Tamoxifen is an effective anti-estrogen for treatment of women with hormonedependent breast cancer but acquired drug resistance limits its therapeutic benefit. We have previously reported that expression of active Akt3 in MCF-7 breast cancer cells results in estrogen-independent tumors that are actually stimulated to grow after tamoxifen treatment. We hypothesize that this tamoxifen resistance may be attributed to binding of different co-regulator proteins and/or different binding affinity of these proteins to the estrogen receptor in M CF-7 cells overexpressing active Akt3 as compared to parental MCF-7 cells. We have immuno-precipitated the estrogen receptor along with bound co-regulator proteins in both cells lines after tamoxifen, estradiol, or vehicle treatment. After 2-D gel electrophoresis separation of these immuno-precipitated proteins and comparing them using PDQuest 2-D analysis software, we identified protein spots that were statistically different under the treatment conditions between the two cell lines. The isolated protein spots were subjected to MALDI-TOF mass spectrometry. By searching protein databases through the MASCOT website for protein identification, we have identified estrogen receptor co-regulator proteins that may play a potential role in tamoxifen resistance. Current studies are focused on addressing the role of differential protein binding as a possible mechanism of tamoxifen resistance in Akt3 over-expressing breast cancer cells.

With the increasing number of breast cancer survivors, there is a need to enhance health education to help survivors make informed decisions about maximizing their quality of life. YouTube is one of the most popular video applications that can be used for public health education. Nonetheless, there is little research on the content of health-related information that is uploaded to YouTube relevant to breast cancer survivors. This study was intended to help fill that gap in knowledge by describing the sources, formats, and content conveyed in the most widely viewed YouTube videos on breast cancer. YouTube was searched with a cleared browsing history using the key search term “breast cancer.” The resulting videos were sorted by view count. Videos were then screened for inclusion and exclusion criteria, yielding a sample of 100 videos with the most views. Video title, link, number of views, and date of upload were coded along with content included in each video. The inter- and intra-rater reliability was acceptable (Kappa’s = .79 and .97, respectively). The sample of 100 videos was collectively viewed 135,311,626 times, suggesting that the subject of breast cancer is a popular topic on YouTube. Nearly half of the sample videos (n = 45) were uploaded by television news/media agencies. Combined/multiple formats were the most popular format (n = 61), followed by still images/text (n = 48). General information on cancer was found to be the most common (n = 71), followed by screening for breast cancer occurrence/ recurrence (n = 62), and cancer treatments/breast cancer treatments (n = 45). Several of the content categories were rarely covered in the most-watched videos—for example, cancer rehabilitation recommendations, returning to work after cancer treatment, and financial burden/management of cancer. Thus, while topics such as breast cancer screening are widely covered, topics for breast cancer survivors regarding maximizing their quality of life are less widely covered. Few videos (n = 3) contained misinformation, but these videos were viewed millions of times, emphasizing the need for ongoing monitoring to identify and remove misinformation. The findings of this study indicated that YouTube videos on breast cancer gained over 135 million views. YouTube can be a great media channel for public health education. Nonetheless, there is significant need for more high-quality YouTube videos to be created to help breast cancer survivors navigate their cancer journey.

Thesis (M.Sc. (Chemistry)) -- University of Limpopo, 2021 / Breast cancer is a malignant tumour that starts in the cells of the breast. Many studies revealed aromatase (CYP19A1) and cyclin-dependent kinase 2 (CDK2) as possible therapeutic targets regarding breast cancer treatment, because they play crucial roles in anti-apoptotic processes during cell proliferation. Quinoxaline derivatives have attracted a great deal of attention due to their biological activities against fungi, virus, bacteria and cancer. Computer modelling was employed in order to reduce time and cost by searching the library of molecules and identifying those which are likely to bind to the drug target. A library of new one hundred (100) nitro and amino quinoxaline alkyne derivatives were successfully designed and screened against target proteins (CYP19A1 and CDK2) using virtual screening technique and thirteen (13) molecules were identified to be hit compounds against both targets with the docking score ranging from -6.143 to -8.372 kcal/mol as a measure of binding affinity. The hit compounds were subjected to IFD in order to identify tight binding through intermolecular interactions with active site residues of the binding pocket of the target proteins. All identified nitro and amino quinoxaline alkyne derivatives were successfully synthesised in a multi-step reaction sequence and their spectroscopic analysis (NMR, FTIR and MS) were in good agreement with the proposed structures in a good to moderate yield. The newly synthesised novel amino and nitro-quinoxaline derivatives were evaluated for anti-proliferative activity against breast cancer (MCF-7). Compound 59 showed to possess good inhibition against MCF-7 with an IC50 of 9.102 μM, whereas compounds 34, 54, 56 and 61 showed promising activity against MCF-7 with an IC50 value of < 50 μM. However, the MTT assay results showed that 59 was found to be toxic with an IC50 value of 0.205 μM against Raw 264.7 cell line. The dose response investigations showed that 31 and 34 have the promising anti-cancer activity against CYP19A and the correlation between molecular modelling (in-silico) and CYP19A inhibition activities (in- vitro), was established as compounds 31 and 34 were identified to bind to the drug target (CYP19A) with the docking score of -8.372 and 7.630 kcal/mol respectively. All the synthesized compounds were evaluated for the antitubercular activity against Mtb H37Rv strain as a secondary study. Compounds 57-62 with nitro-quinoxaline derivatives exhibited stronger inhibitory effects on Mtb H37Rv strain. In addition, compounds 60 and 62 were found to be most active against Mtb H37Rv with the high activity at MIC90 of <0.65 and <0.64 μM respectively. All active compounds are currently investigated for their cytotoxicity which have not been investigated before. / National Research Foundation (NRF) and Sasol Inzalo Foundation

Thesis (M.Med. (Radiation Oncology)) -- University of Limpopo, 2022 / Background Breast cancer is known to be a heterogeneous disease that demands patient centered care. Establishing the clinicopathological characteristics of breast cancer patients is a vital step in an effort to individualize their treatment. Aim The aim is to evaluate the clinicopathologic features of the different subtypes of breast cancer when classified according to immunohistochemistry markers in women attending Pietersburg hospital. Methods A retrospective review of medical records of women treated at Pietersburg hospital between 2010 and 2011 was done. Data collection was extracted on a customized data collection sheet. Chi square was used to determine association between clinicopathologic features and molecular subtypes. Analysis of variants was used to assess association between molecular types and age. Results The mean age of the population was 55.3 years (+/-14 standard deviation). The majority of patients were in stage III (46.9%) and IV (33.5%). The ER, PR, HER2/neu positive rate was 50.6%, 30% and 14,3 % respectively with a negative rate of 13,4%, 19,5% and 23,4% respectively. ER, PR and HER2/neu was unknown in 18%, 19, 5% and 23,4% respectively. The most common molecular subtype was luminal A (53,6%) followed by triple negative (27.2%), HER2/neu (11, 4%) and luminal B (7. 9%).There was no association between the subtypes and tumour stage (p=0.578).The rate of distant metastasis was similar across the subtypes being 37,9%,35%, 32,4% and 31,9% in HER2/neu, luminal B ,luminal A and TNBC, respectively. All four molecular subtypes had high rate of axillary lymph node involvement (p=0.886) Luminal A had the least percentage of high grade tumours with TNBC having the highest. Five-year overall survival for the cohort was 25, 6% with luminal A and B having a better 5 year overall survival of 27,2% and 25% respectively, whereas HER2/neu and TNBC had lower 5 year OS of 24% and 23,3%. Conclusion The findings of this study suggest that luminal A subtype is the most predominant and the majority might benefit from hormonal therapy. However, some patients could not be classified due to missing IHC marker test results. The outcome across all four subtypes is poor and more effort should be put towards improving the diagnosis and treatment individualization and follow-up in these patients.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of different types of cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects to both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. We therefore hypothesize, that the manipulation of the circadian Per2 protein in conjunction with doxorubicin may provide a more effective chemotherapeutic strategy for the treatment of breast cancer. The aims of this project were thus to: (i) Characterize the role of Per2 in normal breast epithelial cells as well as in ER+ and ER- breast cancer cells; (ii) to determine the role of Per2 in doxorubicin-induced cell death, (iii) to determine the role of Per2 in autophagy and finally (iv) to assess whether the pharmacological inhibition of Per2 with metformin, can sensitize chemo-resistant MDA-MB-231 breast cancer cells to doxorubicin-induced cell death. Methods: An in vitro model of breast cancer was employed using the normal MCF-12A breast epithelial, estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER-) MDA-MB-231 breast adenocarcinoma cell lines. Circadian rhythmicity of Per2 protein expression was determined using western blotting, and Per2 cellular localization was assessed using fluorescent confocal microscopy. Per2 was then silenced by means of an endoribonuclease-prepared siRNA, and silencing efficiency was determined with the use of western blotting. The roles of Per2 in doxorubicin-induced cell death and autophagy were assessed by treating MDA-MB-231 breast cancer cells under the following conditions (1) Control, (2) 2.5 μM doxorubicin or 10 nM bafilomycin A1 (3) 30 nM esiPer2 and (4) 30 nM esiPer2 in combination with 2.5 μM doxorubicin or 10 nM bafilomycin A1. Following treatments cell viability was assessed using the MTT assay, western blotting for markers of apoptosis including p-MDM2 (Ser166), p-p53 (Ser15), cleaved caspase-3 and –PARP as well as markers of autophagy (AMPKα, mTOR and LC3). Furthermore, cell cycle analysis, G2/M transition and cell death (Hoechst 33342 and propidium iodide staining) were assessed by means of flow cytometry. The pharmacological inhibition of Per2 was achieved by treating MDA-MB-231 cells with 40 mM metformin as well as in combination with 2.5 μM doxorubicin. MTT cell viability assays, cell cycle analysis (flow cytometry) and western blotting for apoptosis (Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) were assessed. Results and discussion: A circadian pattern of Per2 protein expression was observed in the normal MCF-12A and MDA-MB-231 cancer cells with protein levels peaking at ±700% and ±500% of baseline was observed. However, no rhythmic expression was observed in the MCF-7 cancer cells. Immunostaining for Per2 showed localization OF Per2 in the cytoplasm as well as in the nucleus of both the MCF-12A and MDA-MB-231 cells. Concentration curves showed a significant reduction in cell viability following 2.5 μM doxorubicin treatment for 24 hours. Per2 protein expression was significantly reduced with both esiPer2 and metformin treatment. Silencing of Per2 in combination with doxorubicin treatment resulted in cell cycle arrest with a significant increase in apoptosis, indicating that Per2 silencing effectively sensitized the MDA-MB-231 cancer cells to the anti-carcinogenic properties of doxorubicin. Modulation of Per2 protein expression was effectively achieved with the use metformin although this decrease occurred independently of AMPKα phosphorylation. A significant increase in apoptosis was observed following treatment with metformin in combination with doxorubicin treatment. However, no changes in cell cycle regulation were observed. Per2 appears to be involved in the regulation of autophagy as a significant increase in autophagy flux was observed when Per2 was silenced. Additionally, this increase in autophagic flux resulted in a significant increase in MDA-MB-231 cancer cell death which was enhanced further when autophagy was inhibited with bafilomycin A1 subsequent to Per2 silencing. Conclusions: Per2 protein expression was shown to display a 24 hour circadian rhythm in the MCF-12A cells, and to a lesser extent in the MDA-MB-231 cells. However, the MCF-7 cells failed to show rhythmic changes in Per2 protein expression. Per2 was shown to be located predominantly in the cytoplasm, with nuclear localization observed when cytoplasmic fluorescent intensity was lower. Per2 silencing effectively sensitized the chemo-resistant MDA-MB-231 breast cancer cells to both doxorubicin-induced cell death and autophagic inhibition. / AFRIKKANSE OPSOMMING: Inleiding: Sirkadiese ritmes vorm ‘n integrale fisiologiese sisteem wat die sinkronisasie van alle metaboliese prosesse asook lig/donker siklusse se effektiwiteit optimaliseer. Onderbreking van hierdie sirkadiese ritmes word geïmpliseer in die ontstaan en bevordering van verskillende kankertipes, insluitend borskanker. Verskeie raakpunte bestaan tussen die sirkadiese proteïen, Per2, en die DNA skade-respons. Abnormale Per2 uitdrukking veroorsaak afstroom effekte op beide die selsiklus en apoptotiese teikens wat moontlik aanduidend van ‘n tumor-onderdrukkende rol vir Per2 kan wees. Daar bestaan ‘n groot nood vir meer effektiewe adjuvante terapieë om kankersel vatbaarheid vir chemoterapie te verhoog as gevolg van dosis-beperkende newe-effekte wat geassosieer word met huidige chemoterapeutiese strategieë, insluitende dié van doxorubicin. Ons hipotese is dus dat die manipulering van die sirkadiese Per2 proteïen tesame met doxorubicin ‘n meer effektiewe chemoterapeutiese strategie vir die behandeling van borskanker sal wees. Die doelwitte van hierdie projek was dus om: (i) Die rol van Per2 in normale borsepiteelselle sowel as in ER+ en ER- borsepiteel kankerselle te karakteriseer; (ii) die rol van Per2 in doxorubicin-geïnduseerde seldood te bepaal; (iii) te bepaal of Per2 ‘n rol in autofagie speel en laastens (iv) te bepaal of die farmakologiese inhibisie van Per2 met metformin chemo-weerstandbiedende MDA-MB-231 kankerselle kan sensitiseer vir doxorubicin-geïnduseerde seldood. Metodes: ‘n In vitro model vir borskanker is gebruik wat normale MCF-12A borsepiteelselle, estrogeen reseptor positiewe (ER+) MCF-7 en estrogeen reseptor negatiewe (ER-) MDA-MB-231 bors adenokarsenoomselle insluit. Sirkadiese ritmisiteit van Per2 proteïen uitdrukking is deur middel van die westelike kladtegniek bepaal en die sellulêre ligging van Per2 deur middel van fluoresensie mikroskopie. siPer2 is voorberei deur middel van endoribonuklease-siRNA en die effektiwiteit daarvan is deur middel van westelike kladtegniek getoon. Die rol van Per2 in doxorubicin-geinduseerde seldood en autofagie is bepaal deur MDA-MB-231 borskankerselle onder die volgende omstandighede te toets: (1) Kontrole, (2) 2.5 μM doxorubicin of 10 nM bafilomycin A1 (3) 30 nM esiPer2 en (4) 30 nM esiPer2 in kombinasie met 2.5 μM doxorubicin of 10 nM bafilomycin A1. Na die behandeling, is sellewensvatbaarheid bepaal deur gebruik te maak van ‘n MTT toets; westelike kladtegniek is gebruik om vir merkers van apoptose soos p-MDM2 (Ser166), p-p53 (Ser15), gekliefde caspase-3 en -PARP asook vir merkers van autofagie (AMPKα, mTOR en LC3) te toets. Die selsiklus, G2/M oorgang en seldood (Hoechst 33342 en propidium iodide kleuring) is deur middel van vloeisitometrie bepaal. Per2 is ook farmakologies geïnhibeer deur MDA-MB-231 selle met 40 mM metformin asook in kombinasie met 2.5 μM doxorubicin te behandel. Daarna is sellewensvatbaarheid (MTT) sowel as die selsiklus (vloeisitometrie) en apoptose (westelike kladtegniek vir Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) gemeet. Resultate en bespreking: ‘n Sirkadiese patroon vir Per2 proteïen uitdrukking is in die normale MCF-12A selle asook in die MDA-MB-231 kankerselle waargeneem met proteïenvlakke wat hul piek by ±700% and ±500% onderskeidelik in vergelyking met basislyn bereik het. Geen ritmiese patroon van Per2 proteïen uitdrukking is egter in die MCF-7 kankerselle waargeneem nie. Immunokleuring om Per2 ligging te bepaal het getoon dat Per2 in the sitoplasma sowel as in die nukleus in beide MCF-12A en MDA-MB-231 selle voorgekom het. Konsentrasie kurwes het aangetoon dat daar ‘n insiggewende vermindering in sellewensvatbaarheid voorgekom het na die behandeling van die selle met 2.5 μM doxorubicin vir 24 uur. Per2 proteïen uitdrukking is insiggewend verlaag met beide esiPer2 en metformin behandeling van die selle. esiPer2 aleen of in kombinasie met doxorubicin behandeling het selsiklus staking tot gevolg gehad asook ‘n beduidende toename in apoptose veroorsaak wat dus aangedui het dat esiPer2 effektief was om MDA-MB-231 kankerselle te sensitiseer vir die anti-karsinogeniese doxorubicin behandeling. Modulering van Per2 proteïen uitdrukking was effektief met metformin behandeling, alhoewel die afname onafhanklik van AMPKα fosforilasie plaasgevind het. ‘n Insiggewende toename in apoptose is waargeneem na metformin behandeling in kombinasie met doxorubicin. Geen veranderinge in die selsiklus is egter onder hierdie omstandighede waargeneem nie. Per2 blyk betrokke te wees in die regulering van autofagie aangesien ‘n insiggewende verhoging in autofagie omsetting waargeneem is na esiPer2 behandeling. Die toename in autofagie omsetting is geassosieer met ‘n insiggewende toename in seldood in MDA-MB-231 kankerselle wat verder verhoog is wanneer autofagie met bafilomycin A1 (autofagie inhibitor) in kombinasie met esiPer2 behandel is. Gevolgtrekkings: Per2 proteïen uitdrukking het ‘n 24 uur sirkadiese ritme in die MCF-12A normale selle, en tot ‘n mindere mate in die MDA-MB-231 selle getoon. Die MCF-7 selle het egter geen ritmiese patroon van Per2 proteïen uitdrukking getoon nie. Per2 kom hoofsaaklik in die sitoplasma voor en het slegs in die nukleus voorgekom wanneer die sitoplasmiese fluoresensie intensiteit laer was. esiPer2 was dus effektief om die chemo-weerstandbiedende MDA-MB-231 borskankerselle te sensitiseer vir doxorubicin-geïnduseerde seldood. / National Research Foundation

This study investigated potential apoptotic and anti-proliferative effects of the phytochemicals, genistein and anthocyanin extract, as single and combined treatments in MCF-7 human breast cancer cells. Cells were exposed to single and combined treatments with the phytochemiclas for 48 and 72 hours. Cell viability was assessed using the MTT bioassay. Apoptosis induction was assessed using acridine orange ethidium bromide and rhodamine 123 ethidium bromide fluorescence assays. Both singe and combination treatments induced dose- and time-dependent apoptotic cell death in MCF-7 cells. The percentage of apoptosis was higher in combination treatments than single treatments with either phytochemical, although the difference was not statistically significant. The combination of genistein and anthocyanin extract peaked in efficacy at 48 hours of treatment, to exhibit significantly greater (P<. O5) dose- and time-dependent cell cytotoxicity than single treatments. This study reveals potential chemopreventive implications for the complementary effects of genistein and anthocyanin extract. / by Corine M. Stinson. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

published_or_final_version / Public Health / Master / Master of Public Health

published_or_final_version / abstract / toc / Community Medicine / Doctoral / Doctor of Philosophy

Background: Heart damage from cancer therapy is a significant problem for survivors. Some of the most effective treatments, such as anthracyclines, cause heart toxicity that can lead to significant morbidity and mortality. Cardiotoxicity also contributes to the loss of promising cancer drugs in early development and is notoriously difficult to predict. This translational project employs parallel pre-clinical and clinical studies to explore circulating biomarkers and cardiac magnetic resonance imaging (CMR) during development of anthracycline associated cardiotoxicity with the aim of finding biomarkers to aid clinical decision making and enable forward/back translation. Methods: Pre-clinical work: A rat model of chronic anthracycline-induced cardiomyopathy was developed involving 8 weekly intravenous boluses of doxorubicin followed by a 4 week ‘washout’ period. A time course assessment of cardiac function using multiple MRI parameters was performed alongside a panel of circulating biomarkers measured prior to dosing. Clinical work: In parallel following ethical approval, 30 cancer patients receiving standard anthracycline chemotherapy were recruited. Serial CMR scans were performed using standard and new exploratory techniques before, during and after treatment and blood was taken to evaluate a similar panel of cardiotoxicity biomarkers using multiplex ELISA at corresponding time points. Results: Pre-clinical results: Systolic and diastolic function declined progressively, culminating in left ventricular dysfunction (LVEF < 50%) by 12 weeks. Myocardial electron microscopy revealed myofibrillar and mitochondrial damage after one dose and gross histopathological damage after 5 doses. Myocardial contrast enhancement and troponin I increased significantly after eight doses and preceded LV dysfunction. Extensive fibrosis was seen 1 month after drug cessation. Clinical results: LVEF declined progressively in all patients and 7 patients (23%) had persistent LV dysfunction 12 months after therapy. Troponin I elevations were seen towards the end of therapy and peak troponin I corresponded with LVEF decline. None of the other circulating biomarkers correlated strongly with outcome. Lower baseline extracellular volume (ECV) was associated with greater LVEF decline but little change in ECV was seen over time. Baseline dyssynchrony was associated with worse outcome and deteriorated with time alongside LVEF decline. Conclusions: Results suggest that troponin I and cardiac MRI are sensitive translational tools in drug induced cardiotoxicity. However, troponin I is a relatively late marker, peaking after substantial myocardial damage, too late to halt or change reatment. The imaging suggests that fibrosis and inflammation cannot be detected within a year of chemotherapy but baseline ECV and strain analysis may have a role in risk stratification.

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