ABCG2 polymorphisms, 34G>A and 421C>A in a Korean population: analysis and a comprehensive comparison with other populations

Background and objective: ABCG2, also known as Breast Cancer Resistance Peptide (BCRP) or mitoxantrone‐resistant protein, is the second member of the G‐family of ABC transporters. The frequencies of ABCG2 34G>A and 421C>A polymorphisms in a Korean population were assessed using a newly developed multiplex pyrosequencing method, and compared with the corresponding frequencies seen in other ethnic groups. Method: We designed a multiplex pyrosequencing method to simultaneously detect ABCG2 421C>A and 34G>A polymorphisms and analysed the allele frequencies of these polymorphisms in 250 Korean subjects. Results: The results showed 100% concordance between single and multiplex pyrosequencing methods. We also validated the polymorphisms identified by pyrosequencing with a direct sequencing method using randomly selected samples. The allele frequencies of ABCG2 421C>A and 34G>A in the population tested were 0·298 and 0·190 respectively. The allele frequency of the 421C>A polymorphism is comparable to other Asian populations, including Japanese and Chinese. However, both frequencies are different from those of Caucasians and Africans. Conclusions: The multiplex pyrosequencing method used to detect two ABCG2 polymorphisms concurrently is a rapid and reliable genotyping method for the detection of important ABCG2 genetic polymorphisms. The ABCG2 34G>A and 421C>A polymorphisms are frequently found in the Korean population. The frequencies are similar to those seen in other Asian populations including Japanese and Chinese, but very different to those of Caucasian and African‐American populations.     

Protective mechanisms of resveratrol against methotrexate‐induced renal damage may involve BCRP/ABCG2

Resveratrol (RES) is a well‐known polyphenol antioxidant. We have previously shown that testicular protective effect of RES against the anticancer drug methotrexate (MTX)‐induced toxicity involves transporter‐mediated mechanisms. Here, we investigated the effect of RES on MTX‐induced nephrotoxicity. Rats were administered RES (10 mg/kg/day) for 8 days, with or without a single MTX dose (20 mg/kg i.p.) at day 4 of the experiment. MTX induced nephrotoxicity, as evidenced by a significant increase in serum blood urea nitrogen and creatinine compared to the control, as well as distortion of kidney microscopic structure. MTX also significantly increased renal nitric oxide level along with inducible nitric oxide synthase, fas ligand, and caspase 3 expressions. Administering RES prior to MTX significantly improved kidney function and microscopic picture and also significantly decreased nitrosative and apoptotic markers compared to MTX alone. RES, but not MTX, caused a significant increase in expression of breast cancer resistance protein (BCRP), an apical efflux renal transporter that participates in urinary elimination of both MTX and RES. Interestingly, concomitant MTX and RES caused further upregulation of renal BCRP compared to RES alone. Using Human BCRP ATPase assay, both RES and MTX exhibited a dose‐dependent increase in ATPase activity, with Km values of 0.52 ± 0.03 and 30.9 ± 4.2 μm , respectively. Furthermore, combined RES and MTX caused ATPase activity which was significantly less than maximum ATPase activity attained by the positive control, sulfasalazine (12.5 μm ). In conclusion, RES exerted nephroprotection against MTX‐induced toxicity through antinitrosative and anti‐apoptotic effects, as well as via upregulation of renal BCRP.     

Oral topotecan: Bioavailability,pharmacokinetics and impact of ABCG2 genotyping in Chinese patients with advanced cancers

Oral topotecan (Hycamtin^®) has been recently approved for the treatment of relapsed small cell lung cancer (SCLC) in 2007, however, the bioavailability and pharmacokinetic data of topotecan for Chinese patients is still limited. Xinze^® is a new and the only capsule formulation of topotecan used in China that is similar to Hycamtin^®. The current study aimed to investigate the absolute bioavailability and pharmacokinetics of Xinze^® in Chinese patients with advanced cancers. On day 1, an IV dose of 1.5 mg/m²/d as a 30 min continuous infusion was administered. Patients took the oral topotecan at one of two dose levels: 1.5 mg/m²/d (six patients) or 1.9 mg/m²/d (seven patients) on day 2. Plasma pharmacokinetics of total topotecan and topotecan in the lactone form were performed on both days using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Single-nucleotide polymorphisms (SNPs) identified in exon 5 (421C>A) and in exon 2 (34G>A) in ATP-binding cassette sub-family G member 2 (ABCG2) were analyzed by direct sequencing. Safety assessments were performed throughout the study. The maximum plasma concentration (C_max) reached at 1–2 h and the elimination half-life time (T_1/2) was approximately 4.2 h after oral administration. The absolute bioavailability of total topotecan in the 1.5 mg/m²/d and 1.9 mg/m²/d groups averaged 41.23 ± 11.8% and 36.00 ± 14.8%, respectively. The patients with heterozygous SNPs had essentially the same bioavailability and pharmacokinetics. The bioavailability of topotecan after oral administration illustrates good systemic exposure at dosages of 1.5 mg/m²/d and 1.9 mg/m²/d over a five-day schedule in Chinese patients. On a dose-normalized basis, the values of C_max and AUC_0–t for total topotecan in Chinese patients were higher than in Caucasians following oral and intravenous administration, while the T_1/2 was consistent.     

结直肠癌组织中ABCG2蛋白的表达及其与CPT-11化疗方案疗效的相关性

目的探讨结直肠癌组织中ABCG2蛋白的表达情况及其与CPT-11化疗方案疗效的相关性。方法选取结直肠癌住院患者89例。采用链霉菌抗生物素蛋白-过氧化物酶连结法检测ABCG2表达情况,分析结直肠癌组织中ABCG2蛋白的表达情况与CPT-11化疗方案疗效的相关性,并分析ABCG2蛋白的表达情况与患者年龄、性别等临床资料的相关性。结果89例直肠癌住院患者组织标本中,阳性率达89.89%。52例临近正常肠黏膜组织标本中,阳性率为48.08%,表明结直肠癌组织中ABCG2表达较正常肠黏膜组织显著升高(P0.05)。结直肠癌组织标本ABCG2表达情况与患者年龄、性别、分化程度、病变部位、肝转移无相关性(P0.05)。结直肠癌组织标本ABCG2表达情况与CPT-11化疗疗效无相关性(P0.05)。结论结直肠癌组织中ABCG2蛋白的表达情况与CPT-11化疗方案疗效无相关性。     

PC-3悬浮成球细胞中ABCG2表达及其对化学治疗耐药性的实验研究

目的：探讨人雄激素非依赖性前列腺癌PC-3悬浮成球细胞中ATP结合盒膜转运蛋白G超家族成员2（ABCG2）的表达及其对化学治疗的耐药性。方法：体外无血清悬浮培养PC-3细胞,逆转录定量-PCR检测PC-3悬浮成球细胞中ABCG2 mRNA的表达,采用MTT法检测PC-3悬浮成球细胞对顺铂的耐药性,计算半数抑制浓度（IC50）,并与PC-3贴壁细胞作对比。结果：PC-3悬浮成球细胞可以在无血清培养条件下生存并形成悬浮细胞球,PC-3悬浮成球细胞中ABCG2 mRNA的相对表达水平是PC-3贴壁细胞的33．98倍（P〈0．05）,其IC50是PC-3贴壁细胞的4．26倍（P〈0．05）。结论：人PC-3悬浮成球细胞中ABCG2表达水平较高,该类细胞对化学治疗具有较强耐药性。     

Small-molecule inhibitors of multidrug resistance-associated protein 1 and related processes: A historic approach and recent advances

Multidrug resistance-associated protein 1 (MRP1, ABCC1) is an ATP-binding cassette (ABC) transport protein. This efflux pump uses the energy of ATP hydrolysis to export structurally diverse antineoplastic agents in human cancers. The upregulation of MRP1 (either inherent or acquired) is one major reason for the occurrence of the phenomenon called multidrug resistance (MDR). MDR is characterized by a reduced outcome of chemotherapy due to the active intracellular clearance of cytostatic drugs below the necessary effect concentration. Much effort has been made to overcome MDR, which implied high-throughput screenings of already known pharmacological and natural compounds, modification of intrinsic substrates, as well as design and synthesis of new inhibitors. This review is meant not only to summarize the most recent results over the past 10 years, but also to highlight major achievements regarding reversal of MRP1-mediated MDR, from the time of its discovery until today. The focus lies on small-molecule compounds that feature either direct MRP1 inhibition/transport blockage, toxicity against MRP1-overexpressing cells, inhibition/modification of intracellular processes necessary for MRP1 function, or modification of MRP1-related metabolic and genomic mechanisms. Considering all aspects, this review might be useful to (re)consider possible strategies to overcome MRP1-mediated MDR. Furthermore, it may be the basis for developing new, even better, highly potent, less toxic, and selective (as well as broad-spectrum) MRP1 inhibitors that will enter clinical evaluations in different malignancies and finally conduce to overcome MDR in general.     

联合抑制XIAP和MRP逆转HL-60/ADR细胞耐药

本研究探讨HL-60／ADR细胞的耐药机制，比较单独应用MRP、XIAP反义寡核苷酸或二者联合应用对HL-60／ADR细胞耐药的逆转作用。应用RT-PCR和Western blot分别检测并比较HL-60细胞和HL-60／ADR细胞的BCL-2、XIAP、MDR1、MRP在mRNA和蛋白水平表达的差异。将XIAP和MRP全硫代反义寡核苷酸，以脂质体-反义寡核苷酸复合物的形式单独或联合转染HL-60／ADR细胞，并应用CCK-8实验测定反义寡核苷酸对DNR敏感性影响；用RT-PCR和Western blot检测反义寡核苷酸对XIAP、MRP的mRNA和蛋白表达的影响。结果表明：HL-60／ADR细胞的MRP和XIAP的表达均明显高于HL-60细胞。XIAP和MRP反义寡核苷酸单独应用均能下调XIAP和MRP的表达，增加对柔红霉素的敏感性。二者联合应用与XIAP反义寡核苷酸单独应用比较，并不能增强对XIAP表达的抑制作用（P〉0．05），但使HL-60／ADR细胞对柔红霉素的敏感性明显增加（P〈0．05）；二者联合应用与MRP反义寡核苷酸单独应用比较，并不能提高HL-60／ADR细胞内DNR浓度及增强对MRP表达的抑制作用，却使HL-60／ADR细胞对柔红霉素的敏感性明显增加（P〈0．05）。结论XIAP和MRP都可能参与了HL-60／ADR细胞的耐药机制，MRP、XIAP反义寡核苷酸的联合应用能更大程度逆转HL-60／ADR细胞的耐药性。     

新疆地区维吾尔族和汉族人群ABCG2基因rs2231142位点多态性与高尿酸血症的相关性

目的探讨新疆地区维吾尔族和汉族人群ABC转运蛋白2(ABCG2)基因位点rs2231142的单核苷酸多态性(SNP)与高尿酸血症的关系。方法选取2013年2~12月体检的维吾尔族和汉族人群作为研究对象,并依据尿酸水平以及纳入和排除标准分为高尿酸血症组和健康对照组;采集血液样本,提取全血基因组DNA,PCR扩增各组ABCG2基因rs2231142位点,并对扩增产物用多重高温连接酶检测反应(i MLDR)进行SNP检测。结果总人群中汉族尿酸水平高于维吾尔族(t=10.595,P0.05);不同基因型(χ2=46.007,P0.05)及等位基因(χ2=17.924,P0.05)在两民族间的分布差异有统计学意义;维吾尔族和汉族高尿酸血症组尿酸水平均高于健康对照组(χ2维=7.796,P0.05;χ2汉=9.963,P0.05),两民族高尿酸血症组TT+GT基因型(χ2维=12.02,P0.05;χ2汉=12.28,P0.05)以及T等位基因(χ2维=8.625,P0.05;χ2汉=12.05,P0.05)频率均显著高于健康对照组;Logistic回归分析发现GT+TT基因型分别使得两民族患高尿酸血症的危险性上升了2.45和1.103倍(维吾尔族OR=2.45,95%CI=1.187~5.054,P0.05,汉族OR=1.103,95%CI=1.00~1.026,P0.05)。结论 ABCG2基因rs2231142位点SNP与维吾尔族和汉族高尿酸血症有一定关联,T等位基因可能是高尿酸血症的危险因素,G等位基因则可能是保护因素。     

bcl-2小干扰RNA对HL-60/VCR细胞耐药性逆转的研究

为了探讨以bcl-2基因作为靶分子进行白血病多药耐药基因治疗的可行性,构建bcl-2基因的小干扰RNA载体并将其转导入HL-60/VCR细胞,在G418筛选后应用Western blot进行鉴定;MTT法检测细胞转染前后生长速度和药物敏感性的变化;Western blot检测细胞转染前后凋亡相关蛋白Bax和耐药相关蛋白ZNRD1的表达变化.结果表明：成功构建了bcl-2的小干扰RNA载体并转染HL-60/VCR;经蛋白质水平检测证实成功建立了bcl-2低表达的白血病细胞模型;转染后的细胞对长春新碱和阿霉素的敏感性增强,且低表达耐药相关蛋白ZNRD1.结论：bcl-2小干扰RNA真核表达载体能在一定程度上逆转白血病细胞的耐药性.     

胶质瘤与恶性脑膜瘤患者肿瘤组织ABCG2、IGFBP-2表达及预后的关系分析

目的 探讨胶质瘤与恶性脑膜瘤患者肿瘤组织三磷酸腺苷结合转运蛋白G超家族成员2(ABCG2)、胰岛素样生长因子结合蛋白-2(IGFBP-2)表达及预后的关系.方法 选取资阳市第一人民医院确诊胶质瘤患者96例和恶性脑膜瘤患者38例,采用免疫组化法检测患者肿瘤组织中ABCG2、IGFBP-2阳性细胞的表达,计算其阳性细胞百分...     

CD24、CD133、P75NTR、ABCG2食管鳞癌中表达及与临床病理因素和淋巴结转移的关系

目的控讨CD24、CD133、P75NTR、ABCG2在食管鳞癌（ESCC）中表达及与临床病理因素和淋巴结转移（LNM）的关系。方法应用免疫组化SP法检测40例食管鳞癌（LNM阳性组与阴性组各20例）和20例正常食管标本中CD24、CD133、P75NTR、ABCG2蛋白表达,并对两组病例的检测结果及相关临床病理资料作对比研究。结果CD24、CD133、P75NTR、ABCG2蛋白食管鳞癌表达分别为72．5％、62．5％、60．0％、52．5％,与正常食管表达差异均有统计学意义（P〈0．05）;且与侵袭深度和LNM组与非转移组表达差异均有统计学意义（P〈0．05）;CD24、CD133表达呈正相关（P〈0．05）;这两组在癌侵袭深度（侵袭肌层与穿透肌层的癌）及LNM率分别是41．7％和62．5％有明显差异（P=0．002）。结论CD24、CD133、P75NTR、ABCG2蛋白食管鳞癌高表达,可能与癌侵袭深度和LNM相关,联合检测可能作为预测ESCC LNM和预后的参考指标。     

负载抗原DC与CIK共培养逆转肝癌细胞多药耐药性研究

目的研究负载抗原的树突状细胞(DC)联合细胞因子诱导的杀伤细胞(CIK)对肝癌细胞株HepG2/ADM多药耐药性的逆转作用。方法 HepG2/ADM细胞冻融抗原冲击DC,联合CIK细胞与HepG2/ADM细胞共培养,以未负载抗原的DCCIK作为阴性对照,以未经共培养处理的HepG2/ADM细胞作为空白对照。采用流式细胞仪在细胞处理后不同时间点检测HepG2/ADM细胞内罗丹明-123(R-123)浓度和阿霉素(ADM)浓度,采用Western blot检测细胞内P-糖蛋白(P-gp)水平。结果与DC-CIK共培养相比,负载抗原的DC-CIK共培养能够明显抑制HepG2/ADM细胞内R-123的外排,提高细胞内ADM浓度,同时降低细胞内P-gp水平(P0.05)。结论经抗原冲击的DC和CIK共培养可明显抑制HepG2/ADM细胞内R-123的外排,提高细胞内ADM浓度,抑制P-gp表达,从而有效逆转肝癌细胞多药耐药性。     

葡萄糖神经酰胺合成酶抑制剂PDMP对K562/A02细胞柔红霉素耐药逆转的研究

本研究旨在探讨葡萄糖神经酰胺合成酶(glucosylceramide synthase,GCS)抑制剂D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol(PDMP)hydrochloride对K562/A02细胞株柔红霉素(daunorubicin,DNR)耐药的逆转作用及其逆转机制。采用四甲基偶氮唑蓝(MTT)法检测不同浓度PDMP对K562细胞和K562/A02细胞的增殖抑制效应及其对K562/A02细胞DNR耐药逆转作用;用流式细胞术检测PDMP对K562/A02细胞内DNR浓度及细胞凋亡率的影响;半定量RT-PCR和Western blot方法检测K562及K562/A02细胞mdr1、GCS基因的表达以及PDMP对K562/A02细胞两种基因表达的影响。结果表明:DNR对K562和K562/A02细胞的IC50分别为0.23±0.02和7.15±0.24μg/ml,当PDMP≤20μmol/L时,对K562及K562/A02细胞株无明显生长抑制作用;20μmol/L和10μmol/L PDMP均能增加K562/A02细胞对DNR的敏感性,其耐药逆转倍数分别为2.59和1.69;两种浓度PDMP作用于耐药株48小时后能增加细胞内DNR浓度(p0.05)和细胞凋亡率(p0.01)。20μmol/L PDMP处理K562/A02细胞48小时后可在mRNA和蛋白水平下调GCS和mdr1基因的表达(p0.01)。结论:PDMP可以增强K562/A02细胞对DNR的敏感性,其机制可能与增加细胞内药物浓度及细胞凋亡率、下调GCS和mdr1基因表达有关。     

Association of genotypes and haplotypes of multi-drug transporter genes ABCB1 and ABCG2 with clinical response to imatinib mesylate in chronic myeloid leukemia patients

The introduction and success of imatinib mesylate (IM) has become a paradigm shift in chronic myeloid leukemia (CML) treatment. However, the high efficacy of IM has been hampered by the issue of clinical resistance that might due to pharmacogenetic variability. In the current study, the contribution of three common single nucleotide polymorphisms (SNPs) of ABCB1 (T1236C, G2677T/A and C3435T) and two SNPs of ABCG2 (G34A and C421A) genes in mediating resistance and/or good response among 215 CML patients on IM therapy were investigated. Among these patients, the frequency distribution of ABCG2 421 CC, CA and AA genotypes were significantly different between IM good response and resistant groups (P = 0.01). Resistance was significantly associated with patients who had homozygous ABCB1 1236 CC genotype with OR 2.79 (95%CI: 1.217–6.374, P = 0.01). For ABCB1 G2677T/A polymorphism, a better complete cytogenetic remission was observed for patients with variant TT/AT/AA genotype, compared to other genotype groups (OR = 0.48, 95%CI: 0.239–0.957, P = 0.03). Haplotype analysis revealed that ABCB1 haplotypes (C₁₂₃₆G₂₆₇₇C₃₄₃₅) was statistically linked to higher risk to IM resistance (25.8% vs. 17.4%, P = 0.04), while ABCG2 diplotype A₃₄A₄₂₁ was significantly correlated with IM good response (9.1% vs. 3.9%, P = 0.03). In addition, genotypic variant in ABCG2 421C>A was associated with a major molecular response (MMR) (OR = 2.20, 95%CI: 1.273–3.811, P = 0.004), whereas ABCB1 2677G>T/A variant was associated with a significantly lower molecular response (OR = 0.49, 95%CI: 0.248–0.974, P = 0.04). However, there was no significant correlation of these SNPs with IM intolerance and IM induced hepatotoxicity. Our results suggest the usefulness of genotyping of these single nucleotide polymorphisms in predicting IM response among CML patients.     

COX-2在人肝细胞癌中的表达及其对耐药性的影响

目的探讨环氧合酶-2(COX-2)在肝细胞癌发生、发展中的作用以及COX-2对P-糖蛋白(P-gp)表达的影响。方法选择手术切除的52例肝癌标本为研究对象,应用RT-PCR、免疫组织化学分别检测COX-2 mRNA和COX-2蛋白在肝癌组织和正常肝组织中的表达。同时检测mdr 1mRNA和P-gp蛋白在肝癌组织中的表达情况,并分析COX-2与P-gp在肝癌组织中表达的相关性。结果正常肝组织中未见COX-2表达,中、低分化的肝癌组织表达阳性率高于高分化肝癌组织(P<0.01),在HBsAg阳性的肝癌组织中表达高于HBsAg阴性的肝癌组织(P<0.05),在合并肝硬化的肝癌组织中表达高于无肝硬化的肝癌组织(P<0.01)。mdr 1 mRNA在肝癌组织和正常肝组织中均有表达。同时表达COX-2和mdr1基因为42例,两者相关系数r=0.563(P<0.01)。结论 COX-2参与了以P-gp介导的肝癌的多药耐药机制。     

Platelet hyperprocoagulant activity in Type 2 diabetes mellitus: attenuation by glycoprotein IIb/IIIa inhibition

Summary. Background: Platelets are hyperactive in Type 2 diabetes mellitus (T2DM), and antiplatelet treatment with glycoprotein (GP) IIb/IIIa inhibitors provides better thrombotic protection in DM than in non‐diabetic subjects. Objective: We hypothesized that diabetic platelets are hyperprocoagulant, and that this hyperactivity can be inhibited by GPIIb/IIIa blockade. Methods: Patients with T2DM and gender/age/body mass index‐matched non‐diabetic controls were recruited (n = 12 for both) to study the effect of GPIIb/IIIa blockade on platelet procoagulant activity. Platelet phosphotidylserine (PS), factor (F) Va expression, and platelet‐derived microparticle (PDMP) generation were measured by whole blood flow cytometry. Platelet‐dependent thrombin generation and plasma clotting time were monitored in recalcified platelet‐rich plasma. Results: Compared to controls, basal platelet activation was similar, while thrombin receptor activating peptide stimulated activation was enhanced in patients with T2DM. Diabetic platelets also displayed more profound elevations of platelet PS exposure, FVa binding, and PDMP generation upon stimulation. These alterations resulted in a hyperprocoagulant state, as evidenced by a marked increase in the platelet procoagulant index, enhanced thrombin generation, and a shortened plasma clotting time. GPIIb/IIIa blockade by c7E3 or SR121566 decreased platelet PS exposure and FVa binding, and diminished platelet procoagulant activity in patients with T2DM. Conclusions: Platelets have increased procoagulant activity in patients with T2DM. The hyperprocoagulant activity is counteracted by GPIIb/IIIa blockade.     

小檗胺逆转K562/A02细胞耐药性及其机制

为了探讨钙调素拮抗剂小檗胺 (berbamine,BBM )逆转多药耐药的可能性及其机制 ,采用人红白血病细胞K5 6 2及其阿霉素 (ADR)诱导的耐药细胞K5 6 2 /A0 2与不同浓度ADR和BBM共培养后 ,经MTT比色法计算半数抑制浓度 (IC50 )、耐药倍数和增敏倍数 ;用流式细胞术检测细胞内ADR相对含量和P gp表达水平 ;用半定量RT PCR检测mdr1mRNA和抗凋亡基因survivinmRNA表达。结果显示 ,ADR对K5 6 2和K5 6 2 /A0 2的IC50 分别为1 16± 0 .0 9μmol/L和 37.4 7± 1.76 μmol/L ,K5 6 2 /A0 2对ADR的耐药倍数是K5 6 2的 32 .30倍。 5 μmol/L ,10μmol/L和 2 0 μmol/LBBM增加K5 6 2 /A0 2细胞对ADR的敏感性 ,并呈剂量依赖性 ,增敏倍数分别达 2 .0 1,9.6 8和4 1.18倍 (P值均 <0 .0 1)。5 μmol/L或 10 μmol/LBBM作用 2小时 ,使K5 6 2 /A0 2细胞内ADR相对含量增加到1 4 1和 1 5 2倍 (P <0 .0 1) ;作用 72小时使K5 6 2 /A0 2细胞P gp表达分别下降 4 .12 % ( P <0 .0 5 )和 2 7.0 9% (P<0 .0 1) ,且下调mdr1mRNA和survivinmRNA表达。结论 :BBM提高耐药细胞K5 6 2 /A0 2胞内的ADR浓度 ,下调mdr1mRNA、P gp及survivinmRNA的表达 ,使其对ADR的敏感性显著增高 ,从而逆转耐药性。     

华蟾素对Raji/ADR细胞多药耐药的逆转作用及机制

本研究旨在探讨华蟾素对Raji/ADR细胞多药耐药的逆转作用及其机制.采用MTT法检测华蟾素对Raji及Raji/ADR细胞的生长抑制率、半数抑制浓度（IC50）、逆转阿霉素耐药倍数,并绘制生长抑制率曲线;应用RTPCR法测定MDR-1和MRP-1基因的转录;Western blot法和流式细胞术法测定P-gp、MRP-1蛋白的表达.结果表明：华蟾素对Raji及Raji/ADR细胞72 h增殖抑制率为75.6％和69.3％,IC50分别为3.9 mmol/L和4.6 mmol/L,逆转阿霉素耐药倍数为255.7倍.华蟾素作用于Raji/ADR前后,MDR-1和MRP-1基因转录水平明显下降（P＜0.05）,P-gp和MRP-1蛋白表达明显降低.结论华蟾素可逆转Raji/ADR细胞对阿霉素的耐药,其机制为通过转录途径下调P-gp和MRP-1蛋白的表达.华蟾素是一种有效的肿瘤多药耐药逆转剂.     

Selective COX2 inhibition improves whole body and muscular insulin resistance in fructose‐fed rats

Background The effects of cyclooxygenase‐1 (COX1) and cyclooxygenase‐2 (COX2) inhibition on insulin resistance in subjects with the metabolic syndrome remain elusive. Aims of this study were to examine the effects of COX1 and COX2 inhibitors on whole body and muscular insulin resistance in fructose‐fed rats, an animal model of the metabolic syndrome. Materials and methods The rats on regular or 60% fructose‐enriched diets for 6 weeks were further divided into rats combined with or without piroxicam (a selective COX1 inhibitor) or celecoxib (a selective COX2 inhibitor) treatment for an additional 2 weeks. Euglycaemic hyperinsulinaemic clamp (EHC) with a tracer dilution method was performed at the end of the study. Results The present result showed that fructose‐induced increases in systolic blood pressure and fasting plasma insulin levels were significantly suppressed in rats treated with celecoxib but not piroxicam. In the EHC period, celecoxib significantly reversed fructose‐induced decreases in whole body glucose uptake, mainly by glucose storage. Hepatic glucose production and whole body glycolysis were not significantly changed among groups. Celecoxib but not piroxicam significantly reversed fructose‐induced decreases in glycogen synthase activities in red and white quadriceps muscles and insulin‐stimulated membrane GLUT4 recruitment in soleus muscles. Celecoxib and piroxicam both significantly diminished fructose‐induced increases in plasma thromboxane B2 and 6‐keto prostaglandin (PG) F1α; but only celecoxib treatment significantly attenuated a fructose‐induced increase in 8‐isoprostane levels. Plasma PGE metabolites were not different among groups. Conclusions This study demonstrates that a therapeutic dose of celecoxib, but not piroxicam, could significantly attenuate fructose‐induced whole body and muscular insulin resistance in rats.