茯苓乙醇提取物的分离纯化及抗KBV200细胞多药耐药活性

目的对茯苓的乙醇提取物进行分离纯化,并对所得的单体化合物进行抗KBV200细胞多药耐药活性的检测。方法采用薄层色谱、柱色谱、葡聚糖凝胶分离、高效液相色谱等方法及波谱技术对茯苓的乙醇提取物进行分离纯化和结构鉴定;利用MTT法检测各化合物对KBV200细胞多药耐药性的逆转作用。结果从茯苓乙醇提取物中分离并鉴定出8个三萜类化合物。在浓度为12.5 mg/L时,8个化合物对KBV200的多药耐药性均有一定的逆转作用,其中化合物3（去氢土莫酸）的逆转作用最强,其逆转倍数为12.6。结论茯苓中的三萜类化合物具有逆转KBV200细胞多药耐药性的作用。     

The Effect of Fatty Acids and Analogues upon Intracellular Levels of Doxorubicin in Cells Displaying P-Glycoprotein Mediated Multidrug Resistance

Abstract

Multidrug resistance mediated by overexpression of P-glycoprotein (P-gp) is a major obstacle in the chemotherapeutic management of cancer. The objectives of the current work were to examine if fatty acids affect the intracellular transport and dynamics of doxorubicin in drug-resistant cancer cell lines, and to assess if such effects were mediated through modulation of P-gp efflux pump activity. Among the range of fatty acids tested in this study, eicosapentaenoic acid diester (EPADI) increased doxorubicin accumulation [A] to 137% and retention [R] to 212% in doxorubicin-resistant MCF-7/ADR breast carcinoma cells, and [A] to 147% and [R] to 163% in vinblastine-resistant KBV1 nasopharyngeal carcinoma cells. Consistent with EPADI-induced increases in intracellular doxorubicin concentrations, EPADI (10 μg/ml) sensitized MCF-7/ADR cells to the cytotoxic effects of doxorubicin (1 μg/ml) as assessed by MTT assay (viability < 50% of control), while EPADI itself displayed no cytotoxicity. The combination of EPADI (10 μg/ml) with verapamil (1 μM) resulted in a considerable increase in the [A] and [R] of the model P-gp substrate rhodamine-123 within drug-resistant cells compared to when either agent were used alone. KBV1 cells treated with combination of EPADI (10 μg/ml) and verapamil (1 μM) achieved 160% and 1120% greater [A] and [R] of rhodamine-123, respectively, compared to untreated cells. The P-gp modulatory effects of EPADI either alone, or as part of a combination with more potent inhibitors, should be further investigated.     

Marine sponge-derived sipholane triterpenoids reverse P-glycoprotein (ABCB1)-mediated multidrug resistance in cancer cells

Previously, we reported sipholenol A, a sipholane triterpenoid from the Red Sea sponge Callyspongia siphonella, as a potent reversal of multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp). Through extensive screening of several related sipholane triterpenoids that have been isolated from the same sponge, we identified sipholenone E, sipholenol L and siphonellinol D as potent reversals of MDR in cancer cells. These compounds enhanced the cytotoxicity of several P-gp substrate anticancer drugs, including colchicine, vinblastine and paclitaxel, and significantly reversed the MDR-phenotype in P-gp-overexpressing MDR cancer cells KB-C2 in a dose-dependent manner. Moreover, these three sipholanes had no effect on the response to cytotoxic agents in cells lacking P-gp expression or expressing MRP1 (ABCC1) or MRP7 (ABCC10) or breast cancer resistance protein (BCRP/ABCG2). All three sipholanes (IC₅₀ >50 μM) were not toxic to all the cell lines that were used. [³H]-Paclitaxel accumulation and efflux studies demonstrated that all three triterpenoids time-dependently increased the intracellular accumulation of [³H]-paclitaxel by directly inhibiting P-gp-mediated drug efflux. Sipholanes also inhibited calcein-AM transport from P-gp-overexpressing cells. The Western blot analysis revealed that these three triterpenoids did not alter the expression of P-gp. However, they stimulated P-gp ATPase activity in a concentration-dependent manner and inhibited the photolabeling of this transporter with its transport substrate [¹²⁵I]-iodoarylazidoprazosin. In silico molecular docking aided the virtual identification of ligand binding sites of these compounds. In conclusion, sipholane triterpenoids efficiently inhibit the function of P-gp through direct interactions and may represent potential reversal agents for the treatment of MDR.     

Reversal of P-glycoprotein-mediated multidrug resistance by the novel tetrandrine derivative W6

Overexpression of ATP-dependent efflux pump P-glycoprotein (P-gp) is the main cause of multidrug resistance (MDR) and chemotherapy failure in cancer treatment. Inhibition of P-gp-mediated drug efflux is an effective way to overcome cancer drug resistance. The present study investigated the reversal effect of the novel tetrandrine derivative W6 on P-gp-mediated MDR. KBv200, MCF-7/adr and their parental sensitive cell lines KB, MCF-7 were used for reversal study. The intracellular accumulation with P-gp substrates of doxorubicin was determined by flow cytometry. The expression of P-gp and ERK1/2 was investigated by western blot and real-time-PCR (RT-PCR) analysis. ATPase activity of P-gp was performed by P-gp-Glo^TM assay systems. In comparison with P-gp-negative parental cells, W6 produced a favorable reversal effect in the MDR cells, as determined using the MTT assay. W6 significantly and dose-dependently increased intracellular accumulation of P-gp substrate doxorubicin (DOX) in P-gp overexpressing KBv200 cells, and also inhibited the ATPase activity of P-gp. W6 inhibited P-gp expression in KBv200 cells in a time-dependent manner, but it had no effect on MDR1 expression. In addition, W6 significantly decreased the ERK1/2 activation in KBv200 cells. Our results showed that W6 effectively reversed P-gp-mediated MDR by inhibiting the transport function and expression of P-gp, demonstrating the potential clinical utility of W6.     

赛赓啶对 KBV200细胞多药抗性的逆转作用

研究赛赓啶对KB_V200细胞多药抗性的逆转作用及逆转机制。在KB_V200细胞,采用MTT法,测出赛赓啶对长春新碱、阿霉素和鬼臼乙叉甙耐药的逆转系数分别为5.5,2.0和1.9,而对5-氟尿嘧啶、美法仑的细胞毒性作用无明显影响,表明赛赓啶为多药抗性逆转剂。荧光分光光度法测定表明,赛赓啶可使KB_V200细胞内阿霉素蓄积量增加。流式细胞荧光测定显示赛赓啶可增加罗丹明123的蓄积并减慢其外排。免疫细胞化学及狭缝杂交表明赛赓啶不影响KB_V200细胞的P-糖蛋白染色深度和 mdr1 RNA 表达水平。以上结果提示赛赓啶的多药抗性逆转机制是抑制P-糖蛋白泵的功能。     

Reversal of P-gp mediated multidrug resistance in-vitro and in-vivo by FG020318

Overexpression of P-glycoprotein (P-gp) by tumours results in multidrug resistance (MDR) to structurally and functionally unrelated chemotherapeutic drugs. Combined therapy with MDR-related cytotoxins and MDR modulators is a promising strategy to overcome clinical MDR. This study was performed to explore the MDR reversal activity of a novel compound 2-[4-(2-pyridin-2-yl-vinyl) phenyl]-4,5-bis-(4-N,N-diethylaminophenyl)-1(H)-imidazole (FG020318) in-vitro and in-vivo. Tetrazolium (MTT) assay was used to evaluate the ability of FG020318 to reverse drug resistance in two P-gp-expressing tumour cell lines, KBv200 and MCF-7/adr. Intracellular doxorubicin accumulation was determined by fluorescence spectrophotometry in MCF-7/adr cell line. The effect of FG020318 on P-gp function was demonstrated by rhodamine 123 (Rh123) accumulation in KBv200 cells. KBv200 cell xenograft models were established to study the in-vivo effect of FG020318 on reversing MDR. FG020318 was not cytotoxic by itself against P-gp expressing KBv200 cells and MCF-7/adr cells and their parental drug-sensitive KB cells and MCF-7 cells. FG020318 could significantly increase the sensitivity of MDR cells to antitumour drugs including doxorubicin and vincristine in MCF-7/adr cells and KBv200 cells, respectively. It was much stronger than the positive control verapamil in reversal of MDR. FG020318 also increased the intracellular accumulation of doxorubicin in a concentration-dependent manner in MCF-7/adr cells, but did not affect the accumulation of doxorubicin in drug-sensitive MCF-7 cells. The Rh123 accumulation in resistant KBv200 cells was also increased by the addition of FG020318, but Rh123 accumulation was not affected by FG020318 in drug-sensitive KB cells. FG020318 potentiated the antitumour activity of vincristine to KBv200 xenografts and was an efficacious modulator in-vivo. Our results suggested that FG020318 was a highly potent, efficacious MDR modulator not only in-vitro but also in-vivo. The reversal of drug resistance by FG020318 was probably related to the increased anticancer drug accumulation and its inhibition of P-gp function of MDR tumour cells.     

野生型p53基因促进长春新碱诱导的细胞凋亡

为探讨VCR与p53介导的细胞凋亡的关系,将野生型p53基因导入一个内源性p53基因重组的多药耐药细胞系 KB_V200,经抗性筛选和杂交鉴定获得外源性p53基因稳定表达的转染细胞系KB_V200-p53。VCR处理细胞后,经电镜及荧光显微镜观察表明KB_V200-p53细胞呈现凋亡的典型形态学特征;而流式细胞术结果显示经600nmol·L^-1的VCR作用24h,KB_V200-p53细胞和KB_V200细胞的凋亡百分数分别为42.4%和8.4%。结果提示野生型p53基因能促进VCR诱导的细胞凋亡,肿瘤细胞对VCR的耐药性可能与细胞内p53基因失活有关。     

一种新番荔枝内酯单体atemoyacin—B克服肿瘤多药抗药性

目的 探讨ａｔｅｍｏｙａｃｉｎ－Ｂ（Ａｔｅ）克服肿瘤多药抗药性（ＭＤＲ）作用及机制。方法 Ｂｕｌｌａｔａｃｉｎ（Ｂｕｌ）为阳性对照物,细胞毒测定以ＭＴＴ法,Ｐｇｐ功能测定以Ｆｕｒａ２－ＡＭ法,细胞内药物积累测定以荧光分光光度计法;细胞凋亡测定以流式细胞仪法,结果：Ａｔｅ对ＭＣＦ－７／Ｄｏｘ,ＭＣＦ－７,ＫＢＶ２００和ＫＢ细胞的ＩＣ５０分别为１２２,１２０,１．３４,１．２７ｍｍｏｌ．Ｌ＾－１,Ａｔ     

Farnesiferol A from Ferula persica and galbanic acid from Ferula szowitsiana inhibit P-glycoprotein-mediated rhodamine efflux in breast cancer cell lines

In multidrug resistance (MDR), cancer cells exposed to anticancer agents develop resistance to a wide variety of chemicals and chemotherapeutic agents. Sesquiterpene coumarins are reported to inhibit P-glycoprotein and/or increase cytotoxicity of anticancer drugs in P-gp-overexpressing cell lines. In the current study, we investigated the effects of galbanic acid (from the roots of Ferula szowitsiana) and farnesiferol A (from the roots of Ferula persica) on functionality of the drug transporter P-glycoprotein (P-gp) using a rhodamine 123 efflux assay in a doxorubicin resistant breast cancer cell line (MCF7/Adr). Compared to verapamil, the well-known inhibitor of P-gp, galbanic acid (5, 10, and 25 μg/mL), significantly inhibited the P-gp activity. In inhibition of the P-gp transporter, farnesiferol A (0.5?μg/mL) was more potent than verapamil at 15 min exposure. Our results indicate that the plant derived sesquiterpene coumarins, farnesiferol A and galbanic acid, may be promising candidates to be considered for further studies on the reversal of multidrug resistance phenotype in chemotherapy of cancer patients.     

蛇床子素逆转人膀胱肿瘤T24／ADM细胞耐药作用及其机制

目的研究蛇床子素（osthole,OST）对T24／ADM细胞多药耐药的逆转及对P-gP表达、功能的影响。方法采用MTT法筛选OST对T24／ADM细胞无毒或低毒浓度,以筛选出的浓度作为耐药逆转的实验浓度。分别利用MTF法分别检测阿霉素（adriamycin,ADM）、氟尿嘧啶（fluorouracil,Fu）及顺铂（cisplatin,DDP）对细胞株T24和T24／ADM以及OST处理后的T24／ADM细胞的半数抑制浓度IC50。用流式细胞仪测定OST对T24／ADM细胞上P-gP表达的影响。结果浓度17Ixmol／LOST对T24／ADM无明显细胞毒性,T24／ADM对ADM、FU和DDP均有不同程度的。耐药,耐药倍数分别为18．86、5．09、3．28,使用17μmol／LOST处理后T24／ADM对上述ADM、Fu和DDP的敏感性均有不同程度的提高,ADM对耐药细胞株的IC蜘由（0．962±0．017）μxg／mL降至（0．364±0．031）μg／mL,逆转指数为2．64;FU对耐药细胞株的IC。由（0．723±0．003）μg／mL降至（0．463±0．021）μxg／mL,逆转指数为1．56;DDP对耐药细胞株的IC50由（0．664±0．007）μg／mL降至（0．587±0．016）μg／mL,逆转指数为1．13,使T24／ADM细胞内P-gP蛋白表达明显下降,下降了16．32％（P〈0．05）。结论蛇床子素在体外能部分逆转T24／ADM的耐药性,逆转机制可能是通过抑制P—gP蛋白表达实现的。     

Influence of ceramide metabolism on P-glycoprotein function in immature acute myeloid leukemia KG1a cells

Previous studies have emphasized the role of glucosylceramide (Glu-Cer) synthase in multidrug resistance (MDR) regulation. However, the mechanism by which the inhibition of this enzyme results in increased drug retention and cytotoxicity remains unclear. In this study, we investigated the respective role of ceramide (Cer) accumulation and Glu-Cer derivatives depletion in MDR reversal effect of 1-phenyl-2-decanoylamino-3-morpholino-1-propanolol (PDMP), a Glu-Cer synthase inhibitor. We show here that treatment with PDMP resulted in increased rhodamine 123 (Rh123) retention and potent chemosensitization of P-glycoprotein (P-gp)-expressing cells, including KG1a cells, KG1a/200 cells, K562/138 cells, and K562/mdr-1 cells. Metabolic studies revealed that PDMP induced not only time-dependent Cer accumulation but also reduction of all glycosylated forms of Cer, including Glu-Cer, lactosylceramide (Lac-Cer), monosialo ganglioside (GM3) and disialo ganglioside (GD3). The influence of these metabolites on P-gp function was investigated by measuring Rh123 retention in PDMP-treated cells. P-gp function was found to be stimulated only by the addition of gangliosides in all resistant cell lines, whereas Glu-Cer, Lac-Cer, and Cer had no effect. Moreover, in KG1a/200 cells, GD3 and, to a lesser extent, GM3 were found to phosphorylate P-gp on serine residues. Altogether, these results suggest that, at least in leukemic cells, gangliosides depletion accounts for PDMP-mediated MDR reversal effect, and that gangliosides are important P-gp regulators perhaps through their capacity to modulate P-gp phosphorylation.     

Antibacterial properties of phenolic triterpenoids against Staphylococcus epidermidis

Two phenolic triterpenoids, pristimerol (30 μg/mL) and 8- EPI-6-deoxoblepharodol (20 μg/mL), obtained by catalytic reduction of pristimerin, exhibited bacteriostatic action against Staphylococcus epidermidis. This activity was not dependent on the inoculum size and the growth phase although it showed a stronger effect when cells were growing actively. Addition of phenolic triterpenoids to S. epidermidis cultures in the log-phase of growth led to an inhibitory effect on incorporation and uptake of radiolabeled precursors thymidine, uridine, leucine, and N-acetyl-glucosamine after 30 min of treatment. Furthermore, a clear release of UV-absorbing material and leakage of intracellular potassium were also detected. These findings, coupled with the high lipophilicity of these molecules, shown by high ClogP values, suggest that 8-EPI and pristimerol are able to interact within the lipid bilayer and as a consequence cause functional alterations on the cytoplasmic membrane of S. epidermidis cells.     

鬼臼毒素衍生物CIP-36诱导KBV200细胞凋亡

目的：研究鬼臼毒素衍生物CIP-36对多药耐药人口腔鳞状上皮癌细胞KBV200的抗肿瘤活性及其作用机制。方法：MTT法考察CIP-36对KBV200体外增殖的抑制作用;Giemsa染色、DNA ladder和流式细胞仪等方法进行细胞凋亡检测;免疫荧光法观察CIP-36对细胞骨架的作用;western-blot法检测CIP-36对KBV200细胞P-gp表达的影响。结果：CIP-36对KBV200细胞有明显的抑制作用,IC50值为（2.06±0.38）μmol/L,能够诱导细胞产生凋亡小体和DNAladder。流式细胞检测到了细胞凋亡峰,并观察到细胞周期出现S/G2＋M期阻滞。Western-blot结果显示P-gp表达降低,并且观察到CIP-36可破坏KBV200细胞的细胞骨架。结论：CIP-36可能通过降低P-gp的表达,破坏细胞骨架等多靶点克服KBV200细胞株的多药耐药性。     

Ritonavir decreases the nonrenal clearance of digoxin in healthy volunteers with known MDR1 genotypes

Our objective was to examine the influence of ritonavir on P-glycoprotein (P-gp) activity in humans by characterizing the effect of ritonavir on the pharmacokinetics of the P-gp substrate digoxin in individuals with known MDR1 genotypes. Healthy volunteers received a single dose of digoxin 0.4 mg orally before and after 14 days of ritonavir 200 mg twice daily. After each digoxin dose blood and urine were collected over 72 hours and analyzed for digoxin. Digoxin pharmacokinetic parameter values were determined using noncompartmental methods. MDR1 genotypes at positions 3435 and 2677 in exons 26 and 21, respectively, were determined using PCR-RFLP analysis. Ritonavir increased the digoxin AUC(0-72) from 26.20 +/- 8.67 to 31.96 +/- 11.24 ng x h/mL (P = 0.03) and the AUC(0-8) from 6.25 +/- 1.8 to 8.04 +/- 2.22 ng x h/mL (P = 0.02) in 12 subjects. Digoxin oral clearance decreased from 149 +/- 101 mL/h x kg to 105 +/- 57 mL/h x kg (P = 0.04). Other digoxin pharmacokinetic parameter values, including renal clearance, were unaffected by ritonavir. Overall, 75% (9/12) of subjects had higher concentrations of digoxin after ritonavir administration. The majority of subjects were heterozygous at position 3435 (C/T) (6 subjects) and position 2677 (G/T,A) (7 subjects); although data are limited, the effect of ritonavir on digoxin pharmacokinetics appears to occur across all tested MDR1 genotypes. Concomitant low-dose ritonavir reduced the nonrenal clearance of digoxin, thereby increasing its systemic availability. The most likely mechanism for this interaction is ritonavir-associated inhibition of P-gp. Thus, ritonavir can alter the pharmacokinetics of coadministered medications that are P-gp substrates.     

白藜芦醇通过拮抗hPXR对P-gp的影响

目的研究白藜芦醇通过拮抗hPXR对P-gp基因(MDR1)、蛋白表达及活性的影响。方法在LS174T细胞中,采用瞬时共转染报告基因实验研究白藜芦醇对PXR介导的MDR1的转录调节作用,并进一步应用Real-Time定量PCR和Western blot方法检测白藜芦醇作用24 h后对利福平诱导的P-gp基因和蛋白变化的影响,罗丹明转运实验考察P-gp活性的变化。结果双荧光素酶报告基因检测结果显示,25和50μmol.L-1白藜芦醇可通过拮抗PXR将利福平对MDR1的诱导作用由4.70倍分别降至1.76倍和0.69倍(P<0.01),在过表达hPXR的LS174T细胞中,50μmol.L-1白藜芦醇可以将利福平诱导的MDR1 mRNA水平由1.8倍降至1.3倍(P<0.05),Western blot结果表明白藜芦醇也可降低利福平诱导的P-gp表达。此外,罗丹明转运实验显示,25和50μmol.L-1白藜芦醇可以将利福平抑制的累积量由77.7%升至91.7%和95.1%(P<0.05),表明白藜芦醇可降低利福平诱导的P-gp活性。结论白藜芦醇可以通过拮抗PXR而影响P-gp的基因、蛋白表达及活性。     

三苯氧胺对肺癌A549/DDP细胞多药耐药性的逆转作用及机制

目的探讨三苯氧胺（TAM）逆转肺癌A549/DDP细胞多药耐药性（MDR）的作用及可能机制。方法以噻唑蓝（MTT）比色法测定TAM对A549/DDP细胞的生长抑制率,流式细胞术检测TAM对A549/DDP细胞内Rho123表达的影响,选取最佳的TAM实验浓度。以MTT法检测TAM对阿霉素（ADM）、吉西他滨（GEM）、顺铂（DDP）的逆转效率,即时聚合酶链反应法检测TAM对A549/DDP细胞多药耐药基因1（MDR1）mRNA水平的变化,蛋白质印迹法检测P糖蛋白（P-gp）表达水平的变化。结果 TAM剂量在10.0μmol/L时对A549/DDP细胞毒性小且可增加细胞内Rho123的蓄积,10.0μmol/L的TAM能增加化疗药物的敏感性,对ADM、GEM、DDP的逆转倍数分别为3.0、7.2、2.0倍,使MDR1 mRNA的表达率较用药前下降35%,并可显著抑制P-gp的表达水平。结论 TAM体外可逆转A549/DDP细胞MDR,提高A549/DDP细胞对化疗药物的敏感性,其机制可能与抑制MDR1表达、减少细胞内药物外排有关。     

槲皮素逆转人肝癌MDR相关基因的研究

摘 要 目的: 通过定量PCR芯片技术在基因转录水平上探讨槲皮素逆转人肝癌多药耐药( multi drug resistance, MDR)的作用机制。方法:采用体外培养人肝癌细胞Bel-7402及其耐5-氟尿嘧啶细胞Bel-FU, MTT法测定槲皮素的体外细胞毒性及浓度依赖的逆转耐药作用。定量PCR芯片检测BEL-7402细胞及Bel-FU细胞之间的差异基因,以及槲皮素作用于Bel-FU细胞前后的基因表达差异,实时定量PCR和western-blot法检测细胞中mdr1、H-ras基因以及P-gp蛋白的表达变化以验证基因芯片分析的结果。结果: 槲皮素对Bel-7402、Bel-FU细胞的IC10分别为59.2、58.2μM, IC50分别为226.7、193.9μM。16.5、33.0、49.5 μM 的槲皮素对Bel-FU 的耐药逆转倍数分别为1.14、1.68、2.38倍;与Bel-7402比较, Bel-FU细胞中的基因H-ras、Bcl-2、PDGFA、MDR1、BCRP表达上调（P＜0.05）表达上调。经槲皮素作用后,耐药细胞中基因H-ras、EGFR、FGF、VEGFA、PDGFA、MDR1、Erk1表达下调。实时定量PCR和western-blot证实了槲皮素可下调MDR1、H-ras基因以及P-gp蛋白在耐药细胞中高表达,与芯片检测到的基因水平变化一致。结论：槲皮素可逆转耐药细胞中耐药差异基因的高表达量,从而逆转人肝癌细胞的多药耐药性。     

GCS基因与MDR1基因在肝癌细胞多药耐药中的相关性研究

目的观察葡萄糖神经酰胺合酶(GCS)基因高表达对肝癌细胞HepG2内多药耐药基因(MDR1)表达的影响,探讨GCS基因参与肝癌多药耐药的作用机制。方法脂质体法将GCS基因真核表达质粒pEGFP-GCS导入肝癌细胞株HepG2中,应用G418筛选培养,反转录聚合酶链反应(RT-PCR)检测细胞GCS mRNA和MDR1 mRNA的表达,蛋白印迹法检测细胞GCS蛋白和P-糖蛋白(P-gp)的表达,四甲基偶氮唑蓝(MTT)法检测基因转染前后阿霉素对HepG2细胞半数抑制浓度(IC50)的变化。结果转染后HepG2细胞内GCSmRNA和蛋白的表达水平显著提高(P<0.05),而MDR1mRNA及P-gp的表达水平亦显著提高(P<0.05);同时,MTT法检测结果显示GCS基因转染后HepG2细胞对阿霉素的耐药性显著提高(P<0.05),IC50从(6.2±0.4)μg/ml上升到(11.6±1.0)μg/ml。结论高表达GCS可以上调HepG2细胞中MDR1的表达,使HepG2细胞发生多药耐药。