Dissertations / Theses on the topic 'Anticancer chemotherapy'

The concept of bioreductive prodrug chemotherapy is introduced in chapter 1. Tumour cell hypoxia is a significant factor in limiting tumour growth control with conventional radiotherapy and some chemotherapeutic agents. Following therapy these cells can repopulate and cause a relapse of the cancer. On the other hand, hypoxia is unique to tumours, and is therefore potentially exploitable. Bioreductive prodrugs are compounds in which a oxygen inhibited redox-based bioactivation step triggers a reaction leading to a lethal intermediate. The concept of bioreductive DNA alkylators and DNA topoisomerase 11 inhibitors is discussed. The synthesis of model thiazolylindole compounds based on the natural product BE \0988 are discussed in chapter 2. Two strategies were employed for the construction of the thiazolylindoles: the Hantzsch reaction; and nucleophilic substitution on 2-bromothiazole by an indolyl anion. The synthesis of thiazolylindolequinone compounds are discussed in chapter 3. The quinone C(5) position of the thiazolylindolequinone analogues was elaborated to provide a series of cyclic and acyclic C(5)-amino derivatives. Synthetic strategies towards the synthesis of indole-2-carboxylates are discussed in chapter 4. The Moody-Rees and Cadogan-Sundberg reactions were employed to provide a synthesis of the useful highly substituted indole [154]. The Brederek imidazole reaction (i.e., the reaction of a amidine and a-halo ketone) is discussed in chapter 5. Application of the Brederek reaction was employed towards the construction of the bisindole imidazole natural compounds, the nortopsentins. The biological properties of the compounds of the compounds synthesised are discussed in chapter 6. The compounds were tested for DNA topoisomerase 11 inhibitory activity and cytotoxicity under a hypoxic environment.

Pharmaceutical Sciences
Ph.D.
Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults for which overall prognosis remains poor despite recent treatment advances, thus emphasizing the need for developing effective therapeutic agents. Styryl sulfones belong to a class of non-ATP competitive antineoplastic agents in early stage clinical trials. Detailed investigation of the pharmacokinetics (PKs) and pharmacodynamics (PDs) of novel agents in the preclinical stage plays a pivotal role in drug development that could be applied to guide clinical trials. The main goal of the project was to undertake comprehensive PK and PD evaluation of new agents for brain tumor therapy and in the process establish a PK/PD strategy for the development of such novel agents. The current project was aimed to evaluate the potential of a styryl benzyl sulfone compound, ON01910.Na, as a chemotherapeutic agent for the treatment of GBMs using PK/PD approaches. First, the systemic pharmacokinetics of ON01910 was characterized following single dose intravascular administration of ON01910.Na in healthy mice over a 50-fold dose range of 5 mg/kg - 250 mg/kg. Secondly, an evaluation of the brain and brain tumor disposition of ON01910 was conducted in an orthotopic U87 glioma model in mice using a steady-state dosing regimen, and, in addition, using the same brain tumor model its pharmacodynamic and antiangiogenic activity were determined following multiple dosing. ON01910 exhibited nonlinear pharmacokinetics in the dose range of 50 mg/kg - 250 mg/kg. It showed inadequate brain and brain tumor penetration and insignificant antiangiogenic and antiproliferative activity. The limited brain tumor penetration and activity of ON01910 in the intracerebral glioma model led to the evaluation of ON013105, a prodrug of its more lipophilic anticancer congener, ON013100. A similar PK/PD approach as for ON01910.Na was applied wherein systemic pharmacokinetic properties of ON013105 and its active form, ON013100 in healthy mice, as well as an analysis of their brain and brain tumor distribution following steady-state dosing regimen were determined following administration of prodrug. The active form, ON013100 showed appreciable brain and brain tumor penetration while the prodrug did not. Subsequent pharmacodynamic investigations conducted in vitro identified phosphorylated-ERK (pERK) as a PD marker. To assess time-dependent PK/PD characteristics, mice bearing intracerebral U87 glioma were administered ON013105 at 100 mg/kg intravenously and plasma, brain and brain tumor concentrations of ON013105 and its active form, ON013100 were quantitated as well as tumoral pERK levels. Further, a PK-PD model was developed that characterized plasma, brain and brain tumor concentration-time profiles of ON013105 and ON013100 and tumoral pERK levels. In summary, a PK/PD-driven approach was applied to evaluate and select novel compounds that may have potential in the treatment of brain tumors. The progression of studies yielded one compound, ON013100 that possessed favorable brain tumor distribution and showed PD activity that warrant continued evaluation.
Temple University--Theses

Taxol®, an important anticancer drug, was first isolated in extremely low yield from the bark of the western yew, Taxus brevifolia. The clinical utility of Taxol has prompted a tremendous effort to obtain this complex molecule synthetically. Due to the chemical complexity of Taxol, its commercial production by total synthesis is not likely to be economical. Another natural product, 10-deacetyl baccatin III, is readily available in higher yield. Several methods have been reported for the synthesis of Taxol by coupling baccatin III and the N-benzoyl-β-phenylisoserine side chain. A new method for the synthesis ofTaxol from baccatin III is reported, and this method is compared with other methods.
Master of Science

This dissertation mainly deals with some biochemical aspects regarding the efficacy of novel platinum anticancer compounds, as part of a broader study in which both chemistry and biochemistry are involved. Various novel diamine and N-S donor chelate compounds of platinum II and IV have been developed in which factors such as stereochemistry, ligand exchange rate and biocompatibility were considered as additional parameters. In the first order testing, each of these compounds was tested with reference to their “killing” potential by comparing their rate of killing, over a period of 48 hours with those of cisplatin and oxaliplatin. Some 80 compounds were tested in this way. Although only a few could be regarded as equal to or even better than cisplatin and oxaliplatin, the testing of these compounds on cancer cells provided useful knowledge for the further development of novel compounds. Four of the better compounds, namely Y9, Y14, Y16 and Lt16.2 were selected for further studies to obtain more detailed knowledge of their anticancer action, including some flow cytometric studies. In addition to the above, cisplatin resistant cells were produced for each of the three different cell lines tested, namely, HeLa, HT29 and MCF7 cancer cell lines, by intermittent and incremental exposure to cisplatin (all the cell lines tested became resistant to cisplatin). Each of the selected compounds were exposed to the cells in the same manner, in order to attempt the induction of resistance against these compounds in the three cell lines tested (i.e. whether these cells will become resistant to the various compounds). Each of these selected platinum containing compounds were subsequently tested against the “cisplatin resistant” cell lines in order to determine their efficacy against such cells. One such compound could be singled out, since cervical cancer cells (HeLa cells) do not become resistant to it. This behaviour is similar to that of oxaliplatin against cervical cancer and colon cancer (HT29) cells (oxaliplatin is the number one treatment for colon cancer at present). This compound also proved to be more active against cisplatin resistant cell lines. It was found that all the compounds induced apoptosis in the cell lines tested as well as inhibit the DNA cycle at one or more phase. Finally, an effort was made to evaluate the different compounds by comparing them with respect to their properties relating to anticancer action.

This thesis mainly deals with some biochemical aspects regarding the efficacy of novel platinum anticancer compounds alone and in combination with mangiferin, as part of a broader study in which both chemistry and biochemistry are involved. Various novel diamine and N-S donor chelate compounds of platinum II and IV have been developed in which factors such as stereochemistry, ligand exchange rate and biocompatibility were considered as additional parameters. In the first order testing, each of these compounds was tested with reference to their “killing” potential by comparing their rate of killing, over a period of 48 hours with those of cisplatin and oxaliplatin. Numerous novel compounds were tested in this way, using the MTT cell viability assay and the three cancer cell lines MCF7, HT29 and HeLa. Although only a few could be regarded as equal to or even better than cisplatin, CPA7 and oxaliplatin, the testing of these compounds on cancer cells provided useful knowledge for the further development of novel compounds. Three of the better compounds, namely Yol 25, Yol 29.1 and Mar 4.1.4 were selected for further studies, together with oxaliplatin and CPA7 as positive controls, to obtain more detailed knowledge of their anticancer action, both alone and when applied in combination with mangiferin. In addition to the above, resistant cells were produced for each of the three different cell lines tested and all the selected compounds, both in the presence and absence of mangiferin. The effects of these treatments on the activation of NFĸB when applied to normal and resistant cell lines were also investigated. All the compounds induced apoptosis in the cell lines tested as well as alter the DNA cycle at one or more phase. Additionally, combination of these compounds with mangiferin enhanced the above-mentioned effects. Mangiferin decreases the IC50 values of the platinum drugs by up to 3.4 times and, although mangiferin alone did not induce cell cycle arrest, the presence of mangiferin in combination with oxaliplatin and Yol 25 shows an earlier and greatly enhanced delay in the S-phase, while cells treated with CPA7, Yol 29.1 and Mar 4.1.4 in combination with mangiferin showed a later, but greatly enhanced delay in the S-phase. It was also found that mangiferin acts as an NFĸB inhibitor when applied in combination with these drugs, which, in turn, reduces the occurrence of resistance in the cell lines. Resistance to oxaliplatin was counteracted by the combination with mangiferin in HeLa and HT29, but not in MCF7 cells, while resistance to CPA7 was only counteracted in the MCF7 cell line. Yol 25 and Mar 4.1.4 did not seem to induce resistance in HeLa and MCF7 cells, but did in HT29 cells, whereas Yol 29.1 caused resistance in HeLa and HT29 cells, but not in MCF7 cells. Finally, an effort was made to evaluate the different compounds by comparing them with respect to their properties relating to anticancer action with and without the addition of mangiferin.

The use of metallo-pharmaceuticals, such as the platinum drugs, for cancer treatment illustrates the utility of metal complexes as therapeutic agents. Platinum group metal complexes therefore offer potential as anti-tumour agents to fight cancer. This study was aimed at synthesizing and evaluating the effects of palladium(II) and platinum(II) complexes as anticancer agents.

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.

[Truncated abstract] The trinuclear anti-cancer agent [(trans-Pt(NH3)3Cl)2{μ-trans-Pt(NH3)2(H2N(CH2)6NH2)2}]4+ (BBR3464 or 1,0,1/t,t,t) is arguably the most significant development in the field of platinum anti-cancer agents since the discovery of cisplatin as a clinical agent more than 30 years ago. Professor Nicholas Farrell of Virginia Commonwealth University was responsible for the development of 1,0,1/t,t,t and an entire class of multinuclear platinum complexes. The paradigm shift that was required in the development of these compounds is based on a simple idea. In order to increase the functionality of platinum anti-cancer drugs a new way of binding to DNA must be employed. By increasing the number of platinum centres in the molecule and separating the binding sites, by locating them on the terminal platinum atoms, the result is a new binding motif that does not occur with cisplatin. The work described in this thesis involves the use of [¹H,¹5N] NMR spectroscopy combined with molecular modelling to investigate various aspects of the solution chemistry and DNA binding interactions of BBR3464 and the related dinuclear analogues [{trans-PtCl(NH3)2}2(μ- NH2(CH2)6NH2)]2+ (1,1/t,t) and [{cis-PtCl(NH3)2}2(μ-NH2(CH2)6NH2)]2+ (1,1/c,c). Chapter 2 contains detailed descriptions of the various methodologies used, including the molecular mechanics parameters that were developed for the various modelling studies described in this thesis.... The work described in Chapter 6 employed three duplexes; 5'-d(TCTCCTATTCGCTTATCTCTC)-3'·5'- d(GAGAGATAAGCGAATAGGAGA)-3' (VB12), 5'-d(TCTCCTTCTTGTTCTTCCTCC)- 3'·5'-d(GGATTAAGAACAAGAAGGAGA)-3' (VB14) and 5'- d(CTCTCTCTATTGTTATCTCTTCT)-3'·5'-d(AGAAGAGATAACTATAGAGAGAG)-3' (VB16). Two minor groove preassociated forms of 1,0,1/t,t,t with each duplex were created in which the complex was orientated in two different directions around the central guanine (labelled the 3'→3' and 5'→5' directions). The molecular dynamics simulations of these six systems indicated that each preassociated states was stable within the minor groove and could effectively support the formation of multiple interstrand cross-links. Subsequent investigations into the dynamic nature of the monofunctional adduct were conducted by the assembly of a single monofunctional adduct of the VB14 duplex with 1,0,1/t,t,t. Here it was found that the monofunctionally anchored 1,0,1/t,t,t adopted a position along the phosphate backbone of the duplex in the 5'→5' direction.

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.

The complex natural product paclitaxel (Taxol®), first isolated from Taxus brevifolia, is a member of a large family of taxane diterpenoids. Paclitaxel is extensively used for the treatment of solid tumors, particularly those of the breasts and ovaries. In order to obtain additional information about the mechanism of action of paclitaxel and the environment of the paclitaxel-binding site, several fluorescent analogs of paclitaxel were synthesized, and their biological activities have been evaluated. For the investigation of possible synergistic effects, concurrent modifications on selected positions have been performed and their biological evaluation were studied.
Ph. D.

Application of chemotherapeutic agents in current cancer treatment has been limited by adverse effects as poor selectivity results in systemic toxicity; most chemotherapy approaches also experience inherited or acquired drug resistance which lead to reduced treatment outcome. Research efforts have focused on the discovery of novel chemotherapies that overcome the limitations mentioned above. Ru(II) polypyridyl complexes with anti-cancer properties have been extensively studied as traditional cytotoxic agents and photodynamic therapy agents due to their photophysical and photochemical characteristics. Most research has focused on the design of Ru(II) polypyridyl complexes that have affinities to nucleic acids as inspired by the classic small molecule metal complex cisplatin. Though modifying the structures of ligands on the ruthenium metal center, the hydrophilicity, charge state and photochemical properties can be tuned, resulting to Ru(II) polypyridyl complexes that act through cellular targets other than DNA. Understanding the mechanism of action and identifying functional targets remain the challenging and complex research topic in the design and study of novel medication or candidates. With the development of semi-high throughput cytological profiling in a bacterial system, rapid investigation of the mechanism of action can be achieved to distinguish anti-cancer agents which possess different mechanisms of actions. Ru(II) polypyridyl complexes with different scaffolds have been studied and suggested to have anti-cancer properties through DNA damage response, and/or translational inhibition.

Durant els darrers 30 anys, Ru, Ir, Pd, Fe o Cu han emergit com a alternatives prometedores en substitució als fàrmacs que contenien platí, que presentaven importants efectes secundaris. Especialment a la darrera dècada, els complexos de coure han despertat interès com a agents terapèutics. El coure és un metall essencial, present en moltes de les proteïnes del nostre cos, jugant un paper crucial en els processos bioquímics que es porten a terme. Concretament, dos aspectes han fet del coure un metall d’interès de cara a la teràpia contra el càncer: el fet de ser un metall endogen -fet que hauria de comportar menys efectes secundaris que un d’exogen com el platí- i el seu parell redox Cu(II)/Cu(I) -que s’ha descrit capaç de generar espècies reactives d’oxigen (EROs). Aquestes EROs poden danyar l’ADN i causar la mort cel·lular. A més, el fet que les cèl·lules canceroses tinguin un nivell d’EROs superior a les cèl·lules sanes apareix com una possibilitat per tal d’aconseguir una teràpia més selectiva. La primera part de la tesi es basa en la síntesi, caracterització i l’estudi de l’activitat biològica d’un complex de Cu(II) dinuclear que conté un lligand N,O-donador (L), especialment dissenyat per a promoure una ràpida conversió Cu(II)/Cu(I). Els assajos biològics en cultius cel·lulars mostren una alta producció d’EROs en línies cel·lulars HeLa, i que el complex té més toxicitat en cèl·lules canceroses que en sanes. En aquesta primera part, les interaccions amb ADN i proteïnes també s’han avaluat. A partir d’aquest punt de partida, el lligand L s’ha funcionalitzat amb grups halogenats per tal de modular el potencial redox del parell Cu(II)/Cu(I). La presència de grups electroatraients pretén facilitar la reducció del Cu(II) a Cu(I). De totes maneres, els complexos corresponents mostren certs problemes de solubilitat. En aquest sentit, la segona part de la tesi es centra en la derivatització del lligand L per tal de millorar la seva solubilitat i biodisponibilitat. Per això, s’aborden dues estratègies principals. La primera es basa a derivatitzar el lligand amb grups sulfonat i arginina, mantenint el mateix entorn de coordinació al voltant del coure. La segona estratègia es centra essencialment en millorar la internalització cel·lular, per tal d’incrementar la toxicitat del corresponent complex, amb la funcionalització del lligand amb pèptids rics en arginina, d’alta capacitat penetrant. Finalment, la darrera part d’aquest treball obre la porta a l’ús d’una plataforma dendrítica multimodal com a futur sistema de transport de fàrmacs. S’ha estudiat la seva capacitat de coordinació de coure i la seva potencialitat com a plataforma d’administració de fàrmacs. A partir d’aquí, també s’ha aconseguit ancorar la plataforma al lligand L, i procedir a la seva complexació com a prova de concepte d’aquest sistema de cara a futures teràpies dirigides.
Depuis une trentaine d’années, le Ru, l’Ir, le Pd, le Fe ou le Cu sont apparus comme des alternatives prometteuses pour remplacer les médicaments contenant du platine, et qui ont montré des effets secondaires importants. En particulier au cours des dix dernières années, les complexes de cuivre ont suscité l’intérêt en tant qu’agents thérapeutiques. Le cuivre est un métal essentiel, présent dans de nombreuses protéines de notre corps et joue un rôle crucial dans les processus biochimiques. Plus précisément, deux aspects ont fait du Cu un métal d’intérêt pour le traitement du cancer : le fait qu’il s’agisse d’un métal endogène, et qui devrait par conséquent avoir moins d’effets secondaires que les complexes de métaux exogènes comme le platine ; et le couple redox Cu(II)/Cu(I) rapporté comme pouvant générer des espèces réactives de l’oxygène (EROs). Ces EROs peuvent endommager l’ADN et provoquer la mort cellulaire. Ce fait apparaît comme une possibilité de traitement sélectif. La première partie de la thèse est basée sur la synthèse, la caractérisation et l’étude de l’activité biologique d’un complexe dinucléaire de Cu(II) contenant un ligand N,O-donneur (L), spécialement conçu pour promouvoir une conversion rapide Cu(II)/Cu(I). Les tests en culture cellulaire montrent une forte production d’EROs dans les lignées de cellules HeLa et montrent aussi que le complexe a plus de toxicité au sein des cellules cancéreuses que dans les cellules saines. Dans cette première partie, les interactions avec l’ADN et les protéines ont été également évaluées. À partir de ce point de départ, le ligand L a été fonctionnalisé avec des groupes halogénés afin de moduler le potentiel rédox du couple Cu(II)/Cu(I). La présence de groupes attracteur d’éléctrons vise à faciliter la réduction de Cu(II) en Cu(I). Cependant, tous les complexes correspondants présentent certains problèmes de solubilité. En ce sens, la deuxième partie de la thèse porte sur la dérivatisation du ligand L afin d’améliorer sa solubilité et sa biodisponibilité. deux stratégies principales sont alors abordées : La première est basée sur la fonctionnalisation du ligand avec des groupes sulfonate et arginine, en maintenant le même environnement de coordination autour du cuivre. La deuxième stratégie vise essentiellement à améliorer l’internalisation cellulaire, afin d’augmenter la toxicité du complexe correspondant, avec la dérivatisation du ligand avec des peptides riches en arginine (rapportés pour avoir une haute capacité de pénétration intra cellulaire). Enfin, la dernière partie de ce travail ouvre la porte à l’utilisation d’une plateforme dendritique multimodale pour être utilisé comme futur système d’administration de médicaments. Sa capacité de coordination du cuivre et son potentiel en tant que plateforme d’administration de médicaments ont été étudiés. À partir de là, on a été également capable d’ancrer la plateforme dans le ligand L et de procéder à sa complexation avec cuivre, comme preuve de concept de ce système pour son utilisation dans de futures thérapies dirigées.
During the last 30 years, Ru, Ir, Pd, Fe or Cu have appeared as promising alternatives to overcome the drawbacks encountered with Pt anticancer compounds. Beyond all of them, and mainly during the last decade, Cu complexes have awakened strong interest as therapeutic agents. Two features make Cu attractive to be used in chemotherapy: its nature as an endogenous metal —which may imply fewer side effects than other exogenous metals- and its Cu(II)/Cu(I) redox pair —which can promote reactive oxygen species (ROS) generation. The production of ROS is not only reported to cause cellular damage, but also to offer a putative discrimination between healthy and non-healthy cells. On the first part of this thesis work, we report the synthesis, characterization and biological evaluation of a dimeric Cu(II) complex bearing a N,O-donor salphen-like ligand ((E)-N-(2-(2-hydroxybenzylideneamino)phenyl)acetamide, L1) specifically designed to promote a fast Cu(II)/Cu(I) redox interconversion. In vitro assays outline the high potentiality of the complex to undergo ROS generation inside HeLa cells, and that it shows higher cytotoxicity in cancer than in normal cell lines. Besides, its interactions with some proteins have also been tested, showing that the formed protein-complex adducts do not represent any loss of biological activity respect to the complex itself. From this promising starting point, the Cu(II) complex of L1 ([Cu(L1)]2) serves as the backbone for the synthesis of two -chloro and -bromo analogs. The presence of electrowithdrawing groups intend to tune the redox behavior of the corresponding Cu(II) complexes, and concomitantly, their ROS generation capabilities. However, one of the main drawbacks faced with these two halogen-derived complexes was their poor solubility and bioavailability. Therefore, several functionalization strategies have been explored to overcome it. The first strategy aimed at increasing the solubility while maintaining the same Cu(II) coordination environment, i.e., the high redox activity observed for the initial [Cu(L1)]2 complex. In light of this, a sulfonate group and an Arginine residue have been selected based on their pKa and biological relevance. Secondly, and in order to enhance the delivery of the complex and the candidacy as future anticancer drug, specific improvement on the cellular uptake -ergo, on the cytotoxicity- has been attained by derivatizing [Cu(L1)]2 with two specific Arginine-rich Cell-Penetrating Peptides. Finally, the last part of our work opens the gate to the use of a versatile multimodal dendritic platform as a promising drug carrier. Its potentiality in drug delivery and its copper coordination capabilities have been thoroughly demonstrated. The conjugation approach of the [Cu(L1)]2 complex to the platform is also reported as a proof-of-concept of the versatility of this system for future tailor-made anticancer targeted therapies.

no
Inadequate drug delivery to tumours is now recognised as a key factor that limits the efficacy of anticancer drugs. Extravasation and penetration of therapeutic agents through avascular tissue are critically important processes if sufficient drug is to be delivered to be therapeutic. The purpose of this study is to develop an in silico model that will simulate the transport of the clinically used cytotoxic drug doxorubicin across multicell layers (MCLs) in vitro. Three cell lines were employed: DLD1 (human colon carcinoma), MCF7 (human breast carcinoma) and NCI/ADR-Res (doxorubicin resistant and P-glycoprotein [Pgp] overexpressing ovarian cell line). Cells were cultured on transwell culture inserts to various thicknesses and doxorubicin at various concentrations (100 or 50 microM) was added to the top chamber. The concentration of drug appearing in the bottom chamber was determined as a function of time by HPLC-MS/MS. The rate of drug penetration was inversely proportional to the thickness of the MCL. The rate and extent of doxorubicin penetration was no different in the presence of NCI/ADR-Res cells expressing Pgp compared to MCF7 cells. A mathematical model based upon the premise that the transport of doxorubicin across cell membrane bilayers occurs by a passive "flip-flop" mechanism of the drug between two membrane leaflets was constructed. The mathematical model treats the transwell apparatus as a series of compartments and the MCL is treated as a series of cell layers, separated by small intercellular spaces. This model demonstrates good agreement between predicted and actual drug penetration in vitro and may be applied to the prediction of drug transport in vivo, potentially becoming a useful tool in the study of optimal chemotherapy regimes.

[Truncated abstract] The Phase II clinical candidate, [(trans-Pt(NH3)2Cl)2{μ-trans-Pt(NH3)2(H2N(CH2)6NH2)2}]4+ (BBR3464 or 1,0,1/t,t,t) shows a unique binding profile when compared to the anticancer agent cis-[Pt(NH3)2Cl2] (cisplatin) and dinuclear platinum complexes of the general formula [(trans-Pt(NH3)2Cl)2(H2N(CH2)nNH2)]2+. There is evidence that the increased efficacy of 1,0,1/t,t,t results from the presence of the charged central linker, which can alter the mode of binding to DNA. This alternate binding mode may be due to an electrostatic and hydrogen bonding association of the central platinum moiety in the minor groove that occurs prior to covalent binding (termed “pre-association”) . . . This research shows that 0,0,0/t,t,t is an adequate model to study the pre-association process of 1,0,1/t,t,t and that it binds in the minor groove of DNA. Therefore it is likely that 1,0,1/t,t,t pre-associates in the minor groove of DNA prior to covalent binding. This work supports the conclusions reached in NMR studies of the binding of 1,0,1/t,t,t with the 1,4-GG sequence (Qu et al. JBIC. 8, 19-28 (2003)), which showed simultaneous binding in the major and minor groove. The findings of the current work may also explain the observed binding mode of 1,0,1/t,t,t, which can bind to DNA in both the 3',3' and 5',5' directions (Kasparkova et al. JBC. 277, 48076-48086 (2002)). These unique binding characteristics are thought to be responsible for the increased efficacy of 1,0,1/t,t,t, and in light of the current results the observed binding mode most likely stems from the electrostatic pre-association of the central platinum moiety.

15N-based nuclear magnetic resonance techniques are considered very powerful to study the molecular properties of platinum-containing anticancer agents, these properties being responsible for the efficacy of the compounds, but also for the understanding of resistance mechanisms and toxicity. Therefore, the first part of the present work aimed to develop a new method for synthesizing 15N-labeled, chiral platinum compounds. A theoretical discussion on the nucleophilic ring-opening of aziridines has also been envisaged, rationalizing an interesting regiochemistry question. Indeed, a surprising inversion of regiochemistry arose during the development of the above-mentioned synthetic pathway, and density functional theory calculation brought a rational framework to the experimental findings.

Infrared spectroscopy probes the global chemical composition of a sample and has been used to produce a snapshot of cancer cells contents after treatment with platinum coordinates. Indeed, in vitro studies focused here on the use of modern spectroscopic methods to fingerprint the cellular impact of platinum complexes. These drug signatures help to classify and select promising compounds. It makes no doubt that such systemic approaches for compound discovery are helpful technologies. Also, we made the use of the COMPARE algorithm from the NCI, which analyzes similarity between any active compounds previously tested by the NCI large scale in vitro screening program of anticancer agents.

The last chapter aimed to study the interactions between a series of platinum coordinates and DNA. Binding mode to telomeric-like sequences and binding kinetics to genomic-like sequences were assessed to investigate any differences between the compounds and to gain insight into structure-activity relationships.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

La plupart des molécules utilisées en chimiothérapie conventionnelle, bien qu’ayant des cibles moléculaires différentes, induisent dans la majorité des cas une mort cellulaire par apoptose. Or, de plus en plus de chimiorésistances se rencontrent au niveau des cellules cancéreuses vis-à-vis de ce type de molécules. Face à cette situation il devient urgent de trouver des molécules ayant des mécanismes d’action différents et capables de court-circuiter spécifiquement les mécanismes de résistance des cellules cancéreuses.

La stratégie mise en place lors de ce travail a été de partir d’une molécule naturelle issue d’un extrait de la racine de Glycyrrhiza glabra qui présentait déjà une activité anti tumorale marquée. L’intérêt du travail a été de dériver l’acide 18β-glycyrrhétinique de manière originale afin de potentialiser son effet anticancéreux, notamment vis-à-vis de huit lignées cellulaires présentant des résistances plus ou moins marquées aux stimuli pro-apoptotiques. Ainsi après avoir caractérisé la pureté et la stabilité de cette série de nouvelles molécules, nous avons retenu les dérivés les plus intéressants en termes d’inhibition in vitro de la prolifération cellulaire. Sur base de ce premier choix, nous avons investigué des cibles spécifiques décrites dans la littérature pour les hémidérivés de l’acide 18β-glycyrrhétinique :le protéasome 26S et le récepteur nucléaire PPARγ. Cette étude nous a permis de retenir un dérivé en particulier capable d’inhiber à 50% les trois sites catalytiques du protéasome sans toutefois inhiber PPARγ :le N-(2-{3-[3,5-bis(trifluoromethyl)phenyl]ureido}ethyl)-glycyrrhetinamide (6b). Nous avons ensuite évalué ce composé sur un ensemble de 333 kinases afin de déterminer un profil antitumoral plus large pour ce type de molécule.

Le profil pharmacologique in vitro de ce dérivé de l’acide 18β-glycyrrhétinique nous a amenés à étudier son comportement in vivo chez la souris saine. A cette fin, une étude de préformulation nous a permis de définir une formulation galénique optimale pour ce composé, la nanoémulsion qui a servi à déterminer une dose maximale tolérée (indice DMT) par la souris saine. Nous avons ensuite travaillé à une dose non toxique pour déterminer le profil pharmacocinétique plasmatique chez la souris saine, par voie d’administration intraveineuse et par voie orale.

Les conclusions de cette étude nous montrent que le dérivé de l’acide 18β-glycyrrhétinique que nous avons mis au point présente de remarquables caractéristiques pharmacologiques in vitro et un comportement in vivo proche de la molécule naturelle. Des études d’activité in vivo devraient débuter prochainement.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

There is an urgent need to develop reliable and cost-saving methods to select pre-clinically new drug candidates with original mechanism for cancer therapy. Previous results have shown that IR spectra of cancer cells exposed to various drugs provided a global signature of all the metabolic changes induced by the treatments. In this thesis, we attempted to develop a selection criterion – based on FTIR spectroscopy – for potential antitumor compounds according to their mechanism of action.

In chapter III, it was demonstrated that spectral variations in IR spectra of cancer cells induced by a treatment can be correlated to the mechanism of the drug. Human prostate cancer PC-3 cells were exposed to 7 well-described anticancer drugs belonging to 3 distinct classes. Each class is characterized by a unique mode of action. Drugs known to induce similar types of metabolic disturbances appear to cluster when spectrum shapes are analyzed. Chapter IV generalized the results obtained on PC-3 cells with six other cell lines. We showed that the spectral signatures of drug effects are mainly independent of the cell line. Chapter V indicated that, while the cell cycle phase influence IR spectra of cells, the drug spectral signature was dominated by global metabolic modifications and not much by the cell cycle perturbations due to this drug.

Chapter VI and VII focused on lipids. While the precise identification of particular molecules is particularly complex with IR spectroscopy, we attempted to extract more precise information and to assign spectral variations to specific changes in lipids. IR spectra of lipids contain very interesting details on their nature and structure. We achieved to build a tool which quantifies five major lipid classes in complex mixtures such as total lipid cell extracts. However, based on this tool, the treatments used do not induce any variation in the lipid cell composition (for five classes).

Finally, in chapter VIII, we applied the method developed previously on a new potential class of anticancer molecules: the polyphenols. A global method was particularly interesting as the development of therapy using these compounds is hampered by the complexity of the multiple anticarcinogenic mechanisms of these molecules. We have noticed the similarities and discrepancies among 3 very close synthetic molecules and the observations were coherent with previous biological data. We also compared them with 3 natural molecules already in clinical phase for treatment of various cancers.

In conclusion, we developed an objective classifier for potential anticancer drugs based on their global effects on cancer cells. Applied to a larger scale, this method could constitute a first step in the screening method to select drugs with original mode of action.

There is an urgent need to develop reliable and cost-saving methods to select pre-clinically new drug candidates with original mechanism for cancer therapy. Previous results have shown that IR spectra of cancer cells exposed to various drugs provided a global signature of all the metabolic changes induced by the treatments. In this thesis, we attempted to develop a selection criterion – based on FTIR spectroscopy – for potential antitumor compounds according to their mechanism of action.

In chapter III, it was demonstrated that spectral variations in IR spectra of cancer cells induced by a treatment can be correlated to the mechanism of the drug. Human prostate cancer PC-3 cells were exposed to 7 well-described anticancer drugs belonging to 3 distinct classes. Each class is characterized by a unique mode of action. Drugs known to induce similar types of metabolic disturbances appear to cluster when spectrum shapes are analyzed. Chapter IV generalized the results obtained on PC-3 cells with six other cell lines. We showed that the spectral signatures of drug effects are mainly independent of the cell line. Chapter V indicated that, while the cell cycle phase influence IR spectra of cells, the drug spectral signature was dominated by global metabolic modifications and not much by the cell cycle perturbations due to this drug.

Chapter VI and VII focused on lipids. While the precise identification of particular molecules is particularly complex with IR spectroscopy, we attempted to extract more precise information and to assign spectral variations to specific changes in lipids. IR spectra of lipids contain very interesting details on their nature and structure. We achieved to build a tool which quantifies five major lipid classes in complex mixtures such as total lipid cell extracts. However, based on this tool, the treatments used do not induce any variation in the lipid cell composition (for five classes).

Finally, in chapter VIII, we applied the method developed previously on a new potential class of anticancer molecules: the polyphenols. A global method was particularly interesting as the development of therapy using these compounds is hampered by the complexity of the multiple anticarcinogenic mechanisms of these molecules. We have noticed the similarities and discrepancies among 3 very close synthetic molecules and the observations were coherent with previous biological data. We also compared them with 3 natural molecules already in clinical phase for treatment of various cancers.

In conclusion, we developed an objective classifier for potential anticancer drugs based on their global effects on cancer cells. Applied to a larger scale, this method could constitute a first step in the screening method to select drugs with original mode of action.

Afin d’améliorer les thérapies contre le cancer, il devient actuellement cruciale de développer une méthode pour améliorer la sélection préclinique de nouvelles molécules, potentiellement anticancéreuses. Des publications précédentes ont mis en évidence que les spectres infrarouges de cellules cancéreuses exposées à différents agents thérapeutiques fournissent une empreinte globale de l’ensemble des changements métaboliques induit par ce médicament. Dans cette thèse, nous proposons d’utiliser la spectroscopie infrarouge pour mettre au point un critère de sélection basé sur le mode d’action des agents anticancéreux. Plusieurs aspects de la technique ont été investigués. Nous avons d’abord démontré la possibilité d’utiliser les spectres infrarouges de cellules cancéreuses de prostate PC-3 traitées avec 7 drogues pour classer ces molécules selon leur mode d’action. Nous avons ensuite reproduit les résultats obtenus sur PC-3 avec 6 autres lignées cellulaires et montré que la signature spectrale obtenue était largement indépendante de la lignée. Par la suite, nous avons étudié si l’effet sur le cycle cellulaire induit par de nombreuses molécules anticancéreuses, pouvait expliquer certains changements spectraux observés suite au traitement. Nous avons pu montrer que la majorité des variations spectrales n’étaient pas liées à une perturbation du cycle cellulaire. Nous nous sommes ensuite concentrés sur une classe de molécules en particulier: les lipides. Après avoir mis en évidence l’ensemble des informations contenues dans un spectre infrarouge de lipides, nous avons mis au point un modèle permettant de quantifier 5 classes de lipides dans des mélanges complexes tels que des extraits lipidiques provenant de cellules. Néanmoins, aucune variation du contenu en ces 5 classes de lipides n’a été observée pour les traitements utilisés dans cette étude. Enfin, nous avons appliqué la méthode mise au point dans cette thèse à une classe de molécules prometteuses :les polyphénols. Une approche globale s’avère particulièrement intéressante pour ces composés étant donné qu’ils présentent des mécanismes anticancéreux multiples et complexes. Nous avons comparé 3 molécules naturelles en phase clinique pour le traitement de certains cancers et 3 molécules synthétiques présentant une structure très proche. Par notre méthode, nous avons mis en évidence certaines similarités et différences de ces 6 molécules en termes d’effets globaux sur les cellules. En conclusions, nous avons développé un outil objectif de classification pour les molécules anticancéreuses potentielles, basée sur le mécanisme global des composés. Appliquée à plus large échelle, cette méthode pourrait constituer une première étape permettant de sélectionner les molécules avec un mode d’action original.
Doctorat en Sciences agronomiques et ingénierie biologique
info:eu-repo/semantics/nonPublished

SIRT1 and SIRT2 are two NAD⁺-dependent deacetylases which negatively modulate the activity of p53, a protein which is involved in cell cycle arrest, senescence and apoptosis following genotoxic stress. Part I of the thesis describes the exploration of the chemical space around a reported unselective and modest inhibitor of SIRT1 and SIRT2 with the aim of improving the selectivity and potency of the inhibitor against the two isoforms. Particular emphasis is placed upon understanding the mode of binding of the novel analogues within the active site of the enzymes. Chapter 1 reviews the physiological roles of class III NAD⁺-dependent deacetylases, also known as sirtuins. In particular, the application of SIRT1 and SIRT2 inhibitors as potential anticancer agents is described. Amongst these, only cambinol and the tenovins showed in vivo activity in a mouse xenograft model. Previously only one analogue of cambinol had been reported in the literature. Chapter 2 describes the development of a small collection of novel cambinol analogues (First Generation Studies). The role played by different substituents at the phenyl group and at the N-1 of the thiouracil core is discussed. Along with the synthesis and structure activity relationship (SAR) associated with the core structure, in-cell experiments intended to confirm the activity of the most active compounds are reported. Chapter 3 provides a rationalisation for the SAR discussed in Chapter 2. Based on computational molecular modelling studies (GOLD), the activity of the most potent and selective SIRT2 inhibitors is explained. Two series of novel cambinol analogues were designed (Second and Third Generation Analogues) in order to assess further the proposed binding mode. Chapter 4 focuses on the development of the “Second Generation” analogues, characterised by the presence of lipophilic substituents at the sulfur atom and at the N-3 position of the thiouracil core. The synthesis, biological evaluation and SAR are discussed in detail. Chapter 5 reports the development of the “Third Generation” analogues, characterised by either a benzyl group or para-alkoxy-substituted benzyl group at the N-1 position of cambinol. Once again, the synthesis, biological evaluation and SAR data are presented. An improved understanding of the mode of binding of the novel compounds is proposed based on molecular dynamics (MD) studies. Indole-based alkaloids, such as Vincristine and Vinblastine, are well known for their anticancer activity. Recently, the anticancer activity of members of the calycanthaceous family of alkaloids has been discovered. Part II of the thesis focuses on model studies aimed at developing the total synthesis of one of these compounds, perophoramidine. Chapter 7 provides an overview of the calycanthaceous alkaloid family of natural products, including their biological properties. The structural features of perophoramidine, along with the previously reported synthetic studies are outlined. Chapter 8 describes the synthesis of an advanced intermediate in the total synthesis of dehaloperophoramidine, a structural analogue of perophoramidine Problems encountered, optimisation studies and the synthesis of a re-designed intermediate are also reported in this chapter.

Chemotherapy is the first line of treatment used in cancer. Chemotherapy drugs such as cisplatin, carboplatin and oxaliplatin are used in treatment. Cisplatin enters the cell through copper transporter CTR1 by passive diffusion and bind to DNA and proteins. Cisplatin is found to inhibit several enzymes targeting cysteine, histidine and methionine residues, which are expected to be responsible for its anticancer activity. A better understanding of how the size and shape and leaving ligands of platinum complexes affect cysteine protease, papain enzyme are studied. This could give new ways to optimize anticancer activity. The activity of papain enzyme was measured on UV-Visible spectroscopy. The inhibition profile of papain with different platinum (II) complexes, and with different combinations was studied.

Le cuivre est un métal essentiel, présent dans de nombreuses protéines de notre corps et joue un rôle crucial dans les processus biochimiques. Plus précisément, deux aspects ont fait du Cu un métal d’intérêt pour le traitement du cancer : le fait qu’il s’agisse d’un métal endogène, et qui devrait par conséquent avoir moins d’effets secondaires; et le couple redox Cu(II)/Cu(I) – rapporté comme pouvant générer des espèces réactives de l'oxygène (EROs). La première partie de la thèse est basée sur la synthèse, la caractérisation et l’étude de l’activité biologique d’un complexe dinucléaire de Cu(II) contenant un ligand de type salphène N,O-donneur (L1), conçu pour promouvoir une conversion rapide Cu(II)/Cu(I). Les tests cellulaires montrent une forte production d’EROs dans les lignées de cellules HeLa et plus de toxicité au sein des cellules cancéreuses que dans les saines. À partir de ce point de départ, L1 a été fonctionnalisé avec des groupes halogénés afin de moduler le potentiel rédox du couple Cu(II)/Cu(I). Cependant, tous les complexes correspondants présentent certains problèmes de solubilité. En ce sens, la deuxième partie de la thèse porte sur la dérivatisation du ligand L1 avec des groupes sulfonate, arginine et peptides avec capacité de pénetration intra cellulaire, afin d’améliorer sa biodisponibilité et penetrabilité en maintenant le même environnement de coordination autour du cuivre. La dernière partie de ce travail ouvre la porte à l’utilisation d’une plateforme dendritique multimodale pour être utilisé comme futur système d’administration de médicaments, en preuve de concept de ce système pour son utilisation dans de futures thérapies dirigées
Copper is an essential biometal, present in several proteins of our body and plays a crucial role in many biochemical processes. Two features make Cu attractive to be used in chemotherapy: its nature as an endogenous metal –which may imply fewer side effects than other exogenous metals- and its Cu(II)/Cu(I) redox pair –which can promote reactive oxygen species (ROS) generation. On the first part of this thesis work, we report the synthesis, characterization and biological evaluation of a dimeric Cu(II) complex bearing a N,O-donor salphen-like ligand (L1) specifically designed to promote a fast Cu(II)/Cu(I) redox interconversion. In vitro assays outline the high potentiality of the complex to undergo ROS generation inside HeLa cells, and that it shows higher cytotoxicity in cancer than in normal cell lines. From this promising starting point, L1 was functionalized with halogen groups to modulate the redox potential of the Cu(II)/Cu(I) redox pair. However, one of the main drawbacks faced with these complexes was their poor solubility. Therefore, the second part of the thesis was devoted to the functionalization of L1 with several groups/entities (sulfonate, arginine and/or Cell-Penetrating Peptides), in order to increase the solubility, bioavailability and the delivery of the complex inside the cell while maintaining the same Cu(II) coordination environment. Finally, the last part of our work opens the gate to the use of a multimodal dendritic platform as a promising drug carrier, in a proof-of-concept of the versatility of this system for future tailor-made anticancer targeted therapies

In the 1980's, Nicholas Farrell developed a range of structurally distinct multinuclear Pt complexes that form long-range interstrand crosslinks (IXLs) in DNA. The dinuclear complex [{trans-PtCl2(NH3)}2-µ-(H2N(CH2)6NH2)]2+ (1,1/t,t) was the first of this series to show promising results, however, it was the trinuclear complex [{trans-PtCl2(NH3)}2-µ-trans-Pt(NH3)2(H2N(CH2)6NH2)2]4+ (1,0,1/t,t,t or BBR3464) that was chosen for clinical trials based on significantly increased cytotoxicity compared to 1,1/t,t and cisplatin. Molecular biology experiments have shown that 1,1/t,t exclusively forms IXLs in DNA in the 5'¿ 5' direction, whilst 1,0,1/t,t,t can form IXLs in both the 5'¿5' and 3'¿3' directions. Previously, 2D [1H,15N] HSQC NMR has been used to study the formation of 5'–5' 1,4–GG IXLs. The formation of 3'–3' 1,4–GG IXLs have been studied as part of this thesis. More recently, Pt complexes such as [{trans–PtCl2(NH3)}2{H2N(CH2)6(NH2(CH2)2NH2)(CH2)6NH2}]4+ (1,1/t,t–6,2,6) and [{trans–PtCl2(NH3)}2{H2N(CH2)6(NH2)(CH2)6NH2}]3+ (1,1/t,t–6,6), where the charged central Pt moiety of 1,0,1/t,t,t is replaced by a polyamine linker, have been developed in the Farrell group and show increased potency compared to 1,0,1/t,t,t. The complex 1,1/t,t 6,2,6 is a lead candidate currently undergoing Phase I clinical trials. Prior to the work presented in this thesis, little was known about the aquation chemistry or kinetics of DNA binding of these novel complexes. Reported in Chapter 3 is the study of the formation of 3'–3' 1,4–GG IXLs by both 1,0,1/t,t,t and 1,1/t,t in the duplex 5' {d(TATACATGTATA)2} (33–14XL) (pH 5.4, 298K). A combination of 1D 1H and 2D [1H, 15N] HSQC NMR experiments was used to directly compare the results with the stepwise formation of the 5'–5' 1,4–GG IXL with the previously studied duplex, 5' {d(ATATGTACATAT)2} (55–14XL), under the same conditions. Preassociation as well as aquation were similar, however, differences were observed at the monofunctional binding step with evidence for numerous monofunctional adducts. Both reactions did not yield a single 3'–3' 1,4–GG IXL, rather several adducts that could not be characterised. Molecular dynamics simulations of the 3'–3' 1,4–GG IXLs showed highly distorted lesions that may have implication in cellular repair processes.

Doutoramento em Química
The main scope of this work was to evaluate the metabolic effects of anticancer agents (three conventional and one new) in osteosarcoma (OS) cells and osteoblasts, by measuring alterations in the metabolic profile of cells by nuclear magnetic resonance (NMR) spectroscopy metabolomics. Chapter 1 gives a theoretical framework of this work, beginning with the main metabolic characteristics that globally describe cancer as well as the families and mechanisms of action of drugs used in chemotherapy. The drugs used nowadays to treat OS are also presented, together with the Palladium(II) complex with spermine, Pd2Spm, potentially active against cancer. Then, the global strategy for cell metabolomics is explained and the state of the art of metabolomic studies that analyze the effect of anticancer agents in cells is presented. In Chapter 2, the fundamentals of the analytical techniques used in this work, namely for biological assays, NMR spectroscopy and multivariate and statistical analysis of the results are described. A detailed description of the experimental procedures adopted throughout this work is given in Chapter 3. The biological and analytical reproducibility of the metabolic profile of MG-63 cells by high resolution magic angle spinning (HRMAS) NMR is evaluated in Chapter 4. The metabolic impact of several factors (cellular integrity, spinning rate, temperature, time and acquisition parameters) on the 1H HRMAS NMR spectral profile and quality is analysed, enabling the definition of the best acquisition parameters for further experiments. The metabolic consequences of increasing number of passages in MG-63 cells as well as the duration of storage are also investigated. Chapter 5 describes the metabolic impact of drugs conventionally used in OS chemotherapy, through NMR metabolomics studies of lysed cells and aqueous extracts analysis. The results show that MG-63 cells treated with cisplatin (cDDP) undergo a strong up-regulation of lipid contents, alterations in phospholipid constituents (choline compounds) and biomarkers of DNA degradation, all associated with cell death by apoptosis. Cells exposed to doxorubicin (DOX) or methotrexate (MTX) showed much slighter metabolic changes, without any relevant alteration in lipid contents. However, metabolic changes associated with altered Krebs cycle, oxidative stress and nucleotides metabolism were detected and were tentatively interpreted at the light of the known mechanisms of action of these drugs. The metabolic impact of the exposure of MG-63 cells and osteoblasts to cDDP and the Pd2Spm complex is described in Chapter 6. Results show that, despite the ability of the two agents to bind DNA, the metabolic consequences that arise from exposure to them are distinct, namely in what concerns to variation in lipid contents (absent for Pd2Spm). Apoptosis detection assays showed that, differently from what was seen for MG-63 cells treated with cDDP, the decreased number of living cells upon exposure to Pd2Spm was not due to cell death by apoptosis or necrosis. Moreover, the latter agent induces more marked alterations in osteoblasts than in cancer cells, while the opposite seemed to occur upon cDDP exposure. Nevertheless, the results from MG-63 cells exposure to combination regimens with cDDP- or Pd2Spm-based cocktails, described in Chapter 7, revealed that, in combination, the two agents induce similar metabolic responses, arising from synergy mechanisms between the tested drugs. Finally, the main conclusions of this thesis are summarized in Chapter 8, and future perspectives in the light of this work are presented.
Este trabalho teve como principal objetivo estudar os efeitos metabólicos de alguns fármacos (três fármacos convencionais e um em desenvolvimento) em células de osteossarcoma (OS) e osteoblastos, através da medição de alterações dos perfis metabólicos celulares por metabolómica usando espectroscopia de Ressonância Magnética Nuclear (RMN). O Capítulo 1 apresenta um enquadramento teórico deste trabalho, começando por identificar as principais características metabólicas que descrevem o cancro em geral, assim como as famílias e mecanismos de ação dos fármacos usados no seu tratamento. São ainda apresentados os fármacos usados atualmente na quimioterapia do OS, bem como o complexo de Paládio (II) com espermina, Pd2Spm, com potencial atividade anticancerígena. Seguidamente, é explicada a estratégia da metabolómica celular e apresentado o estado da arte de estudos metabolómicos do efeito de agentes anticancerígenos em células. No Capítulo 2, apresentam-se os princípios das técnicas analíticas usadas neste trabalho, nomeadamente ensaios biológicos, espectroscopia de RMN e análise multivariada e estatística dos resultados. Os detalhes e procedimentos experimentais relativos aos métodos usados são descritos no Capítulo 3. O estudo da reprodutibilidade analítica e biológica do perfil metabólico de células MG-63 medido por RMN de alta resolução e rotação segundo o ângulo mágico (HRMAS) é apresentado no Capítulo 4. Avalia-se o impacto de vários fatores (integridade celular, velocidade de rotação da amostra, temperatura, duração e parâmetros de aquisição) nas características e qualidade do espectro de RMN HRMAS de 1H, definindo-se então os parâmetros de aquisição dos espectros a adquirir subsequentemente. Avaliam-se também os efeitos do nº de passagens celulares e do tempo de armazenamento no perfil metabólico de células MG-63. O Capítulo 5 descreve o impacto metabólico de fármacos convencionais usados atualmente na quimioterapia do OS, estudado por metabolómica por RMN de células lisadas e análise de extratos celulares aquosos. Os resultados mostram que as células MG-63 tratadas com cisplatina (cDDP) sofrem um aumento dramático do teor de lípidos, alterações dos níveis de constituintes dos fosfolípidos (compostos de colina) e de indicadores de degradação do DNA, associados a fenómenos de apoptose. Nas células expostas a doxorrubicina (DOX) ou a metotrexato (MTX) foram identificadas alterações metabólicas mais ténues, com a quase total ausência de alterações no teor de lípidos. Foram também detetadas alterações em metabolitos relacionados com o ciclo de Krebs, stress oxidativo e metabolismo de nucleótidos, interpretadas tentativamente à luz dos mecanismos de ação de cada um dos fármacos. O impacto metabólico da exposição de células MG-63 e osteoblastos a cDDP e ao complexo de Pd2Spm é apresentado no Capítulo 6. Os resultados mostram que, apesar de ambos os fármacos poderem ligar ao DNA, as alterações metabólicas que decorrem da sua ação são muito distintas, nomeadamente no que respeita às variações nos teores de lípidos (ausentes para Pd2Spm). Ensaios de medição de apoptose mostraram que, contrariamente ao verificado para células MG-63 expostas a cDDP, a redução do número de células por exposição a Pd2Spm não se deve a fenómenos de morte celular por apoptose ou necrose. Além disso, este último complexo exerce um efeito mais marcado em osteoblastos do que nas células cancerígenas, o inverso parecendo acontecer com a exposição a cDDP. Contudo, os resultados da exposição de células MG-63 a regimes de tratamento combinado com base em cocktails de cDDP ou Pd2Spm, descritos no Capítulo 7, mostram que, em combinação, os dois agentes induzem respostas metabólicas semelhantes entre si, decorrentes de mecanismos de sinergia entre fármacos. Finalmente, sumariam-se no Capítulo 8 as conclusões deste trabalho e apontam-se perspetivas de trabalho futuro.

Le cancer est la deuxième cause de décès dans le monde. Malgré l'existence des traitements standards, le développement et la recherche de stratégies thérapeutiques innovantes et de médicaments est toujours nécessaire. La combinaison des médicaments induisant la mort cellulaire immunogène (ICD) et l'inhibition des points de contrôle immunitaire (ICB), semble être un protocole prometteur. Dans cette thèse, nous avons démontré que la Lurbinectedine, un inhibiteur de la transcription nouvellement approuvé pour le traitement du cancer du poumon récidivant, déclenche les caractéristiques de l'ICD dans quatre différentes lignées cellulaires humaines et murines in vitro. Vaccinée par des cellules de fibrosarcome traitées par la lurbinectedine, les souris immunocompétentes sont protégées du rechallenge des tumeurs syngéniques. La lurbinectedine limite la croissance du fibrosarcome transplanté d'une manière immunodépendante. Dans les souris, le fibrosarcome murin (MCA205) transplanté et le cancer du sein, induit par des hormones en combinaison avec des cancérigènes, ont été sensibilisés par la lurbinectedine aux deux ICB : PD-1 et CTLA-4. Il convient de noter que la mémoire immunologique à long terme a été générée chez des souris guéries. En outre, nous avons évalué la capacité anticancéreuse de LTX-401, un peptide oncolytique conçu pour l'immunothérapie locale. Les injections intratumorales séquentielles de LTX-401 retardent considérablement la croissance des tumeurs sous-cutanées MCA205 et TC-1 chez un hôte immunocompétent, mais montrent un effet thérapeutique limité sur les tumeurs syngéniques abscopales. Une seule administration de LTX-401 augmente l'efficacité de l'ICB anti-CTLA-4 ou anti-PD-1 + anti-CTLA-4. De plus, le traitement séquentiel avec LTX-401 et les deux ICB présente une immunité antitumorale systémique à la fois contre la tumeur traitée et la tumeur abscopale. En conclusion, la lurbinectedine et le LTX-401 induisent la mort cellulaire immunogène des cellules cancéreuses et renforcent les effets anticancéreux des inhibiteurs de points de contrôle immunitaires. Ces résultats jettent les bases expérimentales de traitements combinés et peuvent faciliter les conceptions d'essais cliniques
Cancer is the second leading cause of death worldwide, despite the existence of standard treatment, innovative therapeutic strategies and drugs are still in urgent demand. The combination of immunogenic cell death (ICD) inducing drugs and immune checkpoint blockade (ICB) seems to be a promising modality. In this thesis, we demonstrated Lurbinectedin, a transcription inhibitor newly approved for relapsed lung cancer treatment, triggers hallmarks of ICD in four different human and murine cell lines in vitro. Vaccinated with Lurbinectedin-treated fibrosarcoma cell protects immunocompetent mice from rechallenge with syngeneic tumours. Lurbinectedin restrains transplanted fibrosarcoma growth in an immune dependent manner. Both transplanted MCA205 cancer and hormone/carcinogen induced breast cancer were sensitized by Lurbinectedin to PD-1 and CTLA-4 double ICBs. Of note, long-term immunological memory was generated in cured mice. Further, we evaluated the anticancer capacity of LTX-401, an oncolytic peptide designed for local immunotherapy. Sequential intratumoral injections of LTX-401 dramatically retards subcutaneous MCA205 and TC-1 tumour growth in immunocompetent host, yet shows limited therapeutic effect of anti-CTLA-4 or anti-PD-1/anti-CTLA-4 ICBs. Moreover, sequential LTX-401 treatment with double ICBs exhibits systemic antitumor immunity to both treated and abscopal tumour. In conclusion, lurbinectedin and LTX-401 induce cancer cell immunogenic cell death and enhance the anticancer effects of immune chekcpoint blockade. These results lay the experimental foundation of combination regiments and may facilitate the clinical trial design

多年來，儘可能多的從植物中提取單體化合物一直是開發新型化學防癌劑和化學治療劑藥物的重要來源。
在本课题中，我们活性測試了从刺果紫玉盘（番荔枝科植物）中分离得到的14个番荔枝内酯化合物和7个多氧环己烯化合物，从三叉刺（豆科植物）和黄瑞香（瑞香科植物）中分离得到的4个黄酮化合物，从黄瑞香（瑞香科植物）和了哥王（瑞香科植物）中分离得到的2个香豆素化合物，以及从总状蕨藻（蕨藻科植物）中分离得到的1 个生物碱化合物，對11種人類常見癌症細胞株，如惡性黑色素瘤、肺癌、子宮頸上皮腺癌、肝癌、前列腺癌、結直腸癌的體外抗癌活性，用以建立一個全面的抗癌活性數據庫，為人們更好得了解番荔枝科植物奠定基礎。
在這些被篩選的單體化合物中，番荔枝內酯（ACGs）顯示出卓越的抗癌活性。它們對某些癌細胞株的細胞毒性甚至達到了nmol/l級別。例如番荔枝內酯desacetyluvaricin（Dau），對11條人類癌細胞株具有廣泛的抗增生活性，其半抑制濃度（IC₅₀）範圍從2.3 nM到37.4 μM。其中，Dau對結直腸癌細胞SW480的毒性最甚。Dau不僅具有高的抗癌效力，并對人正常纖維細胞Hs68幾乎沒有細胞毒性，半抑制濃度超過了247.5 μM。進一步的機理研究表明，Dau可導致SW480細胞產生大量過氧化物，進而導致細胞核內DNA斷裂。DNA損傷會讓MEK/ERK信號通路失活，並且影響了細胞週期調控蛋白的正常表達。例如影響細胞S週期的調控蛋白Cyclin A和Cyclin E的表達，以及影響G₁/S檢查點調控蛋白E2F的表達。由此，Dau促進SW480癌細胞穿過G₁/S檢查點，由G₁進入S期並在S期累計。最終被抑制在S週期的SW480細胞發生了壞死。以上機理的研究可為更好的理解ACG的作用機制提供一定的理論基礎。
番荔枝內酯是一系列長鏈脂肪酸的衍生物。它的結構的多樣性引發了我們極大的興趣去研究它的構效關係。我們比較了14個番荔枝內酯在細胞毒性和細胞週期控制方面對兩種不同的前列腺癌細胞LNCaP（p53基因野生型）和PC-3（p53基因缺失型）的影響。實驗結果表明，LNCaP細胞比PC-3更加敏感。番荔枝內酯的這種選擇性大概跟癌細胞中p53抑癌蛋白的表達水平有關。此外，關於構效關係的研究我們還發現：（1）在番荔枝內酯結構的核心系統中，四氫呋喃環的個數越多，化合物的抗癌活性越高；（2）在含有相鄰雙四氫呋喃環結構的化合物中，擁有threo/trans/threo/trans/erythro立體構型的化合物的細胞毒性比擁有threo/trans/threo/trans/threo立體構型的化合物高；（3）含單或雙四氫呋喃環結構的番荔枝內酯都將通過將LNCaP細胞抑制在G₁/G₀週期從而達到抗癌效果，並不會引起細胞凋亡；（4）含單四氫呋喃環結構的番荔枝內酯都將通過引發細胞凋亡從而達到抑制PC-3癌細胞的增長。然而含雙四氫呋喃環結構的番荔枝內酯會引發更多的PC-3細胞凋亡，並且有不同程度的細胞週期抑制；（5）在四氫呋喃環核心體系上，乙酰氧基會比羥基增加番荔枝內酯的細胞毒性；（6）雙鍵的取代基也會增加毒性效果。我們的研究結果印證了一些文獻已報導的關於番荔枝內酯構效關係的結論，同時我們也提出了一些新的假設。
本研究不僅增加了我們對番荔枝內酯強大的抗癌活性更全面的了解，並且通過機理研究還為它的選擇性毒性及構效關係特點提供了有重要的信息。番荔枝內酯是一類具有充滿前景抗癌化合物。在接下來的研究中，我們將致力於體內抗癌活性的研究，并擴大研究範圍，通過對多個ACG化合物的機理研究來證明我們對它的選擇性毒性的機理假設。
For years and years, the discovery of phytochemicals as many as possible has always been an important strategy for the development of novel chemopreventive and chemotherapeutic drugs.
In this studies, we have screened 14 Annonaceous acetogenins and 7 polyoxygenated cyclohexenes isolated from the root of Uvaria calamistrata (Annonaceae), 4 flavonoids isolated from the stems of Trifidacanthus unifoliolatus (Fabaceae) and Daphne giraldii (Thymelaeaceae), 2 cumarins isolated from the stem bark of Daphne giraldii (Thymelaeaceae) and the root of Wikstroemia indica (Thymelaeaceae), and 1 alkaloid isolated from Caulerpa racemosa (Caulerpaceae). The in vitro anticancer effects of these 28 natural compounds on 11 human cancer cell lines, including malignant melanoma, lung carcinoma, cervix epithelial adenocarcinoma, liver carcinoma, prostate adenocarcinoma and colorectal adenocarcinoma, were tested to set up an overall anticancer activity database for better understanding of the biological actions of Annonaceous plants.
Among the screened natural compounds, Annonaceous acetogenins (ACGs) exhibited outstanding anticancer efficacy. The cytotoxicities of ACGs to some cancer cell lines were even at nmol/l level. For instance, desacetyluvaricin (Dau), an ACG, was identified as a novel antiproliferative agent with a broad spectrum of inhibitions against the tested 11 human cancer cell lines with the IC₅₀ values ranging from 2.3 nM to 37.4 μM, and was especially cytotoxic to SW480 human colorectal carcinoma cells. Despite this potency, Dau was virtually nontoxic toward Hs68 human fibroblasts with an IC₅₀ value exceeding 247.5 μM. Further cell death mechanism studies showed that Dau could induce large amounts of superoxide production, which subsequently induced nuclear DNA fragmentation. DNA damage may inactivate the MEK/ERK signaling pathway and disturbed the expressions of cell cycle regulators such as Cyclin A and Cyclin E, which are S phase regulators, and E2F which is the G1/S checkpoint regulator. Thereafter, Dau arrested SW480 cells in S phase by promoting SW480 cells passing through the G₁/S boundary, and then accumulating in S phase. Finally, the SW480 cells underwent necrotic cell death. This mechanism study may provide a better understanding on the action mode of ACGs.
ACGs are derivatives of long chain fatty acids. Its structural diversity kindled our great interests in its structure-activity relationship (SAR). Therefore, we compared the cytotoxicities and cell cycle regulations of the 14 ACG compounds on two different human prostate cancer cell lines, LNCaP (p53 wild-type) and PC-3 (p53 null-type). Results showed that LNCaP cells were more sensitive to ACGs than PC-3 cells. This selectivity may be due to the presence of p53 tumor suppressor gene. Moreover, we found about SAR study that (1) the more THF rings existing in the core structure of ACGs, the more potent anticancer effects of ACGs would be; (2) for the adjacent bis-THF ACGs, stereo-structure with threo/trans/threo/trans/erythro configuration is generally more cytotoxic than the one with threo/trans/threo/trans/threo configuration; (3) both mono-THF ACGs and bis-THF ACGs inhibited LNCaP cells growth by G₁/G₀ phase arrest without any apoptosis induction; (4) mono-THF ACGs inhibited PC-3 cells growth by inducing apoptosis without cell cycle disturbance. However, the bis-THF ACGs could induce more apoptosis in PC-3 cells with partially cell cycle arrest. (5) the -OAc substituent group instead of -OH in the THF system would enhance the cytotoxicity efficacies of ACGs; (6) the double bond substituent would also enhance the anticancer effect. Our studies have proved several reported disciplines about the SAR of ACGs, and also proposed some new hypothesis.
Taken together, this study not only increased our understanding on the potent anticancer effects of ACG, but also provided valuable information on explaining its special cytotoxicities and the SAR properties through underling mechanism study. ACGs are a group of promising anticancer compounds with potent and steady activities. In the future work, we should further examine the in vivo anticancer effects and study more ACGs on their action modes to validate our hypothesis on their sensitivities to certain cancer cell lines.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Xue, Junyi.
Thesis (Ph.D.) Chinese University of Hong Kong, 2014.
Includes bibliographical references (leaves 215-236).
Abstracts also in Chinese.

Yeung Chung Man.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 83-97).
Abstracts in English and Chinese.
Abstract --- p.i
Abstract (Chinese) --- p.iv
Acknowledgements --- p.vii
Table of contents --- p.ix
List of figures --- p.xii
List of tables --- p.xiv
List of abbreviations --- p.xv
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- General Introduction --- p.1
Chapter 1.2 --- Literature Review --- p.5
Chapter 1.2.1 --- Cancer and melanoma --- p.5
Chapter 1.2.2 --- Anticancer drugs from natural products --- p.6
Chapter 1.2.3 --- Challenges in treatment of melanoma --- p.9
Chapter 1.2.4 --- TCM - New source of natural products for cancer therapy --- p.10
Chapter 1.2.6 --- The genus Schefflera --- p.11
Chapter 1.2.7 --- Anticancer activities of triterpenoids --- p.16
Chapter 1.2.8 --- Cancer and apoptosis --- p.17
Chapter 1.2.8.1 --- The Apoptosis Pathways --- p.20
Chapter 1.2.9 --- Studies of anticancer molecules against melanoma --- p.26
Chapter 1.2.9.1 --- In vitro models for studying anticancer molecules --- p.26
Chapter 1.2.9.2 --- In vivo models for studying anticancer molecules --- p.30
Chapter Chapter 2 --- Materials and Methods --- p.34
Chapter 2.1 --- Phytochemicals --- p.34
Chapter 2.2 --- "Chemicals, Cell Lines and Culture Conditions" --- p.34
Chapter 2.3 --- Determination of in vitro antiproliferative effects of HLDA and the ethyl acetate fraction from S. heptaphylla on human cancer cells --- p.36
Chapter 2.3.1 --- MTT assay --- p.36
Chapter 2.4 --- Determination of the in vitro antiproliferative mechanisms of HLDA and the ethyl acetate fraction from S. heptaphylla in human melanoma A375 cells --- p.37
Chapter 2.4.1 --- Flow cytometric analysis --- p.37
Chapter 2.4.2 --- Western blot analysis --- p.38
Chapter 2.5 --- Determination of the in vivo anticancer effects of the ethyl acetate fraction from S. heptaphylla --- p.41
Chapter 2.5.1 --- Determination of cancer chemopreventive effect of the ethyl acetate fraction with DMBA/TPA-induced skin carcinogenesis model --- p.41
Chapter 2.5.2 --- Determination of cancer therapeutic effect of the ethyl acetate fraction with athymic BALB/c nude mice model --- p.42
Chapter 2.6 --- Statistical Analysis --- p.44
Chapter Chapter 3 --- Results --- p.45
Chapter 3.1 --- Effects of HLDA and the ethyl acetate fraction on viability and proliferation of different cancer cell lines by MTT assay --- p.45
Chapter 3.2 --- Effects of HLDA and the ethyl acetate fraction on cell cycle and apoptosis in A375 cells determined by DNA flow cytometry --- p.46
Chapter 3.3 --- Effects of HLD A and the ethyl acetate fraction on apoptosis induction in A375 cells determined by Western blotting --- p.53
Chapter 3.4 --- Effects of HLD A and ethyl acetate fraction on caspases in A375 cells --- p.55
Chapter 3.5 --- Effects of caspase inhibitors on the HLDA- and the ethyl acetate fraction-induced apoptosis in A375 cells --- p.57
Chapter 3.6 --- Effects of HLD A and the ethyl acetate fraction on the expression of Bcl-2 family proteins in A375 cells --- p.62
Chapter 3.7 --- Chemopreventive effect of the ethyl acetate fraction from S. heptaphylla on the DMBA/TPA-induced skin carcinogenesis model --- p.65
Chapter 3.8 --- Chemotherapeutic effect of the ethyl acetate fraction from S. heptaphylla on A375 xenograft in athymic nude mice --- p.70
Chapter Chapter 4 --- Discussion --- p.73
References --- p.83

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.

yes
A series of ruthenium and iridium complexes have been synthesized and characterized with 20 novel crystal structures discussed. The library of β-ketoiminato complexes has been shown to be active against MCF-7 (human breast carcinoma), HT-29 (human colon carcinoma), A2780 (human ovarian carcinoma), and A2780cis (cisplatin-resistant human ovarian carcinoma) cell lines, with selected complexes’ being more than three times as active as cisplatin against the A2780cis cell line. Selected complexes were also tested against the noncancerous ARPE-19 (retinal pigment epithelial cells) cell line, in order to evaluate the complexes selectivity for cancer cells. Complexes have also been shown to be highly active under hypoxic conditions, with the activities of some complexes increasing with a decrease in O2 concentration. The enzyme thioredoxin reductase is overexpressed in cancer cells, and complexes reported herein have the advantage of inhibiting this enzyme, with IC50 values measured in the nanomolar range. The anticancer activity of these complexes was further investigated to determine whether activity is due to effects on cellular growth or cell survival. The complexes were found to induce significant levels of cancer cell death by apoptosis with levels induced correlating closely with activity in chemosensitivity studies. As a possible cause of cell death, the ability of the complexes to induce damage to cellular DNA was also assessed. The complexes failed to induce double-strand DNA breaks or DNA cross-linking but induced significant levels of single-strand DNA breaks, indicating a mechanism of action different from that of cisplatin.
Lord RM, Hebden AJ, Pask CM, Henderson IR, Allison SJ, Shepherd SL, Phillips RM, McGowan PC

Widely used in cancer treatment, chemotherapy still faces hindering challenges, ranging from severe induced toxicity to drug resistance acquisition. As means to overcome these setbacks, newly synthetized compounds have recently come into play with the basis of improved pharmacokinetic/pharmacodynamic properties. With this mind-set, this project aimed towards the antiproliferative potential characterization of a group of metallic compounds. Additionally the incorporation of the compounds within a nanoformulation and within new combination strategies with commercial chemotherapeutic drugs was also envisaged. Cell viability assays presented copper (II) compound (K4) as the most promising, presenting an IC50 of 6.10 μM and 19.09 μM for HCT116 and A549 cell line respectively. Exposure in fibroblasts revealed a 9.18 μM IC50. Hoechst staining assays further revealed the compound’s predisposition to induce chromatin condensation and nuclear fragmentation in HCT116 upon exposure to K4 which was later demonstrated by flow cytometry and annexin V-FITC/propidium iodide double staining analysis (under 50 % cell death induction). The compound further revealed the ability to interact with major macromolecules such as DNA (Kb = 2.17x105 M-1), inducing structural brakes and retardation, and further affecting cell cycle progression revealing delay in S-phase. Moreover BSA interactions were also visible however not conclusive. Proteome profiling revealed overexpression of proteins involved in metabolic activity and underexpression of proteins involved in apoptosis thus corroborating Hoechst and apoptosis flow cytometry data. K4 nanoformulation suffered from several hindrances and was ill succeeded in part due to K4’s poor solubility in aqueous buffers. Other approaches were considered in this regard. Combined chemotherapy assays revealed high cytotoxicity for afatinib and lapatinib strategies. Lapatinib and K4 proteome profiling further revealed high apoptosis rates, high metabolic activity and activation of redundant proteins as part of compensatory mechanisms.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
As ciclodextrinas (CDs) são macromoléculas naturais muito úteis, que são usadas como excipientes nas formulações farmacêuticas, evidenciando um amplo espetro de aplicação na indústria farmacêutica. A capacidade única de agirem como contentores moleculares através do aprisionamento de uma ampla gama de moléculas hóspede na sua cavidade hospedeira faz das ciclodextrinas uma ferramenta valiosa para a melhoria da solubilidade, da estabilidade e da biodisponibilidade de fármacos. Estas características revelam-se especialmente úteis na quimioterapia, visto que a maioria dos fármacos anticancerígenos apresenta baixa permeabilidade e baixa solubilidade em água. Assim, os complexos de inclusão fármaco-CD oferecem várias vantagens, não apenas em formulações farmacêuticas, mas também na clínica com a redução de efeitos secundários preocupantes, e com a possibilidade de uso de vias de administração mais convenientes. Apesar da sua excecionalidade, a combinação destas macromoléculas com outras tecnologias e materiais constitui uma potencial estratégia no desenvolvimento de novos, avançados e multifuncionais nanossistemas de libertação de fármacos. Assim, o design, a execução e o registo adequados dos resultados in vivo são essenciais para tornarem os dados pré-clínicos mais reprodutíveis e transponíveis para a clínica. Nesta revisão, é apresentada uma exposição abrangente sobre o uso dos sistemas de libertação à base de CDs testados in vivo no combate ao cancro. Até agora, têm sido obtidos resultados encorajadores com o uso das CDs, abrindo novas portas para a obtenção de sistemas de libertação inteligentes viáveis com a capacidade de atender às necessidades da terapia do cancro ainda não ultrapassadas.
Cyclodextrins (CDs) are useful natural macromolecules which act as excipients on pharmaceutical formulations, evidencing a large spectrum of applications in the pharmaceutical industry. Their unique ability to act as molecular containers by entrapping a wide range of guest molecules in their internal cavity makes them as a valuable tool to improve the solubility, stability, and bioavailability of drugs. These features are especially useful when it comes to chemotherapy, as most of the anticancer drugs present low permeability and low water solubility. So, the resulting drug-CD inclusion complexes offer several potential advantages, not only in pharmaceutical formulations, but also on the clinic with the reduction of the concerning side effects and the application of more convenient administration routes. Despite their exceptionality, the combination of these macromolecules with other technologies and materials constitutes a potential strategy to the development of new, advanced and multifunctional nanodelivery systems. Thus, suitable design, execution, and reporting of in vivo results resorting to animal models are mandatory to make pre-clinical data more reproducible and translatable to the clinic. In this review, a comprehensive exposition about the use of CDs-based delivery systems to fight cancers tested in vivo is made. Exciting results using CDs have been obtained so far, paving a new path to the attainment of viable intelligent delivery systems to possibly address cancer unmet needs.

Tang Angie.
Thesis submitted in: November 2005.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (leaves 151-172).
Abstracts in English and Chinese.
Acknowledgements --- p.i
Abstract --- p.iii
Abstract in Chinese --- p.vi
Table of Contents --- p.vii
List of Publications --- p.xi
Awards --- p.xii
List of Abbreviations --- p.xiii
List of Tables --- p.xv
List of Figures --- p.xvi
Chapter Chapter 1 --- Introduction --- p.1
Chapter Chapter 2 --- Literature Review --- p.3
Chapter 2.1 --- Gastric cancer-overview --- p.3
Chapter 2.1.1 --- Epidemology --- p.3
Chapter 2.1.2 --- Pathology --- p.3
Chapter 2.1.3 --- Etiologies and Risk Factors --- p.4
Chapter I. --- Environmental factors --- p.4
Chapter a. --- Helicobacter pylori infections --- p.4
Chapter b. --- Epstein-Barr virus (EBV) --- p.6
Chapter c. --- Dietary factors --- p.6
Chapter d. --- Smoking --- p.6
Chapter II. --- Genetic Factors --- p.7
Chapter a. --- Hereditary Gastric Cancer --- p.7
Chapter b. --- Genetic polymorphism --- p.8
Chapter III. --- Cyclooxygenases (COX) enzymes --- p.10
Chapter IV. --- Molecular carcinogenesis --- p.11
Chapter a. --- Activation of proto-oncogenes --- p.11
Chapter b. --- Candidate tumor suppressor genes --- p.12
Chapter 1. --- Gene mutation and deletion --- p.12
Chapter 2. --- Epigenetic Silencing --- p.13
Chapter 2.2 --- Epigenetics --- p.14
Chapter 2.2.1 --- DNA methylation --- p.15
Chapter 2.2.2 --- Histone modification --- p.28
Chapter I. --- Histone acetylation and deacetylation --- p.32
Chapter II. --- Histone methylation --- p.32
Chapter III. --- Histone phosphorylation --- p.34
Chapter IV. --- Histone ubiquitylation --- p.34
Chapter 2.3 --- "HAT, HDAC and HDAC inhibitors" --- p.36
Chapter 2.3.1 --- HAT --- p.38
Chapter 2.3.2 --- HDAC --- p.39
Chapter (a) --- Class I --- p.40
Chapter (b) --- Class II --- p.41
Chapter (c) --- Class III --- p.42
Chapter (d) --- Mammalian HDAC and their mechanism of deacetylation --- p.44
Chapter 2.3.3 --- HDAC inhibitors --- p.45
Chapter I. --- Class I/II natural inhibitors --- p.47
Chapter II. --- Class I/II synthetic inhibitors --- p.48
Chapter III. --- Sirtuins inhibitors --- p.49
Chapter IV. --- Activity of HDAC inhibitors in vitro --- p.50
Chapter a. --- Effect in the gene expression --- p.50
Chapter b. --- Non-transcriptional effects --- p.55
Chapter c. --- Activity of HDAC inhibitors with other agents --- p.57
Chapter d. --- Effects in xenograft tumor models --- p.57
Chapter V. --- Clinical trials of HDAC inhibitors --- p.59
Chapter Chapter 3 --- Aims of the study --- p.63
Chapter Chapter 4 --- Materials and Methods --- p.64
Chapter 4.1 --- Cell culture --- p.64
Chapter 4.2 --- Drug treatment --- p.64
Chapter 4.2.1 --- Suberoylanilide Hydroxamic Acid treatment --- p.64
Chapter 4.2.2 --- Trichostatin A treatment --- p.65
Chapter 4.3 --- Cell proliferation assay --- p.66
Chapter 4.4 --- Apoptotic assay --- p.67
Chapter 4.5 --- Flow cytometry --- p.67
Chapter 4.5.1 --- Cell preparation --- p.67
Chapter 4.5.2 --- Propidium Iodide staining --- p.68
Chapter 4.5.3 --- Annexin V-FITC staining --- p.68
Chapter 4.5.4 --- Flow cytometer analysis --- p.69
Chapter 4.6 --- Total RNA extraction --- p.70
Chapter 4.7 --- DNA extraction --- p.71
Chapter 4.8 --- Protein extraction --- p.72
Chapter 4.9 --- Western blottng --- p.72
Chapter 4.10 --- Microarray analysis --- p.74
Chapter 4.10.1 --- Sample preparation for microarray --- p.74
Chapter 4.10.2 --- Hybridization --- p.75
Chapter 4.10.3 --- Scanning and data processing --- p.75
Chapter 4.10.4 --- Data analysis --- p.76
Chapter 4.11 --- Primer design --- p.77
Chapter 4.12 --- RT-PCR --- p.77
Chapter 4.12.1 --- Reverse transcription --- p.77
Chapter 4.12.2 --- Quantitative RT-PCR --- p.78
Chapter 4.13 --- Methlyation study --- p.79
Chapter 4.13.1 --- Demethylation by 5-aza-2'deoxycytidine --- p.79
Chapter 4.13.2 --- Bisulfite modification --- p.79
Chapter 4.13.3 --- Methylation-specific PCR (MSP) --- p.79
Chapter Chapter 5 --- Results --- p.81
Chapter 5.1 --- Morphological changes in AGS cells --- p.81
Chapter 5.2 --- Anti-cancer effects of HDAC inhibitors --- p.81
Chapter 5.2.1 --- Effect of HDAC inhibitors on cell growth --- p.81
Chapter a. --- SAHA inhibits cell proliferation --- p.82
Chapter b. --- TSA inhibits cell proliferation --- p.82
Chapter 5.2.2 --- Cell cycle analysis --- p.87
Chapter a. --- Effect of SAHA on cell cycle --- p.87
Chapter b. --- Effect of TSA on cell cycle --- p.88
Chapter 5.2.3 --- Induction of apoptosis on AGS cells --- p.92
Chapter a. --- SAHA induces apoptotic cell death --- p.92
Chapter b. --- TSA induces apoptotic cell death --- p.94
Chapter 5.3 --- Induction of histone expression on AGS cells --- p.102
Chapter 5.3.1 --- HDAC inhibitors induced acetylation of histone H3 --- p.102
Chapter 5.3.2 --- HDAC inhibitors induced acetylation of histone H4 --- p.103
Chapter 5.4 --- SAHA- and TSA-induced gene expression profiles --- p.106
Chapter 5.5 --- Verification of gene expression by quantitative RT-PCR --- p.108
Chapter 5.6 --- Methylation study --- p.113
Chapter Chapter 6 --- Discussion --- p.116
Chapter 6.1 --- Improved treatment strategy is needed for gastric cancer. --- p.116
Chapter 6.2 --- HDAC inhibitors as potential anti-cancer agents --- p.117
Chapter 6.3 --- Potential anti-cancer effect of TSA and SAHA on AGS cells --- p.120
Chapter I. --- Morphological changes of AGS gastric cancer cells --- p.120
Chapter II. --- Inhibition of cell proliferation --- p.120
Chapter III. --- Induction of cell cycle arrest --- p.121
Chapter IV. --- Induction of apoptosis --- p.122
Chapter 6.4 --- Expression of acetylated histones upon treatment with TSA and SAHA --- p.124
Chapter 6.5 --- Identify potential target genes upon treatment with TSA and SAHA --- p.125
Chapter 6.5.1 --- Candidate genes involved in cell cycle --- p.126
Chapter a. --- P21WAF1 --- p.126
Chapter b. --- p27kip1. --- p.128
Chapter c. --- Cyclin E & Cyclin A --- p.128
Chapter d. --- Signal-induced proliferation-associated gene 1 (SIPA1) .… --- p.129
Chapter 6.5.2 --- Candidate genes involved in apoptosis and anti-proliferation --- p.130
Chapter a. --- BCL2-interacting killer (apoptosis-inducing) (BIK) (Pro-apoptotic gene) --- p.131
Chapter b. --- Thioredoxin interacting protein (TXNIP) (Proapoptotic gene)
Chapter c. --- Cell death-inducing DFFA-like effector b (CIDEB) (apoptosis induction) --- p.132
Chapter d. --- B-cell translocation gene 1 (BTG1) - (anti-proliferation) --- p.133
Chapter e. --- Quiescin 6 (QSCN6) (anti-proliferation) --- p.133
Chapter f. --- "Cysteine-rich, angiogenic inducer, 61 (CYR61) (anti-proliferative)" --- p.134
Chapter g. --- Metallothionein 2A (MT2A) (apoptosis induction and anti-proliferative) --- p.134
Chapter 6.5.3 --- Other genes reported to be up-regulated with HDAC inhibitors treatment --- p.135
Chapter a. --- Glia maturation factor-gamma (GMFG) --- p.135
Chapter b. --- v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS)
Chapter c. --- Interleukin 8 (IL-8) --- p.136
Chapter d. --- Insulin-like growth factor binding protein- 2 (IGFBP2) --- p.137
Chapter e. --- Integrin alpha chain 7 (ITGA7) --- p.138
Chapter 6.5.4 --- Selected highly up-regulated genes with HDAC inhibitors treatment --- p.139
Chapter a. --- Aldo-keto reductase family 1，member C3 (AKR1C3) --- p.139
Chapter b. --- GPI-anchored metastasis-associated protein homolog (C4.4A) --- p.139
Chapter c. --- "Serine (or cysteine) proteinase inhibitor，clade I (neuroserpin), member 1 (SERPINI1)" --- p.140
Chapter d. --- "Serine (or cysteine) proteinase inhibitor，clade E (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1)" --- p.140
Chapter e. --- Adrenomedullin (ADM) --- p.141
Chapter f. --- Dehydrogenase/reductase (SDR family) member 2 (HEP27) --- p.142
Chapter g. --- Cholecystokinin (CCK) --- p.142
Chapter h. --- Silver homolog (mouse) (SILV) --- p.143
Chapter 6.6 --- Genes regulated by gene promoter hypermethylation in AGS cells --- p.143
Chapter Chapter 7 --- Conclusion --- p.147
Chapter Chapter 8 --- Further Studies --- p.150
References --- p.151
Appendix I --- p.151
Appendix II --- p.III
Appendix III --- p.IV
Appendix IV --- p.VI

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.

A dissertation submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. March 2015
Lung cancer is currently the most deadly form of cancer due to the fact that metastasis occurs in the lymph nodes making it difficult to remove by surgical means. Chemotherapy has been the most successful method of treatment, although it has been harmful to human health as a consequence of non-specific cytotoxicity. There has been, therefore, a growing interest in cancer research to develop alternative cancer treatments, which are less toxic. Currently plant-derived drugs are perceived to be more effective as they display both cytotoxic activity and are less harmful to overall human health. Thus the aim of the study was to determine the cytotoxic effects of the plant Lobostemon fruticosus on A549 cells. The IC50 of the methanol and butanol extracts of L. fruticosus were obtained at 40 μg/ml and 50 μg/ml, respectively. DNA fragmentation was observed after 48 hour exposure to treatments, indicating that the plant extracts induced apoptosis. Cell cycle analysis indicated that the plant extracts inhibited cell cycle progression at the sub-G0 phase, which indicated that the cells had undergone apoptosis. RT-PCR showed that the expression of p53 was down-regulated; however, p21 and Bax were up-regulated in all treatments. LC-MS identified that the compounds from the plant extracts are known apoptotic inducers. The results lead to the conclusion that the extracts of L. fruticosus, induce cell death in A549 cells. The plant extracts induced a p53-independent apoptotic mechanism, which was mediated by Bax and p21. Key words: Lobostemon fruticosus, camptothecin, taxol, Non-small cell lung cancer (NSCLC)

This dissertation describes an introduction, background and research progress in the areas of multitargeted single agents and tubulin inhibitors in cancer chemotherapy and selective Toxoplasma gondii TS inhibitors for the treatment of toxoplasmosis.<br> Tubulin inhibitors are important antitumor agents that disrupt microtubule dynamics. Thymidylate synthase (TS) inhibitors prevent cell division by interfering with de novo thymidylate synthesis. Antiangiogenic agents target tumor angiogenesis crucial for tumor growth and metastasis. Under normal circumstances, angiogenesis is typically limited to tumor cells and is mediated by receptor tyrosine kinases (RTKs). Combination chemotherapies of RTK inhibitors with cytotoxic agents that target either TS or tubulin have shown significant promise and several preclinical and clinical studies with such combinations are in progress. Multitargeted single agents with dual antiangiogenic and cytotoxic mechanisms could avoid the major limitations associated with cancer chemotherapy: multidrug resistance and dose limiting toxicities. This dissertation focuses on the design and synthesis of pyrimido[4,5-b]indoles and furo[2,3-d]pyrimidines as potential single agents with dual antiangiogenic and cytotoxic activities. These efforts led to the identification of structural features that are necessary for inhibition of RTKs and/or tubulin polymerization. Novel synthetic strategies were developed for efficient synthesis of 2,4-diamino-5-thioaryl-pyrimido[4,5-b]indoles and 4-anilino-5-methyl-furo[2,3-d]pyrimidines.<br> Taxanes and vinca alkaloids are widely used tubulin inhibitors in cancer chemotherapy. However, their clinical use is compromised by two major mechanisms of drug resistance: the overexpression of Pgp and bIII-tubulin. This dissertation describes the design and synthesis of pyrimido[4,5-b]indoles as tubulin inhibitors that circumvent Pgp and bIII-tubulin mediated resistance. This work identified the structural features crucial for tubulin inhibition for the pyrimido[4,5-b]indole scaffold.<br> Infection by Toxoplasma gondii can lead to toxoplasmosis in immune compromised patients such as organ transplant, cancer and AIDS patients. Current therapy involving combination of sulfadiazine and pyrimethamine is limited by drug resistance and treatment failures. The thymidylate synthase‒dihydrofolate reductase enzyme is important for thymidylate synthesis in T. gondii, and hence can be targeted to treat T. gondii infection. TS is highly conserved across species and selectivity for tgTS over human TS is significantly more challenging. The present work provides an efficient synthesis of 2-diamino-4-oxo-5-thioaryl-pyrimido[4,5-b]indoles as selective tgTS inhibitors.
Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences;
Medicinal Chemistry
PhD;
Dissertation;

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