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Title: ({plusmn})-Praeruptorin a derivatives : synthesis, structure-activity relationship and mechanisms in reversal of P-glycoprotein-mediated multidrug resistance in cancer cells
Other Titles: Bai hua qian hu jia su yan sheng wu de he cheng yi ji qi dui P-tang dan bai jie dao de zhong liu xi bao duo yao nai yao xing de ni zhuan huo xing, gou xiao guan xi he ni zhuan ji zhi de yan jiu
白花前胡甲素衍生物的合成以及其對 P-糖蛋白介導的腫瘤細胞多藥耐藥性的逆轉活性, 構效關系和逆轉機制的研究
Authors: Shen, Xiaoling (沈小玲)
Department: Dept. of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2007
Publisher: City University of Hong Kong
Subjects: Cancer -- Chemotherapy
Drug resistance in cancer cells
Herbs -- Therapeutic use
Multidrug resistance
Notes: CityU Call Number: RC271.H47 S45 2007
Includes bibliographical references (leaves 92-107)
Thesis (Ph.D.)--City University of Hong Kong, 2007
xi, 107 leaves : ill. (some col.) ; 30 cm.
Type: Thesis
Abstract: Multidrug resistance (MDR) remains a major problem in human cancer chemotherapy. One of the main underlying mechanisms of MDR is the over-expression of P-glycoprotein (Pgp), an ATP-dependent membrane transporter protein encoded by MDR1 gene. Pgp extrudes a spectrum of anticancer drugs from cancer cells and decreases effective intracellular drug concentrations. (±)-Praeruptorin A (PA), a naturally existing 7, 8-pyranocumarin isolated from the Chinese medicinal herb Radix Peucedani (the dried root of Peucedanum praeruptorum Dunn.), reverses MDR in Pgp-overexpressing KB V1 cells. In this study, we studied a total of twenty-eight PA derivatives synthesized through structural modification of PA or directly isolated from EtOH extracts of the herb. Among these compounds, nine were newly synthesized, they were (±)-3', 4'–bis(4-methoxybenzeneacetyloxy) -cis-khellactone (16), (±)-3', 4'–bis (4-methoxycynnamoyloxy)-cis-khellactone (17), (±)-3', 4'–bis(3, 4-dimethoxycynnamoyloxy)-cis-khellactone (18), (±)-3', 4'–bis(3, 4-dimethoxybenzoyloxy)-cis-khellactone (19), (±)-3', 4'–dicynnamoyloxy-trans- khellactone (23), (±)-3', 4'–bis(4-methoxybenzeneacetyloxy)-trans-khellactone (24), (±)-3', 4'–bis (4-methoxycynnamoyloxy)-trans-khellactone (25), (±)-3', 4'–bis(3, 4-dimethoxycynnamoyloxy)-trans-khellactone (26) and (±)-3', 4'–bis(3, 4-dimethoxybenzoyloxy)-trans-khellactone (27). MDR-reversal activity of the twenty-eight PA derivatives had been investigated in drug sensitive human epidemoid carcinoma KB-3-1, human leukemia K562 and human hepatoma HepG2 cells and their Pgp overexpressing MDR sublines KB V1, K562-DR and HepG2-DR. Our results showed that PA derivatives that contains aromatic acyloxy at C-3' and/or C-4' site of 7,8-pyranocoumarin enhanced sensitivity of MDR cells but not drug-sensitive cells to anticancer agents which are Pgp substrates. In HepG2-DR cells compounds 15, 18, 19, 26 and 27 reduced IC50 (doses required to inhibit 50% of cell growth) of anticancer agents vinblastine, doxorubicin, puromycin and paclitaxel by more than 90%. Compounds 15, 18, 19 and 26 showed similar effects in K562-DR cells and compounds 18 and 19 had similar effects in all three MDR lines. Compound 18 was most active and even at 1µM it reduced IC50 of drugs by more than 95% in HepG2-DR cells. These compounds were also less toxic than PA and are good candidates of Pgp modulators. Structure-activity relationship (SAR) studies showed that (1) aromatic acyloxys contributed more than linear or branched aliphatic acyloxy to MDR reversal activity of 7, 8-pyranocoumarins; (2) compounds with aromatic acyloxys at C-3' and C-4' site in cis-configuration exhibited higher MDR reversal potency than their trans-isomers; and (3) 3, 4-dimethoxyl substituted aromatic acyloxys were more suitable than other groups for enhancing MDR reversing activity of 7, 8-pyranocoumarins. Compounds 15, 18, 19, 26 and 27 were selected for further studies. Our result showed that they increased doxorubicin accumulation in Pgp-MDR cells and inhibited Rhodamine 123 or Hoechst 33342 efflux from these cells in a dose-dependent manner. They markedly enhanced doxorubicin-induced G2/M arrest in HepG2-DR cells and enhanced growth inhibitory effect of doxorubicin and vinblastine in HepG2-DR cell soft-agar colony-forming assay. Unlike PA that decreases MDR1 mRNA and Pgp levels in KB V1 cells, all of the five compounds did not affect the expression level of Pgp in MDR cell lines, implying that they directly inhibited Pgp. Compounds 18, 19 and 27 increased Pgp reactivity to Pgp-specific conformation-sensitive mAb UIC2 like a typical Pgp substrate such as cyclosporine A, on the other hand compounds 15 and 26 decreased UIC2 reactivity like the ATPase inhibitor sodium vanadate. These observations suggest that compounds 18, 19 or 27 might directly interact with the relevant transport substrate-site(s) on Pgp, whereas compounds 15 and 26 might interact with allosteric site(s) on Pgp. In Pgp-enriched membrane vesicles, compounds 15, 19, 26 and 27 stimulated the basal Pgp-ATPase activity to some extent, suggesting some transport substrate-like activity. Compound 18 inhibited the basal Pgp-ATPase activity. All five compounds inhibited ATPase activity stimulated by the standard substrates verapamil or progesterone with decreased Vmax and relatively unchanged Km, suggesting a primarily uncompetitive mode of inhibition. Our results suggest that compounds 15, 18, 19, 26 and 27 did not affect MDR1 gene expression but are direct Pgp inhibitors. They directly interact with substrate site(s) or allosteric site(s) on Pgp and consequently hinder drug binding and slow down drug transport and ATP hydrolysis.
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