de pele é o tipo mais frequente, correspondendo a cerca de 25% de todos os tumores malignos registrados no Brasil. Há que se destacar que uma formulação de uso tópico foi desenvolvida pelo grupo para uso contra câncer de pele e o estudo da estabilidade acelerada desta formulação foi avaliado nesse trabalho. Os frutos da espécie S. lycocarpum foram coletados, secos, triturados, submetidos à extração ácido-base e o precipitado foi suspenso em etanol e filtrado. Os heterosídeos alcaloídicos, SS e SM, foram isolados por cromatografia em coluna a vácuo e purificados em CLAE semipreparativa. A formulação contendo extrato alcaloídico armazenada em temperatura ambiente (27 ± 2 oC) apresentou a melhor estabilidade física. Ensaios para avaliação da atividade antifúngica in vitro do extrato alcaloídico, SS, SM e aglicona (SD) foram realizados com cepas de fungos dermatófitos e Candida spp., sendo que a SM apresentou o menor valor de concentração inibitória. No ensaio de citotoxidade em células humanas de carcinoma espinocelular (A431) e células de fibroblastos de camundongos (L929) foi possível observar a toxidade do extrato alcaloídico e das substâncias, SS, SM e SD frente às células cancerígenas. No ensaio in vivo, os animais foram induzidos ao câncer de pele não-melanoma do tipo espinocelular com células A431 e verificou-se que apenas o grupo de animais tratados com a formulação Curaderm BEC 5 apresentou redução tumoral. Em contrapartida, o grupo tratado com formulação contendo extrato alcaloídico se comportou da mesma maneira que os grupos controle da formulação e controle negativo. Com auxílio da histologia confirmou-se que o câncer de pele induzido nos animais foi do tipo espinocelular. No ensaio imunoistoquímico foi utilizado o anticorpo caspase-3 para marcar as células em apoptose nos tumores, sendo que o maior número de células apoptóticas foi verificada no grupo tratado com a formulação Curaderm BEC 5. / Solanum lycocarpum A. Saint-Hilaire (Solanaceae), commonly known as wolf apple or wolf´s fruit, is a plant native of Brazil very common in the Brazilian savanna. High concentrations of steroidal alkaloids are found in S. lycocarpum fruits, and solasonine (SS) and solamargine (SM) are the most important ones. These two alkaloids present potential antifungal and anticancer activities. Dermatophytic fungi are the most common agents responsible for superficial mycoses in humans and animals, by infecting exclusively the stratum corneum of skin, hair and nails. Skin cancer is the most frequent type corresponding to roughly 25% of all the malignant tumors registered in Brazil. A topical formulation for skin cancer treatment was developed by our research group and its stability tests are reported in this work. The S. lycocarpum fruits were collected, dried, milled and submitted to acid-base extraction furnishing a precipitate, which was solubilized in ethanol and then filtered. The heterosidic alkaloids SS and SM were isolated by using vacuum column chromatography and purified by semi-preparative HPLC. The formulation containing the alkaloidic extract stored at room temperature (27 ± 2 oC) was more stable than the ones in other conditions. Antifungal in vitro test of the alkaloidic extract, SS, SM and the aglycone (SD) were performed with Candida spp and dermatophytic fungi strains. The alkaloid SM displayed the lower inhibitory concentration. The toxicity of the alkaloidic extract, SS, SM and SD was verified in a cytotoxicicity test performed with both cultured human cells of spinocellular carcinoma (A431) and mice fibroblast cells (L929). An in vivo cytotoxicicity test was performed by inducing non-melanoma skin cancer in mice with spinocellular cells A431, in which only the animals treated with a commercial formulation Curaderm BEC 5, showed tumor reduction. There was no statistical difference between the group treated with the formulation containing the alkaloidic extract and control groups. Histological analysis confirmed that the skin cancer induced in the animals were spinocellular type. In an immunohistoquimic test, caspase-3 antibody was employed to stain the cells in apoptosis in the tumors, showing that apoptotic cells were more numerous in the group treated with the formulation Curaderm BEC 5.

Les graines de pin (Pinus halepensis Mill., Pinus pinea L., Pinus pinaster et Pinus canariensis) sont les quatre espèces les plus disponibles dans le bassin méditerranéen. Elles sont très utilisées par les populations Nord-africaines en médecine traditionnelle et en gastronomie où elles agrémentent les plats traditionnels (salades, riz, poissons …etc), car elles sont bien connues pour leur excellent goût salé. Cependant, la composition biochimique, les valeurs nutritionnelles, et les mécanismes d'actions cellulaires et moléculaires via lesquels ces graines exercent leurs effets thérapeutiques restent mal élucidés. Le but de notre travail est d'étudier les propriétés physico-chimiques des graines de quatre espèces de pin et la valeur nutritionnelle et pharmaceutique des fractions lipidiques des graines de Pinus halepensis Mill., en utilisant différentes techniques de séparation et d'analyse telles que (DRX, IRTF, CC, LC/MS, GC, GC/MS et RMN) et en examinant la voie principale impliquée dans le développement du cancer qui est l'angiogenèse via des essais biologiques in vitro sur la prolifération et la migration des cellules endothéliales sur Matrigel et in vivo sur une membrane chorioallantoïdienne (CAM) des œufs de poulet ainsi que leurs toxicités sur des cultures cellulaires (Myélome humain HL60, Adénocarcinome du coulon, humain HCT15, Cellules épithéliales A549 et cellules de mélanomes B16F1). Les résultats de la caractérisation physico-chimiques montrent que les quatre graines sont très riches en métabolites primaires (sucres, protéines, protéines de réserve) et secondaires (Phénols totaux et flavonoïdes) comme elles présentent une importante concentration en oligo-éléments (phosphore, potassium, magnésium, Zinc, fer, cuivre et manganèse). Leurs huiles essentielles sont riches en limonène. Les principaux acides gras insaturés pour les quatre espèces sont l'acide linoléique et l'acide oléique. Les propriétés chimiques et physiques de leurs huiles fixes sont dans la norme de qualité agroalimentaire. Les graines de Pinus halepensis Mill. sont les plus riches en lipides totaux qui atteignent un taux de 36% diversifiés chimiquement avec des lipides apolaires (Lipides neutres) et polaires (Quatre classes de glycolipides et six classes de phospholipides). Ces résultats sont de bons indicateurs de la qualité nutritionnelle des graines de pins et impliquent que les lipides neutres, les glycolipides et les phospholipides des graines de Pinus halepensis Mill. dépourvus de toxicité aux concentrations de 1, 10, 25, 50, 100 et 200µg/ml et ayant une activité cytotoxique à 500 et 1000µg/ml et anti-angiogénique in vitro à des concentrations de 100 et 500µg/ml et in vivo à des concentrations de 1mg /ml et 10 mg/ml peuvent être utilisés dans la prévention des maladies liées à l'angiogenèse et à la lutte contre le cancer. / The pine (Pinus halepensis Mill., Pinus pinea L., Pinus pinaster and Pinus canariensis) seeds are the four most available species in the Mediterranean basin. They are widely used by North African populations in traditional medicine and gastronomy where they adorn the traditional dishes (salads, rice, fish ... etc) because they are well known for their excellent taste salty. However, the biochemical composition, nutritional value, and the cellular and molecular mechanisms of action through which these seeds exert their therapeutic effects remain poorly understood. The aim of our study was to investigate the physicochemical properties of pine seed species and nutritional and pharmaceutical value of lipid fractions of Pinus halepensis Mill. Seeds using different separation and analysis techniques such as (XRD, FTIR, CC, LC/MS, GC, GC/MS and NMR) and examining the main pathway involved in the development of cancer which is angiogenesis through biological tests in vitro on the proliferation and migration of endothelial cells on Matrigel and in vivo on a chorioallantoic membrane (CAM) of chicken eggs, thus that their toxicity on healthy cell cultures (human myeloma HL60, Adenocarcinoma of human coulon, HCT15, human epithelial cells, A549 and cells melanoma, B16F1). The results of the physico-chemical characterization showed that four seeds are rich in primary metabolites (sugars, proteins, protein reserves) and secondary (total phenolic and flavonoids) as they have a high concentration of trace elements (phosphorus, potassium, magnesium, zinc, iron, copper and manganese). Their essential oils are rich in limonene. The main unsaturated fatty acids of all species are linoleic acid and oleic acid. The chemical and physical properties of their fixed oils are the in standard food quality. Pinus halepensis Mill. seeds are the richest in total lipids which achieved a rate of 36% chemically diverse with non polar lipids (neutral lipids) and polar lipids (Four classes of glycolipids and six classes of phospholipids). These results are good indicators of the nutritional quality of pine seeds and imply that the neutral lipids, glycolipids and phospholipids of Pinus halepensis Mill. seeds devoid of toxicity at the concentrations of 1, 10, 25, 50, 100 and 200µg/ml and having cytotoxic activity at 500 and 1000µg/ml and anti-angiogenic effect in vitro at the concentrations of 100 and 500 µM and in vivo at the concentrations of 1 mg/ml and 10 mg/ml may be useful in prevention of angiogenesis-related and the fight against cancer diseases.

Anticancer active metal complexes such as cisplatin are routinely used for treating various cancers since 1978. However, the side effects of cisplatin overwhelm its therapeutic potential, especially in the latter stages of treatment. The nonspecific cytotoxicity of drugs could be avoided if targeted delivery to cancer cells is achieved using two different methodologies namely, enhanced permeability and retention in solid tumors (EPR) and receptor mediated endocytosis using a homing agent (RME). Ru(II)-arene complexes which are delivered specifically into cancer cells by the transferrin enzyme are less toxic compared to other metal complexes. The thesis describes the synthesis and use of Ru(II)-η6cymene complexes with different ancillary ligands which modulates the anticancer activity and the utility of two macromolecular vehicles in directed drug delivery. Ru(II)-η6cymene complexes with different heterocyclic ancillary ligands are synthesized and their anticancer activity tested against various cancer cell lines. Ruthenium complexes with mercaptobenzothiazoles are found to be quite active against the H460 cell lines that overexpress transferrin receptors and non-cytotoxic to the normal cell line, HEL299. Biophysical studies show that complexes (H1 and H8) can unwind the pBR322 DNA and inhibit the Topo IIα enzyme. A unique biphasic melting curve of CT DNA is observed in the presence of H1 which is attributed to formation of a dinuclear species (H20). Half-sandwich complexes of 6-thioguanine (6-TG) have also been prepared to improve the delivery and efficacy of 6-TG which is used in spite of a deleterious photoreaction. The Ru complexes cytotoxic to several leukemia cell lines. As they are photostable and anticancer active, they are better than 6-TG. Anticancer activity exhibiting piazselenols are used as ancillary ligands to make Ru(II)-arene complexes. Unfortunately, 1H NMR spectra suggests that piazselenol complexes dissociate in solution. However, the nitro substituted piazselenol and its Ru complex show the greatest cytotoxicity (<0.1 µM) against the A2780 cell line. The utility of PAMAM dendrimers and hyper branched polymers (hybramers) conjugated with a homing agent to target cancer cells by EPR and RME is probed. A cytotoxic copper complex (CuATSM) is covalently attached to the macromolecules through a disulfide linker, cleaved in the presence of GSH. Targeting efficacy of the folic acid-dendrimer conjugates is checked against two glioma cell lines. The folic acid-dendrimer conjugate is more active compared to dendrimer conjugate without folic acid against folate-receptor-overexpressing LN18 cell line. Biotin conjugated dendrimer shows better accumulation in HeLa cells, which require high amounts of biotin for growth. In vivo studies demonstrate that the conjugate can cross the blood-brain barrier. These studies suggest that PAMAM dendrimer can be used as a targeted delivery vehicle for cytotoxic metal complexes. Hyperbranched polymers decorated with propargyl groups and hydrophilic OH terminated TEG groups are attached to biotin and a cytotoxic Cu complex. (CuATSM-SS-CONH-N3) through ‘click’ reactions and tested against the HeLa cell line. On the basis of the studies conducted, it is concluded that targeted delivery of cytotoxic metal complexes are possible in the case of Ru(II) half-sandwich complexes and macromolecular vehicles like dendrimers are suitable for specifically delivering copper complexes into cancer cells.

>Magister Scientiae - MSc / Genetic variability in genes encoding drug metabolizing enzymes, transporters and targets are known to be the main factors of inter-individual differences in therapeutic outcome. Genetic factors are estimated to be responsible for about 15-30% of inter-individual variation in drug disposition and response. Single-nucleotide polymorphisms (SNPs) are the most prevalent class of genetic variation that could explain the variability in drug efficacy and undesired side effects for patients. The aims of this study were to develop and evaluate the performance of robust and high throughput techniques for genotyping ten polymorphisms related to anticancer drugs and ten polymorphisms related to cholesterol lowering drugs. SNaPshot minisequencing and high resolution melt analysis (HRM) genotyping panels were developed, optimized, and their performances were evaluated and compared. SNaPshot minisequencing systems were developed and successfully optimized for the genotyping of ten SNPs associated with anticancer drug therapy, and ten SNPs associated with cholesterol lowering drugs. These systems were used to genotype the selected SNPs in 130 healthy Cape Admixed participants residing in Cape Town, South Africa. Population genetics data obtained for the studied SNPs were analysed using several statistical analysis software tools. Important population genetic parameters were calculated. Among others, allelic and genotypic frequencies were determined and compared with other populations in the world. High resolution melt analysis (HRM) genotyping panels were developed, optimized and their performance were evaluated and compared to the SNaPshot assays. HRM was explored as an alternative inexpensive and rapid methodology to genotype five SNPs related to anticancer therapy and five SNPs related to cholesterol lowering therapy (statins). Unlike the SNaPshot assays, rigorous optimization was required for the detection heterozygous genotypes via HRM. Both assays were validated using direct sequencing and compared to each other. The HRM system is a closed tube, cheap and (theoretically) rapid method for identifying genetic variations. HRM was however found to be more time consuming, needed further optimization, primer redesigning and more evaluation. The developed genotyping systems could be further validated using clinical samples from patients. This could help in optimizing drug therapy for cancer and cholesterol treatment.

Taxol (generic name paclitaxel), a plant‐derived antineoplastic agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. Obtaining taxol from this source requires destruction of trees. It has been used alone or in combination with other chemotherapeutic agents for the treatment of breast, ovarian as well as many other types of cancer, including non‐small cell lung carcinoma, prostate, head and neck cancer, and lymphoma, as well as AIDSrelated Kaposi’s sarcoma. The mode of action of taxol against a number of human cancer cells is by preventing the depolymerization of tubulin during cell division. This molecule increases microtubule stability in the cell and induces apoptosis. From yew trees, the yield of taxol is usually between 0.004 to 0.1% of the dry weight. The commercial isolation of 1 Kg of taxol requires about 6 to 7 tons of T. brevifolia bark obtained from 2000‐3000 well‐grown trees. The limited supply of the drug has prompted efforts to find alternative sources of taxol. Alternative methods for taxol production, such as chemical synthesis, tissue and cell cultures of the Taxus species are expensive and give low yields. A fermentation process involving any microorganism would be the most desirable means to lower the cost and increase availability. The first report on the isolation of taxol‐producing fungi from Taxus brevifolia appeared in 1993 (Stierle, et al., 1993). Several taxol‐producing fungi have been identified since, such as Taxomyces andreanae, Taxodium disticum, Tubercularia sp., Pestalotiopsis microspora, Alternaria sp., Fusarium maire and Periconia sp (Li, et al., 1996, Strobel, et al., 1996a, Strobel, et al., 1996b, Li, et al., 1998b, Ji, et al., 2006, Xu, et al., 2006). This thesis investigates the isolation of an endophytic fungus, isolated from the stem cuttings of Taxus celebica, which produces taxol and related taxanes. We observed morphological and cultural characteristics and analyzed the sequences of rDNA ITS from the strain. The isolated fungus grew on potato carrot agar (PCA) medium at 25 °C and the colonies were white to off‐white, floccose, with irregular margins. The reverse side of the culture was cream in color. The morphology was examined microscopically following staining with cotton blue in lactophenol. Cultures produced macroconidia on slender, 85 μm long phialides. The macroconidia were 25‐40 X 3.75 μm. Cultures also produced round or oval microconidia. Analysis of the ITS and D1/D2 26S rDNA sequence revealed 99 % identity with Fusarium solani voucher NJM 0271. Based on its morphological, cultural characteristics and 26S rDNA sequence, the fungus was identified as F. solani. This fungus is different from the previously reported endophytic taxol‐producing species of Fusarium. Taxol and baccatin III, produced by this fungus, were identified by chromatographic and spectroscopic comparison with standard compounds. The amount of taxol produced by F. solani in potato dextrose liquid medium is low (1.6 μg l‐1) (Chakravarthi, et al., 2008). We further investigated different growth media and various factors of cultivation to select the medium and conditions that maximize production of taxol and other taxanes by this fungus. F. solani was grown in five well‐defined culture media under stationary and shake conditions separately for various time intervals and the amounts of taxol, baccatin III and other taxanes produced were estimated by competitive immunoassay. The modified flask basal medium (MFBM) was shown to yield the highest production of taxol (128 μg l‐1) which is 80 times more than when grown in potato dextrose liquid medium, baccatin III (136 μg l‐1) and total taxanes (350 μg l‐1) under shake conditions. From our results the highest taxol production of F. solani was achieved when cultured in MFBM. The production in MFBM was 80 times higher than that cultured in the potato dextrose liquid medium. In conclusion, it was shown that the culture medium plays a major role in taxol and other taxanes production and fungal growth. MFBM is the best medium, among the media studied, to produce taxol and other taxanes. The higher concentrations of NH4NO3, MgSO4, KH2PO4 and FeCl3 in the FBM medium seem important for production of taxol and other taxanes. These results can be considered as starting‐point for the research directed to improve taxol and baccatin III production by F. solani via different approaches including fermentations, strain improvement and genetic engineering techniques. Finally, in order to get more insights into the mode of action of this fungal taxol and baccatin III (for the first time), their apoptotic activity on different cancer cell lines was determined. We elucidated the biochemical pathways leading to apoptotic cell death after fungal taxol‐ and baccatin III‐ treatment in different cancer cell lines. Experiments are done on various cancer cell lines namely JR4 Jurkat (T‐cell leukemia), J16 Bcl‐2 Jurkat T cells, HepG2 (hepatoma), caspase‐8‐deficient Jurkat T cells, HeLa (human cervical carcinoma), Ovcar3 (human ovarian carcinoma) and T47D (human breast carcinoma) cells. We were able to demonstrate that both fungal taxol and baccatin III can induce apoptosis in all the cell lines tested, by flow cytometric analysis. Hallmarks of apoptosis following the signaling pathway to far more upstream‐located events were investigated using biochemical and cell biological methods. It has shown that during fungal taxol‐ and baccatin III‐induced apoptosis, DNA is degraded resulting in a increased number of hypodiploid cells reaching up to 65‐70% after 48 h. Disruption of mitochondrial membrane potential was examined by flow cytometric analysis using mitochondrial membrane potential sensitive dye JC‐1 and JR4‐Jurkat cells were shown to undergo significant loss of mitochondrial membrane potential loss of mitochondrial membrane potential reaching up to 70% in 6 nM fungal taxol and 65 % in 3.5 μM baccatin III after 36 h. These results were similar to those observed with standard taxol and baccatin III. We further investigated the role of caspases in fungal taxol‐ and baccatin III‐induced apoptosis, caspase‐8‐deficient Jurkat cells, Bcl‐2‐over‐expressed J16‐Jurkat cells and caspase inhibitors were used. Results derived from caspase‐8‐deficient Jurkat cells show that caspase‐8 is not involved in fungal taxol‐ and baccatin IIIinduced apoptosis of Jurkat cells. Using the pan‐caspase inhibitor (Z‐VAD‐FMK), caspase‐9 inhibitor (Z‐LEHD‐FMK), caspase‐3‐inhibitor (Z‐DEVD‐FMK), caspase‐2‐ inhibitor (Z‐VDVAD‐FMK) and caspase 10‐inhibitor (Z‐AEVD‐FMK), it was shown that caspase‐10 is involved in fungal taxol‐ and baccatin III‐ induced apoptosis in JR4‐Jurkat cells. It was also shown that inhibitors of caspases‐9, ‐2 or ‐3 partially inhibited fungal taxol‐ and baccatin III‐ induced apoptosis, whereas the caspase‐ 10 inhibitor totally abrogated this process. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were identified in fungal taxol‐ and baccatin III‐treated JR4‐Jurkat and HeLa cells. DNA fragmentations were shown by agarose gel electrophoresis method. Our work showed that treatment of JR4‐ Jurkat and HepG2 cells with fungal taxol and baccatin III induces apoptosis as shown by DNA ladder formation. Herein it was demonstrated that fungal taxol and baccatin III have a similar mechanism of action, but the efficacy of fungal taxol to induce apoptosis is higher. In summary, fungal baccatin III is found to be effective in inducing apoptosis similar to taxol but at higher concentration and both fungal taxol and baccatin III induce apoptosis via caspase‐10 and mitochondrial pathway in Jurkat cells. In conclusion, the present study describes isolation of a taxol‐producing endophyte F. solani IISc.CJB‐1. The growth requirements of this fungus for production of taxol, baccatin III and other taxanes were studied. The apoptotic activity of taxol and baccatin III (for the first time) was observed. In addition, our results show that the culture medium plays a major role in taxol and other taxanes production and fungal growth. Among the media studied, modified flask basal medium (MFBM) is the best to produce taxol and other taxanes. It is evident from this data that this fungal strain can be promising candidate for large‐scale production of taxol and related taxanes.

Sutherlandia frutescens is a South African herbal remedy traditionally used for various ailments and lately to improve the overall health in cancer and HIV/AIDS patients. Relatively little is known about the mechanisms of action of the constituents present in S. frutescens. The aim of this project was to examine the in vitro influence of crude ethanolic S. frutescens extracts in human breast adenocarcinoma (MCF-7) and non-tumorigenic breast epithelial (MCF-12A) cells after 48 h of exposure. Dose-dependent studies were conducted on cell numbers and metabolic activity by means of spectrophotometry. Morphological changes were determined with light-, fluorescent- and transmission electron microscopy (TEM). Cell cycle progression and apoptosis were analyzed using flow cytometry. The differential effects of S. frutescens extracts on gene expression levels in both the MCF-7 and MCF-12A cells were conducted utilizing micro array analysis. mTOR kinase activity was measured with an ELISA assay. S. frutescens reduced cell proliferation in both the non-tumorigenic MCF-12A and the tumorigenic MCF-7 cell line in a dose-dependent manner. The tumorigenic MCF-7 cells were more susceptible to S. frutescens treatment compared to the non-tumorigenic MCF-12A cells. Morphological characteristics of apoptosis and autophagy, including cytoplasmic shrinking, membrane blebbing and an increase in autophagic vacuoles were observed in both cell lines with the MCF-7 cells being more susceptible to autophagy and the MCF-12A cells less susceptible to autophagy and apoptotic cell death. TEM confirmed ultrastructural characteristics of autophagy in both cell lines. Flow cytometry revealed a G2/M arrest with no increase in apoptosis in MCF-7 cells and a G2/M arrest with an increase in apoptosis in MCF-12A cells treated with 1.5mg/ml S. frutescens extract. Microarray analyses revealed 325 statistically significantly differentially expressed genes in MCF-7 cells and 1467 genes in MCF-12A cells. The majority of S. frutescens-treated genes were down-regulated when compared to the vehicle-treated control in both cell lines. Several genes involved in DNA replication and repair were differentially expressed in response to S. frutescens exposure. These include Poly (ADP-ribose) polymerase family, member 2 (PARP-2) (down-regulated in both cell lines), PCNA (down-regulated in MCF-7 cells) and growth arrest and DNA-damage-inducible beta (GADD45B) (up¬regulated in MCF-12A cells). This suggests that abrogated expression of genes involved in DNA replication and repair play a role in inducing a G2/M cell cycle arrest in S. frutescens-treated cells. ELISA analysis of the mTOR kinase revealed a decrease in mTOR kinase activity in both cell lines after S. frutescens exposure. Therefore, attenuated mTOR kinase activity as a result of S. frutescens treatment in both cell lines is regarded as a central mediator in inducing autophagy suppressing gene expression and inhibiting ribosome biogenesis. Understanding of in vitro molecular mechanisms of S. frutescens enables researchers to focus on affected cellular mechanisms and identify active compounds with subsequent evaluation as possible candidates for use in anticancer therapy. The current study contributes to the unraveling of the in vitro molecular mechanisms and signal transduction associated with 70% ethanolic S. frutescens extracts, providing a basis for further research on this multi-purpose medicinal plant in Southern Africa. / Dissertation (MSc)--University of Pretoria, 2007. / Physiology / unrestricted

In the 1960s, Rosenberg and his colleagues confirmed the anti-cancer activity of cisplatin. Although cisplatin was capable of killing testicular cancer cells there were also serious side effects. It was necessary to find alternate ways of overcoming side effects, and soon many researchers have discovered novel platinum compounds that show similar reactivity. Recently, replacing one chloride group to a heterocyclic amine group showed significant cytotoxicity with a different binding activity than cisplatin. Previously in our lab, [Pt(Me5dien)(NO3)]+ and [Pt(Et2dien)Cl]+ have been synthesized and reacted with NAcetyl- L-methionine (N-AcMet) and Guanosine 5’-monophosphate (5’-GMP) showed unusual reactivity. Unlike most previously studied platinum triamine compounds, Me5dien compound was reacting faster with 5’-GMP than N-AcMet, due to the bulkiness of the triamine ligand. When both N-AcMet and 5’-GMP were reacted with Et2dien, 5’- GMP displaced one amine group of the triamine ligand and replaced that spot to form a bis-adducts, when the pH was kept below 4. Here a new novel platinum compound has been synthesized with a seven-membered chelate ring triamine ligand, Chloro[2-(4- methyl-1,4-diazepan-1-yl)ethanamine]platinum(II) chloride ([Pt(L)Cl]+). The unusual binding activity of [Pt(L)Cl]+ showed a unique pair of products under 1H NMR, 195Pt NMR and LC/MS spectrometry.

>Magister Scientiae - MSc / Nature through billions of years of trial and error has produced an immeasurable amount of natural systems like plants, birds and animals. The intelligence of nature is hidden in these natural systems and researchers are turning towards “Nature’s intelligence” to find inspiration and advance novelty in the development of nanomaterials. Gold nanoparticles (AuNPs) have unique optical, electronic and physicochemical features which has gained them popularity and widespread exploitation in various applications. The conventional methods used for AuNPs synthesis employs toxic chemicals which makes these NPs unsafe for biomedical applications. Hence, there is a search for new, ‘green’ and more cost effective methods for AuNPs synthesis. Plant extracts are regarded as a highly desirable system for nanoparticle synthesis due to their tremendous capability to produce a wide range of phytochemicals that can act as reducing agents. The main goal of this study was to synthesize AuNPs in a cost effective manner without the use of toxic chemicals in the synthesis process. Avocado seeds which are an agricultural waste by-product were used for the biosynthesis of AuNPs. The study reports on the synthesis optimization, characterization and activities of the biogenic AuNPs. The avocado seed extract mediated - AuNPs (AvoSE-AuNPs) were optimized by varying reaction parameters and characterized by UV-visible, Dynamic Light Scattering (DLS) and High Resolution Transmission Electron Microscopy (HRTEM), Zetasizer and Fourier Transform Infrared Spectroscopy (FTIR). The formation of AvoSE-AuNPs had an absorption maximum at 534 nm. HRTEM and DLS confirmed that the NPs were polydispersed and present in different shapes. The presence of phytochemical constituents on the AvoSE-AuNPs were confirmed by FTIR. Their potential antibacterial activity was tested on bacterial strains known to exhibit resistance to a number of current antibiotics. The catalytic activity of AvoSE-AuNPs was also assessed as a means to contribute to the development of new methods aimed at alleviating organic pollutants such as nitrophenols in the environment. The AvoSE-AuNPs demonstrated excellent catalytic activity in the reduction of 4-NP by NaBH4 as shown by the rapid decrease in the nitrophenolate absorption band at 400 nm and the appearance of new absorption band at 298 nm, revealing the formation of the 4-aminophenol. Furthermore, the rate constants calculated demonstrated that the reaction occurs faster in the presence AvoSEAuNPs. The AvoSE-AuNPs showed low significant cytotoxicity. Cell cycle analysis was conducted to further investigate the apparent exhibited toxicity of the AvoSE-AuNPs. The results showed that in both cell lines treated with AvoSE-AuNPs and AvoSE there was a ii | P a g e disruption in the regulation of cell cycle. Cell cycle analysis helped improve understanding of the low cytotoxicity observed by the MTT assay results. The results presented in this study clearly demonstrate the feasibility of using AvoSE for the synthesis of AuNPs. This study demonstrated that AvoSE mediated AuNPs synthesis is a greener alternative as it abides by the green chemistry principles. Furthermore, the study outcomes contributed to minimizing environmental pollution by finding use for agricultural waste and thus ultimately adding value to the field.

Platinum-based anticancer drugs, such as cisplatin, carboplatin, and oxaliplatin, have been approved for clinical use worldwide for decades. Despite their enormous success, their widespread application is hindered by either cross-resistance or toxic side effects, including nephrotoxicity and neurotoxicity. The need to overcome these drawbacks has stimulated the search for new platinum-based drugs. This dissertation will start with the accidental discovery of cisplatin, followed by an introduction of other platinum-based anticancer agents, including the action mechanism, general structures, and development history. Picoplatin is a newer generation of platinum-based anticancer agent. The bulky 2-methylpyridine as a non-leaving group on picoplatin could reduce the detoxification effect caused by thiol-containing species, such as glutathione and metallothionein, thus may grant picoplatin the ability to overcome cisplatin resistance. A convenient synthesis route for picoplatin derivatives has been developed. 11 new picoplatin derivatives have been designed by varying the bulkiness of the non-leaving amine group. All complexes have been characterized by different instrumentations, including MS, 1H NMR, 13C NMR, 195Pt NMR, HMQC, X-ray crystallography, and elemental analysis. Different bioassays, such as DNA binding, cell viability, and cellular accumulation, have been applied to evaluate their efficacy on cisplatin-sensitive ovarian cancer cell line A2780 and cisplatin-resistant ovarian cancer cell line A2780cis. The newly designed picoplatin derivatives show comparable efficacy with that of picoplatin and less resistance compared with cisplatin. The study of picoplatin derivatives laid the foundation toward the research of bifunctional platinum-based anticancer agents by incorporating histone deacetylase (HDAC) inhibition. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are a pair of important enzymes in epigenetic regulation. They work in harmony to acetylate and deacetylate histone lysine residues, resulting in a more relaxed or more condensed chromatin structure, respectively. HDAC has been found to be overexpressed in some cancer cells. Since 2006, 5 HDAC inhibitors (HDACi) have entered clinical use for cancer treatment. 19 new HDACi with additional coordination sites on the phenyl cap have been designed, synthesized, and evaluated. A few of the new HDACi show comparable or even better HDAC inhibition than that of Vorinostat (SAHA, the first FDA approved HDACi). A logical design would involve the installation of HDACi on the platinum center as a non-leaving group ligand. When the bifunctional drug reaches the cancer cell, the synergistic effect could be maintained as the relaxed chromatin structure makes DNA more susceptible to be attacked by the platinum centers, thus increase the anticancer activity and possibly selectivity toward cancer cells. 6 Pt-HADCi conjugates have been designed and synthesized. Dual functions of the new Pt-HDACi have been confirmed by DNA electrophoresis assay and HDAC inhibition assay. One of the Pt-HDACi (CF-101) shows comparable cytotoxicity with cisplatin and less resistance, which could be used as the lead compound for further structural modification and in vivo studies.

Currently available anticancer therapies are inadequate and spur demand for improved technologies. Among others, the utilization of nanocarriers for anticancer drug delivery has shown great potential in cancer treatment. Nanocarriers can improve the therapeutic efficiency of the drugs with minimization of the undesirable side effects. To evaluate potential application of this technology, two forms of nanocarriers have been studied: multi-walled carbon nanotubes (MWCNTs) and apoferritin. The aim of this study was to determine, whether given cytostatics (ellipticine, etoposide and doxorubicin) are bound to these nanotransporters and how are they released from them, especially depending on pH. Since the pH of the tumor cells is lower than the pH of healthy cells it would be preferred that the drugs would release from nanocarriers at the lower pH while at the physiological pH the release of the drug would be eliminated. The results found show that ellipticine is actually released from its MWCNT- and apoferrtin-encapsulated form at acidic pH (5.0), while at pH 7.4 its interaction with nanocarriers is stable. Ellipticine released from MWCNT is activated by microsomal enzymes to reactive metabolites (13- hydroxyellipticine and 12-hydroxyellipticine) forming DNA adducts. The results indicate that both...