mPGES-1抑制剂MK886对白血病HL-60/A细胞凋亡及耐药性的影响

为了探讨膜结合型前列腺素E2合酶1(mPGES-1)抑制剂MK886对耐药白血病HL-60/A细胞诱导凋亡的作用及对其耐药性的影响,采用QT-PCR及Western blot法检测HL-60/A细胞mPGES-1的表达,CCK-8法观察药物对HL-60/A细胞增殖的影响,流式细胞术检测细胞凋亡,ELISA法检测PGE2的合成,Western blot法检测Akt、P-Akt表达,并观察低浓度(10μmol/L)MK886对HL-60/A细胞耐药性及多药耐药基因表达的影响。结果表明,HL-60/A细胞高表达mPGES-1,MK886可时间、浓度依赖性地抑制HL-60/A细胞增殖,诱导其凋亡(r=-0.83,P<0.05),同时,mPGES-1表达及PGE2合成减少,P-Akt表达明显下降。低浓度MK886可下调多药耐药基因mdr-1及蛋白P170表达,增强对化疗的敏感性。结论:MK886可抑制耐药白血病HL-60/A细胞增殖,诱导其凋亡,增强其化疗敏感性,其机制与下调mPGES-1/PGE2合成、抑制P-Akt蛋白及多药耐药基因表达有关。     

Human breast cancer resistance protein: interactions with steroid drugs, hormones, the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and transport of cimetidine

The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter that extrudes xenotoxins from cells, mediating drug resistance and affecting the pharmacological behavior of many compounds. To study the interaction of human wild-type BCRP with steroid drugs, hormones, and the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), we expressed human BCRP in the murine MEF3.8 fibroblast cell line, which lacks Mdr1a/1b P-glycoprotein and Mrp1, and in the polarized epithelial MDCKII cell line. We show that PhIP was efficiently transported by human BCRP in MDCKII-BCRP cells, as was found previously for murine Bcrp1. Furthermore, we show that six out of nine glucocorticoid drugs, corticosterone, and digoxin increased the accumulation of mitoxantrone in the MEF3.8-BCRP cell line, indicating inhibition of BCRP. In contrast, aldosterone and ursodeoxycholic acid had no significant effect on BCRP. The four most efficiently reversing glucocorticoid drugs (beclomethasone, 6alpha-methylprednisolone, dexamethasone, and triamcinolone) and 17beta-estradiol showed a significantly reduced BCRP-mediated transepithelial transport of PhIP by MDCKII-BCRP cells, with the highest reduction of PhIP transport ratio for beclomethasone (from 25.0 +/- 1.1 to 2.7 +/- 0.0). None of the tested endogenous steroids or synthetic glucocorticoids or digoxin, however, were transported substrates of BCRP. We also identified the H(2)-receptor antagonist drug cimetidine as a novel efficiently transported substrate for human BCRP and mouse Bcrp1. The generated BCRP-expressing cell lines thus provide valuable tools to study pharmacological and toxicological interactions mediated by BCRP and to identify new BCRP substrates.     

mdrlshRNA逆转膀胱癌耐药细胞株BIU-87／ADM对ADM的耐药性

目的构建抑制mdrl基因表达的短发卡状RNA（shRNA）真核表达载体,研究shRNA逆转膀胱肿瘤细胞多药耐药的可行性。方法针对mdrl基因已知mRNA序列不同位点,选择2个靶序列,构建靶向mdrlshRNA真核表达载体,通过脂质体介导转染膀胱肿瘤耐药细胞株BIU-87／ADM,利用RT-PCR和免疫细胞化学检测mdrlmRNA及P糖蛋白（P-gP）表达水平的变化,MTT法检测BIU-87／ADM细胞对ADM的敏感性。结果构建的2种重组质粒pshRNA-A、pshRNA-B均明显抑制BIU-87／ADM细胞株mdrl基因表达（P〈0．05）,免疫细胞化学检测显示P-gP表达明显下调（P〈O．05）,MMT法检测显示膀胱癌耐药株BIU-87／ADM对化疗药物ADM的敏感性明显增强,对ADM药物敏感性的相对逆转效率分别为52．02％和54．87％（P％0．05）。结论shRNA可有效抑制BIU-87／ADM细胞mdrlmRNA的转录和P-gP蛋白的表达,恢复BIU-87／ADM细胞对ADM的敏感性,给逆转膀胱肿瘤的多药耐药提供了一种新的方法。     

mdr—1和DHFR双基因共转染入CD^＋34细胞拓宽造血细胞耐药谱的体外研究

目的：探讨将多药耐药基因（mdr-1)和二氢叶酸还原酶基因（DHFR)同时导入人CD^ 34细胞,以拓宽造血细胞耐药谱,改善骨髓耐受联合化疗的可行性,方法：将以造血细胞中高表达的逆转录病毒载体FMCF为基本结构骨架,通过引入IRES序列构建获得含mdr-1和DHFR（L22Y）双耐药基因的塑转录病毒载体pSF－DIM,通过脂质体介导包装,单嗜性和双 包装细胞上清交叉感染提高病毒滴度,低温离心病毒上清转染人脐血CD^ 34细胞,用流式细胞仪检测P－gp的表达,基因组PCR检测外源性耐药基因的整合,CFU－GM培养药性变化,结果：逆转录病毒载体pSF-DIM转人脐血CD^ 34细胞后,P-gp的表达较未转基因组增加了10．98％,基因组PCR同时检测到两种外源性药基因的整合,与未转基因组比较,在48nmol/L甲氢蝶呤和10ng/ml及12ng/ml紫杉醇（商品名Taxol）浓度水平,CFU－GM集落形成显著培养（P＜0．05）。结论：重组双耐药基因逆转录病毒载体pSF-DIM可度水平,CFU－GM集落形成显著增加（P＜0．05）,结论：重组双耐药基因逆转录病毒载体pSF-DIM可以有效介导mdr-1和DHFR双耐药基因进入人脐血CD34^ 细胞并获得共表达,拓宽了造血细胞药谱。     

Gene rearrangement: a novel mechanism for MDR-1 gene activation.

Drug resistance, a major obstacle to cancer chemotherapy, can be mediated by MDR-1/P-glycoprotein. Deletion of the first 68 residues of MDR-1 in an adriamycin-selected cell line after a 4;7 translocation, t(4q;7q), resulted in a hybrid mRNA containing sequences from both MDR-1 and a novel chromosome 4 gene. Further selection resulted in amplification of a hybrid gene. Expression of the hybrid mRNA was controlled by the chromosome 4 gene, providing a model for overexpression of MDR-1. Additional hybrid mRNAs in other drug-selected cell lines and in patients with refractory leukemia, with MDR-1 juxtaposed 3' to an active gene, establishes random chromosomal rearrangements with overexpression of hybrid MDR-1 mRNAs as a mechanism of acquired drug resistance.     

聚乙二醇修饰后mdr—1基因的反义寡核苷酸对K562＼AO2细胞的作用

目的：探讨聚乙二醇对反义寡核苷酸的生物利用度的影响。方法：针对多耐药基因的反义寡聚脱氧核糖核苷酸靶向第二外显子跨越起始密码子ＡＵＧ的－６－９位点，在其５’端连接聚乙二醇的ＡＳ’的５’端连接聚乙二醇；在少１个碱基的ＡＳ’的５’端连接端连接二离。从ｍｄｒ－１ｍＲＮＡ、ｐ１７０、细胞内柔红霉素浓度以及细胞对阿霉素的敏感性等各个水平上比较这几种反义寡核苷酸人耐药白血病系Ｋ５６２／ＡＯ２的作用，结果ＡＰ’能     

The role of paclitaxel in the development and treatment of multidrug resistant cancer cell lines

Paclitaxel (PTX) is used for treatment of wide range of solid tumors, but its efficacy is often limited by appearance of multidrug resistance (MDR). We explored the MDR induced by PTX in human colon cancer DLD1 and glioblastoma U87 cell lines. After confirmation of the cross-resistance to other anticancer agents in newly established DLD1-TxR and U87-TxR, we analyzed the mRNA expression of membrane transporters involved in MDR. The cells had increased levels of mdr1 gene expression, while mrp1 was decreased. Flow cytometry analyzes showed that the accumulation of P-glycoprotein (P-gp) substrates (rhodamine 123 and doxorubicin) was significantly lower in DLD1-TxR and U87-TxR compared to DLD1 and U87, respectively. The significant depletion of gst-π gene expression and glutathione (GSH) concentration was observed in U87-TxR. The analysis of cell cycle kinetics revealed extensive cell death in colon cancer cells that were accumulated in subG0 phase after PTX treatment, while glioblastoma cells died during interphase (G1, S or G2). The secretion of vascular endothelial growth factor (VEGF) was inhibited by single PTX treatment of colon cancer and in continuous treatment of glioblastoma cell lines. In conclusion, continuous PTX treatment caused the over-expression of P-gp and acquisition of MDR in colon cancer and glioblastoma cell lines, while some mechanisms of MDR and tumor progression such as GSH detoxification system and VEGF secretion were suppressed. Hence, the present results implicate that PTX is an important clinical tool for colon cancer and glioblastoma treatment even in the presence of MDR.     

Biological evaluation of cryptophycin 52 fragment A analogues: effect of the multidrug resistance ATP binding cassette transporters on antitumor activity

Cryptophycin 52 (LY355703) is a potent antiproliferative analogue of the marine natural product cryptophycin 1. It has been shown to have a broad range of antitumor activity against human tumor xenografts and murine tumors including tumors resistant to Taxol and Adriamycin. Its mechanism of action involves arresting cells in the G2-M phase of the cell cycle by binding to microtubules and suppressing their dynamics. This 16-membered depsipeptide can be divided into four major subunits or fragments (A-D). We reported previously on our synthetic efforts around fragment A and discovered that this region of the molecule was amenable to a structure-activity relationship study that resulted in highly active antiproliferative agents when evaluated in the CEM leukemia cell line. The synthetic analogues were designed to help improve the efficacy and aqueous solubility of the parent compound; therefore, many in this series contained ionizable functional groups such as an amino group, a hydroxy group, or a carboxylic acid. Although several of these analogues showed improvements in potency over cryptophycin 52 in drug-sensitive tumor xenograft models, many lost their activity against Adriamycin-resistant tumor lines. It was discovered on additional in vitro evaluation that these analogues became good substrates of the multidrug resistance transporter P-glycoprotein.     

Resistance mechanisms associated with altered intracellular distribution of anticancer agents

The resistance of tumor cells to anticancer agents remains a major cause of treatment failure in cancer patients. The term multidrug resistance (MDR) is used to define a resistance phenotype where cells are resistant to multiple drugs with no obvious structural resemblance and with different molecular targets. It is now clear that MDR is always multifactorial. The intracellular drug distribution is modified in many MDR cell lines, leading to increased drug sequestration in acidic vesicles, such as the trans-Golgi apparatus, recycling endosomes, and lysosomes, followed by transport to the plasma membrane and extrusion into the external medium. Since most anticancer agents target DNA or nuclear enzymes, sequestration of drug in cytoplasmic organelles will lead to decreased drug-target interaction and thereby, decreased cytotoxicity. Altered intracellular drug distribution is usually associated with the expression of drug efflux pumps, such as the P-glycoprotein and the multidrug resistance protein. Another common modification in MDR cells is alkalization of the intracellular pH. The relationship between these different resistance mechanisms is reviewed and a model proposed that suggests why these different resistance mechanisms are co-expressed in multiple cell lines.     

Photochemical internalization of therapeutic macromolecular agents: a novel strategy to kill multidrug-resistant cancer cells

Drug resistance is a major problem for chemotherapy. Entrapment of anticancer drugs in endolysosomal compartments or active extrusions by plasma membrane proteins of the ATP-binding cassette (ABC) superfamily are important resistance mechanisms. This study evaluated photochemical internalization (PCI) of membrane-impermeable macromolecules that are not the target of ABC drug pumps for treating multidrug-resistant (MDR) cancer cells. We used the drug-sensitive uterine fibrosarcoma cell line MES-SA and its MDR, P-glycoprotein (P-gp)-overexpressing derivative MES-SA/Dx5 with the photosensitizer disulfonated meso-tetraphenylporphine (TPPS(2a)) and broad spectrum illumination. The PCI of doxorubicin, the ribosome-inactivating protein gelonin and adenoviral transduction were assessed in both cell lines, together with the uptake and excretion of TPPS(2a) and of two fluid phase markers easily detectable by fluorescence [lucifer yellow (LY) and fluorescein isothiocyanate (FITC)-dextran], as a model of gelonin uptake. Both cell lines were resistant to PCI of doxorubicin, but equally sensitive to PCI of gelonin, even though the endocytosis rates of LY and FITC-dextran were significantly lower in the MDR cells. In control studies, MES-SA/Dx5 cells were more resistant to photodynamic therapy (TPPS(2a) + light only). This was not mediated by P-gp, as there were no differences in the uptake and efflux of TPPS(2a) between the cell lines. After adenoviral infection, PCI enhanced gene delivery in both cell lines. In conclusion, PCI of macromolecular therapeutic agents that are not targets of P-gp is a novel therapeutic strategy to kill MDR cancer cells.     

Drug efflux by breast cancer resistance protein is a mechanism of resistance to the benzimidazole insulin-like growth factor receptor/insulin receptor inhibitor, BMS-536924

Preclinical investigations have identified insulin-like growth factor (IGF) signaling as a key mechanism for cancer growth and resistance to clinically useful therapies in multiple tumor types including breast cancer. Thus, agents targeting and blocking IGF signaling have promise in the treatment of solid tumors. To identify possible mechanisms of resistance to blocking the IGF pathway, we generated a cell line that was resistant to the IGF-1R/InsR benzimidazole inhibitors, BMS-554417 and BMS-536924, and compared expression profiles of the parental and resistant cells lines using Affymetrix GeneChip Human Genome U133 arrays. Compared with MCF-7 cells, breast cancer resistance protein (BCRP) expression was increased 9-fold in MCF-7R4, which was confirmed by immunoblotting and was highly statistically significant (P = 7.13E-09). BCRP was also upregulated in an independently derived resistant cell line, MCF-7 924R. MCF-7R4 cells had significantly lower intracellular accumulation of BMS-536924 compared with MCF-7 cells. Expression of BCRP in MCF-7 cells was sufficient to reduce sensitivity to BMS-536924. Furthermore, knockdown of BCRP in MCF-7R4 cells resensitized cells to BMS-536924. Four cell lines selected for resistance to the pyrrolotriazine IGF-1R/InsR inhibitor, BMS-754807, did not have upregulation of BCRP. These data suggest that benzimidazole IGF-1R/InsR inhibitors may select for upregulation and be effluxed by the ATP-binding cassette transporter, BCRP, contributing to resistance. However, pyrrolotriazine IGF-1R/InsR inhibitors do not appear to be affected by this resistance mechanism.     

酪氨酸激酶抑制剂逆转K562/A02细胞多药耐药的研究

本研究旨在比较酪氨酸激酶抑制剂伊马替尼(Imatinib)、尼洛替尼(Nilotinib)对K562/A02细胞多药耐药的逆转作用。用RT-PCR法检测各组mdr-1mRNA、bcr-abl mRNA表达;用Western blot法检测各组P-gp、P210蛋白表达;用流式细胞术检测各组细胞内柔红霉素(DNR)的蓄积。结果表明:0.0625μmol/L伊马替尼、5nmol/L尼洛替尼对K562/A02细胞无明显细胞毒性,伊马替尼和尼洛替尼上述剂量单独作用48小时均下调mdr-1mRNA、bcr-abl mRNA、P-gp、P210蛋白的表达,且尼洛替尼较伊马替尼作用更强。荧光强度检测显示,K562/A02细胞经伊马替尼、尼洛替尼单独处理48小时后,其细胞内DNR浓度分别为K562细胞中的7.85%、12.02%。结论:酪氨酸激酶抑制剂具有很好的逆转细胞耐药作用,且尼洛替尼较伊马替尼逆转作用更强。     

新型逆转录病毒载体介导的人多药耐药基因转移小鼠造血干／祖细胞

为探讨逆转录病毒介导的外源基因转导对骨髓造血重建的影响,我们以人多药耐药基因(mdr-1)为报告基因,采用包含Friend脾灶形成病毒(spleen focus-forming vims)和鼠胚胎干细胞病毒序列的新型逆转录病毒载体SF-MDR,以病毒包装细胞与小鼠造血细胞体外共培养法进行基因转染,并对基因转导后造血细胞的体外耐药能力及体内造血重建能力进行了观测。结果表明,该载体可有效地介导mdr-1基因转导,明显提高小鼠骨髓造血细胞对秋水仙素及紫杉醇的耐受能力,对细胞体内造血重建能力没有影响,体内移植8个月后经紫杉醇体内筛选,7/7的小鼠可于外周血细胞基因组DNA中检出外源mdr-1基因的存在。     

A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle system

The objectives of this study were to evaluate the potential of a polymer-lipid hybrid nanoparticle (PLN) system to enhance cellular accumulation and retention of doxorubicin (Dox), a widely used anticancer drug and an established P-glycoprotein (Pgp) substrate, in Pgp-overexpressing cancer cell lines and to explore the underlying mechanisms. Nanoparticles containing Dox complexed with a novel anionic polymer (Dox-PLN) were prepared using an ultrasound method. Two Pgp-overexpressing breast cancer cell lines (a human cell line, MDA435/LCC6/MDR1, and a mouse cell line, EMT6/AR1) were used to investigate the effect of nanoparticles on cellular uptake and retention of Dox. Endocytosis inhibition studies and fluorescence microscopic imaging were performed to elucidate the mechanisms of cellular drug uptake. Treatment of Pgp-overexpressing cell lines with Dox-PLNs resulted in significantly enhanced Dox uptake and more substantial increases in drug retention after the end of treatment compared with free Dox solutions (p < 0.05). Fluorescence microscopic images showed improved nuclear localization of Dox and uptake of lipid when the drug was delivered in the Dox-PLN form to MDA435/LCC6/MDR1 cells. Endocytosis inhibition studies revealed that phagocytosis is an important pathway in the membrane permeability of the nanoparticles. These findings suggest that some of the Dox physically associated with the nanoparticles bypass the membrane-associated Pgp when delivered as Dox-PLNs, and in this form, the drug is better retained within the Pgp-overexpressing cells than the free drug. The present study suggests a new mechanism for overcoming drug resistance in Pgp-overexpressing tumor cells using lipid-based nanoparticle formulations.     

Postnatal development of organic cation transport and mdr gene expression in mouse kidney.

The apical surface of the proximal tubular epithelium is the site of both P-glycoprotein localization and postulated active secretion of organic cations in the mammalian kidney. P-glycoprotein has been shown to act as a pleiotropic drug efflux pump across the cell membrane of tumor cells expressing the multidrug resistance phenotype, whereas the renal organic anion and organic cation secretory systems serve the function of pleiotropic drug transport across the proximal tubule epithelium. Because most known substrates for P-glycoprotein are organic cations, we tested the hypothesis that the physiological function of this protein in the kidney is to mediate renal organic cation secretion. In one approach, we compared the postnatal development of organic cation transport with that of kidney mdr gene expression. Cimetidine-sensitive uptake of classical substrates for renal secretion (N-methyl nicotinamide and tetraethylammonium) into kidney slices developed gradually in neonate mice, reaching adult capacity in 4 to 6 weeks. P-glycoprotein and its mRNA, as estimated by immunohistochemical methods and RNAse protection analysis, were undetectable at birth and were expressed abruptly at the adult level between 2 and 3 weeks of age. In another approach, classical inhibitors of renal organic cation secretion (cimetidine and cyanine 863) failed to reverse resistance to adriamycin in Chinese hamster ovary and P388 cell lines, which possess the phenotypic traits of multidrug resistance. These results suggest that the cimetidine-sensitive component of organic cation secretion is mediated by a protein other than the P-glycoprotein in the mammalian kidney.     

The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors.

Currently available HIV-1 protease inhibitors are potent agents in the therapy of HIV-1 infection. However, limited oral absorption and variable tissue distribution, both of which are largely unexplained, complicate their use. We tested the hypothesis that P-glycoprotein is an important transporter for these agents. We studied the vectorial transport characteristics of indinavir, nelfinavir, and saquinavir in vitro using the model P-glycoprotein expressing cell lines L-MDR1 and Caco-2 cells, and in vivo after intravenous and oral administration of these agents to mice with a disrupted mdr1a gene. All three compounds were found to be transported by P-glycoprotein in vitro. After oral administration, plasma concentrations were elevated 2-5-fold in mdr1a (-/-) mice and with intravenous administration, brain concentrations were elevated 7-36-fold. These data demonstrate that P-glycoprotein limits the oral bioavailability and penetration of these agents into the brain. This raises the possibility that higher HIV-1 protease inhibitor concentrations may be obtained by targeted pharmacologic inhibition of P-glycoprotein transport activity.