人参皂苷Rh2逆转MCF-7/ADM多药耐药性的研究

目的观察人采用人参苷Rh2对逆转人乳腺癌多药耐药细胞株MCF-7/ADM的分子机制。方法采用MTT比色法对不同浓度下人参皂苷Rh2对MCF-7/ADM的阿霉素(ADM)以及氟尿嘧啶(Fu)耐药逆转指标检测,同时使用多功能酶标仪检测人参皂苷Rh2干预对细胞内罗丹明123荧光强度以反映其对细胞多药耐药蛋白P-gp活性的影响。结果经过人参皂苷Rh2的干预,MCF-7/ADM对ADM以及Fu两种临床常用化疗的敏感性增强。另外,人参皂苷Rh2还对细胞的罗丹明123外排有显著的浓度依赖性抑制作用。结论人参皂苷Rh2能够有效逆转MCF-7/ADM多药耐药性,其作用机制可能主要为抑制了细胞多药耐药蛋白P-gp的活性。     

四氢异喹啉类化合物HZ08逆转白血病K562/DOX细胞多药耐药性的试验研究

目的:探讨四氢异喹啉类化合物HZ08对人白血病多药耐药K562/DOX细胞的逆转作用及其可能的机制。方法:采用MTT法检测HZ08的体外细胞毒性及其对阿霉素(DOX)的增敏作用,采用逆转倍数(RF)值评价其逆转效果;应用流式细胞仪分析细胞内罗丹明123(Rh123)潴留量的变化和DOX浓度,评价P糖蛋白(P-gp)的功能;采用Western blot法及免疫细胞化学法测定mdr1基因产物P-gp的表达;同时以人白血病敏感细胞株K562/S细胞为对照进行比较试验。结果:与K562/S细胞比较,HZ08可明显增强DOX对K562/DOX的细胞毒性,RF值增加;HZ08能浓度相关性地增加K562/DOX细胞对Rh123的摄取以及细胞内Rh123的潴留,明显抑制P-gp介导的Rh123外排;K562/DOX细胞膜上P-gp呈强阳性表达,但HZ08对K562/DOX细胞P-gp表达水平无明显影响;HZ08可显著增加K562/DOX细胞内DOX浓度。结论:HZ08可通过抑制K562/DOX细胞P-gp的功能、增加耐药细胞内DOX的浓度而增强K562/DOX细胞对DOX的敏感性,其可能成为有效的多药耐药逆转剂的候选药物。     

ZNF300基因在肝癌耐药细胞HepG2/VCR中的表达及功能初探

目的建立人肝癌HepG2/VCR耐药细胞株,检测ZNF300基因在HepG2/VCR中的表达并初步分析其在肝癌多药耐药(MDR)中发挥的功能。方法采用体外低浓度梯度递增的诱导方法建立长春新碱(VCR)获得性HepG2/VCR耐药细胞株。MTT法检测确定HepG2/VCR耐药细胞株对VCR的耐药性,用Western blot方法检测人锌指蛋白ZNF300基因编码的ZNF300及多药耐药基因编码的P糖蛋白(P-gp)在HepG2和HepG2/VCR细胞中的表达差异;在HepG2/VCR细胞中转染ZNF300基因正向或反向cDNA质粒后,MTT法检测VCR对耐药细胞IC50值的变化,Westernblot方法检测细胞内P-gp表达的影响。结果 MTT检测确认HepG2/VCR耐药细胞构建成功,Western blot检测发现耐药细胞中ZNF300及P-gp的表达相对于HepG2细胞明显增高。在HepG2/VCR细胞中转染正向ZNF300 cDNA质粒后,MTT和Western blot检测发现ZNF300过表达可使VCR对耐药细胞的IC50值增高,并使细胞内P-gp表达上调;在转染反向cDNA质粒Knockdown ZNF300基因表达后得到相反的结果。结论 ZNF300基因在HepG2/VCR耐药细胞中表达明显增高,并能通过上调耐药蛋白P-gp的表达促进肝癌细胞耐药性,可以作为逆转肝癌多药耐药的分子作用靶点。     

五味子甲素对K562/ADR、HL60/ADR、MCF-7/ADR多药耐药逆转机制的研究

目的探讨五味子甲素(schizandrin A or deoxyschizan-drin,schA)对白血病细胞K562/ADR、HL60/ADR、乳腺癌细胞MCF-7/ADR多药耐药的逆转作用,并初步探讨其逆转机制。方法 MTT法检测schA对耐药细胞的逆转作用;流式细胞仪检测schA对细胞内柔红霉素、罗丹明-123含量和细胞表面P-gp表达水平的变化;用Real-time PCR方法检测schA对细胞内mdr1 mRNA和mrp1 mRNA表达;生化检测法检测schA对细胞内GSH含量的变化。结果耐药逆转实验显示:不同浓度的schA对作用机制不同的化疗药物耐药产生不同的逆转效果;蓄积实验表明schA可增加柔红霉素、罗丹明123在耐药细胞内的蓄积,并且有良好的剂量依赖关系;schA处理K562/ADR、HL60/ADR细胞24 h后,能降低P-gp蛋白和mdr1、mrp1基因的表达;schA处理K562/ADR、HL60/ADR细胞4 h后,可降低细胞内谷胱甘肽含量。结论 schA对耐药机制不同的细胞株K562/ADR、HL60/ADR均有耐药逆转作用,推测可能是与抑制细胞表面的P-gp蛋白功能和表达,降低mdr1、mrp1耐药基因的表达和降低细胞内谷胱甘肽含量有关,schA通过影响上述机制,进而增加细胞内的药物浓度,达到有效杀灭肿瘤细胞的作用。     

尼美舒利对人胃癌SGC7901 VCR细胞耐药逆转作用

目的 研究尼美舒利对人胃癌敏感细胞株SGC7901及多药耐药细胞株SGC7901VCR的影响,初步探讨其逆转人胃癌耐药的机制。方法 MTT法测定细胞生长抑制率、流式细胞仪检测细胞内Rh123浓度和P-170、GST-π表达水平变化。结果 尼美舒利对SGC7901及SGC7901VCR细胞的生长抑制呈明显时间剂量依赖关系。但尼美舒利对SGC7901VCR细胞效应强度明显弱于SGC7901细胞。尼美舒利能提高SGC7901VCR细胞内Rh123荧光强度,下调P-170和GST-π的水平(P＜0.05)。结论 尼美舒利对人胃癌SGC7901VCR耐药有一定逆转作用,作用机制可能与下调P-170和GST-π的水平有关。     

洛美利嗪逆转K562/ADM细胞多药耐药性

目的研究洛美利嗪(lomerizine,Lom)逆转K562/ADM细胞多药耐药性的作用及机制。方法MTT法检测细胞毒作用,流式细胞仪研究Lom对ADM和长春新碱(vincristine,VCR)的K562/ADM细胞凋亡诱导作用的影响及对罗丹明123(rhodamine 123,Rh123)外排和P-糖蛋白(P-glycoprotein,P-gp)表达的作用。结果Lom明显提高ADM对K562/ADM多药耐药细胞的细胞毒作用及ADM和VCR的凋亡诱导作用,3,10和30 μmol·L^-1 Lom使K562/ADM对ADM的IC₅₀值由79.03 μmol·L^-1分别降至28.14,8.16和3.16 μmol·L^-1。Lom增加胞内ADM的蓄积浓度并抑制Rh123外排;但作用72 h后对K562/ADM细胞P-gp表达无影响。结论Lom通过抑制P-gp的活性逆转K562/ADM细胞的多药耐药性。     

三苯氧胺逆转卵巢癌细胞株多药耐药性的研究

目的探讨三苯氧胺(TAM)逆转卵巢癌细胞株的耐药性和逆转机制。方法应用ATP-TCA法检测细胞株的耐药性及TAM的逆转效果,应用流式细胞仪检测TAM对罗丹明123(Rh123)在细胞内积聚和外排的影响,应用免疫荧光技术定量测定TAM对P糖蛋白表达的影响。结果TAM能增加阿霉素对耐药株的细胞毒性作用,能增加耐药株细胞内Rh123的积聚,减少其外排,而对P糖蛋白的表达没有影响。结论TAM能部分逆转卵巢癌细胞株的耐药性,其强度与维拉帕米相当,其作用机理是抑制P糖蛋白的功能,而对其表达水平没有影响。     

洛美利嗪衍生物CJZ3对K562/DOX细胞阿霉素耐药的逆转作用

目的研究洛美利嗪衍生物CJZ3对K562/DOX细胞阿霉素耐药的逆转作用。方法应用流式细胞仪和MTT法观察了CJZ3对K562/DOX细胞P-糖蛋白(P-glycoprotein,P-gp)的抑制作用及对K562/DOX细胞阿霉素耐药的逆转作用。结果CJZ3能剂量相关性地增加K562/DOX细胞对罗丹明123(rhodamine123,Rh123)的摄取以及细胞内罗丹明Rh123的累计,明显抑制P-gp介导的Rh123外排,增强阿霉素对K562/DOX细胞的细胞毒作用,提高阿霉素诱导的K562/DOX细胞凋亡率,提高细胞Caspase-3活性,增加K562/DOX细胞内阿霉素水平。结论洛美利嗪衍生物CJZ3体外能明显抑制P-gp的外排功能,逆转P-gp介导的K562/DOX细胞的多药耐药性。     

CQ11逆转多药耐药人胃癌细胞株对多柔比星的耐药

目的:探讨氯喹衍生物CQ11对耐长春新碱(vincristine,VCR)人胃癌多药耐药(multidrug resistance,MDR)细胞株SGC7901/VCR的耐药逆转作用。方法:将SGC7901和SGC7901/VCR细胞分别与各种浓度的多柔比星(doxorubicin,DOX)和/或CQ11在体外共同培养,采用MTT法检测其细胞毒作用;采用荧光分光光度计测定细胞内DOX蓄积量。结果:SGC7901/VCR细胞对DOX的耐药程度是SGC7901细胞的37.5倍。1.0、2.5和5.0 mol/L的CQ11分别使DOX对SGC7901/VCR细胞的敏感性分别增加到2.2倍(P<0.01)、5.5倍(P<0.01)和14倍(P<0.01)。DOX蓄积实验表明,CQ11能显著增加SGC7901/VCR细胞内DOX蓄积,而对SGC7901细胞内DOX蓄积无明显影响。结论:通过增加细胞内DOX蓄积量,CQ11在体外能有效逆转SGC7901/VCR细胞对DOX的耐药性。     

米尔贝逆转耐阿霉素人乳腺癌细胞多药耐药的作用

目的 探寻米尔贝类化合物尼莫克汀、米尔贝β1逆转人乳腺癌多药耐药细胞株(MCF-7/adr)多药耐药(multidrug resistance,MDR)的作用及机制.方法 采用MTT比色法测定细胞生长抑制率及耐药指数;高效液相色谱(HPLC)检测细胞内阿霉素(ADR)的积累变化;荧光分光光度仪检测罗丹明123(Rh123)在肿瘤细胞内的积累;通过RT-PCR与流式细胞仪检测MDR1基因与P-糖蛋白(P-gp)表达的变化.结果 5 μmol·L-1的尼莫克汀、米尔贝β1可明显增强MCF-7/adr对ADR的敏感性,增加细胞内ADR及Rh123的积累,且呈剂量依赖关系,不同程度降低MDR1和P-gp的表达.结论 尼莫克汀、米尔贝β1对MCF-7/adr的耐药有一定的逆转作用,且尼莫克汀效果好于米尔贝β1.     

阿霉素对裸小鼠人肝癌原位移植瘤多药耐药性的影响

目的　探讨阿霉素对裸小鼠原位移植人肝癌多药耐药性的影响 ,并研究其耐药机制。方法　人肝癌 (BEL 740 2 )裸小鼠原位移植 ,用阿霉素腹腔注射诱导耐药 ,经MTT法检测原代培养的耐药细胞对抗癌药的敏感性 ,以流式细胞仪检测癌细胞表面mdr1基因产物P170的表达及功能。以裸小鼠原位移植人肝癌模型观察阿霉素对耐药组的疗效。结果　移植瘤组织形态及生物学方面符合人肝癌特征 ,耐药细胞表面P170表达为 75 45 %± 5 6 7% ,而对照组表达仅 4 2 5 %± 1 2 8% (P <0 0 1) ,对阿霉素的耐药倍数提高了 16 7倍 ,对羟基喜树碱和表阿霉素具有交叉耐受性(13 7倍和 7 5倍 )。耐药细胞表面P170有较强的药物外排功能。诱导后的肝癌在体内对阿霉素获得了明显的抗性。结论　阿霉素较易诱导原位移植于裸小鼠的人肝癌多药耐药性的产生 ,耐药机制主要与P170的过度表达有关     

洛美利嗪对大鼠脑微血管内皮细胞上P-糖蛋白活力的影响与P-gp及mdr1基因mRNA表达无关

目的：研究洛美利嗪对原代培养的大鼠脑微血管内皮细胞（RBMECs）上P-糖蛋白（P-gP）功能和表达的影响。方法：使用流式细胞术分析洛美利嗪对P-gp底物-罗丹明123（rhodaminel23，Rh123）在RBMECs中外排的影响，使用流式细胞术分析了洛美利嗪对RBMECs上P-gp表达的影响，应用RT-PCR技术分析了洛美利嗪对RBMECs mdr1基因mRNA水平表达的影响，还使用Transwell模型研究了洛美利嗪对Rh123通透RBMECs单层细胞转运的影响。结果：洛美利嗪通过抑制了RBMECs胞内Rh123的外排；洛美利嗪对P—gP功能的影响与RBMECs上P-gp及mdr1基因mRNA表达无关；在Transwell模型中，洛美利嗪还能显著增加Rh123跨RBMECs单层细胞膜的、经上室至下室的转运，并抑制相反方向的Rh123的转运。结论：洛美利嗪能显著地抑制RBMECs上P-gp的活性，并对P-gp底物的跨细胞转运产生影响。     

咯萘啶逆转肿瘤多药耐药及其作用机制

目的:利用mdr1~ 的人白血病和乳腺癌多药耐药(MDR)细胞系K562/A02和MCF-7/ADR研究咯萘啶(pyronaridine,PND)对MDR的逆转作用及其机制.方法:采用MTT法、荧光分光光度法、荧光显微镜法、流式细胞仪法和RT-PCR法分别测定PND单独或与阿霉素(DOX)合用,对肿瘤细胞的生长抑制、诱导凋亡、细胞内药物浓度、mdr1基因表达的影响.结果:PND对敏感及耐药细胞均具有生长抑制作用,半数抑制剂量(IC_(50))根据不同细胞在5.10-18.66μmol/L之间;低毒剂量PND显著增强DOX对耐药细胞的细胞毒和诱导凋亡作用,且增加DOX在耐药细胞内的蓄积及减少罗丹明(Rh123)的外排.RT-PCR结果显示,PND对mdr1基因无下调作用.结论:PND可作为第三代P-糖蛋白(P-gp)抑制剂,通过下调P-gp药物外排泵功能而产生强大的逆转MDR效应.     

Inhibitors of mdr1-dependent transport activity delay accumulation of the mdr1 substrate rhodamine 123 in primary rat hepatocyte cultures

P-glycoproteins (P-gps) encoded by mdr1 (multidrug resistance) genes mediate extrusion of numerous lipophilic xeno- and endobiotics through the plasma membrane. Rhodamine 123 (Rh123), a fluorescent dye which is accumulated by mitochondria, is a mdr1 substrate and a well-established tool to study mdr1 transport activity. Inhibitors of mdr1-dependent transport such as verapamil or cyclosporin A have been found to decrease Rh123 efflux from mdr1-expressing cells. Mdr1b gene expression increases with time in primary rat hepatocyte culture. In hepatocytes cultured for 4 days and expressing high levels of P-gp, intracellular Rh123 accumulation was enhanced in the presence of mdr1 inhibitors (cyclosporin A, 8 and 80 microM, verapamil, 8 and 80 microM, or triton X-100, 8 microM). Surprisingly, in hepatocytes expressing low levels of P-gp (after 1 day of culture), time-dependent Rh123 accumulation was not enhanced, but delayed by cyclosporin A, verapamil or triton X-100. In these cells orthovanadate (50 microM), an inhibitor of P-glycoprotein ATPase activity, suppressed Rh123 accumulation, while tetraethylammonium (200 microM), an organic cation transporter (OCT) substrate, had no effect. The paradoxical delay in Rh123 accumulation by verapamil and cyclosporin A occurred eventhough these compounds decreased dye extrusion from Rh123 pre-loaded cells. These observations suggest that a hitherto unknown mechanism which is sensitive to modulators of mdr1-activity contributes to Rh123 uptake or accumulation in primary rat hepatocytes.     

Altered expression and function of P-glycoprotein in dextran sodium sulfate-induced colitis in mice

P-glycoprotein (P-gp), the product of the multiple drug resistance (mdr) gene, can actively pump toxic drugs out of cells, but its pathophysiologic role is not yet fully understood. In this study, we examined the expression of P-gp in dextran sodium sulfate (DSS)-induced colitis in mice. Eight-week-old Balb/c female mice were given drinking water containing 7% DSS ad libitum for 7 days. Mice receiving DSS were sacrificed on days 3, 5, and 7 for histopathologic study. Tissue samples were examined by hematoxylin and eosin (HE) staining, and immunostained against mdr, CD4+, CD8+, and B220+. RNA was isolated from the large intestine and the expression of mdr1a was determined by RT-PCR. The function of P-gp was evaluated by rhodamine123 efflux using the everted sac method. The induction of colitis in mice was confirmed by body weight changes, HE staining and immunohistological grading of the large intestine with reference to CD4+, CD8+, and B220+ after 7 days of treatment. Severe inflammation was observed in the large, but not the small, intestine on day 7. The expression of mdr1a in the large intestine was reduced on days 3, 5, and 7. In addition, the P-gp function and the expression of PXR were also reduced in the large intestine of DSS-treated mice on day 3. This reduction was consistent with the immunohistologic observations. The expression of the mdr1a gene was reduced before severe symptoms appeared. These results suggest that P-gp expression may be related to the pathology of colitis.     

基因mdr1高表达多药耐药肿瘤细胞对力达霉素的药物敏感性

目的利用经药物诱导获得的mdr1基因高表达细胞株以及通过mdr1基因转染建立的稳定高表达细胞株，研究多药耐药肿瘤细胞对力达霉素(C-1027)的药物敏感性。方法构建mdr1重组真核表达质粒pcDNA3.1/mdr1，利用脂质体转染技术，获得mdr1高表达HepG2肝癌细胞。经RT-PCR、细胞荧光免疫化学及罗丹明外排实验，鉴定了细胞的mdr1表达水平和药物外排活性。MTT方法测定敏感细胞及相对应的多药耐药细胞对力达霉素等多种抗肿瘤药物的药物敏感性。结果mdr1稳定转染细胞株HepG2/mdr1、多药耐药KBv200细胞和MCF-7/ADR细胞对力达霉素的IC₅₀值分别为(0.020±0.011) nmol·L^-1，(0.24±0.20) nmol·L^-1和(0.028±0.011) nmol·L^-1。相对于各自的敏感细胞，多药耐药细胞HepG2/mdr1，KBv200和MCF-7/ADR对力达霉素的抗药倍数分别是1.3，6.8和1.6倍，对阿霉素的抗药倍数分别是8.8， 37.2和181.3倍，对紫杉醇的抗药倍数分别是40.3， 336.8和49.2倍。结论 mdr1高表达的多药耐药肿瘤细胞对力达霉素仍高度敏感，未表现出抗药性。     

Insulin therapy restores impaired function and expression of P-glycoprotein in blood-brain barrier of experimental diabetes

We aimed to investigate effects of insulin on function and expression of P-glycoprotein (P-GP) in the blood-brain barrier of streptozotocin (STZ)-induced diabetic rats. Brain-to-plasma concentration ratio of vincristine (VCR) in rats was used as an indicator of in vivo function of P-GP. Western blot and quantitative real time-polymerase chain reaction were used to determine protein levels of P-GP and its mdr1a/mdr1b mRNA levels, respectively, in cerebral cortex of rats. In vitro effects of insulin on function and expression of P-GP in primarily cultured rat brain microvessel endothelial cells (rBMECs) were evaluated using rhodamine 123 (Rho123) uptakes and Western blot, respectively. The results showed that 3- and 5-week insulin treatment alleviated the impaired efflux function, expression and mdr1a/mdr1b mRNA levels of P-GP in cerebral cortex of diabetic rats. The 3- and 5-week insulin treatments also significantly enhanced P-GP levels and mdr1a/mdr1b mRNA levels in the cerebral cortex of normal rats. Addition of insulin to the insulin-deficient diabetic rat serum normalized the impaired function and expression of P-GP in rBMECs cultured in diabetic rat serum. When incubated with normal culture medium containing different levels of insulin, the rBMECs exhibited the enhanced P-GP levels and the reduced Rho123 uptake in a concentration-dependent manner. So we may conclude that appropriate level of insulin plays an important role in maintaining the normal function of BBB through regulating the function and expression of P-GP in the diabetic and normal rats.     

Reversal of P‐glycoprotein‐mediated multidrug resistance of human hepatic cancer cells by Astragaloside II

Objectives Chemoresistance is the main obstacle encountered in cancer treatment and is frequently associated with multidrug resistance (MDR). Astragaloside is a saponin which is widely used in traditional Chinese medicine. It has been reported that Astragaloside has antitumour effects on hepatocellular carcinoma Bel‐7402 cells in vitro and in vivo. The purpose of this study was to examine the effects of Astragaloside II on the reversal of MDR and its molecular mechanism in vitro. Methods In this study, Bel‐7402 and Bel‐7402/FU cell lines were used as the experimental model. Drug sensitivity was determined using the 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay, accumulation and efflux of Rh123 were analyzed by flow cytometer, the mRNA level of mdr1 was determined by RT‐PCR and the protein levels of P‐glycoprotein (P‐gp) and mitogen‐activated protein kinase were determined by Western blot. Key findings Astragaloside II (0.08 mg/ml) showed strong potency to increase 5‐fluorouracil cytotoxicity toward 5‐fluorouracil‐resistant human hepatic cancer cells Bel‐7402/FU. The mechanism of Astragaloside II on P‐gp‐mediated MDR demonstrated that Astragaloside II significantly increased the intracellular accumulation of rhodamine 123 via inhibition of P‐gp transport function. Based on the analysis of P‐gp and mdr1 gene expression using Western blot and RT‐PCR, the results revealed that Astragaloside II could downregulate the expression of the P‐gp and mdr1 gene. In addition, Astragaloside II suppressed phosphorylation of extracellular signal regulated kinase 1/2, p38 and c‐Jun N‐terminal kinase. Conclusions The results suggested that Astragaloside II is a potent MDR reversal agent and may be a potential adjunctive agent for hepatic cancer chemotherapy.