大蒜素上调TRAIL表达诱导宫颈癌细胞凋亡机制研究

目的观察大蒜素对人宫颈癌Hela、Caski细胞的生长抑制作用,大蒜素作用前后TRAIL及其受体DR4、DR5的表达以及Caspase-3、8、9活性变化,探讨大蒜素诱导宫颈癌细胞凋亡作用及其机制。方法 (1)2009-2010年于河北医科大学第二医院,体外培养Hela、Caski细胞,MTT法检测不同浓度大蒜素对细胞的生长抑制作用。(2)RT-PCR方法检测大蒜素对细胞TRAILmRNA表达的影响。(3)流式细胞仪检测大蒜素作用前后细胞TRAIL及其受体DR4、DR5的表达。(4)检测大蒜素对HeLa、Caski细胞Caspase-3、8、9活性的影响。结果 (1)大蒜素对细胞有不同程度的生长抑制作用,随着药物浓度的增加以及作用时间的延长,细胞毒作用逐渐增强。(2)大蒜素处理细胞12h后TRAILmRNA表达水平明显升高,实验组与对照组比较差异有统计学意义(P<0.05)。(3)大蒜素处理细胞24h后TRAIL蛋白表达水平上调。(4)大蒜素作用48h后,两种细胞半胱氨酸蛋白酶(Caspase)-3、8、9的活性明显增强。结论大蒜素对Hela和Caski细胞具有生长抑制作用;上调TRAIL表达,进而活化Cas...     

TRAIL联合化疗药物对子宫内膜癌细胞增殖及凋亡的影响

目的:观察肿瘤坏死因子相关凋亡诱导配体(TRAIL)联合化疗药物对子宫内膜癌Ishikawa细胞的增殖及凋亡的影响。方法:MTT法检测TRAIL单独或联合应用阿霉素(DOX)、顺铂(c-DDP)对Ishikawa细胞增殖的作用;流式细胞仪分析TRAIL单独或联合应用DOX、c-DDP对Ishikawa细胞凋亡的影响。结果:单独应用TRAIL(50μg/L),DOX(5mg/L),c-DDP(50mg/L)对Ishikawa细胞增殖无明显抑制作用(P>0.05)。TRAIL(50μg/L)联合低浓度DOX(5mg/L)、c-DDP(50mg/L)处理Ishikawa细胞后,细胞增殖抑制率明显高于对照组及单独作用细胞(P<0.01);不同浓度的TRAIL、DOX、C-DDP均能诱导Ishikawa细胞凋亡,但DOX、C-DDP能显著增强TRAIL诱导Ishikawa细胞凋亡(P<0.01)。结论:TRAIL联合低浓度化疗药物DOX、c-DDP显著抑制了Ishikawa细胞增殖,诱导了Ishikawa细胞凋亡。     

HIF-1α与TRAIL-DR4在宫颈癌组织中的表达及与放疗敏感性的研究

目的:研究缺氧诱导因子-1α(HIF-1α)及肿瘤坏死因子相关的凋亡诱导配体(TRAIL)受体在宫颈癌组织中的表达变化,及与放疗敏感性的关系。方法:收集2003年7月至2005年1月宫颈癌活检组织45例,用原位杂交组织化学法检测宫颈癌组织中HIF-1αmRNA及TRAIL-DR4mRNA水平。结果:(1)在45例宫颈癌组织中27例(60%) HIF-1αmRNA呈阳性表达,Ⅰ/Ⅱ期肿瘤HIF-1αmRNA的表达显著低于Ⅲ/Ⅳ期肿瘤的表达(X~2=9．07,P＜0．05),随着组织分化的降低,HIF-1αmRNA的表达增强(X~2=8．03,P＜0．05),放疗敏感患者中HIF-1αmRNA表达明显低于放疗不敏感患者(X~2=10．37,P＜0．05);(2)TRAIL-DR4mRNA阳性表达率为57．8%。Ⅰ/Ⅱ期肿瘤DR4mRNA的表达显著高于Ⅲ/Ⅳ期肿瘤的表达(X~2=9．01,P＜0．05),中高分化宫颈癌的表达明显高于低分化宫颈癌(X~2=18．18,P＜0．05),放疗敏感患者中TRAIL-DR4mRNA表达明显高于放疗不敏感患者(X~2=6．91,PP＜0．05)。结论:HIF-1αmRNA及TRAIL-DR4mRNA与宫颈癌的放疗敏感有相关性。     

肿瘤坏死因子相关诱导凋亡配体及其受体与宫颈癌的关系

宫颈癌是女性生殖系统最常见的恶性肿瘤之一,其发生率呈明显上升且年轻化的趋势。目前,除传统手术及放化疗方式治疗恶性肿瘤外,肿瘤生物学治疗方法的地位逐渐提高。肿瘤坏死因子相关诱导凋亡配体(TRAIL)是近年新发现的肿瘤坏死因子超家族成员之一,与细胞膜表面的特异性受体结合,通过活化天冬氨酸蛋白水解酶或线粒体依赖途径选择性地诱导肿瘤细胞凋亡而正常细胞可凋亡逃逸,这种性质使其被认为是肿瘤治疗中极有潜力的靶向治疗因子,具有十分重要的临床价值。研究发现,TRAIL及其受体在宫颈癌组织中存在异常表达,并成为近年研究的热点。综述TRAIL及其受体的结构、生物学功能,在宫颈癌上皮细胞中的表达情况及其在宫颈癌中的研究进展。     

肿瘤坏死因子相关诱导配体与化疗药物联合诱导抗化疗卵巢癌细胞凋亡的研究

目的探讨肿瘤坏死因子（TNF）相关诱导配体（TNF-related apoptosis inducinglig and，TRAIL）和（或）化疗药物联合使用对人卵巢癌细胞系耐药株A2780与SKOV3细胞凋亡的影响。方法2005-01日本国岐阜大学免疫病理3种化疗药物（顺铂、紫杉醇、阿霉素）和TRAIL分别加入培养的A2780与SKOV3细胞，通过hoechst 33342染色在荧光显微镜下确定细胞凋亡效应；蛋白印记杂交法分析bax，bcl-2，及caspase-3，8蛋白表达量的变化。应用四甲基偶氮唑蓝（MTr）法测定A2780与SKOV3细胞体外增殖活性。结果3种化疗药物与TRAIL对A2780和SKOV3细胞的生长都表现抑制作用，3种化疗药物与TRAIL联合用药组A2780与SKOV3的生长抑制率明显高于单独用药组。结论3种化疗药物与TRAIL联合作用所产生的正效应对卵巢癌临床治疗有潜在的意义。     

干扰素 γ加强肿瘤坏死因子相关凋亡诱导配体抑制神经母细胞瘤细胞的作用

目的探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对人神经母细胞瘤的作用及干扰素 γ(IFN γ)对新型凋亡分子TRAIL抗瘤活性的影响，并探讨其影响机制。 方法应用MTT分析和流式细胞仪研究TRAIL对人神经母细胞瘤细胞的抑制作用及IFN γ对TRAIL抗瘤活性的影响。用RT PCR方法检测IFN γ作用48h后，Caspase 8 mRNA的表达。 结果TRAIL(10,50,100μg/L)单独作用、IFN γ(1000U/mL)单独作用后，对SY5Y细胞的增殖抑制率分别为(438±089)%、(354±166)%、(503±126)%、(533±206)%；凋亡率分別为(518±333)%、(526±364)%、(500±288)%、(814±735)%。IFN γ(1000U/mL)与TRAIL(100μg/L)联合作用后，对SY5Y细胞的增殖抑制率为(4122±709)%；凋亡率为(2129±620)%。RT PCR方法发现IFN γ作用48h后Caspase 8 mRNA的表达明显上调。 结论神经母细胞瘤细胞SH SY5Y对TRAIL不敏感，IFN γ可以明显提高TRAIL对神经母细胞瘤细胞的敏感性，其发生机制可能是通过IFN γ上调Caspase 8 mRNA的表达而实现的。     

自噬基因beclin1对子宫颈癌HeLa细胞生长的作用及其机制

目的 研究自噬基因beclin 1对宫颈癌细胞株HeLa细胞生长的抑制作用,并探讨其可能的机制.方法 实验分为5组:(1)pcDNA3.1(+)-beclin 1组:转染重组载体pcDNA3.1(+)-beclin 1质粒(过度表达beclin 1基因)的HeLa细胞;(2)pSUPER-beclin 1组:转染重组载体pSUPER-beclin 1质粒的HeLa细胞(抑制Beclin 1基因表达);(3)pcDNA3.1(+)组:转染空载体pcDNA3.1(+)质粒的HeLa细胞;(4)pSUPER组:转染空载体pSUPER质粒的HeLa细胞;(5)空白对照组:未转染质粒的HeLa细胞.采用逆转录(RT)PCR技术和蛋白印迹法检测各组细胞中beclin 1 mRNA和蛋白的表达以及凋亡因子--半胱氨酸天冬氨酸蛋白酶9(caspase-9)mRNA及蛋白的表达;四甲基偶氮唑蓝(MTT)比色法检测各组细胞的生长情况;流式细胞仪检测各组细胞的凋亡率;电镜观察和流式细胞仪检测各组细胞的自噬情况.将各组细胞分别接种于裸鼠皮下,观察各组裸鼠体内的成瘤性及肿瘤生长情况.结果 (1)各组细胞中beclin 1、caspase-9 mRNA和蛋白的表达:pcDNA3.1(+)-beclin 1组beclin 1和caspase-9 mRNA的表达水平分别为994.72±468.76和12.88±2.71,pSUPER-beclin 1组分别为0.18±0.63和0.11±0.08,pcDNA3.1(+)组分别为0.57±0.12和4.28±3.25,pSUPER组分别为0.67±0.29和2.77±1.27,空白对照组分别为0.74±0.25和3.67±3.78,pcDNA3.1(+)-beclin 1组均明显高于pcDNA3.1(+)组、pSUPER组和空白对照组,pSUPER-beclin 1组均明显低于pcDNA3.1(+)组、pSUPER组和空白对照组,差异均有统计学意义(P＜0.05).各组细胞中beclin 1和caspase-9蛋白的表达情况与mRNA相似.(2)各组细胞的生长情况:pcDNA3.1(+)-beclin 1组细胞的生长明显慢于空白对照组及pcDNA3.1(+)组,而pSUPER-beclin 1组细胞的生长明显快于空白对照组及pSUPER组,差异均有统计学意义(P＜0.05).(3)各组细胞的凋亡率:pcDNA3.1(+)-beclin 1组为(28.2±2.3)%,pcDNA3.1(+)组为(14.6±4.6)%,pSUPER-beclin 1组为(5.7±2.0)%,pSUPER组为(16.2±3.1)%,空白对照组为(11.2±3.0)%,pcDNA3.1(+)-beclin 1组和pSUPER-beclin 1组分别与另外3组比较,差异均有统计学意义(P＜0.05).(4)各组细胞的自噬情况:pcDNA3.1(+)-beclin 1组细胞内可见自噬体的形成,而其余各组自噬体形成不明显.pcDNA3.1(+)-beclin 1组自噬率为(10.3±1.5)%,pcDNA3.1(+)组为(3.6±0.8)%,pSUPER-beclin 1组为(1.2±0.3)%,pSUPER组为(3.2±1.2)%,空白对照组为(2.2±1.1)%,pcDNA3.1(+)-beclin 1组和pSUPER-beclin 1组分别与另外3组比较,差异均有统计学意义(P＜0.05).(5)各组裸鼠的成瘤情况:pcDNA3.1(+)-beclin 1组裸鼠成瘤时间长于空白对照组、pcDNA3.1(+)组和pSUPER组.pSUPER-beclin 1组裸鼠肿瘤体积第7天开始明显大于空白对照组、pcDNA3.1(+)组和pSUPER组(P＜0.05);pcDNA3.1(+)-beclin 1组第21天开始明显小于空白对照组、pcDNA3.1(+)组和pSUPER组(P＜0.05).接种第28天,pSUPER-beclin 1组肿瘤质量为(0.52±0.08)g,明显高于空白对照组的(0.37±0.12)g和pSUPER组的(0.34±0.24)g(P＜0.05);pcDNA3.1(+)-beclin 1组肿瘤质量为(0.18±0.12)g,明显低于空白对照组和pcDNA3.1(+)组的(0.34±0.18)g(P＜0.05).结论 自噬基因beclin 1可以抑制宫颈癌HeLa细胞体内、外的生长,这种作用不仅与自噬调控通路有关,而且可能通过调控caspse-9基因的表达参与内源性细胞凋亡通路的调节,为宫颈癌基因治疗提供了新途径.

Abstract：

Objective To investigate the inhibitory effects and the mechanism of autophagy gene beclin 1 on cervical cancer HeLa cells. Methods The eukaryotic expression vector and short hairpin RNA (shRNA) expression vector of beclin 1 were transfected via lipofectamine into HeLa cells. Experimental cells were classified into 5 groups: pcDNA3. 1 ( + )-beclin 1 group, pSUPER-beclin 1 group, pcDNA3.1 ( + )group, pSUPER group and HeLa group. Real time-PCR and western blot were used for detecting expression of mRNA and protein of beclin 1 and caspase-9 in transfected cells. Flow cytometry was employed to observe the effect of transfection on the apoptosis, and autophagy of HeLa, while proliferation was analyzed by methyl thiazolyl tetrazolium (MTT) assay. The ultrastructural analysis of autophagic vacuoles was under the electron microscope. Five groups cells were seeded subcutaneously on nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed, and immunohistochemistry was used to detect the protein expression of beclin 1 in tumor tissue. Results ( 1 ) The mRNA expression of beclin 1 and caspase-9: pcDNA3. 1 ( + )-beclin 1 group were 994.72 ±468.76 and 12. 88 ±2. 71, pSUPER-beclin 1 group were 0. 18 ± 0. 63 and 0. 11 ± 0. 08, pcDNA3. 1 ( + ) group were 0. 57 ± 0. 12 and 4. 28 ± 3. 25,pSUPER group were 0. 67 ± 0. 29 and 2. 77 ± 1.27, and HeLa group were 0. 74 ± 0. 25 and 3.67 ± 3.78,respectively. The eukaryotic expression vector pcDNA3. 1 ( + ) -beclin 1 significantly improved the expression of mRNA of beclin 1 and caspase-9 in HeLa cells( P ＜0. 05 ), and the shRNA expression vector inhibited the expression of mRNA of beclin 1 and caspase-9 ( P ＜ 0.05 ). (2) The cell proliferations: pcDNA3.1 ( + ) -beclin 1 vector significantly inhibited the growth of HeLa cells, while pSUPER-beclin 1 vector significantly improved the growth of HeLa cells(P ＜0. 05). (3) The rate of apoptosis: pcDNA3.1 ( + )-beclin 1 group was (28.2 ±2.3)%, pcDNA3. 1( + ) group was(14.6 ±4.6)%,pSUPER-beclin 1 group was(5.7 ±2. 0) %, pSUPER group was( 16. 2 ± 3.1 ) %, and HeLa group was( 11.2 ± 3. 0) %. The pcDNA3. 1 ( + ) -beclin 1 vector significantly increased the apoptosis rate, while the pSUPER-beclin 1 vector significantly decreased the apoptosis rate(P ＜0. 05 ). (4)The activity of autophagy: more autophagy cells were identified in pcDNA3.1( + )-beclin 1 group; the rate of autophagy of five group were( 10. 3 ± 1.5)% in pcDNA3. 1 ( + ) -beclin 1 group, ( 3.6 ± 0. 8 ) % in pcDNA3. 1 ( + ) group, ( 1.2 ± 0. 3 ) % in pSUPER-beclin 1 group, (3.2 ± 1.2)% in pSUPER group and (2.2 ± 1. 1)% in HeLa group, there was statistical significances between test groups and control groups( P ＜ . 05 ). (5)Carcinogenic activity of HeLa cells in nude mice: the duration of tumorigenesis was the longest in pcDNA3.1 ( + )-beclin 1 group and the shortest in pSUPER-beclin 1 group among all groups. The tumor size began to grow larger from 7th day after injection in pSUPER-beclin 1 group than in control groups( P ＜ 0. 05 ). The tumor size was smaller from 21st day after injection in pcDNA3.1( + )-beclin 1 group than in control groups(P ＜0. 05). From 28th day after injection,the tumor weigh was (0. 52 ± 0. 08 )g in pSUPER-beclin 1 group, apparently more than HeLa group (0. 37 ±0. 12) g and pSUPER group (0. 34 ± 0. 24 ) g ( P ＜ 0. 05 ). While in pcDNA3. 1 ( + )-beclin 1 group the tumor weighed (0. 18 ±0. 12) g, which was lower than HeLa group and pcDNA3. 1 ( + ) group (0. 34 ± 0. 18 ) g ( P ＜ 0. 05 ) . Conclusions Autophagy gene beclin 1 overexpression can inhibit proliferation and growth of HeLa cells in vitro and in vivo. Beclin 1 not noly participate in the regulation of autophagy signaling, but also play an important role in the regulation of endogenous apoptosis signaling through caspase-9. So it might be one of the new strategies for gene therapy of cervical carcinoma.     

宫颈癌细胞TRAIL死亡受体和诱骗受体表达的研究

目的:检测宫颈癌细胞HeLa表面TRAIL死亡受体(DR4、DR5)及诱骗受体(DcR1、DcR2)的表达差异,研究HeLa细胞系对TRAIL蛋白诱导的凋亡敏感程度。方法:应用MTT法检测TRAIL蛋白对人宫颈癌细胞HeLa的诱导凋亡率,分析宫颈癌细胞对TRAIL蛋白的敏感程度;应用逆转录聚合酶链反应技术(RTPCR)、流式细胞术,检测人宫颈癌细胞HeLa表面TRAIL受体DR4、DR5、DcR1、DcR2mRNA和蛋白的表达,分析宫颈癌细胞表面死亡受体DR4、DR5与诱骗受体DcR1、DcR2表达的相对程度。结果:HeLa细胞对TRAIL诱导的细胞凋亡反应有较高的敏感性,在较低浓度(100ng/ml)TRAIL的作用下,即有接近50%的细胞杀伤率。HeLa细胞表面DR4、DR5受体mRNA的表达明显高于DcR1、DcR2受体,差异有显著性(P<0.01),DR4、DR5受体之间及DcR1、DcR2受体之间表达差异无显著性(P>0.01)。HeLa细胞表面DcR1,DcR2受体蛋白的表达率(17.7%、5.3%)明显较DR4,DR5受体表达率(99.9%、97.8%)低(P<0.01)。结论:宫颈癌细胞HeLa对TRAIL诱导的细胞凋亡反应有较高的敏感性,而且DcR1、DcR2在宫颈癌细胞表面的表达程度相对低于DR4、DR5,将TRAIL用于治疗宫颈癌具有潜在的应用前景。     

TRAIL对3AO裸鼠移植瘤治疗作用的实验研究

目的 :探讨肿瘤坏死因子相关凋亡诱导配体 (TRAIL)对 3AO裸鼠皮下及腹腔移植瘤的治疗作用。方法 :将 5× 10 6 和 2× 10 7对数生长期的 3AO分别接种于BALB/C裸鼠右下肢皮下和腹腔内 ;待皮下组移植瘤长至 0 .2 5cm3,腹腔移植瘤组移植后 96h ,随机分为 5组。裸鼠皮下和腹腔移植瘤A、B组为TRAIL组 ,剂量分别为 0 .5 μg/ml和 1μg/ml;C组为TRAIL0 .5 μg/ml加cDDP 1.5mg/kg ,D组为cDDP 3mg/kg ;E组为对照组 (将生理盐水分别注射于瘤周和腹腔 ) ,观察移植瘤成瘤率、裸鼠生存期和生存期延长率。同时观察停药后 1d、1周、2周裸鼠移植瘤细胞的细胞凋亡率及CD95、Apo2 .7、P5 3、Bcl 2基因表达的变化。结果 :(1)A、B、C和D组裸鼠皮下移植瘤的抑制率分别为 2 3%、4 9.1%、6 2 .3%和 5 2 .6 % ,差异有显著性 (P <0 .0 5 ) ;(2 )腹腔移植瘤组 :A、B、C、D组裸鼠移植瘤的成瘤率分别为 89.1%、72 .3%、5 1.7%和 6 1.5 % ;差异有显著性 (P <0 .0 5 ) ;(3)TRAIL作用裸鼠移植瘤后CD95与Apo2 .7的表达均升高 ,P5 3基因表达稍升高 ,Bcl 2基因表达降低。结论 :(1)TRAIL对人卵巢癌裸鼠移植瘤有明显的抑制作用 ,TRAIL能明显降低裸鼠的成瘤率及死亡率 ,延长生存期 ;(2 )其机制可能与基因CD95、Apo2 .7、Bcl 2的调节有关     

体外模拟二氧化碳人工气腹对宫颈癌细胞生长的影响

目的 观察二氧化碳（CO2）人工气腹对宫颈癌细胞生长的影响，探讨其与乏氧的关系。方法 2005-10—2006-03华中科技大学同济医学院附属协和医院通过裸鼠右下腹腔注射宫颈癌细胞，注射宫颈癌细胞株（Hela）数量：4×10^9／L：将注射后40只裸鼠随机分成4组，每组10只：阴性对照组（常氧组）：仅给予右下腹腔注射宫颈癌细胞；CO2气腹模拟环境按CO2灌注压分3组：CO2灌注压4mmHg（1mmHg=0．133kPa）组，CO2灌注压10mmHg组，CO2灌注压16mmHg组．4周后观察裸鼠体重改变、切除瘤灶称瘤重、瘤体标本用免疫组化测定增殖细胞核抗原（PCNA）、Western-blot方法检测瘤体的缺氧诱导因子-1α（HIF-1α）表达．结果 与阴性对照组比较，CO2气腹各组裸鼠体重下降明显，而瘤体重量增加相对显著，并随着CO2灌注压的增高这种趋势更为显著（P〈0．05）；CO2气腹各组裸鼠PCNA表达显著高于阴性对照组（P〈0．01）；CO2气腹各组裸鼠HIF-1α蛋白的表达与阴性对照组比较明显增加，差异有统计学意义（P〈0．01）．结论 与阴性对照组比较，CO2人工气腹环境可促进宫颈癌细胞生长，其机制可能与HIF-1α激活了PCNA表达，促进了细胞增殖有关。     

Expression of TRAIL (TNF-related apoptosis-inducing ligand) receptors in cervical cancer

Abstract. Ryu H-S, Chang K-H, Chang C-J, Kim M-S, Joo H-J, Oh KH. Expression of TRAIL (TNF-related apoptosis-inducing ligand) receptors in cervical cancer.
Apoptosis is an intrinsic and fundamental biologic process that plays a critical role in the normal development of multicellular organisms and in the maintainance of tissue homeostasis. Some of the well known regulators of apoptosis are cytokines of the tumor necrosis factor (TNF) ligand family, such as Fas ligand (Fas L) and TNF, which induce apoptosis by activation of their corresponding receptors, Fas and TNFR-1. Recently, a new member of the TNF family known as TRAIL (TNF-related apoptosis-inducing ligand) was identified and shown to induce p53-independent apoptosis in a variety of tumor cell lines but not in normal cells. Four human receptors for TRAIL were also recently identified and designated TRAIL-R1, -R2, -R3, and -R4. The aim of this study is to examine whether TRAIL and TRAIL receptors (-R1, -R2, -R3) are expressed in uterine cervical cancer and whether it is correlated with apoptosis, TRAIL, and TRAIL receptors. The subjects were 20 patients who were diagnosed with cervical cancer. Western blotting was performed in nine cases and immunohistochemical staining for TRAIL and TRAIL receptors (-R1, -R2, -R3) and TUNEL method for detection of apoptosis was performed in 11 cases. There were proteins for TRAIL, TRAIL-R1, -R2, and -R3 in tissues from cervical cancer. All TRAIL receptors were expressed in both normal cervical epithelium and tumor cells, and TRAIL-R1 and -R2 were more strongly expressed in tumor cells than normal epithelium ( P < 0.05). Apoptosis correlated with expression of TRAIL-R1 and -R2 ( P < 0.05). This study suggests that TRAIL induces apoptosis in cervical cancer through its receptors.     

Anti-tumor activity of an anti-DR5 monoclonal antibody, TRA-8, in combination with taxane/platinum-based chemotherapy in an ovarian cancer model

Objective

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis via binding to death receptors and enhances the anti-tumor effect of conventional cancer therapies. We evaluated the efficacy of TRA-8, an agonistic antibody to DR5, combined with docetaxel and carboplatin in vitro in an intraperitoneal (IP) ovarian cancer model.

Methods

Luciferase positive ES2 cells (ES2H) were treated in 96 well plates with TRA-8, carboplatin, docetaxel, and combination therapy. Cell viability was assessed using ATP-lite assay. Apoptosis was confirmed via Western blot analysis. ES2H cells were injected IP into female athymic nude mice. Animals were sorted based on bioluminescent signal with the following treatments: 1) untreated; 2) TRA-8 alone; 3) docetaxel + carboplatin; and 4) docetaxel + carboplatin + TRA-8. Animals receiving TRA-8 antibody were injected IP with 200 μg of TRA-8 twice weekly until death. Animals receiving docetaxel + carboplatin were injected IP with 5 mg/kg and 15 mg/kg respectively every 3 weeks until death. Animals were assessed for tumor burden using bioluminescence imaging and overall survival.

Results

Combination therapy reduced viability of ES2H cells in vitro over single agent therapy. Tumor burden was lowest in the chemotherapy + TRA-8 group at days 23 (p < 0.001) and 30 (p = 0.04). Mean survival was greatest in the chemotherapy + TRA-8 group (41 days) compared to the chemotherapy only group (34 days) and control group (27 days) as determined by Kaplan-Meier analysis (p < 0.001).

Conclusion

Conventional chemotherapy combined with TRA-8 reduced cell-viability via activation of apoptotic pathways, reduced tumor burden and improved survival in this ovarian cancer model.     

阿司匹林对宫颈癌细胞系Caski的生长抑制作用

目的　探讨阿司匹林对宫颈癌细胞系Caski作用。方法　利用细胞计数法对宫颈癌细胞系Caski细胞进行活细胞计数 ,利用流式细胞仪检测细胞DNA含量。结果　阿司匹林对宫颈癌细胞系Caski细胞的抑制呈剂量依赖性 ,在 1- 5mM浓度范围内 ,生长抑制率为 2 0 %～ 86 %。阿司匹林作用后的Caski细胞呈现出G1和G2期细胞数目增加 ,而S期细胞数目减少的趋势。结论　阿司匹林对宫颈癌细胞系Caski有抑制作用     

人正常宫颈脱落细胞及宫颈癌细胞的AFM观察研究

目的:探讨原子力显微镜(AFM)在人宫颈脱落细胞及宫颈癌细胞微观形貌表征方面的应用。方法:利用液基薄层细胞学检测技术制备细胞样品,应用原子力显微镜分别观察人宫颈脱落细胞及宫颈癌细胞的微观形态。结果:癌变宫颈细胞核独立分布,尺寸变大,且易于聚集,细胞质分散在细胞核周围;正常宫颈细胞的细胞核被纳米条状的细胞质覆盖,成为一个整体。结论:采用AFM检测液基薄层细胞学检测技术制备的细胞样品的微观形貌能反映出与宏观光学显微镜检测获得相同的结论,是一种简易的检测方法。癌变宫颈细胞和正常宫颈细胞在微观上存在明显不同:癌变宫颈细胞核的尺寸增大、聚集在一起;而正常宫颈细胞被纳米条状的细胞质所覆盖。     

外源性PUMA表达对人乳头瘤病毒阳性宫颈癌放疗敏感性的影响

目的 探讨外源性PUMA(p53 up-regulated modulator of apoptosis)基因对人乳头瘤病毒(HPV)阳性宫颈癌放疗敏感性的影响.方法 以重组腺病毒为载体,将PUMA基因导入HPV阳性宫颈癌细胞HeLa和caSki.应用MTT法比较携带PUMA的复制缺陷型重组腺病毒(Ad-PUMA)和携带野生型p53基因的复制缺陷型重组腺病毒(Ad-p53)对HeLa和Caski细胞增殖的影响,平板克隆方法检测Ad-PUMA与X射线联合应用后对细胞生长的影响,并通过DAPI染色、流式细胞检术检测细胞凋亡情况.Western Blot法检测x射线处理后HeLa细胞PUMA表达.结果 (1)Ad-PUMA能够明显抑制HeLa和CaSki细胞增殖,增加细胞凋亡,而Ad-p53对细胞生长无明显抑制作用.(2)X射线能够诱导HeLa细胞PUMA表达升高,具有时间和剂量效应.(3)Ad-PUMA与X射线联合应用后可显著增加细胞凋亡,减少平板克隆形成数,但Ad-PUMA对X射线引起的G2期阻滞无明显影响.结论 PUMA在放射线诱导肿瘤细胞凋亡的过程中发挥作用.Ad-PUMA抑制宫颈癌细胞增殖,诱导细胞凋亡.Ad-PUMA与X射线联合应用后显著提高宫颈癌细胞放疗敏感性.     

Progesterone inhibits growth and induces apoptosis in cancer cells through modulation of reactive oxygen species

Objective.?Progesterone (P4) has been implicated as a protective factor for ovarian and endometrial cancers, yet little is known about its mechanism of action. We have shown apoptosis in ovarian and endometrial cancer cells with high doses of P4. Increased generation of reactive oxygen species (ROS) and an altered redox status have long been observed in cancer cells. The goal of this study was to assess the effect of P4 on cell growth, ROS generation, oxidative stress markers, and the expression of antioxidant proteins.

Methods.?All experiments were performed in vitro using cancer cell lines. Cell proliferation was determined using MTS proliferation assay. Production of ROS in cells was measured with the ROS indicator dye, aminophenyl fluorescein. Alterations in expression of antioxidant and apoptotic proteins were assessed by Western blotting.

Results.?The exposure of ovarian and endometrial cancer cell cultures to various doses of P4 caused a dose-dependent decrease in cell viability and the activation of caspase-3. Levels of ROS, markers of oxidative stress, and antioxidant proteins were elevated in cancer cells compared to normal cells and a marked decrease in their expression was seen following P4 treatment. In cancer cells, ROS was elevated while p-53 expression was low. P4 exposure of cells resulted in increased p-53 and BAX and decreased BCL-2 expression.

Conclusions.?The data indicates that P4 has antioxidant effects. It alleviates ROS stress and causes apoptosis by upregulating proapoptotic (p-53 and BAX) and decreasing antiapoptotic (BCL-2) gene expression in cancer cells. These findings could have potential therapeutic implications.     

Notch1 induces cell cycle arrest and apoptosis in human cervical cancer cells: involvement of nuclear factor kappa B inhibition

Notch signaling can serve as a tumor suppressor or tumor promoter in the same kind of cancer, such as human papillomavirus-positive cervical cancer cells. However, the exact mechanisms remain poorly characterized. Our studies demonstrated that constitutively overexpressed active Notch1 via stable transfection with exogenous intracellular domain of Notch1 (ICN) resulted in growth inhibition of the human cervical cancer cell line HeLa by inducing G(2)-M arrest and apoptosis. Moreover, the growth inhibition was correlated with inhibition of nuclear factor kappa B (NF-kappaB) p50 activation, accompanied by a decrease in the nuclear expression of NF-kappaB p50 and an increase in the cytosolic expression of IkappaBalpha. Consistent with these results, downregulation of cyclin D1 and Bcl-2, which are both the downstream genes of NF-kappaB, were observed in ICN-overexpressed cells. Overall, our results suggest that NF-kappaB inhibition may contribute partially to cell cycle arrest and apoptosis induced by Notch1 activation in human cervical cancer cells.     

Susceptibility to cervical cancer: an overview

Cervical cancer is the second most common cancer in females worldwide. It is well-established that Human Papillomavirus (HPV) infections play a critical role in the development of cervical cancer. However, a large number of women infected with oncogenic HPV types will never develop cervical cancer. Thus, there are several external environment and genetic factors involved in the progression of a precancerous lesion to invasive cancer. In this review article, we addressed possible susceptible phenotypes to cervical cancer, focusing on host genome and HPV DNA variability, multiple HPV infections, co-infection with other agents, circulating HPV DNA and lifestyle.