Study on the mechanism of epidermal growth factor-induced proliferation of hepatoma cells

AIM: Many growth factors, such as epidermal growth factor(EGF), are associated with the carcinogenesis. EGF plays itsrole in the proliferation of hepatoma cells through bindingwith EGF receptor (EGFR) and a series of signal transduction.But the postreceptor pathway is still not clear. In the presentexperiment, we studied the effect of tyrosine kinase, proteinkinase C, Na+/H+ exchange, calmodulin and voltage-dependent Ca2+ channel on EGF-induced hepatoma cellproliferation.METHODS: Hepatoma cell line SMMC7721 was cultured inRPMI1640 serum-free medium. In order to study the effectof thyrosine kinase, protein kinase C, Na+/H+ exchange,calmodulin and voltage-dependent Ca2+ channel on humanheptoma cell proliferation induced by epidermal growth factor(EGF), DNA synthesis rate of hepatoma cells was measuredby the method of 3H-TdR incorporation.RESULTS: EGF (10-9 M) stimulated the proliferation of heptomacells significantly (3H-TdR incorporation was 1 880+281 cpm/well, P＜0.05), and this effect was significantly inhibited bytyrosine kinase inhibitor genistein (3H-TdR incorporation was808±209 cpm/well, P＜0.001). Calmedulin inhibitor W-7, proteinkinase C inhibitor H-7 and Na+/H+ exchange inhibitor amilorideindividually had significant inhibiting effect on EGF-inducedproliferation of hepatoma cells (3H-TdR incorporation was978±87.3 cpm/well, 1 241+147 cpm/well, 1 380+189 cpm/well, respectivly, P＜0.001, P＜0.01, P＜0.05), but they allhad no effect on the basal level proliferation of culturedhepatoma cells (3H-TdR incorporation was 1 284+260 cpm/well, 1 179+150 cpm/well, 1 392+152 cpm/well, respectivly,3H-TdR incorporation of the control was 1353+175 cpm/well, P＞0.05). Voltage-dependent Ca2+ channel inhibitorverapamil had no inhibition on EGF-induced proliferation ofhepatoma cells (3H-TdR incorporation was 1 637+133 cpm/well, P＞0.05), it also had no effect on the basal levelproliferation of cultured hepatoma cells (3H-TdR incorporationwas 1196+112 cpm/well,P＞0.05).CONCLUSION: Our data suggest that tyrosine kinase, Ca2+-calmodulin-dependent pathway, protein kinase C and Na+/H+ exchange play a critical role in EGF-induced proliferationof hepatoma cells and that the effect of EGF is independentof voltage-dependent Ca2+ channel.     

转铁蛋白SA阳离子脂质体介导DNA转染肝癌细胞 SMMC-7721的实验研究

目的检测HTf(Fe)2SA阳离子脂质体是否具有靶向转染肝癌细胞的能力.方法在有血清和无血清两种条件下检测HTf(Fe)2SA阳离子脂质体和SA阳离子脂质体介导DNA进入肝癌细胞SMMC-7721和小鼠B16黑色素瘤细胞内的量.结果在有血清和无血清两种转染条件下HTf(Fe)2SA阳离子脂质体介导入SMMC-7721细胞内DNA的量明显高于SA阳离子脂质体,而对小鼠B16黑色素瘤细胞两者无明显差异.结论HTf(Fe)2SA阳离子脂质体在体外具有良好的靶向介导DNA转染肝癌细胞的能力.     

Growth-inhibiting effects of taxol on human liver cancer in vitro and in nude mice

AIM To investigate the effects of taxol on SMMC-7721 human hepatoma and its mechanisms. MLETHODS In vitro cell growth was assessed by trypan blue exclusion method. Experimental hepatoma model was established by seeding SMMC-7721 cells subcutaneously into Balb/c (nu/nu) nude mice. In vivo tumor growth was determined by measurement of tumor diameter with Vernier calipers. The syntheses of DNA,RNA and protein were analyzed by incorporation of 3H-thymidine, 3H-uridine and 3H-leucine respectively. Using light and electron microscopes to observe the morphological changes of cells including mitosis and apoptosis. RESULTS Taxol was effective against SMMC 7721 human hepetoma cell growth in the ranges of 2.5 nmol/L - 10 nmol/L with mitotic arrest and apoptosis in vitro. DNA, RNA and protein syntheses in cells were also obviously suppressed by in vitro treatment of taxol for 72 h. Taxol at 2.5 nmol/L reduced 3H-thymidine uptake to about 34% of the control value (P＜0.05). Increasing the dose of taxol to 20 nmol/L resulted in a greater decrease in 3Hthymidine incorporation to 60% of the control value (P＜0.01). At a concentration of 20 nmol/L, the 3H-uridine and 3H-leucine uptakes were reduced to 52% (P＜0.05) and 63%(P＜0.01), respectively. In vivo, taxol significantly inhibited SMMC-7721 tumor growth at 10 mg/kg, i.p., once daily for 10 d. A more than 90% decrease in tumor volume was observed by day 11 (P＜0.01) similarly with mitotic arrest and cell apoptosis. CONCLUSION Taxol has a marked anticancer activity in SMMC-7721 human hepatoma both in vitro and in nude mice. Its mechanisms might be associated with mitotic arrest, subsequently,apoptosis of the hepatoma cells. No obvious toxicity was observed with in vivo administration of taxol.     

High-density lipoprotein as a potential carrier for delivery of a lipophilic antitumoral drug into hepatoma cells

AIM: To investigate the possibility of recombinant highdensity lipoprotein (rHDL) being a carrier for delivering antitumoral drug to hepatoma cells. METHODS: Recombinant complex of HDL and aclacinomycin (rHDL-ACM) was prepared by cosonication of apoproteins from HDL (Apo HDL) and ACM as well as phosphatidylcholine. Characteristics of the rHDL-ACM were elucidated by electrophoretic mobility, including the size of particles, morphology and entrapment efficiency. Binding activity of rHDL-ACM to human hepatoma cells was determined by competition assay in the presence of excess native HDL. The cytotoxicity of rHDL-ACM was assessed by MTT method. RESULTS: The density range of rHDL-ACM was 1.063-1.210 g/mL, and the same as that of native HDL. The purity of all rHDL-ACM preparations was more than 92%. Encapsulated efficiencies of rHDL-ACM were more than 90%. rHDL-ACM particles were typical sphere model of lipoproteins and heterogeneous in particle size. The average diameter was 31.26±5.62 nm by measure of 110 rHDL-ACM particles in the range of diameter of lipoproteins. rHDL-ACM could bind on SMMC-7721 cells, and such binding could be competed against in the presence of excess native HDL. rHDL-ACM had same binding capacity as native HDL. The cellular uptake of rHDL-ACM by SMMC-7721 hepatoma cells was significantly higher than that of free ACM at the concentration range of 0.5-10 μg/mL (P<0.01). Cytotoxicity of rHDL-ACM to SMMC-7721 cells was significantly higher than that of free ACM at concentration range of less than 5 ug/mL (P<0.01) and IC50 of rHDL-ACM was lower than IC50 of free ACM (1.68 nmol/L vs3 nmol/L). Compared to L02 hepatocytes, a normal liver cell line, the cellular uptake of rHDL-ACM by SMMC-7721 cells was significantly higher (P<0.01) and in a dose-dependent manner at the concentration range of 0.5-10 μg/mL.Cytotoxicity of the rHDL-ACM to SMMC- 7721 cells was significantly higher than that to L02 cells at concentration range of 1-7.5μg/mL (P<0.01). IC50 for SMMC-7721 cells (1.68 nmol/L) was lower than that for L02 cells (5.68 nmol/L), showing a preferential cytotoxicity of rHDL-ACM for SMMC-7721 cells. CONCLUSION: rHDL-ACM complex keeps the basic physical and biological binding properties of native HDL and shows a preferential cytotoxicity for SMMC-7721 hepatoma to normal L02 hepatocytes, HDL is a potential carrier for delivering lipophilic antitumoral drug to hepatoma cells.     

Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells

AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7, -9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation of human hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.     

Downregulation of alpha-fetoprotein siRNA inhibits proliferation of SMMC-7721 cells

AIM: To study the function of α-fetoprotein (AFP) in SMMC-7721 hepatoma cells. METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescenceactivated cell sorter (FACS). RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis. CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.     

中药960诱导人肝癌细胞凋亡的研究

目的：观察９６０抗癌合剂对人肝癌细胞的凋亡作用,揭示其原发性肝癌作用机理。方法：丫啶橙（ＡＯ）染色,琼脂糖凝胶电经丙锭染色细胞周期分析（ＰＩ－ＦＣＭ）和末端转移酶ＤＮＡ断裂点原位标记（ＴＵＮＥＬＦＣＭ）。结果：经９６０药和血清处理后。细胞皱缩,深染,ＡＯ染色,细胞呈致密浓染的黄绿药荧光;ＤＮＡ电位见典型“梯形”带;ＤＮＡ直方图上见典型凋亡峰（０．９５％～２７．７％）,ＴＵＮＥＬ标记率６４．１２％～     

钙调蛋白和电压依赖性钙通道在EGF促肝癌细胞生长中的作用

目的:许多生长因子如表皮生长因子(EGF)与肿瘤的发生密切相关。EGF与表皮生长因子受体(EGFR)结合,通过一系列的信息传导,导致肝癌细胞的增生。但受体后的信息传导机制尚不清楚。本实验探讨酪氨酸激酶、钙调蛋白和电压依赖性钙通道在EGF促肝癌细胞生长中的作用。方法:本研究于无血清RPMI 1640中培养肝癌细胞SMMC7721。采用3H-Thymidine(3H-TdR)掺入的方法,检测肝癌细胞DNA合成速率,研究酪氨酸激酶、钙调蛋白和电压依赖性钙通道在EGF促肝癌细胞生长中的作用。结果:EGF10-9M对肝癌细胞的生长有极显著促进作用,与对照组比较差异有显著意义(P<0.05)。酪氨酸激酶阻滞剂Genistein对EGF的促肝癌细胞生长极显著抑制作用(P<0.001)。钙调蛋白阻滞剂W-7对EGF的促肝癌细胞生长具有极显著抑制作用(P<0.001),而对基础状态细胞的H-TdR掺入值无显著影响(P>0.05)。电压依赖性钙通道阻滞剂Varapamil对EGF的促肝癌细胞生长无显著抑制作用(P>0.05),对基础状态细胞的3H-TdR掺入值亦无显著影响(P>0.05)。结论:结果显示,酪氨酸激酶及依赖钙-钙调蛋白途径在EGF的作用中起关键作用,电压依赖性钙通道与EGF的作用无关。     

5-Aza-CdR对肝癌细胞DAPK基因甲基化的影响

目的探讨去甲基化药物5-氮杂-2’-脱氧胞苷(5-Aza-CdR)对人肝癌细胞株SMMC-7721中死亡相关蛋白激酶(DAPK)基因启动子CpG岛甲基化的影响。方法不同浓度(0.5μmol/L、5.0μmol/L、50.0μmol/L)5-Aza-CdR处理人肝癌细胞株SMMC-772172小时后,用焦磷酸测序法检测处理前后DAPK基因启动子CpG岛甲基化水平。以未经处理的人肝癌细胞株SMMC-7721作为对照组。结果未经5-Aza-CdR处理的肝癌细胞SMMC-7721细胞株DAPK基因启动子高度甲基化,平均水平为76.71%。经3种不同浓度药物处理72小时后,各组间甲基化平均水平分别为78.29%、77.57%和66.00%,各组间甲基化水平差异均有统计学意义(F=39.71,P<0.01)。其中,50.0μmol/L处理组细胞甲基化水平最低,与其他各组差异均有统计学意义(P<0.01)。结论 SMMC-7721细胞株DAPK基因启动子区呈高度甲基化状态;5-Aza-CdR能够逆转人肝癌细胞SMMC-7721DAPK基因启动子区域的甲基化状态。     

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM To test the hypothesis to block VEGFexpression of SMMC-7721 hepatoma cells mayinhibit tumor growth using the rat hepatomamodel.METHODS Amplifiy the 200 VEGF cDNAfragment and insert it into human U6 genecassette in the reverse orientation transcribingsmall antisense RNA which could specificallyinteract with VEGF165, and VEGF121 mRNA.Construct the retroviral vector containing thisantisense VEGF U6 cassette and package thereplication-deficient recombinant retrovirus.SMMC-7721 cells were transduced with thesevirus and positive clones were selected withG418. PCR and Southern blot analysis wereperformed to determine if U6 cassette integratedinto the genomic DNA of positive clone,Transfected tumor cells were evaluated for RNAexpression by ribonuclease protection assays.The VEGF protein in the supernatant of parentaltumor cells and genetically modified tumor cellswas determined with ELISA. In vitro and in vivogrowth properties of antisense VEGF cell clonein nude mice were analyzed.RESULTS Restriction enzyme digestion andPCR sequencing verified that the antisense VEGFRNA retroviral vector was successfullyconstructed. After G418 selection, resistantSMMC-7721 cell clone was picked up, PCR andSouthern blot analysis suggested that U6cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transducedwith U6 antisense RNA cassette could express200bp small antisense VEGF RNA and secretereduced levels of VEGF in culture condition.Production of VEGF by antisense transgene-expressing cells was 65±10 ng/L per 10~6 cells,420±45 ng/L per 10~6 cells in sense group and 495±30 ng/L per 10~6 cells in the negative controlgroup, (P<0.05), The antisense-VEGF cellclone appeared phenotypically indistinguishablefrom SMMC-7721 cells and SMMC-7721 cellstransfected sense VEGF. The growth rate of theantisense-VEGF cell clone was the same as thecontrol cells. When S. C. was implanted intonude mice, growth of antisense-VEGF cell lineswas greatly inhibited compared with controlcells.CONCLUSION Expression of antisense VEGFRNA in SMMC-7721 cells could decrease thetumorigenicity, and antisense-VEGF genetherapy may be an adjuvant treatment forhepatoma.     

5-脱氧杂氮胞苷抑制肝癌细胞株生物学行为的机制

目的 DNA甲基化模式的改变伴随着永生性细胞系的确立，生长凋控基因CpG岛的重新甲基化可能导致了其转录的不活跃，在体外培养的情况下，使得肿瘤细胞选择了有利于生长的条件．我们用DNA甲基转移酶抑制剂5-脱氧杂氮胞苷处理两株肝癌细胞，研究其对肝癌细胞的影响，探讨DNA甲基化异常与肝细胞癌间的相关性及5-脱氧杂氮胞苷对肝癌细胞株恶性生物学行为的影响及其机制。方法 用5-脱氧杂氮胞苷处理肝癌细胞株SMMC-7721和HePG2，然后采用相差显微镜观察药物处理前后细胞的形态变化，采用MTT法观察细胞的生长速度变化，采用流式细胞仪检测细胞周期、细胞凋亡率、P16蛋白表达的变化．采用RT-PCR法比较p16和甲基转移酶mRNA表达量的变化，比较裸鼠致瘤性的大小。结果 5-脱氧杂氨胞苷处理后细胞形态趋于规则，生长速度减慢．SMMC-7721细胞，G1期细胞增加了8.5％，而s期和G2／M期细胞分别减少了47．8％和10.4％．HePG2细胞，G1期细胞增加了3．5％，S期减少了46．2％．G2／M期增加了23．7％．两细胞凋亡率分别增加了91．6％和133．3％．P16蛋白表达分别增加了23．4％和20.9％，p16mRNA表达增高，而DNA甲基转移酶mRNA表达明显降低，裸鼠皮下移植瘤生长减慢。结论 肝细胞癌的发生与DNA甲基化异常有关，甲基化酶抑制剂5-脱氧杂氮胞苷可能通过抑制甲基转移酶抑制抑癌基因启动子区域的高甲基化，恢复其生长调控功能，从而使肝癌细胞株的恶性生物学行为发生逆转。     

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than G1 phase cells [(307.65&#177;92.10)&#215;10^-10N vs (195.42&#177;60.72)&#215;10^-10N, P&lt;0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in G1 and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.     

缺氧环境中黏着斑激酶对SMMC-7721细胞侵袭能力的影响

目的 研究缺氧对人肝癌细胞SMMC-7721黏着斑激酶(FAK)表达的影响以及FAK表达对SMMC-7721细胞侵袭能力的影响.方法 通过1%体积分数O2的低氧培养建立人肝癌细胞SMMC-7721物理缺氧模型,Western blot检测FAK的表达.构建针对FAK mRNA的干扰质粒pshRNA-FAK及阴性对照质粒pGensil-2,并将其转染至SMMC-7721细胞,G418筛选稳定转染细胞株.Western blot检测FAK蛋白表达的变化,细胞迁移和侵袭实验检测缺氧条件下细胞迁移和侵袭能力的改变.在正常条件下将FAK真核表达质粒pcDNA3-FAK转染至SMMC-7721细胞,观测其侵袭能力的改变.根据数所资料的不同分别采用t检验、单因素方差分析,LSD法及Dunnett法进行统计学处理. 结果低氧培养的SMMC-7721细胞FAK蛋白表达水平逐渐升高,24 h后较0 h时明显升高(P<0.01).SMMC-7721细胞稳定转染pshRNA-FAK后,FAK蛋白表达显著下降,抑制率达74.6%±5.1%,在正常及缺氧条件下都对FAK表达有显著抑制作用.细胞迁移实验结果显示,缺氧显著促进SMMC-7721细胞迁移能力(t=18.66,P<0.01),侵袭实验结果与迁移实验结果一致.转染pshRNA-FAK对促进SMMC-7721细胞在缺氧环境中的迁移能力有显著抑制作用,透膜细胞数(353±36)个较对照组(392±31)个明显降低(F=173.983,P<0.05);细胞侵袭实验显示,转染pshRNA-FAK对促进SMMC-7721细胞侵袭能力有显著抑制作用,透膜细胞数(160±12)个较对照组(194±13)个明显降低(F=59.674,P<0.05).同时转染真核表达质粒pcDNA3-FAK显著促进SMMC-7721细胞侵袭能力.结论 缺氧促进SMMC-7721细胞侵袭可能与FAK表达水平升高相关,FAK表达的上调可能是缺氧促进肝癌细胞侵袭转移的机制之一.     

扶正抗癌方药物血清对人肝癌细胞增殖的影响

目的:阐明扶正抗癌方的抗癌机制。方法:以不同剂量灌胃给药后,不同时间采集大鼠血清,处理体外培养的人肝癌细胞SMMC_(7721),用MTT法和氚标胸腺嘧啶(~3H-TdR)掺入法观察细胞增殖能力。结果:扶正抗癌方(1.6g/kg,8.0g/kg)2次给药后1小时和2小时药物血清处理细胞,MTT转化率和~3H-TdR掺入率明显下降。结论:扶正抗癌方具有明显的抑制人肝癌细胞增殖作用。     

Antitumor mechanism of antisense cantide targeting human telomerase reverse transcriptase

AIM: To investigate the anti-tumor mechanism of antisenseoligodeoxynucleotide cantide against hTERT.METHODS: Tumor cells were cultured overnight and grownto 50-60% confluence. HepG2 and SMMC-7721 were treatedwith cantide mixed with lipofectin, or lipofectin alone. Afterinducted for 6 h at 37℃, 10% FCS in DMEM was replacedin each well. After the treatment repeated twice to threetimes in each concentration of cantide, hTERT mRNA andprotein expression were measured by RT-PCR and Westernblot analysis, respectively. Telomerase aclivity was determinedby TRAP-ELISA assay. CPP32- and ICE-like activity was alsoinvestigated using CasPACE assay system at 48 h aftercantide treatment, and apoptosis was evaluated using theDeadEnd assay at 24, 48 and 72 h after cantide treatment.RESULTS: Compared to the control cells, the cells treated with cantide showed a dose-dependent decrease in hTERT mRNA levels at 24 h and in protein levels at 48 h respectively.The telomerase activity was decreased as the concentration of cantide increased at 48 h. At the concentration of 800 nM,the telomerase activity in the treated HepG2 and SMMC7721 cells was only 17.1% (P＜0.01) and 20.3% (P＜0.01)of that in untreated cells. The levels of CPP32-like protease activity in HepG2 and SMMC-7721 increased by 2.8- and 3.0-fold (P＜0.05) at 48 h, and the levels of ICE-like protease activity also increased by 2.6- and 3.2-fold (P＜0.05)respectively. The percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was 63% and 52% (P＜0.01), respectively. By contrast, 8%and 9% of the cells were apoptosis after 72 h treatment with lipofectin alone.CONCLUSION: Cantide can decrease telomerase activity by inhibiting the expression of hTERT gene and has a rapid anti-tumor effect through inducing the Caspase-dependent apoptosis. The rapid inhibitory effect of cantide on tumor growth demonstrates its feasibility in cancer treatment.     

Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC—7721 cells

AIM:Cyclooxygenase-2(COX-2)has been suggested to be associated with carcinogenesis.We sought to investigate the effect of the selective COX-2 inhibitor,Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS:This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line Various concentrations of Nimesulide(0,200μmol/L,300μmol/L,400μmol/L)were added and incubated.Cell proliferation was detected with MTT colorimetric assay,cell proliferation was detected with MTT colorimetric assay,cell apoptosis by electron microscopy,flow cytometry and TUNEL.RESULTS:Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the controla group.The duration lowerst inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%,the highest inhibition rate was 58.49%,After incubation with Nimesulide for 72h,the most highest apotosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%&#177;1.62%,vs2.24%&#177;0.26%and 21.23&#177;1.78vs2.01&#177;0.23(P&lt;0.05).CONCLUSION:The selective COX-2 inhibitor,Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells,The apoptosis rate and the apoptosis index are dose-dependent.Under electron microscope SMMC-7721 cells incubated with 300 μmol and 400μmol Nimesulide show apoptotic characteristics With the clarification of the mechanism of selective COX-2 inhibitors,Thtese COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.     

去甲斑蝥素诱导人肝癌细胞凋亡的研究

目的探讨去甲斑蝥素（NCTD）对人肝癌细胞SMMC-7721凋亡的影响。方法用MTT方法检测人肝癌细胞SMMC-7721的生长抑制率。应用流式细胞仪检测细胞周期和凋亡。结果 NCTD对人肝癌细胞SMMC-7721有生长抑制作用,随浓度升高、时间延长作用增强,呈剂量和时间效应关系。流式细胞仪示SMMC-7721细胞的S期细胞明显增多,G0/G1期细胞减少,凋亡率上升（P〈0.05或〈0.01）。结论 NCTD能显著抑制人肝癌SMMC-7721细胞的生长,其可能与抑制SMMC-7721细胞增殖,诱导细胞凋亡有关。     

脂质体法转染人肿瘤坏死因子-α基因对裸鼠人肝癌移植瘤的抑制效应及其机制

背景：外源性肿瘤坏死因子（TNF）-α联合化疗药物对肿瘤的疗效较单独应用更佳,为肿瘤治疗提供了新的方向。目的：对裸鼠人肝癌移植瘤模型行脂质体介导的TNF-α基因瘤内转染,研究肝癌移植瘤的生长抑制情况及其机制。方法：经脂质体介导,以真核表达质粒pSVK3-TNF-α分别转染人肝癌细胞株SMMC-7721和裸鼠皮下SMMC-7721细胞移植瘤。测定SMMC-7721细胞的TNF-α浓度,甲基噻唑基四唑（MTT）法测定细胞杀伤率,流式细胞仪和原位末端标记（TUNEL）法检测细胞周期和凋亡情况。结果：TNF-α转基因治疗裸鼠人肝癌移植瘤结束第5d,移植瘤体积为（75.28±35.35）mm^3,显著低于对照组的（326.45±103.64）mm^3（P〈0.05）。TNF-α基因体外转染SMMC-7721细胞24、48、72h后,基因转染组每106个细胞的TNF-α表达量分别为（1680±187）pg、（1702±205）pg和（1650±164）pg,细胞杀伤率分别为（37.1±2.4）%、（79.4±4.3）%和（84.2±4.6）%。基因转染72h后,SMMC-7721细胞增殖指数为（30.5±3.2）%,显著低于对照组的（46.1±3.9）%（P〈0.05）;凋亡指数为（10.0±2.1）%,显著高于对照组的（2.7±0.4）%（P〈0.01）。结论：脂质体介导的TNF-α基因转染裸鼠人肝癌移植瘤可明显抑制肿瘤生长,其机制可能为影响肿瘤细胞生长周期以及诱导肿瘤细胞凋亡。     

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721)to human umbilical vein endothelial cells (ECV-304),expression of adhesive molecule integrinβ1 in SMMC-7721cells and its contribution to this adhesive course.METHODS: Adhesive force of SMMC-7721 cells to endothelialcells was measured using micropipette aspiration technique.Synchronous G1 and S phase SMMC-7721 cells wereachieved by thymine-2-deoxyriboside and colchicinessequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronousrates of SMMC-7721 cells and expression of integrinβ1 inSMMC-7721 cells were detected by flow cytometer.RESULTS: The percentage of cell cycle phases of generalSMMC-7721 cells was 11.01% in G2/M phases, 53.51% inG0/G1 phase, and 35.48% in S phase. The synchronous ratesof G1 and S phase SMMC-7721 cells amounted to 74.09%and 98.29%, respectively. The adhesive force of SMMC-7721cells to endothelial cells changed with the variations ofadhesive time and presented behavior characteristics ofadhesion and de-adhesion. S phase SMMC-7721 cells had higheradhesive forces than G1 phase cells [(307.65±92.10)× 10-10Nvs (195.42±60.72)×10-10N, P＜0.01]. The expressivefluorescent intensity of integrinβ1 in G1 phase SMMC-7721cells was depressed more significantly than the values ofS phase and general SMMC-7721cells. The contribution ofadhesive integrinβ1 was about 53% in this adhesive course.CONCLUSION: SMMC-7721 cells can be synchronizedpreferably in G1 and S phases with thymine-2-deoxyribosideand colchicines. The adhesive molecule integrinβ1 expressesa high level in SMMC-7721 cells and shows differences invarious cell cycles, suggesting integrin β1 plays an importantrole in adhesion to endothelial cells. The change of adhesiveforces in different cell cycle SMMC-7721 cells indicatesthat S phase cells play predominant roles possibly whilethey interact with endothelial cells.     

Effects of insulin-like growth factors-IR and -IIR antisense gene transfection on the biological behaviors of SMMC-7721 human hepatoma cells

BACKGROUND AND AIMS: Insulin-like growth factors (IGFs) are closely related to hepatocellular carcinoma growth. The study aim was to investigate the effects of IGF-IR and IGF-IIR antisense gene transfection on the biological behaviors of SMMC-7721 human hepatoma cells. METHODS: 7721-IGF-IR-AS cells (human hepatoma SMMC-7721 cells transfected with IGF-IR antisense gene in our previous study) were transfected with a plasmid vector expressing IGF-IIR cDNA in the antisense orientation by DOTAP liposome.7721-IGF-R-AS cells were obtained by selection with G418 and hygromycin. Morphological changes of the cells were observed with optic and electron microscopes. In vitro growth of the 7721-IGF-R-AS cells was observed with a soft agar test, MTT test and with naked mice inoculation test in vivo. RESULTS: The following changes were found in the SMMC-7721 cells after being transfected with the IGF-IR and IGF-IIR antisense genes: (i) the degree of malignancy of the tumor cells as revealed by cell morphology was ameliorated; (ii) the growth capability of the tumor cells in soft agar and their tumorigenicity in naked mice were significantly depressed. However, in the control groups, the SMMC-7721 cells transfected both with IGF-IR and IGF-IIR sense cDNA and SMMC-7721 cells transfected without any external genes, had no such changes. However, the cell growth curves had no significant differences among these three groups. CONCLUSION: IGF-IR and IGF-IIR antisense genes could significantly restrain the malignant behavior of human hepatoma cells and might be useful in investigating a potential route for hepatocellular carcinoma gene therapy.