多药耐药逆转剂与血脑屏蔽上P—糖蛋白ATP酶活性间的相互作用

目的：进一步探讨P－糖蛋白（P-gp)与其多种耐药逆转剂量ATP依赖性相互作用的机制。方法：从牛脑灰质中分离得到微血管内皮细胞（BCEC）,制成细胞膜,定磷法测定BCEC膜上P-gp ATPas活性,结果：维拉帕米（Ver),长春新碱（VCR）,阿霉素（Dox),粉防己碱（Tet),蝙蝠葛碱（DRC）,小檗胺（BBM）以及蝙蝠葛苏林碱（DRS）增加基础P-gp ATPas活性,其Km值分别约为17,5．9,41,2．3,11,23和22μmol/L,小檗碱（BBR）仅有轻微的激活作用,延胡索乙素（dl-THP)和左旋四氢巴马汀（l-THP)不改变基础P-gp ATPas活性。环孢素A（CsA)抑制基因P-gp ATPas活性;竞争性抑制Ver或VCR激活的P-gp ATPas活性;非竞争性抑制Dox或Tet激活的P-gp ATPas活性。Dox非竞争性抑制Tet-激活的P-gp ATPas活性。结论：各种多药耐药逆转剂与P-gp 相互作用的机制及其对P-gp ATPas活性的影响各不相同,CsA,Ver和VCR在血脑屏蔽P-gp 上的结合部位可能是重叠的或者是有相互联系的,而CsA,Dox和Tet与P－gp的结合是相互独立的,并存在各自不同的结合部位。     

多药耐药逆转剂与血脑屏障上P-糖蛋白ATP酶活性间的相互作用(英文)

目的:进一步探讨P-糖蛋白(P-gp)与其多种耐药逆转剂间ATP依赖性相互作用的机制.方法:从牛脑灰质中分离得到微血管内皮细胞(BCEC),制成细胞膜,定磷法测定BCEC膜上P-gp ATPase活性.结果:维拉帕米(Ver)、长春新碱(VCR)、阿霉素(Dox)、粉防己碱(Tet)、蝙蝠葛碱(DRC)、小檗胺(BBM)以及蝙蝠葛苏林碱(DRS)增加基础P-gpATPase活性,其Km值分别约为17、5.9、41、2.3、11、23和22μmol/L.小檗碱(BBR)仅有轻微的激活作用,延胡索乙素(dl-THP)和左旋四氢巴马汀(l-THP)不改变基础P-gp ATPase活性.环孢素A(CsA)抑制基础P-gp ATPase活性;竞争性抑制Ver或VCR激活的P-gp ATPase活性;非竞争性抑制Dox或Tet激活的P-gp ATPase活性.Dox非竞争性抑制Tet-激活的P-gp ATPase活性.结论:各种多药耐药逆转剂与P-gp相互作用的机制及其对P-gp ATPase活性的影响各不相同.CsA、Ver和VCR在血脑屏障P-gp上的结合部位可能是重叠的或者是有相互联系的,而CsA、Dox和Tet与P-gp的结合是相互独立的,并存在各自不同的结合部位.     

Effects of chemopreventive citrus phytochemicals on human P-glycoprotein and multidrug resistance protein 1

The effects of dietary chemopreventive citrus phytochemicals on the drug efflux transporters P-glycoprotein (ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-glycoprotein-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural chemopreventive citrus phytochemicals, such as auraptene, nobiletin, citral, citronellal, limonene, limonin, and synephrine were examined. The accumulation of daunorubicin, a fluorescent substrate of P-glycoprotein, increased in the presence of auraptene and nobiletin in KB-C2 cells. Nobiletin also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activity of P-glycoprotein was stimulated by auraptene and nobiletin. The ATPase activity of MRP1 was stimulated by nobiletin. These results suggest that chemopreventive citrus phytochemicals, such as nobiletin found in oranges, have inhibitory effects on P-glycoprotein and/or MRP1, and may cause food–drug interactions.     

Disulfiram metabolites permanently inactivate the human multidrug resistance P-glycoprotein

The human multidrug resistance P-glycoprotein (P-gp) uses ATP to transport a wide variety of structurally unrelated cytotoxic compounds out of the cell. The relatively high expression of P-gp in organs such as the intestine, kidney, blood-brain/testes barrier and in some tumor cells can compromise chemotherapy treatments for patients with cancer or AIDS/HIV. It has been difficult to inhibit P-gp during chemotherapy with noncovalent inhibitors because the relatively high levels of inhibitors have severe side effects. An alternative approach to inhibit P-gp would be to covalently modify cysteine residues within the NBDs. In this study, we tested whether metabolites of disulfiram, a drug currently used to treat chronic alcoholism, could inhibit P-gp. We show that the disulfiram metabolites, S-methyl N,N-diethylthiocarbamate sulfoxide and S-methyl N,N-diethylthiocarbamate sulfone inhibited the verapamil-stimulated ATPase activity of P-gp with IC50 values (concentrations that result in 50% inhibition of activity) of 9 and 4.8 microM, respectively. Similarly, S-methyl N,N-diethylthiocarbamate sulfoxide and S-methyl N,N-diethylthiocarbamate sulfone inhibited the activity of aldehyde dehydrogenase with IC50 values of 3.2 and 1.7 microM, respectively. Inhibition of P-gp by the metabolites was not reversed by addition of the reducing compound, dithiothreitol. We then determined which endogenous cysteine residue was responsible for inhibiting P-gp activity after exposure to the disulfiram metabolites. Treatment of P-gp mutants containing a single cysteine residue showed that inactivation was primarily due to modification of Cys1074 in NBD2. These results indicate that metabolites of disulfiram can covalently inactivate P-gp. Covalent modification of drug transporters could be a useful approach for inhibiting their activities during chemotherapy.     

Modulation of multidrug resistance P-glycoprotein 1 (ABCB1) expression in human heart by hereditary polymorphisms

OBJECTIVES: Variable expression of the ABC-type multidrug resistance membrane protein P-glycoprotein (P-gp, MDR1, ABCB1) in human heart is a potential modulator of drug effects or drug-induced cardiotoxicity. Expression of P-gp is known to be affected by single nucleotide polymorphisms in the MDR1 gene. Therefore, genotype-dependent expression of P-gp could be an important modulator of action of cardiac drugs. METHODS: Heart tissue (auriculum) from 51 patients undergoing coronary artery bypass graft surgery was screened for genotype-dependent P-gp expression. P-gp was identified by immunoblotting and localized using immunohistochemistry. MDR1 mRNA was quantified by real-time PCR and immunohistochemistry and related to the MDR1 genotypes G2677T/A (Ala893Ser/Thr) and C3435T. RESULTS: MDR1/18S rRNA mRNA copy numbers in heart auriculum were 3.48 +/- 2.25 x 10(-6) compared to 4.56 +/- 0.58 x 10(-6) in non-failing ventricular samples studied before. While the exon 26 C3435T genotype did not influence MDR1 mRNA expression, we found significantly elevated MDR1 mRNA expression in 10 patients carrying the exon 21 2677 AT or TT genotype as compared to 12 patients carrying the GG-variant with intermediate MDR1 mRNA expression in 29 heterozygous samples. P-gp was detected in the endothelial wall. Quantitative immunohistochemistry of protein expression, however, did not reveal significant influence of the studied SNPs. CONCLUSION: The present study based on auricular samples suggests that genetic factors play a rather limited role in modulating P-gp expression in human heart. Therefore, the substantial interindividual variability in cardiac P-gp expression is likely related to environmental or disease related factors.     

槲皮素对胃癌细胞多药耐药的逆转作用

目的 了解槲皮素对胃癌组织P-糖蛋白(P-gp)和多药耐药相关蛋白1(MRP1)的逆转作用。方法 取27例胃癌手术标本,采用体外细胞培养,测定槲皮素对肿瘤细胞化疗药物敏感性的影响(H^3-TDR法),并分析肿瘤组织P-gp、MRP1表达的关系。结果 P-gp阴性细胞5种化疗药物的敏感性均高于P-gp阳性,其中ADM组差别有显性。而槲皮素可随剂量增加,使P-gp阳性细胞化疗药物敏感性增加,其中MTX、VP-16及ADM组有显性差别。MRP1阴性细胞对5种化疗药物的敏感性均高于MRP1阳性,其中MTX、MMC和5-FU组差别有显性。槲皮素不能使两组细胞化疗敏感性有明显改变。结论 槲皮素能逆转P-gp的多药耐药,特别是对MTX、VP-16及ADM的耐药。但对MRP1引起的耐药,逆转效果不明显。     

Coumarin derivatives with tumor-specific cytotoxicity and multidrug resistance reversal activity

A preliminary exploration of coumarin derivatives as novel multidrug resistance (MDR) modulators was carried out to determine the basic features of the structure responsible for the MDR reversal activity. Among 44 coumarins, 14 compounds moderately induced the reversal of MDR (fluorescence activity ratio (FAR) values > 1). The most active compound, 6-hydroxy-3-(2-hydroxyethyl)-4-methyl- 7-methoxycoumarin [C34], was equally potent as a MDR modulator verapamil. These data show a relationship between the chemical structure and MDR-reversal effect on tumor cells. All coumarins tested were more cytotoxic against tumor cells than normal cells. Several compounds displayed potent cytotoxic activities (CC50 15 - 29 microg/mL in HSC cells), comparable with that of gallic acid (CC50 = 24 microg/mL). Both 6-hydroxy- 7-methoxy-4-methyl-3-isopropylcoumarin [C43] and 3-ethyl-6-hydroxy- 7-methoxy-4-methylcoumarin [C44] showed the highest tumor-specific cytotoxicity (SI value = 4.1 and 3.6, respectively). We conclude that coumarins are potentially potent new MDR modulators with low toxicity against normal cells. A deeper understanding of the relationship between their structures and their potency will contribute to the design of optimal agents.     

Reversers of the multidrug resistance transporter P-glycoprotein

Multidrug resistance can arise from the presence of the membrane-bound pump, P-glycoprotein, in a tumor. Major efforts have been made to develop inhibitors of this pump, and a number of promising blockers have reached late stages of clinical trials. The kinetics of the inhibition of P-glycoprotein is complex, with binding sites that can interact synergistically. Reversers of increased affinity and specificity could, in principle, be developed on the basis of these synergies, and offer some promise in cancer therapeutics.     

Cytotoxicity and reversal of multidrug resistance by tryptanthrin-derived indoloquinazolines

Aim:

To evaluate the effects and elucidate the mechanisms of a series of indoloquinazolines as novel anticancer agents.

Methods:

Condensation of the substituted isatoic anhydride with the substituted isatin was performed to prepare compounds 1–4, followed by adding malononitrile to prepare compounds 5–7. Cytotoxicity was measured by MTT assays. Apoptosis induction was evaluated using DNA fragmentation, cell cycle assay, caspase 3/7 activity and Western blot.

Results:

Compounds 3, 4, and 5 display cytotoxicity against MCF-7, HeLa, SKOV3, and A498 cancer cells. DNA ladders appear in cells treated with compounds 3, 4, and 5. Within those, compound 4 exhibits the greatest activity in regards to sub-G₁ accumulations in the cell cycle and the activation of caspase-3/7. Furthermore, Fas and Fas ligand levels are elevated by compound 4, implying that the apoptosis is in part mediated through the signals. On the other hand, compounds 1 and 7 display chemosensitizing activity since cytotoxicity of doxorubicine and etoposide is enhanced in combination with compound 1 and 7, respectively, in MCF-7/adr (doxorubicin-resistant) and MCF-7/vp (etoposide-resistant).

Conclusion:

The cytotoxicity of indoloquinazolines is structure-dependent rather than cell type-dependent due to the similar degree of cytotoxicity induced by the individual compounds in all four cell lines. Further modification of the tryptanthrin skeleton is important to develop novel anticancer agents bearing either cytotoxicity against MCF-7 cells or drug resistance reversal in MCF-7/adr and MCF-7/vp.     

黄酮类化合物逆转肿瘤多药耐药作用的研究进展

肿瘤多药耐药是肿瘤治疗过程中一个亟待解决的难题,其表现为肿瘤细胞对单一或多种化疗药物同时出现耐药性,从而导致治疗失败,与药物的化学结构和作用机制无关。将化疗药物与肿瘤多药耐药逆转剂联合应用是目前公认的治疗方案之一。黄酮类化合物由于其低毒、高效,具有多种药理作用等优点而受到广泛的关注。在肿瘤治疗期间,黄酮类化合物能通过抑制ABC转运体、诱导凋亡、调节氧化应激等作用逆转肿瘤多药耐药。归纳总结了黄酮类化合物逆转肿瘤多药耐药的主要机制、应用及其纳米剂型改造的进展,为进一步的临床研究提供参考。     

表面活性剂对肿瘤细胞多药耐药逆转作用的体外筛选

目的:从表面活性剂中筛选出能逆转肿瘤细胞多药耐药(MDR)的耐药逆转剂。方法:用MTT法筛选出对多药耐药的细胞系K562／A02耐药的抗肿瘤药物,选择并确定表面活性剂没有细胞毒性的浓度,以维拉帕米作阳性对照,检测不同的表面活性剂在不同的浓度下逆转多药耐药的细胞系K562／A02对不同抗肿瘤药物耐药的能力。结果:硬脂酸聚氧乙烯、泊洛沙姆、蓖麻油聚氧乙烯醚、吐温-60、辛基酚聚氧乙烯醚等几种非离子表面活性剂均能降低抗肿瘤药物阿霉素、柔红霉素、长春新碱、依托泊苷、三尖杉酯碱对K562／A02细胞的IC50。结论:具有一定化学结构的非离子表面活性剂能逆转多药耐药的细胞系K562／A02的耐药性,作为药用辅料在逆转肿瘤多药耐药上将会有广阔的应用前景。     

Dihydro-beta-agarofuran sesquiterpenes: a new class of reversal agents of the multidrug resistance phenotype mediated by P-glycoprotein in the protozoan parasite Leishmania

Leishmaniasis is the most important emerging and uncontrolled infectious disease and the second cause of death among parasitic diseases, after Malaria. One of the main problems concerning the control of infectious diseases is the increased resistance to usual drugs. Overexpression of P-glycoprotein (Pgp)-like transporters represents a very efficient mechanism to reduce the intracellular accumulation of drugs in cancer cells and parasitic protozoans, thus conferring a multidrug resistance (MDR) phenotype. Pgps are active pumps belonging to the ATP-binding cassette (ABC) superfamily of proteins. The inhibition of the activity of these proteins represents an interesting way to control drug resistance both in cancer and in infectious diseases. Most conventional mammalian Pgp-MDR modulators are ineffective in the modulation of Pgp activity in the protozoan parasite Leishmania. Consequently, there is a necessity to find effective modulators of Pgp-MDR for protozoan parasites. In this review we describe a rational strategy developed to find specific Pgp-MDR modulators in Leishmania, using natural and semisynthetic dihydro-beta-agarofuran sesquiterpenes from Celastraceae plants. A series of these compounds have been tested on a MDR Leishmania tropica line overexpressing a Pgp transporter to determine their ability to revert the resistance phenotype and to modulate intracellular drug accumulation. Almost all of these natural compounds showed potent reversal activity with different degrees of selectivity and a significant low toxicity. The three-dimensional quantitative structure-activity relationship using the comparative molecular similarity indices analysis (CoMSIA), was employed to characterize the requirements of these sesquiterpenes as modulators at Pgp-like transporter in Leishmania.     

The multidrug resistant modulator HZ08 reverses multidrug resistance via P-glycoprotein inhibition and apoptosis sensitization in human epidermoid carcinoma cell line KBV200

Previous studies have demonstrated that the multidrug resistance modulator HZ08 has a strong multidrug resistance reversal effect in vitro and in vivo by inhibiting P-glycoprotein and multidrug resistance-associated protein 1 in K562/A02 and MCF-7/ADM cells, respectively. However, there are many other mechanisms responsible for resistance. In this study, MTT assay was used to examine the cytotoxicity and multidrug resistance reversal of HZ08 in KBV200 cells. It was also used to detect Rh123 and adriamycin accumulation in the presence of HZ08 to assess the effect on P-glycoprotein. Caspase-3 activity was analyzed under the incubation of HZ08 per se and in combination with vincristine. Results showed that HZ08 could increase the activity of caspase-3 with P-glycoprotein inhibition. Further studies revealed that HZ08 increased vincristine-induced apoptosis, characterized as an intrinsic apoptosis pathway with enhanced G2/M phase arrest, since HZ08 had an effect on the intrinsic apoptotic regulator Bcl-2 and Bax. Therefore, the outstanding reversal effect of HZ08 occurs not only through suppressing the P-glycoprotein function but also through activating the intrinsic apoptosis pathway.     

Effects of lipopolysaccharide on intestinal P-glycoprotein expression and activity

It is well known that pharmacokinetics is often altered by changing the expression and activity of P-glycoprotein during sepsis. However, there have been few reports about expression and activity of P-glycoprotein in the small intestine during sepsis. We examined the levels of intestinal P-glycoprotein expression and activity using a rat sepsis model induced by lipopolysaccharide (LPS, from Escherichia coli). LPS was administered to male Wistar/ST rats intraperitonealy (i.p.) at 5 mg/kg. The small intestine was excised before and 1, 3 and 7 days after LPS administration, and the intestinal P-glycoprotein expression was determined using Western blot analysis. The activity of P-glycoprotein was evaluated by measuring the efflux of rhodamine-123 (Rho123) in rats using an in situ single perfusion method. The changes of permeability via the paracellular route were evaluated by measuring the amount of fluorescein isothicyanate-dextran 4400 (FD-4) in a similar way. On Day 1 after LPS administration, both the level of P-glycoprotein expression and the total amount of Rho123 excreted into the intestinal lumen decreased significantly, but levels of both AUC2-95 and CLtot were not significantly different as compared with the control group. On Day 3, the total P-glycoprotein, including intestinal P-glycoprotein, might have been induced by sepsis, and then the excretion of P-glycoprotein substrate drugs into the intestinal lumen increased more than that of the control group. On Day 7, all pharmacokinetic parameters returned to the control level. Thus the intestinal P-glycoprotein function recovered within 3 days of LPS administration.     

Efficacies of tea components on doxorubicin induced antitumor activity and reversal of multidrug resistance

Considering of novel biochemical modulation by some foods and beverages, we have performed screening for green tea components that have enhancing effects on doxorubicin (DOX) induced antitumor activity. Components, such as caffeine, theanine, (-)-epigallocatechin gallate (EGCG) and flavonoids have inhibitory effects on the DOX efflux from Ehrlich ascites carcinoma cells. Thus, it is suggested that EGCG and flavonoids may enhance DOX induced antitumor activity and increase the DOX concentrations in tumors through the inhibition of DOX efflux. It is expected that these components in green tea exhibit low toxicity and that there are few side effects of drinking green tea in combination with an antitumor agent. We think that the intake of a favorite beverage favors a positive mental attitude of a patient and increases the efficacy of the chemotherapeutic index, and that this efficacy is useful for improving the quality of life on cancer chemotherapy. In DOX resistant P388 leukemia cell bearing mice theanine increased the DOX induced efficacy through an increase in the DOX concentrations in the tumors. Theanine attacked the same transport process for DOX in both types of cells, elevated the DOX concentration and increased the DOX induced antitumor activity.     

Reversal of P-glycoprotein associated multidrug resistance by new isoprenoid derivatives

To find a drug to overcome P-glycoprotein associated multidrug resistance, we synthesized 43 new isoprenoid derivatives. Ten compounds were effective in an in vitro assay with the human MDR-type resistant carcinoma KB/VJ-300 and MRP-type KB/VP-4 cell lines. One of the most effective compounds, N-5228 [trans-N,N'-bis(3,4-dimethoxybenzyl)-N-solanesyl-1,2-diaminocyclohexane, mol. wt 1100.481, was tested in P388/VCR-bearing mice. It showed a antitumor effect on MDR-type resistant tumor cells. Moreover, N-5228 potentiated the accumulation of [3H]vincristine in drug-resistant cells and blocked [3H]azidopine photoaffinity labeling of P-glycoprotein molecules in MDR-type resistant cell membranes. We think that N-5228 is promising as a lead compound in the screening of resistance reversing drugs for multidrug resistant cancers.     

Effect of curcumin on multidrug resistance in resistant human gastric carcinoma cell line SGC7901/VCR

AIM: To investigate the reversal effects of curcumin on multidrug resistance (MDR) in a resistant human gastric carcinoma cell line. METHODS: The cytotoxic effect of vincristine (VCR) was evaluated by MTT assay. The cell apoptosis induced by VCR was determined by propidium iodide (PI)-stained flow cytometry (FCM) and a morphological assay using acridine orange (AO)/ethidium bromide (EB) dual staining. P-glycoprotein (P-gp) function was demonstrated by the accumulation and efflux of rhodamine123 (Rh123) using FCM. The expression of P-gp and the activation of caspase-3 were measured by FCM using fluorescein isothiocyanate (FITC)-conjugated anti-P-gp and anti-cleaved caspase-3 antibodies, respectively. RESULTS: Curcumin, at concentrations of 5 micromol/L, 10 micromol/L, or 20 micromol/L, had no cytotoxic effect on a parent human gastric carcinoma cell line (SGC7901) or its VCR-resistant variant cell line (SGC7901/VCR). The VCR-IC50 value of the SGC7901/VCR cells was 45 times more than that of the SGC7901cells and the SGC7901/VCR cells showed apoptotic resistance to VCR. SGC7901/VCR cells treated with 5 micromol/L, 10 micromol/L, or 20 micromol/L curcumin decreased the IC50 value of VCR and promoted VCR-mediated apoptosis in a dose-dependent manner. Curcumin (10 micromol/L) increased Rh123 accumulation and inhibited the efflux of Rh123 in SGC7901/VCR cells, but did not change the accumulation and efflux of Rh123 in SGC7901 cells. P-gp was overexpressed in SGC7901/VCR cells, whereas it was downregulated after a 24-h treatment with curcumin (10 micromol/L). Resistant cells treated with 1 mumol/L VCR alone showed 77% lower levels of caspase-3 activation relative to SGC7901 cells, but the activation of caspase-3 in the resistant cell line increased by 44% when cells were treated with VCR in combination with curcumin. CONCLUSION: Curcumin can reverse the MDR of the human gastric carcinoma SGC7901/VCR cell line. This might be associated with decreased P-gp function and expression, and the promotion of caspase-3 activation in MDR cells.     

Bis-pyranobenzoquinones as a new family of reversal agents of the multidrug resistance phenotype mediated by P-glycoprotein in mammalian cells and the protozoan parasite Leishmania

We have synthesized a set of bis-pyranobenzoquinones through a direct and highly efficient approach based on a double intramolecular domino Knoevenagel hetero Diels-Alder reaction. These bis-pyranobenzoquinone derivatives are compounds whose skeletons have similarities to those of some anticancerous and leishmanicidal drugs. Considering that these drugs are substrates for some members of the ATP-binding cassette (ABC) family of proteins that confers a multidrug resistance (MDR) phenotype, we have carried out the biological evaluation of 20 bis-pyranobenzoquinones as modulators of the MDR phenotype in mammalian cell lines overexpressing P-glycoprotein, MRP1, or BCRP. Moreover, we also tested some of these compounds as potential MDR modulators in a Leishmania tropica line overexpressing a P-glycoprotein-like transporter. Compounds 9 and 10 are, in this work, the most promising reversal agents of MDR in human cancer cell lines, while compounds 4 and 20 showed potent reversal activity of MDR phenotype in the protozoan parasite Leishmania.