姜黄素对HL60/ADR细胞多药耐药逆转作用的研究

目的:研究姜黄素对白血病耐药细胞HL60/ADR多药耐药的逆转作用.方法:用不同浓度姜黄素联合阿霉素处理HL60/ADR细胞,MTT法检测药物对细胞的生长抑制作用;流式细胞仪检测细胞内阿霉素的浓度及bcl-2表达水平.结果:HL60/ADR细胞经0.4,0.8μg/rml姜黄素作用24 h后,阿霉素的IC50分别为3.79、2.58 μg/ml,耐药逆转倍数为1.37、2.02,细胞内ADR浓度增加,bcl-2蛋白的表达下降.结论:姜黄素能部分逆转HL60/ADR细胞的多药耐药.     

硒化壳聚糖逆转K562/ADM细胞耐药与PI-3K/Akt信号通路的关系

目的研究硒化壳聚糖对体外培养多药耐药白血病细胞株K562/ADM的耐药逆转作用,探讨其与PI-3K/Akt信号通路的关系。方法 K562/ADM细胞培养于含0.6μg/ml阿霉素的培养液中维持其耐药性,应用MTT法检测硒化壳聚糖对K562/ADM细胞增殖的作用,计算逆转倍数;应用流式细胞法检测细胞凋亡;应用免疫印迹法检测磷酸化Akt(p-Akt)和P糖蛋白(P-gp)表达的改变。结果 K562/ADM细胞对阿霉素(ADM)耐药,硒化壳聚糖可有效抑制K562/ADM细胞增殖,呈一定的剂量和时间效应关系(P0.05,P0.01);硒化壳聚糖能够对K562/ADM细胞耐ADM产生一定的逆转作用,明显增强ADM对K562/ADM细胞的诱导凋亡作用(P0.05,P0.01),下调p-Akt和P-gp水平(P0.01)。结论硒化壳聚糖对耐药白血病细胞株K562/ADM可产生增殖抑制和多药耐药逆转作用,其部分逆转机制可能是通过诱导细胞凋亡,抑制细胞P-gp表达,阻断细胞PI-3K/Akt信号通路而实现。     

川芎嗪逆转骨肉瘤(MG-63/ADM)细胞多药耐药的研究

目的研究川芎嗪与阿霉素合用对人骨肉瘤(MG-63/ADM)耐药细胞的影响。方法用流式细胞仪检测经过川芎嗪处理的(MG-63/ADM)耐药细胞和未经川芎嗪处理的(MG-63/ADM)耐药细胞P170蛋白的表达。结果人骨肉瘤(MG-63/ADM)耐药细胞对阿霉素(ADM)耐药,耐药倍数是11.62,这种耐药细胞的P170有高表达。单用TMP处理和未用TMP处理的MG-63/ADM细胞之间的P170表达率差异有统计学意义(P<0.01)。结论川芎嗪可以部分逆转骨肉瘤(MG-63/ADM)耐药细胞的对阿霉素的耐药性。     

5b对人乳腺癌耐药细胞株MCF-7ADR的耐药逆转作用

目的研究板蓝根活性单体-5b逆转乳腺癌耐药株MCF-7/ADR细胞的作用及逆转机理。方法选取人乳腺癌MCF-7细胞及耐药株MCF-7/ADR细胞作为主要研究细胞,在2种细胞中加入活性单体-5b,采用MTT法观察活性单体-5b对多药耐药(MDR)逆转作用;采用FCM法观察阿霉素对2种细胞的抑制浓度及倍数。结果当活性单体-5b的剂量低于150μmol/L时,其对2种细胞无毒性作用;活性单体-5b能提高MCF-7/ADR细胞的凋亡率;活性单体-5b能增加阿霉素在体内的积累。结论活性单体-5b对MCF-7/ADR细胞的MDR具有一定的逆转作用,是一种有效的乳腺癌化疗增敏剂。     

防己诺林碱联用紫杉醇对耐药卵巢癌SKOV3/ADM细胞作用机制的初步研究

目的评价防己诺林碱调控紫杉醇对耐药卵巢癌SKOV 3/ADM细胞多药耐药的作用。方法采用MTT法和细胞摄取研究防己诺林碱调控紫杉醇对SKOV 3/ADM细胞多药耐药的作用。结果细胞生长抑制试验表明,紫杉醇对SKOV 3/ADM细胞耐药明显。0.5、1和5μg·mL~(-1)浓度的防己诺林碱分别与紫杉醇联用后,紫杉醇对细胞的半数抑制浓度(IC_(50))降为(0.542±0.117)、(0.924±0.153)和(1.931±0.375)μg·mL~(-1),耐药逆转倍数If分别为16.1、9.4和4.5倍,与单用紫杉醇相比差异有统计学意义(P0.05)。细胞摄取试验研究表明,联用防己诺林碱浓度达到0.5μg·mL~(-1)及以上时,能够显著增加紫杉醇的细胞摄取(P0.05)。结论防己诺林碱是潜在的逆转紫杉醇对SKOV 3/ADM细胞多药耐药候选活性化合物。     

中药MBC逆转K562/ADM细胞的体外研究

目的　研究中药MBC提取物在体外对K5 62 /ADM逆转作用及机制。方法　应用MTT法测定应用MBC前后ADM对K5 62 /ADM耐药细胞株的IC50 ,计算其逆转倍数。应用流式细胞仪技术测定应用MBC前后细胞内Rh12 3浓度。结果　 8× 10 -1 μg/mlMBC能增加ADM对K5 62 /ADM的细胞毒作用 ,逆转倍数 6 2 8倍 ,应用MBC后K5 62 /ADM细胞内Rh12 3浓度增加。结论　MBC可提高ADM对耐药细胞K5 62 /ADM的杀伤作用 ,部分逆转了K5 62 /ADM细胞的耐药     

黄体酮对人舌癌耐药细胞系Tca8113/BLM的逆转作用研究

目的　研究黄体酮 (ProgresteroneProg)对人舌癌耐药细胞Tca8113 /BLM的逆转作用和机制。方法　以人舌癌耐药细胞Tca8113 /BLM为研究对象 ,以逆转剂维拉帕米 (VerapamiVera)为对照 ,采用MTT法检测Prog对细胞Tca8113 /BLM逆转 ;流式细胞仪检测Prog对细胞内阿霉素 (ADM)浓度聚积、细胞膜上P -gp的表达 ,以及对细胞周期的影响。结果　Tca8113、Tca8113 /BLM细胞对BLM的IC5 0分别为 10 1和 12 0 1;加用 2umol/LProg作用细胞后Tca8113、Tca8113 /BLM细胞对BLM的IC5 0分别为 9 5 6和 12 1,耐药细胞对BLM的增敏倍数为 9 92 ;ADM浓度在Tca8113 /BLM细胞内明显增加 (P <0 0 1) ,而亲本细胞Tca8113没有明显改变 ;细胞膜上的P -gp的阳性表达率也显著下降 ( P <0 0 1) ;细胞周期发生改变 ,G1细胞减少 ,G2期和S期细胞增多。结论　Prog能显著提高耐药细胞内药物的浓度 ,可逆转人舌癌耐药细胞Tca8113 /BLM对BLM的耐药作用 ,其逆转机制可能与P -gp的结合有关     

苯妥英钠和维拉帕米对胶质瘤多药耐药影响的研究

目的 研究不同浓度苯妥英钠及维拉帕米对化疗耐药胶质母细胞瘤细胞系( 8-MG-BA)化疗耐药逆转的影响.方法 用不同浓度苯妥英钠对8-MG-BA及H4细胞系处理后,以四甲基偶氮唑蓝(MTT)法检测其对上述两种细胞系化疗耐药逆转的影响,并与维拉帕米处理进行对照.结果 用苯妥英钠5μg/ml、10 μ g/ml和维拉帕米5μg/ml处理后,8-MG-BA细胞对卡莫司汀的逆转倍数分别为25.34、34.97和14.27,对替尼泊苷的逆转倍数分别为13.44、21.71和11.10.结论 苯妥英钠逆转作用大于维拉帕米,且呈现剂量依赖关系.     

姜黄素通过Notch途径抑制人宫颈癌细胞株HeLa细胞增殖

目的:探讨姜黄素对人宫颈癌细胞株HeLa细胞体外增殖抑制及凋亡诱导作用及相关机制。方法:MTT法检测不同浓度姜黄素对HeLa细胞的增殖抑制作用;RT-PCR法检测HeLa细胞Notch1、Notch2、Jagged1、Bcl-2、Bax基因表达情况。结果:姜黄素对HeLa细胞具有增殖抑制作用,且呈时间-剂量依赖性。随着姜黄素剂量的增加,Notch1、Notch2、Bcl-2mRNA的表达逐渐下降,Bax mRNA的表达逐渐升高,Bcl-2/Bax亦逐渐减小,Jagged1mRNA无明显变化。结论:姜黄素可能通过Notch信号途径改变凋亡蛋白的表达,抑制HeLa细胞增殖。     

白藜芦醇增加乳腺癌细胞对阿霉素化疗敏感作用研究

目的研究白藜芦醇对乳腺癌MDA-MB-231阿霉素(adriamycin,ADR)耐药细胞株化疗敏感性的影响。方法建立阿霉素耐药细胞株MDA-MB-231/ADR。MTT法检测白藜芦醇作用对MDA-MB-231、MDA-MB-231/ADR细胞阿霉素化疗敏感性的影响,统计半数细胞死亡药物浓度(IC50值)。流式细胞术检测白藜芦醇对阿霉素诱导MDA-MB-231/ADR细胞凋亡的影响,Western blot法检测Bcl-2、Bax、cleaved-caspase3、肝细胞生长因子受体CMet、P-糖蛋白(P-glycoportein,P-gp)的表达。结果成功建立阿霉素耐药株MDA-MB-231/ADR,其IC50值为8.79±0.96μg/ml,较MDA-MB-231细胞IC50值(0.24±0.03μg/ml)有显著差异,P0.01。5μM、15μM白藜芦醇对MDA-MB-231细胞阿霉素敏感性没有影响,但显著增强MDA-MB-231/ADR细胞阿霉素敏感性,15μM组IC50值下降为3.46±0.58μg/ml,有显著性差异,P0.01。5μM、15μM白藜芦醇均增加阿霉素诱导MDA-MB-231/ADR的细胞凋亡;15μM白藜芦醇处理组,Bcl-2表达显著降低,Bax、cleaved-caspase3表达显著升高。5μM、15μM白藜芦醇处理显著降低耐药基因C-Met、P-gp的表达。结论白藜芦醇增加阿霉素耐药株MDA-MB-231/ADR对阿霉素的敏感性,可能的作用机制为降低耐药基因C-Met、P-gp的表达。     

Glucosamine Reverses P-Glycoprotein-Mediated Multidrug Resistance in the Daunorubicin-Resistant Human Gastric Cancer Cells

Abstract

Glucosamine (GlcN) is a natural amino monosaccharide in the human body, and evidence of its anticancer effects is growing. In this study, we aimed to evaluate the effects of GlcN for its cytotoxicity, MDR reversal effects and inhibitory effects on function and expression of P-glycoprotein (P-gp) transporter in the daunorubicin-resistant human gastric cancer cells. Cell viability was measured by MTT assay to evaluate the cytotoxicity and multidrug resistance (MDR) reversal effects of GlcN. The effects of GlcN on function and mRNA expression level of P-gp transporter were assessed by flow cytometry and real-time RT-qPCR, respectively. Our results indicated that GlcN reduced the proliferation of human gastric cancer cell line EPG85-257 and its drug-resistant variant EPG85-257RD in a dose-dependent manner. GlcN (at the concentrations of 0.5 and 1?mM) also enhanced the sensitivity of EPG85-257RDB cells to daunorubicin. The cellular accumulation studies showed that GlcN inhibited efflux activity of P-gp and enhanced the mean fluorescent intensity of Rho123 while ^˙it had no effects on P-gp gene expression in these cells. This study suggested that the inhibition of P-gp activity is a novel mechanism of action by which GlcN could reverse MDR in EPG85-257RDB cells.     

The synthesized novel fluorinated compound (LJJ-10) induces death receptor- and mitochondria-dependent apoptotic cell death in the human osteogenic sarcoma U-2 OS cells

We designed the 6-fluoro-2-(3-fluorophenyl)-4-substituted anilinoquinazoline derivatives as less toxic anti-cancer candidates. Our result demonstrated that LJJ-10 has greater cytotoxicity than that of the other compounds in human osteogenic sarcoma U-2 OS cells. LJJ-10-induced apoptosis was associated with enhancing ROS generation, DNA damage, and an increase of the protein levels of Fas, FasL, FADD, caspase-8, cytochrome c, Apaf-1, AIF, Endo G, caspase-9 and caspase-3 in U-2 OS cells. LJJ-10-triggered growth inhibition was significantly attenuated by N-acetylcysteine, cyclosporine A, anti-FasL monoclonal antibody, and caspase-8, -9 and -3 specific inhibitors in U-2 OS cells. We suggest that LJJ-10-induced apoptotic cell death in U-2 OS cells through death receptor- and mitochondria-dependent apoptotic signaling pathways.     

The role of turmerones on curcumin transportation and P-glycoprotein activities in intestinal Caco-2 cells

The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases.     

Evaluation of Hydro-Alcoholic Extract of Eclipta alba for its Multidrug Resistance Reversal Potential: An In Vitro Study

The development of multidrug resistance (MDR) causes problems in the chemotherapy of human cancer. The present study was designed to evaluate and establish the role of Eclipta alba as MDR reversal agent using multidrug resistant hepatocellular carcinoma cell line (DR-HepG2). To develop DR-HepG2, hepatocellular carcinoma cell line (HepG2) was transfected with 2-Acetylaminofluorene (AAF) and Aflatoxin B1 (AFB). Cytotoxic effects of the Eclipta alba hydroalcoholic extract (EAE) and standard anti-ancer drug Doxorubicin (DOX) were determined in DR-HepG2 and the parental cells HepG2 using MTT assay. The expression level of MDR1 gene and P-glycoprotein (P-gp) level was analyzed by RT-PCR and western blotting. From the present investigation, it was found that EAE (10 and 20 μg/ml) could significantly inhibit cell proliferation in DR-HepG2 whereas DOX (0.5 μg/ml) could not because of enhancement effect of MDR1/P-gp. This study demonstrated for the first time the antiproliferative activities of EAE in multidrug resistant DR-HepG2 cells. The findings revealed that Eclipta alba components are effective inhibitors of MDR1/P-gp.     

PI3K/Akt信号通路对胃癌细胞SGC7901细胞化疗效果的影响

目的运用磷脂酰肌醇3-激酶抑制剂(phosphatidylinositol 3-kinases,PI3-K)LY294002[2-(4-吗啉基)-8-苯基-4氢-1-苯并吡喃-4-酮]作用于胃癌细胞系SGC7901,探讨抑制PI3K/Akt信号转导通路对胃癌细胞化疗敏感性的影响。方法采用MTT比色法,流式细胞术检测5-FU、DDP及ADM单独或联合PI3K抑制剂LY294002对人胃癌细胞SGC7901的抑制率、凋亡率。并分析单独及联合应用LY294002对SGC7901细胞周期的影响。Western-blot检测单独及联合化疗药后P-Akt蛋白在SGC7901细胞中的表达水平。结果单独使用化疗药5-FU、DDP及ADM均可抑制SGC7901细胞增殖、诱导其凋亡。当化疗药与抑制剂联合应用,对细胞的抑制作用明显增强,促凋亡作用增强,与对照组比较(P0.05)。细胞周期同步分析显示,单独用药均可将SGC7901细胞阻滞于G0/G1期。联合使用抑制剂使处于G0/G1期细胞增加。Western blot显示化疗药上调P-Akt蛋白的表达,联合使用抑制剂后SGC7901细胞P-Akt蛋白的表达与未使用抑制剂比较减弱,差异有统计学意义(P0.05)。结论阻断PI3K/Akt信号通路可提高化疗药5-FU、DDP及ADM对胃癌细胞株SGC7901的抑制率,凋亡率并使阻滞于G0/G1期细胞增多;LY294002通过阻断PI3K/Akt信号通路,抑制P-Akt蛋白表达,增强化疗药的敏感性;LY294002阻断PI3K/Akt信号通路对5-FU、DDP、ADM治疗胃癌有一定的协同或增强作用。     

甲基莲心碱对难治/复发急性白血病细胞增殖及P-糖蛋白表达的影响

目的以急性难治/复发白血病患者为研究对象，探讨甲基莲心碱（nefefine）对耐药白血病细胞增殖及P-糖蛋白（P-gp）表达的影响，为临床应用提供实验依据。方法采用MTT法及免疫细胞化学SABC法观察经nefefine处理后耐药白血病细胞的增殖及P-gp表达的改变。结果10μg/ml nefefine干预白血病细胞48h，nefefine＋ADM组对白血病细胞的抑制率比ADM组明显升高（P＜0．01）；与ADM组比较，nefefine＋ADM组和异博定＋ADM组的P-gp阳性表达率及P-gp平均光密度显著下降（P＜0．01）。Nefefine＋ADM组和异博定＋ADM组比较，P-gp的阳性表达率和平均光密度无显著差异（P＞0．05）。结论Neferine能抑制耐药白血病细胞增殖。Nefefine通过降低耐药白血病细胞P-gp的表达逆转MDR。     

铜对人类肠道上皮Caco-2细胞的毒性研究

目的　了解铜对人类肠道上皮Caco 2细胞的毒性影响。方法　应用噻唑蓝 (MTT)实验、P 糖蛋白(P gp)活性检测实验、活性氧检测及克隆形成实验研究铜对肠道上皮Caco 2细胞的毒性及可能作用机制 ,同时利用Caco 2细胞和肠炎沙门氏菌为模型 ,研究铜对Caco 2细胞对细菌侵入和存活易感性的影响。结果　在一定的暴露场景下 ,铜可明显地降低细胞的活力 ,抑制细胞膜表面P gp的活性 ,促进细胞内活性氧自由基的产生 ,降低细胞的克隆形成能力。此外 ,细胞经铜暴露后 ,肠炎沙门氏菌侵入细胞的数量增加 ,但细胞内存活的细菌数量却下降。结论　过量的铜可导致Caco 2细胞氧化损伤 ,从而引起更广泛的细胞毒性效应 ,但其对细胞对细菌侵入和存活易感性的影响有待进一步研究。     

Macelignan: A New Modulator of P-Glycoprotein in Multidrug-Resistant Cancer Cells

The effect of macelignan, a phytoestrogen, on P-gp function was investigated using multidrug resistant cancer cells overexpressing P-gp (NCI/ADR-RES) and the fluorescent P-gp substrates, daunorubicin and rhodamine 123. Macelignan (40 μM) increased the cellular accumulation of daunorubicin by approximately threefold in NCI/ADR-RES cells, whereas it did not alter the cellular accumulation of daunorubicin in MCF-7/sensitive cells. Similarly, the presence of macelignan also enhanced significantly (P < 0.05) the cellular accumulation of rhodamine 123 in a concentration-dependent manner in NCI/ADR-RES cells. Furthermore, cancer cells were more susceptible to the cytotoxicity of vinblastine, a P-gp substrate, in the presence of macelignan. Those results suggest that macelignan has inhibitory effects on P-gp mediated cellular efflux. However, P-gp activity did not affect the cellular accumulation of macelignan itself. Taken all together, macelignan was identified as a novel inhibitor of P-gp activity and may be a promising lead compound for the rational design of more efficacious drugs to reverse multidrug resistance in cancer.