Cancer is a group of diseases in which normal cells are converted to cells capable of autonomous growth and invasion. In the chemotherapeutic control of cancer, drugs are usually given systemically so they reach toxic levels in healthy cells as well as cancer cells. This causes serious side effects. Another important problem with chemotherapy is resistance developed to cytotoxic drugs (multi drug resistance). Doxorubicin (Dox) occupies a central position in the treatment of breast cancer. However doxorubicin induced cardiac toxicity is associated with a high incidence of morbidity and mortality. Resistance of malignant tumors to Dox is another important cause of treatment failure in patients with cancer. One approach to overcome Dox-related toxicity is to use polymeric drug carriers, which direct the Dox away from heart tissue, and allow usage of lower dosages. In this present study two different anti-cancer drug delivery methods were evaluated. Dox was encapsulated in PLGA microparticles by single and double microemulsion solvent evaporation techniques. The highest entrapment of doxorubicin within PLGA microspheres obtained by optimization of process parameters. A sustained release of doxorubicin was obtained for 20 days. Several protein transduction domains are known to have the ability to pass through biological membranes. One such peptide is HIV-1 TAT. In this study TAT was evaluated for its ability to carry Dox into Dox resistant MCF-7 tumor cells. Dox peptide conjugate was more potent than free drug. The concentration of drug in resistant cancer cells was increased indicating a partial reversal of drug resistance.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-113).

published_or_final_version / Pathology / Master / Master of Medical Sciences

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第19092号 / エネ博第316号 / 新制||エネ||64 / 32043 / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 木下 正弘, 教授 森井 孝, 教授 片平 正人 / 学位規則第4条第1項該当

<p>Multidrug tolerance (MDT) is the ability of pathogenic bacteria to survive killing from exposure to multiple antibiotics, and is a major obstacle in the treatment of infectious disease. A small population of bacteria (0.0001%) termed persisters is the culprit that causes MDT and allows these cells to persist. In <italic>Escherichia coli</italic>, the HipBA toxin&ndash;antitoxin pair plays a role in multidrug tolerance. HipA, a 50 kDa serine protein kinase, is the more stable toxin and abrogates cell growth in the absence of the more labile antitoxin HipB. HipB is a transcription repressor that binds to the four conserved (TATCCN₈GGATA) operator sites of the <italic>hipBA</italic> promoter to autoregulate expression of the <italic>hipBA</italic> operon. Delineation of the molecular mechanism of HipB&ndash;<italic>hipBA</italic>operator binding is critical to understand fully the regulation of persistence by HipB. Thus, we determined the equilibrium dissociation constants (K_d) of HipB for each of the four <italic>hipBA</italic> operators and the paired operator sites <italic>O₁O₂</italic> and <italic>O₃O₄</italic>. We found that the affinity of HipB for binding the <italic>O₁</italic> and <italic>O₃</italic> operators is seven to eight times higher than for the <italic>O₂</italic> and <italic>O₄</italic> operators. In addition, the affinity of HipB for the <italic>O₁O₂</italic> and <italic>O₃O₄</italic> operators is at least four times higher than the <italic>O₁</italic> and <italic>O₃</italic> operators. The HipB&ndash;operator complex structures reveal that HipB makes the same key contacts to the conserved TATCC motifs and bends each operator DNA by the same extent between 50&deg; to 70&deg; implying thus the affinity differences are attributed to indirect readout of the 8 base pair spacer (N₈). Mutational studies on residues involved in HipB&ndash;DNA interaction revealed the contribution of a series of selected residues to binding affinity with residues K38 and Q39 contributing greatly to affinity whereas other base contacting residues S29 and A40 contribute less to affinity. Surprisingly residue S43, which is involved in a hydrogen bond to the DNA phosphate backbone contributes more than expected because S43 forms a hydrogen bond network with nearby water molecules. </p><p>HipA was the first described <italic>bona fide</italic> persistence factor. The <italic>hip</italic> locus was discovered through a mutagenesis screen whereby <italic>hipA7</italic> was isolated. Described herein, biochemical and structure&ndash;function studies on HipA7, the gene product of the high persistent mutant allele having two point mutations G22S and D291A, revealed that the D291A mutation weakens the binding affinity for HipB by 3 to 4 fold. The HipA7 structure revealed the conformational heterogeneity of the P&ndash;loop motif (the ATP binding motif), which suggests a dynamic role of the loop in regulation of the kinase activity of HipA. To identify <italic>in vivo</italic> HipA substrates, we developed a mass spectrometry (MS)&ndash;based kinase assay, which led to identification of a novel phosphorylation site (residue S348) on HipA and a proposed consensus phosphorylation motif +&#981;S, where +, &#966; and S designate a positive, hydrophobic and serine amino acid residue, respectively. Phosphorylation of peptides with this consensus motif, including the S150 (EENDF<bold><underline>RIS</underline></bold>VAGAQEK), S348 (TGI<bold><underline>HIS</underline></bold>DLK) and GltX (GK<bold><underline>KLS</underline></bold>KRH), was confirmed subsequently by the MS&ndash;based kinase assay. Further analysis of the HipA7 structure suggested that HipA might undergo pyrophosphorylation on residue S150, and the MS&ndash;based kinase assay confirmed pyrophosphorylation of HipA. </p><p>Thus, our data support that HipA is a persistence factor via its kinase activity and precise <italic>hipBA</italic> gene regulation through HipB binding tightly to <italic>O₁</italic> and <italic>O₃</italic> is critical for the survival of bacteria in the presence of antibiotics. In addition, we propose a consensus motif for HipA substrates.</p> / Dissertation

The emergence of multidrug resistance is a serious medical problem that has significantly affected the treatment of tumor cells and infectious diseases. This multidrug resistance phenotype is mediated by the action of a large family of membrane proteins that act as active transporters or energy driven efflux pumps in both of prokaryotic and eukaryotic cells. Most eukaryotic multidrug efflux pumps belong to the ATP binding cassette (ABC) family of transport proteins that include P-glycoprotein (P-gp1), Multidrug Resistance Associated Protein (MRP1), and Breast Cancer Resistance Protein (BCRP). In prokaryotic cells, Lactococcus lactis LmrA, a homolog of P-gp1, mediates drug resistance to antibiotics and cytotoxic drugs. The transport function of these proteins is facilitated by the hydrolysis of ATP. However, the mechanism by which these proteins bind to, and are able to transport structurally dissimilar drugs across the cell membrane remains poorly understood. In this thesis we have attempted to characterize the interactions of various ABC transporters (MRP1, BCRP, and LmrA) with structurally diverse drugs, using a well characterized photoreactive drug analogue of Rhodamine 123, [125I] iodoaryl azido-rhodamine 123 (IAARh123). In the case of MRP1 interaction with Rhodamine 123, it was of interest to determine the nature of MRP1 drug interactions. In that study, our results show that CHAPS (1-[(3-cholamidopropyl) dimethylamino]-1-propansulfate) and Brij35 inhibited the photolabeling of MRP1 with IAARh123, and this interaction occurred outside the lipid bilayer. These results were unexpected in light of previous results with another ABC transporter which also binds to Rhodamine 123. Consequently, we show that non-toxic concentrations of CHAPS and Brij35 potentiate the toxicity of two MRP1 substrates, vincristine and etoposide (VP16). In the second chapter, we have used IAARh123 to demonstrate for the first time that the BCRP mediates drug resi

P-glycoprotein (Pgp, ABCB1) is a polyspecific efflux transporter implicated in multidrug resistance in human cancers. In this study, tetramethylrhodamine-5-carbonyl azide (AzTMR) was covalently crosslinked to the Pgp drug binding pocket with a stoichiometry of 1. The Pgp-AzTMR adduct was functionally equivalent to unlabelled Pgp and retained its ability to transport Hoechst 33342. The binding site of AzTMR in Pgp was nonpolar, with a similar environment to that of propanol. Pgp-AzTMR could bind a second drug molecule, with a higher affinity for H-site drugs and lower affinity for other R-site drugs. Unlabelled Pgp interacted with dimeric versions of known Pgp modulators, binding them with higher affinity than the monomer. These compounds were also found to either stimulate or inhibit Pgp ATPase activity depending on the concentration. Pgp-AzTMR was able to bind dimeric drugs, indicating that 3 substrate moieties can fit into the binding pocket. / The Canadian Cancer Society

The emergence of multidrug resistant tumours during the course of chemotherapeutic treatment of cancer patients is a major obstacle in cancer chemotherapy. Although several mechanisms may contribute to the appearance of multidrug resistance phenotype (MDR) in tumour cells, reduced drug accumulation and the ability of cells to undergo apoptosis are thought to be very important in expression of MDR. The work in this thesis focuses on the mechanism responsible for the reduced drug accumulation in tumour cells, mainly the multidrug resistance protein (MRP1). / The molecular mechanism underlying the binding and efflux of drugs by the MRP1 is currently not well understood. Several studies have now demonstrated that the cysteinyl leukotriene C$ sb4$ (LTC$ sb4$) and other glutathione (GSH) S-conjugated anions are substrates for the MRP. To learn more about MRP-drug interactions, we characterized the binding of MRP to a non-glutathione photoactive quinoline compound (abbreviated, ASA-AQ) (Chapter II). Since MRP mediated multi-drug resistance can be modulated by the anionic quinoline LTD$ sb4$ cysteinyl leukotriene receptor antagonist (MK571), we speculated that other quinoline-based compounds are likely to interact with MRP. In Chapter III, we show that MDR cells that express MRP1 are more resistant to the antimalarial drug, chloroquine. We also show that. chloroquine is a substrate for MRP1 drug efflux. / Taken together, the results of this thesis describe the interactions of MRP1 with a quinoline-based photoactive drug and the antimalarial drug chloroquine.

<p>Many studies have reported low cure rates for multidrug-resistant tuberculosis (MDRTB) patients and MDR-TB patients co-infected with human immunodeficiency virus (HIV). However, little is&nbsp / known about the effect of HIV infection and antiretroviral therapy on the treatment outcomes of MDR-TB in South Africa. Therefore, the objectives of the study are: to find out whether HIV infection&nbsp / and interactions between ARVs and second line anti-TB drugs have an impact on the following MDR-TB treatment outcomes: cure rate and treatment failure at Brewelskloof Hospital. MDR-TB&nbsp / patients were treated for 18-24 months. The study was designed as a case-control retrospective study comparing MDR-TB treatment outcomes between HIV positive (cases) and HIV negative&nbsp / patients (controls). Patients were included in the study only if they complied with the following criteria: sensitivity to second line anti-TB drugs, MDR-TB infection, co-infection with HIV (for some&nbsp / of them), male and female patients, completion of treatment between 1 January 2006 and 31 December 2008. Any patients that presented with extreme drug-resistant tuberculosis (XDR-TB)&nbsp / were excluded from the study. Data were retrospectively collected from each patient&rsquo / s medical records. There were a total of 336 patients of which 242 (72%) were MDR-TB patients and 94&nbsp / (27.9%) MDRTB co-infected with HIV patients. Out of the 242 MDR-TB patients, 167 (69.2%) were males and 75 (30.7%) were females. Of the 94 patients with MDR-TB co-infected with HIV, 51&nbsp / (54.2%) males and 43 (45.7%) females. Patients with multidrug-resistant tuberculosis co-infected with HIV who qualify for antiretroviral therapy were treated with stavudine, lamivudine and&nbsp / efavirenz while all MDR-TB patients were given kanamycin, ethionamide, ofloxacin, cycloserine and pyrazinamide. The cure rate of MDR-TB in HIV (+) patients and in HIV (-) patients is 34.5%&nbsp / and 30 % respectively. There is no significant difference between both artes (pvalue = 0.80). The MDR-TB cure rate in HIV (+) patients taking antiretroviral drugs and in HIV (+) patients without&nbsp / antiretroviral therapy is 35% and 33% respectively. The difference between both rates is not statistically significant. The study shows that 65 (28.0%) patients completed MDR-TB treatment but&nbsp / could not be classified as cured or failure, 29 (12.5%) patients failed, 76 (32.7%) defaulted, 18 (7.7%) were transferred out and 44 (18.9%) died. As far as treatment completed and defaulted is concerned,&nbsp / there is no significant statistical difference between HIV (+) and HIV (-) The number of patients who failed the MDR-TB treatment and who were transferred out is significantly higher in the HIV (-)&nbsp / group than in the HIV (+) group. Finally the number of MDR-TB patients who died is significantly higher in the HIV (+) group). The median (range) duration of antiretroviral therapy before starting&nbsp / anti-tuberculosis drugs is 10.5 (1-60) months. According to this study results, the MDR-TB treatment cure rate at Brewelkloof hospital is similar to the cure rate at the national level. The study also&nbsp / hows that HIV infection and antiretroviral drugs do not influence any influence on MDR-TB treatment outcomes.</p>

Polychlorinated biphenyls (PCBs) are persistent organic pollutants. Due to their properties, PCBs accumulate in the food-chain and post a threat to the health of human beings and wildlife. Hydroxylated PCBs (OH-PCBs) are oxidative metabolites of PCBs and are more hydrophilic than their parent PCBs. One of the best approaches to break down these contaminants is through bioremediation, which is an environmental friendly process that uses microorganisms to restore natural environment. Towards this goal, we have investigated the toxicity and accumulation of PCBs and OH-PCBs in a Gram-negative bacterium, Escherichia coli. We have also determined the role played by a primary multidrug efflux transporter AcrB on the accumulation of PCBs and OH-PCBs in bacterial cell. We found that one of the PCBs tested was toxic to E. coli, while different OH-PCBs have different levels of toxicity; the acrB knockout strain accumulated significantly more PCBs and OH-PCBs than the wild-type strain, suggesting that these compounds are substrates of the efflux pump; higher cytoplasmic concentrations of OH-PCBs were also observed in the acrB knockout strain using the biosensors. Based on these observations, we conclude that both PCBs and OH-PCBs are substrates of protein AcrB. Therefore the efflux activities of multidrug resistant pumps in Gram-negative bacteria should be considered while designing bioremediation approaches.