耐顺铂人肺腺癌细胞系A549^DDP耐药机理进一步探讨

应用递增浓度的方法建立了一株顺铂（ＣＤＤＰ）耐药细胞系Ａ５４９ＤＤＰ。用溴化乙啶荧光分光光度法测定细胞内Ｐｔ－ＤＮＡ链间交联物（ＩＣＬ）；用无焰原子吸收光谱法测定细胞浆内及细胞核内铂含量；用流式细胞法及荧光显微镜测定铂的外排速度。结果显示：耐药细胞Ａ５４９ＤＤＰ较亲代敏感细胞Ａ５４９对ＣＤＤＰ耐受性增加８．９倍，与亲代敏感细胞Ａ５４９相比，Ａ５４９ＤＤＰ胞浆内及核内ＣＤＤＰ积聚分别为亲代细胞的１６．９％与２４．３％，铂含量与ＣＤＤＰ浓度正相关；Ａ５４９ＤＤＰ外排ＣＤＤＰ的功能较Ａ５４９明显增强，ＩＣＬ形成量低８４．１％，而修复功能增强２倍。结果提示：Ａ５４９ＤＤＰ细胞内ＣＤＤＰ积聚减少和修复功能增强是ＣＤＤＰ的主要耐药机理。     

Resistant mechanisms of cisplatin in human lung adenocarcinoma cell line A549DDP

To study the resistant mechanisms of cisplatin in human lung adenocarcinoma cell line A₅₄₉DDP. A₅₄₉DDP cells was established by stepwise increasing concentration of cisplatin (CDDP) in medium. Interstrand cross-linked DNA (ICL) was measured by ethidium bromide fluorescence assay. The intracellular and intranuclear accumulation of cisplatin was measured by atomic absorption spectrometry. The removal of GS-X was determined by FCM and fluorescence microscopy. Results: The A₅₄₉DDP cell line was 8.9-fold resistance relative to the parental A₅₄₉ cell line. The formation of ICL in A₅₄₉ was 6.28 times higher than that in A₅₄₉DDP cells. The intracellular and intranuclear accumulation of cisplatin in A₅₄₉ cells was 5.9 times and 4.1 times higher than that in A₅₄₉DDP cells, respectively. The ability of GS-X pump pumped GS-X complex (GS-Pt) in A₅₄₉DDP cells was higher than that in A₅₄₉. The repair rate in A₅₄₉DDP cells was 2 times higher than that in A₅₄₉. Conclusions: Decreased accumulation and increased export of cisplatin might be the main mechanism of cisplatin resistant A₅₄₉DDP cells while the enhanced repair capacity of DNA may play a role in CDDP resistance.     

Decreased accumulation as a mechanism of resistance to cis-diamminedichloroplatinum(II) in human non-small cell lung cancer cell lines: relation to DNA damage and repair

The mechanism of resistance to cis-diamminedichloroplatinum(II) (CDDP) is still controversial, although several kinds of processes have been proposed. For elucidation of the mechanism of CDDP resistance in CDDP-resistant human non-small cell lung cancer cells (PC-9/0.5, 7.1-fold resistant), we examined the formation of DNA interstrand cross-links (ICL), one kind of DNA damage induced by CDDP, its repair, and intracellular accumulation of CDDP. We measured the frequency of CDDP-induced ICL by means of the alkaline elution technique and the amount of intracellular platinum for intracellular accumulation of CDDP by means of atomic absorption spectrophotometry in PC-9 (parental cell) and PC-9/0.5 cells. Formation of ICL in PC-9 cells exposed to 5 micrograms of CDDP per ml for 6 h was 5.85 times that in PC-9/0.5 cells. On the other hand, the ability to repair CDDP-induced ICL was identical in both cell lines. Intracellular accumulation studies revealed that PC-9 retained 5.07 times as much platinum as that in PC-9/0.5 after 3-h exposure to CDDP. It was conjectured that the decrease in the intracellular accumulation of CDDP might be the main cause of CDDP resistance in PC-9 cells, since the decreased accumulation was paralleled by the decreased level of ICL.     

Establishment of a cisplatin-resistant gastric carcinoma cell line OCUM-2M/DDP

 A cis-diamminedichloroplatinum (CDDP)-resistant scirrhous gastric cancer cell line, OCUM-2M/DDP, was established by chronic exposure of cells of the parent scirrhous gastric cancer cell line, OCUM-2M, to CDDP at progressively increasing concentrations. The OCUM-2M/DDP cell line had an 11.3-fold higher level of resistance relative to its parent cell line as determined by a succinate dehydrogenase inhibition test. The biological and biochemical characteristics of the resistant and parent cell line were compared. There were differences in the modal chromosome number and DNA index, suggesting that some alterations of the DNA in the CDDP-resistant cells had occurred. Neither the parent nor resistant cell line expressed mdr-1 mRNA. After exposure to CDDP for 4 h, the intracellular platinum content of OCUM-2M cells was significantly higher than that of OCUM-2M/DDP cells (51.9±1.8 vs 16.4 plus 1.0 ng/mg protein, mean±SD, respectively). The GSH levels in OCUM-2M cells and OCUM-2M/DDP cells were 3.5±1.0 μg/mg protein and 16.8±1.2 μg/mg protein, respectively. These levels were also significantly different. These findings suggest that the possible mechanisms of resistance to CDDP in OCUM-2M/DDP cells may be a decrease in intracellular CDDP accumulation and detoxication by GSH. This OCUM-2M/DDP cell line could be used in further investigations of the mechanism of CDDP resistance in gastric cancer. Received: 16 August 1996 / Accepted: 16 November 1996     

基因工程腺病毒H101逆转A549/DDP细胞对顺铂耐药性的实验研究

背景及目的:耐药性产生是化疗失败的主要原因之一,克服耐药性的方法之一是使用逆转剂。目前尚无一种理想的、可应用于临床的顺铂(DDP)耐药逆转剂。本研究探讨基因工程腺病毒H101对DDP耐药性的逆转作用及其机制。方法:体外利用人肺腺癌细胞株A549的DDP耐药模型A549/DDP,采用MTT法检测H101对A549/DDP细胞耐药性的逆转作用。用流式细胞仪(FCM)测定细胞多药耐药蛋白1(multidrugresistantprotein1,MRP1)、谷胱甘肽鄄S鄄转移酶(GST)及TOPO鄄Ⅱ蛋白表达量,荧光鄄考马斯亮蓝法检测细胞内GSH含量,原子吸收法测定细胞内铂浓度。结果:A549/DDP细胞对DDP的耐药指数为14.3。加入H101后,DDP对A549/DDP的半数抑制浓度(IC50)由352.4μmol/L降至61.6μmol/L,逆转倍数为5.7倍。FCM检测结果显示,A549/DDP细胞的GST和TOPO鄄Ⅱ蛋白水平均较A549细胞高,而两种细胞内均未检测到MRP1的表达,经H101处理后的A549/DDP细胞胞内GST蛋白和TOPO鄄Ⅱ蛋白水平均明显下降。A549/DDP胞内GSH含量比A549高2倍多;H101感染A549/DDP细胞后,胞内的GSH含量下降,随着H101剂量的增加,胞内GSH含量逐渐下降,达A549细胞内GSH水平。原子吸收法测定细胞内铂含量的结果显示,A549细胞内铂含量是A549/DDP细胞内铂含量的3倍,加入H101后,A549细胞内铂的含量无明显变化,但A549/DDP细胞内铂的含量明显增加。结论:H101能逆转A549/DDP对DDP的耐药性,H101逆转DDP耐药性的机制可能是减少A549/DDP细胞内GSH和TOPOⅡ蛋白的水平,增加了A549/DDP细胞内铂的蓄积。     

热疗联合 IL-2逆转人肺腺癌细胞 A549/CDDP 的作用及其可能机制

目的：观察热疗联合白介素-2（IL-2）对人肺腺癌细胞株 A549/ CDDP 的耐药逆转作用,并探讨其可能作用机制。方法采用细胞培养技术,分别培养人肺腺癌细胞株 A549及其耐药细胞株 A549/ CDDP。42℃热疗,联合或不联合200 u·mL -1的 IL-2,同时在2μg·mL -1的 CDDP 作用下,分别干预敏感细胞株和耐药细胞株2 h 后,采用 MTT 法检测2种不同细胞对 CDDP 的敏感性,流式细胞仪间接免疫荧光法检测热疗、IL-2等不同条件作用下细胞中 P -糖蛋白（P-gp）、多药耐药蛋白（MRP）、肺耐药蛋白（LRP）的表达差异,以及细胞内荧光药物 CDDP 的聚集量的变化。结果分别联用热疗、IL-2时,较单用 CDDP,A549/ CDDP细胞的抑制率得到提高,A549/ CDDP 细胞 P-gp、MRP 表达下降,细胞内荧光强度增强,差异均有统计学意义（P 均﹤0.05）。热疗联合 IL-2合用 CDDP 时,A549/ CDDP 细胞的抑制率进一步提高,A549/ CDDP 细胞P-gp、MRP 表达明显下降,细胞内荧光强度显著增强,差异均有统计学意义（P 均﹤0.05）,但 LRP 的表达差异无统计学意义（P ﹥0.05）。结论热疗、IL-2分别能部分逆转 A549/ CDDP 细胞对 CDDP 的耐药性;热疗联合 IL-2可进一步增强其耐药逆转效应。其逆转耐药机制,推测可能与抑制 P-gp、MRP 表达,增加细胞内药物 CDDP 的积聚有关。     

Expression and reversion of drug resistance-and apoptosis-related genes of a DDP-resistant lung adenocarcinoma cell line

Resistanceofcancercellstocytotoxicchemotherapyisacommonprobleminpatientswithcancerandamajorobstacletoeffectivetreatmentofdisseminatedneoplasm.Severalmolecularmechanismshavebeenassociatedwithmultidrugresistance(MDR)inexperimentaltumormodels.Theseincludel)enhancedeffluxofdrugbytransporterproteinssuchasp--glycoprotein(Pgp),multidrugresistance-associatedprotein(MRP)andhumanmajorvaultprotein(LRP)whichmightplayanimportantroleinvesicularsequestrationofdrug;['3]2)alterationsofdrugtargetssuchasDNA…     

Schedule-dependent reversion of acquired cisplatin resistance by 5-fluorouracil in a newly established cisplatin-resistant HST-1 human squamous carcinoma cell line

In vitro continuous stepwise exposure of HST-I human squamous carcinoma cell line to cisplatin (CDDP) for 12 months resulted in a 3.5-fold stably resistant subline designated HST-I/CPO.2. Compared with parental cells, this cell line showed a 1.8-fold increase in cellular glutathione (GSH) and a 50% reduction in initial numbers of DNA interstrand cross-links (ICLs), despite similar levels of intracellular platinum accumulation. Evaluation of the kinetics of DNA ICL removal at nearly equivalent levels of DNA ICL formation indicated that HST-I/CPO.2 cells appeared to remove DNA ICLs more rapidly than do HST-I parental cells. Thus, both elevated cellular GSH and increased DNA repair capacity would be the major factors contributing to CDDP resistance. Pretreatment of HST-I/CPO.2 cells with 5-FU, with drug-free intervals of 24 to 48 hr before exposure to CDDP, completely reversed CDDP resistance, or even increased the sensitivity to a level greater than that of parental cells, whereas the opposite sequence had no effect on resistance. In parallel with augmentation of the cytotoxicity, the levels of cellular GSH were significantly reduced over 48 hr by 5-FU pretreatment. However, depletion of cellular GSH using buthionine sulfoximine resulted in partial reversal of CDDP resistance, indicating that reduction of cellular GSH alone is not sufficient for complete reversal of CDDP resistance. Our data, together with evidence that 5-FU modulates the repair of platinum-DNA cross-links, suggest that schedule-dependent, complete reversal of CDDP resistance by 5-FU might be attributed to its inhibitory effects on both GSH levels and the repair of platinum-DNA adducts. Thus, optimization for the drug administration schedule is important when aiming at therapeutic synergy and circumvention of acquired CDDP resistance. © 1996 Wiley-Liss, Inc.     

长链非编码RNA-FENDRR提高非小细胞肺癌细胞对顺铂的化疗敏感性

目的:研究长链非编码RNA-FENDRR基因对非小细胞肺癌(NSCLC)细胞的顺铂(DDP)化疗敏感性的调节作用.方法:实时定量PCR检测FENDRR基因在DDP耐药的NSCLC组织及细胞系中的表达.应用脂质体将FENDRR基因表达载体转染入顺铂耐药的NSCLC细胞系A549/DDP.MTT法明确A549/DDP细胞对DDP的半数抑制浓度(IC50).结果:与DDP化疗敏感的NSCLC组织相比,FENDRR基因在DDP不敏感的NSCLC组织中的表达明显降低.与A549细胞相比,A549/DDP细胞呈现出对DDP的IC50显著增高,其对化疗药物DDP的耐药性更高.FENDRR基因在DDP耐药的A549/DDP细胞中表达显著低于其在A549细胞中的表达.转染表达载体pc-FENDRR能够显著上调A549/DDP细胞中FENDRR基因的表达.FENDRR高表达能够降低A549/DDP细胞对DDP的IC50,提高化疗敏感性.FENDRR高表达能够显著地促进DDP诱导的A549/DDP细胞的凋亡.结论:长链非编码RNA-FENDRR基因与NSCLC的化疗耐药相关,能够提高NSCLC细胞对DDP的敏感性.     

5—氟脲嘧啶对耐顺铂人肺腺癌细胞A549^DDP的程序依赖性逆转作用

用耐顺铂（ＣＤＤＰ）人肺腺癌细胞系Ａ５４９ＤＤＰ作模型，研究了５－ＦＵ对ＣＤＤＰ耐药的程序依赖性逆转作用。５－氟脲嘧啶（５－ＦＵ）预处理Ａ５４９ＤＤＰ２４ｈ后立即给予ＣＤＤＰ，ＣＤＤＰ细胞毒性增加３．９倍；５－ＦＵ预处理Ａ５４９ＤＤＰ后间隔２４或４８ｈ再给予ＣＤＤＰ，其细胞毒性分别增加２０倍和２５０倍，甚至较亲代细胞Ａ５４９更为敏感；如先给ＣＤＤＰ后给５－ＦＵ则细胞毒性仅增加１．８倍。５－ＦＵ对亲代细胞亦有类似效应但细胞毒性增加程度明显低于耐药细胞。５－ＦＵ预处理后间隔２４，４８ｈ，细胞内ＧＳＨ含量逐渐降低，与细胞毒性逐渐增加相一致。如用ＢＳＯ耗竭Ａ５４９ＤＤＰ细胞内ＧＳＨ，ＣＤＤＰ细胞毒性增加６．４倍，仅能部分逆转ＣＤＤＰ耐药。５－ＦＵ明显抑制ＭＲＰ的表达，但对ＧＳＴπ的表达无影响。在５－ＦＵ预处理后的无药间隔时间内，给予无毒浓度的三苯氧胺则有明显的协同效应。结论：程序性给予５－ＦＵ通过降低细胞内ＧＳＨ含量和抑制ＭＲＰ的表达能完全逆转ＣＤＤＰ耐药     

榄香烯乳剂对肺癌A549/DDP细胞株耐药逆转作用及其机制

目的:探讨榄香烯乳（elemene,ELE）逆转人肺腺癌A549/DDP细胞株耐药性及其机制。方法:采用MTT法检测榄香烯乳单用的细胞毒作用及与顺铂（cisplatin,DDP）合用时耐药逆转作用;采用流式细胞术检测榄香烯乳对A549/DDP细胞内罗丹明-123（rhodamine-123,Rh123）蓄积的影响;采用Western blot检测榄香烯乳对耐药细胞A549/DDP细胞膜上P-gp蛋白表达的影响。结果:不同浓度榄香烯对A549/DDP细胞均有一定的抑制作用,呈时间-剂量依赖性效应。单用DDP的IC50为15.46μg/ml,合用20μg/ml榄香烯24h,IC50为4.15μg/ml,逆转耐药倍数为3.63。同时,榄香烯增加A549/DDP细胞内Rh123的蓄集,降低细胞膜上P-gp的表达,且呈剂量依赖性,表明榄香烯能减少A549/DDP细胞对药物的外排和抑制P-gp蛋白的表达。结论:榄香烯在体外逆转肿瘤细胞耐药性可能与其抑制P-gp的功能和表达有关。     

Increased gene specific repair of cisplatin induced interstrand crosslinks in cisplatin resistant cell lines, and studies on carrier ligand specificity

Development of resistance to cisplatin in previously treatment-responsivemalignancies is a major obstacle to successful treatment. EnhancedDNA repair as well as enhanced replicative bypass of DNA adductshave been suggested to play a role in the development of resistanceto cisplatin. However, the relative contribution of these mechanismsis unknown. Second generation platinum compounds containingthe 1, 2-diaminocyclohexane (dach) carrier ligand have beenof particular interest in the studies of resistance mechanismssince they have been effective in treatment of cells resistantto cisplatin. We have investigated the formation and repairof interstrand crosslinks (ICL) in the mouse leukemia cell lineLI 210/0 and its carrier ligand specific resistant derivativesL1210/DDP and L1210/ DACH after treatment with ethylenediamine(en)-Pt and diaminocyclohexane (dach)-Pt compounds. ICL in theoverall genome were examined using a modification of the alkalineelution assay. A Southern blot technique was employed for thestudy of ICL in specific regions of the genome. In the overallgenome we found decreased formation of ICL with either -en or-dach carrier ligands in the two resistant cell lines withoutcarrier ligand specificity. Some carrier ligand specificityof ICL formation was observed in the dihydrofolate reductase(DHFR) gene, but it did not correlate with the carrier ligandspecificity of resistance. At the level of the overall genomethere was no difference in repair of ICL between the sensitiveand the two resistant cell lines. When measured in the DHFRgene, however, there was enhanced repair of ICL in the two resistantcell lines compared with the sensitive cell line. The enhancedrepair at the level of the gene did not display any carrierligand specificity.     

重组人血管内皮抑素逆转人肺腺癌A549/DDP细胞耐药性研究

目的探讨重组人血管内皮抑素(rh-ES)对A549/DDP细胞耐药性的逆转作用.方法采用人肺腺癌细胞株A549及耐药细胞株A549/DDP作为研究对象,以顺铂(DDP)与重组人血管内皮抑素两者联合及单独给药的方式分别作用于人肺腺癌细胞株A549及耐药细胞株A549/DDP,作用时间72 h,观察不同给药方式造成的同株细胞之间耐药性的不同及不同株细胞间耐药性的差别,采用四甲基偶氮唑蓝(MTT)法检测重组人血管内皮抑素对A549/DDP细胞耐药性的逆转作用.结果DDP对A549细胞半数抑制浓度(IC50)为(0.72±0.05)μg/ml,对A549/DDP的IC50为(11.54±0.64)μg/ml.rh-ES联合DDP对A549/DDP的IC50为(2.0±0.1)μg/ml.逆转倍数(RF)为5.77,相对逆转率(RRR)为88.2%.结论重组人血管内皮抑素能逆转A549/DDP对DDP的耐药性.     

榄香烯乳诱导线粒体凋亡途径逆转肺癌A549/DDP细胞株耐药

目的：探讨榄香烯乳（ elemene,ELE）逆转人肺腺癌耐顺铂（ cisplatin,DDP）细胞A549/DDP的耐药性及作用机制。方法：采用MTT法检测榄香烯乳单用的细胞毒作用及与DDP合用时耐药逆转作用。荧光探针JC-1结合激光共聚焦显微镜检测线粒体膜电位的变化。DCFH-DA荧光探针结合流式细胞仪检测细胞内活性氧（ reactive oxygen species,ROS）水平。用谷胱甘肽试剂盒结合分光光度法检测计算GSH/（ GSSG﹢GSH）比值。蛋白质印迹法检测胞质中Cyto C、Pro-caspase-3、Caspase-3和Bcl-2家族蛋白表达情况。结果：不同浓度榄香烯乳抑制A549/DDP细胞株生长,呈时间-剂量依赖性效应,联合顺铂能提高A549/DDP细胞株对顺铂的敏感性而逆转耐药。不同浓度榄香烯乳联合顺铂使A549/DDP细胞株线粒体膜电位下降,ROS浓度增加,GSH/（ GSSG﹢GSH）比值降低,上调胞质中Cyto C、Caspase-3、Bad蛋白表达,下调Pro-caspase-3、Bcl-2蛋白表达。结论：榄香烯乳逆转A549/DDP细胞株耐药性可能与其损伤线粒体膜,活化胞内氧化还原体系,诱导线粒体凋亡路径有关。     

Non-cross resistance of ZD0473 in acquired cisplatin-resistant lung cancer cell lines

ZD0473 is a new generation platinum agent that, in preclinical studies, shows evidence of an extended spectrum of anti-tumor activity and overcomes platinum resistance mechanisms. The drug contains a bulky methylpyridine ligand at its platinum center, which is responsible for its ability to overcome platinum resistance. We examined the growth inhibitory effects of ZD0473 in human lung cancer cell lines resistant to cisplatin in vitro. Four cisplatin resistant human lung cancer cell lines (PC-14/CDDP, SBC-3/CDDP, PC-9/CDDP, H69/CDDP) showed the expected resistance to cisplatin but were non-cross, or much less, resistant to ZD0473, as determined by an MTT assay. A reduction in the intracellular accumulation of cisplatin, but not of ZD0473, was observed in the PC-14/CDDP cells compared with the levels in PC-14 parental cells. The reduction in cisplatin accumulation is considered a major mechanism of the acquired cisplatin resistance in PC-14/CDDP cells. Therefore, the increase in platinum accumulation is considered a possible mechanism underlying the activity of ZD0473 in cisplatin-resistant cells. Glutathione-mediated resistance to cisplatin was also overcome by ZD0473 in PC-14/CDDP cells. In addition, we showed that the intraperitoneal administration of ZD0473 at its maximum tolerable dose in mice produced a marked in vivo antitumor activity against cisplatin-resistant PC-14/CDDP tumors. These results suggest that ZD0473 may be a potent agent in human lung cancer cells with multifactorial cisplatin resistance.     

沉默CXCR4的表达逆转肺癌细胞顺铂耐药

目的:探讨沉默CXCR4对肺癌细胞顺铂耐药的影响,并研究其分子作用机制。方法:利用RT-qPCR测定肺癌耐药（A549/DDP）及药物敏感细胞株（A549）中CXCR4 mRNA的表达水平;利用LipofectamineTM 2000将siRNA-CXCR4转染至肺癌耐药细胞株中,RT-qPCR及蛋白质印迹法（Western blot）验证siRNA对CXCR4基因的靶向沉默效率;利用CCK-8法检测沉默CXCR4后肺癌耐药细胞的增殖活性;利用流式细胞仪测定沉默CXCR4后肺癌耐药细胞的凋亡率;利用MTT法测定沉默CXCR4后肺癌耐药细胞对顺铂的敏感性;利用Western blot实验测定沉默CXCR4后CYP1B1蛋白的表达水平。结果:RT-qPCR实验结果显示,肺癌耐药细胞株A549/DDP中CXCR4 mRNA的表达量显著高于药物敏感细胞株（P＜0.01）;体外转染siRNA-CXCR4可明显下调A549/DDP细胞株中CXCR4的表达（P＜0.001）;CCK-8实验结果显示,沉默CXCR4的表达抑制了A549/DDP细胞的增殖活性（P＜0.01）;流式细胞仪实验结果显示,沉默CXCR4的表达促进了A549/DDP细胞凋亡（P＜0.01）;MTT实验结果显示,沉默CXCR4的表达增加了A549/DDP细胞对顺铂的敏感性（P＜0.01）;Western blot实验结果显示,沉默CXCR4后CYP1B1蛋白的表达水平显著降低（P＜0.05）。结论:沉默CXCR4的表达抑制肺癌耐药细胞增殖、促进凋亡,逆转肺癌细胞顺铂耐药,CXCR4正向调控CYP1B1的表达,可能是CXCR4逆转肺癌细胞顺铂耐药的作用机制。     

细胞因子诱导的杀伤细胞逆转耐顺铂肺腺癌细胞系A549/DDP耐药性的研究

  目的  观察细胞因子诱导的杀伤细胞(cytokine induced killer, CIK)对耐顺铂(DDP)人肺腺癌细胞系A549/DDP的耐药逆转作用及其逆转A549/DDP耐药的可能机制。  方法  A549/DDP与CIK细胞采用Transwell非接触共培养。四氮甲唑兰比色法(MTT法)验证A549/DDP的DDP耐药性及检测共培养前后A549/DDP对DDP耐药性的变化。RT-PCR法筛选A549与A549/DDP有差异表达的基因作为检测耐药性变化的观察指标, 并检测共培养前后A549/DDP中基因表达水平的变化; Western blot检测A549及共培养前后A549/DDP中基因蛋白水平的变化。  结果  A549/DDP的耐药系数为14.5, 具有较强的DDP耐药性。RT-PCR筛选出A549与A549/DDP表达有差异的耐药相关基因为谷胱甘肽转移酶(glutathione-S-transferase, GST-π)基因、人类铜离子转运蛋白(human copper transporter1, hCTR1)基因, A549/DDP中GST-π表达量明显增加, hCTR1表达量明显降低。与CIK细胞共培养后, A549/DDP对DDP的耐药性明显下降, 共培养20h后耐药逆转倍数约为4.93倍, 细胞内GST-π基因及蛋白水平的表达明显降低(P < 0.05)。  结论  CIK细胞对A549/DDP有逆转DDP耐药的作用, 其机制可能与下调GST-π基因及蛋白水平的表达有关。      

The Mechanism of the Difference in Cellular Uptake of Platinum Derivatives in Non-small Cell Lung Cancer Cell Line (PC-14) and Its Cisplatin-resistant Subline (PC-14/CDDP)

A cisplatin-resistant non-small cell lung cancer cell line, PC-14/CDDP, was established from PC-14 by stepwise escalation of CDDP concentrations in vitro . PC-14/CDDP cells were 11.4-fold more resistant to CDDP compared with PC-14 cells. This resistant cell line was cross-resistant to platinum analogues, such as carboplatin (CBDCA) (X 3.5), cis -diammine(glycolate-O, O')platinum(II) (254-S) (X 5.6) and cis -dichloro(ethylenediammine)platinum(II) (cis-DEP) (X 4.2). On the other hand, relative resistance value to ormaplatin was only 1.4-fold. To elucidate the mechanism(s) of CDDP resistance and of its circumvention by ormaplatin, we investigated the characteristics of this cell line. Total sulfhydryl content was slightly elevated in PC-14/CDDP cells compared with PC-14 cells. There was no significant difference in the DNA repair ability between the two cell lines. Cellular accumulations of CDDP, CBDCA, 254-S, and cis-DEP in PC-14/CDDP cells were markedly decreased to 23%, 27%, 29%, and 32% of those in PC-14 cells, respectively. However, the accumulation of ormaplatin in PC-14/CDDP was almost the same as that in PC-14. To elucidate the mechanisms of uptake of these platinum analogs in the cells, we studied the effects of ouabain, an Na⁺, K⁺ -ATPase inhibitor, on cellular drug uptake in both cell lines. Preincubation with 300 n M ouabain for 1 h inhibited approximately 60% of CDDP accumulation in PC-14. However ouabain preincubation at any concentration up to 300 n M did not affect CDDP accumulation in PC-14/CDDP. The accumulation of ormaplatin was not inhibited by ouabain in either of the cell lines. These data suggest that the mechanism of the uptake of ormaplatin is different from that of CDDP, and that ormaplatin exerts a cytotoxic effect in CDDP-resistant cells which have defective cisplatin accumulation.     

Role of cystine transport in intracellular glutathione level and cisplatin resistance in human ovarian cancer cell lines

Transport system x(c)(-) is a member of plasma membrane heterodimeric amino-acid transporters and consists of two protein components, xCT and 4F2hc. This system mediates cystine entry coupled with the exodus of intracellular glutamate and regulates the intracellular glutathione (GSH) levels in most mammalian cultured cells. We studied the activity of system x(c)(-) and GSH content in human ovarian cancer cell line (A2780) and its cisplatin (CDDP)-resistant variant (A2780DDP). The rate of cystine uptake was approximately 4.5-fold higher in A2780DDP cells than in A2780 cells and the cystine uptake in A2780DDP cells was mediated by system x(c)(-). Intracellular GSH content was much higher in A2780DDP cells but it fell drastically in the presence of excess glutamate, which inhibited the cystine uptake competitively. xCT and 4F2hc mRNAs were definitely expressed in A2780DDP cells, but far less in A2780 cells. Expression of system x(c)(-) activity by transfection with cDNAs for xCT and 4F2hc made A2780 cells more resistant to CDDP. Similar results on the cystine uptake were obtained in human colonic cancer cell lines. These findings suggest that the system x(c)(-) plays an important role in maintaining the higher levels of GSH and consequently in CDDP resistance in cancer cell lines.     

肺腺癌细胞系耐药及凋亡相关基因的表达和逆转

目的 探讨顺铂耐药肺腺癌细胞系Ａ５４９＾ＤＤＰ的耐药,凋亡相关基因的表达及其与亲代细胞Ａ５４９的差异,观察差异表达基因的反义寡核苷酸的逆转作用。方法 将人工合成的反义,正义硫代脱氧寡核苷酸（Ｓ－ｏｌｉｇｏｄｌｅｏｘｙｎｕｃｌｅｏｔｉｄｅ,Ｓ－ＯＤＮ）经脂质体包后转染Ａ５４９＾ＤＤＰ细胞,应用ＲＴ－ＰＣＲ检测耐药及凋亡相关基因ｍＲＮＡ水平,免疫细胞化学及流式细胞－抗体检测相应蛋白及增殖抗原Ｋｉ－６７