PPIs抑制P13K/Akt／mTOR信号通路逆转胃癌细胞的化疗多药耐药

背景：前期实验显示质子泵抑制剂（PPIs）可抑制空泡型质子泵（V-H＋-ATPases）和多药耐药蛋白P—gP、MRP1表达,增强胃癌细胞的化疗敏感性。目的：探讨PPIs抑制空泡型质子泵逆转胃癌细胞化疗多药耐药与P13K／Akt／mTOR信号通路的关系。方法：应用不同浓度埃索美拉唑或泮托拉唑预处理人胃腺癌细胞敏感株SGC7901和多药耐药株SGC7901／MDR,或以V—H＋-ATPasessiRNA干扰SGC7901／MDR细胞内的V-H＋-ATPases表达,或以雷帕霉素阻断mTOR表达,以蛋白质印迹法检测经不同方式处理的细胞内V—H＋ATPases、P—SP、MRPl蛋白表达以及P13K／Akt/mT0R／HIF—1α信号通路及其信号旁路TSCl／2-Rheb中的相关蛋白表达;以免疫荧光法检测经埃索美拉唑预处理的SGC7901／MDR细胞内的V-H＋ATPases、P—gP蛋白表达和定位。结果：PPIs可呈浓度依赖性地抑制SGC7901／MDR细胞内的V—H＋ATPases、P13K、Akt、roTOR、HIF-1仅、TSCI、TSC2、Rheb、P—gP、MRP1表达以及Akt底物和TSC2磷酸化,改变V-H＋ATPases、P—gP的胞内定位,对SGC7901细胞则无上述影响。以V—H＋-ATPasessiRNA抑制SGC7901／MDR细胞内的V—H＋-ATPases表达,作用与PPIs预处理相似。以雷帕霉素阻断mTOR可呈浓度依赖性地抑制SGC7901／MDR细胞内的HIF-1α、P—gP表达。结论：PPIs抑制空泡型质子泵逆转胃癌细胞化疗多药耐药的机制与抑制P13K／Akt／mTOR信号通路有关。     

Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways

AIM： To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action.
METHODS： The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MI-I-） assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential （A~Pm） were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases （PI3K）.
RESULTS： Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time- and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 pmol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901 cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt.
CONCLUSION： Alisol B acetate exhibits an antiproliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways.     

核抗原Mina53在人胃癌细胞中的作用及其意义探讨

背景:核抗原Mina53基因为原癌基因Myc的下游直接靶基因之一,在一些消化系统恶性肿瘤中呈高表达,并与肿瘤增殖、侵袭、转移或患者生存期相关。目的:研究Mina53在人胃癌细胞中的作用及其对胃癌发生、发展的意义。方法:选择Mina53表达水平较高的人胃癌细胞株SGC7901和AGS,应用RNA干扰技术下调其Mina53表达,以转染无关序列siRNA的细胞作为对照组。采用CCK-8实验检测细胞增殖,流式细胞术检测细胞周期和细胞凋亡,细胞侵袭和迁移实验检测细胞侵袭、迁移能力。结果:与相应对照组相比,Mina53 siRNA转染组SGC7901、AGS细胞增殖受抑(96 h相对增殖率:60%和68%),并发生明显细胞周期G1期阻滞(SGC7901细胞G1/G2:2.76±0.12对1.86±0.06,P<0.05;AGS细胞G1/G2:1.78±0.13对1.34±0.05,P<0.05),细胞凋亡率分别增加9.8%±1.2%和10.6%±1.5%(P<0.05),穿透Transwell小室基质胶细胞数(SGC7901细胞:11.67±0.88对24.33±1.45,P<0.05;AGS细胞:8.00±1.15对20.33±1.73,P<0.05)和穿透Transwell小室微孔膜细胞数(SGC7901细胞:7.00±1.53对14.67±2.03,P<0.05;AGS细胞:8.00±1.16对15.33±1.45,P<0.05)均显著减少。结论:Mina53对人胃癌细胞的增殖、细胞周期、细胞凋亡以及侵袭、迁移能力具有调控作用,可能影响胃癌的生长、浸润和转移,有望作为胃癌基因治疗的靶点。     

环氧合酶-2反义核酸抑制胃癌细胞的恶性表型

目的　通过环氧合酶 2 (COX 2 )反义核酸基因转染 ,逆转COX 2高表达的人胃癌细胞系中其表达水平 ,观察细胞生物学行为的变化情况 ,以初步探讨COX 2表达在胃癌发生中的某些具体机制。方法　使用脂质体介导的方法 ,用反义重组质粒及空载体分别转染COX 2异常高表达的人胃癌细胞系SGC 790 1(转染细胞分别命名为 790 1 AS及 790 1 P细胞 )。通过免疫细胞化学及RNA斑点杂交试验检测反义核酸转染的细胞中COX 2的蛋白及mRNA表达水平。四唑盐 (MTT)比色试验检测转染细胞的体外增殖速度。应用裸鼠成瘤试验比较转染前后细胞体内成瘤能力的差别。结果　免疫细胞化学及RNA斑点杂交试验证实 :在反义核酸转染的 790 1 AS细胞中COX 2的蛋白及mRNA水平均显著下调。MTT比色试验显示 790 1 AS细胞的增殖速度低于亲本SGC 790 1细胞。裸鼠成瘤试验表明 ,反义核酸转染细胞成瘤潜伏期延长 ,成瘤体积减小。 3组细胞接种裸鼠 30d后 ,瘤体的平均重量( x±s)分别为 (82 6 6 7± 77 6 7)mg(SGC 790 1细胞 ) ,(776 6 7± 30 0 0 6 )mg(790 1 P细胞 )和 (486 6 7±15 2 8)mg (790 1 AS细胞 )。转染反义核酸的细胞成瘤性显著低于未转染细胞及转染载体对照细胞(P <0 0 1)。结论　胃癌细胞中过表达的COX 2与细胞的恶性表型相关。用     

乳香通过PTEN/Akt/COX-2通路诱导胃癌细胞SGC7901凋亡

目的研究乳香对人胃癌细胞SGC7901增殖、凋亡和迁移的影响及其可能的作用机制。方法不同浓度乳香处理SGC7901细胞不同时间,噻唑兰比色法检测乳香对SGC7901细胞增殖的影响,流式细胞术分析乳香对SGC7901细胞凋亡的影响,蛋白质印迹法(Western blotting)分析乳香对SGC7901细胞中同源性磷酸张力蛋白(PTEN)、磷酸化蛋白激酶B(p-Akt)、蛋白激酶B(PKB,亦称Akt)和环氧化酶2(COX-2)蛋白表达影响,划痕实验检测细胞迁移能力,裸鼠移植瘤实验验证乳香在裸鼠体内抑制肿瘤的效果。采用Graphpad Prism 6.0统计软件对数据进行分析。结果乳香能够抑制SGC7901细胞的增殖和迁移能力,呈现剂量依赖和时间依赖性。乳香诱导SGC7901细胞的凋亡,呈现剂量依赖性。乳香能够诱导SGC7901细胞中PTEN的表达,抑制p-Akt和COX-2的蛋白表达。此外,乳香能够抑制裸鼠体内胃癌移植瘤的生长。结论乳香可以抑制胃癌细胞SGC7901的增殖和迁移,诱导细胞凋亡,这可能与PTEN/Akt/COX-2信号通路相关。     

阿霉素诱导PI3'K/Akt/FKHRL1通路激活与胃癌细胞化疗耐药性的关系

目的 探讨阿霉素诱导胃癌细胞磷脂酰肌醇3’-激酶（PI3’K）／Akt／FKHRL1通路的激活对胃癌细胞SGC-7901化疗效果的影响及二者的关系。方法 阿霉素及PI3’K／Akt抑制剂Wortmannin分别作用于胃癌细胞SGC-7901，MTT比色法检测胃癌细胞的生存率，Western印迹法检测FKHRL1磷酸化表达水平。结果 阿霉素抑制胃癌细胞SGC-7901的生长，呈时间依赖性诱导FKHRL1磷酸化。Wortmannin可明显增强阿霉索的细胞生长抑制作用，同时下调阿霉素诱导的磷酸化FKHRL1表达。结论 阿霉素可能通过激活SGC-7901细胞的PI3’K／Akt通路诱导FKHRL1磷酸化，从而影响胃癌细胞的化疗耐药性。Wortmannin可以阻断PI3’K／Akt／FKHRL1通路而提高胃癌的化疗敏感性。     

PI3K/Akt抑制剂LY294002对胃癌细胞化疗增敏作用的探讨

目的 探讨PI3K/Akt特异性抑制剂LY294002与化疗药物5-Fu及奥沙利铂联合使用对3种胃癌细胞系(MGC803、BGC823和SGC7901)化疗效果的影响.方法 将PI3K/Akt特异性抑制剂LY294002联合化疗药物5-Fu及奥沙利铂作用于3种胃癌细胞系,MTT法检测单独使用5-Fu、奥沙利铂及联合LY294002对体外培养的3种胃癌细胞系的增殖抑制作用,流式细胞术检测细胞凋亡.结果 联合LY294002作用后,5-Fu、奥沙利铂对3种胃癌细胞系的增殖抑制作用明显增强(P<0.05),且凋亡率显著提高(P<0.05).结论结果 LY294002能有效提高化疗药物5-Fu、奥沙利铂体外对胃癌细胞的增殖抑制作用,抑制PI3K/Akt信号转导通路可显著提高胃癌的化疗疗效.     

重组反义c-myc 腺病毒对人胃癌细胞的体内及体外分子治疗

目的研究重组反义ｃｍｙｃ腺病毒对胃癌细胞的体内外生物学作用．方法采用ＬａｃＺ基因Ｘｇａｌ染色、ＭＴＴ,ＤＮＡ梯度降解试验、原位末端标记、流式细胞仪、ＰＣＲ分析、裸鼠致瘤性、裸鼠皮下移植瘤模型实验等方法,对反义ｃｍｙｃ重组腺病毒在人胃癌ＳＧＣ７９０１细胞系中的作用进行体内外研究．结果ＡｄＡＳｃｍｙｃ对ＳＧＣ７９０１细胞能产生明显的生长抑制作用,ＭＴＴ显示生长抑制率为４４１％．ＤＮＡ梯度降解试验、原位末端标记、流式细胞仪显示ＡｄＡＳｃｍｙｃ诱导了ＳＧＣ７９０１细胞凋亡．经ＡｄＡＳｃｍｙｃ处理的ＳＧＣ７９０１细胞裸鼠致瘤性消失．ＡｄＡＳｃｍｙｃ对裸鼠皮下移植瘤模型瘤内注射能有效降低肿瘤的生长速度,生长抑制率为６８９％．结论ＡｄＡＳｃｍｙｃ对胃癌细胞具有显著的体内外生长抑制及凋亡诱导作用     

The therapeutic effects of recombinant adenovirus RA538 on human gastric carcinoma cells in vitro and in vivo

AIM:To evaluate the potential of RA-538 gene therapy for gastric carcinoma.METHODS:Human gastric carcinoma cell line SGC7901 treated with Ad-RA538 or Ad-LacZ were analysed by X-gal stain, MTT, DNA ladder, Tunel, flow cytometric analysis, PCR, and Western Blot in vitro. The tumorigenicity and experimental therapy in nude mice model were assessed in vivo.RESULTS:Ad-LacZ could efficiently transfer the LacZ gene into SGC7901 cells. X-gal-positive cells at MOI 25, 50, 100, and 200 were 90%, 100%, 100%, and 100% respectively. Ad-RA538 could strongly inhibit cell growth and induced apoptosis in SGC7901 cells.The proliferation of the Ad-RA538-infected SGC7901 cells was reduced by 76.3%.The mechanism of killing of gastric carcinoma cells by Ad-RA538 was found to be apoptosis by DNA ladder,Tunel and flow cytometric analysis.The tumorigenicity in nude mice using Ad-RA538 showed that all three mice failed to form tumor from 7 to 30 days compared with Ad-LacZ and parent SGC7901 cells. Experimental therapy on the nude mice model bearing subcutaneous tumor of SGC7901 cells showed that intratumor instillation of Ad-RA538 inhibited the growth of the tumors. Ad-RA538-treated tumors were inhibited by 60.66%, compared with that of the tumor injected with Ad-LacZ and mock.CONCLUSION: The expression of Ad RA538 can inhibit growth and induce apoptosis of gastric cancer cell in vitro and in vivo. Ad RA538 can be used potentially in gene therapy for gastric carcinoma.     

19‐peptide,a fragment of tumstatin,inhibits the growth of poorly differentiated gastric carcinoma cells in vitro and in vivo

Background and Aim: This study investigated whether 19‐peptide, a fragment of tumstatin, inhibited the growth of gastric tumor cells in vitro and in vivo. Methods: 19‐peptide was expressed in bacteria and purified with Sephadex G‐15. SGC7901 gastric carcinoma cells and human umbilical‐vein endothelial cells (HUVECs) were exposed to 19‐peptide in vitro, and their viability was evaluated by biochemical and histopathological analysis. In vivo, pieces of solid tumor derived from SGC7901 cells were inoculated into the gastric serosa of 36 nude mice, with a biological glue to hold them in place. Twenty‐eight days after injection of 19‐peptide, the mice were killed. The tumors were measured and examined by western blotting, histopathology, and terminal deoxynucleotidyl transferase biotin‐dUTP nick end labeling assay. Results: 19‐peptide induced apoptosis of many SGC7901 cells but few HUVECs in vitro. In vivo, after the application of 19‐peptide, significant tumor cell apoptosis was observed in the center of the tumors, tumor volume was reduced significantly (P < 0.001), and the invasion and migration of cancer cells was reduced. PTEN was increased in the treatment group and phospho‐Akt (pAkt) was decreased in the control group. Conclusions: These results suggest that 19‐peptide inhibits the growth and metastases of poorly differentiated gastric carcinoma cells, primarily by inducing apoptosis. The apoptotic mechanism could be related to anoikis and the PTEN/Akt pathway.     

阿司匹林对人胃癌细胞株的作用及机制探讨

目的 观察阿司匹林对人胃癌细胞株SGC7901生长及人胃癌裸鼠移植瘤生长的作用,并初步探讨其作用机制。方法 采用MTT法及流式细胞术检测不同浓度阿司匹林对胃癌细胞增殖及细胞周期的影响;Westem blot法检测阿司匹林对胃癌细胞COX-2、VEGF表达的影响;建立人胃癌裸鼠移植瘤模型,给予阿司匹林20天,观察肿瘤大小,免疫组化检测阿司匹林对肿瘤组织中COX-2、VEGF表达及MVD的影响。结果 阿司匹林对胃癌细胞的增殖具有抑制作用,且呈一定的时间、剂量依赖性;细胞周期分析表明阿司匹枳主要使细胞阻滞在G0/G1期;western blot检测表明阿司匹林能降低胃癌细胞COX-2、VEGF蛋白的表达;阿司匹林对裸鼠移植瘤的生长有抑制作用,免疫组化显示阿司匹林能降低移植瘤组织中(COX-2、VEGF的表达及MVD。结论 阿司匹林对胃癌细胞及裸鼠移植瘤均具有一定的抑制作用,其机制可能是通过对COX-2和VEGF等血管生成相关因子的抑制起作用。     

Antiproliferative effect of octreotide on gastric cancer cells mediated by inhibition of Akt／PKB and telomerase

AIM: To investigate the antiproliferative effect of octreotide,a long-acting analogue of somatostatin, on gastric cancer cell line SGC7901 and its possible molecular mechanisms.METHODS: Gastric cancer cell line SGC7901 employed in the study was treated with 0.008, 0.04, 0.2, 1, 5 and 25μg@ml-1 of octreotide respectively for 24 h to evaluate the antiproliferative effect of somatostatin analog on the tumor cells by MTT assay method. To elucidate the underlying mechanism, the cells were exposed to 1 μg@ml-1 of octreotide for 0, 12, 24 and 48 h, when their Akt/PKB and telomerase activities were respectively determined using PCR-ELSIA and nonradioactive protein kinase assay protocols. The same experimental procedures were also performed in the control cells that were treated with corresponding vehicles instead of somatostatin analog.RESULTS: After exposed to octreotide for 24 h at the concentrations of more than 1 μg@ml-1 SGC7901 cells exhibited a dose-dependent inhibition of growth with the inhibiting rate to be as high as 34.66 % when 25 μg@ml-1 of octreotide was applied. The Akt/PKB and telomerase activity of SGC7901 cells was significantly inhibited when the cells were exposed to 1 μg@ml-1 of octreotide for 12, 24 and 48 h compared with that of their control counterparts (P＜0.01),both of which exhibited in a time-dependent manner.CONCLUSION: The antiproliferative effect of octreotide on SGC7901 cells might be mediated by the inhibition of Akt/PKB and telomerase.     

Expression and function of classical protein kinase C isoenzymes in gastric cancer cell line and its drug-resistant sublines

AIM：To investigate the expression and function of classical protein kinase C(PKC)isoenzymes in inducing MDR phenotype in gastric cancer cells.METHODS:Two cell lines were used in the study:Gastric cancer cell SGC7901 and its drug-resistant cell SGC7901/VCR stepwise-selected by vincristine 0.3,0.7and 1.0mg.L^-1,respectively.THe expression of classical PKC(cPKC) isoenzymes in SGC7901 cells and SGC7901/VCR cells were detected using immunofluorescent cytochemistry,laser confocal scanning microscope and Western blot .The effects of anti-PKC isoenzymes antibody on adriamycin accumulation in SGC7901/VCR cells were setermined using flow cytometric analysis.RESULTS:(1) SGC7901 cells exhibited positive staining of PKC-αSGC7901／VCR cells exhibited stronger staining of PKC-αthan SGC790-1 cells.The higher dosage vincristine selected,the much stronger staining of PKC-α was observed on SGC7901/VCR cells.(2)BOth SGC7901 and SGC7901/VCR cells exhibited positive staining of PKC-βI and PKC-βⅡwith no significant difference.(3) Compared with SGC7901,SGC7901/VCR cells had decreased adriamycin accumulation and retention.Accumulation of adriamycin in SGC7901 was 5.21&#177;2.56mg.L^-1,in SGC7901/VCR0.3was 0.85&#177;0.29mg.L^-1,in SGC7901/VCR0.7was0.81&#177;0.32mg.L^-1,and in SGC7901/VCR 1.0 was 0.80&#177;0.33mg.L^-1;Retention of adriamycin in SGC 7901 was 2.51&#177;1.23mg.L^-1,in SGC7901/VCR0.3 was 0.47&#177;0.14mg.L^-1,in SGC7901/VCR0.7 was 0.44&#177;0.15mg.L^-1,and in SGC 7901/VCR 1.0 was 0.41&#177;0.11mg.L^-1.(4)Fluorescence intensity presented adriamycin accumulation in SGC7901/VCR cells was increased from 1.14&#177;0.35to 2.71&#177;0.94when cells were co-incubated with antiPKC-αbut not with anti-PKC-βI,PKC-βⅡand PKCγ antibodies. CONCLUSION:PKC-α.but not PKC-βⅠ,PKC-βⅡor PKCγ,may play a role in multidrug resistance of gastric cancer cells SGC7901/VCR.     

胃肠富集Krüppel样因子基因转染对人胃癌细胞株SGC-7901及裸鼠移植瘤生长的影响

目的 探讨外源性胃肠富集Krüppel样因子(GKLF)基因转染对人胃癌细胞株SGC-7901在体内外的抗肿瘤效应.方法 荧光实时定量PCR和Western印迹法检测GKLF基因转染前后人胃癌细胞株SGC-7901中GKLF mRNA和蛋白的表达.应用四甲基偶氮唑盐(MTT)法、流式细胞技术、克隆形成实验和细胞侵袭实验分别检测GKLF基因转染后SGC-7901细胞增殖和侵袭力的变化.观察裸鼠移植瘤生长情况和应用免疫组织化学法检测移植瘤组织中微血管密度(MVD).结果 GKLF基因转染后,SGC7901-pcDNA3.1-GKLF组中GKLF mRNA(0.1216±0.0061)和蛋白(1.0547±0.0172)的表达明显高于SGC-7901组[分别为(0.0029±0.0012)和(0.6240±0.0177)]和SGC7901-pcDNA3.1组[分别为(0.0037±0.0013)和(0.5627±0.0510)],P＜0.05.与SGC-7901组和SGC7901-pcDNA 3.1组相比,从48 h开始,SGC7901-pcDNA 3.1-GKLF组细胞生长速度减慢、细胞出现G0/G1期部分阻滞、克隆形成率低、细胞侵袭力明显减弱(P值均＜0.05).SGC7901-pcDNA3.1-GKLF组皮下移植瘤生长速度减慢、瘤重轻、MVD低(P值均＜0.05).结论 GKLF基因转染可导致人胃癌细胞株SGC-7901的增殖活性和侵袭力降低,抑制裸鼠移植瘤生长和血管生成.

Abstract：

Objective To investigate the antitumour effects of transfected gut-enriched Krüppellike factor(GKLF) on human gastric carcinoma cell line SGC-7901 in vitro and in vivo. Methods The expression of GKLF mRNA and protein in human gastric carcinoma cell line SGC-7901 were detected before and after transfection by real-time fluorescence quantitative PCR and Western blot,respectively. Proliferation and invasion in SGC-7901 were measured respectively by MTT assay, flow cytometry, colony formation assay and cell invasion assay after transfected with GKLF. The growth of xenograft was observed, the microvessel density(MVD) of xenograft tissue was determined by immunohistochemistry. Results The GKLF mRNA and protein in SGC-7901 were overexpressed after transfected with GKLF(P＜0.05). The proliferative speed of SGC7901-pcDNA3.1-GKLF group was markedly lower than that of SGC-7901 and SGC7901-pcDNA3.1 groups (P＜0.05). Transfected with GKLF caused part of the G0/G1 arrest, decreased clone formation rate and the invasion ability (P＜0.05). The growth speed of xenograft in SGC7901-pcDNA3.1-GKLF group was lower, the weight and MVD of xenograft tissue in SGC7901-pcDNA3. 1-GKLF group were less (P＜ 0. 05).Conclusion Transfected with GKLF maysuppress proliferation and invasion in human gastric carcinoma cell line SGC-7901, inhibit the growth and the angiogenesis of xenograft in nude mice.     

Blockade of Rho/Rho-associated coiled coil-forming kinase signaling can prevent progression of hepatocellular carcinoma in matrix metalloproteinase-dependent manner

Aim:  There is growing evidence that the Rho/Rho-associated coiled coil-forming kinase (ROCK) signaling pathway is upregulated in tumors and plays a key role in cancer invasion and metastasis. Our aim was to test the anticancer effects of Rho/ROCK inhibitor, Y-27632, including possible mechanisms in a highly-metastasizing hepatocellular carcinoma (HCC) mouse model on its secretion of matrix metalloproteinase (MMP) and tumor progression.
Methods:  Following orthotopic implantation of CBO140C12 HCC tumor fragments into the liver of mice, the mice were randomly assigned to a Y-27632-treated group or control group. After treatment for 4 weeks, specimens were obtained to evaluate tumor size, metastases, and immunohistochemical findings. In vitro , we examined the effects of Y-27632 and RhoC siRNA on MMP-2 and -9 expressions, invasiveness, and apoptosis in cultured tumor cells.
Results:  Both RhoA and RhoC were upregulated in HCC-bearing livers, and Y-27632 significantly inhibited not only tumor growth and intrahepatic metastasis ( P  < 0.05), but also tumoral MMP-9 expression. Moreover, Y-27632 treatment resulted in large necrotic areas in tumors. In vitro , Y-27632 and RhoC siRNA reduced MMP-2 and -9 expressions, as well as the chemotactic migration of tumor cells dose-dependently, and increased apoptosis eight times.
Conclusion:  Y-27632 suppresses progression and limits the intrahepatic metastasis of established HCC. This could be linked to the decreased MMP expression and induction of apoptosis in tumor cells. Rho signaling may prove to be a productive target in anticancer therapy.     

Aging is associated with decreased pancreatic acinar cell regeneration and phosphatidylinositol 3-kinase/Akt activation

BACKGROUND & AIMS: The effects of aging on pancreatic acinar cell proliferation have not been clearly defined. Phosphatidylinositol 3-kinase (PI3K)-mediated phosphorylation of Akt is a critical step for proliferation of various cell types and insulin secretion from pancreatic endocrine cells; however, its role in acinar cell proliferation is not known. The purpose of this study was to (1) delineate the effects of aging on pancreatic regeneration after partial pancreatectomy (Px) and (2) define the involvement of the PI3K/Akt pathway in pancreatic regeneration. METHODS: Following partial Px, pancreatic regeneration and activation of the PI3K pathway were compared in young and aged mice. Activation of the PI3K/Akt pathway was evaluated by Akt phosphorylation (pAkt). The role of the PI3K pathway in pancreatic regeneration after partial Px was assessed by effects of a pharmacologic PI3K inhibitor wortmannin or small interfering RNA (siRNA) to the p85alpha regulatory subunit. To confirm further the critical role of the PI3K/Akt pathway in pancreatic acinar cell proliferation, IGF-1-mediated cell proliferation was determined in cultured acinar cells pretreated with wortmannin or p85alpha siRNA. RESULTS: Pancreatic regeneration and pAkt expression after partial Px were significantly decreased with aging. Treatment with wortmannin or p85alpha siRNA reduced pancreatic regeneration after partial Px. The IGF-1-mediated cell proliferation in vitro was completely blocked by wortmannin or p85alpha siRNA but not by the MEK/ERK inhibitor PD98059. CONCLUSIONS: PI3K/Akt activation plays a critical role in the regeneration of pancreatic acini after resection. Furthermore, pancreatic regeneration is markedly attenuated in the aged pancreas most likely because of decreased PI3K/Akt activation.     

血管内皮生长因子小干扰核糖核酸联合双自杀基因抗胃癌作用的体外研究

目的 探讨联合应用靶向血管内皮生长因子(VEGF)小干扰RNA(siRNA)与双自杀基因yCDglyTK对人胃癌细胞的体外杀伤作用。方法 以磷酸钙纳米颗粒为载体介导空白质粒pcDNA3.1（-）null(空白质粒组)、靶向VEGF的干扰质粒pGenesil-shVEGF(干扰质粒组)、双自杀基因质粒pcDNA3.1（-）CV-yCDglyTK(双自杀基因组)及联合基因质粒pcDNA3.1（-）shVEGF-yCDglyTK（联合基因组）转染胃癌SGC7901细胞,未转染的胃癌细胞为空白对照组,经G418筛选稳定转染的胃癌细胞株,采用RT-PCR和免疫印迹法验证目的基因表达。给予前体药物5-氟胞嘧啶(5-FC)后,通过四甲基偶氮唑盐(MTT)生长曲线、旁观者效应实验、Hoechst 33258染色及流式细胞术,观察各组细胞的生物学特性变化、凋亡细胞形态及凋亡率。采用SPSS 13.0统计学软件进行数据处理分析,组间多重比较采用LSD检验。结果成功建立4种转染不同质粒的胃癌细胞株,联合基因组及双自杀基因组均可检测到双自杀基因yCDglyTK的表达。MTT生长曲线显示5-FC作用24 h后,与空白对照组和空白质粒组相比,干扰质粒组、双自杀基因组及联合基因组吸光度值明显降低(P＜0.01)。当稳定转染联合基因的SGC7901细胞占60％、80％和100％时,细胞相对存活率分别为13.09％±2.40％、9.74％±2.83％及5.68％±1.03％。荧光显微镜下见双自杀基因组及联合基因组大量细胞呈现凋亡形态改变。流式细胞检测结果示干扰质粒组、双自杀基因组以及联合基因组的凋亡率分别为16.40％±4.68％、57.63％±4.96％及69.07％±4.69％,与空白对照组相比,差异有统计学意义(P＜0.01)。结论采用靶向VEGF siRNA与双自杀基因联合治疗可有效杀伤胃癌SGC7901细胞,诱导凋亡是其杀伤瘤细胞的重要机制之一。     

Antisense angiopoietin-1 inhibits tumorigenesis and angiogenesis of gastric cancer

AIM: To investigate the effect of angiopoietin-1 (Ang-1) on biological behaviors in vitro and tumorigenesis and angiogenesis in vitro of human gastric cancer cells. METHODS: Human full-length Ang-1 gene was cloned from human placental tissues by RT-PCR method. Recombinant human Ang-1 antisense eukaryotic expression vector was constructed by directional cloning, and transfected by lipofectin method into human gastric cancer line SGC7901 with high Ang-1 expression level. Inhibition efficiency was confirmed by semi- quantitive PCR and Western blot method. Cell growth curve and cell cycle were observed with MTT assays and flow cytometry, respectively. Nude mice tumorigenicity test was employed to compare in vitro tumorigenesis of cells with Ang-1 suppression. Microvessel density (MVD) of implanted tumor tissues was analyzed by immunohistochemistry for factorⅧstaining. RESULTS: Full-length Ang-1 gene was successfully cloned and stable transfectants were established, namely 7Angl- for antisense, and 7901P for empty vector transfected. 7Angl- cells showed down-regulated Ang-1 expression, while its in vitro proliferation and cell cycle distribution were not significantly changed. In contrast, xenograft of 7Angl- cells in nude mice had lower volume and weight than those of 7901P after 30 days' implantation (p<0.01, 293.00±95.54 mg vs. 624.00±77.78 mg) accompanied with less vessel formation with MVD 6.00±1.73 compared to 7901P group 8.44±1.33(p<0.01). CONCLUSION: Ang-1 may play an important role in tumorigenesis and angiogenesis of gastric cancer, and targeting its expression may be beneficial for the therapy of gastric cancer.     

Reversal of multidrug resistance in vincristine-resistant human gastric cancer cell line SGC7901/VCR by LY980503

AIM： To investigate the reversal effect of LY980503, a benflumetol derivative, on multidrug resistance in vincristine （VCR） -resistant human gastric carcinoma cell line SGC7901/VCR.
METHODS： Cells of a human gastric cancer cell line, SGC7901, and its VCR-resistant variant, SGC7901/VCR, were cultivated with LY980503 and/or doxorubicin （DOX）. The cytotoxicity of drugs in vitro was assayed by M-IF method. Based on the flow cytometric technology, the uptake of DOX was detected in these cells by measuring DOX -associated mean fluorescence intensity （MFI）.
RESULTS： SGC7901/VCR cells were 23.5 times more resistant to DOX in comparison with 5GC7901 cells. LY980503 at the concentrations of 2.0 μmol/L -10 μmol/ L had no obvious cytotoxicity to SGC7901 and SGC7901/ VCR cells. After simultaneous treatment with LY980503 at the concentrations of 2.0, 4.0 and 10 μmol/L, the ICso of DOX to SGC7901/VCR cells decreased from 1.6 ± 0.12 μmol/L to 0.55 ± 0.024, 0.25 ± 0.032 and 0.11 ± 0.015 μmol/L, respectively, thus, increasing the DOX sensitivity by 2.9-fold （P 〈 0. 05）, 6.4-fold （P 〈 0. 01） and 14.5-fold （P 〈 0. 01）, respectively. In the uptake study of DOX, simultaneous incubation of SGC7901/VCR cells with LY980503 significantly increased the DOX -associated MFI in SGC7901/VCR cells. No such results were found in parental SGC7901 cells.
CONCLUSION： LY980503 at non-cytotoxic concentrations can effectively circumvent resistance of SGC7901/VCR cells to DOX by increasing intracellular DOX accumulation.