吉西他滨联合卡培他滨治疗蒽环类与紫杉类耐药转移性乳腺癌的临床观察

为了探讨吉西他滨联合卡培他滨方案治疗蒽环类与紫杉类药物耐药的晚期转移性乳腺癌的近期疗效及不良反应,选取37例耐药的晚期乳腺癌患者,接受吉西他滨1 000 mg/m2,静脉滴入,d1、d8;卡培他滨950 mg/m2,2次/d,口服,d1～d14.每3周重复.2个周期化疗后,总有效率(CR+PR)为29.7%(11/37),疾病控制率(CR+PR+SD)为70.3%(26/37).中位进展时间为6.9个月,中位生存时间为15.5个月.最常见的毒性为骨髓抑制、手足综合征及胃肠道反应.初步研究结果提示,吉西他滨联合卡培他滨是治疗蒽环类和紫杉类耐药的晚期乳腺癌的有效方法,不良反应可以耐受.     

核受体PXR对乳腺癌耐药性影响的研究

目的:探讨上调核转录因子受体PXR的表达对人乳腺癌细胞MCF-7和MDA-MB-231化疗效果的影响。方法:用蛋白质印迹法检测PXR蛋白在2株乳腺癌细胞中的表达,通过PXR激动剂SR12813上调PXR受体的表达;用实时荧光定量PCR技术检测SR12813预处理前后2株细胞中耐药基因MDR1和BCRP表达的变化;CCK-8方法检测2株细胞耐药性的变化;用流式细胞术研究PXR对2株细胞凋亡的影响。结果:MCF-7和MDA-MB-231均有PXR表达;经SR12813 0.3μmol/L预处理后,2株细胞中PXR蛋白表达明显增强,耐药基因MDR1和BCRP表达显著升高(P=0.000),4-羟基他莫昔芬对MCF-7细胞48及72h的IC50分别为(11.57±0.83)和(9.70±0.68)μmol/L,多西他赛对MDA-MB-231细胞24、48和72h的IC50分别为(0.67±0.091)、(0.53±0.056)和(0.46±0.040)μg/mL,与对照组相比,均明显增加,P值均<0.05。经SR12813预处理后,药物对2株细胞诱导的凋亡率分别为(17.00±0.74)%和(7.69±0.54)%,与单药组相比,差异有统计学意义,P<0.05。结论:核转录因子受体PXR表达对PXR阳性乳腺癌的化疗敏感性具有重要影响,PXR抑制细胞凋亡可能是乳腺癌耐药的机制之一。     

PKD2 mediates multi-drug resistance in breast cancer cells through modulation of P-glycoprotein expression

Multi-drug resistance (MDR) represents a major obstacle for chemotherapeutic treatment of a wide variety of human cancers. Increased expression of drug efflux pumps, such as the P-glycoprotein (P-gp) have been linked to development of MDR. Herein, we have identified protein kinase D isoform 2 (PKD2) as an important regulator of MDR and P-gp expression in paclitaxel-treated breast cancer cell lines. PKD2 was expressed with the highest phosphorylated activation status in the MDA-MB-231 cell line. MDA-MB-231 cells were also found to exhibit the highest level of resistance to an array of chemotherapeutic drugs. To further characterize the relationship between PKD2 activation and MDR, we next focused on the effects of the chemotherapeutic agent paclitaxel in MDA-MB-231 cells. Treatment with paclitaxel was shown to induce both PKD2 phosphorylation and P-gp expression in a time-dependent manner. Importantly, shRNA-mediated knockdown of PKD2 in MDA-MB-231 cells resulted in a significant decrease in resistance to paclitaxel, evident as significant decreases in both the IC₅₀ value and the resistance index (RI). Concurrent with the decrease in drug resistance, paclitaxel-induced expression of P-gp was also significantly reduced in PKD knockdown cells. These results indicate that PKD2 is required for paclitaxel-induced MDR and expression of P-gp. Therefore, modulation of PKD2 activity represents an attractive therapeutic strategy for improvement of the clinical effectiveness of chemotherapy.     

Establishment of drug resistance in human gastric and colon carcinoma xenograft lines

We established multidrug-resistant human gastric and colon xenograft lines by means of intratumoral injections of four agents, doxorubicin (DXR), cisplatin (CDDP), 5-fluorouracil (5-FU) and mitomycin C (MMC), into subcutaneous SC1NU and SW480 tumors once a week or less. Such intermittent drug exposure is commonly used in clinical chemotherapeutic protocols. All xenograft lines acquired resistance to the injected drugs as evaluated by in vivo drug-resistance tests. Many of the drug-resistant lines showed various patterns of cross resistance to other drugs. In order to analyze the mechanism of resistance in vivo, we investigated the expression of drug resistance gene, which has been extensively studied in vitro. We used four complementary DNAs (cDNAs) for multidrug resistance (MDR1), glutathione S-transferase-pi (GST-pi), thymidylate synthase (TS) and dehydrofolate reductase (DHFR), as probes. We observed GST-pi, DHFR and TS mRNA expression at various levels, but MDR1 mRNA expression was found only in SW480/DXR by the method of poly (A+) RNA selection. Four resistant SW480 lines had higher TS mRNA expressions. Six resistant lines had stronger GST-pi mRNA expression. Five resistant lines had higher DHFR mRNA expression. Drug resistance genes related to the treated drug were also expressed in this in vivo model; MDR1 in SW480/DXR, GST-pi in SW480/CDDP and in SC1NU/CDDP and TS in SW480/5-FU. In contrast to in vitro resistant lines which have been reported as models of drug resistance, the expression of drug resistance genes in vivo was not always correlated to the acquisition of cross resistance. These resistant xenograft lines and the methods developed to induce drug resistance in vivo should be useful for studies on the mechanism of drug resistance in the clinical setting.     

结肠癌耐药细胞株LoVo/L-OHP的建立及其耐药机制

目的：建立获得性奥沙利铂（L-OHP）耐药的结肠癌细胞模型LoVo/L-OHP,并初步研究其耐药机制。方法：采用L-OHP浓度递增法建立人结肠癌细胞耐药模型LoVo/L-OHP,观察其生长规律并绘制细胞生长曲线;用MTT法鉴定耐药细胞株耐药性并计算耐药指数（RI）;用半定量RT-PCR方法对部分耐药相关基因在耐药细胞及其亲本细胞中的表达情况进行分析。结果：成功建立了耐药的结肠癌细胞模型LoVo/L-OHP,LoVo/L-OHP细胞与LoVo细胞相比,生长缓慢,触角增多。通过RT-PCR半定量分析,P-gp、bcl-2、ERCC-1在LoVo/L-OHP中的表达上调,而p53基因表达下调。结论：LoVo/L-OHP细胞株耐药性稳定,耐药机制可能与P-gp、bcl-2、ERCC-1基因上调、p53基因下调多因素有关。     

结肠癌耐药细胞株LoVo/L-OHP的建立及其耐药机制

目的:建立获得性奥沙利铂(L-OHP)耐药的结肠癌细胞模型LoVo/L-OHP,并初步研究其耐药机制。方法:采用L-OHP浓度递增法建立人结肠癌细胞耐药模型LoVo/L-OHP,观察其生长规律并绘制细胞生长曲线;用MTT法鉴定耐药细胞株耐药性并计算耐药指数(RI);用半定量RT-PCR方法对部分耐药相关基因在耐药细胞及其亲本细胞中的表达情况进行分析。结果:成功建立了耐药的结肠癌细胞模型LoVo/L-OHP,LoVo/L-OHP细胞与LoVo细胞相比,生长缓慢,触角增多。通过RT-PCR半定量分析,P-gp、bcl-2、ERCC-1在LoVo/L-OHP中的表达上调,而p53基因表达下调。结论:LoVo/L-OHP细胞株耐药性稳定,耐药机制可能与P-gp、bcl-2、ERCC-1基因上调、p53基因下调多因素有关。     

P-糖蛋白和肺耐药蛋白在浸润性乳腺癌中的表达及其临床意义

目的探讨P-糖蛋白(P-gp)和肺耐药蛋白(LRP)在浸润性乳腺癌中的表达情况及其临床意义。方法采用搓网法获取乳腺癌手术切除标本制成的完整单细胞悬液,再用流式细胞技术对40例乳腺癌组织及相应癌旁组织中P-gp、LRP的表达情况进行定量分析。结果乳腺癌组织P-gp、LRP表达与相应癌旁组织比较,差异均有统计学意义(P-gp:t=-7.777,LRP:t=-9.861,P均0.050)。在不同年龄、肿瘤大小、病理类型、临床分期、分化程度和雌、孕激素受体状态的患者间,两种耐药相关蛋白的表达差异均无统计学意义(P0.050)。有淋巴结转移组P-gp表达与无淋巴结转移组比较,差异无统计学意义(t=0.102,P=0.919)。有淋巴结转移组LRP的表达为(41.2±18.5)%,无淋巴结转移组为(30.1±15.4)%,有淋巴结转移组LRP表达显著高于无淋巴结转移组(t=2.074,P=0.045)。乳腺癌组织P-gp与LRP的表达间存在正相关性(r=0.698,P0.001)。结论本研究结果可为寻找浸润性乳腺癌的有效辅助化疗药物提供参考。     

Reversal of LRP-associated drug resistance in colon carcinoma SW-620 cells

Resistance to multiple drugs is mediated by lung resistance-related protein (LRP) as well as P-glycoprotein (P-gp) and multidrug resistance protein (MRP). The levels of expression of LRP mRNA and LRP in a human colon carcinoma cell line, SW-620, were increased by the differentiation-inducing agent, sodium butyrate (NaB). Treatment of SW-620 cells with NaB for 2 weeks conferred resistance to adriamycin (ADM) and VP-16. The resistance was almost completely reversed by PAK-104P, a pyridine analog, but not by cepharanthine. ADM accumulated mainly in the nuclei of SW-620 cells not treated with NaB and in the cytoplasm of SW-620 cells treated with NaB. When the NaB-treated SW-620 cells were incubated with ADM in the presence of PAK-104P, the accumulation of ADM in nuclei was substantially increased. Isolated nuclei from untreated cells accumulated more ADM than nuclei from NaB-treated cells. Efflux of ADM from the nuclei isolated from NaB-treated cells was enhanced. PAK-104P and an antibody against LRP increased the accumulation of ADM in the isolated nuclei from NaB-treated cells, and inhibited the enhanced efflux of ADM from the nuclei. These findings suggest that at least in part, PAK-104P reverses LRP-mediated drug resistance by inhibiting the efflux of ADM from nuclei. PAK-104P may be useful for reversing MDR in tumors that overexpress LRP.     

P-糖蛋白与肺耐药蛋白在乳腺癌组织中的表达及其与预后的关系

背景与目的:肿瘤细胞的多药耐药(multidrugresistance,MDR)现象是化疗失败的常见原因。P-糖蛋白(P-glycoprotein,P-gp)和肺耐药蛋白(lungresistanceprotein,LRP)的过度表达在乳腺癌的MDR中起着重要作用。本研究探讨P-gp与LRP在乳腺癌组织中的表达及其与预后的关系。方法:采用免疫组织化学LSAB法检测151例乳腺癌石蜡包埋组织和40例乳腺正常组织的P-gp和LRP表达水平,并将结果与临床病理资料和预后结合起来进行分析。结果:在151例乳腺癌组织中P-gp阳性62例(40.8%),40例正常乳腺组织中P-gp阳性1例(2.5%),两组间差异有显著性(χ2=21.27,P<0.01);151例乳腺癌组织中LRP阳性121例(80.1%),40例正常乳腺组织中LRP阳性22例(55.0%),两组间差异有显著性(χ2=10.62,P<0.01)。P-gp表达与患者年龄、肿块大小、组织学类型、淋巴结转移、组织学分级、临床分期、ER状态和预后等因素无关。在有淋巴结转移的70例乳腺癌组织中LRP阳性62例(88.6%),无转移的81例中LRP阳性59例(72.8%),两组间差异有显著性(χ2=4.891,P<0.05)。在LRP阳性的121例乳腺癌组织中中P-gp阳性55例(45.5%),LRP阴性的30例中P-gp阳性7例(23.3%),两组间差异有显著性(χ2=3.990,P<0.05)。LRP的表达与淋巴结转移(r=0.197,P<0.05)和P-gp表达(r=0.179,P<0.05)呈正     

外周血P糖蛋白功能检测及其与乳腺癌多药耐药性的关系

目的 建立人外周血NK细胞P糖蛋白功能检测的方法,分析P糖蛋白功能与乳腺癌患者原发性多药耐药之间的关系.方法 选择经含蒽环类和紫杉类药物联合化疗的初治乳腺癌患者16例,治疗后化疗敏感和耐药者各8例.采集患者外周血标本,分离NK细胞,与荧光物质罗丹明123(Rh123)孵育,应用流式细胞仪检测不同时间点外周血NK细胞内Rh123的荧光强度;以Rh123的荧光强度(F)和相应时间点(t)作图,通过曲线估计回归分析,构建P糖蛋白药泵功能检测的最佳数学模型,计算每例患者相应的外排速率常数;分析化疗敏感组和耐药组患者速率常数的差异,以及以速率常数预测乳腺癌原发性多药耐药的可行性.结果 所有患者的NK细胞在撤离Rh123后60min,基本不再外排Rh123,NK细胞内Rh123的蓄积量、排出量和残留量均与化疗敏感性之间无明显相关性.通过回归分析建立了P糖蛋白功能检测的最佳数学模型F1=F0·e-kt(F1为t时间点的荧光强度,k为外排速率常数),化疗敏感组与耐药组患者外排速率常数差异有统计学意义(P=0.025).以k>3.9为标准,诊断乳腺癌原发性多药耐药,其诊断敏感性为75.0%,特异性为100%.准确率87.5%.结论 外周血NK细胞P糖蛋白药泵外排功能与乳腺癌患者原发性多药耐药密切相关,外排速率常数可能是预测乳腺癌化疗敏感性的理想指标.     

紫杉类药物在乳腺癌治疗中的耐药机制

目前研究提示紫杉类耐药可能与多药耐药相关蛋白(MRP)表达增高、Ⅲβ-微管过表达、微管相关蛋白tau的过表达有关,而与HER2的表达成负相关.针对这些耐药靶点可逆转或者延缓紫杉类药物耐药的发生,为临床上更好地使用紫杉类药物提供一定参考.     

结肠癌HT-29细胞中PIDD蛋白的表达及其对细胞耐药性的影响

目的:以结肠癌HT-29细胞株为载体,研究PIDD(p53-induced protein with a death domain)蛋白在结肠癌耐药中的作用机制.方法:构建并转染PIDD基因的小干扰RNA(small interfering RNA,siRNA)片段,用Western印迹法检测其干扰效果.通过MTT比色法检测细胞对化疗药物的敏感性,并用凝胶阻滞分析(electrophoretic mobility shift assay,EMSA)法检测核因子NF-κB活性的变化.结果:RNA干扰后给予细胞药物刺激,细胞核中PIDD表达下降,核因子NF-κB活性明显降低,细胞耐药性下降.结论:转染siRNA后,HT-29细胞对5-氟尿嘧啶(5-fluorouracil, 5-FU)的敏感性增加,此作用可能与细胞核PIDD复合体减少、核因子NF-κB活性减低及细胞凋亡增多有关.     

LRRK2在乳腺癌细胞增殖和耐药中的作用及其机制研究

目的:探讨LRRK2在乳腺癌组织中的表达及其对乳腺癌细胞增殖、凋亡及耐药的作用和分子机制.方法:采用实时荧光定量PCR(qRT-PCR)和Western blot检测了48例乳腺癌组织及其配对正常组织中LRRK2表达;并检测人正常乳腺上皮细胞MCF-10A、乳腺癌细胞株MCF-7、MDA-MB-231、BT-483及阿...     

转移性乳腺癌P 糖蛋白表达与化疗疗效的关系

背景与目的临床研究表明,乳腺癌P 糖蛋白(P glycoprotein,P gp)表达与化疗效果、缓解率、生存期及估计预后有关.但有关P gp表达与乳腺癌转移部位及化疗效果的临床研究报道较少,本研究的目的是探讨乳腺癌P gp表达及其与不同转移部位乳腺癌化疗疗效的关系.方法应用SABC法检测46例转移性乳腺癌术后组织中P gp表达.43例使用联合方案化疗.环磷酰胺600mg/m2ivd1,吡喃阿霉素60mg/m2ivd1,5 氟尿嘧啶600mg/m2ivd1、8,每3周重复,至少2个周期,对比分析P gp表达与疗效的关系.结果P gp阳性表达率56 5%,肝和肺转移者阳性表达明显高于皮肤和淋巴结转移者(P=0 049).可评价疗效的43例患者化疗有效率58 1%,P gp阴性组疗效明显优于P gp阳性组(P<0 01).有肝和肺等内脏转移者有效率明显低于皮肤和浅表淋巴结转移者(P<0 05).术后曾接受辅助性CAF或CMF方案化疗者复发或远处转移后化疗疗效无显著性差异(P>0 05).结论P gp表达可作为评估转移性乳腺癌多药耐药,指导临床用药及判断预后的参考指标.     

Clinical significance of P-glycoprotein expression in metastatic breast carcinoma

Multidrug resistance(MDR)is one of the importantfactors,limiting the chemotherapeutic efficacy[l].Thereare various resistance related-proteins overexpression inthe tissue of breast carcinoma[2].MDR phenotype isassociated with overexpression of mdr-1gene andP-glycoprotein,encoding product of mdr-1,which is oneof the major mechanisms of MDR in breast carcinoma[3].We have had partly understanding about the mechanismof breast carcinoma associated with P-gp expression bystudying the mdr-1gene,t…     

乳腺浸润性微乳头状癌耐药机制研究

目的探讨miRNAs在紫杉醇耐药的乳腺浸润性微乳头状癌中的影响。方法收集2011年1月至2014年12月年天津市肿瘤医院11例乳腺浸润性微乳头状癌(invasive micropapillary carcinoma,IMPC)患者的组织切片,实时荧光定量PCR法检测其中miRNAs的表达。采用Target Scan和mi RDB预测miRNAs下游靶基因。结果根据前期研究结果筛选出13个耐药相关的miRNAs,其中与紫杉醇敏感的IMPC相比,miR-744-5p在紫杉类耐药IMPC中的表达水平升高了(t=-2.650,P=0.029),差异有统计学意义。通过生物信息学分析预测发现,miR-744-5p可能通过调节CPFS4,Upf1和PP2A等靶基因影响紫杉醇耐药。结论 miR-744-5p可能参与调节IMPC的紫杉醇耐药,并可能成为其分子标记物。     

Selective modulation of P-glycoprotein-mediated drug resistance.

Multidrug resistance associated with the overexpression of the multidrug transporter P-glycoprotein is a serious impediment to successful cancer treatment. We found that verapamil reversed resistance of CEM/VLB(100) cells to vinblastine and fluorescein-colchicine, but not to colchicine. Chlorpromazine reversed resistance to vinblastine but not to fluorescein-colchicine, and it increased resistance to colchicine. Initial influx rates of fluorescein-colchicine were similar in resistant and parental cells, whereas vinblastine uptake was about 10-fold lower in the resistant cells. These results provide indirect evidence that fluorescein-colchicine is transported from the inner leaflet of the membrane and vinblastine from the outer membrane leaflet. Verapamil inhibited fluorescein-colchicine transport in inside-out vesicles made from resistant cells, whilst chlorpromazine was found to activate the transport of fluorescein-colchicine. The chlorpromazine-induced activation of fluorescein-colchicine transport was temperature-dependent and may reflect its interaction with phospholipids localised in the same bilayer leaflet. Conversely, chlorpromazine localisation in this leaflet may be responsible for its allosteric inhibition of vinblastine transport from the opposing membrane leaflet. The proposed relationship between the selectivity of modulation of P-glycoprotein and the membrane localisation of the cytotoxic drug substrates and modulators may have important implications in the rational design of regimes for the circumvention of multidrug resistance clinically.     

PSC-833, a frontier in modulation of P-glycoprotein mediated multidrug resistance

The expression of drug efflux mechanisms by cancer cells during chemotherapy leads to multidrug resistance (MDR) and constitutes a major obstacle in the effective treatment of cancer. The most widely characterized drug efflex pump is P-glycoprotein (P-gp) and efforts are being directed towards identifying agents that reverse P-gp mediated drug resistance. PSC-833 is a non-immunosuppressive cyclosporin derivative that potently and specifically inhibits P-gp. The current review focuses on the elucidation of the mechanism of action of PSC-833 as a potential MDR reversing agent, using syngeneic multidrug resistant sublines of MDA435 human breast adenocarcinoma cell line that express increasing levels of P-gp. In vitro experiments indicate that PSC-833 interacts directly with P-gp with high affinity and probably interferes with the ATPase activity of P-gp. Studies in multidrug resistant tumor models confirm P-gp as the in vivo target of PSC-833 and demonstrate the ability of PSC-833 to reverse MDR leukemias and solid tumors in mice. Presently, PSC-833 is being evaluated in the clinic.     

Obviation of drug resistance and affinity purification of P-glycoprotein by isoquinolinesulfonamides.

A newly synthesized isoquinolinesulfonamide named H-85; N-[2-[N-formyl-N-[[3-(4-chlorophenyl)-2-propenyl] amino] ethyl]-5-isoquinolinesulfonamide was found to reverse drug resistance in multidrug resistant P388 murine leukemic cells (P388/ADR). The energy-dependent extrusion of [3H]vinblastine from P388/ADR-cells was significantly suppressed by 10 microM H-85 but not so the efflux from the sensitive P388 cells. A 140-kDa protein overexpressed in P388/ADR cells was photoaffinity labeled with a vinblastine analog; N-(P-azid-[3-125I]salicyl-N'-(beta-aminoethyl) vindesine and H-85 selectively inhibited photolabeling of the 140-kDa protein. This 140-kDa protein was purified to apparent homogeneity by succeeding steps of phosphocellulose, DEAE-cellulose, and W-66 (a derivative of H-85)-coupled sepharose chromatography. The purified 140-kDa protein proved to be immunopositive with the P-glycoprotein-specific monoclonal antibody, C219.