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.     

槲皮素恢复柔红霉素在白血病耐药细胞K562/ADM和HL-60/ADM中的分布

背景与目的：槲皮素是一种天然黄酮类中药成分,儿有多种生理活性,最近发现其有逆转白血病细胞耐药的作用,本研究旨在探讨槲皮素恢复柔红霉素在白血病耐药细胞的分布从而达到逆转耐药的机制：方法：通过MTT体外药敏法检测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、HL-60／ADM细胞及相应敏感株K562和HL-60,借助激光共聚焦显微镜观察槲皮素怍用前后柔红霉素在亚细胞水平的分布变化：结果：20～40μmol／L槲皮素在体外能日月显提高柔红霉素对K562／ADM和HL-60／ADM的敏感性,恢复柔红霉素在亚细胞水平的分布,使其回归细胞核内,从而逆转多药耐药。结论：黄酬类中药槲皮素能够成为蒽环类药物治疗白血病中有效的化疗增敏剂。     

MRP1介导肿瘤的多药耐药

目的:探讨多药耐药相关蛋白基因MRP1介导肿瘤耐药机制,寻找逆转耐药的方法.方法:建立肝细胞癌单基因耐药细胞株HepG2/MRP1,体外转录合成目的siRNA片断,脂质体介导转染单基因肝细胞癌耐药细胞株,MTT法检测细胞对化疗药的敏感性,流式细胞仪检测药物浓度及细胞表面MRP1蛋白表达.结果:成功建立多药耐药细胞株,并筛选出靶向MRP1基因高效的siRNA分子.转染HepG2/MRP1细胞后,MRP1 mRNA及MRP1表达水平明显下调,细胞内DNR蓄积量增加,对ADM的IC50下调,相对逆转率达90％.转染前后细胞对化疗药敏感性、细胞内药物浓度、细胞表面MRP1蛋白质表达均有显著差异.结论:肿瘤多药耐药与MRP1高表达密切相关,RNAi技术能有效逆转由MRP1介导的肿瘤细胞的多药耐药.     

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。     

环孢霉素A和Genistein逆转MRP多药耐药的研究

研究发现多药耐药除与多药耐药基因mdr1及其产物糖蛋白(P gp)有关外,多药耐药相关蛋白MRP与多药耐药也有密切关系.本文主要研究环孢霉素A(cyclosporinA,CsA)及酪氨酸激酶抑制剂Genistein对柔红霉素(daunorubicin,DNR)在MRP介导的白血病多药耐药细胞株HL 60/ADR内分布、蓄积的影响,探讨逆转剂逆转MRP耐药的机理.     

水杨酸钠对HL—60／VCR细胞多药耐药的部分逆转

目的：研究水杨酸钠(Na-Sal对HL-60／VCR细胞多药耐药的部分逆转作用。方法：采用噻唑蓝(MTT)法、流式细胞术等方法，观察非甾体抗炎药Na-Sal对HL-60／VCR细胞多药耐药的部分逆转作用。结果：Na-Sal对HL-60／VCR细胞的生长具有抑制作用并表现出剂量依赖性，1mmol/L的非细胞毒剂量的Na-Sal和化疗药物联合应用可以提高多种化疗药物的细胞毒性作用，逆转倍数为1．01-3．26，相对逆转效率为0．52％-73．62％。流式细胞仪测定细胞内柔红霉素(DNR)浓度发现，Na-Sal并不增加HL-60／VCR细胞内DNR浓度。结论：Na-Sal能有效地部分逆转HL-60／VCR细胞的多药耐药性。     

超声波体内逆转肿瘤多药耐药基因表达的实验研究

目的：探讨超声波体内逆转肿瘤多药耐药的有效性及其逆转机制。方法：70只HepG2／ADM移植瘤裸鼠随机分成对照组（10只），ADM组（20只），超声组（20只），ADM联合超声组（20只），治疗28天后收集标本。应用RT-PCR法和免疫组织化学法检测多药耐药相关基因和蛋白（MDR1，MRP和LRP）表达，、结果：超声治疗组能显著逆转裸鼠HepG2／ADM移植瘤多药耐药基因的表达。免疫组化结果显示，超声治疗组和ADM联合超声组的P-gp，MRP阳性表达显著降低；与对照组相比，在超声治疗组和ADM联合超声治疗组P-gP，MRP水平明显降低。．RT-PCR结果显示，超声组和ADM联合超声组能明显降低HepG2／ADM移植瘤细胞MDR1 mRNA和MRPmRNA的表达。结论：多种机制参与超声波逆转肿瘤多药耐药，超声波通过下调MDR1 和MRPmRNA和蛋白表达水平，增加细胞内药物浓度等途径逆转肿瘤多药耐药。，     

HL-60/VCR多药耐药细胞株耐药机制的研究

背景与目的：白血病细胞对化疗药物的耐药是白血病治疗失败的主要原因,多药耐药细胞株为白血病多药耐药机制和逆转多药耐药性的研究提供了良好的模型。为探讨药物诱导产生多药耐药机制,我们对HL-60/VCR细胞的耐药机制进行了研究。方法：应用流式细胞术和一组抗体,对药物敏感细胞株HL-60和多药耐药细胞株HL-60/VCR细胞的耐药相关蛋白P-gp、MRP、LRP、BCRP、GST-π,以及凋亡调节蛋白bcl-2、bax、bcl-x、bad的表达进行分析。结果：在HL-60/VCR细胞株中,耐药相关蛋白P-gp、MRP、BCRP、GST-π分别是其在HL-60细胞株中的18.62、1.19、1.50、1.32倍,而LRP无变化。凋亡抑制蛋白bcl-2、bcl-x分别是其在HL-60细胞株中的2.48、1.25倍,凋亡调节蛋白bad是HL-60细胞株中的1.08倍;而凋亡诱导蛋白bax反而降低,是HL-60细胞株中的0.88倍。结论：多种机制参与HL-60/VCR的多药耐药,涉及耐药蛋白P-gp、MRP、BCRP和GST-π的表达增强,而且凋亡调节蛋白bcl-2、bax、bcl-x、bad均可能参与其耐药机制的形成。     

鲎素抑制多药耐药细胞HL-60/VCR生长的研究

目的：探讨鲎血细胞来源的多肽鲎素是否能够抑制白血病多药耐药细胞HL-60／VCR细胞的生长。方法：HL-60细胞采用持续低浓度和逐渐增加长春新碱（VCR）浓度间歇诱导，建立多药耐药细胞HL-60／VCR；MTT法检测细胞耐药性及鲎素对HL-60／VCR细胞和HL-60细胞生长的影响；流式细胞仪检测HL-60／VCR细胞P-糖蛋白（P-glycoprotein，P-gP）和多药耐药相关蛋白（multidrugresistance-associatedprotein，MRP）的表达水平。结果：HL~0／VCR细胞对多种化疗药物产生耐药；HL-60／VCR细胞表面P-gP、MRP表达上调，分别为78．3％和58．3％（P〈0．01）。鲎素无论对HL-60／VCR细胞还是对HL-60细胞均有抑制作用，作用呈剂量依赖性。HL-60／VCR细胞和HL-60细胞对鲎素具有相似的敏感性。鲎素能增强HL-60／VCR细胞对VCR的敏感性，但不影响HL-60／VCR细胞表达P-gP和MRP。结论：鲎素具有抗HL-60／VCR细胞耐药性并增加其对VCR敏感性的作用。     

体外培养人肝癌细胞对阿霉素耐药机理及电镜形态研究

目的应用人肝癌细胞株SMMC-7721,研究多药耐药（MDR）的产生机理。方法通过不断提高培养液中阿霉素（ADM）的浓度,长期筛选培养,得到人肝癌多药耐药株SMMC-7721／ADM。用MTT法检测常用6种化疗药物对肝癌细胞敏感株SMMC-7721／S和耐药株SMMC-7721／ADM的毒性;用流式细胞技术检测肝癌细胞MDRI基因产物—P—精蛋白（P-gp）的表达以及细胞内柔红霉素（DNR）浓度。结果SMMC-7721／ADM细胞对阿霉素的敏感性下降了59.33倍,同时对表阿霉素（EPi）、柔红霉素（DNR）、丝裂霉素（MMC）也具有显著的交叉抗药性,对顾铂（CDDP）、氯甲喋吟（MTX）无抗药性。耐药株P-gp较敏感株有非常明显升高,耐药细胞内柔红霉素相对该区明显少于其敏感细胞。结论SMMC-7721／ADM对结构和作用不同的亲脂类化疗药物都表现出交叉抗药性。P-gp过度表达引起的细胞内药物浓度减少是SMMC—7721／ADM细胞抗药性的主要原因。     

Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells

STI571, an Abl-specific tyrosine kinase inhibitor, selectively kills Bcr-Abl-containing cells in vitro and in vivo . However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P-glycopro-tein (P-gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [¹²⁵l]azidoagosterol A-photolabeling of P-gp, but not that of MRP1. K562/MDR cells that overexpress P-gp were 3.67 times more resistant to STI571 than the parental Philadelphia-chromosome-positive (Ph+) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB-G2 cells that overexpress P-gp, but not Bcr-Abl, 2.5 μM STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP-16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB-G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P-gp, but is less efficiently transported by P-gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P-gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/ MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P-gp-expressing KB-G2 cells.     

Alteration of the daunorubicin-triggered sphingomyelin-ceramide pathway and apoptosis in MDR cells: influence of drug transport abnormalities

We have previously shown that in myeloid leukemic cells, daunorubicin (DNR) induces apoptosis via the activation of the sphingomyelin-ceramide pathway. We have now investigated sphingomyelin (SM) hydrolysis, ceramide generation, and apoptosis in vincristine-selected multidrug resistant (MDR) HL-60 cells (HL-60/Vinc), compared with their parental counterparts. We show that DNR triggers the SM cycle (stimulation of neutral sphingomyelinase, SM hydrolysis, and ceramide generation) and apoptosis in both parental and MDR cells, when used at isotoxic doses (ie., 1 and 100 microM for HL-60 and HL-60/Vinc, respectively). However, in MDR cells treated with either 10 microM DNR or 1 microM DNR in association with the P-glycoprotein (P-gp) blocker verapamil (treatment conditions which yield an intracellular DNR concentration similar to that achieved with 1 microM in the parental cells), we were unable to detect SM hydrolysis, ceramide generation and apoptosis. This implies that inhibition of the DNR-induced SM cycle in MDR cells is not directly related to P-gp. We have also investigated the influence of intracellular drug localization on the DNR-induced SM-cycle in HL-60/Vinc cells. In these cells, DNR at 10 microM is mainly localized in cytoplasmic vesicles, while the drug is diffusely distributed when used at 100 microM. A diffuse distribution pattern was also observed when MDR cells were treated with 1 microM DNR in association with the cyclosporine derivative PSC-833, but not with verapamil. In parallel, PSC-833, but not verapamil, restored the induction of the SM cycle and the apoptotic potential of DNR, and markedly increased drug cytotoxicity in MDR cells. Our results suggest that altered intracellular drug transport plays an important role in limiting ceramide generation and cell death in MDR cells.     

Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells.

In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects.     

蛋白激酶C抑制剂逆转肾癌多药耐药机制的探讨

目的探讨蛋白激酶C（PKC）抑制剂对肾癌细胞多药耐药（MDR）逆转作用的机制。方法应用荧光显色法、RT-PCR和Western blot方法,检测PKCα cDNA转染肾癌786-0细胞前后,细胞中多药耐药基因1（MDR1）、多药耐药相关蛋白1（MRP1）和肺耐药蛋白（LRP）表达的变化。采用MTT方法,分别测定阿霉素（ADM）与PKC激动剂以及与PKC抑制剂协同作用后,786-0细胞和肾癌转染细胞系PKCα／786-0耐药性的变化。结果RT—PCR结果显示,肾癌转染细胞系PKCα／786-0的MDR1表达水平高于肾癌786-0细胞。ADM与PKC抑制剂协同作用的PKCα／786-0细胞系耐药性明显降低。786-0细胞对ADM的IC50为7．8015e^-7（5．7046e^-7～1．0669e^-6）;PKCα／786-0对ADM的IC50为1．6588e^-6（1．1621e^-6～2．3677e^-6）。PKC激动剂佛波醇酯（PMA）联合ADM处理PKCα／786-0的IC50为2．6794e^-6（2．0521e^-6-3．4983e^-6）;PKC抑制剂Calphostin C联合ADM处理PKCα／786-0的IC50为9．2506e^-8（5．9337e^-8～1．4422e^-7）。结论PKC抑制剂可以逆转入肾癌细胞的多药耐药性,其途径可能与改变MDR1的表达有关。     

Reversal of P-glycoprotein-mediated multidrug resistance with small interference RNA (siRNA) in leukemia cells

The multidrug resistance (MDR) mediated by P-glycoprotein (P-gp), the MDR1 gene product, is one of the major obstacles in leukemia treatment. The present study was designed to explore a MDR1-targeted small interfering RNA (si-MDR1) approach for reversal of P-gp-mediated MDR in the MDR human leukemia cell line k562/A02. It was found that si-MDR1 significantly inhibited MDR1 expression at both mRNA and protein levels. Depletion of MDR1 by si-MDR1 correlated with the increased sensitivity of the cells to cytotoxic agents and with the enhanced intracellular retention of daunorubicin (DNR). One base-pair mutated control (si-MDR1-Mut) lost the effect of si-MDR1 on both the degradation of mdr1 mRNA and the reduction of P-gp expression. These findings indicate that siRNA specifically and efficiently interferes with the expression of mdr1 and could be used as a molecularly defined therapeutic approach for MDR in the treatment of leukemia.     

马钱子碱对白血病K562/A02细胞多药耐药性的逆转作用

目的：研究马钱子碱（vauqueline）对人白血病K562/A02细胞多药耐药性的逆转作用。方法：采用噻唑蓝（MTT）法检测马钱子碱的细胞毒作用;采用半定量逆转录聚合酶链反应（RT-PCR）和免疫印迹（Western blot）分别检测非细胞毒浓度（IC10）的马钱子碱对K562/A02细胞MDR1 （multidrug resistance gene 1）、多药耐药相关蛋白（multidrug resistance-associated protein,MRP）、拓扑异构酶Ⅱ（topoisomeraseⅡ,TopoⅡ）、谷胱苷肽-S-转移酶（glutathione s-transferase,GST-π）mRNA及其蛋白表达的影响。结果：非细胞毒浓度（IC10）的马钱子碱作用后,K562/A02细胞中MDR1mRNA及P-gp表达降低（P<0.01）。而MRP、TopoⅡ、GST-π mRNA及其蛋白的表达无明显变化（P>0.05）,同时马钱子碱能增加化疗药物在白血病细胞内的积累。结论：马钱子碱能部分逆转K562/A02细胞的耐药性,其作用机制可能与下调K562/A02细胞MDR1 mRNA的表达,导致细胞膜上P-gp的表达量减少,化疗药物从细胞内溢出减少有关。