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Title: Pharmacology of dextran-conjugated doxorubicin in multidrug resistant KB cells
Other Titles: Pu ju tang (zuo xuan tang hang) lian jie shang e mei su zai duo kang yao xing KB xi bao de yao li xue yan jiu
葡聚糖(左旋糖酐)連接上阿霉素在多抗藥性 KB 細胞的藥理學硏究
Authors: Lam, Wing (林嶸)
Department: Dept. of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 1999
Publisher: Dept. of Biology and Chemistry, City University of Hong Kong
Subjects: Doxorubicin
Drug resistance in cancer cells
Multidrug resistance
Notes: CityU Call Number: RM666.D68 L36 1999
Includes bibliographical references (leaves 153-184)
Thesis (Ph.D.)--City University of Hong Kong, 1999
xxii, 190 leaves : ill. ; 30 cm.
Type: Thesis
Abstract: One of the major problems in cancer chemotherapy is the development of tumor resistance to drug treatment. In in vitro experiments, step selections of cancer cells resistant to a single anti-neoplastic agent may lead to resistance to multiple agents (multidrug resistance). One of the well known mechanisms leading to multidrug resistance is the over-expression of the MDRl gene product, the 170 kDa membrane P-glycoprotein (P-gp) which is an ATPdriven efflux pump of xenobiotics. Some reports indicate that using macromolecules as drug carriers may improve drug delivery and overcome multidrug resistance. This thesis reports our studies on the effects of dextran conjugated doxorubicin in multidrug resistant KB cells in combination with other free drugs and under the condition of energy starvation (ATP depletion). We also delineated effects of varying dextran size of conjugates' cytotoxicity. First we studied the effects of large "immobile" unreduced dextranconjugated doxorubicin (200 kDa with unreduced Schiffs base, unable to enter nuclei without degradation) in combination with colchicine, vinblastine and free doxorubicin respectively on the killing of human KB-3-1 carcinoma cells and its multidrug resistant subclone KB-V1 cells. Cell survival was quantified by the tetrazolium salt MTT assay. Cytotoxicity studies were designed so that data could be analyzed by the medium-effect principle and the calculated Combination Indices at different cell survival levels. When added alone conjugated doxorubicin was not as effective as the free doxorubicin in cell killing. When conjugated doxorubicin was combined with free doxorubicin or colchicine and at high cell killing rates (over 75%), a significant degree of synergism was observed on the killing of multidrug resistant KB-Vl cells. This synergism was not observed in non-resistant KB-3-1 cells nor when conjugated doxorubicin was combined with vinblastine. We observed that 2-deoxyglucose/Na azide treatment and free or "immobile" (70-500 kDa) unreduced conjugated doxorubicin are synergistic in cell killing. We studied subcellular fate of drugs by tracing fluorescence emitted by doxorubicin. As demonstrated by fluorescence and laser confocal microscopy, KB-V1 cells retained more conjugates than free doxorubicin. Pglycoprotein inhibitors verapamil or 2-deoxyglucose/Na azide enhanced only the retention of the free drug and the smaller (<70 kDa) conjugates, indicating that P-gp is not effective against large conjugates. These "immobile" conjugates were excluded from nuclei. Initially both free and conjugated doxorubicin accumulated in cytoplasmic organelles. Upon 2-deoxyglucose/Na azide treatment, fluorescence labeling of organelles dissipated. Prolong (24- hour) incubation of conjugate-preloaded cells resulted in redistribution of some of the organelle-associated fluorescence to nuclei, suggesting degradation or decoupling. The appearance of free doxorubicin was confirmed by capillary electrophoresis. 2-Deoxyglucose/Na azide treatment retarded decoupling. Our results suggest that energy starvation, in addition to increasing cellular retention of P-gp substrates, may affect cellular fate of conjugated drugs with possible enhancing effect in cancer cell killing. The above studies were performed with larger "immobile" unreduced conjugates (>70 ma). We also investigated the size-effect of "mobile" (0.34 - 10 kDa, able to enter nuclei) dextran as carriers of doxorubicin. Two opposite effects of increasing dextran size must be considered. Large size hinders DNA interaction but may be beneficial by impeding the action of multidrug resistance pumps. Cell penetration and accumulation of "mobile" conjugates (0.34 - 10 kDa with Schiff's base reduced) was monitored by fluorescence emission, and DNA interaction by equilibrium DNA binding constant (KA) as determined by fluorescence titration method. It was observed that "mobile" conjugates could quickly interact with nuclear DNA. In drug sensitive KB-3-1 cells cytotoxicity of "mobile" conjugates decreased with an increase in size. This was due at least partly to a 2.5-fold reduction of KA (less stable DNA complex) and partly to decreased drug accumulation, both are ensured by increased size of dextran. The relative resistance (Dm ratio) between KB-Vl and KB-3-1 cells was reduced from 359 (for free doxorubicin) to 4.3 as the size of dextran increased. Most importantly, we observed that for KB-V1 cells, cytotoxicity of the 10 kDa conjugate was stronger than that of free doxorubicin. This could largely be attributed to the greatly improved accumulation and retention of the conjugate in the multidrug resistant KB-V1 cells. By analyzing results on relative cytotoxicity, accumulation and retention of conjugates in KB-3-1 and KB-V1 cells, we suggest that the optimal size of conjugates for all killing is in the range of 10-100 kDa. Our observations have important implications in R&D of macromolecular drug carriers in cancer chemotherapy.
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