Inhibitory effect of emodin on human hepatoma cell line SMMC-7721 and its mechanism

BackgroundDa Huang (Radix et Rhizoma Rhei) is the dried root or rhizome of Rheum palmatum L., Rheum tanguticum Maxim ex Balf. or Rheum officinale Braill of family Polygonaceae. It has heat clearing, damp drying, fire purging and toxin removing effects. Because of its definite curative efficacy, it has been widely applied in clinical settings.ObjectiveTo study the inhibitory effect of emodin on human hepatoma cell line SMMC-7721 and its mechanism.MethodsMTT assay, flow cytometry and electron microscopy were used to investigate the inhibitory effect of different concentrations of emodin on human hepatoma cell line SMMC-7721.Results12 h, 24 h and 48 h after the action of 20, 40 and 80 umol/L emodin on SMMC-7721 cells, the proliferation of human hepatoma SMMC-7721 cells was inhibited; the inhibitory effects showed time-and concentration-dependence. 48 h after the action of different concentrations of emodin on SMMC-7721 cells, cells in G2/M phase increased significantly, while the proportion of S phase cells gradually declined.ConclusionEmodin can inhibit human hepatoma cell line SMMC-7721.     

苦参素注射液联合顺铂对人肝癌SMMC-7721细胞端粒酶活性及wtp53、hTERT mRNA表达的影响

目的:观察苦参素注射液联合顺铂(DDP)对人肝癌SMMC-7721细胞端粒酶活性、野生型p53(wtp53)、端粒酶逆转录酶(h TERT)m RNA表达的影响。方法:将Dulbecco改良Eagle培养基培养的人肝癌SMMC-7721细胞分为苦参素组、DDP组、联合组和对照组。苦参素组低、中、高质量浓度分别为0.75、1.5、3.0 mg/ml,DDP组低、中、高质量浓度分别为1、2、4 mg/ml,联合组浓度按两药联合用药1∶1的比例应用,对照组加入200μl磷酸缓冲盐溶液(PBS),不给予药物干预。观察4组人肝癌SMMC-7721细胞凋亡率及药物间的相互作用,端粒酶活性吸光度差值(ΔA),wtp53和h TERTm RNA相对表达量。结果:联合组人肝癌SMMC-7721细胞凋亡率>苦参素组>DDP组>对照组,4组比较差异有统计学意义(P<0.01),q值提示苦参素与DDP存在单纯相加或协同作用。苦参素组、DDP组及联合组SMMC-7721细胞端粒酶活性ΔA、h TERTm RNA均显著低于对照组,且联合组DDP组>苦参素组,差异均有统计学意义(P<0.05)。结论:苦参素注射液联合DDP可增加人肝癌SMMC-7721细胞的凋亡,其机制可能与wtp53表达上调、h TERTm RNA表达下调,从而影响端粒酶活性有关。     

野生型p53对肝癌细胞POLD1基因表达及细胞恶性表型的影响

目的:探讨野生型p53对人肝癌细胞SMMC-7721细胞增殖和细胞恶性表型影响的一种可能机制,方法:设计并构建p53特异性小干扰shRNA绿色荧光真核表达质粒(p53-siRNA)和表达EGFP-p53融合蛋白的p53绿色荧光真核增强表达质粒(pEGFP-p53),通过脂质体Lipofection-2000介导转染,将...     

TAZ/WWTR1影响肝癌细胞SMMC-7721的侵袭、迁移能力

目的 探讨转录共激活子TAZ/WWTR1对肝癌细胞株SMMC-7721侵袭、迁移的影响,为寻找原发性肝细胞癌的防治提供理论基础.方法 TAZ基因靶向干扰RNA的设计合成:从TAZ的信使RNA(mRNA)中挑选出合适的siRNA靶点序列作为候选,序列通过基因BLAST,得出三对siRNA;TAZ过表达质粒的构建:从SMMC-7721细胞中提取TAZ基因,使用pCMV5-HA为载体构建人TAZ过表达质粒;细胞转染:以LipofectaminTM2000为载体,将TAZ siRNAs转入SMMC-7721细胞中,应用实时RT-PCR和Western blotting检测TAZ的表达情况,筛选出于扰效果最佳的siRNA,并进一步比较转染了TAZ siRNA组与转染了阴性对照组的SMMC-7721细胞侵袭、迁移能力的变化;构建稳定过表达TAZ基因的人肝癌细胞(SMMC-7721)系:用LipofectaminTM2000将TAZ过表达质粒转染入SMMC-7721细胞中,使用G418筛选出稳定高表达TAZ的细胞系,应用Transwell侵袭实验观测TAZ稳定过表达的SMMC-7721细胞的侵袭、迁移能力.结果 三对siRNA均可使TAZ表达下调,其中siRNA-1效果最佳;与阴性对照组相比,转染了TAZ siRNA-1的SMMC-7721细胞的侵袭、迁移能力下降;经过鉴定确定为稳定过表达TAZ的SMMC-7721细胞的侵袭、迁移能力相比于阴性对照组显著升高.结论 本研究通过靶向干扰和稳定过表达TAZ基因的方法证实TAZ能够影响人肝癌细胞株SMMC-7721的侵袭、迁移能力,为揭示原发性肝癌恶性转移的发病甚至启动机制提供一个崭新思路.     

加热对人肝癌细胞-7721生长规律的影响

目的观察加热对人肝癌细胞-7721生长规律的影响.方法应用人肝癌细胞株-7721(以下简称7721细胞),常规法培养;采用水浴加温法(42.5℃)热疗和热化疗处理细胞后,继续常规法培养;计数1-7d的细胞数,绘制生长曲线、计算细胞倍增时间;观察细胞的电镜下表现.结果加热处理的细胞倍增时间较未处理细胞明显延长,加热处理的细胞发生空泡变性,热化疗处理的细胞胞膜破坏严重、胞浆细胞器外溢明显.结论加热处理可以明显抑制人肝癌细胞-7721的生长能力,原因是加热引起该细胞的某些细胞器变性.     

人源肝癌细胞系中甲胎蛋白基因表达及甲基化的研究

目的:了解不同人源肝癌细胞系甲胎蛋白(AFP)基因表达及不同区域甲基化状态,探讨肝癌发生的表达遗传学调控机制。方法:采用亚硫酸氢盐-基因组测序(bisulfite-assisted genomic sequencing,BAGS)技术,检测人源HepG2、SMMC-7721肝癌细胞系和人成纤维细胞中AFP基因启动子区及CpG岛区的甲基化水平,RT-PCR检测基因的表达,并分析相关性。结果:HepG2细胞高表达AFP,SMMC-7721细胞低表达AFP,成纤维细胞不表达AFP;在启动子区,HepG2细胞的非甲基化修饰位点发生率较高,尤其是第一个和第二个CG位点,SMMC-7721细胞的甲基化程度较之高,而成纤维细胞甲基化程度整体很高;在CpG岛区,成纤维细胞的非甲基化修饰位点发生率较高,主要是第6个和第7个CG位点,SMMC-7721、HepG2细胞的甲基化程度较之高。结论:AFP基因启动子甲基化程度与基因表达水平存在负相关,AFP基因CpG岛甲基化程度与基因是否表达有关,AFP基因启动子低甲基化可能是AFP高表达的分子机制。     

siRNA靶向沉默fbxl20基因对肝癌SMMC-7721细胞生物学行为的影响

目的:通过沉默fbxl20基因,以观察基因沉默后对SMMC-7721细胞的增殖、迁移和周期分布的影响。方法:通过脂质体Lipofectamine2000介导转染SMMC-7721细胞,靶向fbxl20基因的siRNA片段阻低肝癌SMMC-7721细胞株fbxl20 mR-NA表达。结果:siRNA能有效沉默SMMC-7721细胞fbxl20 mRNA的表达,沉默效率为71.49%。MTT试验实验显示:siRNA转染SMMC-7721细胞后,细胞增殖能力显著降低。Transwell小室和划痕愈合实验表明,fbxl20基因被沉默后,SMMC-7721细胞在迁移能力明显减弱,迁移抑制率高达62.03%。流式细胞术检测细胞周期结果显示:转染48 h后,SMMC-7721实验组细胞G1/G0期比率(81.21%)显著高于阴性对照组(71.49%)和空白对照组(67.90%)。结论:本实验运用RNA干扰技术成功抑制了SMMC-7721细胞中fbxl20基因的表达,并且发现fbxl20基因可能与肝癌细胞的增值和迁移相关。     

Holotransferrin enhances selective anticancer activity of artemisinin against human hepatocellular carcinoma cells

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. （the blue-green herb） in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling （TUNEL） and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells （P〈0.05）, likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells （P〈0.01）. Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.     

天然二萜类化合物的抗肿瘤活性

采用ＭＴＴ快速比色法，以长春碱（Ｖｉｎｂｌａｓｔｉｎｅ，ＶＬＢ）为阳性对照，研究了３６种从中草药中分离提取的天然二萜类化合物对三种不同癌细胞（肝癌ＳＭＭＣ－７７２１、肺腺癌Ｌ（３４２）、胃腺癌ＭＣｃ８０－３）生长的抑制活性。结果表明：６种二萜类化合物抗肿瘤活性强度与其化学结构密切相关；其中Ｅ（１８）、Ｅ（２１）和Ｅ（１３）的抑瘤活性强度比长春碱强，顺序为：Ｅ（１８）～Ｅ（２１）＞Ｅ（１３）＞ＶＬＢ＞Ｅ（１９）＞Ｖ８＞Ｅ（１２）；酯酰化取代基是影响其抗肿瘤活性强度的主要官能团。     

雷氏大疣蛛毒素对人肝癌细胞SMMC-7721细胞增殖及细胞周期的影响

目的 探讨雷氏大疣蛛Macrothele raveni毒素对人肝癌细胞SMMC-7721增殖及细胞周期的抑制作用，进一步探讨其作用的分子机制。方法 采用MTT法测定雷氏大疣蛛毒素对SMMC一7721细胞增殖作用的影响；采用[^3H]TdR掺入法检测雷氏大疣蛛毒素作用前后SMMC7721细胞DNA合成的变化；采用流式细胞术（FCM）探讨雷氏大疣蛛毒素对SMMC-7721细胞凋亡率和细胞周期的影响；采用Western Blot方法研究雷氏大疣蛛毒素对细胞周期相关c-myc蛋白表达的影响。结果 MTT方法表明雷氏大疣蛛毒素对SMMC-7721细胞增殖有较强的抑制作用（P〈0．05），时效和量效关系良好。雷氏大疣蛛毒素可以抑制SMMC-7721细胞DNA的合成。流式细胞仪检测结果发现雷氏大疣蛛毒素作用后SMMC-7721细胞凋亡率增加，细胞周期阻滞在G2／M期。Western Blot方法进一步检测到雷氏大疣蛛毒素作用SMMC7721细胞72h后，c-myc蛋白表达减弱。结论 雷氏大疣蛛毒素可以抑制人肝癌细胞SMMC-7721的增殖和DNA的合成，其机制可能是诱导细胞凋亡，使细胞周期相关c-myc蛋白表达减弱，导致细胞周期的变化。