP is essential for normal transactivation function of ER. In order to understand the global influence of E6-AP in ER-dependent gene transcription, we used gene expression microarrays under E6-AP knockdown conditions to identify ER target genes which are regulated by E6-AP. Our microarray analysis revealed 455 genes which are differentially regulated by E6-AP. Pathway analysis revealed that E6-AP regulated genes were involved in cell cycle. Cell cycle profiling at various time points of estrogen treatment reveals that under E6-AP knockdown conditions, breast cancer cells progress slowly through S phase and eventually fail to proliferate. Knockdown of E6-AP has no effect on ovarian and uterine cells, suggesting that E6-AP has cell specific roles. Our analysis suggests that knockdown of E6-AP reduces the levels of early (C-Myc and Cyclin-D1), mid (E2F1, E2F2 and E2F7) and late (BUB1, BUBR1, MAD2, NDC80, NUF2 and CASC5) estrogen-dependent cell cycle genes. Overall our data indicate that E6-AP is a major regulator of cell cycle in breast cancer cells. E6-AP also acts as a coactivator for androgen receptor (AR) and we studied the role of E6-AP in prostate gland development. We report the generation of transgenic mice which specifically over expresses E6-AP in the prostate gland. Prostate glands in these mice are larger when compared with its wild-type litter mates, corroborating our observations that knockout of E6-AP in mice leads to impaired prostate gland development. E6-AP transgenic mice also develop prostatic intra epithelial neoplasia after 18 months of age. In addition to these observations, we also show that over expression of E6-AP in the prostate gland leads to increased Akt signaling. In order to understand the mechanism by which E6-AP regulates prostate gland growth, we generated LNCaP cells that stably overexpress E6-AP protein. Data from these cell lines show that the levels of phosphatidylinositol 3-kinase, total Akt, phosphorylated Akt (active Akt) and its down-stream target protein, GSKβ are elevated, suggesting that E6-AP regulates the PI3K-Akt signaling pathway. We further show that E6-AP modulates PI3K-Akt signaling by regulating the protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased proliferation. Overall our data suggests that E6-AP regulates the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and tumorigenesis.

Sex steroid exposure constitutes a risk factor for breast cancer, but little is known about the effects of sex steroids on factors mediating angiogenesis, the development of new blood vessels, in normal and malignant breast tissue. In this thesis we have investigated the effects of estradiol, progesterone, and the nonsteroidal anti-estrogen tamoxifen on vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2) in normal human breast tissue, endothelial cells, and breast cancer. We have applied the technique of microdialysis to provide in situ sampling of estradiol and VEGF in tumors and normal breast tissue of breast cancer patients in vivo. Furthermore, we present a novel method of culturing normal human breast tissue ex vivo. Our results suggest a pro-angiogenic effect of estradiol and an anti-angiogenic effect of tamoxifen in the breast. Estradiol increased extracellular levels of VEGF in normal human breast tissue and breast cancer cells in vitro. In addition, estradiol decreased sVEGFR-1 in breast cancer cells and indirectly increased VEGFR-2 in endothelial cells. Compared to estradiol treatment alone, estradiol + tamoxifen increased sVEGFR-1 and decreased VEGF in breast cancer cells in vitro. Furthermore, estradiol + tamoxifen decreased tumor VEGF levels and tumor vasculature in human breast cancer xenografts in vivo. In breast cancer patients, a significant correlation was found between in vivo levels of estradiol and VEGF sampled by microdialysis in normal human breast tissue, suggesting that estradiol may be a potent regulator of VEGF in the breast in vivo. Tumor levels of VEGF were significantly higher than in normal breast tissue in vivo, supporting the role of VEGF in tumor angiogenesis. For studies of normal human breast, whole breast tissue may be cultured in vitro for up to one week with preserved morphology. Using this method, estradiol, and not progesterone, appears to be the main sex steroid regulator of extracellular VEGF in normal breast tissue. In conclusion, the data suggest that sex steroids and tamoxifen exert pro- and anti-angiogenic effects in normal breast tissue and breast cancer.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a phytoestrogenic component of Rheum plant extracts which has been used for medical treatment since ancient times. It has been shown to have anti-inflammatory and anti-cancer effects. In our research, we aimed to study the biological effects of emodin on MCF-7 and MDA-231 cell lines. Cytotoxicity assays showed that emodin treatment for 48hours caused a concentration dependent decrease in viable cell numbers of both cell lines. As determined by cell counting with tryphan blue, IC50 values were 8.40 and 12.17 &micro / g/ml for MCF-7 and MDA-231 cells, respectively. Apoptotic effects of emodin was investigated by measuring the changes in apoptotic and antiapoptotic gene expressions by qRT-PCR. In MCF-7 cells, Bax expression increased with increasing emodin concentrations, while Bcl-2 expression was downregulated. Bax/Bcl-2 ratio was calculated as 9.2 fold at 10&micro / g emodin/ml treatment for 48 hours, indicating stimulation of apoptosis. However, Bax/Bcl-2 ratio was found 1.6 fold for MDA-231 cells. These results were in accordance with the results obtained from microarray analysis of related gene expressions. MCF-7 cells were more apoptotic in comparison to MDA-231 cells. DNA fragmentation was observed in MCF-7 cells by TUNEL method. GST enzyme activity that was measured using CDNB as substrate, was increased 100% with respect to control up to 5&micro / g emodin/ml treatment of MCF-7 cells for 48 hours. However, effect of emodin on GST activity in MDA-231 cells was found insignificant. According to microarray analysis results, emodin affected the gene expressions of GST isozymes in MCF-7 cells much more than in MDA-231 cells.

One of the main problems of current cancer chemotherapy is the lack of selectivity of anti-cancer drugs to tumor cells which leads to systemic toxicity and adverse side effects. In order to overcome these limitations, researches on controlled drug delivery systems have gained much attention. Nanoscale based drug delivery systems provide tumor targeting. Among many types of nanocarriers, superparamagnetic nanoparticles with their biocompatible polymer coatings can be targeted to an intented site by an external magnetic field. Thus, the drug can be carried to the targeted site safely. The aim of this study is to prepare poly(dl-lactic-co-glycolic acid) (PLGA) coated magnetic nanoparticles and load anti-cancer drug, doxorubicin to them. For this purpose, magnetite (Fe3O4) iron oxide nanoparticles were synthesized as a magnetic core material (MNP) and then coated with oleic acid. Oleic acid coated MNP (OA-MNP) was encapsulated into PLGA. Effects of different OA-MNP/PLGA ratios on magnetite entrapment efficiency were investigated. Doxorubicin loaded magnetic polymeric nanoparticles (DOX-PLGA-MNP) were prepared. After the characterization of prepared nanoparticles, their cytotoxic effects on MCF-7 cell line were studied. PLGA coated magnetic nanoparticles (PLGA-MNP) had a proper size and superparamagnetic character. The highest magnetite entrapment efficiency of PLGA-MNP was estimated as 63 % at 1:8 ratio. Cytotoxicity studies of PLGA-MNP did not indicate any notable cell death between the concentration ranges of 2 and 250 &mu / g ml-1. It was observed that DOX-PLGA-MNP showed significant cytotoxicity on MCF-7 cells compared to PLGA-MNP. The results showed that prepared nanoparticles have desired size and superparamagnetic characteristics without serious toxic effects on cells. These nanoparticles may be suitable for targeted drug delivery applications. The findings obtained from drug studies may contribute to further work.

Conventional cancer chemotherapies cannot differentiate between healthy and cancer cells, and lead to severe side effects and systemic toxicity. In the last decades, different kinds of controlled drug delivery systems have been developed to overcome these shortcomings of chemotherapeutics. Magnetic nanoparticles (MNP) are potentially important in cancer treatment since they can be targeted to tumor site by an externally applied magnetic field. In this study, it is aimed to synthesize folic acid conjugated / polyethylene glycol (PEG) coated magnetic nanoparticles with appropriate size, surface chemistry, magnetization and biocompatibility to be used in biomedical applications. First MNP were synthesized, then covered with oleic and PEG / and finally conjugated with folic acid. A detailed characterization of synthesized nanoparticles was done by TEM, XRD, FTIR, VSM and XTT analyses. MNP synthesized by the rapid addition of ammonium hydroxide exhibited more spherical nanoparticles with a narrower size distribution. Agglomeration tendency of naked nanoparticles was prevented by oleic acid addition during the synthesis. Both naked and surface treated MNP have been found to exhibit superparamagnetic behavior both at room temperature (23

Abstract Peroxisome proliferator-activated receptors (PPARs) are transcription factors that belong to the family of nuclear hormone receptors and exist as three isoforms namely PPARα, PPARβ and PPARγ. PPARs function as key regulators of glucose and lipid metabolism and are potential targets for drugs used in the treatment of glucose and lipid metabolism dysregulation. PPARs also regulate the expression of genes involved in the process of cellular proliferation and differentiation. Since it was discovered that PPAR ligands cause liver tumourigenesis in rodents, PPARs and their modulators have been investigated widely in in vitro and in vivo studies of carcinogenesis of the liver, colon, prostate, lung and skin. PPARα and PPARγ are the most studied PPAR isoforms in relation to cancer, while the association of PPARβ with cancer is increasingly being investigated. Some studies suggest that PPARβ and its ligands may have anticancer activity, while other studies identify a role for PPARβ in tumour promotion and progression. Breast cancer is one of the most common types of cancer in women with the majority caused by non-hereditary mechanisms. The activation of PPARα in breast cancer cells is associated with an increase in proliferation, while PPARγ activation in breast cancer cells is related to differentiation and an inhibition of cell proliferation. The role of PPARβ and its modulators in breast cancer is uncertain, as there have been limited studies addressing the effects of PPARβ modulation in breast cancer cell lines. Environmental contaminants such as the phthalate plasticizers and alcohol are putative risk factors for breast cancer. The phthalates di(2-ethylhexyl)phthalate (DEHP) and di-n-butyl phthalate (DBP) are plasticizers that are used in a range of common household, medical and beauty products and as a consequence humans are exposed to significant levels of these compounds. DEHP and DBP are known teratogens in rodents and DEHP induces hepatocarcinogenesis in a process thought to be mediated via PPARα. DEHP and DBP are metabolized in vivo by esterases to the monoesters, mono-(2-ethylhexyl) phthalate (MEHP) and mono-n-butyl phthalate (MBP), and these compounds have been identified in human biological samples. MEHP and MBP modulate PPARs in various tissues and cell types, but their ability to modulate PPARs in human breast cancer cells is not known. Like phthalates, ethanol is another modulator of PPARs and alcohol consumption is associated positively with breast cancer development, but the molecular mechanisms involved are unknown and there are no studies that examine the effects of ethanol and its metabolite acetaldehyde on PPARs in breast cancer cell lines. This thesis describes studies establishing and validating a breast cancer cell line that conditionally expresses human PPARβ under the control of a tetracycline regulator. Using this model, the ability of PPARβ over-expression and/or activation by the PPARβ specific ligand GW0742 to promote breast cancer cell proliferation was studied. Furthermore, putative PPARβ regulated genes were examined for alterations in expression in the presence of the PPARβ ligand. This work determined that over-expression of PPARβ and/or its activation by GW0742 does not promote proliferation in MCF-7 breast cancer cells. This thesis also investigated the effects of the phthalate monoesters MEHP and MBP on PPARs in MCF-7 breast cancer cells. It was found that MEHP activated both PPARα and PPARγ but was unable to activate PPARβ, whereas MBP could not activate any of the PPAR isoforms. MBP was an antagonist for both PPARγ and PPARβ. Using breast cancer cell lines, studies were conducted addressing the effects of an increasing concentration of ethanol (0-300 mM) on the transcription and transactivation of PPARα and PPARβ isoforms. Estrogen receptor positive MCF-7 breast cancer cells were more sensitive to the effects of ethanol than estrogen receptor negative MDA-MB-231 cells, with changes in PPARα mRNA more pronounced than PPARβ mRNA. Studies in MCF-7 cells conditionally expressing either PPARα or PPARβ in the presence of their respective specific ligands, GW7647 and GW0742, revealed that ethanol concentrations of 20 mM and 100 mM suppressed the maximal response to ligand-mediated activation for PPARα. Studies using the ethanol metabolism enzyme inhibitors 4-methylpyrazole and cyanamide, suggested that while ethanol was responsible for the modulation of PPARβ transactivation, the primary metabolite acetaldehyde was responsible for the effects on PPARα transactivation. Lastly, it was determined that ethanol and/or GW0742 did not increase the proliferation of MCF-7 Tet-off cells. The findings in this thesis suggest that given the different consequences of MEHP, MBP and ethanol on PPARs, PPAR expression and activation by ligands may have tissue specific consequences and that PPARβ may have a complex role in mammary gland tumourigenesis.