Early infiltration of renal allografts with 27E10-positive macrophages and graft outcome

Abstract Recently, we have demonstrated that acute cellular rejection is correlated with a massive infiltration of 27E10-positive macrophages. To examine the distribution of macrophage differentiation markers in the infiltrate in the very early post-transplantation period, two biopsies were taken intraoperatively, approximately 3 h following reperfusion, in each of 16 renal transplant recipients. One biopsy was taken for conventional histology and the other biopsy was snap-frozen. The sections were stained using an ABC indirect immunoperoxidase technique. A panel of monoclonal antibodies against three macrophage differentiation markers (27E10, 25 F 9 and RM3/1) was used to stain the sections. Using the early inflammation macrophage marker 27E10, there was an unexpected strong staining in 3 out of 16 biopsies. This severe infiltration of 27E10-positive macrophages with 10–20 macrophages per high power field (compared to 0–2 in others) was correlated in all cases with a poor outcome of the graft. All seven kidneys with no 27E10-positive infiltration showed a good function 6 weeks post-transplantation. The other macrophage markers, 25 F 9 and RM3/1, showed a less marked correlation with graft outcome. In conclusion, a massive infiltration of renal allografts with 27E10-positive macrophages 1 h post-transplantation may be a very early predictor of poor graft outcome.     

肺癌化疗耐药分子标志及其逆转研究

目的:探讨肺癌化疗耐药分子标志及其逆转的临床与基础应用评价。方法:采用国内外相关文献,结合我们在这一领域长期研究,深入归纳与分析。结果与结论:肺癌化疗耐药主要分子标志有多药耐药基因(MDR1)表达增加,多药耐药相关蛋白(MRP)基因表达增加,谷胱甘肽解毒酶系统活性增强和线粒体凋亡途径异常,综合采用逆转耐药分子,耐药基因siRNA和中医药结合的逆转耐药策略,为彻底攻克肺癌耐药奠定坚实研究基础。     

耐药相关蛋白P-gp、MRP、LRP在非小细胞肺癌组织中的表达及意义

Objective： To explore the expression and significance of the multidrug resistance-related proteins P-glycoprotein （P-gp）, multidrug resistance-related protein （MRP）, lung resistance protein （LRP）in human non-small cell lung cancer （NSCLC） tissues and paratumor tissues. Methods： Immunohistochemistry （IHC） was used to examine the expression level of proteins P-gp, MRP and LRP in 43 samples of NSCLC and 15 samples of paratumor tissues. Results： The expression rates of P-gp, MRP and LRP in 43 tumor tissues were 74.42% （32/43）, 67.44% （29/43） and 88.37% （38/43）, respectively, while in 15 paratumor tissues were 13.33% （2/15）, 20.00% （3/15） and 6.67% （1/15）, respectively. There was significant difference in the expression of proteins （P-gp, MRP and LRP） between lung cancer tissues and paratumor tissues （P 〈 0.05）. The expression of proteins P-gp, LRP in lung adenocarcinoma were higher than that in other pathological carcinomas （P 〈 0.05）. The expression of protein MRP was not related to pathological type, clinical stage and classification of histodifferentiation （P 〉 0.05）. Conclusion： Multidrug resistance is more common in NSCLC. The proteins of P-gp, MRP and LRP participated in the formation of multidrug resistance in lung cancer. Detection of multidrug resistance-related proteins in lung cancer tissues may be useful to choice drugs.     

葛根素注射液对K562/Adr细胞化疗药物敏感性及机理研究

目的：通过观察葛根素注射液协同化疗药物对K562／Adr白血病耐药细胞的抑制作用，探讨葛根素增加白血病耐药细胞对化疗药物的敏感性及作用机理。方法：选K562／Adr耐药细胞株作为靶细胞，采用MTT法观察不同浓度的葛根素注射液对K562／Adr耐药细胞增殖的影响，及细胞对化疗药物的敏感性。RT—PCR的方法检测葛根素对耐药相关基因（MDR1／P-gp、MRP）表达的影响。结果：①MTT法结果显示低到中等浓度的葛根素（25～100μg／mL）对15562／Adr耐药株的生长与对照组相比无显著差异（P〉0.05），但当葛根素浓度提高至250μg／mL以上时，对细胞则出现增殖抑制作用，且随浓度增加抑制作用增强。②用不同浓度的葛根素和化疗药物（Adr 1μg/mL）合用时结果显示，低浓度（25～50μg／mL）的实验组与对照组无显著差异（P〉0．05）；随着葛根素浓度增加（100～500μg／mL），Adr对细胞的抑制作用明显，即K562／Adr对Adr的敏感性与葛根素呈剂量依赖吴系。③K562／Adr细胞经葛根素（100μg/mL）处理1周后，MDR1基因表达受到抑制，是未经处理细胞的2．5倍，MRP基因表达在处理前后无明显变化；当处理1个月后，细胞中MDR1、MRP两基因表达均明显低于未经处理过的细胞，分别是未经处理细胞的2．6倍和3．5倍。结论：葛根素注射液在一定浓度下，能直接抑制白血病耐药细胞增殖，并能提高耐药细胞对化疗药物的敏感性；其机制可能通过对耐药相关基因（MDR1、MRP）表达抑制来起作用。     

阿司匹林对顺铂抗卵巢癌作用增敏效应的体外实验研究

目的 观察阿司匹林对顺铂抗卵巢癌效应的影响并对其相关机制进行初步探讨。方法 MTT法检测阿司匹林＋顺铂与单纯顺铂或阿司匹林对卵巢癌顺铂耐药细胞株（COC1／DDP）细胞生长曲线的影响，流式细胞仪分别检测不同组别多药耐药相关蛋白P—gp、MRP、GSTπ及核转录因子NF—κB的表达变化。结果 单纯阿司匹林和顺铂对COC1／DDP细胞生长的影响较弱（P〉0．05），阿司匹林＋顺铂显著抑制COC1／DDP细胞生长（P〈0．05）。P—gp在COC1／DDP细胞系呈低表达状态，MRP、GSTπ表达率在各组间差异无显著性（P〉0．05），阿司匹林＋顺铂组核转录因子NF-κB的表达量降低（P〈0．05）。结论 阿司匹林可以增强顺铂的抗卵巢癌作用，其作用可能与核因子NF—κB的表达量降低有关。     

多药耐药相关蛋白-2在乳腺癌组织中的表达及与预后关系

目的:研究多药耐药相关蛋白-2(Multidrug resistance-associated protein2,MRP2)在乳腺癌组织中的表达,评估其在乳腺癌预后中的作用。方法:采用免疫组织化学方法(I mmuno Histo Chemistry,I HC)检测47例手术切除的乳腺癌组织中MRP2的表达,并分析其与临床、病理特征的关系及对预后的影响。结果:MRP2在乳腺癌组织中的阳性表达率为85.1%(40/47),其中高表达者25例(53.2%);MRP2表达与月经状况、肿瘤大小、腋淋巴结转移、组织分级和激素受体状况均无关(P>0.05);Kaplan-Meier生存分析结果表明MRP2表达与无病生存期和总生存期均无关(P>0.05);COX多因素分析显示只有肿瘤大小和腋淋巴结转移和无病生存期和总生存期明显相关(P<0.05)。结论:MRP2在乳腺癌组织中具有一定的表达水平,但与乳腺癌患者预后无关。     

Homocamptothecin-daunorubicin association overcomes multidrug-resistance in breast cancer MCF7 cells

The multidrug-resistance (MDR) status of a novel camptothecin analogue, homocamptothecin (hCPT), was investigated in human colon adenocarcinoma HT29 cells, myelogenous leukemia K562 cells and breast carcinoma MCF7 cells. The cytotoxicity of hCPT was not sensitive to the MDR status in K562 cell lines. However, its cytotoxicity was altered by MRP1, but not Pgp, in naturally MRP1-expressing HT29 cells, and etoposide- and doxorubicin-resistant MCF7/VP and MCF7/DOX cells, respectively. These cells were sensitized to hCPT in presence of MK571, probenecid but not verapamil. These results led to consider hCPT as a substrate for MRP1 and a potential modulator of MRP1 activity. The relationship between the cytotoxic effect of anthracyclines and their nuclear localization had been previously demonstrated. We show that MRP1 mediated the daunorubicin (DNR) efflux in MCF7/VP and MCF7/DOX cells. The combination of sub-toxic doses of hCPT with DNR resulted in the potentiation of DNR activity, well-correlated with an increase in its nuclear accumulation in MCF7/VP cells. Simultaneous pattern was shown to provide higher cytotoxic response than sequential one. In agreement, hCPT increased also the DNR nuclear accumulation in low MRP1-expressing MCF7/DOX cells. However, the enhancement of cytotoxicity in the DNR-hCPT combination was poorly correlated with the nuclear concentration of DNR in MCF7/DOX cells. In addition to the increase in DNR accumulation, the potentiation of DNR activity by hCPT in MCF7/DOX cells implied a synergistic mechanism between both drugs. These data suggest that the present topoisomerase I/II inhibitors combination may be of clinical interest to overcome MDR phenotype in DNR-treated breast cancer patients.     

The influence of P-glycoprotein on morphine transport in Caco-2 cells. Comparison with paclitaxel

In vitro monolayer studies using Caco-2 cells were employed here to explore P-glycoprotein mediated transport of morphine. Bi-directional transport studies of 10-75 microM morphine showed efflux to be twofold higher than influx (4 x 10(-6) compared to 2 x 10(-6) cm/s) and cellular accumulation in the efflux direction was eightfold higher. The cyclosporin analogue (PSC-833) equilibrated morphine transport in both directions. Depletion of intracellular glutathione had a greater effect on increasing cellular morphine accumulation than P-glycoprotein inhibitors, suggesting a role for glutathione in morphine transport. P-glycoprotein had a substantially greater effect on paclitaxel accumulation, efflux and bi-directional transport than for morphine. Paclitaxel transport was below detection (<0.1 x 10(-6) cm/s) in the influx direction, yet efflux was very high (18.4 x 10(-6) cm/s) and P-glycoprotein inhibition increased accumulation >100-fold. These results reinforce the substantial role P-glycoprotein has in paclitaxel transport. Conversely, P-glycoprotein regulated morphine transport is weak. Nevertheless, morphine transport rates could be doubled when administered with P-glycoprotein substrates. Therefore, increased analgesia through P-glycoprotein inhibition should be possible.     

Coordinate regulation of UDP-glucuronosyltransferase UGT1A6 induction by 3-methylcholanthrene and multidrug resistance protein MRP2 expression by dexamethasone in primary rat hepatocytes

Concentration-dependent regulation of 3-methylcholanthrene (MC) inducibility of UDP-glucuronosyltransferase UGT1A6 by the synthetic glucocorticoid, dexamethasone (DEX) was studied. Treatment of cultured rat hepatocytes with MC, 0.1, 1, and 10 microM DEX, and MC combined with DEX, resulted in different induction patterns measured in the intact cells compared to that observed in the microsomes prepared from the same cells. DEX treatment in various concentrations caused a concentration-dependent increase in p-nitrophenol (p-NP) conjugation in intact cells (3-, 4-, and 5-fold over control, respectively), and it positively regulated MC induction (4-, 5-, and 6-fold over control, respectively). In contrast, DEX had smaller effect on microsomal p-NP conjugation (115, 200, 220% of control, respectively) and although MC induction was increased significantly by 0.1 microM DEX (520% of control), but higher concentrations of DEX (10 microM) decreased the degree of induction to 410%. Similar results obtained from in vivo experiments showed that at high DEX concentration (100mg/kg), the rate of MC induction (540%) decreased (420%). Permeabilization of the plasma membrane resulted in a 15-fold increase of p-NP conjugation indicating the importance of transport in the rate of overall p-NP elimination, and the induction pattern was similar to that observed in microsomes isolated from cells. Hyper-osmolarity (405 mOsmol/L) led to a 3-fold decrease of p-NP conjugation, the loss of DEX inducibility and reduction of the MRP2 protein level. Our results suggest coordinated regulation of UGT1A6 inducibility and substrate or product transport by DEX.     

Increased rate of glutathione synthesis from cystine in drug-resistant MCF-7 cells

The rate of glutathione synthesis was determined in drug-sensitive and -resistant MCF-7 cells by monitoring the rate of label uptake from [3,3'-13C(2)]-cystine using NMR spectroscopy and mass spectrometry. Compared with the wild-type human mammary adenocarcinoma cell line (MCF-7wt), the isotope incorporation rate was increased 1.6-, 2.4-, and 5.3-fold in the etoposide-resistant MCF-7 cell line (MCF-7vp), doxorubicin-resistant MCF-7 cell line (MCF-7adr), and 4-hydroperoxycyclophosphamide-resistant MCF-7 cell line (MCF-7hc), respectively. The increase in glutathione metabolism in the MCF-7hc line correlated with steady-state levels as determined by biochemical assay. In contrast, both the MCF-7vp and MCF-7adr lines showed increased metabolic synthesis of glutathione but displayed lower steady-state levels compared with the MCF-7wt line. The increased synthetic rates of all resistant lines reflected, in part, contributions from the increased activities of both gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase. These results emphasize the importance of monitoring glutathione metabolic rates, rather than steady-state levels of enzymes or substrates, for assessing drug resistance.