Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype

AIM: To investigate the inhibitory effect of specialized human telomerase antisense oligodeoxyribonucleotides on the growth of well (MKN-28), moderately (SGC-7901) and poorly (MKN-45) differentiated gastric cancer cell lines under specific conditions and its inhibition mechanism, and to observe the correlation between the growth inhibition ratio and the tumor pathologic subtype of gastric cancer cells. METHODS: Telomerase activity in three gastric cancer cell lines of variant tumor pathologic subtype was determined by modified TRAP assay before and after the specialized human telomerase antisense oligodeoxyribonucleotides were dealt with under specific conditions. Effect of antisense oligomer under specific conditions of the growth and viability of gastric cancer cell lines was explored by using trypan blue dye exclusion assay, and cell apoptosis was detected by cell morphology observation, flow cytometry and TUNEL assay. RESULTS: Telomerase activity was detected in well, moderately and poorly differentiated gastric cancer cell lines (the quantification expression of telomerase activity was 43.7TPG, 56.5TPG, 76.7TPG, respectively). Telomerase activity was controlled to 30.2TPG, 36.3TPG and 35.2TPG for MKN-28, SGC-7901 and MKN-45 cell lines respectively after treatment with human telomerase antisense oligomers at the concentration of 5 μmol/L, and was entirely inhibited at 10 μmol/L, against the template region of telomerase RNA component, whereas no inhibition effect was detected in missense oligomers (P<0.05). After treatment with antisense oligomers at different concentrations under specific conditions for 96 h, significant growth inhibition effects were found in MKN-45 and SGC-7901 gastric cancer cell lines (the inhibition ratio was 40.89% and 71.28%), but not in MKN-28 cell lines (15.86%). The ratio of inactive SGC-7901 cells increased according to the prolongation of treatment from 48 to 96 h. Missense oligomers could not lead to the same effect (P<0.05). Apoptosis of SGC-7901 and MKN-45 cells was detected not only by morphology and TUNEL assay but also by flow cytometry. The apoptotic rate reached 33.56% for SGC-7901 cells and 44.75% for MKN-45 cells. CONCLUSION: The viability and proliferation of gastric cancer cells can be inhibited by antisense telomerase oligomers. The growth inhibition of gastric cancer cells is correlated with concentration, time and sequence specialty of antisense oligomers. The inhibition mechanism of antisense human telomerase oligomers depends not only on the sequence specialty but also on the biological characteristics of gastric cancer cell lines.     

端粒酶hTR反义寡核苷酸对K562细胞端粒酶活性的影响

目的：探讨端粒酶RNA反义寡核苷酸（antisense oligodeoxynucleotide,ASODN)对人白血病K562细胞端粒酶活性及细胞生长的影响。方法：应用与人端粒酶RNA组分模板区互补的硫代ASODN处理K562细胞，用端粒酶重复扩增分析（telomere repeat amplification protocol,TRAP)-PCR-ELISA检测法观察端粒酶活性的变化；用Annexin V分析法及流式细胞术检测凋亡细胞。结果：经ASODN作用后，细胞端粒酶活性明显受到抑制，并与ASODN的浓度和处理时间相关。作用5d后，凋亡细胞明显增加。结论：端粒酶RNA模板区ASODN可明显抑制白血病K562细胞端粒酶活性，抑制细胞生长和增殖，诱导细胞凋亡，可能成为白血病基因治疗新靶点。     

胰腺癌MiR-224的表达与细胞增殖和凋亡

目的:分析miR-224在胰腺癌组织中的表达,探讨其在胰腺癌细胞增殖、细胞周期及凋亡中的意义.方法:采用TagMan MGB探针法定量分析40例原发性胰腺癌及对应的癌旁组织MiR-224的表达;利用反义技术降低胰腺癌细胞(Aspc-1和Bxpc-3)中miR-224的表达;采用MTT比色法检测细胞增殖的改变,利用流式细胞技术检测胰腺癌细胞周期和凋亡情况.结果:在40例胰腺癌病例中,43%(17/40)的胰腺癌组织miR-224表达明显高于癌旁组织(P<0.05);反义miR-224转染胰腺癌细胞Aspc-1和Bxpc-3后,miR-224的表达明显降低,Aspc-1和Bxpc-3胰腺癌细胞生长受到明显抑制,其生长主要停滞在G0/G1期,而S期和G2/M期细胞的比例下降;另外降低miR-224的表达,Aspc-1和Bxpc-3胰腺癌细胞早期凋亡明显增加.结论:miR-224在胰腺癌组织中表达上调,降低其表达能明显抑制Aspc-1和Bxpc-3细胞的生长和诱导细胞早期凋亡增加,miR-224有可能成为胰腺癌基因表达调控的新靶点.     

顺铂对食管癌细胞周期及端粒酶活性的影响

目的 研究顺铂对食管癌细胞周期和端粒酶活性的影响，为其临床应用提供理论依据。方法 用2μg/ml顺铂处理食管癌EC9706细胞，流式细胞仪分析细胞周期的改变，TRAP—ELISA法检测细胞端粒酶活性的改变。结果 顺铂可使食管癌细胞阻滞于S期，诱导细胞凋亡；同时降低食管癌端粒酶活性。结论 顺铂对食管癌有治疗作用，端粒酶活性可作为观察食管癌化疗疗效的一个指标。     

脂质体介导的反义寡核苷酸抑制人胃癌细胞端粒酶活性

目的：探讨特异性反义寡脱氧核苷酸（ａｎｔｉｓｅｎｓｅ ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅｏｔｉｄｅ,ＡＳＯＤＮ）对人胃癌细胞端粒酶活性的影响。方法应用脂质体包裹与端粒酶ＲＮＡ模板区互补的ＡＳＯＤＮ,处理人胃癌细胞系ＦＧＣ８５。用端粒重复扩增分析（ｔｅｌｏｍｅｒｅ ｒｅｐｅａｔ ａｍｐｌｉｆｉｃａｔｉｏｎ ｐｒｏｔｏｃｏｌ,ＴＲＡＰ）－ＰＣＲ－ＥＬＩＳＡ检测法观察端粒酶活性的变化,结果经脂质体包裹ＡＳＯ     

端粒酶反义寡聚脱氧核苷酸诱导人胃癌细胞凋亡过程加端粒酶活性的测定

目的：观察端粒酶反义寡聚脱氧核苷酸对人胃癌细胞株的作用。方法：用端粒酶反义寡聚脱氧核苷酸与人胃癌细胞株共同孵育一定时间后,观察细胞形态变化,观察细胞DNA含量的分布,测定端粒酶活性。结果：端粒酶反义寡聚脱氧核苷酸能诱导人胃癌细胞凋亡,抑制端粒酶活性。在形态学上表现为细胞膜起泡、染色质固缩、核碎裂、凋亡小体形成。电冰呈凋亡特征性Ladder带;流式细胞仪分析显示,在G1期前出现亚2倍体凋亡峰;端粒酶活性抑制。结论：端粒酶反义寡聚脱氧核苷酸能诱导胃癌细胞凋亡,抑制端粒酶活性,抑制端粒形成,从而抑制胃癌细胞的生长。     

端粒酶逆转录酶基因反义寡核苷酸抑制肺癌细胞端粒酶活性和诱导细胞凋亡

目的 研究端粒酶逆转录酶（hTERT)基因反义寡核苷酸（ASODN）对肺癌细胞端粒酶活性和细胞凋亡的影响。方法 实验分为ASODN组、正义寡核苷酸（SODN）组和空白对照组,所用ASODN和SODN的浓度分别为10μmol/L,脂质体为16mg/L,采用端粒重复扩增法、逆转录－聚合酶链反应、Western Blot及流式细胞术分别观察各组端粒酶活性、hTERP mRNA和蛋白质表达以及细胞凋亡。结果 ASODN组显著下调或抑制肺癌细胞端粒酶活性和hTERT表达,但直到第21天才出现细胞凋亡增多。结论 端粒酶活性与hTERT表达密切相关。     

Effect of antisense oligodeoxynucleotide of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer

AIM:To explore the effect of antisense oligocleoxynucletide (As-ODN) of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer.RESULTS:The telomerase activity in SW480 cells transfected with 1.0μmol/L of As-ODN for 2-5days, was significantly decreased in a time-dependent manner, and the cells underwent apoptosis.The missense ODN (Ms-ODN) and the control group transfected with SW480 cells did not show these changes.CONCLUSION:As-ODN can specifically inhibit the telomerase activity of SW480 cells and induce apoptosis.     

硫代修饰端粒酶反义核酸联合顺铂、阿霉素对胃癌细胞作用的研究

目的：探讨应用硫代修饰人端粒酶RNA（hTR)反义核酸后胃癌细胞对顺铂（DDP）和阿霉素（ADM）敏感性的变化。方法：采用脂质体将针对hTR模版区设计的13相碱基硫代磷酸修饰的反义寡核苷酸CAGTTAGGGTTAG导入胃癌细胞SGC7901，应用四甲基偶氮唑蓝（MTT）法，流式细胞仪和TRAP－PCR－ELISA法测定联合应用化疗药后对细胞增殖，凋亡和端粒酶活性的影响。结果：化疗药物ADM和DDP地端粒酶活性抑制作用不同，而且有明显依赖趋势。hTR反义PS－ODN可增加ADR和DDP抑制胃癌细胞系SGC7901端粒酶活性，诱导细胞凋亡和抑制细胞增殖的作用。结论：hTR反义PS－ODN在体外能增加胃癌细胞系SGC701对化疗药ADR、DDP敏感性，其机制可能与其抑制细胞端粒酶活性，诱导细胞凋亡有关。     

叠氮胸苷对胃癌端粒酶活性及细胞增殖的影响

目的探讨反转录酶抑制剂叠氮胸苷对端粒酶活性的影响及抑制端粒酶活性以控制胃癌生长的可能性。方法采用端粒重复片段扩增法（ｔｅｌｏｍｅｒｅｒｅｐｅａｔａｍｐｌｉｆｉｃａｔｉｏｎｐｒｏｔｏｃｏｌ,ＴＲＡＰ）和流式细胞术等检测胃癌细胞经叠氮胸苷处理前后的端粒酶活性和细胞增殖能力。结果经叠氮胸苷处理后,培养的胃癌细胞端粒酶活性较未处理组降低８４．１％,出现衰老形态的细胞,处理１４和２１天后生长抑制率分别为５９．３％和８０．９％,细胞存活分数明显降低,分别为０．５４和０．３３,并对细胞周期具有一定影响。结论叠氮胸苷可明显抑制胃癌细胞端粒酶活性,诱导细胞衰老和增殖能力的降低,临床上应用端粒酶抑制剂限制端粒扩充治疗胃癌可能具有广阔的前景。     

The Retrovirus-mediated Antisense Human Telomerase RNA (hTR) Gene Limits the Growth of Hepatocellular Carcinoma Growth in Cell Culture and Animals

Aim To investigate the inhibitory effect of retrovirus-mediated antisense human telomerase RNA (hTR) gene therapy on hepatocelluar carcinoma. Methods We first constructed the sense and antisense hTR vectors and then transfected these into HepG2 cells. Telomerase activity, cell growth curves, proliferating cell nuclear antigen expression (PCNA), cell cycle distribution, and cell apoptosis were detected by the means of telemere repeat amplification protocol (TRAP), MTT assay, immunofluorescence, flow cytometric analysis, and transferase-mediated nick end labeling (TUNEL), respectively. In order to further confirm the therapeutic effect of this gene therapy, we developed an experimental line of HepG2 tumor-bearing nude mice by and directly injected these with retrovirus expressing the antisense hTR gene. Tumor growth was determined by tumor volume, and cell apoptosis was analyzed by TUNEL. Results The antisense hTR gene was shown to be successfully integrated into the target cells’ genome. HepG2 cells transfected with the antisense hTR gene showed down-regulated telomerase activity, inhibited cell growth, decreased PCNA expression, and increased apoptotic rate. Moreover, flow cytometry revealed a decrease of cells in the S phase with cell cycle arrest at the G2/M phase. In the antisense hTR-treated group, tumor growth was significantly reduced and showed an increase of apoptotic cells. Conclusion The results indicate that the specific inhibitor of the hTR template is likely to be a very efficient tool for hepatocellular carcinoma research and may possess potential therapeutic significance in the future clinical practice.     

Inhibition of human telomerase in MKN—45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.     

多发性骨髓瘤中端粒酶的动态研究

目的研究端粒酶在多发性骨髓瘤(MM)发生发展中所起的作用及探讨端粒酶与细胞周期的相关性。方法将38例不同阶段的MM患者分为癌前组(MGUS)、活动组(初治和复发难治)与稳定组(缓解并达到平台期),10例缺铁性贫血患者设为对照组。用流式细胞术(FCM)提纯CD13+8的MM细胞后,以荧光素标记的端粒重复序列扩增法检测端粒酶的表达,并通过FCM测定细胞周期。结果(1)MM活动组端粒酶阳性率为90.5%,显著高于对照组(10.0%)(P<0.01)。其中初治MM(84.6%)与复发难治MM(100.0%)之间差异无统计学意义(P>0.05)。MM稳定组端粒酶阳性率为13.3%,与对照组相比差异无统计学意义(P>0.05),与活动组差异有统计学意义(P<0.01)。癌前组2例MGUS端粒酶表达均为阳性;(2)细胞周期测定显示端粒酶表达阳性的MM细胞S期比例为(18.78±8.02)%,阴性为(5.64±4.03)%,两者差异有统计学意义(P<0.05)。结论(1)端粒酶对MM的发生发展具有重要意义,可作为监测病情发展、评价疗效及预测预后的指标;(2)端粒酶阳性的细胞更多处于增殖期,端粒酶和细胞周期调控存在相关性。     

Relationship between telomerase activity and its subunit expression and inhibitory effect of antisense hTR on pancreatic carcinoma

AIM: To directly investigate the relationship between telomerase activity and its subunit expression and the inhibitory effect of antisense hTR on pancreatic carcinogenesis. METHODS: We examined the telomerase activity and its subunit expression by cell culture, polymerase chain reaction (PCR), PCR-silver staining, PCR-ELISA, DNA sequencing, MTT and flow cytometry methods. RESULTS: PCR-silver staining and PCR-ELISA methods had the same specificity and sensitivity as the TRAP method. Telomerase activity was detected in the extract of the 10(th),20(th) and 30(th) passages of P3 cells,while it was absent in fibroblasts. Furthermore, after the 30th generation, the proliferation period of fibroblast cells was significantly prolonged. Telomerase activity and hTERTmRNA were detected in two pancreatic carcinoma cell lines, but were found to be negative in human fibroblast cells. Telomerase activity and hTERTmRNA were tested in pancreatic carcinoma specimens of 24 cases. The telomerase activity was positive in 21 of the 24 cases (87.5 %), and the hTERTmRNA in 20 cases (83.3 %). In adjacent normal tissues positive rates were both 12.5 %. There was a significant difference between the two groups. This indicated a significant correlation between the expression level of telomerase activity and histologic differentiation, metastasis and advanced clinical stage of pancreatic carcinoma. Our findings showed that the expressions of hTR and TP1mRNA were not correlated with the activity of telomerase but the expression of hTERTmRNA was. After treatment with PS-ODNs, telomerase activity in P(3) cells weakened and the inhibiting effect became stronger with an increase in PS-ODNs concentration. There was a significant difference between different PS-ODN groups (P<0.05). Inhibition of telomerase activity occurred most significant with PS-ODN1.The results of the FCM test of pancreatic cancer P(3) cells showed an increase in the apoptotic rate with increasing PS-ODN1 and PS-ODN2 concentrations. CONCLUSION: The expression of telomerase activity has a significant relationship to carcinogenesis. A strong correlation exists between telomerase activity and hTERTmRNA expression. The up-regulation of hTERTmRNA expression may play a critical role in human carcinogenesis. The expression of telomerase activity and its subunit level in pancreatic carcinoma significantly correlate with the clinical stage of pancreatic carcinoma and hence, may be helpful in its diagnosis and prognosis. The anti-hTR complementary to the template region of hTR is sufficient to inhibit P3 cell telomerase activity and cell proliferation in vitro, and can lead to a profound induction of programmed cell death.     

全硫代反义寡核苷酸对乳腺癌MDA-MB-231细胞的影响

目的 本研究探讨全硫代反义寡核苷酸( PS-ASODN)对乳腺癌MDA-MB-231细胞端粒酶活性及细胞生长、迁移、侵袭的影响.方法 本实验将PS-ASODN经脂质体转染作用于乳腺癌MDA-MB-231细胞,将实验分为空白对照组、对照正义全硫代寡核苷酸(PS-SODN)组和不同剂量的PS-ASODN组;脂质体介导的PS-ASODN和PS-SODN作用于乳腺癌MDA-MB-231细胞后,分别采用四甲基偶氮唑蓝比色法(MTT)、酶联免疫吸附法(EUSA)、流式细胞术、Transwell小室迁移、侵袭实验检测细胞的体外增殖、端粒酶活性、细胞凋亡、细胞迁移、侵袭能力的影响.结果 终浓度为1、3及5 μmol/L的PS-ASODN对MDA-MB-231细胞端粒酶活性及细胞的增殖均有抑制作用,与空白对照组比较差异显著(P＜0.01),并呈一定剂量依赖性;ELISA法检测结果,1、3及5μmol/L的PS-ASODN对MDA-MB-231细胞作用72 h后端粒酶皆为阴性,表明端粒酶PS-ASODN能够抑制端粒酶活性,对照组端粒酶皆为阳性.1、3及5μmol/L的PS-ASODN作用24h可以明显抑制MDA-MB-231细胞的迁移和侵袭能力(P＜0.01).结论 PS-ASODN能有效抑制人乳腺癌MDA-MB -231细胞的生长、促进凋亡、抑制其侵袭和迁移能力,其机制可能是通过PS-ASODN降低端粒酶活性而实现.     

二甲双胍对人胰腺癌细胞迁移的抑制作用

目的:研究二甲双胍对胰腺癌细胞迁移的影响,并初步探讨可能机制.方法:体外培养人胰腺癌细胞株Bxpc-3,予二甲双胍进行干预作为实验组(M组),无药物组作为对照组(C组).MTT检测二甲双胍对Bxpc-3细胞存活率的影响,细胞划痕实验检测划痕愈合率,RT-PCR检测MMP-2、MMP-9 mRNA的表达,Elisa检测细胞培养上清液MMP-2、MMP-9蛋白的分泌量.结果:与对照组相比,MTT结果示二甲双胍可以抑制人胰腺癌细胞株Bxpc-3的增殖,并呈时间-浓度依赖性(F=8.991,124,114.61,P<0.01);划痕实验示二甲双胍干预组12、24、48h与对照组相比划痕愈合率显著下降(t=7.683,9.013,10.471,P<0.01);RT-PCR示二甲双胍干预组MMP-2、MMP-9mRNA的表达显著降低(t=16.563,28.494,P<0.01);Elisa示二甲双胍干预组MMP-2、MMP-9蛋白分泌明显下降(t=9.428,13.542,P<0.01).结论:二甲双胍能显著抑制人胰腺癌细胞株Bxpc-3的增殖及迁移,其主要机制可能与抑制MMP-2、MMP-9活性有关.