Cell Line QBC939 Research Articles

Effects of bile acids on expression of interleukin-6 and cell viability in QBC939 cell line

To research the effects of bile acids on the expression of interleukin-6 (IL-6) and the cell viability in QBC939 cell line. Human cholangiocarcinoma cells were stimulated with 800 µmol/L bile acid (CA), 100 µmol/L deoxycholate (DCA), 100 µmol/L chenodeoxycholic acid (CDCA), 1200 µmol/L gly acid (GCA), 200 µmol/L glycodeoxycholic acid (GDCA) and 300 µmol/L gly chenodeoxycholic acid (GCDCA).MTT assay and ELISA were used to detect the cell viability and the expression of IL-6 at 24 h, 48 h and 72 h. Treated by DCA, CDCA and GCDCA for 48 hours, the cell viability ratios changed to 0.61, 0.58 and 1.26, which were significant differences between control group and treated groups. And after 72 hours, the viability ratios of group CA, group DCA, group CDCA, group GCA, group GDCA and group GCDCA turned into 0.48, 0.50, 0.42, 1.29, 1.30 and 1.41. The differences of cell viability between bile acid-treated groups and control group were significant (P < 0.05). The expression of IL-6 in control group at 48 h and 72 h was (198 ± 32) ng/L and (323 ± 34) ng/L, while treated by CA, DCA, CDCA, GCA, GDCA and GCDCA respectively for 48 hours, the expression of IL-6 altered to (106 ± 33) ng/L, (88 ± 29) ng/L, (116 ± 54) ng/L, (413 ± 21) ng/L, (587 ± 32) ng/L and (366 ± 30) ng/L. After 72 hours, the expression of IL-6 of each bile acid-treated groups as above was (123 ± 66) ng/L, (45 ± 21) ng/L, (74 ± 45) ng/L, (792 ± 13) ng/L, (1310 ± 22) ng/L and (845 ± 18) ng/L, respectively. The differences between each bile acid-treated group and control group were significant (P < 0.05). Free bile acids (CA, DCA and CDCA) can inhibit the expression of IL-6 and the cell viability, while glycine conjugates (GCA, GDCA and GCDCA) can promote the expression of IL-6 and the cell viability. Bile acids can change tumor cell viability via IL-6 pathway.

Epithelial-Mesenchymal Transition Induced by Hepatitis C Virus Core Protein in Cholangiocarcinoma

Cholangiocarcinoma (CC) is associated with chronic hepatitis C virus (HCV) infection. We investigated the effect of hepatitis C virus core protein (HCVc) on epithelial-mesenchymal transition (EMT) in CC and tried to identify its target trigger. First, we examined expression of HCVc and epithelial and mesenchymal markers in CC tissues. Then we transient-transfected HCVc gene into a CC cell line and examined expression of lysyl oxidase-like 2 (LOXL2) and epithelial and mesenchymal markers by quantitative real-time polymerase chain reaction (PCR) and Western blotting. Finally, LOXL2 gene silencing was shown in QBC939/HCVc cells by RNA interference (RNAi), and we further examined expression of epithelial and mesenchymal markers by quantitative real-time PCR and Western blotting. Through immunohistochemical staining, the present study showed that HCVc is significantly associated with CC invasion and metastasis. In vitro study showed that HCVc expression induces EMT in CC cell line QBC939, and a mechanism through LOXL2 pathway is suggested. Expression of HCVc was significantly correlated with greater migratory and invasive potential of CC cells. These observations indicate that HCVc plays a critical role in promoting invasion and metastasis of CC cells.

Therapeutic effect of photodynamic therapy using hematoporphyrin monomethyl ether (HMME) on human cholangiocarcinoma cell line QBC939

Photodynamic therapy (PDT) is an effective local cancer treatment when aphotosensitizer is administered and the tumor is irradiated with light. We examined the effect of PDT using HMME as the photosensitizer, and the 630nm diode laser on human cholangiocarcinoma cell line QBC939. Cell viability was determined by MTT assay. The percentage of apoptotic cell was determined by flow cytometry following annexin V/PI staining. Two methods were used for the determination of apoptosis: terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay and laser scanning confocal microscope detection. The procaspase-3 and cytochrome cwere measured by western blot. In vitro PDT showed excellent cytotoxicity that was afunction of laser energy and drug concentration to the QBC939 cell lines. PDT-mediated cell death occurred predominantly by apoptosis in vitro. Furthermore, this treatment initiates early cytochrome crelease, followed by late procaspase-3 activation. Our study demonstrates that PDT using HMME and the diode laser induces apoptosis that is mediated by cytochrome crelease and caspase activation in human cholangiocarcinoma cell lines. It is expected that this therapy would be clinically useful for the treatment of patients with cholangiocarcinoma. Hematoporphyrin monomethyl ether, photodynamic therapy, apoptosis, cholangiocarcinoma.

Transfection of the wild-type xeroderma pigmentosum group D gene alters the biological behavior of human cholangiocarcinoma cell line QBC939

Transfection of the wild-type xeroderma pigmentosum group D gene alters the biological behavior of human cholangiocarcinoma cell line QBC939

Effect of XIAP gene interference on proliferation and apoptosis of human QBC939 cells

Objective To study the inhibitory effects of antisense oligodeoxynucleotide (ASODN) on X-linked inhibitor of apoptosis protein (XIAP) gene expression in QBC939 cell line,and the effects on proliferation and apoptosis of QBC939 cells. Methods ASODN was transfected into cell line QBC939 by LipofectamineTM 2000,and transfection efficiency at the 12th,24th,and 48th h after transfec-tion was observed by fluorescence microscopy. Proliferation inhibition rate, XIAP mRNA expression,cell cycle and apoptosis rate of QBC939 cells treated with 1. 11 μmol/L ASODN were assayed by MTT,RT-PCR and flow cytometry (FCM). Results Transfection efficiency was about 95%. In contrast with the control group,the inhibition rate of cell proliferation was about (27.63 ±1.15)% (24 h) and (42.95 ± 1.07) % (48 h) respectively after treatment with 1.11 μmol/L ASODN. The gene expression level of XI-AP in ASODN-transfected group was decreased by 23% (P<0.05). The apoptosis rate and the percent-age of QBC939 cells in G_0/G_1 were increased by 27. 34% and 9. 94% in ASODN-transfected group as compared with control group (P<0. 05). Conclusion The liposomal transfection of ASODN can down-regulate the expression of XIAP gene, which can inhibit proliferation and induce apoptosis of QBC939 cells. Key words: Carcinoma of bile duct; Gene therapy; Apoptosis

Expression of cancer stem cells markers CD24, ESA, CD44, and CD34 in human cancer of biliary tract

Objective To investigate the expression of cancer stem cells markers CD24,ESA, CD44, and CD34 in human cancer of biliary tract, and to definite whether these markers can be used to sort the cancer stem cells of the biliary tract cancer. Methods The expression rate of these cancer stem cell makers in cell lines and tissue of human cancer of biliary tract was detected by using immunohistochemistry and flow cytomytry. Results The protein expression of CD24, CD44, and ESA could be detected in both cell lines and the tissue of human cancer of biliary tract. The mean expression rate of CD24~+ CD44~+ESA~+ cells in eholangiocacinoma cell line QBC939 and carcinoma of gallbladder cell line GBC-SD was 86.1% and 92. 7% respectively, and that in tissue of cholangiocacinoma and gallbladder cancers was 0.62% and 0. 83% respectively. CD34 could not be detected in the biliary tract cancer. Conclusion CD24,CD44 and ESA are the markers of the pancreatic cancer stem cells and the breast cancer stem cells. The expression rate of these markers in human biliary tract cancer is similar to that in the pancreatic and breast cancers. CD24, CD44, and ESA may be the markers of human biliary tract cancer stem cells. Key words: Tumor stem cell; Biliary tract tumor; Marker

Influence of human cholangiocarcinoma cells on normal endothelial cells

Objective To study the influence of human cholangiocarcinoma cells on normal endothelial cells in a transwells co-culture system. Methods Normal endothelium cells and human cholangiocarcinoma cells of the line QBC939 transwells co-culture system were established. Normal endothelial cells cultured were used as control. Expression of F-actin, pp125FAK, MMP-2, MMP-9 and uPA was analyzed with im-munofluorescent method. Expression of the genes pp125FAK, MMP-2, MMP-9 and uPA was analyzed by real-time PCR and western blot. Results The expression of F-actin and the genes pp125FAK, MMP-2, MMP-9 and uPA in the ECs co-cultured with QBC939 cholangiocarcinoma cells were all up-regulated in com-parison with those in the controls (P<0.01). Conclusion Cholangiocarcinoma cells promote the expression of protcolytic enzyme of ECs by paracrine action and induce the changes of ECs cytoskeleton system. Key words: Bile duct neoplasms; Coculture; QBC939 cell line; Endothelial cell; Proteo-lyric enzyme; F-actin

The growth-inhibition effect of tamoxifen in the combination chemotherapeutics on the human cholangiocarcinoma cell line QBC939

In the individual application of adriamycin, mitomycin, vindesine and their combined application with tamoxifen for the pre-treatment of the human cholangiocarcinoma cell line QBC939, QBC939 was determined by MTT assay to investigate the inhibitive effect and its initial mechanism of TAM on cell growth. Growth cycle and apoptosis of each group were determined by flow cytometry. Concentration of ADM in QBC939 was detected by flow cytometry. The levels of their P-glycoprotein were detected by immunohistochemistry. The mRNA and protein levels of apoptotic-associated genes Bcl-2 and Bax were determined by western blot and real-time PCR. The inhibitive rates of adriamycin, mitomycin, vindesine to QBC939 and the apoptosis rates of QBC939 were enhanced after the pre-treatment of tamoxifen. Influence of tamoxifen in their growth cycle was not so obvious except vindesine group because of the increasing cell numbers of G (2)/M phase in which cells may be blocked. The contents of adriamycin in cells rose after the pre-treatment of tamoxifen. Expression level of the multi-drug resistant protein on cell surface was shown as (+). Furthermore, real-time PCR and Western blot analysis revealed an upregulation of Bcl-2 and a downregulation of Bax in QBC939 after the pre-treatment of tamoxifen. Therefore, tamoxifen may have the ability to enhance the relative sensitivity of QBC939 to chemotherapeutics.

Effects of α1-adrenergic receptor on the proliferation of cholangiocarcinoma cells.

Objective To investigate the correlation between α1-adrenergic receptor and the pathological behavior of cholangiocarcinoma, and the effects of norepinephrine (NE) on the proliferation of cholangiocarcinoma cell line QBC939. Methods Thirty-six samples of cholangiocarcinoma were resected in Southwest Hospital from August 2002 to March 2008. The expression of α1-adrenergic receptor in the 36 samples of cholangiocarcinoma tissue and 4 samples of normal bile duct tissue were detected by SABC technique. The proliferation of cholangio-carcinoma cell line QBC939 was detected after processing the cells with NE, phentolamine and prazosin. All the data were analyzed by chi-square test. Results The high positive expression rate of α1-adrenergic receptor was 68% (17/25) in patients with lymph node metastasis, which was significantly higher than 9% (1/11) in patients without lymph node metastasis (χ2=10.604, P<0.05). The high positive expression rate of α1-adrenergic receptor was 85% (11/13) in patients with middle and low positioned cholangiocarcinoma, which was significantly higher than 30% (7/23) in patients with hilar cholangiocarcinoma (χ2=9.753, P<0.05). NE promoted the proliferation of cholangiocarcinoma cell line QBC939 by stimulating the expression of α1-adrenergic receptor, and in a concentration-dependent manner. The proliferative effect was weakened as time passed by, and it was eliminated by phentolamine and prazosin. Conclusions The expression of α1-adrenergic receptor is diverse due to lymph node metastasis and the location of the tumor, α1-adrenergic receptor with high expression may play an important role in the proliferation and metastasis of cholangiocarcinoma. Key words: α1-adrenergic receptor; Bile duct neoplasms; Metastasis; Cell proliferation

Antiproliferation and apoptosis induced by tamoxifen in human bile duct carcinoma QBC939 cells via upregulated p53 expression

Tamoxifen (TAM) is a nonsteroidal antiestrogen that has been used in the treatment of breast cancer for over 30 years. Recently, it was shown that TAM also has efficacy on gastrointestinal neoplasms such as hepatocarcinoma and pancreatic carcinoma, and that the chemopreventive activities of TAM might be due to its abilities to inhibit cell growth and induce apoptosis. In the present study, we investigated the effects of tamoxifen on growth and apoptosis in the human bile duct carcinoma (BDC) cell line QBC939 using MTT assay, inverted microscopy, fluorescence microscopy, transmission electron microscopy, classic DNA fragmentation agarose gel electrophoresis assay, PI single- and FITC/PI double-staining flow cytometry, and Western blotting. Our data revealed that TAM could significantly inhibit growth and induce apoptosis in QBC939 cells. Increased expression of p53 was observed in TAM-treated cells, indicating that p53 might play an important role in TAM-induced apoptosis in QBC939 cells. These results provide significant insight into the anticarcinogenic action of TAM on BDC.

Study of inhibition of GABA on proliferation of cholangiocarcinoma cell line QBC939 and its mechanism

Objective To explore the effect of gamma-aminobutyric acid (GABA) on the growth, apoptosis and telomerase activity of Cholangiocarcinoma cell line QBC939. Methods Cholangiocarcinoma cell line QBC939 was cultured by routine method, and then treated with different concentrations of GABA (1-1000 μmmol/L). The proliferation, apoptosis and cell cycle of QBC939 cells was investigated by MTT, Flow cytometry and transmission electron microscopy. The telomer-ase activity of QBC939 cells was examined by modified PCR-ELISA assay. Radioimmunoassay was used to measure the intracellular cyclic adenosine monophosphate(cAMP) content. Results The dif-ferent concentrations of GABA inhibited the growth of QBC939 cells and promoted the apoptosis. The apop-totic rate of QBC939 cells was increased from 4. 8% to 28. 03%, which had significant difference (P<0.05). It had no effect for distribution of cell cycle. Cell nuclear condensation and apoptotic bodies were seen by transmission electron microscopy. Telomerase activity was inhibited by GABA(0. 82±0. 048 vs 0.56±0. 054, P<0.05). The content of intracellular cAMP was increased with the increase of GABA concentration in a dose-dependent manner [(0. 59±0. 049) nmol/L vs (0. 82±0. 033)nmol/L, P<0. 05]. Conclusion GABA can inhibit the proliferation of QBC939 cells by promoting apoptosis and inhibiting telomerase activi-ty, which may be mediated by the information transmission of post-receptor. Key words: Bile duct neoplasm; Gamma-aminobutyric acid; Cell proliferation; Apoptosis; Telomerase

Suppressing effects of down-regulating DNMT1 and DNMT3b expression on the growth of human cholangiocarcinoma cell line

Hypermethylation in the promoter region is an important epigenetic mechanism for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferases are considered to be the main cause of promoter hypermethylation. In order to explore the roles of two methyltransferase members (DNMT1 and DNMT3b) in the cholangiocarcinoma tumorigenesis, antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene was constructed respectively, and were co-transfected into the human cholangiocarcinoma cell line QBC-939 to observe their biological effects on the cell growth and proliferation ability, apoptosis, cell cycle alteration, and the tumorigenesis ability in the subcutaneous tissue of nude mouse. The results demonstrated that co-transfection with antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene and single transfection with antisense eukaryotic expression plasmid of DNMT1 gene can suppress the growth and proliferation of QBC-939, block the cell cycle at G1 phase, increase the apoptosis rate, minimize the tumor size in the subcutaneous tissue of nude mouse. The suppressing biological effect of co-transfection is stronger than single transfection with antisense DNMT1. Meanwhile, single transfection with antisense eukaryotic expression plasmid of DNMT3b gene has no effects on the biological characteristics of QBC-939. This study suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function in inactivation of tumor suppressor genes. Combination DNMT1 and DNMT3b will increase their biological effects and have the synergistic effect on suppressing the growth of human cholangiocarcinoma cell line QBC-939.

Effect of histone deacetylase inhibitor on proliferation of biliary tract cancer cell lines

To explore the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the growth of biliary tract cancer cell lines (gallbladder carcinoma cell line and cholangiocarcinoma cell line) in vivo and in vitro, and to investigate the perspective of histone deacetylase inhibitor in its clinical application. The survival rates of gallbladder carcinoma cell line (Mz-ChA-l cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) treated with various doses of TSA were detected by methylthiazoy tetrazolium (MTT) assay. A nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-l cell line) was successfully established, and changes in the growth of transplanted tumor after treated with TSA were measured. TSA could inhibit the proliferation of gallbladder carcinoma cell line (Mz-ChA-l cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) in a dose-dependent manner. After the nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-l cell line) was successfully established, the growth of cancer was inhibited in the model after treated with TSA. TSA can inhibit the growth of cholangiocarcinoma and gallbladder carcinoma cell lines in vitro and in vivo.

Effect of Bcl-2 antisense oligonucleotide on expression of Bcl-2 in human cholangiocarcinoma cell line QBC939

胆管癌对放疗、化疗不敏感,手术效果差,如何提高其治疗效果,一直是困扰临床医师的难题[1].作者既往的实验[2,3]显示胆管癌组织中Bcl-2高表达,胆管癌细胞株QBC939中Bcl-2阳性表达,Bcl-2反义寡核苷酸(ASODN)可抑制QBC939细胞增殖.本研究将Bcl-2 ASODN作用于人胆管癌细胞株QBC939,旨在探索Bcl-2 ASODN作用的分子机制,为胆管癌反义基因治疗提供实验依据。

Re-expression of RASSF1A by 5-Aza-CdR induced demethylation of the promoter region in human biliary tract carcinoma cells

Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 micromol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylation status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1 kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

Inhibitory effects of mediated PTEN gene in combination with L-OHP on human cholangiocarcinoma cell line

目的 探讨脂质体介导PTEN基因转染人胆管癌细胞(QBC939),联合化疗药物奥沙利铂(L-OHP),分别进行人胆管癌细胞体外培养和体内接种生长,观察分析该基因对胆管癌细胞生长的抑制情况,探索人类胆管癌的生物治疗的可行性和方法.方法将携带PTEN基因的真核表达载体pBP-PTEN和不含该基因的空载体.转胆管癌QBC939细胞,嘌呤霉素抗性筛选克隆、扩增培养.绘制细胞生长曲线及MTT细胞活性观察;利用免疫组化检测转染前后PTEN阳性表达率;透射电镜扫描观察转染前后及联合奥沙利铂后细胞的超微结构变化;流式细胞分析细胞的周期变化和细胞凋亡情况;体外细胞侵袭力抑制试验;裸鼠体内肿瘤生长,病理学及电镜扫描的观测.结果PTEN基因转染后QBC939细胞稳定表达,阳性表达率升高(P＜0.05);肿瘤细胞活性下降(P＜0.05),细胞周期G1～S期抑制,细胞凋亡率增加(P＜0.01);透射电镜细胞较成熟、分化好;细胞侵袭力明显抑制(P＜0.05);裸鼠体内肿瘤未转染组生长早,加入奥沙利铂后生长受抑制(P＜0.05),病理证实为腺癌.结论1.脂质体成功将PTEN基因转染人胆管癌QBC939细胞,并稳定表达.2.PTEN基因转染后细胞生长速度无明显变化;MTT实验活性细胞数有所下降.3.转PTEN基因后的胆管癌细胞超微结构变化显示线粒体增多,细胞较成熟、分化好;流式细胞议分析,细胞周期G1期被抑制,细胞凋亡多,侵袭力受到抑制;4.接种转PTEN基因裸鼠的体内成瘤显著抑制;6.转基因的生物治疗联合奥沙利铂(L-OHP)对人胆管癌细胞生长具有显著的抑制作用。

In vitro killing effects of adenovirus-mediated double suicide gene on human cholangiocarcinoma cell line QBC939

目的 观察腺病毒介导的胞嘧啶脱氨酶/单纯疱疹病毒-1-胸苷激酶(CD/TK)双自杀基因对人胆管癌细胞QBC939的体外杀伤作用.方法CD/TK克隆入穿梭载体成为pAdtrack-CMV-CD/TK,与骨架载体pAdeasy-1在细菌内同源重组为pAd-CD/TK,经Pac Ⅰ酶切,293细胞包装、扩增、纯化后,体外转染人胆管癌细胞QBC939,并给予前药5-FC或GCV,观察其体外杀伤效果.结果含CD/TK基因的重组腺病毒鉴定正确,扩增纯化后,病毒滴度为1×1011颗粒/ml.重组腺病毒对QBC939细胞在感染倍数(m.o.i)为100时的转染效率为90%,在m.o.i50感染时,0.1 mmol/L的5-FC及10μmol/L的GCV对QBC939细胞的杀伤率为80%,明显高于单用5-FC与GCV的效应.结论双自杀基因以腺病毒为载体对人胆管癌细胞转染效率高,体外杀伤效应明显.腺病毒介导的双自杀基因治疗有望成为治疗胆管癌的有效方法。

Effects of recombinant DNMT1 eukaryotic expression plasmids on mRNA expression level of DNMT1 gene and growth curve of cells in human cholangiocarcinoma cell line

目的 观察重组人DNMT1真核表达质粒转染对胆管癌细胞DNMT1基因mRNA表达水平和细胞生长曲线的影响.方法将构建好的正义和反义DNMT1真核表达质粒用脂质体转染入人胆管癌细胞株QBC-939,然后用G418进行筛选,得到阳性克隆细胞株后,用半定量RT-PCR检测DNMT1基因的mRNA水平的变化,用MTT法观察细胞生长曲线.结果正义DNMT1真核表达质粒能使QBC-939细胞中的DNMT1基因的mRNA水平增加,而反义DNMT1真核表达质粒则使其mRNA水平降低;正义质粒可使QBC-939细胞增殖速度加快,反义质粒使其增殖速度减慢.结论重组人DNMT1真核表达质粒转染能影响胆管癌细胞DNMT1基因的mRNA表达水平和细胞生长曲线。

944. Inhibiting Survivin Expression Enhances Trail-Induced Apoptosis in Human Hepatocellular Carcinoma

Background: The fact that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces profound apoptosis in a wide range of tumor cell lines but lacks cytotoxicity to mosnormal cells raises hope that TRAIL might become a novel potential anticancer agent. Our previous studies shown that hepatoma cells are not sensitive to TRAIL treatment . Overexpression of survivin reduced sensitivity of hepatoma cells to TRAIL.The aim of this study is to investigate whether tumor cells escape TRAIL-killing through survivin experssion and how to reverse this resistance. Methods: Human hepatoma cell lines (HepG2, SMMC 7721), cholangiocarcinoma cell line QBC939, no-small-cell lung cancer cell line A549, colorectal adenocarcinoma cell lines (SW480 and HCT-15), and malignant hematopoietic cell line Jurkat, were treated with or without TRAIL protein and survivin antisense oligodeoxynucleotide (ODN) in culture. Survivin mRNA levels in treated cells were detected by semiquantitative RT-PCR. Synchronization of hepatoma cells was used to test the sensitivity of hepatoma cells to TRAIL. Apoptosis and cell cycle were examined by flow cytometry. Cell viability and proliferation assay were evaluated by MTT. In vivo effects of TRAIL plasmid and /or survivin antisense ODN on tumor growth were investigated in a nude mouse HCC model of HepG2 cell grafts in the liver. Results: Survivin mRNA levels varied in cell lines evaluated and negatively correlated to TRAIL-induced apoptosis (r=|[minus]|0.856,p<0.05).

Effect of antisense MBD1 gene eukaryotic expression plasmid on expression of MBD1 gene in human biliary tract carcinoma cells

Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912 +/- 0.022 to 0.215 +/- 0. 017, and the protein level of MBD1 gene also decreased from (80.19 +/- 5.05) % to (35.11 +/- 4.05) %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group (P < 0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.