The aldehyde dehydrogenase (ALDH) superfamily is gaining momentum in regard to stem cell and cancer research. However, their regulation and expression in the cancer microenvironment is poorly understood. The aim of this work was to understand the role of selected ALDH isoforms (1A1, 1A2, 1A3, 1B1, 2, 3A1 and 7A1) in colorectal cancer (CRC) and explore the impact of hypoxia on their expression. CRC cell lines (HT29, DLD-1, SW480 and HCT116) were grown under normoxic or hypoxic conditions (0.1% O2) and HT29 and DLD-1 in spinner flasks to generate multicellular spheroids (MCS). Hypoxia was demonstrated to have an impact on the ALDH expression, which appeared cell-specific. Notably, ALDH7A1 was induced upon exposure to hypoxia in both HT29 and DLD-1 cells, shown to be expressed in the hypoxic region of the MCS variants and in 5/5 CRC xenografts (HT29, DLD-1, HCT116, SW620, and COLO205). ALDH7A1 siRNA knockdown studies in DLD-1 cells resulted in significant reduction of viable cells and significant increase in ROS levels, suggesting ALDH7A1 to possess antioxidant properties. These findings were further supported using isogenic H1299/RFP and H1299/ALDH7A1 lung cancer cell lines. ALDH7A1, however, was found not to be involved in inhibiting the pharmacological effect or causing resistance to different cytotoxic and molecularly targeted anticancer drugs. To unravel the functional role of ALDH7A1, 9 compounds obtained from a virtual screening of 24,000 compounds from the Maybridge collection of compounds were used to probe ALDH7A1 functional activity. One compound, HAN00316, was found to inhibit the antioxidant properties of ALDH7A1 and thus could be a good starting point for further chemical tool development. Although this study underpins a potential important role of ALDH7A1 in hypoxic CRC, further work is required to fully validate its potential as a biomarker and/or pharmacological target. / Jordan University of Science and Technology

The development of new anticancer drugs is necessary in order deal with the disease and with the drawbacks of currently applied drugs. Epigenetic dysregulations are a central hallmark of cancerogenesis and histone deacetylases (HDACs) emerged as promising anticancer targets. HDAC inhibitors are promising epigenetic anticancer drugs and new HDAC inhibitors are sought for in order to obtain potent drug candidates. The new HDAC inhibitor SF5-SAHA was synthesized and analyzed for its anticancer properties. The new compound SF5-SAHA showed strong inhibition of tumor cell growth with IC50 values similar to or lower than that of the clinically applied reference compound vorinostat/SAHA (suberoylanilide hydroxamic acid). Target specific HDAC inhibition was demonstrated by Western blot analyses. Unspecific cytotoxic effects were not observed in LDH-release measurements. Pro-apoptotic formation of reactive oxygen species (ROS) and caspase-3 activity induction in prostate carcinoma and hepatocellular carcinoma cell lines DU145 and Hep-G2 seem to be further aspects of the mode of action. Antiangiogenic activity of SF5-SAHA was observed on chorioallantoic membranes of fertilized chicken eggs (CAM assay). The presence of the pentafluorothio-substituent of SF5-SAHA increased the antiproliferative effects in both solid tumor and leukemia/lymphoma cell models when compared with its parent compound vorinostat. Based on this preliminary study, SF5-SAHA has the prerequisites to be further developed as a new HDAC inhibitory anticancer drug candidate.

No description available.

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.

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.

Paclitaxel is one of the most successful anti-cancer drugs, particularly in the treatment of breast cancer and ovarian cancer. For the investigation of the interaction between paclitaxel and MD-2 protein, and development of new antagonists for lipopolysaccharide, several C10 A-nor-paclitaxel analogs have been synthesized and their biological activities have been evaluated. In order to reduce the myelosuppression effect of the paclitaxel, several C3â ² and C4 paclitaxel analogs have been synthesized and their biological evaluation have been studied. / Master of Science

The thesis entitled “New DNA-Targeting Small Molecules as Potential Anticancer Agents and for in vivo Specificity toward Enhanced Silk Production” encompasses design, computational calculations, and syntheses of diverse small molecular scaffolds to explicitly target duplex and higher order DNA morphologies (G-quadruplex DNA). Some of these molecules have a potential as anticancer agents. Besides, an attempt has been made elucidate the importance of novel oligopyrrole carboxamides in the enhancement of silk yield, hence proving to a boon in the field of sericulture. The work has been divided into six chapters. Chapter 1. DNA Binding Small Molecules as Anticancer Agents Figure 1. DNA targeting by small molecules. Cancer has always been a dreadful disease and continues to attract extensive research investigations. Various targets have been identified to restrain cancer. Among these DNA happens to be the most explored one. A wide variety of small molecules, often referred to as “ligands”, has been synthesized to target numerous structural features of DNA (Figure 1). The sole purpose of such molecular design has been to interfere with the transcriptional machinery in order to drive the cancer cell toward apoptosis. The mode of action of the DNA targeting ligands focuses either on the sequence-specificity by groove binding and strand cleavage, or by identifying the morphologically distinct higher order structures like that of the G-quadruplex DNA. Chapter 2. Ligand 5, 10, 15, 20-tetra(N-methyl-4-pyridyl)porphine (TMPyP4) Prefers the Parallel Propeller-type Human G-Quadruplex DNA over its other Polymorphs The binding of ligand 5, 10, 15, 20-tetra(N-methyl-4-pyridyl)porphine (TMPyP4) with telomeric and genomic G-quadruplex DNA has been extensively studied. However, a comparative study of interactions of TMPyP4 with different conformations of human telomeric G-quadruplex DNA, namely parallel propeller-type (PP), antiparallel basket-type (AB), and mixed hybrid-type (MH) G-quadruplex DNA has not been done. We considered all the possible binding sites in each of the G-quadruplex DNA structures and docked TMPyP4 to each one of them. The resultant most potent sites for binding were analyzed from the mean binding free energy of the complexes. Molecular dynamics simulations were then carried out and analysis of the binding free energy of the TMPyP4-G-quadruplex complex showed that the binding of TMPyP4 with parallel propeller-type G-quadruplex DNA is preferred over the other two G-quadruplex DNA conformations. The results obtained from the change in solvent excluded surface area (SESA) and solvent accessible surface area (SASA) also support the more pronounced binding of the ligand with the parallel propeller-type G-quadruplex DNA (Figure 2). Figure 2. Ligand TMPyP4 prefers parallel propeller-type G-quadruplex DNA morphology. Chapter 3. A Theoretical Analysis on the Selective Stabilization of Intermolecular G-quadruplex RNA with a bis-Benzimidazole Ligand EtBzEt over TMPyP4 in K+ Environment Ever since the discovery of G-quadruplex RNA, a constant urge exists to target these higher order RNA conformations. These structures play a significant role in the transcriptional and translational mechanism. Herein we have determined the mode and extent of association of certain G-quadruplex DNA binding bisbenzimidazole ligand (EtBzEt) in comparison to a known porphyrin ligand (TMPyP4). We have performed docking studies of the known G-quadruplex DNA binding ligands with the parallel propeller G-quadruplex RNA (PPR) to determine the most potent binding conformation which showed EtBzEt to be a better RNA binder than others. Furthermore, a molecular dynamics (MD) simulation (6 ns) was performed for the most stable docked complex in explicit solvent environment. The role of K+ ions, Hoogsteen hydrogen bond formation and backbone dihedral angle between the tetrads were carefully analyzed during the entire simulation run to determine the stability of each ligand associated PPR complex. All the analyses conclusively showed that while TMPyP4 merely stabilized the PPR, the ligand EtBzEt stabilized PPR very efficiently (Figure 3). Figure 3. Stabilzation and destabilization by EtBzEt and TMPyP4, repectively. Red and green ovals represent EtBzEt and TMPyP4, repectively. Chapter 4A. Design and Synthesis of New DNA Binding Fe(III) and Co(II) Salen Complexes with Pendant Oligopyrrole Carboxamides Extensive research on these oligopyrrole carboxamides has shown their specificity toward AT-rich sequences with high binding affinity. Here we have designed and synthesized Fe (III)-and Co (II)-based salen complexes attached with minor groove targeting oligopyrrole carboxamide side-chains (Figure 4). While the ligands showed excellent activity toward DNA damage, they also exhibited high affinity toward the minor grooves of the ds-DNA. This was also reflected in the high efficiency of the ligands toward cancer cell cytotoxicity. Further studies revealed that the ligands resulted in prominent nuclear condensation and fragmentation thereby driving the cells toward apoptosis. The presence of metal coordinated salen moiety conjugated with positively charged pendants ending with minor groove binding oligopyrrole carboxamides might have resulted in the increased activity of the ligands toward DNA targeting and cancer cell death. Figure 4. Chemical structures of the ligands used in this study. Chapter 4B. Design and synthesis of novel oligopyrrole based salen metal complexes and their efficiency toward stabilization of G-quadruplex DNA DNA targeting has been the key strategy toward the restriction of cancer cell proliferation. In a similar effort, we have designed and synthesized novel salen based Ni(II) and Pd(II) metal complexes with positively charged flanking side-chains comprising attached N-methylpyrrole carboxamides of varying lengths (Figure 5). The ligands showed efficient stabilization of the G-quadruplex DNA morphologies, with specificity over the duplex DNA. Sufficient inhibition of the telomerase activity was observed by the TRAP-LIG assay which was ascertained by the prominent restriction of cancer cell proliferation in the long-term cell viability assay. The ligands exhibited condensation and fragmentation of the nucleus when observed under confocal microscopy which is indicative of the cells undergoing apoptosis. Further annexin V-FITC and PI dual staining showed apoptosis to be the mechanistic pathway underlying the cancer cell cytotoxicity by the ligands. Modeling studies clearly showed the stacking of the salen moiety over the G-tetrads with the association of the pendant oligopyrrole carboxamide units to the grooves. Figure 5. Chemical structures of the ligands used in this study. Chapter 5A. Role of Metal Ions in Novel Fluorescein based Salen and Salphen Complexes toward Efficient DNA Damage and their Effect on Cancer Cells Metal ions play an important role toward DNA damage and numerous ligands have been synthesized for their use in anticancer therapy. Herein, we have designed and synthesized Fe(III) and Co(II) based salen/salphens by bridging two fluorescein moieties with varying spacers (Figure 6). Although the ligands exhibit dual binding mode, the more flexible salen ligands prefer to associate to the minor groove of the DNA while the relatively rigid salphen ligands show greater intercalation. The biophysical experiments reveal better binding affinity of the salphens toward duplex DNA as compared to the salen ligands. The metal coordination resulted in efficient DNA cleavage of plasmid at low ligand concentrations. The ligands also showed cancer cell cytotoxicity, cellular internalization with apoptosis as the proposed mechanism for cell death. Figure 6. Chemical structures of the salen and salphen ligands used in this study. Chapter 5B. Fluorescein based Salen and salphen Complexes as stabilizers of the Human G-quadruplex DNA and Promising Telomerase Inhibitors Metal based salen complexes have been considered as an important scaffold toward targeting of DNA structures. In the present work we have designed and synthesized nickel(II)-and palladium(II)-salen and salphen ligands by using fluorescein as the backbone to provide an extended aromatic surface (Figure 7). The ligands exhibit sufficient affinity toward the human telomeric G-quadruplex (G4) DNA in preference to the duplex DNA and also exhibit promising inhibition of telomerase activity. This is ascertained by their potency in the long-term cell viability assay which shows significant cancer cell cytotoxicity in presence of the ligands. Confocal microscopy showed cellular internalization followed by nuclear localization. Considerable population at the sub-G1 phase of the cell cycle showed cell death via apoptotic pathway. Figure 7. Chemical structures of the ligands used in this study. Chapter 6. Knockdown of Broad-Complex Gene Expression of Bombyx mori by Oligopyrrole carboxamides Enhances Silk Production Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5′-TTAGG-3′ sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5′-TTAGG-3′ sequences in controlling the gene expression in B. mori (Figure 8). The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The compounds caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production. Figure 8. Chemical structures of the ligands used in this study (top) and increased cocoon size on ligand treatment.

La plupart des médicaments anticancéreux sont des substances à marge thérapeutique étroite, les erreurs médicamenteuses peuvent avoir des conséquences majeures pour les patients. Il est de la responsabilité pharmaceutique de garantir le bon médicament à la bonne dose par la mise en place d’un contrôle qualité des préparations avant administration. Par ailleurs, ces molécules potentiellement cancérogènes, mutagènes et reprotoxiques présentent un risque pour les personnes exposées notamment le personnel de santé. L’objectif de ce travail de thèse a été de développer des outils permettant d’optimiser la sécurité du circuit de ces médicaments anticancéreux à l’hôpital tant pour le patient que pour le personnel de santé. Des outils analytiques associés à des méthodes d’interprétation des données issues de la chimiométrie et de la gestion de risque ont été mis en œuvre afin de répondre à cette problématique.Dans le cadre de la sécurisation du personnel, la recherche de traces de molécules anticancéreuses dérivées du platine a permis de mettre en évidence les zones les plus contaminées. Sur la base de ces contaminations et des conditions de travail, une méthodologie d’analyse de risque multicritère a été développée pour évaluer le risque d’exposition du personnel. Face au risque encouru, différentes mesures correctives ont été envisagées et des études évaluant plus spécifiquement l’efficacité détergente des opérations de décontamination des surfaces et des flacons ont été menées.En parallèle, des essais visant à sécuriser les préparations avant administration ont été conduits sur deux molécules anticancéreuses : le 5-fluorouracile et la gemcitabine. Au regard de leur caractère non destructif, non invasif et de ce fait, plus sécurisé, les spectroscopies vibrationnelles Raman et proche infrarouge ont été explorées. Les données spectrales (zones spectrales et prétraitements) ont été optimisées par des analyses multivariées ComDim pour développer des modèles de régression PLS prédisant la concentration en principe actif en solution. Les résultats ont montré la faisabilité et la complémentarité des techniques de spectroscopie Raman et proche infrarouge pour la détermination quantitative des molécules anticancéreuses. / Most of the anticancer drugs are defined by a narrow therapeutic margin; therefore medical errors can have major consequences on patients. Thus, it’s necessary to guarantee the good drug at the good dose by the implementation of a quality control of the preparation before administration. These potentially carcinogenic, mutagenic or teratogenic drugs present a risk for exposed people especially healthcare workers.The aim of this study was to develop tools which can optimize the safety of the cytotoxic medication circuit in hospitals, for the patient as much as for healthcare workers. In order to respond to these problematics, analytical tools have been associated with different methods of data interpretation of chemometric and risk management.To improve healthcare workers’ safety, environmental monitoring looking for traces of platinum compound cytotoxic drugs were performed to identify the most contaminated areas. Based on these contaminations and working conditions, a methodology of multi-criteria risk analysis has been developed to quantify the risk of exposure of healthcare workers. Regarding the risk, various corrective measures were considered. Thus, studies based on the detergent efficiency of decontamination protocols used to clean workplace surfaces and cytotoxic vials were conducted.In parallel, assays were performed on two anticancer molecules to secure cytotoxic preparations before administration: 5-fluorouracile and gemcitabine. Regarding their non-destructive, non-invasive properties and therefore, more secured handling, Raman and near infrared spectroscopy were explored. Spectral data (spectral zones and pretreatments) were optimized by multivariate analyses ComDim to develop models of regression PLS predicting the concentration of the active ingredient in solution. Results showed the feasibility and the complementarity of these two spectroscopies in the quantitative determination of the cytotoxic drugs.These works participate in the continuous approach of quality assurance implemented in numerous health institutions. We hope that they will contribute to durably decrease risks associated to cytotoxic drugs for both patients and healthcare workers.

Chemotherapy is one of the most important treatments for cancer. However, systemic toxicity, drug resistance and unstable kinetics of the drug in the blood are serious problems of chemotherapy. The use of biodegradable polymers for controlled release of anticancer drugs has gained popularity in recent years. Controlled release of drugs from polymeric carriers has some advantages such as improvement in the efficiency of treatment, reduction in systemic toxicity and prevention of the drug resistance that is developed by the cancer cells. In this study, poly(D,L-lactide-co-glycolide) microparticles were used as carriers for the controlled release of all-trans-Retinoic acid, tamoxifen, tamoxifen citrate and idarubicin. It was aimed to prepare a drug carrier system for controlled release of hydrophobic anticancer drugs. The empty and drug loaded poly (D,L-lactide-co-glycolide) microparticles were prepared by solvent extraction/evaporation technique with single emulsion (oil/water). Optimized microparticles were characterized by using inverted light microscopy and scanning electron microscopy to examine their morphology and sizes. Drug content of microparticles and the amount of released drug were determined spectrophotometrically. In vitro toxicity of the microparticles on MCF-7 human breast cancer cells was investigated. It was revealed that the microparticles were smooth and spherical in shape. Their sizes differed in the range of 2-20 &micro / m. atRA-loaded microparticles showed approximately 90% encapsulation efficiency and it was confirmed that changing in drug/polymer ratio affected the extend of drug content. Increase in drug content caused a slower release pattern. Moreover, although the empty microparticles caused some toxicity, atRA-loaded PLGA microparticles showed slight cell growth inhibition.

Breast cancer is the most frequent type of cancer seen in woman. Chemotherapy is one of the most important treatments for breast cancer. However, systemic toxicity, drug resistance and unstable kinetics of the drug in the blood are serious problems of chemotherapy. The use of biodegradable polymers for controlled release of anticancer drugs has gained popularity in recent years. Controlled release of anticancer drugs from polymeric carriers has some advantages such as improvement in the efficiency of treatment, reduction in systemic toxicity and prevention of the drug resistance that is developed by the cancer cells. In this study, it was aimed to prepare such a controlled release system for anticancer drugs which are used in breast cancer treatment by using biodegradable copolymer poly(D,L-lactide-co-glycolide) and to characterize in terms of morphology, size, drug content and drug release rate. In the first part of this study / empty and drug loaded poly (D,L-lactide-co-glycolide) microspheres were prepared. Two sets of empty poly(D,L-lactide-co-glycolide) microspheres were prepared by solvent evaporation technique with single emulsion (oil/water) to determine the effect of stirring rate on size of microspheres. Increase in stirring rate caused decrease in size of microspheres. Drug loaded poly(D,L-lactide-co-glycolide) microspheres were prepared for controlled release of anticancer drugs which are used in breast cancer treatment namely / 5-fluorouracil, methotrexate and tamoxifen by using solvent evaporation technique either with double emulsion (water/oil/water) or single emulsion (oil/water). In the second part of this study / empty and drug loaded microspheres were characterized. Inverted light microscopy and scanning electron microscopy were used to examine morphology and size of microspheres. Drug content of microspheres and amount of released drug were determined and drug release profile was obtained for each anticancer drug separetely.