Inhibitory effects of antisense phosphorothioate oligodeoxynucleotides on pancreatic cancer cell Bxpc-3 telomerase activity and cell growth in vitro

AIM: To investigate the effect of telomerase hTERT gene antisense oligonucleotide (hTERT-ASO) on proliferation and telomerase activity of pancreatic cancer cell line Bxpc-3. METHODS: MTT assay was used to detect the effect of different doses of hTERT-ASO on proliferation of Bxpc-3 cell for different times. To study the anti-tumor activity, the cells were divided into there groups: Control group (pancreatic cancer cell Bxpc-3); antisense oligonucleotide (hTERT-ASO) group; and nosense oligonucleotide group decorated with phosphorothioate. Telomerase activity was detected using TRAP-PCR-ELISA. Cell DNA distribution was examined using flow cytometry assay. Cell apoptosis was observed by transmission electron microscope in each group. RESULTS: After treatment with 6 mmol/L hTERT-ASO, cell proliferation was inhibited in dose- and time-dependent manner. The telomerase activity decreased after treatment with hTERT-ASO for 72 h. Flow cytometry showed the cell number of G0/G1 phase increased from 2.7% to 14.7%, the cell number of S phase decreased from 72.7% to 51.0%, and a sub-Gl stage cell apoptosis peak appeared in front of G1 stage. CONCLUSION: Telomerase antisense oligodeoxy-nucleotide can inhibit the proliferation of pancreatic cancer cell line Bxpc-3 and decrease the telomerase activity and increase cell apoptosis rate in vitro.     

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.     

Development and characterization of multidrug resistant human hepatocarcinoma cell line in nude mice

INTRODUCTION Tumor cell drug resistance represents a signif icant obstacle to successful chemotherapy. Cells which have acquired resistance to one anti-tumor drug usually show resistance to other anti-tumor drugs[1]. This cellular resistance is known as m…     

RNAi特异性抑制FoxO3a对软脂酸诱导HepG2.2.15细胞凋亡的影响

目的:观察FoxO3a基因干扰对软脂酸诱导HepG2.2.15细胞凋亡的影响.方法:HepG2.2.15细胞分五组:mock组(加脂质体)、FoxO3a siRNA组、FoxO3a siRNA+软脂酸组、阴性siRNA对照组、阴性siRNA+软脂酸组;Western blot法检测细胞的FoxO3a蛋白表达水平.MTT...     

肝癌细胞对5-氮杂-2'-脱氧胞苷的敏感性与细胞总DNA甲基化水平无关

目的 比较不同肝癌细胞株对5-氮杂-2'-脱氧胞苷(5-aza-dC)的敏感性,探讨肝癌细胞对5-aza-dC的敏感性是否与细胞总DNA甲基化水平有关.方法 用不同剂量(0.5、5.0、10.0μmol/L)的5-aza-dC处理肝癌细胞株(HepG2、QGY7701和HepG2.2.15细胞)及正常肝细胞株L02,比较不同浓度处理前后的细胞增殖抑制率,比较10 μmol/L 5-aza-dC处理前后的Caspase-3活性及细胞DNA片段化水平(5-溴脱氧尿嘧啶核苷掺入率),比较不同细胞总DNA甲基化水平.组间检测结果比较采用t检验.结果 5-aza-dC对HepG2、QGY7701、HepG2.2.15、L02细胞的半数抑制浓度分别为0.5、0.5、4.5、11.4μmol/L,与HepG2细胞和QGY7701细胞相比,HepG2.2.15绌胞和L02细胞对5-aza-dC不敏感.HepG2和QGY7701细胞中Caspase-3的活性升高较L02和HepG2.2.15细胞明显(P值均＜0.05),QGY7701细胞中5-溴脱氧尿嘧啶核苷掺入率升高较L02细胞明显(P＜0.05).L02、HepG2、QGY7701和HepG 2.2.15细胞的DNA总甲基化水平分别为11.7%±0.9%、10.9%±1.3%、11.7%±1.7%和12.2%±1.0%,差异无统计学意义(P值均＞0.05).结论 细胞对5-aza-dC的敏感性与细胞总DNA甲基化水平无关.     

Inhibitory effect of antisense oligodeoxynucleotides complementary to HBVon HepG2.2.15 cell line

AIM To explore the therapeutic potential of antisense oligodeoxynucleotides on hepatcellular cacinoma(HCC).METHODS Four antisense phosphorothioated oligodeoxynucleotides (asON), complementary to differentsites of HBV, were synthesized and assayed for their anti-HBV activity in HepG22. 2.15 cells with ELISA.The most effective asON was chosen for the following study: FACSCAN, TRAP and immuno-staining wereused respectively for checking apoptosis, telomerase activity and expression of oncogene p21ras and p62C-myc inHepG2.2.15 cells after treated by asON.RESULTS The oligomer directed against the initiator of pre-S2 was the most effective one with aninhibitory rate of 66% on HBsAg and 91% on HBeAg (P<0.02). Two inhibitory peaks (bimodal)appeared. Telomerase activity as well as the expression of p21fas and p62C-myc decreased drastically 3 days afterasON-HBpreS2 treatment. Meanwhile, apoptosis appeared in the experiments.CONCLUSION The inhibitory effects of as-preS2 on the HBV gene expression and the reversion of somemalignant behaviour in HepG2.2.15 cells were the significant, effective therapy against HBV infection andhepatocellular carcinoma.     

槲芪癥消汤对人肝癌细胞株HepG2.2.15增殖和凋亡的影响

目的:研究槲芪癥消汤对体外培养的人肝癌细胞株HepG2.2.15的增殖、凋亡、迁移能力、细胞内p53基因及蛋白表达量的影响,揭示该方可能的抗肿瘤机制。方法:应用不同浓度的槲芪癥消汤处理细胞,MTT法测定细胞增殖抑制率,Transwell检测细胞侵袭能力、流式细胞术检测细胞凋亡情况、rt-qPCR及Western blot检测细胞内p53的RNA及蛋白的表达变化。结果:槲芪癥消汤对HepG2.2.15细胞增殖有明显的抑制作用,使细胞的侵袭能力明显下降,并可促进细胞凋亡,同时细胞内p53的RNA及蛋白表达量明显增加,与对照组比较,差异均有统计学意义(均P<0.05)。结论:槲芪癥消汤能抑制肝癌HepG2.2.15细胞的增殖、侵袭,促进其凋亡,可能与促进细胞内的p53基因表达有关。     

Effects of allicin on both telomerase activity and apoptosis in gastric cancer SGC-7901 cells

AIM: To investigate bhe effects of allicin on both telomerase activity and apoptosis in gastric cancer SGC-7901 cells.METHODS: The gastric cancer SGC-7901 adenocarcinoma cells were treated with allicin and the cell cycle, inhibitory rate, apoptosis, telomerase activity and morphoiogic changes were studied by MTT assay, flow cytometry (FCM), TRAP-PCR-ELISA assay, light microscope, electron microscope respectively. Results were compared with that of AZT (3′-Azido-3′deoxythymidine).RESULTS: SGC-7901 cells were suppressed after exposure to allicin of 0.016 mg/ml, 0.05 mg/ml, and 0.1 mg/ml for 48 h. Compared with the control, the difference was significant (P&lt;0.05). Allicin could induce apoptosis of the cells in a dose-dependent and non-linear manner and increase bhe propo~on of cells in the G2/M phase. Compared with the control, the difference was significant in terms of the percentage of cells in the G2/M phase (P&lt;0.05). Allicin could inhibit telomerase activity in a time-dependent and dose-dependent pattern. After exposure to allicin at 0.016mg/ml for 24 hours, SGC-790t cells showed typical morphologic change.CONCLUSION: Allicin can inhibit telomerase activity and induce apoptosis of gastric cancer SGC-7901 cells. Allicin may be more effective than AZT.     

端粒酶检测在良恶性腹水鉴别诊断中的价值

目的探讨脱落细胞中端粒酶活性在良恶性腹水鉴别诊断中的价值。方法应用端粒酶TRAP-PCR-ELISA法分别检测60例腹水患者脱落细胞中的端粒酶活性,并与细胞学及相关肿瘤标志物进行对比分析。结果恶性腹水中的端粒酶活性水平明显高于良性腹水,在良性腹水中端粒酶活性的阳性率为10%,明显低于恶性腹水中端粒酶活性的阳性率90%,同时恶性腹水中端粒酶活性的阳性率同脱落细胞病理学检查、腹水综合指标测定(癌胚抗原、甲胎蛋白等)以及乳酸脱氢酶腹水/血清检测结果相比较,差异亦具有显著性(P<0·01)。结论脱落细胞中端粒酶活性检测可作为良恶性腹水的鉴别诊断标志。     

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。     

丙型肝炎病毒核心区蛋白对 HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响

目的　研究丙型肝炎病毒核心区 (HCV C)蛋白对肝癌细胞HepG2细胞周期、细胞凋亡和细胞端粒酶活性的影响。方法　首先运用基因重组技术构建含有HCV C基因的真核表达质粒pcDNA3.1( ) ,然后利用脂质体介导将重组真核表达质粒转染HepG2 ,经G4 18筛选获得稳定转染HepG2细胞 (HCV C转染HepG2细胞 ) ,经逆转录 聚合酶链反应技术 (RT PCR)和间接免疫荧光法证实其中有HCV C蛋白表达。然后进行如下实验 :( 1)利用四甲基偶氮唑蓝比色 (MTT)法检测HCV C转染HepG2细胞、空白质粒转染HepG2细胞和未转染HepG2细胞的生长增殖率 ;经流式细胞术(FACS)检测 3组细胞的细胞周期 ;( 2 )经流式细胞术检测细胞凋亡率 ;( 3)经端粒重复扩增 酶联免疫吸附试验 (TRAP ELISA)法检测上述 3组细胞端粒酶活性表达情况。结果　 ( 1)HCV C转染HepG2细胞增殖率显著高于空白质粒转染HepG2和未转染HepG2细胞增殖率 ;HCV C转染HepG2细胞S期所占百分率高于未转染HepG2细胞S期所占百分率 ;( 2 )HCV C转染HepG2细胞凋亡率显著低于无HCV C转染细胞凋亡率 ;( 3)上述 3组细胞端粒酶活性之间差异无显著性。结论　 ( 1)HCV C蛋白具有抑制细胞凋亡的作用 ;( 2 )HCV C蛋白促进HepG2从G0 /1期进入S期 ,从而可能促进细胞生长增殖 ,抑制细胞凋亡 ;( 3)HCV     

HepG2与HepG2．2．15细胞中P53表达及活性的比较

目的探讨HBV对抑癌基因P53表达及活性的影响。方法选用HepG2及转染了HBV表达质粒的HepG2．2．15细胞，采用Western blotting法检测两种细胞P53的表达状况；磷酸钙法将报告基因质粒PG13-CAT和P21-LUC分别转染细胞，通过检测报告基因的表达观察各细胞中P53的活性。结果HepG2．2．15细胞中P53蛋白表达水平高于HepG2细胞，而且两种报告基因的表达在HepG2．2．15细胞中也较高。结论HBV在肝癌细胞内的复制对P53的表达及功能具有一定的增强作用。     

VP22 fusion protein-based dominant negative mutant can inhibit hepatitis B virus replication

AIM: To investigate the inhibitory effect of VP22 fusion protein-based dominant negative (DN) mutant on Hepatitis Bvrus (HBV) replication. METHODS: Full-length or truncated fragment of VP22 was fused to C terminal of HBV core protein (HBc), and subcloned into pcDNA3.1 (-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the supernatant HBsAg concentration determined by RIA and HBV-DNA content by fluorescent quantification-PCR (FQ-PCR). Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry. RESULTS: VP22-based DN mutants and its truncated fragment were expressed in HepG2.2.15 cells, and had no toxic effect on host cells. DN mutants could inhibit HBV replication and the transduction ability of mutant-bearing protein had a stronger inhibitory effect on HBV replication. DN mutants with full length of VP22 had the strongest inhibitory effect on HBV replication, reducing the HBsAg concentration by 81.94%, and the HBV-DNA content by 72.30%. MTT assay suggested that there were no significant differences in cell metabolic activity between the groups. CONCLUSION: VP22-based DN mutant can inhibit HBV replication effectively.     

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.     

肝脏肿瘤细胞诱导T淋巴细胞PD-1表达及功能研究

目的研究肝脏肿瘤细胞系细胞HepG2细胞与HepG2．2．15细胞对T淋巴细胞程序性死亡分子1（PD-1）表达的影响及PD-1的作用。方法HepG2细胞、HepG2．2．15细胞分别和Jurkat细胞共同培养后,标记流式抗体,流式细胞检测仪检测Jurkat细胞PD-1的表达,ELISA检测并比较阻断组与对照组培养上清中细胞因子含量,单核细胞直接细胞毒性测定法检测并比较阻断组与对照组T淋巴细胞杀伤能力即OD值的改变。结果肝脏肿瘤细胞能诱导T淋巴细胞PD-1的表达,共培养2d时表达率分别为16．17％±2．5％（HepG2细胞）和17．43％±2．2％（HepG2．2．15细胞）;阻断组培养上清中IL-2、IFN-γ、IL-10浓度分别为202．9±53．0pg/ml、88．6±4．6pg/ml和63．7±13．4pg／ml,均明显高于对照组（102．9±53．0pg/ml、39．3±4．2pg/ml和34．6±13．7pg/m1）,P〈0．05;阻断组Jurkat细胞对HepG2．2．15细胞杀伤的OD值为0．29±0．06,明显高于对照组（0．19±0．09）,P〈0．05。结论肝脏肿瘤细胞能诱导T淋巴细胞PD-1的表达;阻断PD-1／PD—L1能提高T淋巴细胞分泌细胞因子的量和杀伤能力。     

Anti-HBV activity of TRL mediated by recombinant adenovirus

AIM: To investigate the inhibitive effect of hepatitis B virus (HBV)-TRL on HBV replication. METHODS: Based on previously constructed pcDNA3.1 (-)/TRL, TR, TRmut, HBV core protein (HBVc) and hEDN, interest gene sequences TRL, TR, HBVc and hEDN were inserted into adenovirus shuttle plasmid pDC316 respectively and co-transfected HEK293 cells with rescue plasmid pBHGIox(delta)El,3Cre to acquire RAd/TRL, TR, HBVc and hEDN. And then RAds were identified, amplified and the titers in HEK293 cells were determined. RAd/TRL and TR were named as the experimental groups, and others were control ones. After HepG2.2.15 cells were infected, RAd/TRL expression was identified by indirect immunofluorescence staining. Supernatant HBV-DNA content was determined by fluorescent quantification PCR. Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry. RESULTS: RAd vectors with distinct interest gene sequence were successfully constructed. Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of supernatant HBV-DNA content compared to RAd/TR (0.63±0.14 vs1.60±0.47, P= 0.0266, <0.05) and other control groups (0.63±0.14 vs8.50±2.78,8.25±2.26, 8.25±2.29, 8.50±1.51, 8.57±1.63, P<0.01). MTT assay suggested that there were no significant differences in cell metabolic activity between groups (P>0.05). CONCLUSION: The construction and expression of RAd/TRL has been achieved and it could inhibit HBV replication successfully, which has laid the foundation for further research on anti-HBV activity in vivo.