槲皮素逆转白血病细胞株K562/ADM多药耐药的研究

目的探讨槲皮素逆转白血病细胞多药耐药在膜转运蛋白方面的机制.方法通过MTT体外药敏法证明槲皮素对柔红霉素的增敏作用并确定逆转的浓度范围,作用于K562/ADM耐药株及相应敏感株K562/S,运用逆转录多聚酶链式反应(RT-PCR)和流式细胞术检测多药耐药基因(Mdr1)及其膜蛋白产物P-170糖蛋白(P-gp)的表达情况,在激光共聚焦显微镜观察柔红霉素在亚细胞水平的分布变化.结果 20～40 μmol/L终浓度槲皮素在体外能明显提高柔红霉素对K562/ADM耐药株的敏感性,并能下调Mdr1基因及其膜蛋白产物P-gp的表达,恢复柔红霉素在亚细胞水平的异常分布,回归其作用靶点--细胞核,从而逆转多药耐药,且有效浓度范围的药物对细胞本身无毒性作用.结论槲皮素有可能成为蒽环类药物治疗白血病中有效且低毒的化疗增敏剂.     

槲皮素逆转白血病细胞株HL-60/ADM多药耐药的研究

目的探讨槲皮素(Que)逆转白血病细胞多药耐药在膜转运蛋白方面的机制。方法通过四唑蓝体外药敏法,检测Que对柔红霉素(DNR)的增敏作用,并以不同浓度作用于HL-60／ADM耐药株及相应敏感株HL-60;运用逆转录多聚酶链式反应和流式细胞术,检测HL-60／ADM和HL-60细胞株多药耐药相关基因1(MRP1)及其膜蛋白产物MRP1蛋白的表达情况;借助激光共聚焦显微镜,观察DNR在亚细胞水平的分布变化。结果20～40μmol／L终浓度的Que在体外能明显提高DNR对HL-60／ADM耐药株的敏感性,并能下凋MRP1基因及其膜蛋白产物MRP1的表达,使DNR回归于细胞核内,从而逆转多药耐药,而且对细胞本身无明显毒性作用。结论Que有可能成为蒽环类药物治疗白血病的有效且低毒的化疗增敏剂。     

人类白血病多药耐药细胞株K562/ADM、HL60/ADM的耐药性及耐药逆转的研究

目的：研究K562／ADM、HL60／ADM的耐药性及CsA对其耐药的逆转作用。方法：MTT比色法检测细胞耐药性及CsA对细胞耐药性的影响，流式细胞仪检测Pgp、MRP的表达及细胞内柔红霉素(DNR)的浓度。结果：K562／ADM、HL60／ADM对DNR、ADM、VCR、Har、VP16、MIT交叉耐药，对Acla和Ara—C基本不耐药。K562／ADM细胞Pgp过度表达，HL60／ADM细胞的MRP过度表达。CsA使K562／ADM、HL60／ADM细胞内药物浓度增加，逆转了K562／ADM、HL60／ADM的耐药性，而对K562、HL60的耐药性基本无影响。结论：两种不同耐药机制的细胞株对8种常用化疗药物的耐药谱相似，对Acla基本不产生耐药性，故在防止和克服多药耐药的基础上，Acla可能取代DNR、ADM。环孢菌素A(CsA)对两种不同耐药机制的细胞株的耐药性均有逆转作用，主要是通过增加胞内药物浓度来达到逆转作用。     

干扰素和环孢霉素A 协同逆转K562/ ADM细胞对阿霉素的耐药性

  目的 观察干扰素(α-Interleron，α-IFN)和环孢霉素A(Cyclosporine A,CsA)对白血病K562/ADM细胞耐药性的协同逆转效应。方法 以多药耐药基因／P-糖蛋白(Muhidrug resistance gene／P-glycoprotein，mdrl／P-gp)超表达的K562／ADM细胞为靶细胞，MTT比色法检测药物的细胞毒效应；流式细胞仪检测细胞P-糖蛋白(P-glycoprotein，P-gp)表达水平；激光共聚焦显微镜观察细胞内阿霉素含量变化。结果 K562／ADM细胞对阿霉素呈高度耐药性，并与柔红霉素和鬼臼乙叉甙交叉耐药，但与CsA无交叉耐药。CsA和α-IFN单独或联合应用均对K562／ADM细胞的耐药性有较强的抑制效应。流式细胞仪和激光共聚焦显微镜分析发现α-IFN和CsA单独或联合均不能下调细胞mdrl/P-gp的表达，反而应激性地刺激耐药细胞P-gp的合成增加，但可抑制P-gp的功能、增加K562／ADM细胞内阿霉素的积聚。结论 α-IFN和CsA联合可协同逆转耐药白血病细胞的耐药性，其作用机制为抑制P-gp的功能而非下调mdrl／P-gp的表达水平。     

siRNA逆转白血病K562/ADM细胞对抗癌药物耐受性的研究

目的:研究小干扰RNA(small interfering RNA, siRNA)对多药耐药细胞K562/ADM耐药性的逆转效应.方法:以K562/ADM细胞为靶细胞,针对mdrl基因mRNA不同靶点设计合成4对siRNA分子,脂质体介导转染K562/ADM细胞,RT-PCR和实时荧光定量PCR检测mdrl基因mRNA的表达水平,流式细胞术(FCM)测定P-糖蛋白(P-gp)表达;MTT比色法检测K562/ADM细胞对多柔比星(ADM)、柔红霉素(DNR)和依托泊苷(Vp-16)的敏感性.结果:选择mdrl mRNA的4个靶点设计合成的siRNA中,针对333靶点的siRNA(si-mdrl/333)对mdrl基因的表达具有较好的沉默效果,RT-PCR和实时定量PCR检测,mdrl mRNA表达分别降低69%和63%;FCM检测P-gp的表达强度(MFI)降低约50%.si-mdrl/333可提高K562/ADM细胞对ADM、DNR和Vp-16的敏感性,耐药逆转倍数分别为3.45、1.70和2.91倍.结论:siRNA能特异性地阻抑白血病多药耐药细胞K562/ADM细胞mdrl基因的表达,逆转其对抗癌药物的耐受性.     

CRKL活性与白血病细胞多药耐药的关系

 目的　探讨CRKL活性在白血病细胞多药耐药中的作用,发现与耐药相关的新因素。方法　应用流式细胞术比较CRKL在K562、HL-60及其耐药细胞中的活性变化;对敏感细胞用柔红霉素处理后,检测CRKL活性与化疗作用时间的关系以及撤药后CRKL活性的改变。结果　与K562、HL-60敏感细胞比较,在K562、HL-60耐药细胞株中,CRKL的磷酸化水平明显升高;在用柔红霉素处理72 h后,K562和HL-60细胞CRKL的磷酸化水平与化疗药物作用呈时间依赖性,未发现Jurkat细胞CRKL磷酸化水平的改变。结论　CRKL活性变化与白血病细胞多药耐药的形成有关,是白血病细胞多药耐药的又一个新的影响因素。     

二甲亚砜对K562/ADM耐药细胞株耐药性的逆转作用

目的：研究二甲亚砜对K562／ADM耐药细胞的逆转作用。方法：通过加入二甲亚砜后耐药细胞的生长情况，逆转倍数的计算，细胞内过氧化物染色的阳性程度以及电镜下观察耐药细胞诱导前后的变化。结果：二甲亚砜诱导K562／ADM耐药细胞前后生长情况有显著差异且逆转借故为4．0，Pox染色阳性程度明显递增，电镜下可观察诱导后K562／ADM耐药细胞有向成熟分化趋势。结论：二甲亚砜对逆转K562／ADM的耐药性是有益的。     

中药提取物逆转K562/ADM细胞的耐药作用及其分子机制的探讨

目的 观察两种中药提取物黄芩苷、京尼平苷对多药耐药(MDR)K562/ADM细胞的耐药逆转作用,探讨其分子机制.方法 建立阿霉素(ADM)诱导K562/ADM细胞耐药模型,分别将不同浓度黄芩苷、京尼平苷作用于K562/ADM细胞,用四甲基偶氮唑盐(MTT)法检测其对化疗药物的敏感性,用逆转录聚合酶链反应(RT-PCR)检测多药耐药基因-1(mdr-1)、拓扑异构酶Ⅱ(TopoⅡ)基因在mRNA水平的表达变化.结果 两种中药提取物黄芩苷、京尼平苷,可增加K562/ADM细胞对ADM的敏感性,K562/ADM细胞增殖受到明显抑制,耐药逆转倍数分别为1.95倍及1.46倍;RT-PCR检测mdr-1基因表达减弱,Topo Ⅱ基因表达增强(P<0.01).结论 黄芩苷、京尼平苷可通过降低K562/ADM细胞mdr-1基因表达和提高TopoⅡ的表达来逆转白血病细胞MDR.     

siRNA抑制K562/ADM细胞mdr1基因表达并逆转其耐药性

背景与目的：白血病耐药性是白血病治疗中的难点，RNAi技术具有特异、高效、毒性小的特点，可高效、特异地抑制特定基因的过度表达。本文研究小干扰RNA分子（siRNA）对白血病多药耐药K562／ADM细胞mdr1基因表达和耐药性的影响。方法：设计、筛选和合成针对mdr1基因的siRNAs（si—mdrl—1，si—mdr1—2），脂质体介导转染K562／ADM细胞；RT—PCR法检测mdr1 mRNA的转录；流式细胞术测定P-糖蛋白（P—gP）表达水平；MTT法检测K562／ADM细胞对多柔比星（阿霉素，ADM）的敏感性。结果：si—mdr1—1、si-mdr1-2转染24h和48h，si—mdr1—1的抑制率分别为55．5％和22．5％，而si—mdr1—2则分别为16．0％和57．6％。si—mdr1—1和si—mdr1—2作用72h时，P—gP的表达强度分别下降74％和85％。si—mdr1—1和si—mdr1—2均可提高K562／ADM细胞对多柔比星的敏感性、逆转其耐药性，逆转倍数分别为2．52倍和1．96倍。结论：siRNA可特异性地沉默mdr1基因的表达，逆转P—gP介导的白血病细胞耐药性。     

汉防己甲素逆转白血病细胞株K562/ADM多药耐药性机制研究

目的 研究汉防己甲素(TTD)对慢性粒细胞白血病急性变白血病细胞株K562／ADM多药耐药(MDR)逆转的机理。方法 采用流式细胞仪检测细胞内化疗药物的浓度及细胞表面P糖蛋白(P-gp)的表达；荧光定量PCR法检测MDR1 mRNA；通过流式细胞仪检测Anexin—V判断凋亡细胞的数量。结果 10μmol/L的TTD处理K562／ADM细胞后，细胞内阿霉素(ADM)的浓度明显提高；K562／ADM细胞MDR1 mRNA／P-gp的表达下降；TTD能增强ADM致细胞凋亡的作用。结论 汉防己甲素的耐药逆转机制除了下调MDR1 mRNA／P-gp的表达引起细胞内抗癌药物的积聚外，增加抗癌药物致细胞凋亡也是耐药逆转的重要原因。．     

MDR1基因下调逆转人白血病阿霉素耐药细胞株K562/ADM的耐药性

目的：探讨RNA干扰（RNAi）人MDR1基因对人白血病阿霉素耐药细胞株K562/ADM耐药性的影响。方法：应用针对人MDR1基因的RNAi质粒pENTRTM/U6-MDR1转染人白血病阿霉素耐药细胞株K562/ADM和亲本细胞株K562,48 h后实时荧光定量PCR检测MDR1 mRNA表达,流式细胞术检测P-gp蛋白表达和P-gp功能,MTT法检测细胞对ADM的耐药性。结果：与未转染细胞相比,K562/ADM耐药细胞pENTRTM/U6-MDR1组的MDR1 mRNA和P-gp蛋白表达和功能均显著下降（ P <0.05）,对阿霉素的耐药性显著降低（ P <0.05）。结论：MDR1基因下调可逆转人白血病阿霉素耐药细胞株对阿霉素的耐药性。     

miR-139过表达降低急性髓系白血病TRAIL耐受性

目的:探讨miR-139过表达对急性髓系白血病肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis inducing ligand,TRAIL)耐受性的影响。方法:采用浓度为100、200、300和400 ng/ml TRAIL处理人急性髓系白血病K562、HL-60细胞及相对应的多药耐药K562/A02和HL-60/ADM细胞,采用CCK8法计算IC50值,以IC50法评价急性髓系白血病细胞对TRAIL的敏感性。RT-PCR检测miR-139在 TRAIL敏感细胞和耐药细胞中的表达。采用脂质体2000将miR-139 mimics转染至TRAIL耐药细胞株,RT-PCR检测转染效果,CCK8法检测细胞活力及IC50值,流式细胞仪检测细胞凋亡率。结果:在K562、K562/A02、HL-60和HL-60/ADM细胞中,HL-60/ADM细胞对TRAIL最为敏感,而K562细胞对TRAIL的耐药性最强。与敏感细胞株HL-60/ADM相比,miR-139在耐药细胞株K562中表达显著下降。转染miR-139 mimics后,K562细胞中miR-139表达和细胞凋亡率明显升高,而细胞存活率及IC50值明显降低。结论:miR-139在TRAIL耐药细胞株K562中低表达,过表达miR-139可能通过促进TRAIL诱导的细胞凋亡降低K562细胞对TRAIL的耐受性。     

FGFR抑制剂BGJ398对白血病细胞生物学行为及耐药的影响

背景与目的:成纤维细胞生长因子(fibroblast growth factor,FGF)家族成员能够调节细胞增殖、迁移和分化,在肿瘤性疾病的发生和转移等过程中发挥重要作用,FGF受体(FGF receptor,FGFR)抑制剂具有抗肿瘤活性.探讨FGFR抑制剂BGJ398对白血病耐药细胞株K562/ADM增殖、迁移、...     

柔红霉素在耐药细胞株K562/ADR内的异常分布

目的 了解化疗药物在糖蛋白（Pgp)耐药细胞株K562/ADR亚细胞结构的异常分布及其与耐药的关系。方法 采用共聚焦激光扫描显微镜、荧光法和RT-PCR等方法,研究柔红霉素（DNR）在K562/ADR细胞亚细胞结构的分布及其与耐药的关系。以及维拉帕米尔（verapamil)、布雷菲尔得菌素（brefeldinA)、氯喹对细胞内DNR异常分布的影响,将3种特异染色线粒体、高尔基体、溶酶体的荧光物质Rh123、DBD-ceramide、中性红作为探针、鉴定分隔储留DNR的细胞器。结果 在K562ADR细胞中,DNR荧光主要集中在核周和周边胞浆,核内及胞浆其他部位荧光很少,这种分布方式与Rh123荧光在该细胞的分布极其相似,与DNR在敏感细胞K562/S细胞核、浆均匀分布不同,verapamil可逆转DNR在K562/ADR的异常分布,而氯喹、brefeldinA无此作用。结论 DNR在耐药细胞的异常分布参与了肿瘤细胞耐药的形成,Pgp在过程中发挥了重要作用。     

P13-K抑制剂LY294002逆转P-gP介导的白血病和胃癌细胞耐药

背景与目的：磷脂酰肌醇3-激酶／蛋白激酶B[phosphatidylinositol-3-kinase（P13-K）／proteinkinaseB（Akt）,P13-K／Aktl通路是调控细胞生存的重要信号转导通路之一。本研究旨在探讨P13-K抑制剂LY294002对P-gP过表达的人类白血病K562／DNR和胃癌SGC7901／ADR细胞多药耐药性的逆转作用。方法：将细胞分为单纯药物组和LY294002预处理组,单纯药物组分别以柔红霉素（daunorubicin,DNR）、阿霉素（adriamycin,ADR）、长春新碱（vincristine,VCR）和依托泊甙（etoposide,VP-16）处理,LY294002预处理组在加药前以LY294002进行预处理。用台盼蓝拒染法及MTT法检测药物敏感性及LY294002对细胞耐药性的影响。Westernblot检测K562／DNR和SGC7901／ADR细胞中P．gP及p-Akt的表达。流式细胞术检测细胞内药物浓度。结果：2．5μmol／LLY294002预处理显著降低DNR、ADR、VCR和VP-16对K562／DNR细胞的IC50,相对逆转效率分别为72．4％、64．9％、60．4％和52．8％。此外,LY294002部分逆转SGC7901／ADR对ADR的耐药性,相对逆转效率为31．0％。LY294002预处理可部分抑制p-Akt和P-gP的表达。随着处理时间的延长．K562／DNR、SGC7901／ADR细胞内DNR、ADR的蓄积效应有增强的趋势。结论：LY294002通过抑制P13-K／Akt信号转导通路,部分逆转P-gp介导的白血病和胃癌细胞的多药耐药。     

Sufficient levels of quinine in the serum circumvent the multidrug resistance of the human leukemic cell line K562/ADM

Reversal of multidrug drug resistance (MDR) has been achieved in vitro by a variety of agents including verapamil, quinidine, cyclosporine A, and amiodarone. The toxicity of these agents precludes the achievement of sufficient levels in the serum to circumvent efficiently the MDR in vivo. The authors previously demonstrated that quinine, the widely used antimalarial agent, is able to reverse primary resistance of rat colon cancer cells to anthracyclines. In this report, the efficiency of quinine formiate in reversing the doxorubicin (ADM) (Adriamycin, Adria Laboratories, Columbus, OH) resistance of the well-defined MDR human leukemic cell line K562/ADM was demonstrated. In culture medium, quinine is slightly less effective than verapamil in increasing the cytotoxicity and uptake of ADM when both drugs are used at the same concentration. A nontoxic dose of 5 micrograms/ml is necessary to reverse the MDR in K562/ADM cells. In patients receiving quinine formiate in a continuous intravenous infusion, a significant correlation (r = 0.84) was found between the serum levels of quinine and the ability of sera to increase ADM uptake in K562/ADM cells. When quinine is administered at a conventional dose (25 to 30 mg/kg/d), serum levels consistently reach more than 8 micrograms/ml without severe side effects; ear noises and vertigo are the dose-limiting side effects. At these concentrations, quinine induces a more than double increase in ADM uptake in K562/ADM cells. Pharmacokinetic data indicate that quinine should be administered 24 to 36 hours before anti-cancer drugs in clinical trials that test its efficiency as a modifier of MDR in human hematologic malignant neoplasms.     

Inhibition of P-glycoprotein by flavonoid derivatives in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells.

We investigated the effects of natural flavones, quercetin and morin, and their pentamethyl, pentaethyl, pentapropyl, pentabutyl and pentaallyl ethers, on the function of P-glycoprotein (P-gp) assessed by an increase in the uptake of [3H]vincristine by human myelogenous leukemia (K562) cells and adriamycin-resistant human myelogenous leukemia (K562/ADM) cells. Pentamethyl, pentaethyl, pentapropyl and pentaallyl ethers of morin and quercetin (20 microM) all increased the uptake of [3H]vincristine by K562/ADM cells, while quercetin, morin and their pentabutyl ethers had no effect. Pentamethylquercetin, pentaallylquercetin and pentaethylmorin remarkably increased the uptake of [3H]vincristine by K562/ADM cells by 10.6, 10.8 and 14.4-fold, respectively. These inhibitory potencies for P-gp were more potent than typical P-gp inhibitors, cyclosporine A and verapamil. Taking into consideration that these flavonoid derivatives possess antitumor promoter activity, they may become candidates of effective multidrug resistance-reversing agents in cancer chemotherapy.     

Experimental Study on the Mechanism of Reversal of Leukemia Multidrug Resistance by Proteasome Inhibitor Bortezomib

OBJECTIVE In this study, we applied multidrug resistant leukemia cell line expressing mdr1-mRNA to observe changes in mdr1-mRNA, the P-gp, cell cycle and apoptosis before and after bortezomib was used, in order to explore the mechanism of reversal of leukemia multidrug resistance by the proteasome inhibitor bortezomib.METHODS Flow cytometry (FCM) was used to detect the intracellular drug concentration, expression of P-gp, cell apoptosis and cell cycle status of K562/DNR cells before and a er treatment with different concentrations of bortezomib. Fluorescence quantitative PCR was applied to detect the mdr1-mRNA expression in K562/DNR and K562/S cells.RESULTS Bortezomib could increase the intracellular DNR content in K562/DNR cells, but showed no e. ect in K562/S cells.5-100 nmol/L bortezomib could significantly reduce the P-gp/mdr1-mRNA expression in K562/DNR cells in vitro, and showed a dose-dependent effect. There was a statistically significant di. erence (P < 0.05) between di. erent concentration groups and the control group. P-gp/mdr1-mRNA expression was negatively correlated with cell apoptosis (r = -0.912 and P < 0.01). After treatment with different concentrations of bortezomib for 24 h,K562/DNR cells in G2 + M phases were significantly increased,while cells in G0 + G1 phases and S phase were significantly decreased, accompanied by an increased apoptotic rate.CONCLUSION Bortezomib can induce G0 + G1 phase to G2 + M phase, and thereby enhance the chemosensitivity of leukemia, and may also reverse the multidrug resistance in leukemia mediated by P-gp overexpression encoded by mdr1 gene. This confi rms that bortezomib can reverse leukemia multidrug resistance at the levels of nucleic acid and protein molecules.