In general, ionizing radiation promotes cytoprotective autophagy in a majority of breast tumor cells. Previous studies from our laboratory indicated that radiation (5x2 Gy) induces cytoprotective autophagy in MCF-7 cells. In the current work, inhibition of autophagy by silencing of Beclin-1 in MCF-7 cells resulted in an increase in sensitivity to radiation based both on cell number and clonogenic survival; however, there was no increase in apoptosis and the basis for this sensitization is currently under investigation. Unexpectedly, enhancement of autophagy by silencing of Bcl-2 also led to an increase in sensitivity to radiation, possibly through the conversion of cytoprotective to cytostatic autophagy. In contrast to the MCF-7 cells, radiation (5x2 Gy) induces non-protective autophagy in Hs578t cells. Interference with autophagy through silencing of Beclin-1 or induction of Bcl-2 did not alter radiation sensitivity in the Hs578t cells. Since the induction of cytoprotective autophagy can represent an impediment to radiation therapy, it is important to understand the types of autophagy that occur in response to radiation in specific cellular settings and whether interference with autophagy can increase sensitivity to different forms of cancer treatment.

Resistance to chemotherapeutics is a widespread phenomenon in cancer cells that may counteract the successful therapy of many patients. In resistant cells, higher level of ABC transporters, among others, often can be detected. This high level of ABC transporters represents a suspected mechanism of acquired cancer resistance. We studied the molecular mechanism of resistance to taxanes in cancer cells using SK-BR-3 and MCF-7 breast cancer cell lines. We analyzed the effect of paclitaxel on apoptosis induction in the originally sensitive cells of these lines as compared to their counterpart resistant cells, developed by gradual adaptation to paclitaxel. In resistant cells of the SK-BR-3 and MCF-7 lines, we did not detected ongoing induction of apoptosis but we did detect significantly increased expression of ABCB1 transporter after paclitaxel application. By silencing the expression of the transport via employment of small interfering RNA (siRNA), we tested the role of the ABCB1 transporter in cells resistant to paclitaxel. We found that resistant cells with silenced expression of the ABCB1 transporter had a statistically significant increase of sensitivity to paclitaxel as compared to control resistant cells with high expression of this transporter. Along with increased sensitivity, we demonstrated...

Le chloroacétaldéhyde (CAA), un des principaux produits du métabolisme hépatique de l'ifosfamide (IFO), est considéré comme responsable de la néphrotoxicité de ce médicament. Les mécanismes exacts de cette néphrotoxicité ne sont pas complètement élucidés. Dans la première partie de cette étude, nous avons essayé de préciser les mécanismes physiopathologiques de la toxicité du CAA sur un modèle de tranches de cortex rénal de rat, puis, dans la deuxième partie, nous avons recherché un effet anticancéreux éventuel du CAA sur des cellules de cancer du sein humain (MCF-7). La néphrotoxicité du CAA, utilisé à des concentrations proches de celles mesurées chez les patients traités par l'IFO, soit 0 - 75 µM, s'est manifestée par une chute d'ATP et du glutathion ainsi que par une inhibition du métabolisme du lactate. Certaines enzymes de la néoglucogenèse, notamment la glyceraldéhyde 3-phosphate déshydrogénase, ont été inhibées par le CAA. Le complexe I de la chaîne respiratoire mitochondriale ainsi que l'oxydation du lactate ont été également inhibées par le toxique. D'autre part, le CAA (10 et 25 µM) a inhibé la prolifération des cellules MCF-7 sans que cette inhibition soit accompagnée d'une chute d'ATP cellulaire. Le transport cellulaire et le métabolisme du glucose ainsi que certaines enzymes de la glycolyse ont été également inhibés par le CAA. Parmi celles-ci, l'hexokinase semble être l'enzyme qui catalyse l'étape limitante de la voie de la glycolyse. En conclusion, le CAA est bien impliqué dans les mécanismes de la néphrotoxicité de l'IFO, mais de plus, il pourrait, via l'inhibition de la glycolyse, contribuer à l'effet thérapeutique de l'IFO.

Sex steroids are inevitable in women. However, long-term exposure to sex steroids increases the risk of breast cancer. A complete understanding of sex steroid control of the breast and how it relates to breast cancer risk is still lacking. Angiogenesis and proteolytic enzyme activity are crucial for the process by which tumors evolve into a vascularized, invasive phenotype. Matrix metalloproteinases are potent matrixdegrading enzymes that affect several steps in tumor progression including angiogenesis. In the female reproductive organs, sex steroids regulate angiogenesis and MMP activity, yet little is known how sex steroids affect these crucial events in normal and malignant breast tissue. This thesis elucidates a link between sex steroids, MMP activity, and angiogenesis. It is shown that estradiol down-regulates while tamoxifen up-regulates the protein expression and activity of MMP-2 and MMP-9 in human breast cancer cells in vitro and in human breast cancer xenografts in vivo. The results further suggest that a biological consequence of this regulation may be modulation of tumor angiogenesis. The net effect of adding tamoxifen to estradiol treatment was an increase in extracellular levels of the endogenous angiogenesis inhibitor endostatin and decreased levels of the tumor promoter TGF-β1 compared to estradiol treatment only. This was accompanied by reduced vasculature and decreased tumor growth. Similarly, a regulatory effect of estradiol and tamoxifen on endostatin generation was observed in normal human breast tissue by whole-tissue culture and microdialysis in human breast tissue in situ. In conclusion, the results presented in this thesis suggest previously unknown mechanisms of action of estradiol and tamoxifen in breast cancer and in normal human breast tissue, and novel means by which estradiol may tip the scale to favor angiogenesis. This knowledge may be important for the understanding of sex steroid dependent breast carcinogenesis and in the future development of tissue-specific preventive as well as therapeutic strategies against breast cancer.

Chemotherapy is one of the common treatments in cancer therapy. The effectiveness of chemotherapy is limited by several factors one of which is the emergence of multidrug resistance (MDR). MDR is caused by the activity of diverse ATP binding cassette (ABC) transporters that pump drugs out of the cells. There are several drugs which have been used in treatment of cancer. One of them is doxorubicin that intercalates and inhibits DNA replication. However, doxorubicin has been found to cause development of MDR in tumors. It has been reported that there is a correlation between multidrug resistance and invasiveness of cancer cells. Vimentin is a type III intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and also poor prognosis of cancer. There are several studies that have shown the connection between expression level of vimentin and invasiveness. In this study, MCF-7 cell line (MCF-7/S), which is a model cell line for human mammary carcinoma, and doxorubicin resistant MCF-7 cell line (MCF-7/Dox) were used. The resistant cell line was previously obtained by stepwise selection in our laboratory. The main purpose of this study was to investigate changes of metastatic behaviour in MCF-7/Dox cell line, after transient silencing of vimentin gene by siRNA. In conclusion, down-regulation of vimentin gene expression in MCF-7/Dox cell lines was expected to change the characteristics in migration and invasiveness shown by migration and invasion assays.

Resistance to broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. MCF-7 cells were selected in the presence of paclitaxel and vincristine by stepwise dose increments. The cell viability and growth profiles of resistant sublines were examined. As the resistance indices increased, the growth rates of sublines were found to decrease. Gene and protein expression levels of the basic drug resistance proteins P-gp and MRP1 were studied in sensitive and drug resistant MCF-7 cells. It was shown that P-gp overexpression is significantly contributing to the developed drug resistance phenotype. Mutation analysis of beta tubulin gene which encodes the target of paclitaxel and vincristine was performed. Single histidine to proline mutation was identified near GTP binding site of beta tubulin in vincristine resistant subline which was not reported before. Apoptosis related BCL-2 and BAX were examined at both gene and protein expression levels and they were not found to be significantly related to the developed resistance in the sublines. The reversal of drug resistance by various inhibitory agents of P-gp and MRP1 was investigated by using flow cytometry. Synthetic silicon compounds were found to be the most effective MDR reversal agents. The effects of various combinations of anticancer drugs and reversal agents on cell proliferation were examined by checkerboard microplate method. ALIS409-paclitaxel and paclitaxel-doxorubicin pairs seem to have highest antiproliferative effects on resistant sublines. The microarray expression profiling of sensitive and resistant MCF-7 cells was performed for a much detailed and comprehensive analysis of drug resistance. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of paclitaxel and vincristine drug resistance. Additionally up regulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant down regulation of apoptotic genes (i.e. PDCD2/4/6/8) and alterations in expression levels of genes related to invasion and metastasis (MMPs, ADAMs, COL4A2, LAMA etc.) were detected. Upregulation of some oncogenes (i.e. ETS, RAS) and cell cycle regulatory genes (CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Further studies will demonstrate the relationship between the components contributing to drug resistance phenotype in breast cancer cells.

Orientador: Prof. Dr. Anderson Orzari Ribeiro / Tese (doutorado) - Universidade Federal do ABC. Programa de Pós-Graduação em Ciência e Tecnologia/Química, 2017. / No presente trabalho apresentamos a sintese e caracterizacao do fotossensibilizador hipericina e alguns de seus sais hipericinato, bem como o estudo de sua atividade fotodinamica in vitro em celulas de adenocarcinoma mamario humano. A hipericina sintetizada foi caracterizada por espectroscopia 1H RMN, espectrometria de massas e espectroscopia no infravermelho. As propriedades fotofisicas e fotoquimicas da hipericina e dos hipericinatos foram determinadas por analises dos espectros de absorcao na regiao do UV-Vis, espectros de excitacao e emissao, coeficiente de absortividade molar (¿Ã) e capacidade de geracao de especies reativas de oxigenio atraves da degradacao do DPBF (1,3-difenilbenzofurano). Para os estudos in vitro utilizou-se a linhagem de adenocarcinoma mamario humano (MCF-7) avaliando a toxicidade e fototoxicidade da hipericina e hipericinatos, localizacao celular, capacidade mutagenica e genotoxica, capacidade clonogenica, tempo de captacao celular e identificacao de via de morte celular. Os resultados apresentam indicativos de que os hipericinatos possuem eficiencia relativa superior ao da hipericina na producao de oxigenio singlete, bem como uma menor taxa de agregacao em meio biologico. Ainda, nos ensaios in vitro, foi verificado que a atividade fotodinamica foi maior para os hipericinatos em comparacao com a hipericina, demonstrando que as modificacoes moleculares no composto promovem alteracoes na sua interacao com a linhagem celular estudada. / In this study we present the synthesis and characterization of photosensitizers like hypericin and hypericinates and its in vitro photodynamic ativity in human mammary adenocarcinoma cells. The synthesized hypericin was characterized by 1H NMR spectroscopy, mass spectrometry and infrared spectroscopy. The hypericinates were characterized by analysis of absorption spectra in the UV-vis region, excitation and emission spectra, molar absorptivity (å) and reactive oxygen species generation capacity through the degradation of DPBF (1,3-diphenylbenzofuran). For in vitro studies, human mammary adenocarcinoma (MCF-7) lineage was used to evaluate the toxicity and phototoxicity of hypericin and hypericinates, as well as their cell colocalization, mutagenic and genotoxic capacity, clonogenic capacity, time of cellular uptake and path of cell death identification. The results indicated that hypericinates have higher relative efficiency in the production of singlet oxygen than hypericin and lower rate of aggregation in biological medium. Furthermore, in in vitro assays, it was verified that the effectiveness of the photodynamic activity was higher for the hypericinates than hypericin, demonstrating that the molecular modifications in the hypericin macrocycle promotes changes in their interaction with cell line studied.

Philosophiae Doctor - PhD / Globally, breast cancer is the most common cancer affecting women and it is predicted that in 2030 about 12 million deaths will occur with approximately 21.7 million new cases [2]. Genetic risk factors as well as race and ethnicity, account for about 5-10% of all breast cancer occurrences. Triple negative breast cancer (TNBC), tumors that tested negative for oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), contribute to 10-20% of all breast carcinomas [3,4] and is known to be a more aggressive type of cancer with varying degree of response to chemotherapeutic and radiation therapy [5,6] / 2022-02-24

Introduction: In South Africa, breast cancer has the highest prevalence with a life time risk of 1 in every 9 women being diagnosed annually. There are four sub-types of breast cancer and according to the stage of the cancer, various treatment regimens are prescribed. A major obstacle is that majority of cancers have developed multi-drug resistance and new treatment regimens need to be developed in order to obtain therapeutic efficacy. Cancer cells use aerobic glycolytic metabolism for energy generation and inhibition of this pathway increases sensitivity of the cells to anti-neoplasic treatments. 2-Deoxyglucose (2-DG) competes with and inhibits glucose uptake inhibiting the glycolytic pathway which can result in depolarisation of the mitochondrial membrane potential releasing cytochrome c. Two 2-Methoxyestradiol (2-ME) derivatives, ESE- 15-ol and ESE-16 have shown to be promising anti-cancer agents and combination therapy could allow the use of these compounds with a decreased side effect profile. The combination of these compounds with 2-DG was therefore investigated. Aim: To investigate combinations of two oestrone analogues and the glycolysis inhibitor 2- deoxyglucose for potential synergistic effects using a cell enumeration assay, mitochondrial membrane potential and cell cycle analysis, on breast cancer cells in an in vitro setting. Cell apoptosis, necrosis and autophagy pathways were assessed to indicate the mechanism of cytotoxicity. Methods: The breast cancer MCF-7 and non-tumorigenic MCF-12A cell line were used. Cells were exposed to ESE-15-ol, ESE-16 and 2-DG alone and in combination. Mechanistic studies were performed using the various research methodologies including the sulforhodamine B assay for cell enumeration, Annexin-V FITC and propidium iodide labeling for apoptosis/necrosis studies, PlasDIC and light microscopy for morphological analysis, propidium iodide staining for cell cycle progression, JC-1 for mitochondrial membrane potential studies, transmission electron microscopy and western blotting for the analysis of autophagy. Results: A GI50 of 34.1 nM was reported for MCF-7 cells after treatment with ESE-15-ol, 141 nM for ESE-16 and 1.3 mM 2-DG. The GI50 of ESE-15-ol treated MCF-12A cells was 141 nM, 140.1 nM for ESE-16 treated cells and 1.7 mM for 2-DG. ESE-16 had the greatest effect on cell viability in MCF-7 cells and a shift from an inhibitory effect to the initiation of cell death was evident after treatment of 100 nM of ESE-15-ol and ESE-16. 2-DG had a lower cytotoxic effect than the oestrone analogues. The MCF-12A cell line was less susceptible to the experimental compounds. The combination of the oestrone analogues with 2-DG elicited a greater effect on cell enumeration than each of the compounds alone with a less pronounced effect on the MCF- 12A cell line in comparison to the MCF-7 cells. The experimental compounds initiated apoptosis with ESE-16 eliciting a greater effect than ESE-15-ol. The combination of the oestrone analogues with 2-DG resulted in increased apoptosis in contrast to the compounds alone. ESE-16 alone and in combination with 2-DG lead to the most prominent morphological changes, with ESE-15-ol decreasing cell density slightly. The combination of ESE-15-ol with 2-DG decreased cell density with membrane blebbing apparent. The MCF-12A cell line was less susceptible to morphological changes after treatment of ESE-15-ol with 2-DG however ESE-16 and the combination with 2- DG resulted in similar attributes seen in MCF-7 treated cells. ESE-15-ol resulted in accumulation of cells in the G2 cell cycle phase which was further amplified after the combination of 2-DG. A sub-G1 accumulation was observed after treatment with ESE-16 with a shift to a G2 accumulation after the combined treatment of ESE-16 with 2-DG. After 48 hours, ESE-15-ol alone and in combination with 2-DG on MCF-7 cells resulted in depolarisation of the mitochondrial membrane. A slight decrease in the membrane potential was observed after treatment with ESE-16 and this was further increased after the combined treatment of ESE-16 with 2-DG. The MCF-12A were less susceptible after 24 hour treatment than 48 hour exposure of the experimental compounds. The presence of autophagic-like vacuoles were apparent in all treatment groups as well as the increased expression of LC3-II. Conclusion: The combined treatment of synthetic oestrone analogues with 2-DG displayed greater therapeutic efficacy than each of the compounds alone. As a result, the apoptotic and autophagic pathways were induced and a shift in cell cycle progression was observed. Mitochondrial involvement was apparent and the compounds significantly affected cell viability. This suggests that the combinations between the antimitotic oestrone analogues and glycolysis inhibitor 2-DG act synergistically to induce apoptosis and autophagy in MCF-7 breast cancer cells. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

Utilization of potential synergistic interactions among different bioactive agents is a promising approach to inhibit complex diseases such as cancer. Nobiletin (NBT) and tangeretin (TAN) are major polymethoxyflavones (PMFs) found in citrus fruits. Herein, we studied NBT and TAN in combination with atorvastatin (ATST, Lipitor, a cholesterol-lowering drug) in MDAMB231 and MCF-7 human breast cancer cells. Both NBT/ATST and TAN/ATST combinations at low doses produced much stronger inhibitory effect on cancer cell viability in comparison to those produced by NBT, TAN, or ATST alone at much higher doses. Isobologram analysis confirmed that both NBT/ATST and TAN/ATST combinations produced strong synergy in inhibiting the growth of two breast cancer cell lines. Flow cytometry analysis showed that both NBT/ATST and TAN/ATST combinations caused significant cell cycle arrest at G0/G1 phase in MDAMB231 cells (ER+). Consistent with these results, PMFs and ATST combinations decreased expression levels of phospho Rb, cyclin D1, and CDK4. Further experiments showed that the combination treatment induced autophagy and late apoptosis in MDA-MB-231 cells. Meanwhile, co-treatment of PMFs and ATST induce G2/M phase in MCF-7 (ER+) cells.. The combination of PMFs and ATST also caused autophagy in MCF-7 cells, which was evidenced by activation of LC3B and P62. In conclusion, our result demonstrated strong synergy between two major citrus PMFs (NBT and TAN) and ATST in inhibiting human breast cancer cell growth.