Dissertations / Theses on the topic 'Anticancer drug treatment'

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.

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 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.<br>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.

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.

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.

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<br>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

Thesis (PhD)--Stellenbosch University, 2003.<br>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.<br>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.

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.

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

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.

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.<br>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.

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<br>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

The non-selective nature of anticancer drugs leads to harmful side-effects. This work investigates the branched polymer, poly(acrylic acid), PAA, and its salt, poly(sodium acrylate), PNaA, for their potential as an anticancer drug carrier for better targeted delivery. The binding between the anticancer drug cisplatin and PNaA was investigated using capillary electrophoresis in the critical conditions (CE-CC). The average degree of branching (DB) was also quantified using NMR spectroscopy. The influence of the synthetic parameters on the DB was also investigated. The precision and accuracy of the DB quantification via NMR spectroscopy were estimated. An empirical formula was derived to assess the errors originating in the low signal to noise ratio and the user-dependent data treatment. Solubility, viscosity and moisture content were shown to influence the accuracy of DB quantification. Dissolution for most branched PAAs/PNaAs were incomplete obtaining only a maximum of 54 % for the extent of the dissolution. A set of guidelines on what should be considered for DB quantification was developed not exclusively for PAAs/PNaAs but also for other branched polymers. The DB in PAAs/PNaAs synthesized by conventional radical polymerization (CVRP) and reversible-deactivation radical polymerization (RDRP) were quantified by 13C NMR spectroscopy. The type of polymerization (CVRP or RDRP) does not play a role for the DB in PAAs/PNaAs unlike what was observed in poly(alkyl acrylates). The presence of ethanol or of chain transfer agents resulted in a decreased DB due to patching of the mid-chain radical. The influence of synthetic parameters is important for the design of branched PAAs/PNaAs. Lastly, the binding of the anticancer drug, cisplatin to PNaAs with different branching topologies (linear and hyperbranched) was monitored for the first time using CE-CC. An increase in the electrophoretic mobility (μ) of PNaA was observed over time which suggests the formation of PNaA-cisplatin copolymers. Furthermore, the dispersity of the μ distributions in PNaA was determined to assess the heterogeneity of the composition of the copolymers formed through the binding reaction. Through the dispersity of the μ distributions, the heterogeneity of branching can be represented through which hyperbranched PNaA was observed to be more heterogeneous in branching than linear PNaA. The knowledge obtained from this work enables synthetic chemists to choose polymerization conditions to produce branching architectures of PAA/PNaA that are tailored for desired applications such as drug delivery. The characterization can be further improved by coupling CE-CC to other techniques such as size exclusion chromatography (SEC). With the enhanced knowledge on the branching in PAA/PNaAs, better drug carriers can be designed. The information on the binding process via CE-CC enables optimization of drug loading for effective targeting and delivery in the future. Additionally, the enhanced knowledge on the binding reaction and how the drug binds will be beneficial for clinical applications as CE-CC may be used as a novel approach to monitor the drug activity inside the body. As a result, the information obtained from this work can allow comprehensive knowledge on the dosage relationships specified to an individual patient’s needs therefore, bringing PAA/PNaA closer to being an effective anticancer drug carrier.

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

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.

碩士<br>嘉南藥理大學<br>生物科技系<br>103<br>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.

The thesis “Ancillary Ligand Effects on the Anticancer Activity of Ruthenium (II) Piano Stool Complexes” is an effort to design better antitumor metallodrugs based on ruthenium(II) complexes with various H-bond donor/acceptor ligands and to understand their mechanism of action. Chapter 1 presents a brief review of metallodrugs and their mechanism of action. Different classes of metallodrugs are discussed. A short discussion on ruthenium based anticancer drugs and their established mechanism of action is also included in this chapter. Chapter 2 deals with the synthesis, characterization and anticancer activity of Ru(II) complexes with P(III) and P(V) ligands. The effect of a strong hydrogen bond acceptor on the cytotoxicity of the complexes has been investigated which allows comparison of complexes with ligands possessing a strong hydrogen bond donor or hydrogen bond acceptor. Partial oxidation of the tertiary phosphine ligands leads to a decrease in cytotoxicity of the ligand, while coordination to ruthenium resulted in a significant increase in the cytotoxicity. A molecular mechanism of action for these complexes was suggested on the basis of various biophysical studies. These complexes bind DNA through non-intercalative interactions which lead to the destabilization of the double helix of the DNA and also unwinding of the negatively supercoiled DNA. Results show that the presence of a hydrogen bond acceptor on the ligand is not capable of enhancing interactions with DNA in comparison with hydrogen bond donor groups. Cellular studies of these complexes showed that inhibition of DNA synthesis and apoptosis occur on treatment with these complexes. Interestingly, these complexes are found to be not only cytotoxic but also antimetastatic. Chapter 3 deals with the synthesis, characterization and anticancer activity of Ru(II) complexes with biologically active S containing heterocyclic ligands and their mechanistic study. Complexation of ruthenium with mercaptobenzothiazole (MBT) gave the most cytotoxic complex (H3) in the series. Heterocyclic Ru(II) complexes behave differently as evidenced by cellular and biophysical studies. Unlike phosphine complexes, H3 shows biphasic melting of DNA at higher concentrations which suggests two different types of interaction with DNA. Chapter 4 deals with synthesis and characterization of water soluble multiruthenated hydrophilic ruthenium(II) complexes with urotropine. An increase in cytotoxicity and binding affinity has been observed with increase in the number of ruthenium atoms per molecule. The complex with three ruthenium atoms showed the best activity. However cytotoxicity of the complexes decreases with decrease in the lipophilicity of the complexes. Chapter 5 describes studies on the interaction of Ru complexes with water, ss-DNA, AMP, GMP and GSH by various spectroscopic techniques. Hydrolysis of Ru-Cl bond in the complexes correlates with the cytotoxicity. Chapter 6 reports the summary of the observations of the thesis and the future prospects of metallodrugs.

Les glycosides cardiotoniques sont des inhibiteurs des pompes sodium / potassium utilisés pour le traitements des insuffisances cardiaques, qui détiennent également des activités anticancéreuses et épigénétiques récemment caractérisées. Toutefois, dans l’objectif de repositionner ces médicaments comme traitement anticancéreux, les mécanismes sousjacents aux activités anticancéreuses et épigénétiques des glycosides cardiotoniques restent à être déterminés. Dans nos travaux, nous révélons que la proscillaridine A est le glycoside cardiotonique qui détient des activités anticancéreuses et épigénétiques les plus puissantes dans des lignées de cancer du côlon, de leucémies et de sarcomes pédiatrique. De plus, nous avons identifié que l’activité anticancéreuse de la proscillaridine A corrèle positivement avec le niveau d’expression protéique du proto-oncogène MYC dans un panel de 14 lignées cellulaires cancéreuses. Dans les lignées cellulaires exprimants un haut niveau de MYC telles que les lignées leucémiques, la proscillaridine A agit comme un inhibiteur de MYC et module sa stabilité protéique ainsi que la régulation transcriptionnelle et translationnelle de ces cibles. Cette inhibition est induite par la baisse significative de l’expression des enzymes épigénétiques les lysines acétyltransférases (KATs), qui contrôlent l’ajout des résidus d’acétylcoenzyme A sur les histones et sur d'autres protéines dont MYC. La baisse d’expression des KATs résultent à une baisse de l’acétylation des résidus de l’histone 3 et à une reprogrammation de l’acétylome des cellules cancéreuses surexprimant MYC. Ces changements au niveau de la chromatine induisent une reprogrammation transcriptionnelle et phénotypique des cellules surexprimant MYC, qui se traduit par une perte de la transcription des programmes oncogéniques et l’induction des programmes associés à la différenciation cellulaire. Pour finir, nous avons évalué le potentiel synergique anticancéreux et épigénétique de la proscillaridine A avec le médicament épigénétique la décitabine dans des lignées cancéreuses exprimants des niveaux différentiels de MYC. Dans une lignée résistante à la proscillaridine A et exprimant de faible niveau de MYC (lignée de cancer de côlon), la décitabine et la proscillaridine A démontrent des activités épigénétiques synergiques tandis que dans une lignée sensible à la proscillaridine A et surexprimant MYC (lignée de sarcome pédiatrique), la décitabine et la proscillaridine A démontrent des activités antiprolifératives synergiques. Dans ces travaux, nous avons donc démontré le potentiel de repositionner la proscillaridine A dans les cancers surexprimant MYC. Également, nous démontrons le potentiel synergique anticancéreux et épigénétique de la proscillaridine A avec la décitabine et nous suggérons d’étudier cette combinaison de médicaments dans les cancers plus résistants à la proscillaridine A.<br>Cardiac glycosides are sodium/potassium pomps’ inhibitors used for the treatment of heart failure, and whose anticancer and epigenetic activities have been recently characterized. However, in order to repurpose cardiac glycosides as anticancer drugs, mechanistic studies are required to identify the anticancer and epigenetic mechanism of actions. In our experiments, proscillaridin A exhibited the most powerful anticancer and epigenetic activities in colon cancer, leukemia, and sarcoma cell lines. Moreover, we demonstrated that in a panel of 14 cancer cell lines, proscillaridin A anticancer activities positively correlated with MYC protooncogene expression level. In high MYC expressing cell lines such as leukemia, proscillaridin A inhibited MYC expression through protein destabilization and through transcriptomic and translational regulation of MYC targets. Theses inhibitions are induced by the loss of lysine acetylatransferase (KAT) expressions, which are epigenetic enzymes controlling the addition of acetyl-coenzyme A on histones and other proteins such as MYC. KAT inhibitions are responsible for the global loss of histone 3 acetylation and acetylome reprogrammation in high MYC expressing cancer cells. These chromatin changes induced transcriptomic and phenotypic reprogrammation, defined by a loss of the transcription of oncogenic programs and the induction of cell differentiation. To finish, we evaluated the anticancer and epigenetic synergic potential of proscillaridin A in combination with the epigenetic drug the decitabine in cancer cell lines expressing different MYC levels. In a cancer cell line resistant to proscillaridin A treatments and expressing low MYC level (colon cancer cell line), the combination of decitabine and proscillaridin A demonstrated synergistic epigenetic activity although, in a cell line sensitive to proscillaridin A treatments and expressing high MYC level (sarcoma cell line), the combination of decitabine and proscillaridin A exhibited synergistic anti-proliferative activity. To conclude, we highlighted the potential of repurposing proscillaridin A as an anticancer treatment in high MYC expressing cells. Furthermore, we demonstrated the anticancer and epigenetic synergistic potential of proscillaridin A in combination with decitabine and we propose to study the drug combination in cancers that are resistant to proscillaridin A treatment.