C421 allele-specific ABCG2 gene amplification confers resistance to the antitumor triazoloacridone C-1305 in human lung cancer cells

The A421 ABCG2 genotype is a frequent polymorphism encoding the K141 transporter, which is associated with a significant decrease in transporter expression and function when compared to the wild type (wt) C421 allele encoding the Q141 ABCG2. Here we show that during the acquisition of resistance to the novel triazoloacridone antitumor agent C-1305 in lung cancer cells harboring a heterozygous C421A genotype, a marked C421 allele-specific ABCG2 gene amplification occurred. This monoallelic C421 ABCG2 gene amplification brought about the overexpression of both C421 ABCG2 mRNA and the transporter at the plasma membrane. This resulted in the lack of cellular drug accumulation due to increased efflux of both C1305 and C-1311, a fluorescent imidazoacridone homologue of C-1305, as well as marked resistance to these antitumor agents and to established ABCG2 substrates including mitoxantrone and SN-38. Consistently, the accumulation and sensitivity to these drugs were restored upon incubation with the potent and specific ABCG2 transport inhibitors Ko143 and fumitremorgin C. Moreover, upon transfection into HEK293 cells, the wt Q141 ABCG2 allele displayed a significantly decreased accumulation of C-1311 and increased resistance to C-1305, C-1311 and mitoxantrone, when compared to the K141 ABCG2 transfectant. Hence, the current study provides the first evidence that during the exposure to anticancer drugs, an allele-specific Q141 ABCG2 gene amplification occurs that confers a drug resistance advantage when compared to the K141 ABCG2. These findings have important implications for the selection and expansion of malignant anticancer drug resistant clones during chemotherapy with ABCG2 drugs.     

黄酮类化合物对肿瘤多药耐药调节作用的研究进展

多药耐药是临床上化疗失败的重要原因之一。黄酮类化合物存在于多种植物中,具有广泛的药理活性,对P-糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、乳腺癌耐药蛋白(BCRP)等外向转运蛋白的抑制作用使其可能成为肿瘤多药耐药调节剂。文中分别对黄酮类化合物对ABC家族转运蛋白抑制作用的研究概况、作用机制以及构效关系进行综述,为肿瘤多药耐药抑制剂的开发和应用提供重要信息。     

中药逆转肿瘤多药耐药的研究进展

目的综述逆转肿瘤多药耐药(MDR)的研究进展。方法检索中药提取物或中药单体联合化疗药物逆转肿瘤MDR的研究。结果一些中药提取物或中药单体能使耐药株对化疗药物变得敏感,使耐药蛋白的转运活性下降。结论中药具有逆转肿瘤MDR的活性,其作用机制大多是与耐药相关蛋白相关的。     

Resistance to purine and pyrimidine nucleoside and nucleobase analogs by the human MDR1 transfected murine leukemia cell line L1210/VMDRC.06

Overexpression of human MDR1 P-glycoprotein [Pgp] is associated with cellular resistance to bulky amphipathic drugs, such as taxol, anthracyclines, vinca alkaloids, and epipodophyllotoxins by actively effluxing drugs from cells. We have found that human MDR1 transfected murine L1210/VMDRC.06 leukemia cells exhibit relatively large amounts of Pgp and high levels of resistance to 6-mercaptopurine [6-MP] and other purine and pyrimidine nucleobase and nucleoside analogs. L1210/VMDRC.06 cells accumulated 6-MP as the nucleotide in vitro at only about one-third of that formed by parental L1210 cells in normal medium; however, under conditions of ATP-depletion, the amount of 6-MP nucleotide formed was essentially the same in both cell lines. The findings support active efflux of 6-MP in L1210 cells, suggesting involvement of Pgp in 6-MP resistance even though it is generally believed that Pgp does not transport such agents. The resistance pattern observed in L1210/VMDRC.06 cells was not duplicated in P388/VMDRC.04 leukemia cells transfected with the same MDR1 cDNA, even though a similar amount of Pgp was present in both cell lines. Immunofluorescent staining of surface membrane Pgp showed that L1210/VMDRC.06 cells contained at least three-fold more surface Pgp than P388/VMDRC.04, implying that P388/VMDRC.04 cells are unable to actively efflux 6-MP and other antimetabolites as effectively as L1210/VMDRC.06, because of significantly lower membrane Pgp. The findings suggest that the exceedingly large concentration of overexpressed Pgp in the surface membrane of L1210/MDRC.06 cells is responsible for resistance to 6-MP and other purine and pyrimidine analogs, even though these agents usually are not considered to be substrates for Pgp.     

Reduction of bFGF-induced smooth muscle cell proliferation and endothelin receptor mRNA expression by mevastatin and atorvastatin

The anti-atherosclerosis mechanisms of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) occur via both cholesterol-dependent and cholesterol-independent mechanisms. The present study used aortic and cerebral vascular smooth muscle cells (SMC) from rat to investigate whether atorvastatin and mevastatin affect basic fibroblast growth factor (bFGF)-induced SMC proliferation and the mRNA expression of endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Cell proliferation was assessed by MTT and real-time PCR was used to quantify ET(A) and ET(B) receptor mRNA. bFGF-induced concentration and time dependent SMC proliferation and up-regulation of the mRNA expression of ET(A) and ET(B) receptors. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors inhibited bFGF-induced proliferation of SMC (P<0.01). In aortic SMC atorvastatin and mevastatin significantly inhibited bFGF-induced mRNA expression of endothelin ET(A) and ET(B) receptors (P<0.05). Although in cerebral SMC the inhibitory effect of the statins was comparable in size with that seen in aortic SMC, only reached borderline significance (P=0.06) for ET(A) receptor mRNA but not for ET(B). The findings suggested a direct effect of statins on the vascular wall beyond their well-known lipid lowering effect in anti-atherosclerosis. Furthermore, the specific antagonists of ET(A) and ET(B) receptors (FR139317 and BQ788, respectively) significantly inhibited bFGF-induced SMC proliferation (P<0.001). The results suggested that endothelin receptors and the mevalonate pathway were involved in bFGF-induced SMC proliferation.     

洛伐他汀对氧化修饰低密度脂蛋白刺激U937细胞缺氧诱导因子-1α表达的影响

目的：研究氧化修饰低密度脂蛋白（ox—LDL）刺激U937细胞表达缺氧诱导因子-1α（HIF-1α）的变化,并观察洛伐他汀对其的影响。方法：用油红染色的方法检测泡沫细胞的形成,采用实时定量PCR（Real—timePCR）及蛋白印记杂交技术（Westernblotting）检9n,4细胞HIF-1α表达的变化。结果：未加ox—LDL刺激的U937细胞未见泡沫细胞形成;U937细胞加入ox-LDL刺激24h后,全部转化为泡沫细胞。ox—LDL刺激下,U937细胞HIF-1α表达量显著升高,加入洛伐他汀后,HIF-1α的表达量显著降低,随剂量增加,抑制作用显著增强,有明显的剂量依赖关系。结论：ox-LDL氧化损伤导致巨噬细胞HIF—1α高表达,洛伐他汀可抑制U937细胞HIF-1α的表达,抑制泡沫细胞的形成。     

Flavonoid conjugates interact with organic anion transporters (OATs) and attenuate cytotoxicity of adefovir mediated by organic anion transporter 1 (OAT1/SLC22A6)

Flavonoids are conjugated by phase II enzymes in humans to form glucuronidated and sulfated metabolites that are excreted in urine via the kidney. In this study, we examined the interaction between metabolites of quercetin and isoflavonoids found in vivo with human organic anion transporters 1 (OAT1) and 3 (OAT3) and their potential in attenuating OAT-induced cytotoxicity of adefovir. Accumulation of flavonoid conjugates was studied in human embryonic kidney 293H cells overexpressing OAT1 or OAT3. OAT1-overexpressing cells exhibited an increased uptake of the sulfated conjugates, genistein-4′-O-sulfate and quercetin-3′-O-sulfate. OAT3-overexpressing cells demonstrated enhanced uptake of glucuronide conjugates, such as daidzein-7-O-glucuronide, genistein-7-O-glucuronide, glycitein-7-O-glucuronide and quercetin-3′-O-glucuronide. Position of conjugation was important since quercetin-3-O-glucuronide and quercetin-7-O-glucuronide were poorly transported. Kinetic analysis revealed high affinity uptake of quercetin-3′-O-sulfate by OAT1 (K_m = 1.73 μM; V_max = 105 pmol/min/mg). OAT3 transported isoflavone glucuronides with lower affinity (K_m = 7.9–19.1 μM) but with higher V_max (171–420 pmol/min/mg). Quercetin-3′-O-sulfate strongly inhibited OAT1-mediated p-aminohippuric acid uptake with an IC₅₀ of 1.22 μM. Transport of 5-carboxyfluorescein by OAT3 was potently inhibited by quercetin-3-O-glucuronide, quercetin-3′-O-glucuronide and quercetin-3′-O-sulfate (IC₅₀ = 0.43–1.31 μM). In addition, quercetin-3′-O-sulfate was shown to effectively reduce OAT1-mediated cytotoxicity of adefovir, an antiviral drug, in a dose-dependent manner. These data suggest that OAT1 and OAT3 are responsible for basolateral uptake of flavonoid conjugates in kidney, and flavonoid conjugates inhibit OAT1 and OAT3 activity at physiologically relevant concentrations. Interaction with OATs limits systemic availability of flavonoids and may be a mechanism of food–drug interaction via inhibition of renal uptake.     

紫杉醇诱导的人白血病CCRF-CEM多药耐药细胞模型的建立

目的建立稳定的紫杉醇(paclitaxel)诱导的人肿瘤多药耐药细胞系,并对其生物学特点进行评价。方法用亚致死浓度(1/50IC50值)的紫杉醇连续诱导对化疗药物敏感的人急性T淋巴细胞白血病CCRF-CEM细胞,使其成为对紫杉醇耐药的CCRF-CEM/paclitaxel细胞,同时观察CCRF-CEM/paclitaxel细胞对柔红霉素(daunorubicin)和长春碱(vinblastine)的交叉耐药;用相应的单抗和FITC标记的二抗与细胞孵育后,用流式细胞仪分别检测细胞表面和细胞总P糖蛋白(P-gp)、多药耐药相关蛋白1(MRP1)和肺癌耐药相关蛋白(LRP)等表达水平的变化,并用流式细胞仪检测细胞周期和细胞凋亡率的变化。结果CCRF-CEM/paclitaxel细胞传至第90代时,对紫杉醇产生了耐药,耐药倍数约为256.4倍;同时对柔红霉素和长春碱也产生了耐药,耐药倍数约为9.8和2.0倍。与CCRF-CEM细胞比较,CCRF-CEM/paclitaxel细胞表面P-gp和细胞总P-gp表达分别升高了18.8和14.4倍,MRP1和LRP分别升高了2.1和1.2倍;S期细胞比例由(48.3±1.2)%降低至(23.8±0.5)%,细胞凋亡百分率由(7.82±0.19)%减少至(2.47±0.37)%。结论成功建立了稳定的紫杉醇诱导的人CCRF-CEM/paclitaxel多药耐药细胞模型,该模型具有一般肿瘤多药耐药细胞的生物学特点。     

Identification of gene expression profiles predicting tumor cell response to L-alanosine

The methylthioadenosine phosphorylase (MTAP) gene gained considerable interest as therapeutic target for tumors with the 9p21 deletion. This gene maps to 9p21 and loss of this chromosomal region in tumors offers an unique opportunity for chemoselective treatment, since MTAP is an important salvage enzyme for the formation of adenine that is needed for DNA synthesis. L-Alanosine, an antibiotic from Streptomyces alanosinicus, blocks the common de novo purine biosynthesis pathway and, thereby, inhibits tumor cells with MTAP deficiency. Normal cells escape the detrimental effects of L-alanosine due to their proficiency in the MTAP salvage pathway. The present analysis was undertaken to gain insights into the molecular architecture of tumor cells that determines the response to L-alanosine apart from the MTAP gene. Analysis of cell doubling times and IC(50) values for L-alanosine showed that slowly growing cell lines were more resistant to L-alanosine than rapidly growing ones. Mining the database of the National Cancer Institute (N.C.I.), for the mRNA expression of 9706 genes in 60 cell lines by means of Kendall's tau-test, false discovery rate calculation, and hierarchical cluster analysis pointed to 11 genes or expressed sequence tags whose mRNA expression correlated with the IC(50) values for L-alanosine. Furthermore, we tested L-alanosine for cross-resistance in multidrug-resistant cell lines which overexpress selectively either the P-glycoprotein/MDR1 (CEM/ADR5000), MRP1 (HL-60/AR), or BCRP (MDA-MB-231-BCRP) genes. None of the multidrug-resistant cell lines was cross-resistant to L-alanosine indicating that L-alanosine may be suitable to treat multidrug-resistant, refractory tumors in the clinic. Finally, the IC(50) values for L-alanosine of the 60 cell lines were correlated to the p53 mutational status and expression of p53 downstream genes. We found that p53 mutated cell lines were more resistant to L-alanosine than p53 wild type cell lines.     

Down-regulation of deoxycytidine kinase in human leukemic cell lines resistant to cladribine and clofarabine and increased ribonucleotide reductase activity contributes to fludarabine resistance

Mechanisms of acquired resistance to three purine analogues, 2-chloro-2'-deoxyadenosine (cladribine, CdA), 9-beta-D-arabinofuranosyl-2-fluoroadenine (fludarabine, Fara-A), and 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (clofarabine, CAFdA) were investigated in a human T-lymphoblastic leukemia cell line (CCRF-CEM). These analogues are pro-drugs and must be activated by deoxycytidine kinase (dCK). The CdA and CAFdA resistant cell lines exhibited increased resistance to the other nucleoside analogues activated by dCK. This was also the case for the Fara-A resistant cells, except that they were sensitive to CAFdA and guanosine analogues. The CdA and CAFdA resistant cells displayed a deficiency in dCK activity (to <5%) while the Fara-A resistant cells showed only a minor reduction of dCK activity (20% reduction). The activity of high K(m) 5'-nucleotidase (5'-NT) (cN-II) using IMP as substrate, was 2-fold elevated in the resistant cell lines. The amount of the small subunit R2 of ribonucleotide reductase (RR) was higher in the Fara-A resistant cells, which translated into a higher RR activity, while CdA and CAFdA cells had decreased activity compared to the parental cells. Expression of the recently identified RR subunit, p53R2 full-size protein, in CAFdA cells was low compared to parental cells, but a protein of lower molecular weight was detected in CdA and CAFdA cells. Co-incubation of Fara-A with the RR inhibitor 3,4-dihydroxybenzohydroxamic acid (didox) enhanced cytotoxicity in the Fara-A resistant cells by a factors of 20. Exposure of the cells to the nucleoside analogues studied here also caused structural and numerical instability of the chromosomes; the most profound changes were recorded for CAFdA cells, as demonstrated by SKY and CGH analysis. We conclude that down-regulation of dCK in cells resistant to CdA and CAFdA and increased activity of RR in cells resistant to Fara-A contribute to resistance.     

Functional studies on the activity of efflux transporters in an ex vivo model with chicken splenocytes and evaluation of selected fluoroquinolones in this model

The efflux proteins P-glycoprotein (P-gp), BCRP and members of the MRP-family (MRPs) are increasingly recognized as determinants of the absorption, tissue distribution and excretion of numerous drugs. A widely applied in vitro screening method, to assess the effect of these efflux transporters in transmembrane transport of drugs is based on the use of peripheral blood mononuclear cells (PBMC), in which the efflux of fluorescent dye Rhodamine 123 (Rh-123) can be easily measured. In avian species, the isolation of PBMCs is compromised by the presence of thrombocytes having approximately the same size. As an alternative, we validated the use of isolated splenocytes to assess Rhodamine 123 transport in the presence and absence of specific inhibitors for P-gp, MRPs and BCRP. Rh-123 efflux was concentration-dependent with the percentage of efflux that decreased with increasing concentrations. P-gp inhibitors, PSC833 and GF120918, significantly inhibit Rh-123 efflux, whereas inhibitors for MRPs and BCRP, MK571 and Ko-143, respectively, have a limited inhibitory effect. However, the effect of GF120918 was more pronounced as compared to PSC833, suggesting an additional role for BCRP next to P-gp in Rh-123 efflux. Moreover, fluoroquinolones were selected to test the applicability of the described model. None of these fluoroquinolones significantly inhibit P-gp function at concentrations up to 50 microM, with exception of danofloxacin and danofloxacin mesylate that were found to reduce Rh-123 efflux by approximately 15%.     

The imidazoline-like drug S23515 affects lipid metabolism in hepatocyte by inhibiting the oxidosqualene: lanosterol cyclase activity

Imidazoline-like drugs are centrally-acting antihypertensive agents that inhibit the activity of the sympathetic nervous system by interacting with the alpha2-adrenoreceptor and also with a non-adrenergic imidazoline binding site called the imidazoline 1 receptor. Recently, these molecules were proposed to play an additional role in cardiovascular diseases by acting on glucose and lipid metabolism. We used S23515, a potent imidazoline-like molecule acting selectively on blood pressure through the imidazoline 1 receptor, to decipher the effects of these drugs on lipid metabolism. We found that S23515 inhibited specifically and dose-dependently cholesterol synthesis in cultured rodent and primate hepatocytes. This hypocholesterolemic effect was likely due to the inhibition of the oxido:lanosterol cyclase (OSC), a rate-limiting enzyme in the cholesterol biosynthetic pathway. Partial OSC inhibition induced by S23515 led to the generation of 24(S),25-epoxycholesterol, a potent ligand for the liver X receptor (LXR). Furthermore, S23515 increased in human macrophages the expression of both ABCA1 and G1, the 2 ATP binding cassette transporters, which play a pivotal role in the reverse cholesterol transport. Thus, these results suggest that S23515, and potentially other imidazoline-like drugs, could exert hypolipidemic effects in addition to their hypotensive activities.     

卡维地洛对氧化低密度脂蛋白诱导血管内皮细胞ADMA-DDAH系统的影响

目的 观察卡维地洛对氧化低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)二甲基精氨酸-二甲基赖氨酸水解酶(DDAH)活性及表达的影响,以探讨卡维地洛对内源性一氧化氮合酶抑制物不对称二甲精氨酸(ADMA)代谢机制的影响.方法 采用改良的Jaffe法培养原代人脐静脉内皮细胞(HUVECs),取生长良好的3～6代HUVECs用于实验,分为(1)空白对照组:加DMEM培养液;(2)ox-LDL组:加入ox-LDL(100 mg/L,150 mg/L);(3)ox-LDL 卡维地洛组:同时加入150 mg/L ox-LDL及卡维地洛(10 μmol/L)共孵24h后,检测上清液中NO、NOS活性、ADMA含量、L-胍氨酸(L-cit)浓度,采用Western blotting测定细胞裂解液中二甲基精氨酸-二甲基赖氨酸水解酶(DDAH)的蛋白表达.结果 ox-LDL条件培养下,内皮细胞的代谢产物ADMA、ET的量均较空白对照组高,而NO的量及NOS的活性减少;反应DDAH酶活性的L-cit浓度显著降低,且有浓度依赖性,而DDAH的表达无明显变化.卡维地洛干预后,ADMA、ET的量较ox-LDL组降低,NOS活性及NO增加,L-cit浓度明显升高.结论 ox-LDL诱导下,内皮损伤ADMA的增加与DDAH的活性减弱有关,而与DDAH的表达无关.卡维地洛通过增加DDAH活性促进ADMA代谢,使NOS活性增高,抑制ox-LDL对内皮功能的损伤.     

Influx and efflux transport as determinants of melphalan cytotoxicity: Resistance to melphalan in MDR1 overexpressing tumor cell lines

There is a considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines (r² = 0.47, P = 0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan (r² = 0.56, P = 0.03 and r² = 0.62, P = 0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1 overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non-MDR1 expressing one. Again, the resistance was reversed by cyclosporine and by MDR1-specific shRNA.LAT1 was the major influx transporter in tumor cell lines with 4000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.