磷脂酰肌醇3激酶通路在胰岛素调节子宫内膜癌细胞增殖凋亡中的作用

目的 探讨磷脂酰肌醇3激酶(PI3K)信号通路在胰岛素调节子宫内膜癌细胞生长中的作用.方法 2006年5月至2006年10月于中国医学科学院血液病学研究所临床药物室完成试验,即将子宫内膜癌Ishikawa3-H-12细胞进行无血清饥饿培养,分成不同处理组,即空白对照组、胰岛素刺激组(Ins组)、PI3K抑制剂--LY294002预处理后再以胰岛素刺激组(LY Ins组).以四甲基偶氮唑蓝(MTT)试验和流式细胞仪观察细胞增殖和凋亡情况.不同处理组分别培养24h、48h和72h,观察各组570nm波长吸光度(OD570nm).结果 Ins组细胞OD570nm值高于空白对照组(P＜0.01);LY Ins组细胞OD570nm低于Ins组(P＜0.01),LY294002拮抗了胰岛素对内膜癌细胞的增殖促进作用.流式细胞仪检测凋亡发现Ins组细胞凋亡率低于空白对照组[(2.02±0.39)%对(14.45±2.70)%,P＜0.01];LY Ins组细胞凋亡率高于Ins组[(18.73±2.11)%对(2.02±0.39)%,P＜0.01].结论 PI3K信号通路在胰岛素引起的促内膜癌细胞增殖、抑制凋亡中起重要作用,胰岛素对子宫内膜癌细胞增殖、凋亡的调节作用是PI3K依赖性的.     

GOLPH3通过PI3K/Akt信号通路调控上皮性卵巢癌细胞增殖与凋亡的研究

目的:探讨GOLPH3基因对上皮性卵巢癌细胞增殖及凋亡的影响及作用机制。方法:siRNA干扰技术沉默SKOV3细胞株中GOLPH3基因表达,MTT法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Western blot法检测PI3K/Akt信号通路相关基因Akt、p-Akt 473、p-Akt 308及Bcl-2表达。PI3K/Akt信号通路抑制剂LY294002处理卵巢癌SKOV3细胞后,检测SKOV3细胞的增殖能力及凋亡情况。结果:siRNA干扰SKOV3细胞株中GOLPH3基因,GOLPH3表达下调,细胞增殖减少,凋亡增多;PI3K/Akt信号通路相关基因p-Akt 473、p-Akt 308、抗凋亡基因Bcl-2蛋白表达水平均下降,Akt蛋白水平表达无显著变化。LY294002处理SKOV3细胞后,细胞增殖率降低,凋亡率升高。结论:GOLPH3基因可能通过激活PI3K/Akt信号通路促进卵巢癌细胞增殖,并抑制其凋亡的作用。     

TFF3激活PI3K/Akt通路参与宫颈腺癌的发病机制

目的:探讨TFF3通过激活PI3K/Akt信号通路调控宫颈腺癌发生发展的机制。方法:TFF3细胞因子和PI3K/Akt信号通路抑制剂(LY294002)或两者共同处理Hela细胞,CCK-8法检测Hela细胞增殖能力,Transwell小室观察细胞迁移能力,流式细胞学方法检测细胞凋亡,Western blot检测PI3K/Akt信号通路关键蛋白Akt及其激活状态蛋白p-Akt的表达。结果:与对照组相比,TFF3处理后Hela细胞的增殖和迁移能力明显提高,细胞凋亡减弱,差异有统计学意义(P0.05);LY294002处理后Hela细胞的增殖及迁移能力明显降低,细胞凋亡增多,差异有统计学意义(P0.05);TFF3+LY294002处理后Hela细胞的增殖、迁移能力及细胞凋亡与TFF3和LY294002处理组比较,差异均有统计学意义(P0.05)。Western blot结果表明,TFF3能激活p-Akt基因表达。结论:TFF3能通过提高p-Akt蛋白表达激活PI3K/Akt信号通路,从而参与调节宫颈腺癌细胞的恶性行为。     

抵抗素促进子宫内膜癌细胞生长作用机制的探讨

目的:研究抵抗素对子宫内膜癌细胞增殖的影响及其对子宫内膜癌细胞PI3K/Akt信号传导通路的作用。方法:CCK-8法检测不同浓度抵抗素(0ng/ml、10ng/ml、50ng/ml、100ng/ml、200ng/ml)对Ishikawa和KLE两种子宫内膜癌细胞系增殖的影响。Western blot法检测不同浓度抵抗素作用下PI3K/Akt信号通路的变化及PI3K/Akt信号通路抑制剂对抵抗素活化PI3K/Akt信号通路作用的影响。结果:抵抗素能促进两种子宫内膜癌细胞系Ishikawa和KLE的生长,并呈时间和浓度依赖性。抵抗素可促进两种子宫内膜癌细胞系中PI3K/Akt信号通路的激活。PI3K/Akt信号通路抑制剂LY29004可减弱抵抗素诱导的Akt磷酸化水平。结论:抵抗素能促进子宫内膜癌细胞增殖,这种作用可通过激活PI3K/Akt信号通路来实现。     

双酚A激活PI3K/AKT通路促进子宫内膜癌细胞增殖的研究

目的:研究双酚A(bisphenol A,BPA)活化磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)促子宫内膜癌细胞Ishikawa和RL952增殖的机制。方法:CCK8试剂盒检测Ishikawa和RL952细胞的增殖情况,蛋白质印迹(Western blotting)法检测p-AKT蛋白的表达。结果:在Ishikawa和RL952细胞中,BPA作用48 h时,细胞增殖呈浓度依赖性,1μmol/L的BPA促细胞生长效应最显著,Ishikawa和RL952细胞增殖率分别为0.758±0.023和0.692±0.042。BPA浓度超过1μmol/L后,促细胞增殖的作用下降。BPA作用24 h时促增殖效应不明显,BPA作用72 h时表现出细胞毒作用。1μmol/L BPA处理的Ishikawa和RL952细胞48 h后p-AKT蛋白的表达较对照组升高(P<0.05),加入PI3K抑制剂(LY294002),p-AKT的蛋白表达比对照组降低,但差异无统计学意义(P>0.05)。结论:BPA通过激活PI3K/AKT通路促进子宫内膜癌细胞增殖。     

双酚A激活PI3K/AKT通路促进子宫内膜癌细胞增殖的研究

目的:研究双酚A(bisphenol A,BPA)活化磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)促子宫内膜癌细胞Ishikawa和RL952增殖的机制。方法:CCK8试剂盒检测Ishikawa和RL952细胞的增殖情况,蛋白质印迹(Western blotting)法检测p-AKT蛋白的表达。结果:在Ishikawa和RL952细胞中,BPA作用48 h时,细胞增殖呈浓度依赖性,1μmol/L的BPA促细胞生长效应最显著,Ishikawa和RL952细胞增殖率分别为0.758±0.023和0.692±0.042。BPA浓度超过1μmol/L后,促细胞增殖的作用下降。BPA作用24 h时促增殖效应不明显,BPA作用72 h时表现出细胞毒作用。1μmol/L BPA处理的Ishikawa和RL952细胞48 h后p-AKT蛋白的表达较对照组升高(P0.05),加入PI3K抑制剂(LY294002),p-AKT的蛋白表达比对照组降低,但差异无统计学意义(P0.05)。结论:BPA通过激活PI3K/AKT通路促进子宫内膜癌细胞增殖。     

SDF-1对卵巢癌细胞增殖和侵袭能力作用的相关通路研究

目的:探讨PI3K/Akt信号通路抑制剂LY294002、MEK通路抑制剂PD98059对基质衍生因子-1(SDF-1)作用下卵巢癌细胞SKOV3的增殖、侵袭能力的影响,初步探讨SDF-1在卵巢癌中的相关作用通路。方法:细胞免疫化学法检测SDF-1不同作用时间及不同作用浓度对卵巢癌细胞中磷酸化Akt(p-Akt)和ERK(p-ERK)表达的影响。选取对数生长期细胞,分别加SDF-1 100ng/ml、CXCR4中和抗体10μg/ml、CXCR4抑制剂AMD3100 1μg/ml、LY294002 50μmol/L、PD98059 50μmol/L。MTT、Transwell细胞侵袭实验检测细胞的增殖、侵袭能力的变化。结果:随着SDF-1作用时间的延长,p-Akt、p-ERK蛋白表达水平增高;Akt、ERK蛋白的最佳活化时间为30min。作用30min时,随着SDF-1作用浓度的增加,p-ERK1、p-Akt蛋白表达水平增高,差异具有统计学意义(P0.05)。SDF-1可明显促进SKOV3细胞的增殖、侵袭能力(P0.05);但加入CXCR4中和抗体、CXCR4抑制剂AMD3100、通路抑制剂PD98059、LY294002后,SKOV3细胞的增殖、侵袭能力无明显变化(P0.05)。结论:SDF-1对卵巢癌SKOV3细胞的增殖、侵袭能力的增强作用可被PI3K/Akt信号通路抑制剂、MEK通路抑制剂所阻断。SDF-1可能通过Ras/ERK信号转导通路、PI3K/Akt信号通路发挥作用。     

PI3K抑制剂拮抗雌激素介导的子宫内膜癌细胞Ishikawa增殖的研究

目的:探讨磷脂肌醇3-激酶(PI3K)抑制剂LY294002对雌激素(E2)介导下的子宫内膜癌Ishikawa细胞增殖和凋亡的影响。方法:CCK-8法检测E2对Ishikawa细胞的增殖作用,以及LY294002对Ishikawa细胞增殖的抑制作用;流式细胞仪检测Ishikawa细胞在E2与LY294002作用下的细胞凋亡情况;划痕实验检测细胞迁移能力。结果:E2促进Ishikawa细胞增殖,LY294002抑制E2介导下的Ishikawa细胞增殖,且呈时间浓度依赖性。LY294002作用下,Ishikawa细胞失去了迁移侵袭能力,细胞大量凋亡。结论:E2促进Ishikawa细胞的增殖,具有时间和浓度依赖性,LY294002对E2介导下的Ishikawa细胞增殖具有抑制作用。     

过表达MicroRNA-195抑制子宫内膜癌细胞系Ishikawa的增殖、迁移与侵袭能力

目的:观察MicroRNA-195对子宫内膜癌细胞增殖、迁移与侵袭能力的影响。方法:取对数生长期人子宫内膜癌细胞系Ishikawa,分别转染MicroRNA-195模拟物(MicroRNA-195过表达组)、MicroRNA-195抑制物(MicroRNA-195低表达组)及阴性对照质粒MicroRNA-NC(空白质粒组)。转染48h后,qRT-PCR法检测细胞中MicroRNA-195表达,CCK-8法检测细胞增殖,Transwell小室实验检测细胞迁移与侵袭能力,Western blot法检测Akt、p-Akt蛋白表达。将转染MicroRNA-195模拟物、MicroRNA-195抑制物及阴性对照质粒MicroRNA-NC的人子宫内膜癌细胞系Ishikawa分别注射至裸鼠皮下,每天检测肿瘤体积,实验周期为3周。结果:MicroRNA-195过表达可抑制人子宫内膜癌细胞系Ishikawa的增殖、迁移与侵袭能力(P<0.05),提高p-Akt蛋白表达(P<0.05);MicroRNA-195低表达可促进人子宫内膜癌细胞Ishikawa的增殖、迁移与侵袭能力(P<0.05),降低p-Akt蛋白表达(P<0.05)。MicroRNA-195过表达可抑制裸鼠体内人子宫内膜癌细胞Ishikawa的增殖(P<0.05),MicroRNA-195低表达可促进裸鼠体内人子宫内膜癌细胞Ishikawa的增殖(P<0.05)。结论:MicroRNA-195可抑制子宫内膜癌细胞Ishikawa的增殖、迁移与侵袭能力,其作用机制可能与激活PI3K/Akt信号通路有关。     

抑制磷脂酰肌醇-3-羟基激酶/蛋白激酶B信号传导通路对宫颈癌放疗增敏的作用

目的:探讨抑制磷脂酰肌醇-3-羟基激酶/蛋白激酶B(PI3K/AKT)信号传导通路对宫颈癌的放疗增敏作用。方法:选用LY294002为PI3K阻滞剂。培养PI3K功能活性和AKT磷酸化水平稳定的人宫颈癌HeLa细胞株,皮下注射于BALB/C裸鼠体内,建立裸鼠宫颈癌移植瘤模型。随机将荷瘤裸鼠分为空白对照组、单纯放疗组、单纯药物组(LY294002)和联合治疗组(放疗+LY294002)。应用Western blotting检测各组AKT磷酸化(phospho-Ser473AKT)水平。通过克隆形成实验检测LY294002在体内的放射增敏作用。长期观察肿瘤体积的变化并绘制肿瘤生长曲线。结果:Western blotting显示,联合治疗组的AKT去磷酸化现象最显著。克隆形成实验显示,LY294002与放射治疗对肿瘤细胞生长的抑制在各剂量水平均表现出协同作用(P0.05)。肿瘤生长曲线表明联合治疗组的肿瘤体积增长最缓慢。结论:PI3K阻滞剂显著增强宫颈癌对放射治疗的敏感性。     

Mitogenic and anti-apoptotic effects of insulin in endometrial cancer are phosphatidylinositol 3-kinase/Akt dependent

Objective

To determine serum insulin levels, expression and phosphorylation of InsR, IRS-1 and Akt in endometrial cancer (EC) tissues, and to explore the correlation between them. To investigate if insulin-induced mitogenic and anti-apoptotic effects are PI3K-dependent in EC cells.

Methods

Serum insulin levels were measured by radioimmunoassay. We performed RT-PCR and western blotting to evaluate the expression and activation of key proteins of PI3K/Akt pathway in 63 EC tissues. The proliferation and apoptosis rates were determined with MTT, BrdU and annexin V/PI assays.

Results

Serum insulin levels and InsR, IRS-1 and Akt expression and phosphorylation were significantly elevated in patients with EC compared to those without EC. Additionally, levels of p-InsR, p-IRS-1, and p-Akt were significantly higher in patients with high-grade, advanced stage, deep myometrial invasion, and lymph-node metastasis. The expression and activation of InsR, IRS-1, and p-Akt were positively related with the levels of serum insulin. The insulin-induced mitogenic and anti-apoptotic effects in EC cells were blocked when cells were pre-incubated with LY294002. Ishikawa 3-H-12 cells showed increased p-Akt levels after treatment with insulin at 10^− 8 M for 15 min. The insulin-induced Akt activation was inhibited by LY294002 in a dose-dependent manner.

Conclusion

Insulin played an essential role in EC tumorigenesis. Activation of InsR, IRS-1, and Akt was associated with features of aggressive EC. Insulin was a mitogenic and anti-apoptotic agent for EC cells, and these effects were dependent on PI3K/Akt pathway. Decreasing insulin level and blocking the InsR-IRS-PI3K-Akt pathway could be viable preventive and therapeutic strategies for EC.     

胰岛素对子宫内膜癌细胞增殖和凋亡的影响

目的 探讨胰岛素对子宫内膜癌细胞系Ishikawa3-H-12细胞增殖、凋亡和细胞周期的影响。方法 应用免疫细胞化学方法和RT-PCR技术检测Ishikawa3-H-12细胞胰岛素受体（INSR）蛋白和mRNA的表达。以不同浓度胰岛素作用Ishikawa3-H-12细胞不同时间,采用四甲基偶氮唑蓝比色法、流式细胞仪检测细胞增殖、凋亡和细胞周期。结果 （1）Ishikawa3-H-12细胞INSR蛋白呈棕黄色阳性表达,并可见INSR基因的表达。（2）胰岛素以浓度和时间依赖的方式促进子宫内膜癌细胞增殖,1×10^-4mol／L胰岛素作用48h时促增殖作用最显著,增殖率为（340．2±15．9）％,与对照细胞（以胰岛素浓度为0作为对照,为100％）比较,差异有统计学意义（P〈0．05）。（3）胰岛素以浓度和时间依赖的方式使Ishikawa3-H-12细胞中G0／G1期细胞比例减少,S期细胞比例增加,1×10^-4mol／L胰岛素作用72h时最显著,G0／G1期细胞比例为（27．7±2．5）％,S期细胞为（55．2±1．4）％,分别与对照细胞[分别为（67．6±1．5）％、（15．7±1．0）％]比较,差异均有统计学意义（P〈0．05）;胰岛素对G2／M期细胞无影响（P〉0．05）。（4）随着胰岛素浓度的增加,Ishikawa3-H-12细胞凋亡率逐渐下降。1×10^-4mol／L胰岛素作用最显著,作用24、48、72、96h时的细胞凋亡率分别为（1．76±0．16）％、（1．70±0．15）％、（1．56±0．20）％、（1．31±0．24）％,分别与对照细[分别为（9．81±0．61）％、（9．93±1．44）％、（9．10±0．66）％、（10．30±1．20）％]比较,差异均有统计学意义（P〈0．05）。结论 胰岛素对子宫内膜癌Ishikawa3-H-12细胞具有促进增殖、抑制凋亡的作用。     

17β-雌二醇对子宫内膜癌细胞磷脂酰肌醇3激酶/蛋白激酶B信号传导通路的激活作用

目的　探讨 17β 雌二醇 (E2 )对子宫内膜癌细胞系Ishikawa细胞磷脂酰肌醇 3激酶 /蛋白激酶B(PI 3K/PKB)信号传导通路的激活作用以及PI 3K抑制剂对其的影响。方法　应用免疫印迹杂交技术 ,检测一定浓度 (1× 10 -6mol/L) 17β E2 作用于Ishikawa细胞不同时间 (分别为 0、5、15、30、6 0、12 0min)后和不同浓度 (分别为 0、1× 10 -10 、1× 10 -8、1× 10 -6、1× 10 -4mol/L) 17β E2 作用Ishikawa细胞一定时间 (30min)后PKB的活化情况 [以磷酸化PKB和总PKB比值 (p PKB/PKB)表示 ],以及用同法检测PI 3K抑制剂LY2 94 0 0 2对 17β E2 活化PKB的影响。结果　 1× 10 -6mol/L 17β E2 作用于Ishikawa细胞 15min时 ,PKB的活化水平 (0 5 33± 0 0 2 9)已明显高于对照 (0 36 1± 0 0 2 9,P <0 0 5 ) ,30min时最明显 (0 6 6 6± 0 0 2 1,P <0 0 0 1) ,而且持续至少达 12 0min时 ;随着 17β E2 浓度 (分别为 1× 10 -10 、1×10 -8、1× 10 -6、1× 10 -4mol/L)的增加 ,PKB的活化水平逐渐增高 ,与对照比较 ,差异有显著性 (除 1×10 -10 mol/L外 ,P值分别为 >0 0 5、<0 0 5、<0 0 5、<0 0 0 1)。随着LY2 94 0 0 2作用浓度 (分别为 0 1、10、5 0、10 0 μmol/L)的增加 ,17β E2 作用Ishikawa细胞后P     

雷公藤内酯醇联合紫杉醇对COC1/DDP细胞凋亡的影响及其对PI3K/AKT/GSK3β信号通路的调控

目的：通过研究雷公藤内酯醇（TP）联合紫杉醇对耐顺铂人上皮性卵巢癌细胞（COC1/DDP细胞）凋亡的影响,以探讨两药联合对COC1/DDP细胞作用的机制。方法：将对数生长期COC1/DDP细胞随机分为6组：空白对照组、紫杉醇组（3.13μg/ml）、LY294002组（10μmol/ml）、TP组（10ng/ml）、LY294002＋紫杉醇组（10μmol/ml LY294002＋3.13μg/ml紫杉醇）、TP＋紫杉醇组（10ng/ml TP＋3.13μg/ml紫杉醇）。采用MTT法检测各组的细胞增殖活性;普通光镜及AO/EB染色荧光显微镜观察细胞形态的变化;Annexin V-FITC/PI双染法流式细胞仪检测细胞凋亡率;Western blot法检测各组细胞中Akt、pAkt、GSK3β、p-GSK3β蛋白的表达变化。结果：（1）细胞增殖抑制率：TP＋紫杉醇组显著高于TP、紫杉醇组（P〈0.05）;LY294002＋紫杉醇组显著高于LY294002、紫杉醇组（P〈0.05）。联合用药组中,TP＋紫杉醇组显著高于LY294002＋紫杉醇组（P〈0.05）;单用药组中,TP组〉紫杉醇组〉LY294002组（P〈0.05）。各组抑制率均呈时间依赖性（P〈0.05）。（2）普通光镜及AO/EB染色荧光显微镜下观察,除空白组外,各用药组的细胞形态均有凋亡样改变。两联合用药组的细胞凋亡数显著高于各自单药组,TP＋紫杉醇组高于LY294002＋紫杉醇组;单用药组的细胞凋亡数：TP组〉紫杉醇组〉LY294002组。（3）流式细胞仪检测发现,两联合用药组的细胞凋亡率大于各自单药组（P〈0.05）。联合用药组中,TP＋紫杉醇组凋亡率显著高于LY294002＋紫杉醇组（P〈0.05）;单药组的细胞凋亡率为：TP〉紫杉醇〉LY294002（P〈0.05）。（4）p-Akt蛋白和p-GSK3β蛋白的表达从空白对照组→紫杉醇组→LY294002组→TP组→LY294002＋紫杉醇组→TP＋紫杉醇组,呈逐渐减少的趋势,而总Akt、GSK3β蛋白的表达不变。结论：TP可协同紫杉醇促进COC1/DDP细胞凋亡,其机制可能是通过抑制PI3K/Akt/GSK3β信号通路,从而下调了耐药相关蛋白p-Akt、p-GSK3β的表达。     

Stromal cell-derived factor 1alpha stimulates human endometrial carcinoma cell growth through the activation of both extracellular signal-regulated kinase 1/2 and Akt

OBJECTIVE: The aim of study was to investigate the proliferative effects of stromal cell-derived factor-1alpha (SDF-1alpha) on endometrial carcinomas cell lines with different estrogen receptors (ER) and PTEN protein profiles. METHODS: MTT assays was used to detect the proliferation of HEC-1A and Ishikawa cells, and Western blotted analysis was used to detect activation of Akt and ERK1/2 in both cell lines after exposure to various concentrations of SDF-1alpha, MAPK-specific inhibitor PD98059 or PI3K-specific inhibitor LY294002. RESULTS: Low concentrations of SDF-1alpha (50 ng/ml) induced proliferation in both cell lines. ERK1/2 was significantly activated for more than 2 h by SDF-1alpha at 20 ng/ml in HEC-1A cells, but not in Ishikawa cells. In contrast, Akt was significantly activated for over 2 h in Ishikawa cells but remained unchanged in HEC-1A cells. High concentrations of SDF-1alpha activated Akt and ERK1/2 pathways in both cell lines in a dose-dependent manner, which was primarily inhibited by LY294002 for pAkt and by PD98059 for pERK 1/2. CONCLUSIONS: SDF-1alpha could stimulate the cell proliferation of endometrial carcinoma with different expression status of ER and PTEN in vitro, likely through the activation of both Akt and ERK1/2 signaling pathways.     

Elevated phosphatidylinositol 3-kinase activation and its clinicopathological significance in cervical cancer

OBJECTIVE: The objective was to study the role of PI3K signaling in the development of cervical cancer and the antitumor effect of PI3K inhibitors. STUDY DESIGN: PI3K protein and mRNA expression of cervical cancer and non-neoplastic tissues were analyzed by Western blotting and RT-PCR. PI3K/Akt/mTOR pathway components in HeLa cells were assessed by immunocytochemistry and Western blotting. The inhibitive effect of LY294002 on HeLa cells was studied using MTT assay and flow cytometry. RESULTS: PI3K protein expression was detected in 25 out of 31 tumor specimens. Compared with non-neoplastic tissues, significant overexpression was observed in tumor tissues. For PI3K overexpression with all clinicopathological features, a decreasing trend in adenocarcinoma, advanced stage, and grade was observed. PI3K inhibitor LY294002 efficiently inhibited HeLa cell growth with IC50 of 20.77 microM, and induced apoptosis. The apoptotic rate was 36% at 3h after LY294002 treatment. These pharmacological roles of LY294002 might be played through the PI3K/Akt/mTOR signaling pathway. CONCLUSIONS: The PI3K signaling pathway was implicated in the development of cervical cancer. The activation of its signaling molecules might have clinical implications. Novel targeted therapies for the PI3K/Akt/mTOR signaling pathway components could provide a useful adjuvant therapeutic strategy for cervical cancer.     

信号传导通路抑制剂PD98059和LY294002对子宫内膜癌细胞和裸鼠移植瘤的抑制作用

目的 观察有丝分裂原活化蛋白激酶(MAPK)信号传导通路抑制剂PD98059和磷脂酰肌醇3激酶(PI3K)信号传导通路抑制剂LY294002对子宫内膜癌细胞株Ishikawa细胞和子宫内膜癌裸鼠移植瘤的抑制作用.方法 (1)体外实验:实验分为4组,即PD组(加入浓度分别为0、1、50、100 μmol/L的PD98059)、LY组(加入浓度分别为0、1、50、100 μmol/L的LY294002)、PD+LY组(加入浓度均为50 μmol/L的PD98059和LY294002)、对照组[仅加入二甲基亚砜(DMSO)],分别培养24、48和72 h.应用四甲基偶氮唑蓝(MTT)比色法检测各组Ishikawa细胞的增殖情况,流式细胞仪检测各组Ishikawa细胞的细胞周期比例和细胞凋亡率.(2)体内实验:建立子宫内膜癌裸鼠移植瘤模型,随机分为4组(每组6只):PD组(注射PD98059 50 mg/kg)、LY组(注射LY294002 50 mg/kg)、PD+LY组(注射PD98059 50 mg/kg和LY294002 50 mg/kg)、对照组(注射等量的生理盐水),每周2次,共3周;观察移植瘤的生长情况,并计算抑瘤率;用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸标记(TUNEL)法检测移植瘤组织的细胞凋亡情况,以细胞凋亡指数表示;免疫组化法检测移植瘤组织内磷酸化细胞外信号调节激酶(p-ERK)和磷酸化蛋白激酶B(p-Akt)蛋白的表达.结果 (1)PD98059和(或)LY294002处理后,PD组、LY组Ishikawa细胞增殖的抑制作用均呈明显的时间和浓度依赖性(P＜0.05),且PD+LY组细胞增殖的这种抑制作用明显高于PD组、LY组(P＜0.05).LY组细胞S期、G0/G1期比例的变化呈明显的时间和浓度依赖性(P＜0.05);PD组细胞各期比例的变化均无时间依赖性(P＞0.05),但G0/G1期、S期比例的变化呈明显的浓度依赖性(P＜0.05);PD+LY组与PD组、LY组比较,G0/G1期比例明显增加、S期比例明显减少(P＜0.05).PD+LY组细胞凋亡率为(63.3±0.5)%,明显高于PD组、LY组[分别为(30.7±20.1)%和(40.8±1.3)%,P＜0.01].(2)注射PD98059和(或)LY294002后,随着时间的延长,PD组、LY组、PD+LY组裸鼠移植瘤体积增长相对缓慢,对照组移植瘤体积增长明显,PD组、LY组、PD+LY组分别与对照组比较,差异均有统计学意义(P＜0.05);PD+LY组分别与PD组、LY组比较,差异也均有统计学意义(P＜0.05),而PD组与LY组比较,差异无统计学意义(P＞0.05).LY组、PD组、PD+LY组抑瘤率分别为(32±16)%、(38±17)%、(68±9)%,PD+LY组明显高于LY组、PD组(P＜0.05).LY组、PD组、PD+LY组细胞凋亡指数分别为(13.7±1.5)%、(14.1±1.2)%、(29.0±1.8)%,PD+LY组明显高于LY组、PD组(P＜0.01).LY组、PD组、LY+PD组裸鼠移植瘤组织中p-ERK和p-Akt蛋白的表达强度均明显弱于对照组.结论 信号传导通路抑制剂PD98059、LY294002能够抑制体外及体内子宫内膜癌细胞的生长,并促进其凋亡.

Abstract：

Objective To investigate the effects of signal pathway inhibitors PD98059 and LY294002 on cell proliferation, apoptosis, expressions of phosphorylated extracellular signal-regulared kinase (p-ERK) and phosphorylated protein kinase B ( p-Akt) in endometrial carcinoma xenografts. Methods Human endometrial carcinoma Ishikawa cells were cultured in vitro. The effects of PD98059 and LY294002 on proliferation, apoptosis, and cell cycle distribution of endometrial cancer cells were detected by monotetrazolium ( MTT) assay and fluorescence-activated cell sorting technique. The models of xenografted tumor were established by the subcutaneous inoculation in 24 nude mice, and then they were randomly divided into 4 groups ( n = 6) , normal saline group, PD98059 group (PD group) , LY294002 group ( LY group) or PD98059 + LY294002 group ( PD + LY group) by intraperitoneal injections, respectively. The anti-tumor efficacy was evaluated by measuring tumor volume and tumor growth status. The histopathological change of tumor specimens was observed using HE staining and terminal deoxynucleotidyl transferasemediated dUTP-digoxigen in nick and labeling method (TUNEL) testing and the expression levels of p-ERK and p-Akt were detected by immunohistochemistry method. Results ( 1) The proliferation of Ishikawa cells were suppressed after treated by PD98059 and ( or) Y294002, in which A570 values of cells decreased showing both time-dependent and concentration-dependent manner ( LY294002: Fgroup = 9. 801, P = 0. 002; Ftime = 10. 398, P = 0. 001. PD98059: Fgroup= 8. 213, P = 0. 015; Ftime = 6. 839, P = 0. 036). Cell cycle distribution analysis revealed that percentage of Ishikawa cells at G0/G1 phase(Ftime =35.049, P= 0.004; Fgroup = 32. 024, P ＜0. 01) increased and percentage of S phase cells (Ftime = 7. 789, P = 0. 049; Fgroup = 30. 132, P ＜0. 01) decreased significantly. The percentage of apoptotic cells increased significantly among PD group, LY group and PD + LY group, in which there were significant difference [(63. 3 ±0.5)% vs (30. 7 ± 20. 1) % vs(40. 8 ± 1. 3) % ; F = 621. 059, P ＜ 0. 01]. (2) Compared with the control group, the increasing of transplanting tumor volume in the treated groups were obviously ( F = 23. 545 , P ＜ 0. 01) , and the inhibited rate of the tumor was higher in PD + LY group than that in PD group or LY group [(68 ± 9 ) % vs ( 32 ± 16 ) % or ( 38 ± 17 ) % ; F = 10. 283 , P ＜ 0. 05]. ( 3 ) HE staining shown that there were different degrees of necrosis for endometrial carcinoma cell in different groups. The apoptosis of tumor cells were significantly increased in treated groups by TUNEL testing [(13. 7 ± 1. 5)% , ( 14. 1 ± 1. 2)% , (29. 0 ± 1. 8 ) % ; F = 320. 344, P ＜ 0. 01]. Immunohistochemistry results demonstrated that the expressions of p-ERK and p-Akt in treated groups were lower than that in control group, of which LY + PD group was the lowest one. Conclusion The signal pathway inhibitors PD98059 and LY294002 could inhibit the growth of human endometrial carcinoma in vivo and in vitro, in which may induce cell apoptosis.