Thesis (Ph. D.), Faculty of Health Sciences, University of the Witwatersrand, 2009 / With the high prevalence and high mortality rate of cancer in the global community, it is increasingly essential to accelerate our understanding of the disease, to identify new genetic targets for therapy, and to pursue avenues for improving on the therapies in development and in current use. The aim of this study is to identify cell cycle-related genes whose expression is influenced by the chemotherapeutic drugs curcumin, SAHA, lycopene and thalidomide in breast and colon cancer and normal cell lines. These drugs are currently not in clinical use for cancer in South Africa, and while there have been investigative studies of these chemotherapeutic agents, this study aims to identify the specific genes that are influenced by the drugs. The result of this is that several genes that were not previously documented as targets of these drugs are highlighted. The cell cycle pathway is the area of focus as loss of regulation in the cell cycle is one of the important factors involved in promoting cancer initiation and progression. In the first instance, flow cytometry was used to identify optimal drug concentrations relative to the cell cycle stages. Following this, alterations in gene expression were assessed using a PCR-based differential display after each drug treatment. Subsequently, a more focussed approach was taken in a PCR-array analysis of panels of cell cyclerelated genes. A subset of genes is identified that is implicated in oncogenic transformation in breast cancer. This has the potential to inhibit the genetic pathways involved in breast malignancy by providing targets that perhaps may not be manipulated in current therapies. The gene expression studies here suggest that lycopene and thalidomide function in inhibiting this transformation, and play significant roles in suppressing the oncogenic state of breast cancer. Curcumin and SAHA also exhibit important functions in inhibiting tumourigenesis in colon cancer. While the results propose that the drugs have clear roles in inhibiting breast and colon cancer, they are also implicated in promoting cancer. This research has defined the genes that must be carefully monitored during drug administering as they may promote these and other cancers. The availability of these results to researchers will aid in selecting the criteria for assessing the success rate of these drugs.

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Class of 2012 Abstract / Specific Aims: This study was aimed to evaluate inpatient clinical treatment characteristics associated with the use of intracranial implantation of chemotherapeutic wafers for malignant brain neoplasms within United States, and assess inpatient mortality and total charges regarding treatment with wafer versus without. Methods: A retrospective cohort investigation was conducted utilizing inpatient discharge records from the Agency for Healthcare Research and Quality (AHRQ) Healthcare Cost and Utilization Project (HCUP) Nationwide Inpatient Sample from 2005 to 2009. From this nationally- representative sample, 9,455 adults aged 18 years or older were identified with malignant neoplasms of the brain treated with implantable chemotherapeutic wafers. Outcomes of inpatient mortality and charges were assessed via multivariate regression analysis, controlling for patient characteristics, hospital structure, comorbidities, and clinical complications. Main Results: The average age of patients with brain neoplasms was 56.6 (±16.5) years, and of those patients, 42.9% were female. The odds ratio for inpatient mortality of patients treated with implantable chemotherapeutic wafers was OR=0.380 (P<0.001), and patients that received wafer treatment had increased charges exp(b)=2.147 (P<0.001). Conclusions: Multiple factors were associated with inpatient mortality and charges among the 247,829 patients that were diagnosed with malignant brain neoplasms from 2005-2009. With regards to these patients, implantable chemotherapeutic wafers were associated with increased inpatient survival and increased charges.

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TAZ (Transcriptional co-activator with PDZ-binding motif) is a WW-domain containing protein recently identified as a downstream component of the Hippo tumor suppressor pathway and mediator of biologically important processes (mesenchymal stem cell differentiation, embryonic stem cell renewal, mechanotransduction). Recently, loss of LATS1/2, a negative regulator of TAZ, has been observed in ~50% of breast cancers. However, whether and how TAZ is also involved in breast cancer has not been investigated. Therefore, this study explores the cellular functions of TAZ in breast cancer and the underlying molecular mechanisms. The cellular functions of TAZ were investigated using overexpression studies in an immortalized mammary epithelial cell line (MCF10A). Compared to control vector-only expressing cells (MCF10A-WPI) TAZ overexpression (MCF10A-TAZ) promotes enhanced cell proliferation, cell migration and cell-ECM adhesion, induces transformation and the epithelial-mesenchymal transition, and confers resistance to chemotherapeutics (paclitaxel, cisplatin). Together, these findings strongly suggest TAZ functions as an oncogene in the development, progression and drug resistance of breast cancer. As a transcriptional co-activator, TAZ likely mediates these cellular functions through the transcriptional activation of downstream genes. By screening a 44K human genome microarray we have identified and characterized Cyr61 and CTGF, mediators of paclitaxel resistance, and BMP4, a regulator of cell migration. Through stable shRNA-mediated knockdown, we show that loss of Cyr61/CTGF expression in MCF10A-TAZ cells can rescue TAZ-induced paclitaxel resistance. Similarly, shRNA-mediated knockdown of BMP4 can significantly attenuate TAZ-induced cell migration. Therefore, these findings demonstrate that Cyr61/CTGF and BMP4 are functionally significant mediators of TAZ-induced paclitaxel resistance and cell migration, respectively. The clinical relevance of our in vitro findings were also validated by immunohistochemistry using tissue microarrays containing human breast cancer samples. TAZ levels were highly expressed in 66.6% of clinical samples further suggesting TAZ may be an important oncogene in breast cancer. Our study has characterized TAZ as an oncogene in breast cancer and elucidated two novel mechanisms underlying paclitaxel resistance and cell migration. These findings highlight the importance of TAZ during the development, progression and drug resistance of breast cancers and the potential use of TAZ as a therapeutic target to treat TAZ-expressing breast cancers. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-08-01 23:59:08.68

Doctor of Philosophy / Department of Chemistry / Sundeep Rayat / Sundeep Rayat / This dissertation focused on the synthesis of 1-(2-ethynylphenyl)-4-phenyl tetrazole-5-thione derivatives 1 and evaluating their potential as a new class of photoactivated DNA cleaving prodrugs. We hypothesized that light activation of 1 would cause the decomposition of the tetrazolethione ring system to enyne-carbodiimide 2 with simultaneous loss of dinitrogen and sulfur via 1,3-triplet biradicals 1′, which would be spontaneously followed by Schmittel cyclization to indoloquinolines 3 via benzofulvene type biradicals 2′. The biradicals 1′ and 2′ would have the potential to cause DNA cleavage by abstracting hydrogens from its sugar phosphate backbone, analogous to the mechanism of action of naturally occurring enediyne antitumor antibiotics. Note that our proposed prodrugs contained a 1,4-diaryl tetrazolethione functionality and a direct synthetic route for their construction was lacking in the literature despite their wide spread applications. Therefore, our initial efforts were directed towards developing a general strategy to obtain these ring systems. We employed a highly versatile and efficient copper mediated N-arylation to first obtain a series of 1,4-diaryl tetrazol-5-ones which were thionated with Lawesson’s reagent to afford the corresponding 1,4-diaryl tetrazole-5-thiones in moderate yields. Specifically, the synthesis of 1 involved Sonogashira coupling of the obtained 1-(2-bromophenyl)-4-phenyl-1H-tetrazole-5(4H)-thione with the appropriate ethynyl compounds (Chapter 2). Since the tetrazole ring is an important structural component in many biologically and medicinally relevant compounds, we were interested in evaluating the anticancer activity of these compounds in the absence of photochemical activation. The moderate IC50 values against leukemia and breast cancer cell lines showed that the anticancer activity of these compounds prior to photoirradiation was minimal (Chapter 3). Independent studies have shown that the photodecomposition of tetrazolethiones gives carbodiimides via biradicals, and photocyclization of enyne-carbodiimide forms indoloquinoline also via biradicals. However, it was not known whether these two photoreactions could happen sequentially in one pot with one light source from a substrate like 1, generating biradicals 1′ and 2′ which could later be employed for DNA photocleavage as hypothesized. Therefere, we photolysed 1 in acetonitrile, and our results show clean formation of a mixture of enyne-carbodiimides and indoloquinolines via biradicals 1′ and 2′ (Chapter 4). Finally, we investigated DNA photocleavage by 1 at 350 nm and our results showed significant DNA cleavage in concentrations as low as 100 μM (Chapter 5).

Polymer carriers composed of poly(methacrylic acid – grafted – ethylene glycol) (P(MAA-g-EG)) hydrogels modified with poly(butyl acrylate) (PBA) to form IPNs or photopolymerized in the presence of poly(methyl methacrylate) (PMMA) nanoparticles were investigated for their use in the oral delivery of therapeutic agents for cancer treatment. The P(MAA-g-EG) hydrogel provided pH-responsive and hydrophilic properties while PBA or PMMA polymers provided hydrophobic properties. An inulin- doxorubicin conjugate was also synthesized to provide local, direct targeting for the treatment of colon cancer. The pH-responsive behavior of these polymer systems was investigated using equilibrium and dynamic swelling experiments. In gastric conditions (low pH) all materials were in a collapsed state and in intestinal conditions (neutral pH) these material were swollen. The equilibrium swelling ratios decreased with increasing hydrophobic content for both IPNs and compositions of P(MAA-g-EG) containing nanoparticles. The loading efficiencies of doxorubicin, a chemotherapeutic drug, were as high as 56% for IPNs and the IPN structure and hydrophobicity influenced the loading efficiency values. The loading efficiency of doxorubicin using P(MAA-g-EG) containing nanoparticles was as high as 64% and increased with increasing weight percent of PMMA nanoparticles in the P(MAA-g-EG) hydrogel. In gastric conditions (low pH), IPNs released a majority of the encapsulated doxorubicin (up to 70%) as compared to the P(MAA-g-EG) containing nanoparticles (up to 27%). P(MAA-g-EG) containing nanoparticles was used to load and release the inulin-doxorubicin conjugate. Loading efficiency was 54% and release profiles behaved similarly as doxorubicin. Both polymer systems were biocompatible with Caco-2, HT29-MTX, and SW620 cell models over concentration ranging from 1 mg/mL to 5 mg/mL and exposure times lasting from 2 hr to 24 hr. The 75/25 IPN exhibited the highest degree of mucoadhesion and the P(MAA-g-EG)-5.0NP the lowest. Using the same cell lines and cytotoxicity assays, the inulin-doxorubicin conjugate was determined to be more toxic than free doxorubicin at equal doxorubicin concentrations. Doxorobuicin and inulin-doxorubicin conjugate were tested for transport across Caco-2/HT29-MTX cell monolayers with and without the presence of unmodified P(MAA-g-EG) or P(MAA-g-EG)-5.0NP microparticles. The presence of the microparticles did not increase transport across the cell monolayer which is advantageous for local, direct delivery to the colon. / text

Orientador: Edson Luis Maistro / Banca: Mario Sergio Montovani / Banca: Luis Fernando Barbisan / Resumo: Os anabolizantes esteróides têm sido amplamente utilizados por profissionais e atletas de elite para melhorar sua aparência e habilidades atléticas. Além disso, eles apresentam um importante papel quimioterapêutico no tratamento de vários tipos de distúrbios metabólicos, homeostáticos e sexuais, em ambos os sexos. Tendo em vista que muitas drogas esteróides têm apresentado diferentes resultados considerando efeitos genotóxicos e mutagênicos, o objetivo desse trabalho foi avaliar o potencial genotóxico do hormônio nandrolona (deca-durabolin®) in vivo em células da medula óssea e do sangue periférico de camundongos, usando o teste do micronúcleo e o ensaio do cometa, respectivamente. Os animas receberam injeção intradérmica de 3 concentrações do hormônio esteróide (1.0, 2.5 e 5.0 mg/kg peso corporal). As células foram coletadas 24 h após o tratamento hormonal para o teste do micronúcleo (avaliação da clastogenicidade) e o teste do cometa (avaliação da genotoxicidade). O teste do micronúcleo evidenciou que as duas maiores doses testadas da nandrolona induziram aumentos estatisticamente significativos de células micronucleadas e o teste do cometa não evidenciou aumento significativo de danos no DNA nos linfócitos do sangue periférico. Sob estas condições experimentais, conclui-se que o hormônio esteróide nandrolona apresentou efeito clastogênico e/ou aneugênico e, por outro lado, não foram observados efeitos genotóxicos quando o mesmo foi administrado intradermicamente em camundongos / Abstract: Anabolic androgenic steroids have been widely used by professional and elite athletes to improve their appearance and athletic abilities. Besides, they have an important place in the chemotherapeutic treatment of various types of metabolic, homeostatic, and sexual disorders in both sexes. Since many steroidal drugs have been found to be different results considering genotoxic and mutagenic effects, the aim of this study was to evaluate the genotoxic potential of nandrolone (deca-durabolin®) in vivo in bone marrow and peripheral blood cells of mice, using micronucleus and comet assays, respectively. The animals received intradermal injection of the 3 concentrations of the steroid (1.0, 2.5 and 5.0 mg/kg body weight). The cells were collected 24 h after the hormone-treatment for the micronucleus (clastogenicity endpoint) and comet assays (genotoxicity endpoint). Micronucleus test showed that the two higher tested-doses of the nandrolone induced statistically significant increase of the micronucleated cells and comet assay no evidenced significant increase in the DNA damage of the lymphocytes from peripheral blood. Under our experimental conditions, the nandrolone steroid hormone showed clastogenic and/or aneugenic effects and, on the other hand, no genotoxic effects when administered intradermally to mice / Mestre

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