调节细胞自噬增强卵巢癌顺铂敏感性的体外研究

目的:探讨细胞自噬与上皮性卵巢癌顺铂耐药的关系及调节细胞自噬对卵巢癌顺铂耐药的影响。方法:应用MTT法、MDC荧光染色法及激光共聚焦显微镜技术,观察SKOV3、SKOV3/DDP细胞株自噬现象;通过自噬特异性抑制剂3-MA作用,了解3-MA对卵巢癌细胞胞浆内自噬体的抑制作用及抑制细胞自噬对卵巢癌顺铂敏感性的影响。结果:SKOV3细胞株顺铂IC50值为3.330μg/ml,SKOV3/DDP细胞株顺铂IC50值为14.152μg/ml。3-MA联合顺铂作用后SKOV3细胞株的顺铂IC50值为3.312μg/ml,其对顺铂的敏感性无明显影响(P=0.221),SKOV3/DDP细胞株顺铂IC50值为6.386μg/ml,提示3-MA可提高上皮性卵巢癌顺铂耐药细胞对顺铂的敏感性(P=0.000)。MDC荧光染色及激光共聚焦显微镜观察SKOV3、SKOV3/DDP细胞株胞浆自噬体的形成情况,顺铂能够诱导自噬体的生成,在3-MA作用下,SKOV3/DDP细胞株顺铂诱导的自噬体荧光强度变小,而SKOV3细胞株自噬体的荧光强度变化不大。结论:细胞自噬活性增强与上皮性卵巢癌顺铂耐药有关,抑制细胞自噬活性可以部分逆转上皮性卵巢癌SKOV3/DDP顺铂耐药,但不改变顺铂敏感型细胞SKOV3对顺铂的敏感性。     

亚砷酸与托泊替康等对人多药耐药卵巢癌细胞NIH-OVCAR-3的协同抑制作用及对比性研究

目的:研究亚砷酸(As2O3)与托泊替康(TPT)、顺铂(DDP)联合应用对人多药耐药卵巢癌细胞NIH-OVCAR-3的协同效应,并进行对比研究。方法:采用MTT法比较亚砷酸、托泊替康单独或联合及与一定浓度的顺铂联合应用,对人卵巢癌多药耐药细胞系NIH-OVCAR-3的抑制作用。结果:0.1μg/ml、1μg/ml、10μg/ml亚砷酸与0.5μg/ml、5μg/ml、50μg/ml托泊替康单用和联合应用均表现出细胞毒作用,联合应用时细胞毒作用分别为单用的1.10~1.53倍。相应浓度的托泊替康与0.4μg/ml、4μg/ml4、0μg/ml顺铂联用时,其细胞毒作用为与相应浓度亚砷酸联用时的84%~97%。结论:亚砷酸与托泊替康联合应用对于人多药耐药卵巢癌细胞系NIH-OVCAR-3有更强的抑制作用,并且亚砷酸与托泊替康联合应用的杀伤效应强于顺铂与托泊替康的联合作用。     

TWEAK激活自噬增强上皮性卵巢癌顺铂耐药细胞株A2780/DDP顺铂敏感性的研究

目的:观察TWEAK对上皮性卵巢癌顺铂耐药细胞株A2780/DDP的顺铂(DDP)耐药性的影响,并初步探讨其机制。方法:CCK8法测定不同浓度TWEAK刺激后A2780/DDP的顺铂半数细胞致死量(IC50)。Western blot法检测TWEAK刺激A2780/DDP后Beclin-1、微管结合蛋白轻链3(LC3)及成纤维细胞生长因子诱导早期反应蛋白14(Fn14)的表达水平。激光共聚焦显微镜检测绿色荧光-LC3蛋白(GFP-LC3)在A2780/DDP细胞内的分布情况。用TWEAK刺激敲除Fn14表达的A2780/DDP细胞,Western blot法检测Beclin-1、LC3的表达。结果:CCK8检测结果显示,10、100ng/mlTWEAK组的顺铂IC50分别为(31.940±0.118)μg/ml、(28.769±0.090)μg/ml,均显著低于0ng/ml组(36.358±0.106)μg/ml(P均<0.05)。Western blot法结果显示,TWEAK可呈剂量和时间依赖性诱导Beclin-1、LC3及Fn14表达上调,而特异性敲除Fn14表达后,Beclin-1、LC3表达明显下调。激光共聚焦显微镜检测结果显示,TWEAK可诱导GFP-LC3致密斑形成。结论:TWEAK可增强人卵巢癌细胞耐药株A2780/DDP的顺铂敏感性;呈剂量和时间依赖性激活A2780/DDP的自噬活性,且主要通过与其受体Fn14结合介导。     

谷胱甘肽-S-转移酶P1启动子调控的胞嘧啶脱氨酶和尿嘧啶磷酸核糖转移酶融合基因对卵巢癌顺铂耐药细胞的体外杀伤作用

目的　观察谷胱甘肽 S 转移酶P1(GSTP1)启动子调控的胞嘧啶脱氨酶和尿嘧啶磷酸核糖转移酶融合基因 (CD UPP ,即自杀基因 )表达的腺病毒载体 ,对卵巢癌顺铂耐药细胞的体外杀伤作用。方法　采用细菌内同源重组法构建腺病毒载体 ,设立卵巢癌顺铂敏感细胞株A2 780和以其诱导的耐药细胞株A2 780 /DDP ,在体外用重组腺病毒转染两组细胞并联合应用前药 5 氟胞嘧啶 (5 FC) ,观察两组卵巢癌细胞对 5 FC的敏感性。将CD UPP阳性和CK UPP阴性的A2 780 /DDP细胞按不同比例混合后给予 5 FC ,观察 5 FC对混合细胞的杀伤作用。结果　当重组腺病毒滴度为 10 0感染复数(MOI)、5 FC浓度为 2 5 0 μg/ml时 ,对顺铂耐药的A2 780 /DDP细胞的存活率为 (3 6± 1 0 ) % ,对顺铂敏感的A2 780细胞的存活率为 (76 5± 2 8) % ,两者比较 ,差异有显著性 (P <0 0 5 )。此外 ,2 0 ? UPP阳性A2 780 /DDP细胞 ,即可引起 80 3%的混合细胞死亡 ,CD UPP / 5 FC系统表现出明显的旁观者效应。结论　由腺病毒介导的含有GSTP1调控的CD UPP/ 5 FC系统 ,对卵巢癌顺铂耐药细胞具有特异性杀伤作用 ,为临床上克服卵巢癌化疗耐药 ,提供了一条安全、高效的治疗途径。     

S100A4与卵巢癌细胞顺铂耐药相关性的研究

目的:探讨钙结合蛋白S100A4与卵巢癌细胞顺铂耐药的关系。方法:MTT法测定顺铂对卵巢癌顺铂敏感细胞株A2780和耐药株CP70的半效抑制浓度(IC50);分别应用免疫细胞化学、Western blot和实时荧光定量PCR检测A2780和CP70细胞株中S100A4的差异表达,观察顺铂处理CP70细胞后S100A4在蛋白水平及基因水平的动态变化。结果:顺铂对A2780和CP70的IC50分别为20.88μmol/ml和57.10μmol/ml。S100A4蛋白在二者中均以胞浆表达为主;CP70中S100A4 mRNA水平和蛋白水平的表达均显著高于A2780;经顺铂处理CP70后,S100A4的表达呈剂量-时间依赖性。结论:S100A4的高表达与卵巢癌顺铂化疗耐药有关。     

KDM5B基因在卵巢癌中的表达及其对卵巢癌耐药能力的影响

目的:检测组蛋白去甲基化转移酶KDM5B(JARID1B/PLU-1)在正常卵巢、卵巢癌组织及细胞株中的表达,以及其对卵巢癌细胞株耐药能力的影响。方法:RT-q PCR法检测KDM5B在36例卵巢癌、15例正常卵巢、6例术后化疗耐药复发患者组织及卵巢上皮细胞株IOSE和5株卵巢癌细胞中的表达情况;构建p MSCVpuro-KDM5B过表达质粒和p GIPZ-sh1、p GIPZ-sh2干扰质粒,并筛选得到稳定过表达的SKOV3和稳定干扰的Caov3细胞株,RT-q PCR和Western blot法鉴定调控效果;用浓度梯度递增的顺铂处理SKOV3过表达和Caov3干扰细胞株,CCK-8法检测顺铂半抑制浓度(IC50)变化。结果:RTq PCR结果示,正常卵巢组织中KDM5B相对表达量(1.950±0.3003)低于卵巢癌组织(4.924±0.2989),差异有统计学意义(t=5.909,P0.0001)。KDM5B表达量与卵巢癌患者的FIGO分期、病理分级、转移和耐药发生明显相关,与患者年龄、CA125水平不相关。6例复发患者中,5例KDM5B表达明显升高(P0.001)。KDM5B的mRNA和蛋白水平在卵巢上皮性细胞株IOSE中最低,在卵巢癌细胞株SKOV3、HO-8910、ES-2、A2780、Caov3中表达逐渐增加。RT-q PCR结果示,SKOV3过表达组的KDM5B表达量为对照组的27.4倍,Caov3干扰sh1和sh2组分别为对照组的0.38和0.41倍,Western blot也显示过表达和干扰有效。用浓度梯度递增的顺铂处理Caov3细胞株,KDM5B表达量随顺铂浓度增加而逐渐升高;检测SKOV3细胞株KDM5B过表达组IC50为3.666(95%CI为3.067~4.382)μg/ml高于对照组[1.676(95%CI为1.553~1.810)μg/ml],Caov3细胞株干扰sh1组和sh2组分别为3.359(95%CI为2.875~3.925)μg/ml、2.881(95%CI为2.49~3.324)μg/ml,均低于对照组[6.972(95%CI为6.182~7.864)]。结论:组蛋白去甲基化转移酶KDM5B在卵巢癌中表达升高,并且具有促进卵巢癌细胞株耐药的作用。     

Caspase-3活性改变与人卵巢癌细胞系COC1/DDP耐药的关系

目的 探讨人卵巢癌顺铂耐药细胞株COC1/DDP中抗凋亡蛋白Bcl-XL、Bcl-2、细胞色素C的表达及半胱天冬氨酸蛋白酶-3(caspase-3)活性与人卵巢癌顺铂耐药的关系。方法:采用RT-PCR和Western Blot分析人卵巢癌顺铂敏感细胞株COC1和顺铂耐药株COC1/DDP中Bcl-XL、Bcl-2、细胞色素C的表达和caspase-3的活性。并用流式细胞术测定顺铂作用后COC1和COC1/DDP细胞株的凋亡率。结果:在COC1/DDP细胞中,Bcl-XL和Bcl-2的表达明显高于COC1细胞;顺铂作用后,在COC1/DDP细胞株中细胞色素C的表达明显减少,caspase-3活性明显降低(P<0.05);其凋亡率也明显低于COC1细胞株(P<0.05)。结论:人卵巢癌细胞株COC1/DDP对顺铂产生耐药可能与细胞内Bcl-XL、Bcl-2过度表达抑制了线粒体细胞色素C的释放及caspase-3活性有关。     

顺铂抑制SKOV3细胞转移的动态监测及相关机制探讨

目的:在细胞水平实时动态监测顺铂抑制SKOV3细胞黏附和转移特性,探讨其相关机制.方法:CCK8法检测顺铂对SKOV3细胞增殖的抑制;采用RTCA监测仪24h实时动态观察顺铂对SKOV3细胞的黏附、迁移和侵袭的抑制作用;细胞组化法检测顺铂作用SKOV3细胞24h后细胞表面CD44v6的表达情况.结果:(1)顺铂对SKOV3细胞增殖的抑制作用呈浓度和时间依赖性,24、48h的IC50分别为24.065±1.031、5.083±0.128μg/ml;(2)在第50min时点,25 μg/ml顺铂组与对照组对SKOV3细胞黏附力的抑制作用差异最显著(P＜0.05).第2h时点,12.5、25 μg/ml顺铂组对SKOV3细胞迁移的抑制作用显著高于对照组(P＜0.05).第10h 15min时点,25μg/ml顺铂组对SKOV3细胞侵袭的抑制作用显著高于对照组(P＜0.05).(3)与对照组比较,25 μg/ml顺铂组细胞表面表达CD44v6明显减少(P＜0.05),而5μg/ml顺铂组减少不明显.结论:顺铂抑制SKOV3细胞转移特性首先表现为抑制细胞的黏附和迁移,这与顺铂减少细胞表面CD44v6蛋白的表达密切相关.CD44v6将成为上皮性卵巢癌治疗的新靶点.     

IDO与卡铂诱导卵巢癌耐药的免疫学研究

目的:通过卡铂诱导卵巢癌耐药的细胞系对免疫细胞功能影响的研究,从免疫学角度探讨耐药卵巢癌转移或复发的机制。方法:建立卵巢癌SKOV3细胞对卡铂耐药的细胞系SKOV3/CBP,ELISA法检测SKOV3和SKOV3/CBP细胞吲哚2,3双加氧酶(IDO)表达;将两组细胞分别与淋巴细胞、NK细胞及C8~+T细胞培养,MTT法检测淋巴细胞的增殖能力、LDH检测NK细胞的杀伤能力及ELISPOT检测C8~+T细胞的杀伤能力。结果:与SKOV3细胞株相比,耐药细胞株SKOV3/CBP内IDO表达明显增强(P0.05),且与其共培养的淋巴细胞的增殖能力、NK细胞的杀伤能力及CD8+T细胞的杀伤能力较SKOV3组降低,差异有统计学意义(P0.05)。结论:卡铂耐药SKOV3细胞内IDO表达增加,其可通过降低免疫细胞作用的敏感性而发生免疫逃逸促进耐药肿瘤的转移和复发,IDO可作为卵巢癌卡铂耐药治疗新的靶点。     

米非司酮对卵巢上皮性癌耐药细胞DNA修复基因表达和顺铂敏感性的影响

目的 研究米非司酮对卵巢上皮性癌(卵巢癌)耐药细胞DNA修复基因表达的调控,及对顺铂的增敏作用.方法 应用四甲基偶氮唑蓝比色法检测2.5、5.0、10.0 μmol/L米非司酮作用后卵巢癌顺铂耐药细胞株COC1/DDP的顺铂半数抑制浓度(IC50);用RT-PCR技术、流式细胞仪检测米非司酮联合顺铂作用后COC1/DDP细胞ERCC1、BRCA1、hMLH1 mRNA的表达水平及细胞周期和凋亡率的变化;建立COC1/DDP细胞裸鼠皮下移植瘤模型,分为顺铂组、米非司酮组、联合组和对照组,观察米非司酮在裸鼠体内对顺铂的增敏作用.结果 2.5、5.0、10.0 μmol/L米非司酮分别使COC1/DDP细胞对顺铂的IC50下降为(3.18±0.46)、(1.95±0.14)和(0.64±0.18)μg/ml,2.5 μmol/L米非司酮联合顺铂作用后的IC50与单用顺铂者[(3.71±0.38)μg/ml]比较,差异无统计学意义(P=0.076),其他浓度之间两两比较,差异均有统计学意义(P＜0.01).米非司酮下调ERCC1、BRCA1、hMLH1 mRNA表达水平,3种基因mRNA的相对表达量随米非司酮浓度的升高均呈下降趋势.2.5、5.0、10.0 μmol/L米非司酮联合顺铂对COC1/DDP细胞作用后,G0/G1,期细胞比例升高(分别为51.68%、53.74%、55.08%).2.5、5.0、10.0 μmol/L米非司酮与10 μg/ml顺铂联合作用使细胞凋亡率分别增加为5.11%、9.13%和12.24%.米非司酮联合顺铂治疗裸鼠皮下移植瘤的生长抑制率为70.1%,与顺铂组、米非司酮组(分别为22.5%、6.5%)比较,差异均有统计学意义(P＜0.05).结论 米非司酮通过下调ERCC1、BRCA1、hMLH1基因的表达及阻滞G0/G1期细胞、增加细胞凋亡率来增强顺铂对卵巢癌细胞的抗肿瘤敏感性.     

肝素结合表皮生长因子在顺铂耐药卵巢癌中的表达及相关性研究

目的检测肝素结合表皮生长因子（HB-EGF）在顺铂耐药卵巢癌中的表达水平并探讨HB-EGF与卵巢癌对顺铂耐药性的关系。方法①体外实验：蛋白印迹法检测卵巢癌亲本细胞株A2780和顺铂耐药株A2780/CDDP中HB-EGF蛋白的表达。分别予以HB-EGF抑制剂CRM197治疗,四甲基偶氮唑蓝（MTT）比色法检测细胞的增殖能力,酶标仪检测加药前后caspase-3蛋白的活性;②体内实验：建立A2780组和A2780/CDDP组卵巢癌裸鼠移植瘤模型,免疫组化法检测2组移植瘤中HB-EGF蛋白的表达。分别予以CRM197治疗观察移植瘤的生长情况并计算CRM197抑瘤率。结果体内外实验结果表明,与A2780组相比,HB-EGF在A2780/CDDP组中高表达。CRM197可有效地降低A2780/CDDP细胞的增殖能力和caspase-3的表达,抑制A2780/CDDP组裸鼠移植瘤的生长（P〈0.001）。结论 HB-EGF在顺铂耐药卵巢癌中高表达,并与卵巢癌对顺铂的耐药性相关。     

Gene therapy of adenovirus mediated CD ::upp/5-FC directed by GSTP1 promoter in cisplatin-resistant ovarian cancer

PURPOSE: To evaluate the specific killing effect of the adenoviral vector in which CD ::upp genes were directed by the GSTP1 promoter on cisplatin-resistant ovarian cancer cells. EXPERIMENTAL DESIGN: Cisplatin-sensitive (A2780) and cisplatin-resistant (AD6) ovarian cancer cells were infected with recombinant adenoviral plasmid carrying the CD ::upp gene driven by the GSTP1 promoter and followed with 5-FC administration. RESULTS: In vitro, when MOI was 100 and 5-FC was 250 microg/ml, relative survival rate of the AD6 cells was only 3.63 +/- 1.01%, while under the same conditions, A2780 cells were 76.50 +/- 2.81%. Significant bystander effect was caused by the CD ::upp gene and 20% of gene-transferred AD6 cells caused death to 80.3% of the total cells. Furthermore, a significant anti-tumor effect of the Ad.GST-CD ::upp/5-FC was observed in nude mice bearing tumors of AD6 cells. CONCLUSIONS: These results indicated that adenovirus-mediated Ad.GST-CD ::upp/5-FC directed by GSTP1 promoter is an effective approach to overcome cisplatin-resistant ovarian cancer.     

顺铂耐药卵巢癌细胞鼠肾包膜下动物模型的建立及其临床意义

目的 :建立顺铂耐药卵巢癌细胞鼠肾包膜下动物模型 ,探讨其临床意义。方法 :采取肾包膜下纤维蛋白细胞凝块移植法 ,用环磷酰胺免疫抑制小鼠建立顺铂耐药卵巢癌模型。结果 :移植后 7d肿瘤体积明显增大 ,顺铂耐受细胞组与非耐受细胞组体积无明显差异 (P >0 .0 5) ;移植后细胞形态学特征无改变 ;在用顺铂治疗后 ,耐药细胞组肿瘤体积较非耐药细胞组体积大 (P <0 .0 5)。结论 :该动物模型可作为短期体内实验模型 ,能保持细胞的耐药性     

Regulation of HtrA2/Omi by X-linked inhibitor of apoptosis protein in chemoresistance in human ovarian cancer cells

OBJECTIVE: Development of cisplatin resistance by cancer cells is a major hurdle in successful treatment of human ovarian cancer. However, the mechanism of cisplatin resistance in ovarian cancer cells remains unclear. In this study, we have examined the possible role of X-linked inhibitor of apoptosis protein (Xiap) in the development of cisplatin resistance in ovarian cancer cells and investigate if suppressed cytosolic high temperature required protein A (HtrA2/Omi) level is an important factor in cisplatin resistance in ovarian cancer. METHODS: Cisplatin-sensitive (A2780 and COC1) and -resistant (A2780/DDP and COC1/DDP) ovarian cancer cells were cultured for different durations (0-24 h) and with different cisplatin concentrations (0-20 muM). Xiap content and cytosolic HtrA2/Omi content were analyzed by Western blot. Antisense oligonucleotides were used to downregulate Xiap content in ovarian cancer cells. RESULTS: Cisplatin decreased Xiap content and increased cytosolic HtrA2/Omi content and caspase-3 activity in cisplatin-sensitive ovarian cancer cell lines (A2780 and COC1), but not in the resistant variants (A2780/DDP and COC1/DDP). Downregulation of Xiap by antisense Xiap oligonucleotides increased caspase-3 activity and sensitized cisplatin-resistant cells to cisplatin treatment. Cytosolic HtrA2/Omi level increased while Xiap was downregulated in cisplatin-resistant ovarian cancer cells. CONCLUSION: Development of cisplatin resistance may be due to Xiap neutralizing caspase-3 activation and lower cytosolic HtrA2/Omi level in response to cisplatin in human ovarian cancer cells. Cytosolic HtrA2/Omi level is partly regulated by Xiap in ovarian cancer cells.     

卵巢肉瘤样癌细胞系的生物学特性及对化疗药物的敏感性

目的 研究人卵巢肉瘤样癌永生化细胞系BUPH；OVSC-2的生物学特性，筛选卵巢肉瘤样癌敏感的化疗药物。方法 以手术切除的卵巢肉瘤样癌腹壁转移组织为材料，进行体外培养。将永生化基因——SV40T抗原基因转染第10代细胞，经筛选，抗性克隆扩大培养，得到人卵巢肉瘤样癌永生化细胞系。通过光学显微镜、电子显微镜、生长曲线测定、染色体分析、双层软琼脂培养、裸鼠接种、免疫组化等，研究其生物学特性。用MTT的方法检测该细胞系对顺铂、阿霉素、足叶乙甙、拓扑替康四种化疗药物的敏感性。结果 人卵巢肉瘤样癌永生化细胞系，BUPH：OVSC-2，现已传至100余代。其生物学特性为，形态学观察细胞呈肉瘤样细胞形态，超微结构证实为上皮起源；细胞生长旺盛；具有恶性细胞的特征，恶性度高。该细胞在体外对阿霉素和顺铂非常敏感，对足叶乙甙尚敏感，对拓扑替康不敏感。结论 BUPH：OVSC-2为一株恶性度高的人卵巢肉瘤样癌永生化细胞系，保留了其来源细胞的生物学特性，阿霉素与顺铂联合治疗可能是卵巢肉瘤样癌较敏感的化疗方案。     

SV40 T基因诱导的人卵巢癌细胞永生化

目的 建立人卵巢癌永生化细胞系，为卵巢癌体外研究提供实验模型。方法 利用基因重组技术构建猴肾病毒40（SV40）T抗原基因的高效真核表达载体pcD2-SV40。将其转染10例原代培养的早期传代细胞，经G418筛选，抗性克隆扩大培养，建立永生化细胞系。免疫组化检测细胞的上皮起源，用PCR、RT－PCR和Southern Blot方法鉴定SV40 T基因在转染细胞中的表达。观察所建立的永生化细胞系的染色体核型。结果 10例转染细胞中，6例筛选出抗性克隆。角蛋白免疫组化证实3例为上皮起源。其中2例上皮起源细胞得以扩大培养成系，长期稳定传代，分别命名为BUPH：OVSC－2和BUPH：OVCA－3。经鉴定两种细胞系中存在SV40 T在基因水平及转录水平的表达。其染色体核型均符合人体恶性细胞特征。结论 利用SV40T抗原基因进行人卵巢癌原代细胞的 生化是一种可行的、可重复的建系方法，可提高卵巢癌细胞成系机率。     

Increased efficiency of cisplatin-resistant cell lines to DNA-mediated gene transfer with cationic liposome

AIM: Because of its effectiveness against many gynecologic malignancies, chemotherapy including cisplatin is mainly used as the first-line chemotherapy for epithelium ovarian cancer. However, one of the major problems that is well recognized is that tumor cells can easily acquire resistance to cisplatin. Various trials were carried out in order to establish treatment against cisplatin-resistant tumor cells. METHOD: Using both in vivo and in vitro studies, we examined whether or not the newly developed liposome could be used to demonstrate sufficient transfection activity as the anticancer reagent for cisplatin-resistant tumor cells. RESULT: With our newly developed liposome, GTE 319 and GTE 321, the lac-Z gene was more efficiently transfected in cisplatin-resistant variant cells, mEIIL-R, KF-ra and KF-rb, than in parental cells, mEIIL and KF, using X-gal staining. In cytotoxic assay, transfection of herpes simplex thymidine kinase (HSV-tk) gene conjugated with GTE319 or GTE 321, and cultivation with aciclovir for 5 days revealed accelerated tumor-inhibition activity in all of the cisplatin-resistant tumor cells compared with that in the naive parental cells. In addition, the high anti-tumor effect was obtained from intratumoral local injection of the tk gene conjugated with GTE-321 liposome following aciclovir administration against KF-rb-transplanted tumor formed in nude mouse hypodermic. CONCLUSION: These results suggest that gene therapy using a newly developed liposome-conjugated suicide gene can be an attractive approach for treatment against cisplatin-resistant ovarian cancer cells.     

Profiling of proteins associated with cisplatin resistance in ovarian cancer cells

Resistance to cisplatin is a major impediment to the successful treatment of ovarian cancer, but the precise nature of the resistance is still unclear. In the current study, we aimed to investigate and compare the protein expression profiles in cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines. We employed the recent development of surface-enhanced laser desorption/ionization ProteinChip technology to measure protein expression in three human ovarian cancer cell lines (KF-1, MN-1, and A2780) and their sublines (KF-r, MN-r, and A2780cp) resistant to cisplatin. The ProteinChip Arrays were analyzed using the ProteinChip Reader. We did not find any regularity in protein expressions in secretions of cisplatin-sensitive and cisplatin-resistant cells. But on the IMAC3 array, we captured 12 identical expressions which represent a subset of proteins whose expression levels are different between parent ovarian cancer cells and their cisplatin-resistant cells. In particular, at the molecular weight of 7829 d, three kinds of parent cell lines exhibited an elevated expression and their cisplatin-resistant sublines revealed a lowered expression. At the molecular weight of 6881 d, for KF and MN cell lines, opposite protein expressions were seen in the parent cell line and its cisplatin-resistant subline. We think the interesting protein expressions perhaps suggest some mechanisms involved in cisplatin resistance.     

A high nuclear basal level of ERK2 phosphorylation contributes to the resistance of cisplatin-resistant human ovarian cancer cells

OBJECTIVE: The aim of this study was to elucidate the role of ERK1/2 on cisplatin resistance in human ovarian cancer cells. METHODS: The relationship between nuclear levels of ERK2 and cisplatin-induced apoptosis in human ovarian carcinoma cell line, OVCAR-3, and in cells of the cisplatin-resistant subclone, OVCAR-3/CDDP, was examined using immunoblotting and immunocytochemistry. RESULTS: Cisplatin treatment resulted in the activation of ERK2, both in OVCAR-3 and OVCAR-3/CDDP cells. However, considerable levels of activated ERK2 existed in the nuclei of OVCAR-3/CDDP cells during serum starvation and in the early period (1-3 h) after cisplatin treatment. Conversely, phospho-ERK2 was marginally detected in the nuclei of OVCAR-3 cells prior to cisplatin treatment. These phenomena were confirmed by immunofluorescence staining of the phosphorylated ERK2 in the nuclei of both cells. High basal phospho-ERK2 in the nuclei of OVCAR-3/CDDP cells contributed to cisplatin resistance, and was supported by several observations; (1) treatment of U0126, an inhibitor of MEK/ERK signaling pathway, partially sensitized OVCAR-3/CDDP cells to cisplatin; (2) pretreatment of OVCAR-3 cells with phorbol 12-myristate 13-acetate (PMA), an activator of ERK, induced nuclear translocation of activated ERK2, which led to the suppression of cisplatin-induced apoptosis. CONCLUSIONS: These results collectively indicate that prelocalization of activated ERK2 in the nuclei contribute to cisplatin resistance in OVCAR-3/CDDP cells.