Establishment and characterization of adriamycin-resistant human colorectal adenocarcinoma HCT-15 cell lines with multidrug resistance

The multidrug resistance (MDR) phenotype, either intrinsic and/or acquired, is discussed in relation to several MDR-associated markers such as P-glycoprotein (P-gp) encoded by mdr1, multidrug-resistance-associated protein (MRP) encoded by MRP and lung-resistance-associated protein (LRP) encoded by LRP. Well-characterized in vitro models are required to elucidate the mechanisms of MDR. The aim of the present study is the establishment of a drug-resistant subline from human colorectal adenocarcinoma HCT-15 that intrinsically expresses moderate levels of P-gp, MRP and LRP. Three adriamycin-resistant sublines (HCT-15/ADM1, HCT-15/ADM2 and HCT-15/ADM2-2) were established by stepwise exposure in growth medium that was supplemented with 25-200 ng/ml adriamycin-resulting in a 2.2- to 7.8-fold increase in IC(50) values by using the XTT assay. They were cross-resistant to MDR-related drugs, epirubicin, mitoxantrone, vincristine, etoposide and taxol, but not the MDR-unrelated drug, mytomycin C. The resistance to adriamycin was confirmed in vivo by a lack of sensitivity in athymic nude mice. Gene expression data for mdr1/P-gp, MRP/MRP and LRP/LRP on both mRNA and protein levels demonstrated that the molecules contributing to MDR in resistant sublines are mainly P-gp and partially MRP. The newly established adriamycin-resistant sublines of HCT-15 will provide clinically relevant tools to investigate how to overcome drug resistance and elucidate possible mechanisms of acquired MDR in human colon cancer.     

Catfish egg lectin causes rapid activation of multidrug resistance 1 P-glycoprotein as a lipid translocase

Rhamnose-binding lectin from catfish (Silurus asotus) eggs (SAL) has the ability to induce externalization of phosphatidylserine (PS), followed by cell shrinkage in globotriaosylceramide (Gb3)-expressing Burkitt's lymphoma Raji cells. Because phospholipid scramblase and aminophospholipid translocase did not participate in SAL-induced PS externalization, we examined the relationship of ATP-binding cassette (ABC) transporters, such as multidrug resistance (MDR) 1 P-glycoprotein (MDR1 P-gp) and MDR-associated protein 1 (MRP1), for translocation of PS. Since cyclosporin A (MDR1 P-gp inhibitor) but not MK571 (MRP1 inhibitor) inhibited SAL-induced PS externalization, it was suggested that MDR1 P-gp is involved in this phenomenon. On the other hand, SAL activated both of the ABC transporters for efflux of rhodamine123 (MDR1 P-gp substrate, Rho123) and 5-carboxyfluorescein diacetate (MRP1 substrate, 5-CFDA) in Raji cells. In contrast, SAL did not activate these two transporters in Gb3-negative cell lines, such as K562 and doxorubicin-resistant K562 cells, involving not only PS externalization but also efflux of Rho123 or 5-CFDA. Since Gb3 and both transporters in Raji cells are located in the glycosphingolipid-enriched microdomain (GEM), it is suggested that the binding of SAL to Gb3 localized in the GEM specifically induces MDR1 P-gp activation in Raji cells.     

Expression of multidrug resistance proteins and accumulation of cisplatin in human non-small cell lung cancer cells

In order to understand and overcome multidrug resistance (MDR) of human non-small cell lung cancer (NSCLC), mRNA and protein expression levels of P-glycoprotein (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung resistance-related protein (LRP) were investigated and compared with the chemosensitivity and the intracellular/intranuclear cisplatin accumulation of three NSCLC cell lines (Ma-10, Ma-31, and Ma-46). Ma-31 was more resistant than Ma-10 and Ma-46 to cisplatin, carboplatin, etoposide, and paclitaxel. The mRNA level of MDR1 was extremely low, and MDR1 protein was not detected in all cell lines. MRP1 mRNA expression was highest in Ma-31 and lowest in Ma-10, but there was no notable difference between the MRP1 protein expression in three cell lines. LRP mRNA/protein was equally expressed in Ma-10 and Ma-31, but was nominal in Ma-46. The intracellular/intranuclear cisplatin accumulation of the cells was determined to be Ma-31>Ma-46>Ma-10. Thus, MDR1, MRP1, and LRP mRNA and protein expression levels were not correlated with the chemosensitivity or the intracellular/intranuclear cisplatin accumulation of each cell line. The present results indicate that MDR proteins (MDR1, MRP1, and LRP) may not play an important role in the chemoresistance and drug efflux of NSCLC cells.     

Both P-gp and MRP2 mediate transport of Lopinavir, a protease inhibitor

Polarized epithelial non-human (canine) cell lines stably transfected with human or murine complementary DNA (cDNA) encoding for various efflux transporters (P-gp/MDR1, MRP1, MRP2, and Bcrp1) were used to study transepithelial transport of Lopinavir (LVR) and compare results with the MDCKII-wild type cells. These transmembrane proteins cause multidrug resistance by decreasing the total intracellular accumulation of drugs. Lopinavir efflux was directional and was completely inhibited by MK-571, a selective MRP family inhibitor in the MDCKII-MRP2 cell line. Similarly, LVR efflux was also inhibited by P-gp inhibitors P-gp-4008 and GF120918 in the MDCKII-MDR1 cell line. The efflux ratios of LVR in the absence of any efflux inhibitors in the MDCK-wild type, MDCKII-MDR1, MDCKII-MRP1 and MDCKII-MRP2 cell monolayers were 1.32, 4.91, 1.26 and 2.89 respectively. The MDCKII-MDR1 and MDCKII-MRP2 cells have significantly increased LVR efflux ratio relative to the parental cells due to the apically directed transport by MDR1 and MRP2 respectively. The efflux ratios in MRP2 and MDR1 transfected cell lines were close to unity in the presence of MK-571 and P-gp-4008, respectively, indicating that LVR efflux by MRP2 and P-gp was completely inhibited by their selective inhibitors. MDCKII-MRP1 cells did not exhibit a significant reduction in the LVR efflux relative to the parental cells, indicating that LVR is not a good substrate for MRP1. Transport studies across MDCKII-Bcrp1 cells indicated that LVR is not transported by Bcrp1 and is not a substrate for this efflux protein. In conclusion, this study presents direct evidence that LVR is effluxed by both P-gp and MRP2 which may contribute to its poor oral bioavailability and limited penetration into the CNS.     

RNAi逆转淋巴瘤LRP和P-gp介导的多药耐药研究

目的 应用RNA干扰(RNA interference,RNAi)技术逆转淋巴瘤中P-糖蛋白(P-glycoprotein,P-gp)和肺耐药蛋白(lung resistance protein,LRP)介导的多药耐药(multidrug resistance,MDR).方法 采用细胞毒性试验、荧光定量PCR、Western blot等方法观测双靶向干扰载体对P-gp和LRP介导的MDR的逆转作用.结果 采用干扰质粒转染耐药淋巴瘤细胞后,该细胞的耐药指数降低,外排米托蒽醌的能力降低,细胞中P-gp、LRP的mRNA和蛋白表达水平也显著降低,且差异均有统计学意义(P＜0.05).结论 构建的双靶向干扰载体能在体外逆转淋巴瘤细胞中P-gp和LRP介导的非典型性多药耐药.     

复方当归注射液逆转K562/A02细胞多药耐药的研究

目的:研究复方当归注射液对人红白血病多药耐药(MDR)细胞株K562/A02MDR1、多药耐药相关蛋白(MRP)、拓扑异构酶Ⅱ(TopoⅡ)、谷光甘肽-S-转移酶-π(GST-π)基因及其相应蛋白表达的影响。方法:采用台盼蓝染色法检测复方当归注射液的细胞毒作用;采用半定量逆转录聚合酶链反应(RT-PCR)和免疫印迹法分别检测复方当归注射液在非细胞毒浓度(IC10)作用下对K562/A02细胞上述基因及其表达的影响。结果:复方当归注射液作用后,K562/A02细胞中MDR1基因及P-gp表达降低(P<0.01);TopoⅡ蛋白表达增高(P<0.01),TopoⅡ基因表达无显著变化(P>0.05);MRP、GST-π基因及其蛋白的表达无明显变化(P>0.05)。结论:复方当归注射液能部分逆转K562/A02细胞的耐药性,其作用机制可能与下调K562/A02细胞MDR1mRNA的表达,导致细胞膜上P-gp的表达量减少及在蛋白水平上调K562/A02细胞TopoⅡ的表达有关。     

Low-level doxorubicin resistance in benzo[a]pyrene-treated KB-3-1 cells is associated with increased LRP expression and altered subcellular drug distribution

The P-glycoprotein (P-gp)-negative epidermoid pharyngeal carcinoma cells KB-3-1 were grown in 0.25 mM benzo[a]pyrene (BaP) for 3 months and increased resistance to doxorubicin, but not to vinblastine, colchicine, or cisplatin, was found. Doxorubicin resistance was not altered by cyclosporin, the P-gp inhibitor. Intracellular accumulation of BaP or calcein, a substrate for P-gp and multidrug resistance protein (MRP), was not altered by inhibitors of the P-gp and MRP. The expression of cytochrome P450 (CYP) 1A1, lung-resistance-related protein (LRP), P-gp, and MRP was investigated. Overexpression of CYP1A and LRP, on the mRNA and protein levels, was found. BaP-treated KB-3-1 cells remained P-gp negative while the level of MRP was not altered. Subcellular accumulation of BaP was found to be localized in the cytoplasm and minimal in the nuclei in BaP treated cells. In contrast, even penetration of BaP to the nuclei and cytoplasm was found in untreated cells. Subcellular distribution of doxorubicin was altered following BaP treatment with localized accumulation of the cancer drug in cytoplasmic organelles but not in the nuclei. Our data suggested that LRP might play a protective role against toxic compounds. The correlation of increased expression of LRP, but not P-gp nor MRP, with decreased doxorubicin accumulation in the nuclear target suggests a pivotal role of this perinuclear transporter in the MDR phenotype of P-gp-negative cancer cells. These results also propose an alternative mechanism of cancer drug resistance emergence, namely, induction of LRP activity following treatment with BaP, an environmental toxicant and a carcinogen.     

多药耐药基因在大肠癌组织中的表达和临床研究

目的探讨大肠癌组织中7种多药耐药因子的表达、共表达及与患者临床病理特征的相关性。方法采用微波EnVision免疫组织化学法联合检测63例大肠癌患者癌组织中P-糖蛋白(P-gp)、谷胱甘肽-S转移酶-π(GST-π)、DNA拓扑异构酶(TopoⅡ)、胸苷酸合成酶(TS)、甲基鸟嘌呤甲基转移酶(MGMT)、肺耐药蛋白(LRP)及多药耐药相关蛋白(MRP)的表达水平。结果P-gp、GST-π、TopoⅡ、TS、MGMT、LRP和MRP在大肠癌组织中的阳性表达率分别为54%(34例)、71%(34例)、62%(39例)、67%(42例)、57%(36例)、79%(50例)和30%(19例)。所有的多药耐药因子表达均与患者的年龄、性别无相关。MGMT、LRP的表达与淋巴结转移相关;P-gp、GST-π、TopoⅡ、MRP的表达与组织学分级相关;TS、MGMT、LRP的表达与临床分期相关;P-gp、MGMT的表达与生存期明显相关。结论大肠癌组织表达多种耐药因子,各耐药因子间共表达具有明显相关性。大肠癌耐药由多种耐药基因共同参与,联合检测多项耐药因子有重要的临床意义。     

屈洛苷芬对K562耐阿霉素细胞株耐药性的逆转作用

目的：研究屈洛昔芬（DRO）对耐阿霉素（ADR）K562细胞株（K562／A02)多药耐药性（MDR）的逆转作用及逆转机制。方法：用DRO分别处理K562／A02和K562敏感株。MTT法观察DRO影响K562／A02对ADR化学敏感性的变化。DRO 10μmol/L处理K562／A02前后,通过RT－PCR和免疫细胞化学染色,分析MDR1、GSTπ基因表达的变化,采用流式细胞技术测定细胞内ADR浓度的变化。结果：DRO显著逆转K562／A02的MDR,在20,10和5μmol/L浓度时,对ADR的化学敏感性分别增加到14、13和4倍,逆转活性与维拉帕米相当。MDR1和GSTπ的mRNA和蛋白表达在DRO 10μmol/L处理后第2天开始下降,第5天明显降低。用20、10和5μmol/L浓度的DRO处理两株细胞,K562／A02细胞内ADR积累分别增加到2．9、2．3和1．5倍。但DRO不能明显增加K562细胞内的ADR的浓度。结论：DRO对K562／A02的MDR有较强的逆转活性,逆转强度与维拉帕米相当,其逆转机制有多种不同的途径。     

肺癌中多药耐药因子的表达相关性及临床意义的研究

目的 探讨多药耐药因子在肺癌中表达与共表达的水平、相关性及临床意义.方法 采用免疫组化SP技术检测60例肺癌患者组织芯片中P-糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、肺耐药蛋白(LRP)、谷胱苷肽-S转移酶-π(GST-π)等多药耐药因子的表达水平.结果 ①P-gp、MRP、LRP、GST-π阳性表达率分别为53.3%(32/60)、63.3%(38/60)、70.0%(42/60)、80.0%(48/60).②耐药因子在不同病理类型中表达差异有统计学意义(P＜0.05),在NSCLC中表达高于SCLC;在不同TNM分期、不同分化程度间表达差异无统计学意义(P＞0.05).③各耐药因子之间阳性共表达的结果分别为P-gp+MRP:41.6%,P-gp+LRP:35.0%,MRP+LRP:53.3%,MRP+GST-π:50.0%,LRP+GST-π:58.3%,P-gp+GST-π:45.0%,P-gp +MRP+LRP+GST-π:20.0%.其中P-gp与MRP间具有相关性(r=0.756,P＜0.01),P-gp与LRP间具有相关性(r=0.689,P＜0.01),MRP与LRP间具有相关性(r=0.669,P＜0.01),MRP与GST-π间具有相关性(r=0.546,P＜0.01),LRP与GST-π间具有相关性(r=0.848,P＜0.01),P-gp与GST-π具有相关性(r=0.535,P＜0.01).结论 肺癌的多药耐药现象是由多个耐药因子共同参与作用的结果,肺癌多药耐药的发生在肿瘤细胞的病理分型间有差异性,在不同分化程度、不同TNM分期间无差异性.

Abstract：

Objective To study the expression,co-expression, and clinical significance of four multi-drug resistance factors in lung cancer. Methods The P-glycoprotein (P-gp), mullidrug resistance-associated protein ( MRP, lung resistance protein ( LRP), glutathione-S-transferase (GST-π) of 60 lung cancer patients were detected by immunohistochemical methods. Results The positive drug resistance rate of P-gp, MRP, LRP, GST-π was 53.3% (32/60) ,63.3% (38/60) ,70.0% (42/60) ,80.0% (48/60) respectively. Patients with NSCLC had significantly higher expression of the drug resistance factors than those with SCLC. No relation was observed among the expression of drug resistance factors and TNM stage and cell differentiation. The-expression rate was as follows: Pgp + MRP :41.6%, P-gp + LRP :35.0%, MRP + LRP :53.3%, MRP + GST-π:50.0%, LRP + GST-π: 58.3%, Pgp + GST-π:45.0%. P-gp + MRP + LRP + GST-π:20.0%. Among them, significant relation was detected between P-gp and MRP ( rs = 0.756, P ＜ 0. 0 ), between P-gp and LRP ( rs = 0.689, P ＜ 0.01 ), between MRP and LRP (rs = 0.669, P ＜ 0.01 ), between MRP and GST-π( rs = 0.546, P ＜ 0.01 ), between LRP and GST-π ( rs = 0.848, P ＜0.01 ), between P-gp and LRP( rs =0.535 ,P ＜0.01 ). Conclusions The Multi-drug resistance in lung cancer patients is affected by various multidrug resistance factors. The drug resistance factors' expression is related to histology, but not to TNM stage end cell differentiation.     

温下方逆转A549/DDP细胞的多药耐药及对膜表面蛋白表达的影响

目的运用血清药理学方法,体外研究温下方对A549/DDP细胞多药耐药的逆转作用及机制。方法正常血清及不同浓度的温下方含药血清作用于A549/DDP细胞,四甲基偶氮唑蓝(MTT)法检测温下方含药血清对A549/DDP细胞的逆转作用;运用免疫荧光技术,激光共聚焦显微镜及流式细胞仪检测肺耐药相关蛋白(LRP)、多药耐药相关蛋白(MRP)和P-糖蛋白(P-gp)的表达。结果温下方含药血清能明显逆转A549/DDP细胞的多药耐药,增敏倍数为2～2.5倍。并可明显降低P-gp、LRP、MRP蛋白表达。结论温下方通过降低P-gp、LRP、MRP的表达以增强A549/DDP对化疗药的敏感性,逆转肺腺癌细胞的多药耐药。     

酪氨酸激酶抑制剂伊马替尼耐药K562细胞系的建立及其耐药特征

目的建立酪氨酸激酶抑制剂伊马替尼(STI-571)耐药K562细胞系并研究其耐药特征。方法采用含递增浓度伊马替尼的培养基培养K562细胞,诱导耐伊马替尼K562细胞系(K562R)。通过细胞生长曲线、细胞形态和细胞凋亡分析确定耐药细胞是否形成;采用MTT法观察耐药细胞的耐药谱;通过半定量PCR和Western印迹法检测相关基因及蛋白的表达,并通过基因测序分析K562R细胞B细胞受体-c-Abelson(BCR-ABL)激酶区基因序列。结果成功地将伊马替尼高敏感的K562细胞诱导成K562R,伊马替尼抑制K562和K562R细胞存活的IC50值分别为0.01±0.00和(2.35±0.01)μmol·L-1,耐药倍数为235.0倍。K562R具有一定的交叉耐药性,对高三尖杉酯碱、长春新碱和对柔红霉素的耐药倍数分别为13.2,63.2和11.8倍。K562R可对抗伊马替尼诱导的细胞凋亡,伊马替尼1.0μmol·L-1培养24 h K562和K562R细胞凋亡率分别为72.1%和18.2%。耐药形成机制研究表明,与K562细胞相比,K562R细胞BCR-ABL基因、多药耐药基因(MDR)和p-糖蛋白基因(p-GP)的表达均明显增加。此外,K562R细胞BCR-ABL基因的第696位发生A→C点突变,该位点突变导致激酶区第231位氨基酸由赖氨酸取代原有的天冬酰胺。结论成功建立了耐伊马替尼细胞系K562R。K562R细胞具有交叉耐药性和对抗伊马替尼诱导的细胞凋亡等特征。K562R细胞BCR-ABL基因序列发生点突变,MDR和p-GP等基因表达亦明显升高。     

肺癌组织及外周血多药耐药基因相关蛋白的表达及其相关性研究

目的 检测多药耐药相关蛋白(MRP)和肺耐药蛋白(LRP)在肺癌组织及外周血中的表达及其相关性,探讨其临床意义.方法 采用逆转录一聚合酶链反应(RT-PCR)检测47例患者肺癌组织及外周血的MRP、LRP mRNA的表达水平.结果 肺癌组织及外周血,MRP mRNA的阳性表达率分别为74.5％(35/47)和70.2％...     

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。     

耐阿霉素人骨肉瘤细胞株的建立及其耐药机制探讨

目的诱导并建立耐阿霉素(ADM)的人骨肉瘤细胞株并探讨其耐药机制。方法采用逐步增加药物剂量冲击诱导方法诱导人成骨肉瘤原代Saos-2细胞株;MTT法检测原代与耐药细胞株对ADM、顺铂(DDP)、甲氨蝶呤(MTX)、异环磷酰胺(IFO)、表柔比星(EPI)、比柔比星(THP)、紫杉醇(Paclitaxel,PTX)药物敏感性;利用光学显微镜、透射电镜观察细胞形态及超微结构变化;RT-PCR和IHC法分别检测多药耐药基因1(MDR1)、多药耐药相关蛋白(MRP)基因及其相应蛋白(P-gp)、MRP的表达。结果经167d的诱导,建立Saos-2/ADM1、Saos-2/ADM4细胞株,其对ADM的耐药指数分别为原代细胞株的49.8和74.6倍;耐药细胞株对MTX、EPI、THP、PTX亦产生不同程度的交叉耐药(P<0.05),对DDP仍然敏感(P>0.05);光镜观察Saos-2/ADM1、Saos-2/ADM4细胞株细胞体积增大,多核现象较原代Saos-2细胞株明显增加;透射电镜显示Saos-2/ADM1、Saos-2/ADM4细胞株表面突起较原代Saos-2细胞株减少、且核仁增大增多;细胞生长曲线显示耐药细胞株增殖能力下降。MDR1mRNA、MRPmRNA和P-gp、MRP在各耐药细胞株表达阳性。结论MDR1 mRNA、MRP mRNA及其相应蛋白参与了耐药细胞株耐药的形成,这些骨肉瘤耐药细胞株为进一步研究骨肉瘤耐药特征及逆转方法打下了基础。     

Inhibit multidrug resistance and induce apoptosis by using glycocholic acid and epirubicin.

Cancer-cell resistance to chemotherapy limits the efficacy of cancer treatment. The primary mechanisms of multidrug resistance (MDR) are "pump" and "non-pump" resistance. We evaluated the effects and mechanisms of glycocholic acid (GC), a bile acid, on inhibiting pump and non-pump resistance, and increasing the chemosensitivity of epirubicin in human colon adenocarcinoma Caco-2 cells and rat intestine. GC increased the cytotoxicity of epirubicin, significantly increased the intracellular accumulation of epirubicin in Caco-2 cells and the absorption of epirubicin in rat small intestine, and intensified epirubicin-induced apoptosis. GC and epirubicin significantly reduced mRNA expression levels of human intestinal MDR1, MDR-associated protein (MRP)1, and MRP2; downregulated the MDR1 promoter region; suppressed the mRNA expression of Bcl-2; induced the mRNA expression of Bax; and significantly increased the Bax-to-Bcl-2 ratio and the mRNA levels of p53, caspase-9 and -3. This suggests that GC- and epirubicin-induced apoptosis was mediated through the mitochondrial pathway. We conclude that simultaneous suppression of pump and non-pump resistance dramatically increased the chemosensitivity of epirubicin. A combination of anticancer drugs with GC can control MDR via a mechanism that involves modulating P-gp and MRPs as well as regulating apoptosis-related pathways.     

Modulation of P-glycoprotein-mediated multidrug resistance in K562 leukemic cells by indole-3-carbinol

Resistance to chemotherapeutic drugs is one of the major problems in the treatment of cancer. P-glycoprotein (P-gp) encoded by the mdr gene is a highly conserved protein, acts as a multidrug transporter, and has a major role in multiple drug resistance (MDR). Targeting of P-gp by naturally occurring compounds is an effective strategy to overcome MDR. Indole-3-carbinol (I3C), a glucosinolates present in cruciferous vegetables, is a promising chemopreventive agent as it is reported to possess antimutagenic, antitumorigenic, and antiestrogenic properties in experimental studies. In the present investigation, the potential of I3C to modulate P-gp expression was evaluated in vinblastine (VBL)-resistant K562 human leukemic cells. The resistant K562 cells (K562/R10) were found to be cross-resistant to vincristine (VCR), doxorubicin (DXR), and other antineoplastic agents. I3C at a nontoxic dose (10 x 10(-3) M) enhanced the cytotoxic effects of VBL time dependently in VBL-resistant human leukemia (K562/R10) cells but had no effect on parent-sensitive cells (K562/S). The Western blot analysis of K 562/R 10 cells showed that I3C downregulates the induced levels of P-gp in resistant cells near to normal levels. The quantitation of immunocytochemically stained K562/R10 cells showed 24%, 48%, and 80% decrease in the levels of P-gp by I3C for 24, 48, and 72 h of incubation. The above features thus indicate that I3C could be used as a novel modulator of P-gp-mediated multidrug resistance in vitro and may be effective as a dietary adjuvant in the treatment of MDR cancers.     

Mitochondrial localization and activity of P-glycoprotein in doxorubicin-resistant K562 cells

It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170 kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.