siRNA阻断NF-κB信号通路联合5-FU对食管鳞癌细胞凋亡的促进作用

目的: 利用RNAi技术特异性的抑制NF-κB亚单位p65的表达,观察其对p65表达的抑制作用及联合5-FU对食管鳞癌细胞Eca109和EC9706的影响.方法:将终浓度为50 nmol/L的p65 siRNA转染到食管鳞癌细胞EC9706和Eca109中,通过RTPCR检测0、24、48和72 h时段p65 mRNA的表达水平.Western blotting法检测p65和Bcl-2蛋白表达,Annexin V/PI复染结合流式细胞仪检测细胞凋亡,显微镜下观察p65 siRNA与5-FU单独或联合应用对食管鳞癌细胞形态学特性的影响.结果:EC9706和Eca109细胞转染p65 siRNA 24、48和72 h后,p65 mRNA的表达水平随时间的延长逐渐下调,在72 h的阻断效率最为明显,与0 h相比,差异有显著性(0.12±0.01 vs 0.28±0.05,0.1±0.01 vs 0.38±0.04,均P<0.05),转染72 h后,p65和Bcl-2蛋白表达水平下调.EC9706和Eca109转染p65siRNA后,细胞凋亡指数明显升高(6.65%±0.27% vs 2.03%±0.08%,8.03%±0.06% vs 2.66%±0.25%,均P<0.05);p65 siRNA转染72 h后,EC9706和Eca109细胞增殖较慢;当p65 siRNA与5-FU联合作用,细胞增殖明显受到抑制.结论:p65 siRNA可阻断NF-κB信号通路,下调NF-κB下游基因中抗凋亡蛋白Bcl-2的表达,表明活化的NF-κB信号通路可成为食管鳞癌基因治疗中一个重要的分子靶点.     

TGF-β1反义寡核苷酸对食管鳞癌细胞EC9706增殖及凋亡的影响

目的:探讨转染TGF-β1反义寡核苷酸(TGFβ1-ASODN) 对食管鳞癌细胞EC9706 增殖及凋亡的影响. 方法:将化学合成的TGF-β1-ASODN 转染食管鳞癌细胞EC9706,采用RT-PCR 和流式细胞术检测转染效率;观察TGF-β1-ASODN 转染后细胞形态学的改变;采用MTT 和流式细胞术检测TGF-β1-ASODN 转染后对细胞增殖及凋亡的影响. 结果:转染TGF -β1-ASODN 可有效抑制EC9706 细胞中TGF-β1的活性,其mRNA 及蛋白的表达均明显低于转染前的水平(0.25 ±0.07 vs 0.43±0.09;35.35% vs 41.38%,均P<0.05);TGF-β1-ASODN 可明显促进细胞增殖、抑制细胞凋亡,转染后细胞生长拥挤失去正常形态,细胞存活率高于转染前(109.4% vs 100.0%,P <0.05),G1期、S期细胞百分比低于转染前,G2期细胞百分比则高于转染前,细胞凋亡率低于转染前(62.9% vs 66.5%;21.3% vs 23.7%;14.8% vs 9.8%;0.69% vs 0.96%,均P<0.05). 结论:TGF -β1-ASODN 可高效特异地将TGF-β1基因沉默,并解除其抑制细胞增殖、阻滞细胞周期、促进细胞凋亡的作用.     

p15INK4B和p21WAF1基因联合转染对人食管鳞癌细胞系EC109的协同抑制作用

目的:探讨p15INK4B和p21WAF1基因联合转染对人食管鳞癌细胞系EC109细胞增殖和凋亡的影响.方法:脂质体介导PcDNA3.1(+)-p15和pcDNA3.1(+)-p21转染EC109细胞,稳定筛选后用RT-PCR检测转染细胞p15与p21基因mRNA表达,Western blot检测转染细胞P15和P21蛋白的表达.用MTT法和透射电镜检测p15和p21基因分别及联合转染对EC109细胞增殖与凋亡的影响,流式细胞仪检测EC109细胞周期分布与凋亡率.结果:p15和p21转染组EC109细胞生长速度低于空载体组与未转染组,联合转染组与二者单独转染组相比.亦明显抑制EC109细胞体外生长速度.p15和p21转染组EC109细胞发生G1/S阻滞,G1期细胞比例显著高于空载体组和未转染组,S期则显著降低(G1期:60.52%±3.75%,63.12%±2.89% vs 42.17%±5.30%.41.38%±6.54%;S期:22.67%±1.25%,17.96%±2.03% vs 30.96%±3.33%,36.05%±1.78%,均P<0.01),并出现凋亡峰,透射电镜亦发现p15和p21转染组发生细胞凋亡,联合转染组发生更为明显的G1/S阻滞,G1期比例显著升高、S期比例明显降低(G1期:72.83%±2.31% vs60.52%±3.75%,63.12%±2.89%:S期:13.59%±2.59% vs 22.67%±1.25%,17.96%±2.03%.均P<0.05),凋亡率明显升高(21.21%±1.78%vs 4.32±1.74%,10.83%±2.40%,均P<0.01).结论:p15和p21基因联合转染在体外可以进一步增强对人食管鳞癌EC109细胞的抑制与诱导凋亡作用.     

转基因细胞模型L02/HBx的构建及HBx对细胞周期的影响

目的:构建L02/HBx转基因细胞模型并研究HBx对肝细胞周期的影响.方法:运用脂质体转染和G418筛选获得L02/ HBx阳性克隆,并分别用RT-PCR和Western blot鉴定HBx mRNA与蛋白的表达.进一步用四唑蓝(MTT)比色试验、流式细胞仪检测L02/HBx的增殖、凋亡和细胞周期.结果:RT-PCR和Western blot分别检测到L02/ HBx细胞中HBx mRNA和蛋白的表达.MTT比色试验显示L02/HBx生长速度加快,流式细胞仪检测发现L02/HBx凋亡率低(0.09%±0.13% vs 3.74%±1.29%,P<0.05),G1期细胞比例减少(61.35%±0.82% vs 67.80±6.84%,P<0.05),S期细胞比例相应增加(36.59%±2.54% vs 22.37%±2.17%,P<0.05).经阿霉素(ADM)培养后,L02/HBx的凋亡率显著增加(34.91%±5.85% vs 0.09%±0.13%,P<0.05),G1期细胞比例明显增加但低于对照组(82.81%±6.48% vs 61.35%±0.82%,P<0.05;82.81%±6.48% vs 87.19%±1.92%,P<0.05),S期细胞比例降低但较对照组高(13.84%±6.16% vs 36.59%±2.54%,P<0.05;13.84%±6.16% vs 2.22%±1.26%,P<0.05).结论:L02/HBx构建成功,HBx能促进细胞周期进程,加快细胞的生长并抑制细胞的凋亡;转染HBx基因的肝细胞凋亡更易受凋亡因子所触发,表明HBx可能会增加正常肝细胞对诱导凋亡因素的敏感性.     

珠子参对小鼠H22肝癌抑制作用及机制

目的:观察珠子参对小鼠移植性肝癌(H_(22))的增殖抑制作用及其作用机制.方法:建立H_(22)小鼠肝癌实体瘤模型,观察珠子参对荷瘤小鼠肿瘤生长的抑制作用,放免法(RIA)检测血清中肿瘤坏死因子(TNF)-α含量,光镜(HE染色)观察肿瘤细胞生长增殖状态,并在透射电镜下观察肿瘤细胞超微结构形态,流式细胞仪技术(FCM)检测细胞凋亡并进行周期分析.结果:与模型对照组相比,珠子参低浓度和高浓度组和5-FU组小鼠平均肿瘤质量均显著降低(1.66±0.96g,1.49±0.52 g,1.56±0.19 g vs 2.70±0.90 g,均P<0.01);珠子参低浓度和高浓度组小鼠胸腺平均质量和胸腺指数均高于模型对照组(胸腺平均质量:74.18±20.51 mg,80.25±15.73 mg vs 61.82±12.26 mg,P>0.05,P<0.05;胸腺指数:3.17±1.28 mg/g,3.36±1.17 mg/g vs 2.35±0.60 mg/g);与5-FU组(59.51±24.74 mg,2.18±1.18 mg/g)比较,珠子参高浓度组平均胸腺质量和胸腺指数均增高,且结果有显著差异(均P<0.01);血清中TNF-α浓度珠子参低浓度和高浓度组高于正常对照组(12.89±1.10 pmol/L.12.15±2_35 pmol/L vs 10.97±1.16 pmol/L),而比模型对照组低(15.31±3.45 pmol/L,均P<0.05);光镜观察到珠子参组小鼠肿瘤组织周围有大量的吞噬细胞和淋巴细胞浸润,电镜下可见珠子参组癌细胞胞质浓染,核固缩,线粒体明显减少;FCM分析显示,珠子参低浓度和高浓度组与模型组相比G_0-G_1期(35.73%±5.90%,33.78%±6.12% vs 36.59%±2.61%)和S期细胞(42.51%±9.85%,50.48%±3.28% vs 52.46%±2.76%)减少,而G_2-M期细胞(21.76%±7.02%,15.73%±6.91% vs 10.96%±2.75%,均P<0.01)和凋亡细胞(26.88%±1.22%,27.69%±0.21% vs 9.45%±0.14%,均P<0.01)增加.结论:珠子参对H_(22)肝癌小鼠具有良好的抑瘤作用,其作用机制可能与珠子参阻止G_2-M期细胞转换而影响癌细胞在细胞周期中的进程,干扰S期DNA合成以及诱导小鼠肝癌细胞凋亡有关.     

中药淫羊藿苷逆转肝癌HepG2.2.15细胞恶性表型及诱导分化研究

目的: 探讨淫羊藿苷(ICA)诱导HepG2.2.15细胞分化凋亡的作用机制.方法:MTT法检测细胞增殖;流式细胞术(FACS)检测细胞周期分布;RT-PCR方法检测P27基因、FLIP基因mRNA表达水平的变化;AnnexinⅤ-FITC/PI双染法检测ICA处理后HepG2.2.15细胞凋亡的变化;电化学发光法和速率散射比浊法分别检测ICA处理后HepG2.2.15细胞上清液中甲胎蛋白(AFP)和转铁蛋白(Tf)水平变化.结果: ICA作用HepG2.2.15细胞增殖呈抑制作用, 具有时间依赖性. ICA处理后, HepG2.2.15细胞周期各时相分布与对照组相比发生变化, G0/G1期升高, S期减小, 与对照组相比有显著性差异(56.26±1.56% vs 49.68±1.34%, 19.95±1.24% vs 28.02±1.03%;P<0.01). ICA分别上调HepG2.2.15细胞P27和下调FLIP基因mRNA的表达水平(0.78 vs 0.27, 0.54 vs 0.90);HepG2.2.15细胞凋亡率增加, AFP下降, Tf水平升高, 与对照组相比均有显著性意义(7.09% vs 0.59%, 156±46 mg/L vs 285±58 mg/L, 152.1±26 mg/L vs 67.1±24 mg/L;P＜0.05).结论:ICA可能通过升高G0/G1期, 减少S期抑制HepG2.2.15细胞的增殖;上调P27 mRNA和Tf水平, 下调FLIP mRNA的表达和AFP合成的水平来诱导细胞分化.     

拓扑替康对食管癌EC9706细胞自噬和凋亡的影响

目的探讨拓扑替康(TPT)对食管鳞癌细胞株(EC9706)自噬和凋亡的影响。方法使用20 mg/L TPT和(或)5 mmol/L 3-甲基腺嘌呤(3-MA)处理人食管鳞癌细胞EC9706,用四甲基偶氮唑蓝(MTT)法检测TPT对食管癌EC9706细胞生长的抑制作用,单丹磺酰尸胺(MDC)法检测TPT对EC9706细胞自噬水平的影响,采用流式细胞仪技术检测TPT对EC9706细胞凋亡的影响,Western印迹测定TPT对EC9706细胞自噬相关蛋白Beclin1和凋亡相关蛋白Caspase-3表达的影响。结果流式细胞仪检测结果显示,TPT作用于EC9706细胞48 h后,TPT组、3-MA组及TPT+3-MA组细胞凋亡结率分别为(32.45±2.79)%、(7.58±1.12)%及(48.69±3.51)%。MDC染色结果显示TPT能够诱导细胞内自噬囊泡的出现,而3-MA能显著抑制这一现象。Western印迹结果显示对照组、TPT组、3-MA组及TPT+3-MA组Beclin1和Caspase-3蛋白的相对灰度值分别为0.62±0.06、0.91±0.12、0.47±0.04、1.04±0.08和0.54±0.05、0.84±0.11、0.42±0.03、0.71±0.07。结论 TPT康可以诱导食管鳞癌细胞自噬和凋亡的发生,而3-MA能够通过抑制自噬上调Caspase-3的表达增强对食管癌细胞的杀伤作用。     

siRNA阻断NF-κB信号通路联合5-FU对食管鳞癌细胞凋亡的促进作用

目的: 利用RNAi技术特异性的抑制NF-kappaB亚单位p65的表达, 观察其对p65表达的抑制作用及联合5-FU对食管鳞癌细胞Eca109和EC9706的影响. 方法: 将终浓度为50 nmol/L的p65 siRNA转染到食管鳞癌细胞EC9706和Eca109中, 通过RT-PCR检测0、24、48和72 h时段p65 mRNA的表达水平. Western blotting法检测p65和Bcl-2蛋白表达, Annexin V/PI复染结合流式细胞仪检测细胞凋亡, 显微镜下观察p65 siRNA与5-FU单独或联合应用对食管鳞癌细胞形态学特性的影响. 结果: EC9706和Eca109细胞转染p65siRNA 24、48和72 h后, p65 mRNA的表达水平随时间的延长逐渐下调, 在72 h的阻断效率最为明显, 与0 h相比, 差异有显著性(0.12±0.01 vs 0.28±0.05, 0.1±0.01 vs 0.38±0.04, 均P<0.05), 转染72 h后, p65和Bcl-2蛋白表达水平下调. EC9706和Eca109转染p65siRNA后, 细胞凋亡指数明显升高(6.65%±0.27% vs 2.03%±0.08%, 8.03%±0.06% vs 2.66%±0.25%, 均P<0.05); p65 siRNA转染72 h后, EC9706和Eca109细胞增殖较慢; 当p65 siRNA与5-FU联合作用, 细胞增殖明显受到抑制. 结论: p65 siRNA可阻断NF-kappaB信号通路, 下调NF-kappaB下游基因中抗凋亡蛋白Bcl-2的表达, 表明活化的NF-kappaB信号通路可成为食管鳞癌基因治疗中一个重要的分子靶点.     

丹参酮ⅡA联合5-FU对低氧下人胃癌SGC7901细胞增殖、凋亡的影响及与HIF-1α和突变型P53表达的关系

目的:观察低氧环境下不同浓度的丹参酮ⅡA(Tan Ⅱ A)联合5-氟尿嘧啶(5-FU)对人胃癌SGC7901细胞增殖、凋亡的影响及与HIF-1α和突变型P53的表达.方法:用氯化钴(CoCl<,2>)创建低氧模型,采用0、0.5、1.0、2.0、5.0、10.0 mg/L的TanⅡA联合10.0 mg/L的5-FU分别作用于低氧SGC7901细胞24、48和72 h,MTT法检测细胞活力:用上述同样方式作用于低氧SGC7901细胞24、48和72 h后,Hoechst染色法检测细胞凋亡:TanⅡA(0、0.5、2.0、10.0 mg/L)联合10.0 mg/L的5-FU作用于低氧SGC7901细胞48h后,免疫细胞化学二步法检测IHIF-1α及突变型P53的表达.结果:低氧环境下,不同浓度的Tan Ⅱ A联合10.0 mg/L 5-FU呈时间、剂量依赖性地抑制SGC7901细胞的增殖(均P<0.01),10.0 mg/LTan Ⅱ A联合5-FU作用细胞72 h后,其抑制率为67.46%.0.5-10.0 mg/L TanⅡA联合5-FU作用细胞24、48、72 h.TanⅡA呈时间、剂量依赖性地促进SGC7901细胞凋亡(均P<0.01).HIF-1α及突变型P53表达明显高于常氧组(t=22.786,13.914,均P<0.01),不同浓度的Tan Ⅱ A联合10.0 mg/L 5-FU作用细胞48 h后,HIF-1α、突变型P53蛋白表达明显降低(F=182.234,130.062,均P<0.01),且二者呈高度正相关(n=5,r=0.995,P<0.01).结论:在低氧环境下,TanⅡA可能通过抑制HIF-1α及突变型P53蛋白表达从而增强5-FU抑制SGC7901细胞增殖及诱导凋亡作用.     

曲古菌素A对食管癌EC1细胞增殖和细胞周期的影响及其分子机制

目的:探讨不同浓度曲古菌素A对食管癌细胞系EC1 细胞增殖、细胞周期的影响及其对细胞周期调控基因p21 WAF1/CIP1 表达的影响. 方法:用0.3,0.5,1.0 μmol/L 的TSA 处理EC1 细胞,MTT 检测TSA 作用24 、48 h 对EC1 细胞的抑制作用,流式细胞仪检测0.3,0.5,1.0 μmol/L 的TSA 作用24 h 后EC1 细胞周期的改变,Western blot 法检测p21 WAF1/CIP1 变化. 结果:TSA 在0.5 μmol/L 以上时对EC1 细胞有抑制作用;0.3 μmol/L TSA 处理细胞后细胞周期与对照组相比,无明显变化;0.5 μmol/L TSA 处理EC1 细胞后,G0/G1期细胞较对照组明显增加,S 期细胞较对照组明显减少(74.56% ±1.34% vs 62.12%±0.52%;14.52%±1.81% vs 27.50%±0.66%,均P <0.05);0.5,1.0 μmol/L TSA 处理细胞后p21 WAF1/CIP1 表达明显增加(均P<0.05). 结论:一定浓度的TSA 对人食管癌细胞EC 具有的增殖抑制作用,引起EC1 细胞发生G0/G1 期阻滞,其部分机制与p21 WAF1/CIP1 上调有关.     

Antiproliferation and apoptosis induction of paeonol in HepG2 cells

AIM To investigate the antiproliferative effect of paeonol (Pae) used alone or in combination with chemotherapeutic agents [cisplatin (CDDP), doxorubicin (DOX) and 5-fluorouracil (5-FU)] on human hepatoma cell line HepG2 and the possible mechanisms.METHODS The cytotoxic effect of drugs on HepG2 cells was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) assay.Morphologic changes were observed by acridine orange (AO) fluorescence staining. Cell cycle and apoptosis rate were detected by flow cytometry (FCM). Drug-drug interactions were analyzed by the coefficient of drug interaction (CDI).RESULTS Pae (7.81-250 mg/L) had an inhibitory effect on the proliferation of HepG2 cells in a dose-dependent manner, with the IC50 value of (104.77 7.28) mg/L. AO fluorescence staining and FCM assays showed that Pae induced apoptosis and arrested cell cycle at S phase in HepG2 cells. Further, different extent synergisms were observed when Pae (15.63, 31.25, 62.5 mg/L) was combined with CDDP (0.31-2.5 mg/L), DOX (0.16-1.25 mg/L), or 5-FU (12.5-100 mg/L) at appropriate concentrations. The IC50 value of the three drugs decreased dramatically when combined with Pae (P ＜0.01). Of the three different combinations, the sensitivity of cells to drugs was considerably different.CONCLUSION Pae had a significant growth-inhibitory effect on the human hepatoma cell line HepG2,which may be related to apoptosis induction and cell cycle arrest. It also can enhance the cytotoxicity of chemotherapeutic agents on HepG2 cells, and the S phase arrest induced by Pae may be one of the mechanisms of these interactions.     

己酮可可碱对人食管癌细胞EC9706的5-Fu化疗增敏作用

目的探讨己酮可可碱对人食管癌细胞EC9706的5-Fu化疗增敏作用。方法采用MTT法测定5-Fu及己酮可可碱联合5-Fu对EC9706细胞的抑制作用。结果不同浓度的己酮可可碱联合5-Fu作用于EC9706 12、24、48、72 h,同一时间各组抑制率差异有统计学意义(P均<0.01),同一5-Fu浓度,随着己酮可可碱药物浓度的增加,5-Fu对己酮可可碱细胞的抑制作用也增强(P均<0.01)。25μg/mL 5-Fu联合己酮可可碱(0.5 mg/mL、1.0mg/mL)与50μg/mL 5-Fu对EC9706细胞抑制作用,在24、48、72 h无统计学差异(P均>0.05)。结论己酮可可碱可以增加EC9706细胞对5-Fu的敏感性,减少5-Fu的剂量。     

Effect of resveratrol and in combination with 5-FU on murine liver cancer

AIM: To study the anti-tumor effect of resveratrol and in combination with 5-FU on murine liver cancer. METHODS: Transplantable murine hepatoma22 model was used to evaluate the anti-tumor activity of resveratrol (RES) alone or in combination with 5-FU in vivo. H22 cell cycles were analyzed with flow cytometry. RESULTS: Resveratrol could inhibit the growth of murine hepatoma22, after the mice bearing H22 tumor were treated with 10 mg/kg or 15 mg/kg resveratrol for ten days, and the inhibition rates were 36.3% (n = 10) and 49.3% (n = 9), respectively, which increased obviously compared with that in control group (85+/-22 vs 68+/-17, P<0.01). RES could induce the S phase arrest of H22 cells, and increase the percentage of cells in S phase from 59.1% (n = 9) to 73.5% (n = 9) in a dose-dependent manner (P<0.05). The enhanced inhibition of tumor growth by 5-FU was also observed in hepatoma22 bearing mice when 5-FU was administered in combination with 10 mg/kg resveratrol. The inhibition rates for 20 mg/kg or 10 mg/kg 5-FU in combination with 10 mg/kg resveratrol were 77.4% and 72.4%, respectively, compared with the group of 20 mg/kg or 10 mg/kg 5-FU alone, in which the inhibition rates were 53.4% and 43.8%, respectively (n = 8). There was a statistical significance between the combination group and 5-FU group. CONCLUSION: RES could induce the S phase arrest of H22 cells and enhance the anti-tumor effect of 5-FU on murine hepatoma22 and antagonize its toxicity markedly. These results suggest that resveratrol, as a biochemical modulator to enhance the therapeutic effects of 5-FU, may be potentially useful in cancer chemotherapy.     

体外研究腺苷酸活化蛋白激酶对食管鳞癌EC9706细胞增殖及凋亡的影响

目的通过体外研究观察腺苷酸活化蛋白激酶(AMPK)的激活剂AICAR对人食管癌EC9706细胞的增殖及凋亡影响。方法 AICAR以不同浓度作用于人食管癌EC9706细胞,通过光学显微镜观察不同时间后EC9706细胞的生长状态,并用MTT方法检测其吸光度值及细胞存活率的变化,流式细胞术检测其细胞凋亡率情况。结果随着AICAR浓度的增加,细胞的死亡数目明显增加。MTT法检测结果表明,AICAR能够抑制EC9706细胞增殖,并呈现出良好的浓度依赖性。流式细胞术检测结果显示,不同浓度的AICAR干预24 h后早期凋亡率、晚期凋亡率及总凋亡率均高于对照组,但仅有总凋亡率有统计学意义(P0.01)。结论 AMPK可以抑制人食管癌EC9706细胞的生长增殖,并可诱导EC9706细胞凋亡。     

Construction of pETNF-P16 plasmid and its expression properties in EC9706 cell line induced by X-ray irradiation

AIM: Recombined plasmid pETNF-P16 was constructed to investigate its expression properties in esophageal squamous carcinoma cell line EC9706 induced by X-ray irradiation and the feasibility of gene-radiotherapy for esophageal carcinoma. METHODS: Recombined plasmid pETNF-P16 was constructed and transfected into EC9706 cells with lipofectamine. ELISA,Western blot, and immunocytochemistry were performedto determine the expression properties of pETNF-P16 in EC9706 after transfection induced by X-ray irradiation. RESULTS: Eukaryotic expression vector pETNF-P16 was successfully constructed and transfected into EC9706 cells. TNFα expressions were significantly increased in the transfected cells after different doses of X-ray irradiation than in those after 0Gy irradiation (1 192.330-2 026.518 pg/mL,P&lt;0.05-0.01), and the TNFα expressions and P16 were significantly higher 6-48 h after 2 Gy X-ray irradiation (358.963-585.571 pg/mL, P&lt;0.05-0.001). No P16 expression was detected in normal EC9706 cells. However, there was strong expression in the transfected and irradiation groups. CONCLUSION: X-ray irradiation induction could significantly enhance TNFα and P16 expression in EC9706 cells transfected with pETNF-P16 plasmid. These results may provide important experimental data and therapeutic potential for gene-radiotherapy of esophageal carcinoma.     

Expression of heparanase mRNA in anti-sense oligonucleotide-transfected human esophageal cancer EC9706 cells

AIM: To investigate the effects of anti-sense oligonudeotides (ASODNs) on mRNA expression of heparanase in human esophageal cancer EC9706 cells. METHODS: One non-sense oligonucleotide (N-ODN) and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied by in situ hybridization. RESULTS: The expression of heparanase mRNA could be inhibited by ASODNs.There was no significant difference among five ASODNs (P>0.05), but there was a significant difference between ASODNs and N-ODN or non-transfected group (ASODN1: 2.25±0.25, ASODN2: 2.21±0.23, ASODN3: 2.23±0.23, ASODN4: 2.25±0.24 vs N-ODN: 3.47±2.80 or non- transfected group: 3.51±2.93 respectively,P<0.05). CONCLUSION: The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.     

1,3-Bis(2-chloroethyl)-1-nitrosourea enhances the inhibitory effect of Resveratrol on 5-fluorouracil sensitive/ resistant colon cancer cells

AIM:To study the mechanism of 5-fluorouracil(5-FU)resistance in colon cancer cells and to develop strategies for overcoming such resistance by combination treatment.METHODS:We established and characterized a 5-FU resistance(5-FU-R)cell line derived from continuous exposure(25μmol/L)to 5-FU for 20 wk in 5-FU sensitive HCT-116 cells.The proliferation and expression of different representative apoptosis and anti-apoptosis markers in 5-FU sensitive and 5-FU resistance cells were measured by the MTT assay and by Western blotting,respectively,after treatment with Resveratrol(Res)and/or 1,3-Bis(2-chloroethyl)-1-nitrosourea(BCNU).Apoptosis and cell cycle arrest was measured by 4’,6’-diamidino-2-phenylindole hydrochloride staining and fluorescence-activated cell sorting analysis,respectively.The extent of DNA damage was measured by the Comet assay.We measured the visible changes in the DNA damage/repair cascade by Western blotting.RESULTS:The widely used chemotherapeutic agents BCNU and Res decreased the growth of 5-FU sensitive HCT-116 cells in a dose dependent manner.Combined application of BCNU and Res caused more apoptosis in5-FU sensitive cells in comparison to individual treatment.In addition,the combined application of BCNU and Res caused a significant decrease of major DNA base excision repair components in 5-FU sensitive cells.We established a 5-FU resistance cell line(5-FU-R)from 5-FU-sensitive HCT-116(mismatch repair deficient)cells that was not resistant to other chemotherapeutic agents(e.g.,BCNU,Res)except 5-FU.The 5-FU resistance of 5-FU-R cells was assessed by exposure to increasing concentrations of 5-FU followed by the MTT assay.There was no significant cell death noted in5-FU-R cells in comparison to 5-FU sensitive cells after5-FU treatment.This resistant cell line overexpressed anti-apoptotic[e.g.,AKT,nuclear factorκB,FLICE-like inhibitory protein),DNA repair(e.g.,DNA polymerase beta(POL-β),DNA polymerase eta(POLH),protein Flap endonuclease 1(FEN1),DNA damage-binding protein 2(DDB2)]and 5-FU-res     

苦参碱联合5-氟尿嘧啶对人胃癌裸鼠移植瘤的抑制作用

目的　研究苦参碱和5 -氟尿嘧啶(5- FU)联用对人胃腺癌SGC 7901裸鼠移植瘤的抑制作用及骨髓毒性作用。方法　将42只Balb/c小鼠分为阴性对照组(12 只)、5- FU组(6 只)、苦参碱A组(50 mg/kg,6只)、苦参碱B组(100 mg/kg,6只)、联合用药A组(苦参碱50 mg/kg+5 -FU,6只)和联合用药B组(苦参碱100 mg/kg+5 -FU,6只)。观察苦参碱和5 -FU联合用药对胃癌裸鼠移植瘤的抑制作用,计算出相对肿瘤体积(RTV)和肿瘤抑制率(IR);取裸鼠骨髓,计数有核细胞数并进行骨髓集落培养。结果　联合用药B 组抑瘤作用为70. 4%,与其他各组相比疗效明显升高,差异有统计学意义(P<0.01)。与联合用药A组相比,联合用药B组抑瘤作用亦显著增强(P<0.05)。联合用药组对骨髓的抑制作用与5 FU组相比差异无统计学意义(P>0.05)。骨髓集落培养后,与对照组相比,用药组骨髓集落明显生长旺盛。结论　苦参碱和5 -FU联用对人胃癌裸鼠移植瘤的抑制作用明显优于两者单独应用;联合用药对裸鼠骨髓增殖期造血细胞抑制作用有所加重,但不损伤静止期骨髓干细胞。     

Inhibitory effect of ubiquitin-proteasome pathway on proliferation of esophageal carcinoma cells

AIM: To investigate the inhibitory effect of ubiquitin-proteasome pathway (UPP) on proliferation of esophageal carcinoma cells.METHODS: Esophageal carcinoma cell strain EC9706 was treated with MG-132 to inhibit its UPP specificity. Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA synthesis was evaluated by ^3H-thymidine (^3H-TdR) incorporation. Morphologic changes of cells were observed under microscope. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) of PCRELISA. Cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. Expression of p27^kip1 was detected by immunocytochemical technique. RESULTS: After exposed to MG-132, the growth and value of ^3H-TdR incorporation of EC9706 cells were obviously inhibited. Cells became round, small and exfoliative under microscope. TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5 μmol/L of MG-132 for 24, 48, 72 and 96 h (P&lt;0.01). The percentage of cells at G0/G1 phase was increased and that at S and G2/M was decreased (P&lt;0.01). The rate of apoptotic cells treated with 5 μmol/L of MG-132 for 48 and 96 h was 31.7% and 66.4%, respectively. Agarose electrophoresis showed marked ladders. In addition, the positive signals of p27^kip1 were located in cytoplasm and nuclei in MG-132 group in contrast to cytoplasm staining in control group. CONCLUSION: MG-132 can obviously inhibit proliferation of EC9706 cells and induce apoptosis. The mechanisms include upregulation of p27^kip1 expression, G1 arrest and depression of telomerase activity. The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.