Molecular mechanisms of peritoneal dissemination in gastric cancer

Peritoneal dissemination represents a devastating form of gastric cancer(GC) progression with a dismal prognosis. There is no effective therapy for this condition. The 5-year survival rate of patients with peritoneal dissemination is 2%, even including patients with only microscopic free cancer cells without macroscopic peritoneal nodules. The mechanism of peritoneal dissemination of GC involves several steps: detachment of cancer cells from the primary tumor, survival in the free abdominal cavity, attachment to the distant peritoneum, invasion into the subperitoneal space and proliferation with angiogenesis. These steps are not mutually exclusive, and combinations of different molecular mechanisms can occur in each process of peritoneal dissemination. A comprehensive understanding of the molecular events involved in peritoneal dissemination is important and should be systematically pursued. It is crucial to identify novel strategies for the prevention of this condition and for identification of markers of prognosis and the development of molecular-targeted therapies. In this review, we provide an overview of recently published articles addressing the molecular mechanisms of peritoneal dissemination of GC to provide an update on what is currently known in this field and to propose novel promising candidates for use in diagnosis and as therapeutic targets.     

Apoptosis mechanisms of human gastric cancer cell line MKN-45 infected with human mutant p27

AIM: To explore the inducing effect of human mutant p27 gene on the apoptosis of the human gastric cancer cell line MKN-45 and its associated mechanisms. METHODS: The recombinant adenovirus Ad-p27mt was constructed to infect the human gastric cancer cell line MKN-45. Using flow cytometry, TUNEL assay and DNA fragment analysis, we measured the apoptotic effect of Ad-p27mt on the human gastric cancer cells. RESULTS: Ad-p27mt was successfully constructed and the infection efficiency reached 100%. After 18 h of infection, we observed an apoptotic hypodiploid peak on the flow cytometer before G1-S and apoptotic characteristic bands in the DNA electrophoresis. The apoptotic rate detected by TUNEL method was significantly higher in the Ad-p27mt group (89.4±3.12%) compared to the control group (3.12±0.13%, P < 0.01). CONCLUSION: Human mutant p27 can induce apoptosis of the human gastric cancer cells in vitro.     

缬草波春诱导MKN-45胃癌细胞凋亡

目的:研究缬草波春诱导胃癌细胞凋亡,探讨其诱导凋亡与半胱氨酸酶(Caspase)及生存素(Survivin)mRNA、P53蛋白、Survivin蛋白表达的关系.方法:以100 mg/L的缬草波春作用于加Caspase-3抑制剂、Caspase-8抑制剂、Caspase-9抑制剂和未加Caspases抑制剂培养的MKN-45细胞24,48和72 h,用流式细胞仪分别检测细胞凋亡率;不同浓度的缬草波春(5,10,25,50,100 mg/L)作用MKN-45细胞不同时间(24,48,72 h)后,用tripure提取液提取细胞RNA,用RT-PCR法,检测Survivin mRNA的表达.不同浓度缬草波春(50和100 mg/L)作用MKN-45胃癌细胞株24 h后,用免疫组化的方法,检测P53蛋白和Survivin蛋白的表达.结果:单用Caspase抑制剂组,作用24,48和72 h对MKN-45细胞凋亡率无明显影响,与对照组比较差异无显著意义.Caspase-3抑制剂、Caspase-9抑制剂与缬草波春联合应用后24,48和72 h使MKN-45细胞凋亡率高于对照组(24 h:5.73%,5.41% vs 4.38%,P＜0.01;48 h:6.88%,6.32% vs 4.35%,P＜0.01;72 h:7.72%,8.62% vs 4.54%,P＜0.01),低于缬草波春组(24 h:5.73%,5.41% vs 8.14%,P＜0.01;48 h:6.88%,6.32% vs 12.31%,P＜0.01;72 h:7.72%,8.62% vs 26.41%,P＜0.01),与对照组及缬草波春组比较差异均有显著意义(P＜0.01).Caspase-8抑制剂与缬草波春联合应用后24,48和72 h MKN-45细胞凋亡率明显增加,与对照组比较差异有显著意义(8.02% vs 4.38%,P＜0.01;11.05% vs 4.35%,P＜0.01;24.86% vs 4.54%,P＜0.01),与单用缬草波春组比较差异无显著意义.缬草波春降低MKN-45胃癌细胞株Survivin mRNA的表达,并有浓度依赖性和时间依赖性,而且使MKN-45胃癌细胞株P53蛋白表达增加,Survivin蛋白表达降低,均有浓度依赖性.结论:缬草波春可诱导MKN-45细胞凋亡,其作用可部分被Caspase-3,Caspase-9抑制剂所抑制,但不能被Caspase-8抑制剂所抑制.缬草波春诱导MKN-45胃癌细胞株凋亡与P53蛋白表达提高及Survivin mRNA和Survivin蛋白低表达降低有关.     

Molecular therapy with recombinant antisense c-myc adenovirus for human gastric carcinoma cells in vitro and in vivo

BACKGROUND AND AIMS: This study used a recombinant antisense c-myc adenovirus (Ad-ASc-myc) to evaluate how alterations of c-myc expression in the SGC7901 human gastric carcinoma cells could influence the proliferation, apoptosis and the growth of human gastric tumors in nude mice. METHODS: The human gastric carcinoma cell line, SGC7901, treated with Ad-ASc-myc or adenovirus recombinants carrying LacZ gene (Ad-LacZ) were analyzed by using X-gal stain, MTT, DNA ladder, TUNEL assay, flow cytometric analysis, polymerase chain reaction and western blot in vitro. The tumorigenicity and experimental therapy in nude mice models were assessed in vivo. RESULTS: The Ad-ASc-myc could strongly inhibit cell growth and induce apoptosis in SGC7901 cells. The proliferation of the Ad-ASc-myc-infected SGC7901 cells was reduced by 44.1%. The mechanism of killing gastric carcinoma cells by Ad-ASc-myc was found to be apoptosis, which was detected by the use of a DNA ladder, TUNEL and flow cytometric analysis. Infection of Ad-ASc-myc in nude mice showed that all three mice failed to form tumors from the 7 to 30 day period, compared with injection of Ad-LacZ and parent SGC7901 cells. Experimental therapy on the nude mice bearing subcutaneous tumors of SGC7901 cells showed that intratumor instillation of Ad-ASc-myc inhibited the growth of the tumors. Recombinant antisense c-myc adenovirus-treated tumors were inhibited by 68.9%, compared with tumors injected with Ad-LacZ and control (LacZ and phosphate-buffered saline). CONCLUSION: The expression of Ad-ASc-myc can inhibit growth and induce apoptosis of gastric cancer cells in vitro and in vivo and thus is a potential clinical utility in gene therapy for the treatment of gastric carcinoma.     

shRNA沉默干扰HMGA2基因对胃癌细胞株MKN-45的增殖与凋亡的影响

目的:观察小分子RNA(shRNA)沉默后HMG-A2基因在胃癌细胞株MKN-45的表达,并探讨HMGA2基因对胃癌细胞的增殖与凋亡的影响.方法:构建针对人HMGA2基因的shRNA真核表达载体,瞬时转染人胃癌细胞株MKN-45.用细胞免疫组化的方法观察转染72h后HMGA2的蛋白表达水平,以MTT比色法、流式细胞术检测转染后MKN-45细胞的生长增殖、凋亡的情况.结果:转染HMGA2-shRNA组的蛋白表达强度(171.34±19.61)明显弱于scrambled组(143.48±19.04)和空白对照组(141.79±18.09,P<0.05),较之scrambled组(5.66%±0.63%)和空白对照组,HMGA2-shRNA组(39.32%±2.37%)能明显抑制细胞的增殖(P<0.05),HMGA2-shRNA组的凋亡率(39.67%±2.35%)与scrambled组(4.29%±1.33%)和空白对照组(5.05%±1.84%)相比明显增加(P<0.05).结论:靶向HMGA2基因的shRNA可以有效抑制人胃癌MKN-45细胞的生长,并促进细胞的凋亡,HMGA2可能是胃癌治疗的一个潜在靶点.     

外源性FHIT基因表达对长春新碱诱导人胃癌MKN-28细胞凋亡的影响

目的:探讨外源性FHIT基因表达对长春新碱诱导的胃癌细胞凋亡的影响及其分子机制.方法:通过脂质体将重组FHIT基因PRC/CMV质粒和空载体转染到人胃癌细胞MKN-28,Western blot法检测外源性FHIT蛋白的表达;使用不同浓度的长春新碱分别处理各组细胞,MTT法分析细胞增殖,流式细胞术检测细胞的凋亡:Western blot法检测细胞Bcl-2和Bax的表达.结果:转染FHIT基因后,MKN-28细胞检测到FHIT蛋白的表达;长春新碱处理48 h后,转染FHIT基因组细胞、转染空载体组细胞及未转染组细胞的凋亡率分别是30.967%±2.122%、11.033%±1.724%、10.733%±1.021%,转染FHIT基因组细胞凋亡更明显(F=142.045,P<0.05);转染FHIT基因组细胞经长春新碱处理后Bcl-2蛋白表达减少,Bax蛋白表达增加.结论:外源性FHIT基因表达可以增强长春新碱诱导的胃癌细胞凋亡,其机制可能与凋亡相关蛋白Bcl-2和Bax表达有关.     

Ad-p27mt转染重组腺病毒治疗裸鼠内人胃癌的分子机制

目的:研究Ad-p27mt转染重组腺病毒对人胃癌细胞凋亡的作用及机制.方法:Ad-p27mt转染重组腺病毒导入胃癌细胞株SGC-7901内;流式细胞仪检测凋亡染色体亚二倍体峰值,了解Ad-p27mt对人胃癌组织凋亡的作用;TUNEL法检测DNA片断,在Ad-p27mt组和Ad-LacZ组中分析细胞的凋亡.结果:Ad-p27mt成功转入人胃癌细胞SGC-7901内,转化率达100%.流式细胞仪检测发现在感染后18h出现G1-S相前出现凋亡染色体亚二倍体峰值,并且DNA电泳出现凋亡特征性的条带;TUNEL法检测Ad-p27治疗组与对照组的凋亡率分别是92.3%±3.76%和2.01%±0.15%,两组的差异有显著性(P<0.01).结论:重组腺病毒转染的人p27突变基因能诱导裸鼠体内人胃癌细胞SGC-7901的凋亡.     

Effect of trans-retinoic acid and folic acid on apoptosis in human gastric cancer cell lines MKN-45 and MKN-28

Induction of apoptosis has been implicated as an anticarcinogentic mechanism of both folic acid and retinoic acid. The ability of retinoic acid or folic acid to induce gastric cancer cell apoptosis was investigated in the human gastric cancer cell lines MKN-45 and MKN-28, and DNA fragmentation was studied in situ by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and DNA agarose gel electrophoresis. The rates of apoptosis in both the poorly differentiated MKN-45 and the well differentiated MKN-28 cell line were less than 5% after treatment with either retinoic or folic acid. Apoptosis may be induced by the administration of retinoic acid or folic acid, and the apoptosis indices of MKN-45 and MKN-28 cells were related to the doses of these drugs. The induction of gastric cancer cell apoptosis may play a role in the anticarcinogenetic effect of retinoic acid and folic acid, both of which are potential agents for the treatment of human gastric cancer. Received Sept. 16, 1997; accepted Mar. 17, 1998     

Docetaxel plus 5-fluorouracil for terminal gastric cancer patients with peritoneal dissemination

BACKGROUND/AIMS: There are few reports on chemotherapy for poor prognosis terminal patients with peritoneal dissemination of gastric cancer, especially in those with renal dysfunction, because of the possibility of severe toxicity. We conducted a study of the combination of docetaxel and 5-fluorouracil for the treatment of these patients to improve quality of life because of its low toxicity. METHODOLOGY: Five patients were treated in this study. All patients had a large volume of carcinomatous fluid in the abdomen, without liver or distant metastasis. The respective doses of docetaxel and 5-fluorouracil were 60 mg/m2 on day 1 and 370 mg/m2 on days 1 to 5 by intravenous infusion. Patients received this treatment 2-7 times every 2-3 weeks. RESULTS: Grade III/IV toxicity occurred, consisting of neutropenia (100%) and diarrhea (40%). No patients developed renal dysfunction. Carcinomatous fluid volume diminished for 15-96 days. Quality of life questionnaire score and performance status scale was significantly improved from 75-86 to 51-61 (p = 0.041) and 3-4 to 0-1 (p = 0.039), respectively. All patients were able to leave hospital after this treatment. Four of five patients died and median survival time was 223 days. Two of five patients achieved partial response and three patients showed no response (response rate 40%). CONCLUSIONS: This new combination therapy had benefit for terminal patients with peritoneal dissemination of gastric cancer.     

Adenovirus mediated p53 tumour suppressor gene therapy for human gastric cancer cells in vitro and in vivo

BACKGROUND/AIMS: Gastric cancer is one of the most prevalent forms of cancer in East Asia. Point mutation of the p53 gene has been reported in more than 60% of cases of gastric cancer and can lead to genetic instability and uncontrolled cell proliferation. The purpose of this investigation was to evaluate the potential of p53 gene therapy for gastric cancer. METHODS: The responses of human gastric cancer cell lines, MKN1, MKN7, MKN28, MKN45, and TMK-1, to recombinant adenoviruses encoding wild type p53 (AdCAp53) were analysed in vitro. The efficacy of the AdCAp53 treatment for MKN1 and MKN45 subcutaneous tumours in nude mice was assessed in vivo. RESULTS: p53-specific growth inhibition was observed in vitro in two of four gastric cancer cell lines with mutated p53, but not in the wild type p53 cell line. The mechanism of the killing of gastric cancer cells by AdCAp53 was found, by flow cytometric analysis and detection of DNA fragmentation, to be apoptosis. In vivo studies showed that the growth of subcutaneous tumours of p53 mutant MKN1 cells was significantly inhibited by direct injection of AdCAp53, but no significant growth inhibition was detected in the growth of p53 wild type MKN45 tumours. CONCLUSIONS: Adenovirus mediated reintroduction of wild type p53 is a potential clinical utility in gene therapy for gastric cancers.     

靶向ING1基因的miR-622真核表达载体在胃癌细胞MKN-45中的鉴定及其功能

目的:研究构建靶向ING1基因的miR-622真核表达载体并验证其转染人胃癌细胞株MKN-45细胞后对ING1基因的干扰效果及其功能.方法:将外源性重组真核表达载体pSuper/miR-622转染到人胃癌细胞株MKN-45内,经G418筛选并建立高表达miR-622的稳定转染胃癌细胞株.稳定表达该miR-622的胃癌细...     

黄芪、莪术配伍对胃癌细胞COX-2表达的调节作用

目的:观察黄芪、莪术配伍对MKN-45细胞COX-2、PPlAR-Y、NF-KB的影响.方法:以黄芪、莪术配伍作用于MKN-45细胞,并设塞莱希布组、罗格列酮组、黄芪组、莪术组和空白组相对照.用MTT法观察各组药物对胃癌细胞的抑制率,用RT-PCR以及Western blot方法检测给药后人胃癌细胞COX-2、PPARy、NF-KB基因及COX-2蛋白表达的变化.结果:各组对COX-2和NF-KB的mRNA表达均有抑制作用,除莪术外对PPARy mRNA均有促表达作用,其中以塞莱希布组和配伍组对COX-2 mRNA表达的抑制作用最为明显,且配伍组作用明显强于黄芪组和莪术组,以罗格列酮组和配伍组对NF-KB mRNA的表达抑制作用和对PPARy表达的促表达作用都是最明显.结论:黄芪、莪术配伍能起到增效作用,对细胞的抑制效应明显,对COX-2的抑制作用大于黄芪、莪术的单独效应,与塞莱希布相近,其对COX-2的抑制作用可能是通过PPARy/NFKB信号途径发挥作用的.     

Integrin alpha6beta4 as a suppressor and a predictive marker for peritoneal dissemination in human gastric cancer

BACKGROUND & AIMS: Because alterations of integrin expression in cancers contribute to cancer cell biology, we analyzed the association between the potential for peritoneal dissemination and integrin expression. METHODS: The dissemination potential of 10 human gastric cancer cell lines in mice with severe combined immunodeficiency (SCID) was compared with the expression of various integrins. The relationship between integrin expression and peritoneal dissemination was also investigated in surgically resected gastric cancer cases. RESULTS: The level of integrin beta4 subunit expression was inversely correlated with dissemination potential. Introduction of a full-length complementary DNA (cDNA) for beta4 subunit into cancer cells showing negligible beta4 subunit expression markedly suppressed peritoneal dissemination and inhibition of endogenous integrin alpha6beta4 by introduction of a cytoplasmic domain-deleted beta4 subunit cDNA into cells showing high expression of beta4 subunit promoted peritoneal dissemination. Apoptosis, which was histologically evident in peritoneal nodules of SCID mice, was induced in the cells with high beta4 subunit expression by attachment to laminin and stimulation with growth factors in vitro. An immunohistochemical study of specimens from 120 cases of primary gastric cancer showed that patients with beta4 subunit-positive tumors exhibited peritoneal dissemination only infrequently (P < 0.0001) and had a better outcome (P < 0.01). CONCLUSIONS: These results indicate that integrin alpha6beta4 is both a suppressor and a predictive marker for peritoneal dissemination in gastric cancer.     

丹参酮ⅡA磺酸钠对MKN-45胃癌裸鼠移植瘤增殖及血管生成的影响

目的:明确丹参酮ⅡA磺酸钠(TanⅡA)在体内的抗癌作用及其对肿瘤血管生成的影响.方法:建立MKN-45胃癌裸鼠移植瘤模型,随机分成对照组和低、中、高丹参酮ⅡA磺酸钠干预组,每组6只.干预组分别用TanⅡA磺酸钠1、5、10 mg/kg,ip,1次/d,连续14d.对照组等量生理盐水干预.2 wk后处死裸鼠,分离瘤块,观察抑瘤率;TUNEL法测凋亡指数:CD34标志法观察四组瘤块微血管密度(MVD);免疫组化法观察缺氧诱导因子HIF1α、血管内皮生长因子(VEGF)、COX2的蛋白表达.结果:与对照组相比,3组干预组凋亡指数AI明显上升(36.02%±3.41%,34.78%±3.41%,30.05%±3.41% vs 10.83%±0.92%,均P<0.05).低剂量组增殖减慢,抑瘤率为20.69%±1.79%;中、高剂量瘤重反而增加,高剂量组抑瘤率为-(21.4%±2.38%).与对照相比,3组干预组MVD、VEGF表达上调,尤以中高剂量组差异显著(8.11±1.011,10.01±0.89 vs6.56±0.42;63628±611,70957±684 vs 51056 ±410,均P<0.05);3组干预组COX-2表达与对照无明显差异;HIF1α在低剂量组表达略有下调,但未达到统计学差异,中高剂量组表达则有明显上调(29468±204,42227±214 vs15691±106,P<0.01或0.05)结论:丹参酮ⅡA体内的抗肿瘤效果没有体外细胞实验显著,高剂量丹参酮ⅡA的抗癌效应可被HIF的代偿高表达抵消甚至逆转.     

Effects of TS-1 on peritoneal dissemination of gastric cancer in nude mice

BACKGROUND/AIMS: We investigated the effects of TS-1 on the survival of nude mice developing peritoneal dissemination of gastric cancer. METHODOLOGY: MKN-45 cells were injected into the peritoneal cavity of nude mice and a model of peritoneal dissemination was developed. TS-1 was administered orally every day from day 1 to day 10 or day 10 to day 19. RESULTS: Survival time of these treatment groups was significantly longer than untreated controls. In a pharmacokinetic study, TS-1 was administered on day 10 and the 5-fluorouracil levels were retained and maintained for a longer time, in the ascites and tumor than in plasma. The area under the concentration curve for 5-FU in the tumor was higher, than in plasma or ascites. CONCLUSIONS: TS-1 could be effective in treating peritoneal dissemination of gastric cancer, due to the supply of 5-fluorouracil in the tumor by systemic and intraperitoneal circulation.