靶向调控的活性半胱氨酸天冬氨酸蛋白酶3联合细胞周期蛋白依赖性激酶抑制剂flavopiridol对卵巢上皮性癌的治疗作用

目的 观察人端粒酶逆转录酶启动子-二步转录增强系统调控下表达活性半胱氨酸天冬氨酸蛋白酶3(caspase-3)的重组腺病毒AdHTVP2G5-rev-casp3联合细胞周期蛋白依赖性激酶抑制剂flavopiridol对卵巢上皮性癌(卵巢癌)的治疗作用.方法 应用活细胞计数法--细胞计数试剂盒8(CCK-8)、流式细胞仪和蛋白印迹法(western blot)检测AdHTVP2G5-rev-casp3联合flavopiridol作用后卵巢癌AO细胞的细胞存活率、凋亡率、细胞周期和细胞中活性caspase-3片段p17及其底物聚(腺苷二磷酸-核糖)多聚酶的裂解片段p85的表达情况;观测AdHTVP2G5-rev-casp3联合flavopiridol治疗前后荷卵巢癌裸鼠生存情况、移植瘤体积、肝酶水平及各器官组织的病理检查结果.结果 AdHTVP2G5-rev-casp3[感染复数(MOI)=5～20]或flavopiridol(300 nmol/L)单独作用均不引起AO细胞的显著凋亡,凋亡率均＜11%;联合应用能产生显著的协同杀伤作用,当两者分别以MOI=20、300 nmol/L联合作用时,细胞存活率为11.6%,凋亡率为73.5%,该协同作用在AdHTVP2G5-rev-casp3感染细胞后72 h再加入flavopiridol作用48 h最为显著.低剂量(MOI=10)的AdHTVP2G5-rev-casp3能引起细胞的S期比例显著增高(62.5%).两者联合作用后的AO细胞中p17和p85的表达水平也显著高于两者单独作用时.单独应用AdHTVP2G5-rev-casp3或flavopiridol均可延长荷瘤裸鼠的生存时间[分别为(141±14)、(134±10)d]并抑制肿瘤生长(抑瘤率分别为30%、40%),但两者联合应用具有协同作用,裸鼠生存时间可达(286±6)d,抑瘤率为81%.两者联合应用后裸鼠的肝酶水平无显著升高,肝等各器官组织的HE染色病理检查未见异常.结论 低剂量的活性caspase-3可引起卵巢癌细胞在S期的募集,从而增强细胞对flavopiridol的敏感性;低剂量的活性caspase-3联合flavopiridol对卵巢癌细胞具有显著的协同杀伤作用,能显著抑制移植瘤的生长并延长荷瘤裸鼠的生存期,并降低flavopiridol对肝脏的毒性作用.     

吴茱萸碱抑制人卵巢癌细胞SKOV3增殖的实验研究

目的:探讨吴茱萸碱对人卵巢癌细胞SKOV3增殖的抑制作用及其机制。方法:采用噻唑蓝(MTT)法检测不同浓度吴茱萸碱对SKOV3细胞增殖活性的影响;采用Annexin V-FITC/PI染色检测吴茱萸碱对SKOV3细胞凋亡的影响;Western blot法检测吴茱萸碱对SKOV3细胞内caspase-3和PARP活化的影响。构建卵巢癌SKOV3裸鼠移植瘤模型并给予吴茱萸碱灌胃,绘制肿瘤生长曲线、称取瘤重,检测肿瘤组织中caspase-3和PARP活化情况。结果:0.5、1、2、4μmol/L吴茱萸碱处理SKOV3细胞24h后,细胞活性较对照组分别减少(15.95±1.61)%、(33.82±4.03)%、(46.62±5.45%)和(63.82±5.42)%,且呈明显时间和浓度依赖性;0.5、1、2μmol/L吴茱萸碱作用SKOV3细胞24h诱导细胞凋亡率分别为(12.93±3.81)%、(26.27±3.31)%和(36.72±4.73)%,均高于对照组(8.08±1.04,P0.05);吴茱萸碱显著活化SKOV3细胞内caspase-3和PARP,诱导细胞凋亡发生。成功构建卵巢癌裸鼠皮下移植瘤模型,给药14天后裸鼠皮下移植瘤体积明显小于对照组(P0.01),给药21天后移植瘤体积较对照组减少54.01%(P0.01)、瘤重减少42.03%(P0.01),给药组裸鼠移植瘤蛋白内cleaved caspase-3和cleaved PARP小片段活化部分显著增多。结论:吴茱萸碱显著抑制人卵巢癌细胞SKOV3体外增殖和裸鼠皮下移植瘤生长,其作用机制与吴茱萸碱诱导细胞凋亡密切相关。     

人端粒酶逆转录酶启动子-二步转录增强系统调控下的活性半胱氨酸天冬氨酸蛋白酶3的构建及其对卵巢上皮性癌的治疗作用

目的构建含人端粒酶逆转录酶启动子．二步转录增强系统（hTERTp．TSTA）调控下的活性半胱氨酸天冬氨酸蛋白酶3（rev-caspase-3）的重组腺病毒AdHTVP2G5-rev-casp3,并检测其对卵巢上皮性癌（卵巢癌）的治疗作用。方法采用基因重组法构建AdHTVP2G5-rev-casp3,经DNA测序鉴定并证实其序列正确。实验分为4组：分别以AdHTVP2G5-rev-casp3（AdHTVP2G5-rev-casp3组）、含hTERTp调控下的rev—caspase-3的重组腺病毒AdHT-rev-casp3（AdHT-rev-casp3组）和含人巨细胞病毒启动子（CMVp）调控下的rev—caspase-3的重组腺病毒Ad—rev—casp3（Ad-rev-casp3组）转染人卵巢癌细胞系AO细胞及正常人脐静脉内皮细胞系HUVEC细胞,以含hTERTp-TSTA调控下的增强型绿色荧光蛋白（EGFP）的重组腺病毒AdHTVP2G5-EGFP（AdHTVP2G5-EGFP组）为阴性对照。采用蛋白印迹法（westernblot）、细胞计数法（CCK-8）、流式细胞术分别检测各组AO和HUVEC细胞中caspase-3的活性单位p17蛋白及其底物聚腺苷二磷酸-核糖多聚酶的裂解片段p85蛋白的表达强度、细胞存活率、细胞凋亡率和细胞周期的变化;westernblot法检测各组裸鼠移植瘤及肝脏组织中p17蛋白的表达强度,检测各组裸鼠生存率、肿瘤体积、体内肝酶水平及各器官组织的病理检查结果。结果AO细胞中p17蛋白表达强度,AdHTVP2G5-rev—casp3组明显高于Ad-rev-casp3和AdHT-rev-casp3组;而HUVEC细胞中AdHTVP2G5-rev-casp3组则明显低于Ad—rev—casp3,与AdHT-rev-casp3组相似。当病毒感染复数（UOI）为70时,AO细胞的存活率和凋亡率,AdHTVP2G5．rev—casp3组（分别为17．8％和40．2％）与AdHT—rev—casp3组（分别为75．2％和16．1％）比较,差异有统计学意义（P〈0．01）;而HUVEC细胞的存活率和凋亡率,AdHTVP2G5．rev—casp3组（分别为97．7％和2．1％）与AdHT-rev-casp3组（分别为98．5％和1．7％）比较,差异则无统计学意义（P〉0．05）。荷瘤裸鼠肿瘤组织中p17蛋白的表达强度,AdHTVP2G5-rev—casp3组最高,Ad-rev-casp3组次之,AdHT—rev-casp3组最弱;而其肝脏组织中p17蛋白的表达强度,Ad-rev—casp3组最高（与其在肿瘤组织中相似）,其他各组几乎无p17蛋白表达。荷瘤裸鼠的生存时间,AdHTVP2G5-rev-casp3和Ad-rev-casp3组分别为（259±14）和（213±16）d,明显长于AdHT—rev—casp3和AdHTVP2G5-EGFP组的（177±12）和（109±7）d（P〈0．01）。荷瘤裸鼠的肿瘤体积,用药53d后AdHTVP2G5-rev-casp3组（为406mm^3）明显小于AdHT-rev-casp3、Ad—rev—casp3和AdHTVP2G5-EGFP组（分别为990、645、1728mm^3;P〈0．01）。荷瘤裸鼠体内的肝酶水平,Ad—rev—CaS．p3组明显高于其他各组（P〈0．01）。各组荷瘤裸鼠的肝脏及其他器官组织经HE染色显示均无明显变化。结论hTERTp-TSTA系统调控下的rev—caspase-3致细胞凋亡的能力明显强于hTERTp,并同时保证了被作用肿瘤的靶向性,在明显抑制卵巢癌移植瘤的生长并延长荷瘤裸鼠生存时间的同时降低了凋亡基因对肝脏的毒性作用。     

小分子细胞周期素依赖性蛋白激酶抑制剂flavopiridol联合泰素治疗卵巢癌的实验研究

目的:检测flavopiridol联合泰素对人卵巢癌细胞系SKOV3、裸鼠人卵巢癌皮下及腹腔移植瘤模型的治疗作用。方法:应用CCK-8法、流式细胞术和原位细胞凋亡检测法(TUNEL)检测flavopiridol和泰素作用后卵巢癌细胞系SKOV3的细胞存活率和凋亡率;应用逆转录-多聚酶链反应(RT-PCR)检测作用前后细胞中cyclinD1的表达:应用ELISA法检测细胞中活性caspase-3的表达。建立裸鼠人卵巢癌皮下及腹腔移植瘤模型,观测flavopiridol和泰素治疗前后裸鼠肿瘤体积的变化并计算抑瘤率,用TUNEL和免疫组化法分别检测作用前后肿瘤组织中的凋亡情况和微血管密度(MVD)值。结果:flavopiri-dol(300nmol/L)或泰素(1μmol/L)单独应用24h均能显著降低细胞存活率,增强caspase-3活性,诱导细胞凋亡,并下调细胞中cyclinD1的表达。Taxol先作用24h再用flavopiridol作用24h的联合用药对二者的上述作用产生显著的协同效应:联合用药后细胞存活率为(5.2±0.8)%,凋亡率为(51.10±2.52)%,caspase-3活性相对值为0.602±0.008,cy-clinD1相对表达水平为0.264±0.076。Flavopiridol和泰素单独应用均能显著抑制皮下移植瘤生长(抑瘤率分别为84.5%和85.7%),并降低肿瘤组织MVD值(分别为12.4±4.7vs 35.2±10.3和15.2±2.9 vs 35.2±10.3)。二者联合应用抑瘤作用稍差(抑瘤率68.9%),抑制MVD作用(23.0±5.1 vs 35.2±10.3)也不及单药治疗。结论:Flavopiridol和泰素均能通过致凋亡作用显著抑制卵巢癌细胞及移植瘤生长,二者联合应用对体外培养的卵巢癌细胞具有协同杀伤作用,但体内治疗中无协同抑瘤作用。     

细胞周期蛋白依赖性蛋白激酶抑制剂flavopiridol对卵巢上皮性癌细胞及移植瘤干预作用

目的检测细胞周期蛋白依赖性蛋白激酶抑制剂flavopiridol对卵巢上皮性癌（卵巢癌）细胞及移植瘤的干预作用。方法应用流式细胞仪和脱氧核苷酸末端转移酶介导的脱氧尿苷三磷酸标记法（TUNEL）检测flavopiridol作用后卵巢癌细胞系AO的细胞凋亡率和细胞周期,应用实时荧光定量PCR技术检测flavopiridol作用前、后AO细胞中细胞周期蛋白（cyclin）D和活性半胱氨酸天冬氨酸蛋白酶（caspase）3的表达情况。建立卵巢癌裸鼠皮下及腹腔移植瘤模型,观测flavopiridol干预后裸鼠的生存情况及肿瘤体积的变化,TUNEL和免疫组化方法分别检测肿瘤组织中的细胞凋亡情况和微血管密度（MVD）。结果AO细胞在150、300、500nmol／L浓度的flavopiridol作用下,凋亡率分别为4．1％、10．7％和7．6％;G1期细胞比例显著增加,S期比例显著降低（P〈0．05）。flavopiridol作用后AO细胞cyclin D的表达量（0．25）显著下降,活性caspase-3的表达量（2．55）轻度升高,高于flavopiridol作用前的0．69（P〈0．05）、2．49（P〉0．05）。flavopiridol干预后裸鼠的累积生存率显著提高（P〈0．05）;裸鼠的平均生存时间为（141±14）d,显著高于磷酸盐缓冲液（PBS）作用后的（106±11）d,两者比较,差异有统计学意义（P〈0.05）;在flavopiridol干预后53d时抑瘤率为40％。flavopiridol干预后裸鼠的肿瘤组织中有细胞凋亡发生,MVD为（12±5）个,显著高于PBS作用后的（35±10）个,两者比较,差异有统计学意义（P〈0．05）。结论flavopiridol能显著抑制卵巢癌细胞及移植瘤的生长,延长荷瘤裸鼠的生存期。     

米非司酮诱导卵巢癌细胞株3AO细胞凋亡的研究

目的　观测米非司酮对卵巢癌细胞株 3AO和SKOV3细胞体外增殖和凋亡活性 ,雌激素受体 (ER)、孕激素受体 (PR)蛋白表达和细胞形态学的影响。方法　应用四甲基偶氮唑蓝 (MTT)法测定 3AO和SKOV3细胞体外增殖活性。应用流式细胞仪 (FCM )检测米非司酮在不同浓度和培养时间下 ,3AO细胞的ER、PR、p5 3和bcl 2蛋白表达率、增殖率和凋亡率。应用光学和电子显微镜观察3AO细胞的形态学变化。结果　 3AO细胞在不同浓度米非司酮 (5、10、2 0、4 0、80 μmol/L)和不同时间培养 (2 4、4 8、72h)下 ,其生长抑制率由 1 7%增加至 75 0 % ,与剂量和时间呈明显正相关 (P <0 0 1) ,但SKOV3细胞增殖无明显变化 (P >0 0 5 )。 3AO细胞凋亡率与米非司酮剂量和培养时间呈正相关 (P <0 0 1)。米非司酮阻断 3AO细胞周期 ,使其停滞于G0 、G1期 ,降低S期细胞比率。米非司酮诱导 3AO细胞凋亡 ,光学和电子显微镜观察发现 ,经米非司酮培养的 3AO细胞呈现典型的细胞凋亡的特征性变化 ,包括染色体皱缩、细胞核碎裂和凋亡小体的形成。米非司酮显著升调p5 3蛋白表达 ,而降调bcl 2蛋白表达 (P <0 0 1)。米非司酮浓度为 10 μmol/L ,培养 2 4h时 ,3AO细胞 p5 3和bcl 2蛋白表达率分别为(5 4 8± 4 0 ) %和 (10 1± 1 2 ) % ,与对照的 (2     

冻融抗原致敏的树突细胞对裸鼠人卵巢癌移植瘤治疗作用的研究

目的 :研究冻融抗原致敏的树突细胞 (DC)对裸鼠人卵巢癌移植瘤的治疗作用。方法 :联合应用粒性白细胞与巨噬细胞集落刺激因子 (GM CSF)及白介素 4 (IL 4 )从正常足月产妇分娩后新生儿脐血中培养出DC ,以人卵巢癌细胞系 3AO细胞冻融抗原激活DC ,测定其诱导的细胞毒性T淋巴细胞 (CTL)对 3AO的杀伤活性 ;CTL预防性接种于裸鼠皮下 ,观察裸鼠人卵巢癌移植瘤的发生率 ,以DC激活的CTL治疗裸鼠人卵巢癌移植瘤并观察治疗效果。结果 :体外抗原冲击致敏的DC能显著刺激T淋巴细胞增殖 ,其诱导的CTL对细胞系 3AO具有显著的杀伤作用 ,在效靶比为 4 0 :1、2 0 :1、10 :1、5 :1时 72h杀伤率平均分别为 90 .1%、67.4 %、4 0 .4 %、17.8%。DC激活的CTL能预防裸鼠人卵巢癌移植瘤的发生 (预防组 16.6% ,对照组 10 0 % ,P <0 .0 0 1) ,并能抑制移植瘤生长 ,对照组、治疗组移植瘤的大小分别为 (5 .6± 1.1)cm3 、(2 .7± 0 .78)cm3 (P <0 .0 1)。结论 :人卵巢癌细胞冻融抗原体外冲击致敏的DC可作为一种抗癌疫苗在免疫治疗卵巢癌患者中发挥重要作用     

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。     

整合素及纤粘连蛋白在调控卵巢癌细胞失巢凋亡过程中的作用

目的研究整合素及纤粘连蛋白(fibronectin,FN)在卵巢癌细胞逃避失巢凋亡过程中的作用及其机制。方法2005年1月至2006年5月于山东省立医院采用间接免疫荧光染色检测人卵巢癌SKOV3细胞高转移亚克隆SKOV3-S1细胞中整合素蛋白αv、β1、α5β1的表达;建立细胞悬浮分离培养导致凋亡的模型,流式细胞仪检测细胞凋亡;抗体封闭法检测不同整合素蛋白在凋亡过程中的作用;逆转录PCR检测p53、bcl-2、bax基因表达的变化;比色法检测半胱氨酸天门冬氨酸蛋白酶-3(caspase-3)活性改变。结果(1)S1细胞表面普遍表达整合素αv、β1、α5β1。(2)FN使细胞凋亡率由63.7%降至29.0%,αv及β1抗体使凋亡率上升,α5β1抗体对凋亡没有影响。(3)FN使S1细胞中bcl-2基因表达上升,bax表达下降。αv封闭抗体能抑制FN的基因表达调节作用。(4)在悬浮生长状态下,caspase-3被激活;FN使caspase-3活性减弱;加入αv及β1封闭抗体后,酶活性增强。结论SKOV3-S1细胞表面整合素受体蛋白αv通过与FN结合,提高了细胞凋亡调节基因bcl-2/bax的比率,使caspase-3活性减弱,这是SKOV3-S1细胞避免失巢凋亡的重要原因之一。     

带hTERT启动子的腺病毒干扰ERCC1基因表达逆转卵巢癌顺铂耐药的研究

目的:研究带hTERT启动子的腺病毒通过干扰ERCC1基因表达而逆转卵巢癌顺铂耐药的作用。方法:以1、10、50、80感染复数(MOI)的重组腺病毒分别感染卵巢癌细胞株SKOV3、卵巢癌细胞顺铂耐药株SKOV3/DDP和脐静脉内皮细胞ECV304,同时以相同滴度的空载体腺病毒感染细胞,将感染前的细胞作为对照,以RT-PCR方法检测重组腺病毒转染细胞中ERCC1 mRNA表达,以Western blot方法检测重组腺病毒转染细胞中ERCC1蛋白表达,并以MTT法检测肿瘤细胞对顺铂化疗敏感性的影响。结果:(1)在SKOV3及SKOV3/DDP细胞中,不同剂量Ad-hTERT-ERCC1 shRNA转染组与转染前相比,ERCC1 mRNA及ERCC1蛋白表达水平下调,差异有统计学意义(P<0.01)。而在空载体腺病毒转染组及ECV304细胞中,ERCC1 mRNA及ERCC1蛋白表达水平无改变,差异无统计学意义(P>0.05);(2)重组腺病毒转染后,SKOV3/DDP细胞对顺铂的敏感性显著增加(P<0.01),并呈剂量依赖性。结论:带hTERT启动子的腺病毒能够通过干扰ER-CC1基因表达而逆转卵巢癌顺铂耐药,并且具有剂量依赖性。     

Sequential combination therapy with flavopiridol and autocatalytic caspase-3 driven by amplified hTERT promoter synergistically suppresses human ovarian carcinoma growth <Emphasis Type="Italic">in vitro</Emphasis> and in mice

Background

Induction of cell apoptosis and regulation of cell cycle are very attractive for treatments of tumors including ovarian carcinoma. Flavopiridol is a potent small molecular cyclin-dependent kinase(cdk) inhibitor, but its antitumor efficacy is not satisfied yet. Caspase-3 play a major role in the transduction of apoptotic signals and the execution of apoptosis in mammalian cells. We have successfully constructed the recombinant adenovirues AdHTVP2G5-rev-casp3 containing autocatalytic caspase-3 (rev-caspase-3) driven by amplified hTERT promoter system (TSTA-hTERTp). In this study, we applied it with flavopiridol to investigate their antitumor effect on ovarian cancer in vitro and in vivo.

Methods

Cell viabilities were determined using Cell Counting Kit 8 and flow cytometry. RT-PCR and immunoblotting assays were used to detect cellular apoptotic activities. Tumor growth and survival of mice bearing tumors were studied.

Results

Flavopiridol or AdHTVP2G5-rev-casp3 at low dosage alone was mildly cytotoxic in vitro with a viability rate of 86.5?±?4.7% for 300 nM flavopiridol and 88.9?±?5.4% for AdHTVP2G5-rev-casp3 (MOI 20). By contrast, significant synergism of their sequential combination was observed, and the treatment of AdHTVP2G5-rev-casp3 (MOI 20) infection for 72 h, followed by flavopiridol (300 nM) for 48 h, can result in the most synergistic cell death, with cell survival rate and apoptotic rate of 11.6% and 69.7%, respectively. The sequential combination showed synergistic tumor suppression rate of 77.8%, which was significantly higher than that of AdHTVP2G5-rev-casp3 (33.6%) or flavopiridol (40.1%) alone. The mean survival of mice treated with the combination was 286?±?8 d, which was synergistically longer than that of mice treated with AdHTVP2G5-rev-casp3 (141?±?14d), flavopiridol (134?±?10 d) or controls (106?±?11 d) (P?<?0.01).

Conclusions

The sequential combination of rev-caspase-3 and flavopiridol result in significant synergistic cell killing effects, significant tumor growth suppression and extended survival of mice bearing OVCAR3 cells. The combination should be further explored as a potential clinically useful regimen against ovarian cancer.

     

Irregularly shaped inclusion cysts display increased expression of Ki67, Fas, Fas ligand, and procaspase-3 but relatively little active caspase-3

Human ovarian cancers are thought to arise from sequestered ovarian surface epithelial (OSE) cells that line the wall of inclusion cysts. Nevertheless, the early events toward neoplasia are not well understood. In this study, immunoreactivity for apoptotic proteins in human OSE of control and tumor ovarian sections was examined. Ki67, a marker for cell proliferation, was generally absent in the flat-to-cuboidal OSE cells on the ovarian surface and in regularly shaped inclusion cysts. Fas, Fas ligand, and caspase-3, components of the apoptotic pathway, were also largely absent. Ki67, Fas, Fas ligand, and procaspase-3 expression, though not active caspase-3 expression, was more frequently observed in epithelial cells lining irregularly shaped inclusion cysts, particularly in the columnar and Müllerian-like OSE cell types that resembled ovarian tumor OSE cells. Immunoreactivity for these factors as well as active caspase-3 was found frequently in ovarian tumors. We postulate that the appearance of the Fas system and its related proteins in sequestered columnar OSE cells of irregularly shaped inclusion cysts may contribute to balance cell growth with cell death, although little active caspase-3 expression was observed. Further studies are required to identify whether inhibition of apoptosis in inclusion cysts is an early event in ovarian carcinogenesis.     

Over-expression of PTEN sensitizes human ovarian cancer cells to cisplatin-induced apoptosis in a p53-dependent manner

OBJECTIVE: Resistance to cisplatin-centered chemotherapy is a major cause of treatment failure in human ovarian cancer. Whereas PTEN, a tumor suppressor gene product, is believed to promote apoptosis primarily via inactivation of the PI3K/Akt cell survival pathway, recent evidence suggests that PTEN may function independently of this pathway. Activation of p53 is a key determinant of sensitivity to cisplatin-induced apoptosis. Whether PTEN can facilitate cisplatin sensitivity, and this involves the activation of p53, remains unclear. In this study, we determined whether and how PTEN over-expression sensitizes ovarian cancer cells to CDDP-induced apoptosis. METHODS AND RESULTS: Using pairs of chemosensitive and chemoresistant ovarian cancer cell lines (OV20028 vs. C13* and A2780-s vs. A2780-cp) as an in vitro model, we have examined the influence of PTEN over-expression in regulation of cisplatin-induced apoptosis. Apoptosis was assessed morphologically by Hoechst staining and confirmed by the detection of cleaved products of caspase-3 and PARP by Western blot. Over-expression of PTEN by PTEN cDNA transfection up-regulates p53 content and increases the sensitivity of chemoresistant cells to cisplatin-induced apoptosis without detectable changes in the levels of phosphorylated Akt and FKHR as well as FasL mRNA abundance as determined by Western blot and RT-PCR, respectively. PTEN-mediated chemosensitization was attenuated by p53 down-regulation by siRNA in C13*, a chemoresistant wild-type p53 cell. Moreover, PTEN over-expression failed to sensitize the chemoresistant p53 mutant ovarian cancer cell line A2780-cp to cisplatin-induced apoptosis, unless wild-type p53 was reconstituted by adenoviral p53 infection. CONCLUSION: Taken together, these data suggest that PTEN over-expression may represent a novel therapeutic approach for chemoresistant human ovarian cancer and that this may involve a p53-mediated apoptotic cascade independent of the PI3K/Akt pathway.     

Curcumin enhances Apo2L/TRAIL-induced apoptosis in chemoresistant ovarian cancer cells

OBJECTIVE: Curcumin, the active component of turmeric (Curcuma longa), exhibits growth inhibitory activity against prostate, colon, and breast cancer; however, the effect of curcumin on ovarian cancer cells is not known. We hypothesized that curcumin could induce cell death in ovarian cancer cells, and enhance apoptosis induced by tumor necrosis factor-related apoptosis inducing Apo2 ligand/TRAIL. METHODS: Chemoresistant ovarian cancer cell lines SKOV3 and ES-2 were used. The cytotoxic effect of curcumin, Apo2L/TRAIL, and curcumin+Apo2L/TRAIL in combination was determined by sulforhodamine assay. Apoptotic fraction was determined by staining cells with propidium iodide followed by analysis of the sub-G0 DNA content of cells by flow cytometry. Caspase activation was determined by immunoblotting. RESULTS: Curcumin alone had a cytotoxic effect in cisplatin-resistant cells at 25 microM. Curcumin at low doses (5-15 microM) or Apo2L/TRAIL alone was not significantly cytotoxic to the cell lines tested. Preincubating cells with curcumin at low doses prior to treating with Apo2L/TRAIL resulted in markedly enhanced cell death. The combined treatment of curcumin and Apo2L/TRAIL resulted in activation of both the extrinsic, receptor-mediated apoptotic pathway (cleavage of caspase-8) and the intrinsic, mitochondria-mediated apoptotic pathway (cleavage of caspase-9). CONCLUSIONS: Combined curcumin and Apo2L/TRAIL treatment results in enhanced induction of apoptotic cell death. Because curcumin and Apo2L/TRAIL together can activate both the extrinsic and intrinsic pathways of apoptosis, they may circumvent chemoresistance to conventional chemotherapeutic agents.     

Differential induction of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand in human ovarian carcinoma cells

OBJECTIVES: In this study, we examine the sensitivity of a panel of ovarian carcinoma cells, which includes four primary ovarian cancer cell samples, and four normal ovarian epithelium samples to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We also examine the intracellular regulation of TRAIL-mediated apoptosis. METHODS: The sensitivity to TRAIL was determined by short-term survival assays on seven ovarian carcinoma cell lines, four primary samples of ovarian cancer, and four normal ovarian epithelium samples. We assessed the activation of the apoptotic pathway in TRAIL-resistant and -sensitive tumor cells. The expression of TRAIL receptors was determined by flow cytometry. The protein expression of FADD, XIAP, caspase-8, caspase-3, BAX, and c-FLIP were determined by immunoblot analyses. RESULTS: We show that ovarian cancer cells display variable sensitivity to TRAIL-induced apoptosis although most cell lines have similar sensitivity to cisplatin. Normal ovarian epithelium samples were mostly sensitive to TRAIL. In sensitive cells, TRAIL induced caspase-8-dependent apoptosis, which subsequently led to activation of caspase-3. Both sensitive and resistant cells expressed caspase-8, caspase-3, FADD, XIAP, and c-FLIP at similar levels. A significant enhancement in cell death was observed in TRAIL-resistant cells when c-FLIP(L) levels were downregulated by RNA interference. CONCLUSIONS: These data suggest that sensitivity to TRAIL and chemotherapy does not necessarily correlate in human ovarian cancer cells. Cancerous cells isolated from patients with ovarian cancer show variable sensitivity to TRAIL but most normal ovarian epithelial cells are sensitive. In human ovarian cancer cells, c-FLIP(L) may participate to the regulation of the TRAIL signaling cascade.     

Recombinant adenovirus-p53 gene transfer and cell-specific growth suppression of human cervical cancer cells in vitro and in vivo

PURPOSE: We investigated the time-course expression patterns of p53 and E6 on cervical cancer cells to obtain a molecular level understanding of cell-dependent tumor growth suppression effects of recombinant adenovirus expressing p53 in vitro and in vivo. METHODS: Four human papillomavirus (HPV)-infected human cervical cancer cell lines (HPV 16-positive cells, CaSki and SiHa cells; and HPV 18-positive cells, HeLa and HeLaS3 cells) were used. Also, HPV negative C33A and HT3 cell line that has a mutation on p53 gene were used. After infection with AdCMVp53, the cell growth inhibition was studied via cell count assay, MTT assay, and Neutral red assay. After transfecting AdCMVp53 and AdCMVLacZ into the cancer cells-xenografted nude mice, antitumor effects were investigated for 1 month, respectively. RESULTS: For each cervical cancer cell, IC50 was as follows; CaSki (68.5 multiplicity of infection, or MOI), SiHa (43.5 MOI), HeLa (31 MOI), HeLaS3 (42 MOI), C33A (21 MOI), and HT3 (62 MOI). In particular, complete inhibition of cell growth was observed at 125 MOI in both CaSki and SiHa cells. However, the complete inhibition was detected at 62.5 MOI in HeLa and HeLaS3. In contrast, at these MOI, no suppression of cell growth was observed when cells were infected with recombinant adenovirus expressing beta-gal as a negative control. The levels of p53 protein were notably expressed in CaSki and HeLa more than in SiHa and HeLaS3 on days 2 and 4. However, the p53 was only detected in HeLaS3 on day 6. In contrast, p53 expression was continually maintained in C33A and HT3 during the same periods. After transfection AdCMVp53 into CaSki- and SiHa-xenografted nude mice, the size of tumor was remarkably decreased in SiHa cells as compared to AdCMVLacZ transfection. CONCLUSION: The adenovirus-mediated p53 gene transfection was done effectively in vitro and in vivo. Also, the antitumor effects were accomplished via differential role of p53-specific apoptotic cell death, which is dependent upon the cervical cancer cell line.