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.     

人端粒酶逆转录酶干扰增强肿瘤坏死因子相关的凋亡诱导配体诱导肝癌细胞凋亡的机制

目的探讨人端粒酶逆转录酶(hTERT)干扰增加肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导肝癌HepG2和SMMC 7721细胞凋亡的分子机制。方法采用膜联蛋白V/碘化丙锭染色的流式细胞术方法检测细胞凋亡;采用Western blot方法检测凋亡相关蛋白Procaspase-8、9、-3及Bax、Bcl-2和hTERT表达;采用端粒重复扩增法和端粒数量和长度测定法检测端粒酶活陛和端粒长度。结果hTERT干扰显著增加TRAIL诱导的肝癌细胞凋亡。100 ng/ml TRAIL作用24 h后,HepG2细胞凋亡率由5.53%增加至10.35%;SMMC 7721细胞凋亡率由14.73%增加至77.24%。hTERT干扰明显增加Procaspase-8、-9和Bcl-2表达,显著降低Bax表达,明显促进TRAIL作用后Procaspase-8、-9、-3活化,并且hTERT干扰后端粒酶活性显著降低,端粒长度明显缩短,然而对照细胞与未转染细胞相比各指标均无明显变化。结论hTERT干扰明显增加TRAIL诱导的肝癌细胞凋亡,其机制可能与Procaspase-8、-9表达增加,端粒酶活性降低和端粒长度缩短有关,而与Bcl-2和Bax表达无关。     

人参皂甙单体Rh2对人脑胶质瘤细胞U251增殖和凋亡的影响及其机制探讨

目的观察人参皂甙单体Rh2（GS-Rh2）对人脑胶质瘤U251细胞增殖和凋亡的影响,并探讨其机制。方法用GS-Rh2处理人脑胶质瘤U251细胞,采用MTT法检测U251细胞增殖抑制率,流式细胞仪和Annexin V-FITC/PI双染法观察U251细胞凋亡情况,TRAP-ELISA法检测U251细胞端粒酶活性,RT-PCR法检测U251细胞人端粒酶逆转录酶（hTERT）mRNA的表达。结果 5、10、20、40、80μg/ml GS-Rh2组U251细胞增殖抑制率分别为20.42%、32.19%、45.09%、57.37%、73.82%。根据细胞增殖抑制曲线计算出IC30、IC50、IC70值分别为9.7、25.2、68.4μg/ml。FITC和PI荧光双参数点图显示实验组细胞分布区域与对照组相比出现明显改变,随着药物浓度的增加,早期凋亡、晚期凋亡或坏死的区域细胞数量逐渐增多。9.7、25.2、68.4μg/ml GS-Rh2处理12、24、48、72 h后U251细胞端粒酶活性随GS-Rh2浓度增加和作用时间的延长而降低。浓度为25.2μg/ml GS-Rh2作用24、48、72 h的U251细胞hTERTmRNA与β-actin灰度值比为0.964 9±0.004 2、0.401 8±0.001 4、0.297 8±0.001 8,对照组为0.976 4±0.005 1。作用48、72 h U251细胞hTERT mRNA与β-actin灰度值比与对照组相比P均〈0.05;作用48、72 h者相比P〈0.05。结论 GS-Rh2能够诱导人脑胶质瘤U25l细胞凋亡,抑制U25l细胞增殖。其机制与其抑制hTERT基因表达,降低端粒酶活性有关。     

DNA损伤诱导胃癌细胞端粒酶活性和TRF2表达增高

目的:检测在化疗药引起胃癌细胞DNA损伤过程中端粒酶、TRF1和TRF2的表达.方法:用不同浓度足叶乙甙处理胃癌细胞SGC7901和MKN28,分别在3,6,12,24和36h进行检测.采用实时定量TRAP分析检测端粒酶活性;用实时RT-PCR检测hTERTmRNA表达;用Westernblot和实时RT-PCR检测TRF1和TRF2表达.结果:两种细胞端粒酶活性及TRF2mRNA表达均在药物处理的早期明显增高(P<0.05),且显示明显的药物浓度依赖性(P<0.05);TRF1表达稍增高,但无统计学意义(P>0.05).hTERTmRNA表达无明显改变(P>0.05).TRF2表达在蛋白和mRNA水平均增高(P<0.05).结论:端粒酶和TRF2参与化疗药引起的胃癌细胞DNA损伤反应.     

人端粒酶逆转录酶干扰对肿瘤坏死因子相关凋亡诱导配体诱导肝癌细胞凋亡的影响

目的研究人端粒酶逆转录酶(hTERT)干扰对肝癌．HepG2、SMMC-7221细胞生物学形为的影响和对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)诱导凋亡的影响。方法将HepG2细胞和SMMC-7721细胞分为转染组 (转染重组质粒真核表达载体)、对照组(转染空载体质粒)和未转染组。采用聚合酶链反应方法检测hTERT干扰序列, 逆转录聚合酶链反应方法检测hTERT表达,HE染色、生长曲线和流式细胞术方法分别检测细胞形态、增殖情况和细胞周期,β-半乳糖苷酶染色方法检测细胞状态,Armexin V／PI染色流式细胞术检测细胞凋亡。结果转染组细胞内均存在hTERT干扰序列,HepG2和SMMC 7221细胞hTERT干扰率分别为100％和43．3％;与未转染组细胞相比, 转染细胞核质比明显缩小,增殖率下降差异有统计学意义(P<0．05),老化细胞和G2-M期细胞明显增加(P<0．05)。细胞老化率分别由未转染组的0增加到转染组的20．4％,由3．60%,增加到10．O％;G2-M期分别由未转染组的7．1％、6．9％增加到转染组的10．6％、7．9％。hTERT干扰显著增加肝癌细胞凋亡和TRAIL诱导凋亡敏感性(P<0．05)。两株肝癌细胞凋亡率分别由未转染组的3．5％、4．8％增至转染组的5．2%、7．9％;100 ng／ml TRAIL作用24 h后两株肝癌细胞凋亡率分别由未转染组的5．3％、13．9％增加到转染组的10．4％、77．2％,而对照组细胞各指标均无显著变化。结论 hTERT干扰明显影响肝癌细胞的生物学行为,显著增加细胞凋亡和TRAIL诱导凋亡的敏感性。     

端粒酶逆转录酶激活脐静脉内皮细胞端粒酶活性

目的　探讨外源性端粒酶逆转录酶 (hTERT)能否激活人脐静脉内皮细胞端粒酶活性 ,为建立体外人脐静脉内皮细胞系奠定基础。方法　用逆转录病毒转染法 ,将编码hTERT片段逆转录病毒载体与VSV G共转染 2 93 GP2 细胞 ,产生病毒上清液 ,感染原代分离的人脐静脉内皮细胞 (hUVEC) ,以嘌呤霉素筛选。观察细胞生长曲线、第Ⅷ因子、CD34、β 半乳糖苷酶染色、逆转录 聚合酶链反应 (RT PCR)等指标。 结果　细胞生长曲线显示培养至 30代的转染细胞生长速度基本与原代培养脐静脉内皮细胞一致但快于原代培养传至第 10代内皮细胞 ;RT PCR结果转染细胞有特异扩增 ;衰老指标 β 半乳糖苷酶染色显示转染后细胞胞浆内着染阳性细胞数明显少于第 10代内皮细胞。结论　外源性hTERT转入原代培养人脐静脉内皮细胞 ,可重现其端粒酶活性 ,从而阻止人脐静脉内皮细胞老化     

Telomerase inhibition and telomere lossin BEL—7404 humen hepatoma cells treated with doxorubicin

AIM：To study the effects of doxorub icin on telomerase activity and telomere length in hepatocellular carcinoma.METHODS:Telomerase activity was assayed with a non-radioisotopic quantitative telomerase repeat amplification protocal-based method.The effect of doxorubicin(DOX) on the growth of BEL-7404 human hepatoma cells was the growth of BEL-7404human hepatoma cells was determined by microculture tetrazoloum assay.Mean telomere length(terminal restriction fragment)was detected by Southern blot method.The expression of telomerase subunits genes was investigated by RT-PCR,Cell apoptosis and cell cycle distribution were evaluated by flow cytometry.RESULTS:Telomerase activity was inhibited in a dose and time-depandent manner in BEL-7404human hepatoma cells treated with DOXfor24,48or72h in concentrations from 0.156to 2.5μMwhich was crrelated with the inhibition of cell growth.No changes were found in the mRNA expression of three telomerase subunits(hTERT,hTR and TP1)after drug exposure for 72h with indicated concentrations.The cells treated with DOX showed shortened mean telomer length and accumulated at he G2/M phase.However,there was almost no effects on cell apoptosis by DOX.CONCLUSION:The telomerase inhibition an d the telomere shortening by ODXmay contribute to its efficiency in the treatment in hepatocellular carcinoma.     

Antisense oligonucleotide targeting at the initiator of hTERT arrests growth of hepatoma cells

AIM:To evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT) on the growth of hepatoma cells.METHODS:The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with ashTERT at the concentration of 10μmol/L. After 72h, these cells were obtained for detecting growth inhibition,telomerase activity using the methods of MTT,TRAP-PCR-ELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline.Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA in vitro and in vivo.RESULTS: Growth inhibition was observed in cells treated with as-hTERT in vitro. A significant different in the value of A570-A630 was found between cells treated with as-hTERT and control (P&lt;0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced,the value of A4so nm was 0.42 compared to control (1.49) with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21.12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg in vivo and in vitro.CONCLUSION: As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors.     

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。     

Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell

AIM: To search for the biomarker of cellular immortalization, the telomere length, telomerase activity and its subunits in cultured epithelial cells of human fetal esophagus in the process of immortalization. METHODS: The transgenic cell line of human fetal esophageal epithelium (SHEE) was established with E(6)E(7) genes of human papillomavirus (HPV) type 18 in our laboratory. Morphological phenotype of cultured SHEE cells from the 6th to 30th passages, was examined by phase contrast microscopy, the telomere length was assayed by Southern blot method, and the activity of telomerase was analyzed by telomeric repeat amplification protocol (TRAP). Expressions of subunits of telomerase, hTR and hTERT, were assessed by RT-PCR. DNA content in cell cycle was detected by flow cytometry. The cell apoptosis was examined by electron microscopy (EM) and TUNEL label. RESULTS: SHEE cells from the 6th to 10th passages showed cellular proliferation with a good differentiation. From the 12th to the 16th passages, many senescent and apoptotic cells appeared, and the telomere length sharply shortened from 23kb to 17kb without expression of hTERT and telomerase activity. At the 20th passage, SHEE cells overcame the senescence and apoptosis and restored their proliferative activity with expression of telomerase and hTERT at low levels, but the telomere length shortened continuously to the lowest of 3kb. After the 30th passage cells proliferation was restored by increment of cells at S and G2M phase in the cell cycle and telomerase activity expressed at high levels and with maintenance of telomere length. CONCLUSION: At the early stage of SHEE cells, telomeres are shortened without expression of telomerase and hTERT causing cellular senescence and cell death. From the 20th to the 30th passages, the activation of telomerase and maintenance of telomere length show a progressive process for immortalization of esophageal epithelial cells. The expression of telomerase may constitute a biomarker for detection of immortalization of cells.