Dissertations / Theses on the topic 'Anticancer treatment'

Pharmacogenetics (PGx) aims at the definition of predictive and prognostic genetic biomarkers that can help clinicians in treatment tailoring. In this thesis, we explored two emerging topics in PGx: SNPs affecting the activity and maturation of a class of non coding RNAs, microRNAs (miRNAs), and immunogenetics. Genetic analyses were performed by a medium throughput technology, Veracode technology (Illumina). The clinical model of this study is represented by the locally advanced rectal cancer (LARC). Specifically, the main two aims of this study were: 1. the identification of predictive biomarkers of pathological response to neoadjuvant treatment in LARC patients, defined in terms of tumour regression grade (TRG); 2. the identification of biomarkers of disease free survival (DFS) and overall survival (OS) in LARC patients. To find an answer to these questions, we analyzed two panels of SNPs, selected according to bioinformatic analysis and literature data, on a group of 280 LARC patients homogeneously treated with fluoropyrimidines-based chemoradiotherapy in neo-adjuvant setting. In the first part of this project, we analyzed a panel of 144 SNPs potentially involved in miRNA maturation and activity. With a quite complex statistical strategy, we identified 5 new predictive biomarkers of response to neoadjuvant treatment. Specifically, DROSHA-rs10719 and SMAD3-rs17228212 were unfavourable predictive biomarkers (p=0.0274, p=0.0049), while SMAD3-rs744910, SMAD3-rs745103, and TRBP-rs6088619 showed an opposite effect (p=0.0153, p=0.0471, p=0.0125). Moreover, in patients with complete pathological response (TRG1), SMAD3-rs745103 was significantly associated with DFS (p=0.011). This study underlines the potential key role of SMAD3, factor involved not only in miRNA maturation but also in inflammation and in particular in TGFβ pathway, that is crucial in cancer progression and treatment response. Bearing in mind this interesting results and the huge amount of literature data addressing the high potentiality of immunogenetics in oncology, we analyzed a panel of 192 SNPs in genes involved in immune response in the same group of 280 LARC patients. We investigated another clinical end-point, the 2-year disease-free survival (2yDFS), because it is a strong prognostic biomarker of OS. Firstly, we identified 4 SNPs significantly associated with the 2yDFS. Two of them are located in IL17F (rs641701: OR=5.84, 95% CI=1.52-22.45, p=0.010; rs9463772: OR=3.56, 95% CI=1.22-10.35, p=0.020) and the other ones in STAT3 (rs8069645: OR=0.36, 95% CI=0.13-0.99, p=0.048; rs9867701: OR=3.00, 95% CI=1.09-8.30, p=0.034). Secondly, we studied the potential association of these 4 SNPs with the 10 years overall survival (OS). Interestingly, 3 SNPs remained significant and two of them are located in IL17F gene (IL17F-rs641701: OR=3.23, 95% CI=1.50-6.95, p=0.003; IL17F-rs9463772 OR=2.89, 95% CI=1.49-5.61, p=0.002, and STAT3-rs8069645 OR=0.50, 95% CI=0.25-0.98, p=0.044). We tested these associations in a validation group of 63 LARC patients who underwent radical surgery and adjuvant treatment based on fluoropyrimidines. Surprisingly, IL17F-rs9463772 is still significantly associated with OS (p=0.045), thus we can conclude that this is really a strong prognostic biomarker. To conclude, we performed 2 different PGx projects that led us to identify different predictive and prognostic biomarkers in LARC patients. These data, if confirmed in larger studies, will help clinicians to personalize patients treatment and management.

Modifications of the potent anticancer agent taxol were carried out in order to gain an understanding of the chemical reactivity of the drug and the factors which contribute to its biological activity. The C-2' and/or the C-7 hydroxyl groups of taxol were substituted with acetyl, ßalanyl, silyl, succinyl, trichloroethyloxycarbonyl or carbonate linked dibenzylidene protected glucosyl groups. The C-7 position was selectively epimerized under free radical conditions and a 2'-epiacetyltaxol was produced via base catalysed epimerization. The C-2' amide became nucleophilic in the presence of base and could attack a C-2; acyl substituent. The C-13 ester side chain was selectively reduced by borohydride. The 7 position of taxol was selectively oxidized by Jones reagent and longer reaction times also oxidized the 2' position. The D rings of the 7 oxotaxols were readily opened via beta elimination; hydrogenation of the double bond in the enone of the D seco products produced a product in which the C ring was opened. The D ring was also susceptible to electrophilic attack. Reaction of taxol with triethyloxonium tetrafluoroborate or acetyl chloride/HC1 produced D seco taxols. C-7 deoxygenation was not achieved due to steric hindrance at C-7 and the instability of taxol under free radical conditions. Biological testing of modified taxols showed that substitution of the C-2' hydroxyl removed biological activity but that C-2' acyl groups were readily removed vivo. The water soluble 2'-βalanyItaxol possessed in vivo activity equal to that of taxol. Substitution of the C-7 hydroxyl did not inhibit the ability of a taxol derivative to polymerize tubulin but did decrease in vivo activity; epimerization of C-7 decreased in vivo activity slightly. A 2'-oxotaxol was found to be less active than, but still comparable to, its nonoxidized analogue. All taxol derivatives having a 7-oxo group and/or not possessing a D ring lost almost all biological activity.
Ph. D.

The mechanism of resistance of tumor cells to chemotherapeutic agents is explored using probabilistic methods where it is assumed that resistant cells arise spontaneously with a defined frequency. The resistance process is embedded in a discrete time Markov branching process which models the growth of the tumor and contains three seperate cell types: stem, transitional and end cells. Using the asymptotic properties of such models it is shown that the proportion of each type of cell converge to constants almost surely. It is shown that the parameters relating to stem cell behaviour determine the asymptotic behaviour of the system. It is argued that for biologically likely parameter values, cure of the tumor will occur if, and only if, all stem cells are eliminated. A model is developed for the acquisition of resistance by stem cells to a single drug. Probability generating functions are derived which describe the behaviour of the process after an arbitrary sequence of drug treatments. The probability of cure, defined as the probability of ultimate extinction of the stem cell compartment, is characterised as the central quantity reflecting the success of therapeutic intervention. Expressions for this function are derived for a number of experimental situations. The effects of variation in the parameter values are examined. The model is extended to the case where two anticancer drugs are available and formulae for the probability of cure are developed. The problem of therapeutic scheduling is examined and under situations where drugs are of "equal" effectiveness, but may not be given together, it is shown that the mean number of tumor cells is minimised by sequential alternation of the drugs. The models are applied to data collected on the L1210 leukemia treated by the drugs Cyclophosphamide and Arabinosylcytosine. In both cases the analysis of the data provide evidence that resistant cells arise spontaneously with a frequency of approximately 10⁻⁷ per division. When applied to human breast cancer, the model indicates that neoadjuvant therapy is unlikely to greatly influence the likelihood that the patient will die from the growth of drug-resistant cells.
Science, Faculty of
Statistics, Department of
Graduate

This dissertation is an overview on the functionalization of known anticancer compounds in order to form different drug-conjugates able to self-assemble in water to form nanoparticles. This approach is useful to improve the drug delivery properties and pharmacokinetic profile of anticancer drugs. All the described conjugates, except for the ones illustrated in chapter 5, have the same general structure: the anticancer drug is connected to the self-assembly inducer trough a linker. Chapter 1 regards a general introduction about nanomedicine, the advantages of the use of nanotechnology-based systems in cancer treatment and the benefits of nano- formulated drugs in the improvement of drug-delivery. Furthermore, nanoparticles are presented with a focus on their classification, characterization and preparation techniques. Chapter 2 regards the preparation of different types of self-assembled nanoparticles using various anticancer compounds and dyes but with the same lipophilic tail as self- assembling inducer: squalene. Different natural anticancer compounds such as paclitaxel, cyclopamine and doxorubicin and dyes, as fluorescein and tetramethylrhodamine, were functionalized to obtain squalene-based conjugates. Both hetero-nanoparticles composed by two drug-conjugates and drug- and dye-conjugates were prepared and tested. Chapter 3 is focused on the importance of the self-assembly inducer and describes the preparation of new conjugates containing an active moiety as self-assembly inducer. In particular, in this section, is described the preparation of conjugates composed by aloin or podophyllotoxin as active compounds and 4-(1,2-diphenylbut-1-en-1-yl) aniline, an analog of the know anticancer compound tamoxifen, as self-assembly inducer. Chapter 4 highlights the influence of the linker between the active compound and the self-assembly inducer to obtain an effective release of the free drug. In particular, it is described the synthesis of a new self-immolative linker able to trigger the drug release in particular conditions, specifically in the presence of a lipase. This linker was used for the preparation of two conjugates containing the known anticancer compound N- desacetylthiocolchicine. Chapter 5 concerns the preparation of dual drug-conjugates able to form nanoparticles without the presence of a self-assembly inducer. These conjugates have a symmetrical structure: two molecules of the same drug are linked by a chain able to guarantee the drug release in particular conditions. The natural anticancer compounds involved in the preparation of this type of conjugates are paclitaxel, epothilone A, podophyllotoxin and camptothecin and the linker used contain a disulfide moiety able to be cleaved in cellular environment.

Biological screening of extracts of various bryophytes showed that the species Dicranum fulvum gave extracts with activity in both in vitro and in vivo bioassays. This plant was thus selected for extraction and fractionation, monitored by iin vitro bioassays.

Isolation was guided by a combination of bioassay and chemical methods, and led to the isolation of three compounds, betulin, 9,l9- cyclolanostâ 23â eneâ 3,25â diol, and B-sitosterol. Purification was achieved by open column, flash column, gel filtration, thin layer chromatography, the chromatotron and crystallization.

The isolated compounds were identified by comparisons of spectroscopic data with those of authentic samples and the matching of experimental and literature melting points and optical rotations.

Master of Science

Nowadays, the search for new drugs against cancer is one of the major goals to improve the quality of life of patients. The development of more selective treatments against cancer cells may lead to a significant reduction of the side-effects, being one of the most important topics in current research. In this regard, cell-penetrating peptides (CPPs) have been described to efficiently transport therapeutic molecules across the cell membrane. Furthermore, some metal complexes based on platinum (cisplatin and derivatives) are used in current chemotherapy. Despite their efficacy, these drugs display high toxicity. Thus, the design of novel complexes based on non-toxic metals (iron or manganese) is currently under way. These complexes could induce an irreversible oxidative stress through reactive oxygen species (ROS) at the subcellular level. Consequently, this might result in the alteration of the redox homeostasis and in cell death via apoptosis.The main objective of this thesis is the transport of iron or manganese complexes based on aminopyridine ligands into cancer cells through their conjugation to a non-toxic cell-penetrating peptide. Towards this aim, in one hand an undecapeptide (BP16) has been identified as a novel cell-penetrating peptide. On the other hand, it has been developed a straightforward synthetic methodology to conjugate the tetradentate aminopyridine ligands Me2PyTACN and (S,S’)-BPBP to peptide derivatives. Using this methodology, conjugates incorporating the aforementioned ligands and a tetrapeptide sequence have been prepared. These conjugates have been metallated with iron, manganese and other metals, and the resulting metallopeptides have been characterized and studied as DNA nucleases. Next, following the previous synthetic methodology, the Me2PyTACN and (S,S’)-BPBP ligands have been conjugated to the cell-penetrating peptide BP16 with the aim of being efficiently internalized into cancer cells. The resulting conjugates exhibit high cytotoxic activity similar to that observed by well-known anticancer drugs. Besides, BP16 displays high efficient drug delivery properties since it is able to enhance the uptake of the anticancer agent chlorambucil, improving its cytotoxicity against cancer cells between 6- to 10-fold. These high cytotoxic activities correlated with the high cellular uptake observed by the resulting conjugates, which it has been attributed to BP16. Based on the sum of these results, we have shown that BP16 is able to significantly enhance the cellular uptake of redox-active complexes and other well-known drugs. In brief, this study establishes that the synergy between the cytotoxic activity provided by metal complexes and their efficient transport into the cells could be useful to develop more selective therapies as well as novel anticancer treatments.
La recerca de nous fàrmacs per combatre el càncer representa un factor clau per millorar la qualitat de vida dels pacients. El desenvolupament de tractaments més selectius per les cèl•lules canceroses pot donar lloc a una reducció significativa dels efectes secundaris, essent aquest el tema principal de molts projectes de recerca. En aquest sentit, s’han descrit pèptids capaços de transportar els fàrmacs allà on es requereix que actuïn. Aquests pèptids es coneixen com a cell-penetrating peptides (CPPs). D’altra banda, en quimioteràpia s’utilitzen complexos metàl•lics basats en metalls com el platí (cisplatí i derivats). Tot i ser efectius, aquests fàrmacs presenten una elevada toxicitat, per la qual cosa s’està estudiant el disseny de complexos de metalls no tòxics, com el ferro o el manganès. Aquests complexos metàl•lics poden induir un estrès oxidatiu irreversible a nivell cel•lular gràcies a l’alta producció d’espècies reactives d’oxigen. En conseqüència, la pròpia alteració de l’homeòstasi redox de la cèl•lula pot causar la seva mort cel•lular per apoptosi.L’objectiu principal d’aquesta tesi doctoral és el transport de complexos de ferro o manganès basats en lligands de tipus aminopiridina dins les cèl•lules canceroses mitjançant la seva conjugació a un cell-penetrating peptide. Per assolir aquest objectiu, en primer lloc, s’ha identificat el pèptid d’onze aminoàcids BP16 com a cell-penetrating peptide. En segon lloc, s’ha desenvolupat una metodologia sintètica per a conjugar els lligands tetradentats de tipus aminopiridina Me2PyTACN i (S,S’)-BPBP a un derivat peptídic. Mitjançant aquesta metodologia s’han preparat conjugats incorporant aquests lligands i una seqüència de quatre aminoàcids. Aquests conjugats s’han metal•lat amb ferro, manganès i altres metalls, i els metal•lopèptids obtinguts s’han caracteritzat i estudiat com a nucleases de DNA. A continuació, seguint la metodologia sintètica anterior, els lligands Me2PyTACN i (S,S’)-BPBP s’han conjugat al cell-penetrating peptide BP16 amb l’objectiu de que siguin transportats dins les cèl•lules canceroses. Els conjugats resultants presenten activitat citotòxica elevada, comparable a la de fàrmacs anticancerígens utilitzats avui en dia. A més, paral•lelament, també s’ha comprovat que el pèptid BP16 és capaç de transportar de manera efectiva el fàrmac anticancerigen clorambucil, incrementant entre 6 i 10 vegades la seva activitat citotòxica enfront les cèl•lules canceroses. Aquestes altes activitats citotòxiques estan directament correlacionades amb una major internalització cel•lular dels conjugats resultants, proporcionada pel pèptid BP16. Així doncs, el pèptid BP16 permet millorar significativament la internalització cel•lular de complexos redox actius i d’altres fàrmacs. Amb caràcter general, aquest estudi estableix que la sinèrgia entre l’activitat citotòxica de complexos anticancerígens i el seu transport eficient a nivell cel•lular pot ser útil per desenvolupar teràpies més selectives i nous tractaments contra el càncer.

On the basis of clinical studies, some researchers have advocated that the neurohormone and antioxidant melatonin, shown to possess intrinsic anticancer properties, be used as co-therapy in cancer patients being treated with the antineoplastic agent 5-fluorouracil, as increased patient survival times and enhanced quality of life have been observed. The focus of this research was thus to investigate the mechanisms of this seemingly beneficial drug interaction between 5-fluorouracil and melatonin. Metabolism studies were undertaken, in which it was established that there is no hepatic metabolic drug interaction between these agents by cytochrome P450, and that neither agent alters the activity of this enzyme system. Co-therapy with melatonin is thus unlikely to alter plasma levels of 5-fluorouracil by this mechanism. Novel mechanisms by which 5-fluorouracil is toxic were elucidated, such as the induction of lipid peroxidation, due to the formation of reactive oxygen species; decreases in brain serotonin, dopamine and norepinephrine levels, possibly leading to depression; hippocampal shrinkage and morphological alterations and lysis of hippocampal cells, which may underlie cognitive impairment; and a reduction in the nociceptive threshold when administered acutely. All these deleterious effects are attenuated by the co-administration of melatonin, suggesting that the agent exhibits antidepressive and analgesic properties, in addition to its known antioxidative and free radical-scavenging abilities. This suggests that melatonin cotherapy can significantly decrease 5-fluorouracil-induced toxicity, but this may also exert a protective effect on cancer cells and thus compromise the anticancer efficacy of 5-fluorouracil. It was, furthermore, found that stimulation of indoleamine 2,3-dioxygenase activity, mediated by increases in superoxide anion and interferon-γ levels, may underlie resistance to 5-fluorouracil therapy. Melatonin was shown to increase superoxide anion levels in vivo, and this is believed to be by conversion to the metabolite and known oxidant 6- hydroxymelatonin. This highlights that the possible deleterious effects of melatonin metabolites should be studied further. Serum corticosterone levels and cytokine profiles are unaltered by both 5-FU and melatonin, suggesting that these agents may be used by HIV infected individuals without promoting the progression to AIDS. It can thus be concluded that melatonin co-therapy is potentially useful in countering 5-fluorouracil toxicity.

Neoplastic diseases hold the second place of the most common causes of death worldwide. Available treatments include various combinations of surgery, chemotherapy, radiation, hormone therapy, immune therapy and targeted therapy. The emphasis is currently laid on nanomedicine, where new nanosized complexes are developed and applied for the targeted treatment and chemotherapy. The aim is to significantly improve the anticancer effect and decrease the damage to organism. In this thesis, ruthenium nanoparticles with a size of 12–14 nm were synthesized and their surface modified with polyvinylpyrrolidone. Furthermore these were subsequently modified with polyoxyethylene(40)stearate for binding of doxorubicin. These nanoparticles were tested on breast carcinoma cells (MDA-MB-231), ovarian carcinoma cells (A2780) and neuroblastoma cells (UKF-NB-4). Apoptosis and necrosis testing showed 60—64 % increase in apoptosis when comparing ruthenium nanoparticles modified with doxorubicin to nonmodified ruthenium nanoparticles. The modification increased level of oxidative stress in tumorous cells and slightly a genotoxicity to non-tumorous cells, nevertheless the hemocompatibility was significantly improved. Testing has proven with IC50 0.98 g/ml, 3.91 g/ml and 1.95 g/ml higher sensitivity to these cells and confirmed expected anticancer activity. Compared to one of the most common chemotherapeutic agents cisplatin the modified ruthenium nanoparticles are significantly more toxic to cell lines A2780 (IC50=21 µg/ml), MDA-MB-231 (IC50=9 µg/ml) and UKF-NB-4 (IC50=4 µg/ml).

In the last decades colloidal decorated gold nanoparticles (GNPs) have been studied as platform for drug and gene delivery, for diagnostic and other biomedical applications. These metal nanoparticles are intriguing because of their unique physico-chemical properties that can be exploited for multimodal and combined treatment of cancer. In the present thesis work gold nanoparticles were decorated with a targeting ligand (Folate-PEG) to combine an active and a passive targeting aiming to enhance the selective accumulation within the tumour site. Deep studies have been done to investigate the effect of surface Folate density on the internalization efficiency of gold nanoparticles. Afterwards intracellular trafficking studies were performed to clarify the uptake mechanism and investigate lysosomal delivery. Confocal microscopy and TEM analysis showed in good agreement that Folate targeted gold nanoparticles are internalized via a clathrin-independent pathway. Another purpose of the project have concerned the exploitation of GNPs as sensitizers in the sonodynamic therapy. This is a non-invasive approach which consists in cancer tissue irradiation with focused ultrasounds (HIFU) to trigger cavitation phenomena leading to irreversible destruction of the target tissue. The combination of the ultrasound exposure and the pre-incubation of cells with Folate targeted particles induced a significant and selective cell death. The concept of multimodal targeting was extended to the development of pH responsive targeted gold nanoparticles, using a pH sensitive polymer able to respond with morphological alterations to environmental pH changes. The cell uptake results confirmed that the “hiding” and “reveal” of targeting agents on GNP surface is modulated by the sensitive polymer. As a result there is an enhanced site-selective GNP accumulation in the cancer tissue, according to a cooperative exploitation of phenotypic and environmental features of the tumour. In conclusion, the present thesis work is proposed as proof-of-concept to show that by finely tuning the surface properties of nanosystems, site-selectivity can be significantly enhanced, thus reducing the disposition of drug nanocarriers in off-target tissues.
Lo scopo del presente progetto di dottorato è stato quello di produrre e caratterizzare dal punto di vista chimico-fisico e biologico un nanocarrier per il direzionamento selettivo di farmaci antitumorali a tumori sovraesprimenti il recettore per l’acido folico. Sono stati compiuti studi approfonditi per verificare come la densità dell’agente di targeting influenzasse l’efficienza d’internalizzazione del sistema. Inoltre studi di trafficking intracellulare hanno verificato come particelle d’oro direzionate con agente di targeting Folato-PEG vengano internalizzate mediante meccanismo clatrina-indipendente. Si è inoltre indagata la capacità di nanoparticelle d’oro come sensibilizzanti alla terapia sonodinamica al fine di poter combinare un trattamento farmacologico ad un approccio fisico. Un ulteriore sviluppo del progetto ha riguardato la modifica di nanoparticelle d’oro direzionate con Folato-PEG con una seconda componente pH responsiva in grado di passare da una conformazione estesa a pH fisiologico di 7.4 ad una forma idrofobica globulare a pH 6.5, condizione tipica del tessuto tumorale. In questo modo é possibile modulare il mascheramento/esposizione dell’agente di targeting e ridurre il bio-riconoscimento aspecifico a favore della sito-specificità. Tra gli sviluppi futuri del progetto, vi è la decorazione di nanoparticelle d’oro con un polimero dotato di gruppi idrazinici coniugati a Doxorubicina mediante legame idrazonico. In virtù delle proprietà del legame idrazonico, la Doxorubicina sarà rilasciata esclusivamente nei comparti endosomiali e lisosomiali, in seguito all'uptake cellulare mediato dal recettore FR per l’acido folico.

The occurrence of pharmaceuticals in wastewater and the wider environment is of growing concern. This thesis focuses on anticancer drugs - a group of biologicallypotent and often recalcitrant set of chemicals whose fate and impact on the wider freshwater environment is poorly studied. The aims of this thesis were to prioritise a group of anticancer drugs for environmental monitoring programmes (from the many drugs in use), based on their consumption and fate during wastewater treatment; to undertake a national and regional survey of two commonly used anticancer drugs, cyclophosphamide (CP) and ifosfamide (IF) in wastewater and river water; to assess the performance of a river-based chemical fate model through comparisons with field observations; and to conduct a mass balance for CP in wastewater treatment plants to assess chemical fate during the different stages of wastewater treatment. Given the large number of anticancer drugs currently in use (>70) a decision support process was developed to ascertain a short list of drugs which are most likely to persist and be released with treated effluent to environmental waters. To do this, accurate consumption data were compiled from a hospital survey in NW England and combined with urinary excretion rates derived from clinical studies. Physical– chemical property data were then compiled along with likely chemical fate and persistence during and after wastewater treatment. A shortlist of 15 chemicals (from 65), including CP and IF, was prioritised based on their consumption, persistency and likelihood of occurrence in surface waters and supported by observational studies where possible. The ecological impact of these ‘prioritised’ chemicals however is uncertain as the measured concentrations in surface waters generally fall below standard toxicity thresholds, although there is evidence that exposure of aquatic organisms to some of these chemicals may induce low-dose genotoxic effects. This prioritised sub-list of anticancer drugs should prove useful for developing environmental screening programmes and targeted toxicity assays. To assess the occurrence of anticancer drugs in wasterwaters both CP and IF were measured in raw influent and final effluent waters from fourteen STPs located across England using a sensitive analytical method. CP was detected in both wastewater influent and effluent with mean (SD) concentration of 4.1 ng/L (4.8) and 6.6 ng/L (6.5), respectively, in agreement to measured ranges from a limited number of studies conducted in Europe and elsewhere. IF was only detected in four wastewater samples with the highest concentration being observed in wastewater effluent at 0.77 ng/L (cv = 24.3% (n=3)) and possibly reflecting the relatively lower consumption of this drug relative to CP. Additional monitoring was conducted in the rivers Calder, Darwen and Ribble (North West UK) with CP present at 5 of the 6 river locations with concentrations ranging from 0.41 to 3.71 ng/L. All these rivers receive treated wastewater effluent from sewage treatment works serving different population sizes, with CP measured in river water some ~20 miles downstream of the nearest STP, indicating the widespread dispersal and persistence of this chemical. CP and IF were measured systematically down the Rivers Aire and Calder in NE England and the results compared to a GIS-based water quality model (LF2000- WQX) used to predict CP and IF distributions in the two rivers, using regional consumption data and subsequent release quantities from STPs. CP was detected in 90% of river samples, apart from rural/uplands sites located at the source of the River Aire and Calder, respectively. CP presented the highest concentration, ranging from 0.17 to 4.53 ng/L (average 1.14 ng/L). IF was seldom detected in the sampled sites and concentrations ranged from < LOD to 1.82 ng/L (average 0.51 ng/L). Model results showed a fair agreement to the measured data for CP in the River Aire, discrepancies arise as the river progressed further downstream where the modelled data was lower than the measured data. A significant input of CP from Leeds STP at A7 (STP-1) saw the continuing rise in CP despite the increase in river flow. At the lower end of the Calder (pre-confluence with the River Aire) a spike in CP is detected far beyond the modelled value. A risk assessment was carried out to establish the potential adverse effects of anticancer drugs in the river catchment. All calculated risk quotients were below 1, showing no significant risk to aquatic organisms. However, long term toxicity studies for these chemicals are needed to define the environmental stress produced by their continuous exposure and induction. The fate and removal efficiency of cyclophosphamide (CP) and ifosfamide (IF) were investigated in two conventional sewage treatment plants (STP-S and STP-C) during different stages of waste water treatment. Overall average concentrations of CP were 1.17±1 ng/L in the two plants, which is lower than recent measurements conducted elsewhere. Grab-samples were coordinated with the hydraulic residence time of wastewater in each of the treatment stages in order to monitor changes in CP concentrations in the same parcel of water as it passed through the STP. Interestingly, concentrations of CP were observed to increase from raw influent to final tertiarytreated effluent and this is likely to be attributable to the degradation of a CPmetabolite and subsequent ‘liberation’ of the parent CP as the metabolite passes through the various sewage treatment processes. This observation, apparent in both studied STPs, has implications for chemical fate modelling of anti-cancer drugs, especially if STP influent loads are used to predict subsequent fluxes to receiving waters rather than final effluent values. Moreover, this increase in concentrations made a mass balance difficult to achieve, but highlighted that elimination/removal of CP in wastewater during primary to tertiary processing is very low (<20%). The calculated fluxes of CP with final effluent discharge were 3.16- 6.48 g/year for STP-S and 4.56 -51.57 g/year for STP-C and highlight that STPs are a continuing source of highly water-soluble, recalcitrant anticancer drugs to the environment.

Thesis (PhD)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The "classic" prostate cell lines, DU145, PC-3 and LNCaP, have served as a valuable cell biological model for research into prostate cancer. However, their relevance may be limited because they derive from metastatic, and not from primary normal and tumour epithelium. The cell lines (1532T, 1535T, 1542T, 1542N and BPH-l) have been derived from primary benign and malignant human tumour prostate epithelium and may be more representative. Using these cell lines I have examined the role of basic cell damage responses (repair, checkpoint activation, apoptosis and associated signalling proteins, and the influence of androgen status) in cell inactivation, and its relevance to treatment. Numerous studies have suggested that loss of p53 function leads to resistance to chemotherapeutic agents and irradiation. It is shown here that the p53-inactive cell lines are, in fact, the most sensitive to chemotherapeutic agents such as etoposide, vinblastine and estramustine, whilst the p53 wild-type cell line, LNCaP, is the most radiosensitive. Notwithstanding the effects of p53 degradation by the HPV -16 E6 viral protein, the results on chemosensitivity raises the possibility that different chemotherapeutic agents may have different p53-dependent effects in different tumour cells. Androgen deprivation is demonstrated to sensitise prostate cancer cells to chemotherapeutic agents and it is shown that the hormone independent cell lines are the most chemosensitive. The LNCaP cell line displayed an increased resistance to apoptosis induced by etoposide and gamma irradiation, suggesting that androgens are capable of protection against both these DNA damaging agents. The major factors determining radiosensitivity in human tumour cell lines are known to be DNA double-strand break (dsb) induction and repair. In the prostate cell lines I find that cellular radiosensitivity correlates with the number of DNA double-strand breaks measured within 2 hours of irradiation, and that the more radioresistant cell lines show better repair competence. Conclusions as to the influence of androgen dependence on radiosensitivity and repair are not possible at this stage since only the LNCaP cell line was androgen sensitive. The fact that the 2 hour repair period can separate radiosensitive from radioresistant cells in 2 groups of human tumour cell lines highlights the role of non-homologous end-joining repair. This has implications for therapy, and is consistent with the clinical observation that prostate tumours can be successfully controlled by low dose rate-brachytherapy. To evaluate the role of apoptosis, cells were exposed to TD50 concentrations of chemotherapeutic drugs, and 60Co y-irradiation. Apoptosis was found to be low, overall, and ranged from 0.1% - 12.1%,3.0% - 6.0% and 0.1% - 8.5% for etoposide, estramustine and vinblastine, respectively. The percentage of cells undergoing druginduced apoptosis was, on average, higher in the tumour cell lines than in the normal cell lines. Gamma irradiation-induced apoptosis levels ranged from 1.3% - 7%. The LNCaP cell line yielded the lowest percentage of apoptotic cells after exposure. The l532T cell line yielded the highest percentage of apoptotic cells after exposure. Apoptotic propensity did not rank the cell lines according to their radiosensitivity. Immunoblotting demonstrated that the apoptosis-associated proteins, bax and bcl-2, are expressed at a basal level in all the cell lines tested, but no increase was detected after exposure to TD50 doses of etoposide, vinblastine and estramustine. The ratio of bax and bcl-2 also was not altered by DNA damage. No evidence was found that a correlation may exist between reproductive cell death and the expression of genes which control apoptosis. My results show that apoptosis is not a major mechanism of drug- or radiation-induced cell death in prostate cell lines. In conclusion, loss of p53 function and loss of androgen dependence was not found to be correlated with resistance of tumours to chemotherapeutic drugs. Cellular radiosensitivity was found to be correlated with the number of DNA double-strand breaks remaining after 2 hours of repair. The more radioresistant cell lines showed better repair competence. Apoptosis and genes affecting apoptosis, such as p53 and members of the bcl-2 family, do not seem to contribute significantly to the sensitivity of prostate cancer cells to anticancer drugs and irradiation.
AFRIKAANSE OPSOMMING: Die klassieke prostaat sellyne, DU145, PC-3 en LNCaP, het 'n waardevolle bydrae gemaak in die sel biologiese model in prostaat kanker. Die toepaslikheid daarvan mag egter beperk wees, aangesien hierdie sellyne afkomstig is van metastatiese, en nie van primêr normale en tumor epiteel nie. Die sellyne 1532T, 1535T, 1542T, 1542N en BPH-I is afkomstig van primêre benigne en maligne menslike prostaat tumor epiteel en mag moontlik meer verteenwoordigend wees. Deur van hierdie sellyne gebruik te maak, is die rolondersoek van die reaksie op basiese selskade (d.w.s. herstel, beheerpunt aktivering, apoptose en verwante sein proteïene, en die invloed van androgeen status) tydens die proses van sel inaktivering, asook die toepaslikheid ten opsigte van behandeling. Volgens verskeie studies lei die verlies aan p53 funksie tot weerstandigheid teen chemoterapeutiese middels en bestraling. Die resultate van hierdie studie toon dat die p53-onaktiewe sellyne egter die sensitiefste is vir chemoterapeutiese middels, soos etoposied, vinblastien en estramustien, terwyl die p53 natuurlike-tipe sellyn, LNCaP, die meeste radiosensitief is. Ten spyte van die invloed van p53 afbraak deur die HPV -16 E6 virale proteïen, dui die resultate van chemosensitiwiteit op die moontlikheid dat verskillende chemoterapeutiese middels verskillende p53-afhanklike effekte op verskillende tumorselle mag hê. Dit is bewys dat onttrekking van androgeen prostaat kankerselle sensitiseer teen chemoterapeutiese middels en dat hormoon-onafhanklike sellyne die hoogste chemosensitiwiteit vertoon. Die LNCaP sellyn vertoon 'n verhoogde weerstandigheid teen apoptose wat deur etoposied en y-bestraling geïnduseer is, wat 'n aanduiding is dat androgene beskerming kan bied teen beide hierdie DNA beskadigingsfaktore. Die belangrikste faktore wat die radiosensitiwiteit in menslike tumorselle bepaal, IS bekend dat dit die dubbelbande van DNA verbreek en herstel. Hierdie studie het aangetoon dat in prostaat sellyne die sellulêre radiosensitiwiteit korreleer met die aantal DNA dubbelband verbrekings binne 2 uur na bestraling, en dat die meer radioweerstandige sellyne beter herstelvermoë vertoon. Gevolgtrekkings oor die invloed van androgeen se afhanklikheid van radiosensitiwiteit en herstel kan egter nie op hierdie stadium gemaak word nie, aangesien slegs die LNCaP sellyn androgeenafhanklik was. Die feit dat die 2 uur herstelperiode 'n skeiding kan maak tussen radiosensitiewe en radioweerstandige selle in twee groepe menslike tumor sellyne, onderstreep die rol van herstel van nie-homoloë endverbindings. Dit hou implikasies in vir terapie, en stem ooreen met die kliniese waarnemings dat prostaat tumore suksesvol gekontroleer kan word deur lae intensiteit dosis bragiterapie. Ten einde die rol van apoptose te ondersoek, is selle blootgestel aan TD50 konsentrasies chemoterapeutiese middels, asook 60Co y-bestraling. Apoptose was oor die algemeen laag, en het gestrek van 0.1% tot 12.1%,3.0% tot 6.0% en 0.1% tot 8.5% vir etoposied, estramustien en vinblastien onderskeidelik. Die persentasie selle wat middel geïnduseerde apoptose ondergaan het, was gemiddeld hoër in tumor sellyne as in normale sellyne. Die waardes van apoptose geïnduseer deur y-bestraling het gewissel van 1.3% tot 7.0%. Die LNCaP sellyn het die laagste persentasie apoptotiese selle na bestraling gelewer, terwyl die 1532 r sellyn die hoogste persentasie gelewer het. Die volgorde van die radiosensitiwiteit van die sellyne was nie waarneembaar in hulle geneigdheid tot apoptose nie. Immunoblots het aangetoon dat die apoptose-geassosieerde proteïene, bax en bcl-2, uitgeskei word teen 'n basisvlak in al die sellyne wat getoets is, maar dat geen verhoogde uitskeiding waarneembaar was na blootstelling aan TD50 dosisse etoposied, vinblastien en estramustien nie. Die verhouding van bax en bcl-2 is ook nie beïnvloed deur DNA beskadiging nie. Dit blyk daarom dus onwaarskynlik dat daar 'n korrelasie bestaan tussen reproduktiewe seldood en die uitskeiding van gene wat apoptose beheer. Die resultate dui daarop dat apoptose me 'n belangrike meganisme vir middel- of bestralingsgeïnduseerde seldood in prostaat sellyne is nie.

It has been estimated that between 3000 and 4000 plant species are used for their medicinal properties throughout South Africa, with approximately 27 million South Africans making use of traditional medicines. Of this 27 million, 3 million South Africans rely on traditional medicine as their primary source of health care. Of the 250 000 to 500 000 known plant species, very few have been investigated for their pharmacological qualities, and compounds of significant medicinal value may still remain undiscovered in many plant species. The aims of this study included investigating the antimicrobial properties of Geranium incanum and Artemisia afra, both plants traditionally used for their medicinal properties, and comparing the antimicrobial activity of the latter to that of Artemisia absinthium, as well as investigating the anticancer properties of G. incanum and A. afra, and comparing the anticancer activity of the latter to that of A. absinthium. Infusions, aqueous-, methanol- and acetone extracts of the three plants were prepared and used for anticancer and antimicrobial screening. Plant specimens used to prepare extracts for antimicrobial activity were collected and extracted over three seasons, while extracts used for anticancer screening were prepared from plants collected during the summer only. Considerable variation existed in the percentage crude extract yields obtained when different extractants were used, while the season in which the plants were harvested and extracted also appeared to play a significant role in the amount of extract obtained. The plant extracts were screened for antimicrobial activity against various strains of Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus and Bacillus cereus, using an agar dilution method. G. incanum and A. afra possessed activity for C. albicans, while all three plants showed activity for S. aureus and B. cereus. Activity was largely dependent on the extraction method used. iii The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was used to screen for anticancer activity of the respective extracts, at varying concentrations, against MCF-7 (human breast adenocarcinoma) cells, HT-29 (human colonic adenocarcinoma) cells and HeLa (human cervical cancer) cells. All of the extracts showed cytotoxic activity in all three cell lines to varying extents, depending on the extract used and cell line screened. The acetone extract of A. afra proved to be the most effective inhibitor with the lowest IC50 (2.65 ± 1.05 μg/ml) having been shown in MCF-7 cells. A. afra and A. absinthium showed similar inhibitory patterns, with the methanol- and acetone extracts having been the most potent inhibitors of each of the respective cell lines in general. Fluorescence microscopy employing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and propidium iodide (PI) staining indicated that the acetone extract of A. afra induces apoptosis in MCF-7 cells as apposed to necrosis, and the results were comparable to those obtained for cells exposed to cisplatin. Screening of the A. afra acetone extract for toxicity in normal human cells using the CellTiter-Blue® assay indicated the extract to be toxic to peripheral blood mononuclear cells (PBMC’s) at concentrations comparable to that for MCF-7 cells, while cell cycle analysis of MCF-7 cells exposed to the A. afra acetone extract indicated the extract’s ability to induce apoptosis comparable to that of cisplatin, with the extract exerting its activity at a point during or just prior to the S phase of the cell cycle.

This project is a step forward in understanding the mode of action of phytochemicals in the prevention and therapy of cancer by using tissue engineered in vitro and in vivo models. In this thesis, the natural compound lycopene, primarily found in tomatoes and tomato based products, was tested as a treatment option especially for ovarian cancer patients. The study revealed the distinct potential of lycopene to prevent the onset of ovarian cancer and its ability to reduce tumor and metastatic load in an already established disease status, as tested in an intraperitoneal humanized animal model.

The problem of finding targeted medicine is a central problem in chemotherapy. From this point of view the ubiquitin-proteasome system is a highly promising object in the pharmaceutical approach. Proteasome plays a critical role in cellular protein degradation, cell cycle and apoptosis regulation. Proteasome inhibitors are substances blocking the actions of proteasome. Cancer cells are more sensitive to inhibition of the ubiquitin-proteasome system than normal cells. Therefore proteasome inhibitors have the potential to be successfully used in the cancer treatment. The study aimed to test various substances to identify possible proteasome inhibitors with the IncuCyteTM FLR image system and fluorometric microculture cytotoxicity assay. Using the IncuCyte FLR method allows for detecting changes in the molecular processes of living cells. To make proteasome inhibition visible the model cell line MelJuSoUbG76V-YFP is used which helps to detect alterations in proteasome activity by means of the yellow fluorescent protein enrichment in cells as a response to proteasome inhibition. Fluorometric microculture cytotoxicity assay is a method for the determination of cytotoxicity in human tumor cells. The study showed that substance #25 possessed a proteasome inhibitory capacity in a dose-dependent manner as demonstrated with the IncuCyte FLR image system. According to the fluorometric microculture cytotoxicity assay, substance #1 was the most stable and toxic. Substances #2 and #185 had selective toxicity against cancer cells and lower effects against normal cells. Combining IncuCyte FLR and fluorometric microculture cytotoxicity assay allows finding substances which act as proteasome inhibitors with high toxic effect.

In summary, this thesis has explored the anti-cancer mechanisms of novel medicinal compounds via targeting calpains or S100A4 in the treatment of colon cancer, which could facilitate future establishment of effective medicinal compounds in the treatment of metastatic colon cancers with known molecular targets.;The incidence of colon cancer in Hong Kong and worldwide is on a rising trend, while its metastatic development is the leading cause of cancer-related deaths. Understanding the molecular mechanisms of how tumors progress and metastasize to secondary sites, at both biological and genetic levels, could enable us to identify potential molecular targets in drug development. In the present study, we explored how manipulation of signaling pathways by targeting calpains and S100A4 could facilitate the development of anti-tumor and anti-metastatic drugs.;The study investigating drug targeting on S100A4 in both in vitro and in vivo models had shown that the pharmacological store-operated calcium channel blocker would suppress S100A4-mediated migration by weakening extracellular S100A4-mediated calcium responses. The effects on S100A4-induced metastasis formation were confirmed in vivo with reduced splenic tumor volume and decreased number of liver metastases. These results have provided new insights to correlate between S100A4 and calcium signaling, making an important step forward in characterizing the dependence of calcium homeostasis in the process of metastasis, providing a novel strategy for S100A4-mediated metastasis.;With respect to the targeting on calpains, it was discovered that total Astragalus saponins (AST) and cryptotanshinone (CPT) are effective anti-cancer agents that elicit the endoplasmic reticulum (ER) stress response. They act by upregulating the expression of glucose-regulated protein (GRP) 78, leading to the initiation of apoptosis when the ER recovery process begins to fail. In particular, CPT caused rapid and sustained increase in cytosolic calcium in colon cancer cells that was accompanied by early GRP78 overexpression. The increase in cytosolic calcium was blocked by pre-treatment of BAPTA-AM through depletion of the ER calcium store. In consistent with these, we also confirmed that CPT significantly increased calpain activity, which could be blocked by calcium chelator or calpain inhibitors. Furthermore, a dynamic interaction between GRP78 and calpain under ER stress was unveiled during AST or CPT exposure. The degree of association was increased following prolonged ER stress, and suppressed either as the ER recovery process failed or with the presence of calpain inhibitors. Besides, inhibition of calpain activity suppressed NF-κB activation (a consequence of ER stress) and substantially enhanced the effects of CPT to promote apoptosis. More importantly, it was confirmed that the effects of calpain inhibitors to sensitize colon cancer cells to ER stress-associated apoptosis are p53-dependent. The anti-tumorigenetic effects of CPT were further demonstrated in vivo in xenografted nude mice by trageting calpains and in combination with calpain inhibitors.

Pancreatic cancer is known to be one of the most life-threatening cancers characterized by aggressive local invasion and distant metastasis. The high basal level of autophagy in pancreatic cancer may be responsible for the low chemotherapeutic drug response rate and poor disease prognosis. However, the clinical application of autophagy inhibitors was unsatisfactory due to their toxicity and minimal single-agent anticancer efficacy. Hence, oncologists begin to consider the tumor microenvironment when exploring new drug targets. In the present study, the anti-tumorigenic mechanisms of two major phytochemicals derived from Chinese medicinal herbs had been investigated against pancreatic cancer development. Calycosin is a bioactive isoflavonoid of the medicinal plant Astragalus membranaceus. Our results have shown that calycosin inhibited the growth of various pancreatic cancer cells both in vitro and in vivo by inducing cell cycle arrest and apoptosis. Alternatively, calycosin also facilitated MIA PaCa-2 pancreatic cancer cell migration in vitro and increased the expression of epithelial-mesenchymal transition (EMT) biomarkers in vivo. Further mechanistic study suggests that induction of the Raf/MEK/ERK pathway and facilitated polarization of M2 tumor-associated macrophage in the tumor microenvironment both contribute to the pro-metastatic potential of calycosin in pancreatic cancer. These events appear to be associated with calycosin-evoked activation of TGF-β signaling, which may explain the paradoxical drug actions due to the dual roles of TGF-β as both tumor suppressor and tumor promoter in pancreatic cancer development under different conditions. Isoliquiritigenin (ISL) is a chalcone obtained from the medicinal plant Glycyrrhiza glabra, which can be a precursor for chemical conversion to form calycosin. Results have shown that ISL decreased the growth and EMT of pancreatic cancer cells in vitro, probably due to modulation of autophagy. ISL-induced inhibition of autophagy subsequently promoted reactive oxygen species (ROS) production, leading to induction of apoptosis in pancreatic cancer cells. Such phenomenon also contributed to the synergistic growth-inhibitory effect in combined treatment with the orthodox chemotherapeutic drug 5-fluorouracil. In addition, ISL-induced tumor growth inhibition in vivo was further demonstrated in a tumor xenograft mice model of pancreatic cancer. ISL promoted apoptosis and inhibited autophagy in the tumor tissues. Study on immune cells indicates that ISL could reduce the number of myeloid-derived suppressor cells (MDSCs) both in tumor tissue and in peripheral blood, while CD4+ and CD8+ T cells were increased correspondingly. In vitro test has revealed that ISL inhibited the polarization of M2 macrophage along with its inhibition of autophagy in M2 macrophage. These immunomodulating effects of ISL had reversed the pro-invasive role of M2 macrophage in pancreatic cancer.In conclusion, calycosin acts as a "double-edged sword" on the growth and metastasis of pancreatic cancer, which may be related to the dual roles of TGF-β and its influence on the tumor microenvironment. Alternatively, ISL consistently inhibited the growth and metastatic drive of pancreatic cancer through regulation of autophagy and reprogramming of the immune system. The differential modes of action of these compounds have provided new insights in the development of effective pancreatic cancer treatment adjuvants.

Although chemotherapy still represents the gold-standard treatment option for cancer cure, it often results in several long-term negative side effects, including cancer relapse and acquired resistance to the therapy. Docetaxel is the preferred anticancer drug for several tumor types, including metastatic castrate-resistant prostate cancer. However, it exhibits only modest efficacy and many patients who received Docetaxel treatment experience tumor progression and chemotherapy insensitivity. Therefore, identifying the molecular mechanisms that associate anticancer therapy with its negative bystander effects is an urgent need in order to develop novel strategies targeting chemotherapy vulnerabilities. Interestingly, a possible link is provided by therapy-induced cellular senescence. Indeed, a recent body of evidence highlights that several antineoplastic agents promote the senescent phenotype in both stromal and tumor cells. Despite it has been originally identified as a tumor-suppressive mechanism, many findings underline that cellular senescence sustains tumor growth and dissemination. The tumor-promoting effects of cellular senescence are mainly mediated by secreted factors, that exert profound paracrine effects through the generation of a pro-inflammatory and immunosuppressive microenvironment. For instance, it is well known that cancer progression and aggressiveness are strongly supported by the bi-directional crosstalk between cancer cells and the surrounding tumor microenvironment, that favors cancer cell migration and invasion, neo-angiogenesis and homing of distant organs. In this scenario, stromal cells exert a promoting role in cancer progression providing tumor with energy sources, growth factors and cytokines. In this study, we found that Docetaxel treatment strongly promotes the senescent phenotype in stromal prostate fibroblasts, as revealed by a sharp increase in numerous senescence markers, including SA-β-Galactosidase activity, γ-H2AX nuclear foci, and p53 expression. We also provided evidence that Docetaxel treatment induces the senescence-associated secretory phenotype (SASP) in prostate stromal fibroblasts, by increasing the levels of pro-inflammatory cytokines (IL-6, IL-8), growth factors (VEGF-A) and matrix-metalloproteinases (MMP-2 and MMP-3). The clinical relevance of the senescence-inducing effects of Docetaxel in the stromal compartment of prostate cancer was determined analyzing the accumulation of lipofuscin aggregates in tissue specimens from 20 patients with prostate cancer, 10 of whom received neo-adjuvant Docetaxel chemotherapy before radical prostatectomy. Remarkably, we found that lipofuscin staining is significantly higher in patients treated with Docetaxel therapy and preferentially accumulates in the stromal compartment of prostate cancer tissues. Besides, this study underlined that Docetaxel-induced senescent stromal cells exhibit a strong mitochondrial dysfunction, characterized by increased mitochondrial mass and oxidative stress, reduced mitochondrial membrane potential, and morphological changes of the mitochondrial structure. Moreover, we observed that the anticancer drug promotes metabolic alterations in senescent prostate fibroblasts, including increased extracellular acidification rate and lactate secretion, suggesting that senescent cells may shift towards a more glycolytic metabolism to meet their energetic demands, as a consequence of impaired mitochondria. We then investigated the role of the paracrine factors and metabolites secreted by senescent fibroblasts on prostate cancer aggressiveness by incubating PC3 tumor cells with conditioned media from control or senescent fibroblasts. We highlighted that therapy-induced stromal senescence supports the increase in the invasive and migratory abilities, and clonogenic and stemness potential of prostate cancer cells. Interestingly, these effects are directly correlated to stromal senescence. Indeed, clearance of senescent fibroblasts through administration of ABT263, a senolytic drug, reverts the malignant phenotype of prostate cancer cells. The results obtained in this study highlight that the long-term adverse effects of Docetaxel therapy could be correlated to its ability to induce the senescent phenotype in the stromal compartment, thus generating a supportive tumor microenvironment, that further promotes prostate cancer progression and aggressiveness. In addition, this study shed new light on the use of senolytic drugs to improve Docetaxel efficacy and overcome its detrimental bystander effects in prostate cancer.

FLIP (FLICE inhibitory protein) est une protéine anti-apoptotique qui a des identités de séquence partagées avec la protéine pro-apoptotique caspase-8. FLIP se trouve en compétence avec caspase-8 pour se fixer sur la protéine adaptatrice FADD, ce qui empêche l’activation de caspase-8 bloquant ainsi l'apoptose. Lors du développement des molécules interférant avec des protéines anti-apoptotiques, la recherche d'inhibiteurs de la protéine FLIP qui est surexprimée dans un très grand nombre de cancers, a échoué. Cela s'explique en partie par le fait que peu d'information structurelle de FLIP est actuellement disponible TRAIL est une cytokine de la famille TNFα. Elle est décrite pour activer des voies de signalisation conduisant à la mort cellulaire par apoptose. TRAIL a montré un grand intérêt dans la thérapie anticancéreuse, grâce à sa capacité d’induire la mort des cellules tumorales sans aucun effet sur les cellules normales. Cependant, l’efficacité de TRAIL est limitée par plusieurs mécanismes moléculaires. Un de ces mécanismes est la surexpression de FLIP qui fait compromettre l’utilisation thérapeutique de TRAIL. Le but principal de ce projet est de développer des nouvelles molécules capables d’inhiber la protéine FLIP dans les cellules tumorales, sans aucun effet sur la protéine homologue caspase-8. Après modélisation des protéines FLIP et caspase-8 sur la base de la structure cristallographique de FLIP viral et FADD respectivement, des premières expériences d’ancrage ou “docking” utilisant une banque de composés chimiques du «National Cancer Institute NCI » ont été réalisées. Les 9 molécules les plus intéressantes, étant comme sélectives pour FLIP et non caspase 8, ont été sélectionnées et testées sur des lignées de cancer de poumons surexprimant la protéine FLIP. Une co-administration de chacune des molécules inhibitrices de FLIP avec TRAIL a été faite pour vérifier la restauration de la voie apoptotique dans les cellules cancéreuses. Un test moléculaire de « Pull down assay » a été fait afin de confirmer l’inhibition de l’interaction de FLIP avec FADD. Finalement, l’évaluation de l’activité enzymatique des caspases a été étudiée pour vérifier la réactivation de la voie apoptotique après la combinaison de TRAIL avec les inhibiteurs de c-FLIP. En conclusion, la combinaison de TRAIL avec les inhibiteurs de FLIP aboutit à la restauration de la voie apoptotique dans des cellules cancéreuses. Ces composés nouvellement identifiés, peuvent servir ultérieurement comme des potentiels éléments des stratégies utilisées dans le domaine du traitement du cancer
FLIP (FLICE Inhibitory Protein) is an anti-apoptotic protein which shares sequencesimilarity with the pro-apoptotic protein caspase-8. FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-associated death domain), thus it inhibits caspase-8 activation, thereby blocking apoptosis. During the development of molecules interfering with anti-apoptotic proteins, searching for inhibitors of FLIP protein which is overexpressed in a very large number of cancers, has failed. This is partly due to the fact that little FLIP structural information is available at present. TRAIL is a member of TNFα superfamily. It has been described to activate the apoptotic signaling pathways. TRAIL showed great interest in anti-cancer therapy, due to its ability to induce tumor cell death without any effect on normal cells. However, the efficacy of TRAIL is limited by several molecular mechanisms. One of these mechanisms is the overexpression of FLIP which is able to compromise the therapeutic use of TRAIL. The main goal of this project is to develop novel inhibitory molecules able to interfere with FLIP in tumor cells without any effect on the homologous protein caspase 8. After the construction of FLIP and caspase-8 proteins on the basis of the crystallographic structure of the viral FLIP and FADD respectively, the first docking experiments using a chemical library of the National Cancer Institute NCI have been carried out. The most interesting molecules, being selective for FLIP versus caspase 8, were selected and tested on lung cancer cell lines that overexpress FLIP protein. Co-administration of FLIP inhibitors with TRAIL was performed to verify the restoration of the apoptotic pathway in cancer cells. A molecular test of "Pull down assay" was done in order to confirm the inhibition of the FLIP/FADD interaction. Finally, the evaluation of caspases activity was carried out to confirm the reactivation of the apoptotic machinery after TRAIL/FLIP-inhibitors combination. In conclusion, the combination of TRAIL with FLIP inhibitors resulted in apoptosis restoration in resistant tumor cells. These newly identified compounds may serve later as potential elements in cancer treatment field

Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in North American males and a leading cause of cancer deaths. The lack of effective treatment options for advanced PCa such as AR-positive castration-resistant PCa (CRPC-AD) and the highly aggressive AR-negative CRPC, e.g. neuroendocrine PCa (CRPC-NE) presents a critical, unmet need for the development of novel therapeutics. Altered metabolism in the form of elevated aerobic glycolysis is a common cancer characteristic. Here we propose a novel conceptual understanding for the central, functional role of excessive cancer-generated lactic acid. In particular, the acidification of the tumor microenvironment via increased MCT4-mediated lactic acid secretion can facilitate multiple crucial cancer-promoting processes, including proliferation, tissue invasion/metastasis, angiogenesis, and suppression of local anticancer immunity. As such, the inhibition of MCT4 could be an effective therapeutic strategy broadly impacting multiple downstream lactate-associated tumour-promoting processes. Experimentally, we were able to confirm the clinical relevance of elevated glycolysis and increased lactic acid production in various advanced PCa patient-derived xenograft (PDX) models and patient tumours using a novel metabolic pathway score. In particular, NEPC tumours appear to rely much more heavily on elevated aerobic glycolysis and MCT4-mediated lactic acid secretion. In a proof-of-concept study using MCT4-specific antisense oligonucleotides (ASOs), reduced MCT4 expression is able to reduce proliferation, invasion/migration, and glucose metabolism of advanced PCa cells in vitro. More importantly, we demonstrated in two distinct in vivo models containing residual functional immune cells that MCT4 inhibition enhanced anticancer immunity. Finally, a state-of-the-art in silico drug discovery pipeline was employed in the first steps towards developing a potent and specific MCT4 small molecule inhibitor. Computer modeling of MCT4 structure, virtual molecular docking, and downstream experimental validation identified a promising hit series based on the chemical scaffold of VPC-25009 as a potential second therapeutic modality for MCT4 inhibition. Taken together, we were able to provide experimental support for our novel hypothesis regarding the central tumour-promoting and immunosuppressive role of cancer-generated lactic acid. A therapeutic approach blocking lactic acid secretion by targeting MCT4 function could thus inhibit multiple downstream lactate-associated processes for effective treatment of advanced PCa and other highly glycolytic cancers.
Medicine, Faculty of
Graduate

The incidence of skin cancer is increasing and conventional treatments such as surgery are not suitable for all patients. This study aimed to develop an elastic liposomal gel to be applied directly to the tumour for the controlled release of anti-cancer agents to the dermal layer. The proposed anti-cancer flavonoids EGCG and naringenin as well as the novel potent cytotoxic agent MTL-004 were loaded into the bilayer of liposomes. Furthermore, aqueous gels HEC and HPMC were investigated as carriers for the liposomes to be applied topically. Liposomes loaded with either Tween 80, Tween 20 or sodium cholate were found to have increased elastic properties, liposomes with an average size of 400 nm were able to pass through a pore size of 100 nm. Release studies from liposomes loaded with either EGCG, naringenin and MTL-004 as well as varying ratios of Tween 20 were carried out. Within 24 hours, EGCG liposomes loaded with 0% or 10% w/w Tween 20 gave a release of 13.7 ± 1.1 % and 94.4 ± 4.9 % respectively; naringenin liposomes loaded with 0% or 10% w/w Tween 20 gave a release of 109.7 ± 5.0 % and 48.5 ± 2.1 % respectively; MTL-004 liposomes loaded with 0% or 10% w/w Tween 20 gave a release of 59.8 ± 1.2 % and 74.0 ± 1.8 % respectively. This indicates a compounds individual physiochemical properties influences release of compound from liposomes. EGCG, naringenin and MTL-004 loaded liposomes added into the aqueous gel HEC or HPMC gels may have had an additive effect in terms of retarding drug release. Release was faster from HEC gels and liposomes formulated with Tween 20. In vitro cellular uptake of liposome uptake into HDFa and HaCat cells was apparent. Thus it appears elastic liposomes are useful in enhancing drug penetration into dermal cells and furthermore may be useful in the development of a controlled release formulation.

Cancer research and development of targeting agents in this field is based on robust studies using preclinical models. The failure rate of standardized treatment approaches for several solid tumors has led to the urgent need to fine-tune more sophisticated and faithful preclinical models able to recapitulate the features of in vivo human tumors, with the final aim to shed light on new potential therapeutic targets. Epithelial Ovarian Cancer (EOC) serous histotype (HGSOC) is one of the most lethal diseases in women due to its high aggressiveness (75% of patients diagnosed at FIGO III-IV state) and poor prognosis (less of 50% in 5 years), whose therapy often fails as chemoresistance sets in. This thesis aimed at using the novel perfusion-based bioreactor U-CUP that provides direct perfusion throughout the tumor tissue seeking to obtain an EOC 3D ex vivo model able to recapitulate the features of the original tumor including the tumor microenvironment and maintaining its cellular heterogeneity. Moreover, we optimized this approach so that it can be successfully applied to slow-frozen tumoral tissues, further extending the usefulness of this tool. We also investigated the effectiveness of Plasma Activated Ringer’s Lactate solution (PA-RL) against Epithelial Ovarian Cancer (EOC) serous histotype in both 2D and 3D cultures using ex-vivo specimens from HGSOC patients. We propose PA-RL as a novel therapy with local intraperitoneal administration, which could act on primary or metastatic ovarian tumors inducing a specific cancer cell death with reduced damage on the surrounding healthy tissues.

L’équilibre rédox entre les niveaux des espèces réactives de l’oxygène et de l’azote (ROS, RNS) et les espèces antioxydantes cellulaires est déterminant pour le fonctionnement normal de la cellule et sa viabilité. Le déséquilibre redox ou « stress oxydant » peut altérer les voies de signalisation cellulaires et générer des dommages sur les protéines, les lipides et l’ADN des cellules. Il est ainsi associé à de nombreuses pathologies, notamment les cancers. Les cellules cancéreuses présentent une dérégulation redox importante et un stress oxydant basal intrinsèque plus élevé par rapport aux cellules normales. Elles sont donc très dépendantes des systèmes antioxydants pour leur viabilité. Ainsi, l’administration de drogues qui i) génèrent des ROS / RNS additionnelles ou ii) inhibent les systèmes antioxydant cellulaires, permet une cytotoxicité sélective contre les cellules cancéreuses. C’est la base biologique de la « thérapie anticancéreuse basée sur la modulation de l’équilibre redox». Dans ce contexte, nos travaux ont pour but de décrypter les mécanismes redox derrière l’activité anticancéreuse de la vitamine C (VitC) et de l’auranofin (AUF), seuls ou en combinaison, dans le modèle du cancer du sein. La VitC à des concentrations pharmacologiques élevées présente des propriétés pro-oxydantes. Dans cette étude, l’activité anticancéreuse de la VitC contre les lignées du cancer du sein est associée à une génération extracellulaire et intracellulaire de peroxyde d'hydrogène (H₂O₂) accompagnée d'une oxydation intracellulaire du glutathion (GSH). L’approche protéomique «redoxome» a révélé que la VitC induit une altération de l'état rédox d’enzymes antioxydantes clés et d'un certain nombre de protéines contenant des cystéines, impliquées dans les métabolismes de l’ARN et l’ADN et dans les processus énergétiques. La VitC est également responsable d’un retard dans la progression du cycle cellulaire et d’une inhibition de la traduction. Finalement, des analyses bioinformatiques ont montré que les niveaux d'expression de la peroxiredoxine 1 (PRDX1) sont corrélés à la cytotoxicité différentielle de la VitC dans les cellules cancéreuses du sein. L'AUF, un antirhumatismal, est un inhibiteur des thiorédoxines réductases qui a reçu une attention croissante pour son activité anticancéreuse. Nos travaux montrent que l’AUF inhibe également le système antioxydant du GSH et que cette inhibition est primordiale pour son activité anticancéreuse. L’AUF modifie l'état redox de nombreuses protéines impliquées dans la prolifération et le cycle cellulaire, et provoque une déplétion des dNTPs et un arrêt du cycle cellulaire. De façon remarquable, nous avons démontré que la combinaison de l’AUF et de la VitC présente une cytotoxicité accrue, synergique, médiée par H₂O₂ dans les cellules MDA-MB-231 et d'autres lignées cellulaires du cancer du sein sans trop affecter les cellules normales. In vivo, l’efficacité de la combinaison AUF/VitC a été validée sur des xénogreffes de MDA-MB-231 chez les souris sans présenter une toxicité notable, tandis que l'administration de l’AUF ou de la VitC en monothérapie n’inhibe pas la croissance tumorale. Enfin, les analyses protéomiques, bioinformatiques et fonctionnelles ont identifié la prostaglandine réductase 1 (PTGR1) comme biomarqueur prédictif de la réponse des cellules cancéreuses du sein à la combinaison AUF/VitC. En résumé, ces résultats contribuent à une meilleure compréhension des mécanismes anticancéreux de la VitC et de l'AUF, seuls et en combinaison. En particulier, la combinaison de ces deux médicaments disponibles et non toxiques pourrait être efficace contre le cancer du sein triple négatif et potentiellement d'autres cancers présentant des propriétés redox similaires. Ainsi, une évaluation préclinique et clinique de ces traitements ouvrira la voie à des nouvelles thérapies anticancéreuses basées sur la modulation de l’équilibre redox cellulaire
Reactive oxygen and nitrogen species (ROS, RNS) homeostasis and intracellular reductive/oxidative (redox) dynamics play a key role in regulating cell fate and are critical for normal cellular functions. Oxidative stress via the disruption of redox homeostasis can lead to aberrant cell signaling and toxic oxidative damage of DNA, lipids and proteins, and is therefore associated with human pathologies such as cancers. Cancer cells experience extensive redox deregulation and generally exhibit higher intrinsic basal oxidative stress than normal cells, as a consequence, they are more dependent on their antioxidant systems for survival. Thus, the administration of a drug generating additional ROS / RNS or inhibiting cellular antioxidant systems will exert a selective cytotoxicity towards cancer cells while sparing their normal counterparts. This is the biological basis for « redox-based anticancer therapy ». The work described here aims to investigate the redox-based anticancer activity of vitamin C (VitC) and auranofin (AUF), as single drugs or in combination, in breast cancer model. VitC at high pharmacological concentrations shows pro-oxidant properties. In this study, we showed that VitC anticancer activity against breast cancer cell lines was associated to extracellular and intracellular generation of hydrogen peroxide (H₂O₂), accompanied by the oxidation of intracellular glutathione (GSH). A “redoxome” proteomics approach revealed that VitC induces alterations of the redox state of key antioxidant enzymes and a number of cysteines-containing proteins including many proteins involved in RNA and DNA metabolisms and energetic processes. Cell cycle arrest and translation inhibition are associated with VitC-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlate with VitC differential cytotoxicity in breast cancer cells. AUF, an antirheumatic drug and known inhibitor of thioredoxin reductases, has been repurposed recently as a potent anticancer drug. We showed that AUF acts on both the thioredoxin and GSH systems and its impact on GSH system is essential for its anticancer activity. AUF alters the redox state of a number of nucleic acid-binding proteins involved in cell proliferation, cell division and cell cycle, triggering dNTP depletion and cell cycle arrest. Importantly, we observed that the combination of AUF and VitC reveals a synergetic and H₂O₂-mediated cytotoxicity towards MDA-MB-231 cells and other breast cancer cell lines without much impact on normal cells, thus decreasing the cytotoxic concentrations of AUF or VitC single drug. The anticancer potential of AUF/VitC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects, while administration of AUF or VitC as a single agent failed to suppress tumor growth. Finally, SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell response to AUF/VitC combination, thus identifying a potential predictive biomarker. Overall, these results provide new insights into the anticancer mechanisms of VitC and AUF, as single drugs and in combination. In particular, this combination of two non-toxic and commonly available drugs could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties. Further assessment in preclinical and clinical studies of these drugs and combinations could open new avenues for redox-based anticancer therapy

L'électrochimiothérapie est un traitement anticancéreux utilisé en routine en Europe dans près de 130 centres de traitement du cancer. Le taux de réponse objective atteint 85 % pour le traitment de tumeurs cutanées et sous-cutanées et des études sont en cours afin d'appliquer ce traitement à des tumeurs profondes. Au cours de ce doctorat, les méchanismes sous-jacents à cette excellente efficacité antitumorale ont été étudiés. Dans un premier temps, l'objectif a été d'évaluer la capacité de l'électrochimiothérapie à induire la mort des cellules souches cancéreuses, considérées comme les racines du cancer. Ensuite, les mécanismes immunologiques à l'origine du développement d'une immunité antitumorale mise en place par le traitement ont été investigués. Cependant, malgré le haut taux de réponse observé, l'électrochimiothérapie reste un traitement local qui n'induit pas de réponse antitumorale à distance, sur les tumeurs non-traitées. Afin de pallier à cette absence d'activité systémique, une collaboration a été mise en place avec une entreprise innovante de biotechnologies, INVECTYS, dans le but de développer une stratégie de vaccination à ADN ciblant la télomérase et basée sur l'électrogènetransfert. Il est attendu que la combinaison de cette immunothérapie avec un traitement local par électrochimiothérapie, détruise non seulement la tumeur primaire, dont les cellules souches cancéreuses, mais également les cellules cancéreuses circulantes et les métastases
Electrochemotherapy is an anticancer treatment used in routine in Europe in 130 cancer treatment centers. The objective response rate reaches 85 % for the treatment of cutaneous and subcutaneous tumors and studies are ongoing to spread the use of electrochemotherapy to deep-seated tumors. In the frame of this doctorate, the mechanisms underlying this excellent antitumor efficiency were investigated. First, the goal was to evaluate the ability of electrochemotherapy to induce the death of cancer stem cells, considered as the roots of cancer. Second, the immunological mechanisms responsible for the development of antitumor immune responses following the treatment were investigated. However, although a very high response rate is observed, electrochemotherapy remains a local treatment which does not induce antitumor responses in distant non-treated nodules. In order to circumvent this lack of systemic activity, a collaborative project was initiated with an innovating biotech company, INVECTYS, in order to develop a DNA vaccination strategy targeting the telomerase and based on electrogenetransfer. It is expected that the combination of this immunotherapy with a local treatment by electrochemotherapy could destroy not only the primary tumor, including cancer stem cells, but also circulating cancer cells and metastases

Dans la majorité des cas, les mitochondries sont nécessaires à la tumorigenèse et à la réponse des cellules cancéreuses aux signaux générés par les facteurs micro-environnementaux (exemples : privation de nutriments, hypoxie) ou par les traitements anticancéreux (exemples : chimiothérapie, radiothérapie). Presque toutes les protéines mitochondriales sont codées par le génome nucléaire et importées dans l'organelle. Des machineries d'import ont donc évolué afin de répondre aux besoins d'import protéique. Dans ce contexte, la machinerie régulée par CHCHD4/Mia40 fonctionne dans l’espace intermembranaire et contrôle l’import d’un groupe de protéines (substrats) qui joue des rôles importants dans la survie et la réponse au stress. Les substrats de CHCHD4/Mia40 sont impliqués dans un vaste panel d’activités mitochondriales qui inclut la biogenèse des complexes de la chaîne respiratoire, l’homéostasie lipidique, le stockage du calcium, ainsi que l'ultrastructure et la dynamique mitochondriale. Ce programme de thèse a été dédié à l’étude de la voie d’import CHCHD4/Mia40 dans les cellules cancéreuses et a porté un intérêt tout particulier à l'un des substrats CHCHD4/Mia40 qui façonne l'ultrastructure mitochondriale. En utilisant des techniques d’ARN interférence et de sur-expression de protéines recombinantes, dans un modèle de cancer du côlon, nous avons montré que l’expression du substrat étudié a un effet crucial sur la prolifération et la croissance tumorale. Nos données ont également impliqué cette protéine dans la réponse aux traitements anticancéreux. Dans l'ensemble, ces travaux ouvrent un nouveau champ d'investigations qui non seulement permettra de mieux comprendre la plasticité métabolique des cellules cancéreuses, mais aidera également à identifier de nouveaux biomarqueurs métaboliques
In the vast majority of cases, mitochondria are required for tumorigenesis and also for the tumoral response to signals generated by the microenvironmental factors (e.g. nutrient deprivation, hypoxia) or to the effects of anti-cancer treatments (e.g. chemotherapy, radiotherapy). As almost all mitochondrial proteins are nuclear-encoded and imported into the organelle, specialized import machineries have evolved in order to meet the need for protein import. Among these machineries, the one that operates in the intermembrane space and is controlled by CHCHD4/Mia40, regulates the import of a group of proteins (substrates) that play important roles in survival and stress response. Substrates of CHCHD4/Mia40 are involved in a broad panel of mitochondrial activities that includes the biogenesis of respiratory chain complexes, lipid homeostasis, calcium storage, as well as ultrastructure and mitochondrial dynamics. This thesis program was dedicated to the study of the CHCHD4/Mia40 import pathway in cancer cells, with a particular interest for one of the CHCHD4/Mia40 substrates that shapes mitochondrial ultrastructure. Using RNA interference approach and recombinant protein overexpression technique, in a colon cancer model, we showed that the expression of this substrate had a crucial effect on proliferation and tumor growth. Our data also involved this protein in the response to anti-cancer treatments. All together, this work opens a new field of investigations that will not only shed new lights on the metabolic plasticity of cancer cells but also help to identify new metabolic biomarkers

Includes abstract.~Includes bibliographical references.
Managing patients whose disease has become unresponsive to anticancer treatment confronts oncologists with major stressors which may range from the management of distressing physical symptoms to complex psychosocial issues. These sets of circumstances prompted the undertaking of this study: An evaluation of the current practices followed by oncologists in private practice in Cape Town, South Africa, in the management of patients with advanced cancer which no longer responds to anticancer treatment and the identification of the needs associated with such management. A descriptive qualitative study was selected for data collection. Cross-sectional, in-depth semi structured face to face interviews were conducted with fifteen radio-oncologists working in five satellite units of a private oncology company in Cape Town. The interviews were conducted with the aid of a topic guide. The process of coding was employed to organise and manage the collected data. The following six themes which had a bearing on the main topic were distilled from the data: Oncologists' experiences pertaining to the management of patients with advanced disease; the difficult discussion necessary when a patient's disease became incurable and when it had to be decided whether anticancer treatment should be stopped; the decision to stop anticancer treatment; advance directives; oncologists’ burnout and the palliative care team approach. A description of challenging aspects associated with the management of terminally ill cancer patients is given. Identified needs include training of staff in palliative care; guidance for oncologists regarding the discussion of and the decision to stop anticancer treatment; implementation of advance directives; the development and employment of a multidisciplinary approach to provide palliative care; and support for oncologists facing burnout. Recommendations were made pertaining to appropriate training in the field of palliative care; the development of guidelines to aid oncologists in the discussion of and decision to stop anticancer treatment and the implementation of advance directives; the provision of palliative care through employment of a multidisciplinary approach led by a palliative care physician; and external support which should be provided by the oncologists' company to prevent and treat burnout.

L'homéostasie des espèces réactives de l'oxygène (ROS) et des espèces réactives de l'azote (RNS), ainsi que la dynamique des processus redox, sont cruciaux pour réguler le destin cellulaire et maintenir une fonction cellulaire normale. Par rapport aux cellules normales, les cellules cancéreuses maintiennent généralement un état pro-oxydant persistant en raison de l'activation d'oncogènes, d'un métabolisme aberrant, de dysfonctionnements de la mitochondrie, et dans certains cas, de défauts des systèmes de défense antioxydants. Cet état pro-oxydant, ainsi que leur dépendance aux systèmes antioxydants pour leur survie, offrent une fenêtre thérapeutique pour un ciblage sélectif des cellules cancéreuses. Les molécules qui génèrent des ROS ou inhibent les systèmes antioxydants peuvent élever les niveaux de ROS au-delà d'un seuil critique incompatible avec la survie des cellules cancéreuses, sans causer de dommages significatifs aux cellules normales. Ce concept constitue la base biologique de la thérapie anticancéreuse basée sur la manipulation de l'équilibre redox. Le travail décrit ici vise à étudier l'activité anticancéreuse, basée sur la modulation redox, de la combinaison de l'auranofine (AUF) et de la vitamine C (VC) dans les cellules de leucémie aiguë myéloïde (LAM). L'AUF, un médicament antirhumatismal et un inhibiteur connu des thioredoxines réductases, a été repositionné comme agent anticancéreux prometteur. La VC, à des concentrations pharmacologiques élevées, présente des propriétés pro-oxydantes. Notre étude a démontré que la combinaison d'AUF et de VC présente une toxicité synergique contre des lignées cellulaires leucémiques LAM, éliminant efficacement ces cellules avec une toxicité moindre pour les cellules stromales normales de la moelle osseuse. Le traitement AUF/VC favorise considérablement la production de ROS dans le cytoplasme et les mitochondries et augmente considérablement les niveaux de PRDX1 et PRDX3 oxydées. De plus, le traitement AUF/VC réduit la teneur totale en ATP et diminue rapidement le potentiel de membrane mitochondrial dans les lignées cellulaires leucémiques. Nos recherches ont également révélé que le traitement AUF/VC a un impact sur diverses voies de signalisation dans différentes lignées cellulaires leucémiques. En particulier, la combinaison affecte rapidement la voie de signalisation mTOR et influence directement la phosphorylation de 4EBP1 et p70S6K, peut-être indépendamment de la voie classique mTOR, conduisant à une inhibition rapide de la synthèse protéique totale dans les cellules. Cette inhibition rapide de la synthèse protéique peut contribuer à la mort cellulaire dans les lignées cellulaires leucémiques. Nous avons évalué l'effet thérapeutique de l'AUF/VC à l'aide de 22 échantillons de LAM prélevés chez des patients. À des concentrations pharmacologiquement atteignables, la combinaison AUF/VC tue efficacement un nombre substantiel de cellules LAM primitives dans la majorité des cas analysés, avec moins de toxicité pour les cellules hématopoïétiques normales. Les tests de clonogénicité ont confirmé que la combinaison AUF/VC a une activité puissante contre les cellules souches/progénitrices de LAM tout en épargnant certaines cellules souches/progénitrices hématopoïétiques normales. Dans l'ensemble, ces résultats apportent de nouvelles perspectives sur l'activité et les mécanismes anticancéreux de la combinaison AUF et VC. Cette combinaison de deux médicaments non toxiques et facilement disponibles pourrait être efficace contre la leucémie et potentiellement d'autres cancers aux propriétés redox similaires. D'autres études précliniques sur cette combinaison pourraient ouvrir la voie à de nouvelles thérapies anticancéreuses basées sur la manipulation de l'équilibre redox
The homeostasis of reactive oxygen species (ROS) and nitric oxide species (NOS), along with the dynamics of redox processes, is crucial for regulating cell fate and maintaining normal cell function. Cancer cells typically maintain a persistent pro-oxidative state compared to normal cells. In fact, in cancer cells, cellular events such as activation of oncogenes, aberrant metabolic stress, mitochondrial dysfunction, and in some cases, defective antioxidant systems, can increase ROS levels, leading to intrinsic oxidative stress. This pro-oxidative state, along with their reliance on antioxidant systems for survival, provides a therapeutic window for selective targeting. Molecules that either generate ROS or inhibit antioxidant systems can elevate ROS levels beyond a critical threshold that is incompatible with cancer cell survival, without causing significant damage to normal cells. This concept forms the biological basis for redox-based anticancer therapy. The work described here aims to investigate the redox-based anticancer activity of the combination of auranofin (AUF) and vitamin C (VC) in acute myeloid leukemia (AML) cells. AUF, an anti-rheumatic drug and known inhibitor of thioredoxin reductases, has been repurposed as a promising anticancer agent. VC, at high pharmacological concentrations, exhibits pro-oxidant properties. Our study demonstrated that the combination of AUF and VC exhibits synergistic toxicity against AML cell lines, effectively eliminating these cells with less toxicity to normal bone marrow stromal cells. The AUF/VC treatment significantly promoted ROS production in both the cytoplasm and mitochondria and markedly increased the levels of oxidized PRDX1 and PRDX3. Additionally, AUF/VC treatment reduced the total ATP content and rapidly decreased the mitochondrial membrane potential in leukemia cell lines. Our research also revealed that AUF/VC treatment impacts various signaling pathways in different leukemia cell lines. In particular, the combination rapidly affected the mTOR signaling pathway and influenced the phosphorylation of 4EBP1 and p70S6K, possibly independent of the classical mTOR pathway, leading to a rapid inhibition of total protein synthesis in cells. This rapid inhibition of protein synthesis may contribute to cell death in leukemia cell lines. We evaluated the therapeutic effect of AUF/VC using 22 AML samples collected from patients. At pharmacologically achievable concentrations, the AUF/VC combination effectively killed a substantial number of primitive AML cells in the majority of samples tested with less toxicity to normal hematopoietic cells. Colony-forming unit assays confirmed that the AUF/VC combination had potent activity against AML stem/progenitor cells while sparing some normal hematopoietic stem/progenitor cells. Overall, these results provide new insights into the anticancer mechanisms of the AUF and VC combination. This combination of two non-toxic and readily available drugs could be effective against AML and potentially other cancers with similar redox properties. Further preclinical studies of this combination could pave the way for new redox-based anticancer therapies

Utveckling av polymer baserade läkemedelsbärare i nanostorlek har blivit allt viktigare för att effektivisera behandling och diagnosering av olika sjukdomar, speciellt cancer. Flera läkemedel som används i kemoterapi har bristfälliga egenskaper som låg löslighet i vatten, oönskad nedbrytbara till dess inaktiva form, och distribution i stora volymer till oönskade organ p.g.a. dess icke-selektiva förmåga. Nanopartiklar är små partiklar med diameter 1-500 nm som genom passiv/aktiv transport kan passera olika biologiska barriärer och transportera läkemedel i optimala mängder till specifika celler. Denna selektiva transport bidrar till ökad terapeutiskt index och minskning av toxiska effekter i övriga delar av kroppen. Hyperförgrenade linjär-dendritiska hybrider är en subgrupp av dendritiska polymer som har stor potential att användas som byggstenar i utvecklingen av läkemedelsbärare. I detta projekt producerades ett bibliotek av hyperförgrenade linjär-dendritiska material via Fischer esterifikation reaktionen som är en snabb, billig och uppskalningsbar produktionsmetod. Vidare post funktionaliserades materialen med allyl grupper för produktion av nano geler genom UV-inducerad korslänkning och vidare funktionalisering. Samtliga producerade hyperförgrenade linjär-dendritiska material hade förmågan att bilda miceller i vatten. Materialen med bäst micelle bildningsförmåga användes för att kemiskt korslänka dem och producera nano geler. Nano gelernas inre del funktionaliserades framgångsrikt med tre olika funktionella grupper; katjoniska, anjoniska och hydrofoba via resterande fria allyler. Detta påvisar att dessa dendritiska nano geler har potential att bära olika material som hydrofobiska läkemedel eller genetiskt material. Dom producerade nano gelerna hade en hydrodynamisk volym inom intervallet 124-200 nm. Detta är fördelaktigt då dem kan transporteras till tumörområdet via ökad permeabilitet och retention, också kallad EPR effekten, utan att initiera ett immunologiskt svar eller filtreras från blodomloppet via njuren.
The development of nano- based drug carriers is of high importance in anti-cancer treatment as anticancer drugs suffers from limitations as low aqueous solubility, non-selective targeting, off-target degradation and low therapeutic concentrations at target site. Hyperbranched polymers are potential candidates as drug carrier due to its unique properties as globular shape, high number of functional groups and high degree of branching. In addition, hyperbranched polymers are synthesized via one-step polymerization reaction with high yields, low costs and good scale-up possibilities. In this project a library of hyperbranched linear-dendritic hybrid materials based of 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) and monofunctional poly (ethylene glycol) (mPEG) was synthesized via the Fischer esterification reaction. The materials were then post functionalised with hydrophobic allyl groups. The materials self-assembled into micelles in water and candidates with best self-assembly ability were used to fabricate dendritic nanogels by UV-induced cross-linking. The formed dendritic nanogels obtained a hydrodynamic volume between 124-200 nm, which indicates that these dendritic nanogels can be used as drug carrier and accumulate at target-site via the enhanced permeability and retention (EPR) effect. The dendritic nanogels inner core was also successfully attached with cationic, hydrophobic and anionic groups respectively. This confirmed that the dendritic nanogels have the potential to encapsulate different types of cargo such as DNA or hydrophobic drugs in the inner core.

L’objectif de ce travail est d’une part (i) étudier le comportement de la biomasse notamment la production d’EPS en présence des composés pharmaceutiques (un agent anticancéreux et cinq antibiotiques); et d’autre part, (ii) étudier les EPS dans le contexte de décantation des boues en présence d’agents fongiques et en situations réelles dans des stations d’épuration. L’étude en présence de l’agent anticancéreux a été réalisée dans des bioréacteurs à membranes. La présence de l’agent anticancéreux a induit l’augmentation de la production d’EPS agissant comme un mécanisme de protection microbienne qui était à l’origine du colmatage des membranes. L’effet de cinq antibiotiques a été évalué en réacteur batch. La famille des macrolides a montré un effet plus important sur l’activité microbienne avec une augmentation significative de la production d’EPS associée à un mécanisme de protection. La décantation des boues en présence des cultures fongiques a été conduite en réacteur pilote. Une amélioration spectaculaire de la décantation a été liée à une meilleure cohésion au sein des flocs imputable en grand partie à l’augmentation de la production d’EPS. Enfin, le diagnostic du procédé de traitement des eaux a été établi sur trois stations d’épuration des papeteries grâce à une double approche d’une part l’analyse physico-chimique des boues et d’autre part, l’exploitation statistique d’analyse en composantes principales (ACP) des paramètres technologiques enregistrées dans chaque station. Nous avons tenté d’exprimer sous forme de régressions linéaires ou polynomiales de deuxième degré, la décantation en fonction d’une quantité réduite des paramètres mesurés
The objective of this work is firstly, i) to study the microbial behaviour of the biomass especially the production of the EPS in the presence of pharmaceutical compounds (an anticancer product and five antibiotics); and secondly, ii) to study EPS in the context of the sludge settling in wastewater treatment plants. The study in the presence of the anticancer product was done in membrane bioreactors. The presence of the anticancer product provoked the production of the EPS as the protection mechanism which is at the origin of the membrane fouling.The effect of five antibiotics was evaluated in batch reactors. The family of macrolides showed the most important effect on the microbial activity with a significant increase of the EPS production which was associated with a protection mechanism.Sludge settling in the presence of fungi was carried out in a pilot reactor. The spectacularly improvement of the sludge settleability was related with a better cohesion inside of the flocs attributed to an increase of the EPS production.Finally, the diagnosis of different wastewater treatment processes was established in three paper mill wastewater treatment plants thanks to the double approach used here, the physico-chemical analysis of the sludge and the statistical analysis by principal components analysis of the different parameters recorded in each plant. We tried to describe the parameter related to the settling behaviour by linear or polynomial regressions of second degree in function of a reduced number of the measured parameters

Since the discovery of anti-tumor activity of cisplatin in 1960, significant progress has been made in treating metastatic or advanced cancer with cisplatin and platinum compounds. Platinum compounds covalently bind to DNA and disrupt DNA function. They are also known to bind with amino acids like methionine, histidine and cysteine to form cisplatin-protein adducts which are responsible for most of its cytotoxicity and side effects. Recent articles on cisplatin-protein have shown that adding bulky adjuncts to cisplatin or using different platinum compounds varies the degree and extent of reaction thus possibly reducing cisplatin resistance and side effects. One of the proteins to study is cytochrome C, which is an intermediate in apoptosis (a controlled form of cell death used to kill cells in the process of development or in response to infection or DNA damage). Cytochrome C activates caspase 9, a cysteine protease, which in turn goes on to activate caspases 3 and 7, which are responsible for destroying the cell from within. In this study, we tried to examine how various platinum compounds like cis-Pt(NH3)2Cl2, cis-Pt(NH3)2(NO3)2, Pt(en)(NO3)2, Pt(Me4en)(NO3)2, Pt(NH3)2 (oxalate), Pt(en)(oxalate),Pt(Me4en)(oxalate), which have different ligands/bulk, react with cytochrome C in different physiological conditions. This research project subsequently focused on three main aspects: 1) to determine whether the concentration of platinum compounds made a difference in the reaction rate, 2) to determine whether the pH of the buffer shows any difference in the reaction rate, 3) to determine how the ligands coordinated to the platinum affected the rate. We used 1) HPLC with vitamin B12 (cyanocobalamin) as an internal standard. 2) Separate samples of platinum compounds with bovine serum albumin were then subjected to dialysis and were then sent to the Materials Characterization Center for analysis by ICP-AES spectroscopy. In summary, the following conclusions are stated: •The leaving group, pH, bulk and the concentration play a very vital role in determining the reaction rate for platinum-cytochrome C interactions. •Chlorides form excellent leaving groups followed by oxalates then nitrates. •Pt(en) reacts faster than Pt(NH3)2 which reacts faster than Pt(Me4en). •Nitrates, Pt(en) and few oxalate form multiple products showing non-specific binding. Only cis-Pt(NH3)2Cl2 and Pt(Me4en)(oxalate) formed predominately a single product showing target specific binding. •cis-Pt(NH3)2Cl2 showed an increased reaction rate at lower pH while cis-Pt(NH3)2(NO3)2 and Pt(Me4en)(NO3)2 showed higher reactions at higher pH. •Despite platinum compound was present in significant molar excess relative to cytochrome C, at the end of 21 hrs there was a significant amount of unreacted cytochrome C left except in case of cis-Pt(en)Cl2 which reacted with the whole cytochrome C in less than ten minutes. •We saw the rate of reaction in order of cis-Pt(en)Cl2 > Pt(en)(oxalate) > cis-Pt(NH3)2Cl2 > Pt(en)(NO3)2 > cis-Pt(NH3)2(NO3)2 > cis-Pt(NH3)2(oxalate) > Pt(Me4en)(oxalate) > Pt(Me4en)(NO3)2

La délocalisation de la chimiothérapie de l’hôpital vers le domicile et l’arrivée des anticancéreux oraux transforment radicalement le schéma thérapeutique des patients atteints de cancer. Ce dernier, inédit dans le domaine de l’oncologie, fait reposer l’efficacité du protocole sur la responsabilité entière du patient, de la délivrance du médicament à la gestion des effets secondaires. Cet éloignement de la sphère hospitalière et des soins de support amène les patients à gérer également seuls les conséquences psychologiques de la maladie. La première étude de cette thèse est transversale avec une finalité comparative entre les représentations de la maladie et des traitements, le sentiment d’auto-efficacité et la qualité de vie des patients traités par anticancéreux oraux et ceux traités par voie périphérique. Les analyses de régression montrent que le sentiment d’auto-efficacité et les représentations sont des variables médiatrices de la relation forme du traitement – qualité de vie. La seconde étude consiste en l’élaboration d’un programme d’éducation thérapeutique et son évaluation. Une étude préliminaire de faisabilité auprès de patients testeurs est réalisée. Puis, la seconde partie de cette étude est exploratoire, l’objectif étant de montrer les effets de l’éducation thérapeutique immédiats et/ou à long terme. Une méthodologie mixte permet d’explorer l’impact psychologique de l’annonce et de l’initiation du traitement oral, de cibler les besoins éducatifs des patients et préciser si ceux-ci sont en adéquation avec les recommandations de la littérature. De plus, les analyses statistiques évaluent l’évolution des représentations de la maladie et des traitements, le sentiment d’auto-efficacité et la qualité de vie des patients. Enfin, la comparaison des résultats des participants à l’éducation thérapeutique et ceux d’un groupe contrôle permet de rendre compte de l’efficacité du programme
The transfer of chemotherapy from the hospital towards the home patient and the increase of oral anticancer drugs prescriptions has radically transformed the therapeutic pattern for cancer patients. It is unprecedented in the oncology field and it increase the patients’ own responsibility for the effectiveness of the protocol from the dispensing to the management of side effects. Being away from healthcare professionals and supportive care leads patients to also manage the psychological consequences of the illness. The first study is transversal with a comparative purpose between the representations of disease and treatments, self-efficacy and the quality of life of patients treated with oral cancer drugs and those treated with intravenous chemotherapy. Regressions analysis indicate the role of self-efficacy and representations as mediating variable in the relationship administration route – quality of life. The second study aim to develop and evaluate a therapeutic patient education. A preliminary feasibility study with test patients is carried out. Then, the second part of the study is exploratory. The aim is to show the immediate and/or long term effects of therapeutic education. The mixed methodology explores the psychological impact of the disease announcement and the initiation of the oral treatment, the educational patients needs and whether the are consistent with the recommendations of the literature. In addition, statistical analysis assess the evolution of disease and treatment representations, the self-efficacy and the quality of life for cancer patients. Finally, comparing the results of participants in therapeutic education with tose of a control group shows the effectiveness of the therapeutic education

Epigenetic transcriptional gene silencing plays a fundamental role in cancer development and has been considered as a target for cancer therapy in the last few years, mainly due to its reversibility by small molecules. Among the several methylated genes investigated, glutathione-S-transferase CGST) PI, a protein belonging to cellular detoxification systems, has been shown to be extensively promoter-methylated in prostate cancer. My study therefore describes a new therapeutic approach against prostate cancer, based on the combination of demethylating agents and brostallicin, a DNA minor groove binding drug, which is activated in the cell by binding to glutathione, a reaction catalyzed by GST. Among the demethylating molecules tested on the prostatic cancer cell line LNCaP in in vitro combinations with brostallicin, zebularine was able to increase brostallicin activity with little toxicity compared to the other tested demethylating drugs. These in vitro results prompted the in vivo testing of zebularine with brostallicin on LNCaP cells transplanted in mice. Prolonged treatment with zebularine was able to significantly improve brostallicin antitumour activity compared to both drugs administrated as single agents. When GSTPI expression was investigated in treated samples versus untreated controls, no protein re-expression was found and this was related to the unchanged levels of GSTPI promoter methylation. In contrast, the demethylating effect of zebularine was clearly evident in the promoter of GSTMI gene, which is also silenced by methylation in LNCaP cells. GSTMI codes for a class of GST. enzymes that has recently been found to be more active on brostallicin than GSTPI. This indicates that the activation of brostallicin cytotoxicity in LNCaP cells by zebularine likely depends on enzymatic activation by GSTMI rather than GSTPI and strengthens the feasibility of this combination as a treatment for prostate cancer in the clinic, and as model for the therapy of other solid tumours.

In response to DNA damage, cell survival can be enhanced by activation of DNA repair mechanisms and of checkpoints that delay cell cycle progression to allow more time for DNA repair. G₂ checkpoint inhibitors can force cells arrested in G₂ phase by DNA damage to enter mitosis thereby enhancing the killing of cancer cells by DNA-damaging approaches. Checkpoint inhibitors represent a promising class of therapeutic agents in the treatment of cancer. The goals of my research were to (i) characterize novel checkpoint inhibitors in terms of their therapeutic potential and, if possible, mode of action, (ii) increase the therapeutic properties of checkpoint inhibitors by combining them with DNA repair inhibitors, and (iii) develop a therapeutic use of the checkpoint inhibitor, isogranulatimide. Two novel checkpoint inhibitors were psilostachyin and cryptofolione. The target of psilostachyin may be Wee1, a kinase which inhibits CDK1. Cryptofolione shares structural and biological properties with leptomycin B, a highly specific inhibitor of Crm1-mediated nuclear export. This suggests nuclear export inhibition may be a novel means to achieve checkpoint inhibition. Inhibiting both repair and the checkpoint with drugs might cause cancer cells to undergo cell division in the presence of lethal amounts of un-repaired DNA. However, interfering with DNA repair via inhibition of DNA-dependent protein kinase (DNA-PK) reduces the ability of checkpoint inhibitors to abrogate G₂ arrest, as well as their radio sensitizing activity. Components of the checkpoint pathway were highly over-activated in this situation. This suggests that combining checkpoint inhibition with targeted DNA repair inhibition may not be therapeutically viable. Varying the time of addition of checkpoint inhibitors following DNA damage revealed that G₂ checkpoint abrogation alone is insufficient for radio sensitization. Instead, checkpoint inhibitors must be present for the duration of S-phase progression to achieve radio sensitization. Taken together, these studies describe the search for and development of checkpoint inhibitors, ideally for future therapeutic use. These results have relevance to the development of G₂ checkpoint inhibitors as experimental therapeutic approaches to the treatment of cancer. This research represents a major step forward in the pre-clinical development of checkpoint inhibitors as anticancer agents.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

Submitted in complete fulfillment for the Degree of Doctorate of Philosophy in Biotechnology, Durban University of Technology, Durban, South Africa, 2016.
Plants have provided a source of medicine from the beginning of human history and are the core of modern medicine. Moreover, plant based drug discovery has led to the development of various anticancer drugs (such as vincristine, vinblastine, etoposide, paclitaxel, camptothecin, topotecan and irinotecan). The use of botanical, phytochemical, biological and molecular techniques have facilitated the discovery of anthraquinones from Ceratotheca triloba that can inhibit the human topoisomerase II enzyme (target for anticancer drugs) and kill cancer cells. However, the C. triloba plant has not been extensively studied for its anticancer activity. Therefore, the aim of this study was to further investigate the anticancer activity of C. triloba and determine the classes of compounds that contributed towards its activity. In this study the leaf and root extracts were prepared by using hexane, DCM, hexane: DCM (1:1), methanol and/or water. These extracts were examined for their growth inhibitory potential on three cancer cell lines (A375 [melanoma], MDA-MB-231[breast] and WHCO1 [esophageal]) by using the MTT assay. Then, different mobile phases were prepared for optimizing the separation of the compounds of the active extract by TLC. Column chromatography was performed with the active extract by using five mobile phases (hexane : DCM [60 : 40, 40 : 60], DCM, DCM : ethyl acetate [90 : 10, 70 : 30, 60 : 40, 50 : 50, 50 : 60, 30: 60, 20 : 80], ethyl acetate and ethyl acetate: methanol [80 : 20, 70 : 30, 50 : 50]). The fractions collected from the column were examined for their growth inhibitory potential on two melanoma cell lines (A375 and UACC-62). The IC50 and TGI (total growth inhibition) values of the active fractions were determined. Also, the apoptosis inducing effects of the active fractions and standards (camptothecin and doxorubicin) were determined by using flow cytometer based assays (FITC annexin assay, PE active caspase 3 assay and BD MitoScreen assay). Subsequently, the chemical structures of the compounds that contributed towards the activity of these fractions were obtained by EI-LC-MS analysis. The results demonstrated that the hexane root extract exhibited the best percentage of growth inhibition (%GI) on all three cancer cell lines. The separation of the compounds of the hexane root extract was optimized on TLC plates by using different ratios of hexane and DCM. Column chromatography allowed for fractionation of this extract. Purified compounds were not obtained due to co-elution. Further research would have to be conducted to obtain purified compounds. This may involve the use of mini-column chromatography and PTLC. Overall a total of ten combined fractions were collected from the column. Four of these fractions (F2, F4, F5 and F8) displayed a high %GI on the A375 and UACC-62 cell lines. Moreover, fraction F4 was the most active fraction as it had the lowest IC50 (0.70 µg.ml-1 [A375] and 0.39 µg.ml-1 [UACC-62]) and TGI (12.50 µg.ml-1[A375] and 25 µg.ml-1 [UACC-62]) values in comparison to the other fractions. All four fractions induced depolarization of the mitochondria membrane potential (ΔΨ), caspase 3 activation, early apoptosis (phospholipid phosphatidylserine exposure) and/or late apoptosis in the melanoma cells. The results also revealed that fraction F4 (25 µg.ml-1) induced depolarization of the ΔΨ in a higher percentage of A375 (78.11%) and UACC-62 (87.4%) cells than the other fractions and standards. This fraction also induced caspase 3 activation in a high percentage of A375 (90.56%) and UACC-62 (96.78%) cells. Therefore fraction F4 was also the most active fraction in terms of apoptosis activity. Based on our results and literature findings we can deduce that the active fractions induced the intrinsic or extrinsic (type II) apoptosis pathway in the melanoma cells. Six classes of compounds were identified from the four active fractions. These were: benzothiophenones, benzopyranones, naphthoquinones, anthraquinones, androstanes and quinazolines. In conclusion, this is the first study that evaluated the growth inhibition potential of the leaf and root extracts of C. triloba on a panel of cancer cells. This research indicated that the hexane root extract displayed the best levels of cell growth inhibition. The active constituents of this extract were isolated into four fractions which elicited apoptosis inducing effects that promoted the extrinsic (type II) or intrinsic apoptosis pathway in the melanoma cells. Furthermore, fraction F4 contained the most active compounds from C. triloba as it had the lowest IC50 and TGI values (in comparison to the other fractions) and induced depolarization of the ΔΨ in the highest percentage of melanoma cells. It was confirmed that six classes of compounds were accountable for the anticancer activity of these fractions. Thus, the C. triloba plant is a rich source of anticancer compounds.
D

Methyl 2-cyano-3,11-dioxo-18b-olean-1,12-dien-30-oate (CDODA-Me) is a synthetic triterpenoid that inhibits growth of Panc1 and Panc28 pancreatic cancer cell lines and activates peroxisome proliferator-activated receptor B (PPARB)-dependent transactivation in these cells. CDODA-Me has also induced p21 and p27 protein expression and downregulated cyclin D1; however, these responses were receptor-independent. CDODA-Me induced apoptosis, which was accompanied by receptor-independent induction of the proapoptotic proteins early growth response-1 (Egr-1), nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), and activating transcription factor-3 (ATF3). Induction of NAG-1 in Panc28 cells was p38-mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3-K)-dependent, but Egr-1-independent, whereas induction in Panc1 cells was associated with activation of p38-MAPK, PI3-K and p42-MAPK and was only partially Egr-1-dependent. Specificity protein (Sp) transcription factors Sp1, Sp3 & Sp4 are overexpressed in multiple tumor types and negative prognostic factors for survival. Since Sp proteins regulate genes associated with survival (survivin), angiogenesis [vascular endothelial growth factor and its receptors] and growth [cyclin D1, epidermal growth factor receptor], research in this laboratory has focused on development of anticancer drugs that decrease Sp protein expression. Arsenic trioxide, curcumin, 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid (CDDO), CDDO-Me, and celastrol exhibit antiproliferative, antiangiogenic and proapoptotic activity in many cancer cells and tumors. Treatment of cancer cells derived from urologic and gastrointestinal tumors with arsenic trioxide decreased Sp1, Sp3 and Sp4 transcription factors and cotreatment with the proteosome inhibitor MG132 did not inhibit downregulation of Sp proteins in these cancer cells. Mechanistic studies suggested that compound-dependent downregulation of Sp and Sp-dependent genes was due to decreased mitochondrial membrane potential and induction of reactive oxygen species, and the role of peroxides in mediating these responses was confirmed using hydrogen peroxide, demonstrating that the mitochondriotoxic effects of these compounds are important for their anticancer activities. Moreover, repression of Sp and Sp-dependent genes by CDDO-Me and celastrol was due to downregulation of microRNA-27a and induction of ZBTB10, an Sp repressor, and these responses were also reversed by antioxidants. Thus, the anticancer activity of CDDO-Me and celastrol is due, in part, to activation of ROS which in turn targets the microRNA-27a:ZBTB10?Sp transcription factor axis to decrease growth inhibitory, pro-apoptotic and antiangiogenic genes and responses.

Thesis submitted to the Faculty of Science under the school of Molecular and Cell Biology, University of the Witwatersrand, in partial fulfilment of the requirements for PhD. Gauteng, Johannesburg, 2015
Cancer is an enormous burden of disease, accounting for millions of deaths annually worldwide. Today, more people are dying from cancer than HIV/AIDS, malaria and tuberculosis combined. According to the American Cancer Society, it is expected that the global cancer burden will double by 2030 if preventative measures are not applied. Breast and gynaecological cancers remains a big scourge in developing countries, with breast cancer being the most common cancer and gynaecological cancers accounting for approximately 25% of all cancers in women in developing countries. Currently, the standard cancer treatments include surgery, radiation and chemotherapy. Adverse toxicities have been associated with these therapies and their effectiveness is also limited to drug resistance. The cost of treatment is another major burden. Limitations associated with these conventional cancer treatments have made discoveries of novel therapeutics which exhibit less toxicity and at a lowered cost of paramount importance. Medicinal plant extracts have recently attracted attention to modern medical science research with their non-lethal activity. Currently, up to 50% of the world drugs including chemotherapeutic drugs such as taxol and camptothecin are made from natural products or their derivatives. In this study we aimed to investigate the anti-cancer properties of the medicinal plant Euphorbia tirucalli. The crude extracts of E. tirucalli extracted using butanol; hexane and methanol solvents were screened for antiproliferative activity in breast (MCF-7 and MDA-MB231), ovarian (RMG-1) and cervical (SiHa) cancer cell lines. MTT assay and Real-Time Cell Analyzer (RTCA), xCELLigence were used to determine cytotoxicity of the extracts and calculate IC50. From MTT and xCELLigence results, we observed that E. tirucalli extracts exhibited dose-dependent inhibition of cell proliferation with RMG-1 and MCF-7 cells being more sensitive than MDA-MB231 and SiHa cells to all three extracts for an unclear reason. The butanol extract appeared to exhibit iv the most cytotoxicity with all cell lines reaching IC50 at low extract concentrations. Most therapies in anticancer treatment, such as chemotherapy, mainly induce cell death by causing either G0/G1 or G2/M cell cycle arrest and then inducing an apoptotic pathway. Therefore, cell cycle arrest and the induction of apoptosis in cancer cells become the major indicators of anticancer effects. Cells were stained with propidium iodide dye to determine if cells were arrested at G0/G1 or G2/M cell cycle stages while annexin V and PI staining were used to determine the type of cell death induced by the extracts. Cell cycle analyses revealed MCF-7, MDA-MB231 and SiHa cancer cells underwent arrest at G0/G1 following treatment with the plant extracts. Annexin V and PI staining revealed different proportions of apoptotic and necrotic populations. The extracts mainly induced apoptosis on MCF-7 and MDA-MB 231 cells, with the butanolic extracts inducing the most apoptosis. RMG-1 and SiHa cells had a high proportion of cells undergoing both late apoptosis and necrosis. The molecular mechanism of cell death induction was investigated using real time PCR and western blot. From the gene expression studies, p21 was observed to be over expressed in all cells following all treatments, in line with the observed cell cycle arrest at G0/G1. The extrinsic pathway of apoptosis was identified as the type of cell death induced with caspase 8 being overexpressed in MDA-MB 231 cells treated with butanol and hexane extracts. Upon further fractionation, flavonoids and especially isorhamnetin were identified as the active compounds in these extracts. Overall, the plant contains compounds that have some activity against cell proliferation and can be a promising tool to treat cancer cells. However, more work needs to be done to verify which compounds are mainly involved.

In recent years, medicinal plants have become popular for the treatment of several diseases due to their efficacy and cost effectiveness. Plant derived therapeutic agents are increasingly sought out as pharmaceuticals for the treatment of life-threatening illnesses. Important contributions have already been made in the recent years to the drug market by the compounds isolated from natural sources or from their derivatives. There is no doubt that, novel lifesaving drugs could be discovered by a systematic evaluation of ethno medicinal information using modern scientific tools. This thesis attempts to systematically combine ethnomedicinal knowledge together with the contemporary scientific methods to evaluate immunomodulatory and anticancer properties of sixteen traditional medicinal herbs with an aim to discover anticancer formulations. The major objective of this project is to isolate and characterise potent anticancer polysaccharides from Traditional Chinese Medicinal (TCM) herbs. In order to achieve these objectives, sixteen Chinese Medicinal herbs have been successfully screened for their antioxidant, immunomodulatory and anticancer activities. Based on their immunomodulatory and anticancer activities, three traditional anticancer TCM herbs, namely, Amauroderma rugosum, Lobelia chinensis and Artemisia annua have been selected for further studies. Isolation and characterisation of polysaccharides from these three medicinal herbs has been carried out. The structural characterization employing FT-IR and NMR spectroscopy has been carried out and presented in Chapters 5 to 7. A total of eight potent immuno-therepuritic polysaccharides have been discovered for the first time from the three important anticancer TCM herbs (A. annua, L. chinensis and A. rugosum). The structure of three of them have been determined. The MRI contrast potentials of Gd(III) complexes of three of the herbal polysaccharides have also been studied. Low molecular weight polysacchro-proteins were found to be T1 agents and high molecular weight polysacchro-proteins were T2 agents. Findings of this thesis have contributed significant scientific knowledge that has offered some suggestions for designing new immuno-therepeutic formulations based on the eight polysaccharides discovered in this thesis. However, further research is required in order to design such formulations.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library.
The aim of the work described in this thesis was initially to screen the ethanol extracts of eleven Indonesian ginger species (Zingiberaceae family) for anticancer activity. MCF-7 breast and HT-29 colon cancer cells were used for the investigations. Extracts of Zingiber aromaticum and Boesenbergia pandurata were found to be the most active species, similar to that of Curcuma langa which has been shown to possess anticancer activity in vitro and in vivo (Aruna and Sivaramakrishnan, 1992; Azuine and Bhide, 1992). These two active species were then further investigated. Bioactive compounds from the species were isolated and identified using various chromatography procedures and nuclear magnetic resonance (NMR) and their anticancer activities were further tested on MCF-7 breast and HT-29 colon cancer cells including cell cycle analysis and measurements of apoptosis. The ethanol extracts of these two active species were also investigated using the AOM-induced colon cancer model in rats. The antiinflammatory activity of the ethanol extract of Z. aromaticum was also investigated using dextran sulfate sodium (DSS) induced ulcerative colitis (UC) in rats. The inhibitory activity of ethanol extracts of rhizomes of 11 ginger species was initially tested against MCF-7 breast and HT-29 colon cancer cells using colorimetric tetrazolium salt (MTT) assay. Ethanol extracts of eight species (Amommum cardamomum, C. longa, C. mangga, C. xanthorrhiza, Boesenbergia pandurata, Zingiber aromaticum, Z. officinale, Z. cassumunar) showed a strong inhibitory effect on the growth of the cancer cells with the IC50 concentrations between 100-100 g/ml. The ethanol extract of Curcuma aeruginosa was less active (IC5o between 100-120 g/ml) and extracts of Kaempferia galangal and K. rotunda had no effect on the growth of either cell lines at concentrations up to 250 g/ml. Ethanol extract of C. longa was used as a comparison since curcumin, an active compound isolated from this species, has had demonstrated its anticancer activity in vitro, in vivo and is currently undergoing clinical trial against colon cancer (Greenwald, et al., 2001; Sharma et al., 2001). Extracts of Z. aromaticum and B. pandurata had very strong inhibitory activity similar to the extract of C. longa. Curcumin was not detectable in either Z. aromaticum or B. pandurata. The ethanol extracts of the active species were not toxic on human skin fibroblast cells (SF 3169). The ethanol extracts of Z. aromaticum and B. pandurata were further fractionated using two different solvents by reversed phase preparative HPLC. Fraction A was eluted with a mobile phase containing 5% vlv aqueous methanol containing 0.025% v/v trifluoroacetic acid (TFA) and fraction B was eluted with 100% methanol. The inhibitory activity of fractions was then investigated against HT-29 colon cancer cells and assayed using the MTT assay. Zerumbone, a sesquiterpenoid compound was isolated from fraction B of the extract of Z. aromaticum and a chalcone derivative, panduratin A was isolated from fraction B of the extract of B. pandurata. Curcumin was in fraction A of extract of C. longa. The anticancer activity of zerumbone and panduratin A was investigated using MCF-7 breast. HT-29 and CaCo-2 colon cancer cells. The inhibitory activity of the active compounds was assessed using the MTT assay. The ICso of zerumbone in each of the cell lines was about 10 uM and of curcumin on HTU29 cells was 25 uM. The IC50 of panduratin A in HT-29 cells was 16 uM and in MCF-7 cells was 9 uM. Zerumbone and panduratin A showed antiproliferative effects by alteration of the DNA distribution in the cell cycle and induction of apoptosis. HT-29 cells treated with zerumbone at concentrations of 10 -25 uM or panduratin A at concentrations of 9 -65 uM for 24 h were stained with propidium iodide (PI) to determine cell cycle distribution and analysed using FACScan flow cytometry. The proportion of cells in the S phase was reduced from 18.7% in untreated cells to 10.2% in HT-29 cells after treatment with zerumbone at 10 uM to 3.1% at 25 uM. Cells in the G2 phase increased from 18.5% at 10 uM to 40% at a concentration of 25 uM. Panduratin A increased the proportion of cells in the GO/G1 phase from 33% of untreated cells to 71% after treatment with 65 uM for 24 h. Panduratin A slightly reduced the proportion of cells in S phase and cells in G2/M phase also decreased from 36,8% in untreated cells to 15.4% at 65 M. Apoptosis was determined using double labelled (Annexin-V-Fluos and PI) and then evaluated using FACScan Flow Cytometry. Morphological features of apoptosis were also examined using DiffQuick stain and fluorescent Hoechst 3355 and 4,6-diamino-2-phenylindole (DAPI). Zerumbone induced apoptosis in HT-29 cells in a dose dependent rnanner, At 48 h, 2% of cells treated with 10 M of zerumbone underwent apoptosis, which increased to 8% when treated with 50 M, Panduratin A at 28 M increased the number of cells undergoing apoptosis from 2,2% to 16.7% when treated with a concentration of 65 M. The ethanolic extracts of Z. aromaticum and B. pandurata were also investigated using the azoxymethane (AOM) induced aberrant crypt foci (ACF) model of colon cancer in rats in a short and long term study. Ethanolic extracts of C. tonga and curcumin were used as comparison. The basal diet used throughout all animal studies in this thesis was a semi-purified AIN-93 G diet (Reeves et aI., 1993). ACF were induced by two doses (15 mg/kg BW) subcutaneously of AOM one week apart and ACF were visualised in the formalin fixed colon using methylene blue stain. The ACF study was run over a short (5 weeks) and long (13 weeks) experiments. Diets containing ethanol extracts prepared from the equivalent of 2% (w/w) dried rhizome of Z. aromaticum, B. pandurate or C. tonga in a short term study did not affect the formation of ACF in rats compared to those in the control diet group. The ACF formation in a short term study was dominated by small numbers of aberrant crypts (1 or 2) per focus. It is suggested that large ACF (4 or more ACs/focus) are better predictors of colon cancer (Uchida et aI., 1997; Jenab et aI., 2001). Diets containing ethanol extracts of the equivalent of 4% by weight of dried rhizomes of Z. aromaticum, B. pandurata, C. longa were investigated over 13 week study, Total ACF were significantly reduced by Z. aromaticum extract (0.34%) in the diet (down 21%, p<0.05) relative to rats fed the control diet. A similar reduction was observed with C, longa extract (0.86%) in the diet (down 24%, p<0.01) and with 2000 ppm curcumin. There was no significant different in small ACFs (1-2 ACs/ focus) between dietary treatments. The number of foci containing 3-4 ACs/focus was significantly reduced (35%, p<0,001) in animals fed the Z. aromaticum extract and 34% (p<0.001) of animals fed the C. tonga extract. The total number of ACF containing 5 or more ACs per focus of animals fed 0.34% Z. aromaticum extract was 41 % lower than control (p<0.05) and for 0.86 % C. tonga extract was 22% (not significant). A diet containing extract (0.56%) of B. pandurata did not significantly affect the formation of ACF compared to the control AIN group. The concentration of zerumbone in the Z.aromaticum extract diet was assayed at 300 ppm, and of curcumin in the C. tonga extract diet was also 300 ppm. The concentration of panduratin A was not assayed in the diet due to late identification of the active compound. The antiinflammatory activity of ethanol extract of Z. aromaticum was investigated using dextran sulfate sodium (DSS) induced ulcerative colitis in rats. Sulfasalazine, a widely used compound to treat inflammatory bowel disease (IBD) in humans was used as the positive control. Diets containing ethanol extracts (0.34% and 0.68%) prepared from the equivalent of 4% and 8% by weight of dried rhizomes of Z. aromaticum were given to the animals throughout the experiment. On day three, rats were given 2% DSS in drinking water for 5 d and then just water for 3 d and then were killed. During the DSS treatment rats were maintained in metabolic cages, body weight, food and fluid intake and clinical symptoms such as consistency of stools and blood in faeces were recorded daily. There was slight but not significant reduction in the body weight of rats fed 0.68% extract of Z. aromaticum in the diet due to reduced food consumption. The extract of Z. aromaticum (0.34%) and sulfasalazine suppressed clinical signs of ulcerative colitis. Eleven percent of the controls were hemoccult positive on day 2 after DSS administration, which progressed further by day three with 67% being hemoccult positive and 100 % on day five. By comparison, blood appeared on day 3 of rats treated with diet containing 0.34% and 0.68% extract of Z. aromaticum and 0.05% sulfasalazine, and only 33%, 67% and 22%, of rats being hemoccult positive on day 5 respectively. The disease activity index (DAI) of rats fed diet containing 0.34% extract of Z. aromaticum was about 0.4 and similar to those which were fed with diet containing sulfasalazine. The DAI of untreated rats was 1.4. The crypt score of rats fed the extract of Z. aromaticum was slightly reduced but it was not significantly different from those of untreated rats. Other histological scores were not significantly different between dietary treatments. Extract of Z. aromaticum significantly decreased the content of PGE-2 in colon tissue compared to that of untreated animals. There was a reduction of TX8-2 content in colonic tissue of rats fed with extracts of Z. aromaticum but this was not significant. The activity of myeloperoxidase (MPO) activity in the colonic tissue of rats fed with sulfasalazine was significantly lower than that of the untreated controls and those which fed with extracts of Z. aromaticum. The results from the studies performed in this thesis showed that extract of Z. aromaticum which contains an active sesquiterpenoid zerumbone have anticancer and antiinflammatory activity suggesting that the extract may have benefits as a chernopreventative agent. However further studies are needed to elucidate their other pharmacological actions. Panduratin A showed potential anticancer activity in cell culture in vitro. However an extract of B. pandurata did not have effect on the AOM-induced colon cancer model. Different cancer models such as breast and prostate cancer could be used to further investigate the anticancer activity of extract of B. pandurata and panduratin A and to elucidate their mechanism.
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Thesis (Ph.D.) -- University of Adelaide, Dept of Medicine, 2003

Chemotherapy is a primary source of treatment for victims of cancer, a globally prominent disease that affects millions. The platinum(II) agents cisplatin, carboplatin and oxaliplatin are used in approximately half of all chemotherapy treatment schemes. These drugs kill cancerous cells by binding covalently to DNA and preventing replication, which leads to apoptosis. Despite the success of these drugs they have many debilitating side effects such as nephrotoxicity, myelotoxicity, nausea and vomiting. Furthermore, many cancers are resistant to treatment from these drugs, often through DNA repair mechanisms. To overcome these issues, researchers are developing platinum complexes (PCs) that kill cancerous cells through different mechanisms of action to cisplatin. A promising series of PCs in this field are those of the type [Pt(PL)(AL)]2+, in which PL is a polyaromatic heterocyclic ligand and AL is a cyclic diamine. Relative to cisplatin, these polyaromatic PCs (PPCs) are more cytotoxic to cancer cells, kill these cells through different mechanisms, and bind to DNA through noncovalent interactions. Due to these characteristics, PPCs have the potential to surpass traditional platinum drugs as chemotherapy candidates. This potential is further amplified when they are oxidised from platinum(II) to platinum(IV), resulting in complexes of the type [Pt(PL)(AL)(X)2]2+, where X is an axial ligand such as hydroxide or succinimide. The PCs can be tuned, through modification of these axial ligands, to target cancer cells selectively, improve bloodstream stability, and to selectively reduce to the active platinum(II) form once inside a cancer cell. In this work, reported in four published journal articles together with some additional experiments, several novel PPCs have been synthesised and tested for their viability as DNA binders and anticancer agents. Several PL and AL combinations were explored, most of which were new to this series of PCs. All PPCs were characterised through nuclear magnetic resonance, elemental microanalysis, ultraviolet (UV) spectroscopy, electrospray ionisation mass spectrometry (ESIMS), and, where applicable, circular dichroism (CD) and X-ray crystallography. Most of the PCs adopted a square-planar coordination geometry, although the geometry of 2-(2ꞌ-pyridyl)quinoxaline complexes was distorted, leading to unusual CD, diffusion and crystal packing activity. All PPCs were produced with desirable purity and yield. The anticancer potential of the PPCs was assessed in several human cancer cell lines, revealing high in vitro cytotoxicity across a wide variety of cancers, often higher than that of cisplatin, oxaliplatin and carboplatin. Most of the PCs were particularly active against Du145 prostate cancer, HT29 colon carcinoma and SJ-G2 glioblastoma cells. It was found that while both the choice of PL and AL affected activity, the AL choice was more impactful. Considering that the PL is the component of the PPCs responsible for DNA binding, this is suggestive that DNA interactions are not the primary mechanism of action of these complexes.

碩士
嘉南藥理大學
生物科技系
103
Hepatocellular carcinoma (HCC) is the third most common malignancy worldwide. More than 75% of HCC cases occur in the Asia-Pacific region. In Taiwan, there are about 3,700 patients a year die from liver cancer. There are many chemical drugs have been used in liver cancer therapy. Antrodia cinnamomea is a medicinal fungus that has been served as a functional food in Taiwan for decades. Previous studies have shown that the secondary metabolites form Antrodia cinnamomea have various activities such as antitumor, protection of liver function, anti-inflammation and antioxidant. However, the safety and the possible interaction of Antrodia cinnamomea extracts with drugs in liver cancer patients have been not studied yet. In the current project, we want to identify the possible role and mechanisms of Antrodia cinnamomea extracts in modulating the activity of drugs in hepatoma cells both in vitro and in vivo. First, Antrodia cinnamomea extracts (EAC) were isolated from culture medium using ethanol. Our preliminary results indicated that the exposure to high concentrations of drugs (5-25 M) or Antrodia cinnamomea extracts (200-250 g/ml) for 48 h caused significant death in Huh-7 and HepG2 cells. In addition, EAC could sensitive tumor cells to low dose of drugs in Huh-7 and HepG2 cells.Considering the cell morphology, the combination of drugs with EAC caused progressive changes in Huh-7 cells from flat to round. Therefore, our preliminary results indicated that EAC could have a positive role in the application of drugs for the treated hepatocellular carcinoma. In this paper, we will further analyze the effect of combination on cell cycle for Huh-7 cells. The percentage of G2/M phase cells notably increased. Then further analysis of cell cycle related proteins on Hun-7 cells, the combination-treated has inhibition. In animal models, the combination significantly inhibition of tumor growth. These results could provide important insight in chemotherapy and safety of Antrodia cinnamomea.

Ph.D.
Ligands bis(pyrazolyl)acetic acid (L1) and bis(3,5-dimethylpyrazolyl)acetic acid (L2) were synthesised by reacting pyrazoles and dibromoacetic acid under phase transfer conditions, by using benzyltriethylammonium chloride as the catalyst. Ligands L1 and L2 were characterised by a combination of 1H, 13C{1H} NMR, IR spectroscopy and microanalysis. Esterification of L1 and L2 led to formation of bis(pyrazolyl)ethyl acetate (L3) and bis(3,5-dimethylpyrazolyl)ethyl acetate (L4). Ligands L3 and L4 were also characterised by a combination of 1H, 13C{1H} NMR, IR spectroscopy and microanalysis. Subsequently, new pyrazolyl palladium(II) and platinum(II) compounds, [PdCl2(L1)] (1), [PdCl2(L2)] (2), [PtCl2(L1)] (3a) and [PtCl2(L2)] (4) were prepared by reacting bis(pyrazolyl)acetic acid ligands (L1-L2) with K2[PdCl4] or K2[PtCl4] respectively. The structures of complex 1 and 2 reveal distorted square planar geometries. The bond angles of N-Pd-N, N-Pd-Cl, N-Pd-Cl, for 1 and 2 are between 85.8(3)o and 90.81(4)o). The platinum compound, K2[Pt4Cl8(L1)2(deprotonated-L1)2].2H2O (3b), crystallised from aqueous solutions containing 3a when such solutions were left to stand overnight. Each platinum coordination environment consists of two cis-Cl ligands and one K2-N^N(L1) unit (L1 = bis(pyrazolyl)acetic acid), with two ligand moieties in 3b that are deprotonated with two K+ counter ions. Reaction of bis(pyrazolyl)acetic acid ligands (L1-L2) with [HAuCl4].4H2O gave gold(III) complexes [AuCl2(L1)]Cl (5a) and [AuCl2(L2)]Cl (6a). The spectroscopic, mass spectroscopy and microanalysis data were used to confirm the formation of the desired complexes. However, attempts to crystallise 5a and 6a led to formation of [AuCl2(pz)(pzH)] (5b) and [AuCl2(3,5-Me2pz)(3,5-Me2pzH)] (6b). This was confirmed by the structural characterisation of 5b, which has a distorted square-planar geometry. When complexes 1-6a were screened for their anti-tumour activity against CHO-22 cells, they showed no appreciable biological activities against CHO-22 cells. Substitution reactions of complexes 1-6a with L-cysteine performed to probe any relationship between the observed antitumour activities and the rates of ligand substitution of these complexes were inconclusive. Dithiocarbamate ligands L5-L8 were synthesised as potassium salts by introducing a CS2 group in positions 1 of pyrazole, 3,5-dimethylpyrazole, indazole and imidazole. The reaction of L5-L8 with [AuCl(PPh3)], [Au2Cl2(dppe)], [Au2Cl2(dppp)] and [Au2Cl2(dpph)], led to isolation of complexes [Au(L)(PPh3)] (13-16), [Au2(L)2(dppe)] (17a-19), [Au2(L)2(dppp)] (20-22) and [Au2(L)2(dpph)] (23-25) (dppe = bis(diphenylphosphino)ethane, dppp = bis(diphenylphosphino)propane, dpph = bis(diphenylphosphino)hexane; L = anions of L5-L8). The mononuclear molecular structure of 15 features a near linear geometry with a P(1)-Au(1)-S(1) angle of 175.36(2) o. The binuclear gold(I) complexes 20-22 and 23-25 have two P-Au-S moieties as evident in the solid state structure of 25. Attempts to crystallise complex 17a led to the formation of a gold(I) cluster complex [Au18S8(dppe)6]2+ (17b) as confirmed by X-ray crystallography. Cluster 17b features weak Au···Au interactions (2.9263(7)-3.1395(7) Å). Complexes 13-16 and 20-25 were tested in vitro for anticancer activity on HeLa cells. The activities of gold(I) complexes 13-16 were comparable to that of cisplatin. Dinuclear gold(I) complexes 20-25 also showed appreciable antitumour activity against HeLa cells. However, the dpph gold(I) compounds (23-25) were highly active, with 24 showing the highest activity against HeLa cells (IC50 = 0.1 μM). The tumour specificity (TS) factors for 23 and 24 were 31.0 and 70.5, respectively.

Yes
Enantiomers of a relatively rigid DNA-binding metallo-helix are shown to have comparable activity to that of cisplatin against the cell lines MCF7 (human breast adenocarcinoma) and A2780 (human ovarian carcinoma) but are ca five times more active against the cisplatin-resistant A2780cis. The cell-line HCT116 p53+/+ (human colon carcinoma) is highly sensitive giving IC50 values in the nM range, far lower than the cisplatin control. The hypothesis that the biological target of such metallohelices is DNA is probed by various techniques. Tertiary structure changes in ct-DNA (formation of loops and intramolecular coiling) on exposure to the compounds are demonstrated by atomic force microscopy and supported by circular/linear dichroism in solution. Selectivity for 50-CACATA and 50-CACTAT segments is shown by DNase I footprinting. Various three- and four-way oligonucleotide junctions are stabilised, and remarkably only the L metallo-helix enantiomer stabilizes T-shaped 3WJs during gel electrophoresis; this is despite the lack of a known helix binding site. In studies with oligonucleotide duplexes with bulges it is also shown for the first time that the metallo-helix binding strength and the number of binding sites are dependent on the size of the bulge. In contrast to all the above, flexible metallo-helices show little propensity for structured or selective DNA binding, and while for A2780 the cancer cell line cytotoxicity is retained the A2780cis strain shows significant resistance. For all compounds in the study, H2AX FACS assays on HCT116 p53+/+ showed that no significant DNA damage occurs. In contrast, cell cycle analysis shows that the DNA binders arrest cells in the G2/mitosis phase, and while all compounds cause apoptosis, the DNA binders have the greater effect. Taken together these screening and mechanistic results are consistent with the more rigid helices acting via a DNA binding mechanism while the flexible assemblies do not.