Drug transporter expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line

Ovarian cancer is characterized by the higher mortality among gynecological cancers. In results of MDR development during chemotherapy cancer cells become resistant to further treatment. Microarray techniques can provide information about MDR development at gene expression level. ABC and SLC transporters are most important proteins responsible for this phenomenon. In this study changes of ABC and SLC genes expression pattern in drugs resistant sublines of the A2780 ovarian cancer cell line were demonstrated. The cytostatic resistant sublines were generated by culture of A2780 cell line with an increasing concentration of the indicated drugs. As screening methods, we used Affymetrix U219 Human Genome microarrays. Independent t-tests were used to determinate statistical significances of results. Genes that expression levels were higher than assumed threshold (upregulated above threefold and downregulated under −3 fold) were visualized using scatter plot method, selected and listed in table. Between the ABC genes increased expression of seven genes and decreased expression of three genes were observed. Expression of two genes was increased or decreased depending on the cell line. We observed significant (more than tenfold) increase in expression of four ABC genes: ABCA8, ABCB1, ABCB4 and ABCG2 and decreased expression of ABCA3 gene. We also observed changes in expression of 32 SLC genes. Between them we observe increased expression of 17 genes and decreased expression of 15 genes. Expression of four genes was increased or decreased dependent on cell line. The expression of nine SLC genes increased or decreased very significantly (more than tenfold). Five genes were significantly upregulated: SLC2A9, SLC16A3, SLC16A14, SLC38A4 and SLC39A8. Four additional genes were significantly downregulated: SLC2A14, SLC6A15, SLC8A1 and SLC27A2. Expression profiles of these genes give strong arguments for assumption of correlation between expression of ABC and SLC genes and drug resistance phenomenon. Identifying correlations between specific drug transporters and cytostatic drug resistance will require further investigation.     

急性肺损伤肺间质巨噬细胞TLR4的表达及意义

目的 观察严重胸部创伤致急性肺损伤肺间质巨噬细胞TLR4基因及蛋白表达水平的变化,并探讨其意义。方法 建立严重胸部创伤致急性肺损伤模型。肺组织机械剪碎,然后用胶原酶、DNA酶消化肺组织,分离、培养肺间质巨噬细胞,利用免疫印迹蛋白、Northern Blot等分子生物学技术检测创伤前以及创伤后2、4、8、16和24 h肺泡巨噬细胞TLR4蛋白及基因表达。结果 利用小型多功能生物撞击机以400 kPa驱动压力对大鼠右侧上胸壁进行致伤,能够建立稳定可靠并且符合临床特点的严重胸部创伤模型;严重胸部创伤可以上调TLR4蛋白及基因在肺间质巨噬细胞内的表达,表达高峰在伤后8和16 h,伤后24 h恢复正常。结论 TLR4的上调参与了创伤后失控性炎症反应的病理生理过程。     

Post-transcriptional regulation of VEGF expression by oxidised LDL in human macrophages

BACKGROUND: Accumulation of oxidised low density lipoproteins (oxLDL) in macrophages and their subsequent transformation into lipid-filled foam cells is generally considered an early event in atherosclerosis. Vascular Endothelial Growth Factor (VEGF) may contribute to atherogenesis through increased vascular permeability, chemoattraction towards monocytes and intraplaque vessel formation. In this study we investigate the effect and regulation of VEGF expression in human macrophages stimulated with oxLDL. MATERIALS AND METHODS: Vascular Endothelial Growth Factor mRNA and protein expression was assayed using RT-PCR and ELISA, respectively. The activity of mitogen-activated protein kinases (MAPKs) was investigated using Western blots. RESULTS: In human monocyte-derived macrophages, oxLDL significantly increased VEGF mRNA expression and subsequent protein secretion in a concentration-dependent manner after 6 h and 18 h, respectively. Using an in vitro mRNA decay assay, we show that this oxLDL-induced VEGF expression partly is regulated through increased stability of the VEGF mRNA. Involvement of MAPKs has previously been implicated in the stabilisation of VEGF mRNA. Activity of the p38 MAPK, but not the c-jun-N-terminal kinase (JNK), increased in macrophages stimulated with oxLDL (50 micro g mL-1) for 5-15 min. Preincubation with SB202190 (20 micro M), a specific inhibitor of p38 MAPK, significantly decreased the oxLDL-induced VEGF mRNA expression by 40%. The prolonged half-life of VEGF mRNA, induced by oxLDL, was not inhibited by SB202190. CONCLUSIONS: OxLDL increases VEGF expression and p38 MAPK activity in human macrophages. The increased VEGF mRNA expression by oxLDL is mediated through at least two intracellular pathways, one involving p38 MAPK and another that independently of p38 MAPK activity increases VEGF mRNA stability.     

腹腔巨噬细胞在创伤后胰岛素抵抗中的作用

目的　研究小鼠创伤后腹腔巨噬细胞对胰岛素、胰岛素敏感性以及骨骼肌葡萄糖摄取量的影响 ;腹腔巨噬细胞在创伤后胰岛素抵抗中的作用。方法　将 4 0只健康C5 7BL/6J小鼠随机分为创伤组、培养液对照组、正常细胞对照组、实验组。使用DMEM培养液培养ANA - 1细胞。取部分ANA - 1细胞 ,装入透析袋中密闭 ,并置入创伤组小鼠腹腔内 ,然后行小鼠背部 15 %～ 2 0 %Ⅲ度烧伤。烧伤后 4h收集透析袋内ANA - 1细胞并注入实验组小鼠腹腔内 ;收集正常培养的ANA - 1细胞同样注入正常细胞对照组 ;培养液对照组腹腔注入同量DMEM培养液。 4h后分别采血和分离比目鱼肌。测定血糖和血清胰岛素以及离体骨骼肌葡萄糖摄取量。结果　正常细胞对照组与培养液对照组的血糖、血清胰岛素、胰岛素敏感指数 (ISI)、骨骼肌基础葡萄糖摄取量和胰岛素刺激下葡萄糖摄取量均无明显差异 ;实验组的血糖、血清胰岛素水平及骨骼肌基础葡萄糖摄取量显著高于正常细胞对照组和培养液对照组 ,而ISI水平及胰岛素刺激下的葡萄糖摄取量与正常细胞对照组和培养液对照组相比显著降低。结论　未激活的腹腔巨噬细胞对机体无明显影响 ,而创伤后激活的腹腔巨噬细胞可使正常小鼠产生明显的胰岛素抵抗 ,胰岛素敏感性和反应性显著降低 ,表明创伤后胰岛素抵抗与腹     

Interferon-gamma, tumor necrosis factor-alpha, and lipopolysaccharide promote chitotriosidase gene expression in human macrophages

Human chitotriosidase (Chit), a chitinolytic enzyme, is a member of the chitinase family. In human plasma, Chit activity has been proposed as a biochemical marker of macrophage activation in several lysosomal diseases. Recently we found that Chit activity is higher in patients affected by Plasmodium falciparum malaria infection, suggesting that Chit may reflect induction of an immunological response. To assess this hypothesis, we evaluated the CHIT1 mRNA levels in human monocytes/macrophages (HMMs) following treatment with interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha), and lipopolysaccharide (LPS). Stimulation of macrophages with INF-gamma, TNF-alpha, and LPS resulted in an increase in Chit activity as well as the levels of CHIT1 mRNA as measured by quantitative real-time PCR. The data presented in this article show that Chit plays a role in the response to the activation of INF-gamma-, TNF-alpha-, and LPS-driven macrophages, suggesting that the production of Chit by macrophages could have biological relevance in the immune-response.     

Modulation of indoleamine-2,3-dioxygenase expression and activity by HIV-1 in human macrophages

Human immunodeficiency virus (HIV) infection is often complicated by the development of acquired immunodeficiency syndrome (AIDS) dementia complex (ADC). Implications of kynurenine pathway (KP) are suggested in ADC and other inflammatories brain diseases. The first and regulatory enzyme of the KP is the indoleamine-2,3-dioxygenase (IDO). IDO activation is known to contribute to the modulation of the immune response during various infectious diseases particularly in AIDS. HIV and viral proteins can activate IDO in immune cells leading to an increase catabolism of tryptophan through the KP; the consequence being the production of immuno-modulative and neuroactive metabolites. This mechanism is likely to favour HIV persistence. The present study analysed concomitantly several parameters involved in IDO regulation and activity associated with HIV-1-infection. We investigated relevant intracellular and extracellular mechanisms involved in the regulation of IDO expression and activity during the HIV infection and replication in human monocyte-derived macrophages (MdM). Using a complementary set of in vitro experiments, we found that HIV-1/Ba-L infection induces IDO expression and increases its activity in MdM. We also showed that IDO activation by HIV-1 is likely to be a direct effect of the infection and seems to be independent of IFN-γ production.     

罗格列酮对肾小管上皮细胞人尿酸盐转运子基因表达的影响

目的：观察不同浓度罗格列酮对肾小管上皮细胞（HK-2细胞）人尿酸盐转运子（hUAT）mRNA表达的影响。方法：根据培养液中所含罗格列酮浓度的不同，将HK-2细胞分为4组，（1）仅使用DMEM组（对照组）；（2）DMEM＋5μmol／L罗格列酮组（R1组）；（3）DMEM＋10μmol／L罗格列酮组（R2组）；（4）DMEM＋15μmol／L罗格列酮组（R3组），每组均培养6瓶细胞。上述细胞在不同培养液中分别培养48h。采用实时荧光定量PCR检测HK-2细胞中hUATmRNA的相对表达量（2^△△O法）。结果：所有标本均能检测到huAT mRNA的表达，且R1、R2、R3组hUAT mRNA表达水平均明显低于对照组．其中R1组（浓度5μmol／L）hUAT mRNA表达水平为对照组的19．6％，R2组（浓度10μmol／L）hUAT mRNA表达水平为对照组的18．8％，R3组（浓度15μmol／L）hUAT mRNA表达水平仅为对照组的9．5％。结论：罗格列酮可能有下调hUAT mRNA表达的作用。[著者文摘]     

蛛网膜下腔出血大鼠脑干组织谷氨酸转运体的表达及尼莫地平的影响

目的探讨谷氨酸转运体（EAATs）在蛛网膜下腔出血（ShH）大鼠脑干表达的动态变化。方法采用枕大池二次注血法建立Wistar大鼠SAH模型。应用颅多普勒超声检测和RT-PCR方法,分别观察蛛网膜下腔出血大鼠基底动脉血流速度和谷氨酸转运体在脑干的变化。结果①超声检测显示与对照组相比,枕大池注血大鼠第1天基底动脉血流加快,第7天达到高峰,第14天血流速度下降,第21天未见明显变化,表明大鼠出现了脑血管痉挛（CVS）。尼莫地平给药组大鼠的基底动脉血流速度均比SAH组血流速度慢。②RT-PCR结果显示在第1天和7天SAH模型组脑干EAAT-1、EAAT-2和EAAT-3mRNA的表达下降,第14天上升,其对应的尼莫地平治疗组EAAT1—3mRNA的表达均高于SAH组;而在第21天SAH模型组与其对应的尼莫地平治疗组均上升,但两者相比未见明显差异。结论尼莫地平可以明显的减轻SAH发生后CVS的程度,并使EAAT-1、EAAT-2和EAAT-3mRNA的表达水平增高,提示EAAT-1、EAAT-2和EAAT-3可能参与了大鼠CVS的形成。     

The Anopheles gambiae ATP‐binding cassette transporter family: phylogenetic analysis and tissue localization provide clues on function and role in insecticide resistance

The role of ATP‐binding cassette (ABC) transporters in conferring insecticide resistance has received much attention recently. Here we identify ABC transporters differentially expressed in insecticide‐resistant populations of the malaria vector, Anopheles gambiae. Although we found little evidence that the orthologues of the multidrug resistance proteins described in other species are associated with resistance in An. gambiae we did identify a subset of ABC proteins consistently differentially expressed in pyrethroid‐resistant populations from across Africa. We present information on the phylogenetic relationship, primary sites of expression and potential role of ABC transporters in mediating the mosquito's response to insecticides. Furthermore we demonstrate that a paralogous group of eight ABCG transporters, clustered on chromosome 3R, are highly enriched in the legs of An. gambiae mosquitoes, consistent with a proposed role for this ABC subfamily in transport of lipids to the outer surface of the cuticle. Finally, antibodies raised against one of the most highly expressed ABC transporters in adult females, ABCG7 (AGAP009850), localized this transporter to the pericardial cells. These data will help prioritize members of this gene family for further localization and functional validation studies to identify the in vivo function of these transporters in the mosquito and determine whether elevated expression of members of this family contribute to insecticide resistance.     

乳腺癌组织中耐药基因蛋白表达的相关研究

目的 探讨多药耐药基因P-gp、LRP、TOPOⅡ在化疗前乳腺癌组织的表达及其与ER、PR、CerbB-2及淋巴结转移之间的相关性。方法 采用免疫组化SP法检测76例乳腺癌组织中ER、PR、C．erbB-2．P-gp、LRP、TOPOI的表达水平。结果 P-gp、LRP、TOPOⅡ在化疗前乳腺癌中的表达率分别为28．9％（22／76）、84．2％（64／76）和80．3％（61／76）。LRP、TOPOⅡ与CerbB-2之间存在正相关关系，LRP、TOPOII与ER、PR之间无相关，P-gp与ER、PR及CerbB-2之间均无相关性。有无淋巴结转移对耐药相关基因LRP、P-gp、TOPOI的表达无显著性影响。结论 P-gp、LRP、TOPOI与乳腺癌原发MDR的发生有相当关系，CerbB-2基因可能也参与了MDR基因的调节机制。     

Multidrug resistance protein gene expression in Trichoplusia ni caterpillars

Many insect species exhibit pesticide‐resistant phenotypes. One of the mechanisms capable of contributing to resistance is the overexpression of multidrug resistance (MDR) transporter proteins. Here we describe the cloning of three genes encoding MDR proteins from Trichoplusia ni: trnMDR1, trnMDR2 and trnMDR3. Real‐time quantitative PCR (qPCR) detected trnMDR mRNA in the whole nervous system, midgut and Malpighian tubules of final instar T. ni caterpillars. To test whether these genes are upregulated in response to chemical challenge in this insect, qPCR was used to compare trnMDR mRNA levels in unchallenged insects with those of insects fed the synthetic pyrethroid, deltamethrin. Only limited increases were detected in a single gene, trnMDR2, which is the most weakly expressed of the three MDR genes, suggesting that increased multidrug resistance of this type is not a significant part of the response to deltamethrin exposure.     

鸟分枝杆菌脂类诱导的人肺泡巨噬细胞抗结核免疫反应性的改变及机制的初步研究

目的研究鸟分枝杆菌脂类(MALs)对人肺泡巨噬细胞抗结核免疫反应性的影响。方法平板计数法评价MALs对人肺泡巨噬细胞杀结核菌效应的影响;ELISA检测肿瘤坏死因子-α(TNF-α)水平;Greisse法检测一氧化氮(NO)水平。结果MALs减弱了人肺泡巨噬细胞对牛结核分枝杆菌(BCG)的杀灭。MALs刺激后,结核分枝杆菌纯蛋白衍生物(PPD)和干扰素γ(IFN-γ)诱导的人肺泡巨噬细胞TNF-α和NO的分泌均有所下降。结论 MALs减弱了人肺泡巨噬细胞对结核分枝杆菌的免疫反应性。     

三种耐药相关基因及其蛋白在乳腺癌组织中的表达和意义

目的 探讨乳腺癌组织和癌旁正常组织中MDR1、BCRP、LRP的mRNA及其相应蛋白P-gp、BCRP、LRP的阳性表达情况及临床意义.方法 采用RT-PCR检测42份乳腺癌组织及其相应癌旁正常组织中MDR1、BCRP、LRP mRNA的相对表达量,同时采用IHC检测126份乳腺癌组织及42份相应癌旁正常组织中P-gp、BCRP、LRP蛋白的阳性率,并观察3种耐药相关基因及其蛋白与乳腺癌患者临床病理因素、腋窝淋巴结转移及5年复发转移与未复发转移之间的关系.结果 RT-PCR检测MDR1、BCRP、LRP mRNA在乳腺癌组织中的相对表达量分别为0.81±0.17、0.78 ±0.14、0.79±0.13,癌旁正常组织中的相对表达量分别为0.33±0.11、0.45±0.09、0.36±0.10;3种耐药基因在2组中的相对表达量的差异均有统计学意义(t值分别为4.613、4.850、8.089,P均<0.01).IHC检测P-gp、BCRP、LRP蛋白在乳腺癌组织中的阳性率分别为41%(52/126)、39%(49/126)和66%(83/126),癌旁正常组织中的阳性率分别为14%(6/42)、17%(7/42)和19%(8/42),3种耐药蛋白在2组中的表达差异有统计学意义(x2值分别为10.147、7.020、27.820,P均<0.01).MDR1 mRNA和P-gp与乳腺癌患者不同临床分期及腋窝淋巴结有无转移有关(r=0.369、0.39 8,P均<0.05),BCRPmRNA及其蛋白与乳腺癌患者腋窝淋巴结有无转移有关(r=0.355,P<0.05);5年内发生复发转移的39例患者与5年内未发生复发转移的32例患者P-gp、BCRP及LRP阳性率比较,P-gp阳性率在2组中的差异有统计学意义(x2=11.771,P<0.01),BCRP及LRP阳性率在2组中的差异无统计学意义(x2=2.261、0.078,P均>0.05).3种耐药基因及其蛋白在乳腺癌组织中阳性表达的一致率分别为90.48%、92.85%和85.71%(Kappa值分别为0.806、0.751和0.697,P均<0.01).结论 乳腺癌中存在着广泛的原发性多药耐药现象,3种耐药基因MDR1、BCRP、LRP的mRNA及其P-gp、BCRP、LRP蛋白均参与了乳腺癌多药耐药的形成;检测3种耐药基因和蛋白的表达可为乳腺癌患者有目的 地选择化疗方案提供一定的理论依据及策略;特别是对于P-gp阳性表达者不宜使用CAF(环磷酰胺+阿霉素+5-氟尿嘧啶)方案化疗.     

某地区分离自呼吸系统感染患者产ESBLs、AmpC酶肠杆菌科细菌耐药性检测

目的 分析肠杆菌科细菌临床分离株产超广谱β-内酰胺酶(ESBLs)和头孢菌素酶(AmpC酶)情况,及其与肠杆菌科细菌耐药性的关系.方法 收集该地区12家医院2009～2010年分离自呼吸系统感染患者呼吸道标本的肠杆菌科细菌1 612株,双纸片增效法检测ESBLs、三维试验检测AmpC酶、K-B纸片法检测菌株耐药性,采用χ2检验进行统计学分析.结果 1 612株细菌中,产ESBLs和AmpC酶菌株检出率分别为45.0%(726/1 612)和11.6%(187/1 612),产酶株对多种抗菌药物的耐药率高于非产酶株;未检出碳青霉烯类抗菌药物耐药菌株.结论 分离自该地区呼吸道感染患者呼吸道标本的产ESBLs和AmpC酶肠杆菌科细菌具有多药耐药性,应加强细菌耐药性监测,采取有效措施防止耐药性的水平传播.     

The complement component C5a induces the expression of plasminogen activator inhibitor-1 in human macrophages via NF-κB activation

BACKGROUND: Atherosclerosis is considered to be a chronic inflammatory disorder. Activation of the complement cascade is a major aspect of chronic inflammatory diseases. Complement components were identified in atherosclerotic plaques, and a correlation between adverse events and C5a plasma levels was found. These findings support the notion that complement activation contributes to development and progression of atherosclerotic lesions. OBJECTIVES: We investigated whether complement components C3a and C5a regulate plasminogen activator inhibitor (PAI-1) in human macrophages. METHODS: Human monocyte-derived macrophages (MDM) and human plaque macrophages were cultured and incubated with the complement component C5a. RESULTS: C5a increased PAI-1 up to 11-fold in human MDM and up to 2.7-fold in human plaque macrophages. These results were confirmed at the mRNA level using real time-polymerase chain reaction. Pertussis toxin or anti-C5aR/CD88 antibody completely abolished the effect of recombinant human C5a on PAI-1 production, suggesting a role of the C5a receptor. Experiments with antitumor necrosis factor (TNF)-alpha antibodies and tiron showed that the effect of C5a was not mediated by TNF-alpha or oxidative burst. Furthermore C5a induced NF-kappaB binding to the cis element in human macrophages and the C5a-induced increase in PAI-1 was completely abolished by an NF-kappaB inhibitor. CONCLUSIONS: We conclude that C5a upregulates PAI-1 in macrophages via NF-kappaB activation. We hypothesize that - if operative in vivo- this effect could favor thrombus development and thrombus stabilization in the lesion area. On the other hand one could speculate that C5a-induced upregulation of PAI-1 in plaque macrophages could act as a defense mechanism against plaque destabilization and rupture.     

COX-2在人肝细胞癌中的表达及其对耐药性的影响

目的探讨环氧合酶-2(COX-2)在肝细胞癌发生、发展中的作用以及COX-2对P-糖蛋白(P-gp)表达的影响。方法选择手术切除的52例肝癌标本为研究对象,应用RT-PCR、免疫组织化学分别检测COX-2 mRNA和COX-2蛋白在肝癌组织和正常肝组织中的表达。同时检测mdr 1mRNA和P-gp蛋白在肝癌组织中的表达情况,并分析COX-2与P-gp在肝癌组织中表达的相关性。结果正常肝组织中未见COX-2表达,中、低分化的肝癌组织表达阳性率高于高分化肝癌组织(P<0.01),在HBsAg阳性的肝癌组织中表达高于HBsAg阴性的肝癌组织(P<0.05),在合并肝硬化的肝癌组织中表达高于无肝硬化的肝癌组织(P<0.01)。mdr 1 mRNA在肝癌组织和正常肝组织中均有表达。同时表达COX-2和mdr1基因为42例,两者相关系数r=0.563(P<0.01)。结论 COX-2参与了以P-gp介导的肝癌的多药耐药机制。     

Regulation of drug transporter mRNA expression by interferon‐γ in primary human hepatocytes

Interferon (IFN)‐γ is known to downregulate expression of drug detoxifying proteins such as cytochromes P‐450 (CYPs) in human hepatocytes. The present study was designed to determine whether IFN‐γ may also impair expression of influx and efflux drug transporters, which constitute important determinants of the liver detoxification pathway. Exposure of primary human hepatocytes to 10 ng/mL IFN‐γ was found to downregulate mRNA levels of sinusoidal influx transporters such as sodium‐taurocholate cotransporting polypeptide, organic anion transporting polypeptide (OATP) 2B1, OATP1B1, and OATP1B3. IFN‐γ concomitantly reduced mRNA expression of drug efflux pumps such as multidrug resistance gene 1, multidrug resistance protein (MRP) 2, MRP3, breast cancer resistance protein and bile salt export pump. Such IFN‐γ‐mediated repression of major hepatic drug transporters may contribute to impaired liver clearance of drugs administrated to patients suffering from inflammation or viral infections associated with increased secretion of IFN‐γ.