No description available.

No description available.

No description available.

No description available.

Azole-resistance in Candida albicans is becoming common and is associated with the widespread prophylactic use of azoles. Resistance to one azole is usually associated with resistance to other structurally dissimilar azoles. C.albicans is also inherently resistant to a wide range of eukaryotic inhibitors such as cycloheximide and gentamycin. Certain studies have shown that azole-resistance in some strains of C.albicans is associated with alterations in the cell membrane. This project has sought to determine whether azole-resistance in C.albicans strain 3302 was due, at least in part, to a multidrug resistance mechanism. An assay was developed using the fluorescent dye Rh123 to measure P-glycoprotein like activity. Active efflux of Rh123 has been shown to correlate with P-glycoprotein activity in a number of organisms. Results from this assay suggest that an energy-dependent efflux mechanism for Rh123 is present in azole-resistant strain 3302 but not in azole-sensitive strain 3153. The P-glycoprotein inhibitor, reserpine, inhibited Rh123 efflux. However, azoles did not appear to compete with Rh123 for efflux in the azole-resistant strain 3302, suggesting that azole-resistance in this strain is not mediated by a P-glycoprotein like mechanism. Southern analysis showed that sequences homologous to MDR genes existed in C.albicans. A PCR strategy was used to clone gene fragments containing the Walker motif which is found in MDR genes.

The search for more effective treatment strategies in melanoma led to many new innovative approaches aiming at different molecular targets. Chemotherapy still remains the most effective treatment and many efforts are put in order to improve targeting and delivery of the chemotherapeutic agents. Among these, peptide conjugates of anticancer drugs were designed to increase stability, cell penetration, specificity and accumulation in cancer cells. We as well as others evaluated such a conjugate, termed PSF (L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine-ethylester) in terms of its cytotoxicity in vitro and in vivo using a human melanoma tumor as a model, its stability, transport, and metabolisation. By comparing the cytotoxicity of PSF and melphalan towards different cancer primary melanoma cell cultures, we noticed some interesting observations: PSF displayed the same toxicity pattern both in short (2h) and long term (24h) cell exposures whereas melphalan and m-sarcolysin needed long term exposure to reach the same toxicity. This could indicate that PSF very quickly penetrates the cells in accordance with what has been shown with red blood cells (RBCs). PSF has shown a much better and quicker penetration into the cells in vitro as compared to melphalan. In this present work, the cytotoxic effect of PSF was further evaluated in vivo using a standardized nude mice tumor model bearing a human melanoma. First, the acute toxicity in rats and mice and the maximum tolerated dose were determined. After a dose-escalation study one dose was singled out and tested as a single dose and as a fractionated dose. PSF was able to reach the tumor site and a dose-response relationship was observed. The IP administration of fractionated doses of PSF had significantly better effect on tumor growth inhibition, regression and regrowth than single dose administration and this without any evidence for general toxicity monitored by animal weight loss. We also compared the efficacy of PSF to its parent drug m-sarcolysin, melphalan and cyclophosphamide and observed that PSF was much more active than both melphalan and m-sarcolysin at the same molar doses. Body distribution of the 14C-labelled PSF revealed ratios of 2.4 and 1.5 compared to muscle tissue for the two melanoma tumors evaluated with no significant and stable accumulation in any vital organ. The amount of tracer was still high in the blood after 24 hours explaining the high radioactivity in the kidney and partly in the liver. Interestingly, the spleen had an unusual high radioactivity uptake reflecting the exceptional binding of the tracer to blood cells (BC), while the pancreas very high load was an indicator of protease-mediated specific delivery and strongly support our hypothesis elaborated on the basis of in vitro results. Our in vitro data point to a particular mechanism of action of PSF based on the transport of PSF through the body by the rapid binding to blood cells and the delivery at the tumor site by the subsequent release of its active metabolites due to cleavage by tumor-associated proteases. Concerning the binding of PSF to membranes and its transport the following observations were made: while PSF was stable in human plasma, it disappeared very quickly in whole blood along with the generation of a main metabolite: m-sarcolysin. The presence of BC membranes was required for both binding and generating the metabolites. Binding to natural or artificial membranes was achieved and only competition with melanoma cells or proteolytic enzymes such as dispase, led to the generation of active metabolites. The different metabolites were isolated using preparative LC and were then identified using Electrospray Ionisation Mass Spectrometry (ESI). Three metabolites, of which m-sarcolysin was the main one, were identified all bearing the chloroethyl alkylating group. Enzymatic catalysis was further supported by a set of experiments where the enzymatic activity was non-specifically and specifically inhibited. In order to look at the effect of extracellular matrix proteases on PSF, three representatives of ECM proteases were incubated with PSF: collagenase A had no effect, but both dispase and trypsine were able to process PSF. The following data indicate the higher processing of PSF in the presence of cells with a higher proteolytic activity and thus the delivery of the blood cell-bound PSF. When comparing BC with melanoma cells (MC), the latter showed a higher ability to bind and process PSF both by membrane-associated and most interestingly soluble proteases. A lot of families of enzymes are reported to be overexpressed by melanoma cells including: metalloproteases, cysteine cathepsins, serine proteases and aminopeptidases. All the melanoma cells and cell lines evaluated were able to generate PSF active metabolites. To identify the families of enzymes expressed on the membrane of melanoma cells that might be involved in the mechanism of action of PSF, we performed 2D-gel electrophoresis on their membrane extracts. The 2D-gels experiments revealed the presence of proteins compatible with enzymes known to be important in melanoma and further work is needed to identify the individual enzymes involved by using mass spectrometry and Western blotting. Both our in vitro and in vivo findings strongly suggest that not only melanoma tumor cells and tumor sites but other types of tumors as well may be targets for the toxic activity of PSF owing to their much higher load in proteolytic enzymes that are closely related to their invasive potential. The transport of PSF by the blood cells and the release of its metabolites at the tumor site result in a low amount of drug in its free soluble form within the blood and this may explain the relatively lower side-effects observed. PSF is thus expected to have a much better therapeutic index than conventional alkylating agents. This original mechanism of drug delivery may well be extended to other cancer and non-cancer drugs than alkylating agents.

Many chemotherapeutic drugs interact with DNA to induce cytotoxicity. Mass spectrometry has become an essential technique in the investigation and identification of anticancer DNA adducts. Traditionally, identification of therapeutic DNA adducts was conducted by P1 enzymatic digestion followed by separation via gel electrophoresis or high performance liquid chromatography (HPLC). Structural information about binding was identified via NMR and x-ray crystallography. These methods are arduous and require significant sample consumption. Mass spectrometry is a high-through put methodology that requires a minimal amount of sample consumption to produce site specific binding information. Anticancer agents may bind directly to DNA via formation of a covalent bond creating a monoadduct, a single covalent bond at one site of double stranded DNA, or a crosslink, two covalent bonds on each strand of duplex DNA. Cytotoxicity of covalent anticancer agents is achieved by effectively blocking replication of DNA, thus preventing proliferation of cancerous cells. Much effort has been directed in the search for new chemotherapies to increase binding specificity for cancerous cells. In designing new drugs it is essential to understand DNA interactive properties--such as differences in the cellular conditions, the number of nucleobases within the binding site, and the tendency to form a monoadduct or a crosslink. These factors can then be exploited to design more selective anti-cancer drugs. Several covalent bond-forming anti-cancer DNA adducts have been investigated using mass spectrometry. These include mitomycin C, nitrogen mustards, cisplatin, psoralen derivatives, a bioreductive prodrug (RH1), and an enediyene. Mitomycin C is an anticancer antibiotic that forms a DNA crosslink at the 2-amino group of guanine. The DNA/mitomycin C adduct was evaluated by tandem mass spectrometry and the results demonstrated that the mitomycin C adduct formed isomeric tetramer nucleotides upon activation for dissociation.. Nitrogen mustards can form DNA crosslinks and monoadducts. The extent of DNA alkylation with a sulfur acridine mustard derivative was evaluated by tandem mass spectrometry using infrared multiphoton dissociation. . Cisplatin is an anticancer therapeutic that crosslinks DNA at N7 guanine residues. The fragmentation pattern of cisplatin/DNA adducts investigated by tandem mass spectrometry confirmed the formation of a crosslink in the platinated diagnostic fragment ions detected. Psoralens, used for centuries to treat psoriasis, form crosslinks preferentially at thymine nucleobases by the sequential absorption of two photons. The results of tandem mass spectrometry were used to identify the sequence selectivity of psoralen derivatives. Recently, a study of a bioreductive prodrug, 2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone (RH1), was shown to form crosslinks with DNA at N7 guanine residues and the fragment ions produced via tandem mass spectrometry confirmed the site of the crosslink. Lastly, enediynes are of therapeutic interest because they exhibit a high cytotoxicity when the drug moiety forms a DNA crosslink through a biradical intermediate across opposing cytosine nucleobases. The tandem mass spectrometry results indicated that the enediyne moiety binds in a somewhat nonselective manner, as it associated with thymine as well as cytosine, and the formation of a covalent crosslink was confirmed by the retention of the enediyne by diagnostic fragment ions. . Other anticancer agents associate with DNA through noncovalent interactions like minor groove binding or intercalation. In both cases, the electrostatic interactions between the chemotherapeutic agent and the DNA double helix interfere with DNA transcription leading to incomplete proteins synthesis and ultimately cell death. Noncovalent anticancer moieties historically suffer from a lack of specificity, as most drug moieties have only two to four base pair binding sites. Thus, current research focuses on increasing the specificity of these small molecules. A novel tetraintercalator, 1,4,5,8-tetracarboxylic naphthalene diimide units connect by peptides (TET), has four intercalation units and a 14 base pair binding site allowing for dramatically greater sequence selectivity. The novel tetraintercalator shows the highest specificity amongst known intercalating moieties. An investigation into the sequence selectivity and binding site affinity compared to well characterized small molecule intercalators, actinomycin D and echinomycin, was assessed by mass spectrometry. The results show that TET preferentially binds to sequences that contain the unmodified binding site and also shows a slight preference to adenine and thymine rich sequences, indicating the peptide linkers play an important role in DNA interactions. Tandem mass spectrometry results demonstrated that TET binds with high affinity to its binding site compared to small molecule intercalators. Upon collision induced dissociation (CID) the predominant species in the mass spectrum was the DNA/TET – G ion peak. Intercalating adducts generally dissociate by strand scission with either strand retaining the drug moiety or by ejection of the drug, as seen with both actinomycin D and echinomycin in this study. Therefore, TET shows promise as a new development toward an anticancer therapeutic with high sequence selectivity and binding affinity with DNA. This workfocuses on reviewing the advancements of covalent bond forming DNA interactive anticancer therapeutics that have been studied by mass spectrometry, and presents a study of the interactions of a novel intercalation drug with DNA explored by mass spectrometry. / text

No description available.

Chlorpromazine (CPZ), a phenothiazine derivative, which has been extensively used in the treatment of schizophrenia for the last 50 years, also has recently been reported to have anticancer potential. Though it is one of the widely publicised compounds the information on the mechanism of its cytotoxicity is limited. Hence, an in vitro investigation was undertaken to study its cytotoxic mechanism. In a time and dose related study in SA3T3 cells the early statistically significant cytotoxicity was measured after a six-hour exposure with 50 uM and above (maximum concentration applied was 100 uM). The cytotoxicity increased with time and after 24 hours even 10 uM produced significant cytotoxicity and the I/C50 concentration was 40 uM. CPZ was found to induce dose-dependent statistically significant DNA single strand breaks in SA3T3 cells two hours before cell viability loss was measured, but no double strand breaks was noticed. The cytotoxic response in the presence of proadifen revealed that CPZ, but not any of its a number of metabolites, as suggested by many authors, is the molecule responsible for the cytotoxicity. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, known to induce N-demethylation and N-polydemethylation of tertiary and quaternary amines potentiated the cytotoxicity of CPZ. Potentiation of CPZ cytotoxicity was seen also in the presence of clorgyline, a monoamine oxidase (A) inhibitor. Increased cytotoxicity in the presence of these agents indicates that the dimethylamino moiety of the dimethylaminopropyl side chain of CPZ plays an important role in the process. Although the importance of dimethylamino moiety in the cytotoxicity of CPZ has not been previously reported, established cytotoxic drugs like dacarbazine, tamoxifen and ethylenimines possess this group(s) in their structure. Flow cytometric analysis and other cytotoxicity measurements have indicated that the cytotoxicity induced by CPZ is biphasic that at 50 uM and above it induces cell death and at 40 uM and below it inhibits cell proliferation. The cytostatic effect of 40 uM CPZ is the same as that induced by 25-150 uM cisplatin, 6-50 juM vinblastin or 12.5- 200 uM doxorubicin. None of these compounds potentiated the cytotoxicity of CPZ but 30 jaM menadione, which causes double strand breaks in SA3T3 cells, increased the cytotoxicity of 70-100 uM CPZ. However, all the cytotoxic effects remained reversible up to the first four hours, for it was possible to restore total cell viability by removing CPZ from the extracellular medium after the said period. A fluorimetric analysis was developed, using 2',7'-dichlorodihydroxy fluorescein diacetate to measure free radical generation, which turned out to be useful in assessing the redox status of cells. Thus CPZ was found to raise the redox status of SA3T3 cells while keeping the cellular glutathione (GSH) concentration unaltered. Involvement of reactive oxygen species in CPZ mediated cytotoxicity was investigated. The OH radical scavengers: sodium benzoate, ot-tocopherol, ascorbic acid or retinol; the iron chelator: deferoxamine; the iron and copper chelator: 1,10-phenanthroline; O2 inhibitors: superoxide dismutase or diphenyline iodonium chloride; H2O2 inhibitors: catalase, or sodium azide; or the singlet oxygen quencher: 2,2,6,6-tetramethyl 4- piperidone gave no protection against CPZ cytotoxicity. There was no nitric oxide generated in CPZ treated SA3T3 cells and the peroxidase inhibitor sodium azide did not give any protection. Thus, the involvement of peroxinitrite or hypochloride anion as cytotoxicity mediators also was excluded. tertiary-Butyl hydroperoxide (t-BH)-mediated cytotoxic response was fully restored by CPZ though the cells died within the next two hours. The cytotoxic effect of CPZ in precision cut kidney slices of Wistar rats was found to be greater than that of liver slices. CPZ (100 uM) abolished lipid peroxidation and decreased lactate dehydogenase leakage induced by 1.5 mM t-BH in both kidney and liver tissue slices. However, in contrast to the findings in SA3T3 cells, CPZ inhibited GSH, though not to the extent of causing cytotoxicity, and the MTT assay did not show any protective effect in the said tissue slices. The relevance of all these findings in establishing the cytotoxic mechanism of CPZ is discussed. Since induction of cytotoxicity is a beneficial effect in anticancer chemotherapy the significance of the mechanisms and cytotoxicity of CPZ in cancer treatment is also discussed.

Chemotherapeutic agents act by targeting rapidly dividing cancer cells. The full extent of their cellular mechanisms, which is essential to balance efficacy and toxicity, is often unclear. In addition, the use of many anticancer drugs is limited by dose-limiting toxicities as well as the development of drug resistance. The work presented in this thesis aims to address the basic biology that underlies these issues through the development and application of chemical genomic tools to probe mechanisms of current and novel anticancer compounds. Chemical genomic screens in the yeast Saccharomyces cerevisiae have been used to successfully identify targets and pathways related to a compound’s mode of action. I applied these screens to examine the mode of action of potential anticancer drugs: a class of platinum-acridine compounds and the apoptosis-inducing compound elesclomol. By analogy to the yeast screens, I developed an RNAi-mediated chemical genomic screen in human cells which has the potential to reveal novel targets and drug mechanisms. This screen was applied to further understand doxorubicin’s mode of action. In parallel with the loss-of-function assays, our lab developed a human ORF overexpression screen in human cells. I applied this gain-of-function screen to identify those genes that, when overexpressed, are toxic to cells. Characterization of such genes that cause toxicity can provide insight into human diseases where gene amplification is prevalent.