Human Cholangiocarcinoma Cell Line QBC939 Research Articles

TGR5 promotes cholangiocarcinoma by interacting with mortalin

Takeda-G-protein-receptor-5 (TGR5) is a G-protein-coupled receptor (GPCR) activated by bile acids, and mortalin is a multipotent chaperone of the HSP70 family. In the present study, TGR5 was detected by immunohistochemistry (IHC) in extrahepatic cholangiocarcinoma (ECC) specimens, and TGR5 expression in ECC tissues and adjacent tissues was compared. In vitro TGR5 was overexpressed and knocked down in human intrahepatic cholangiocarcinoma (ICC) cell line RBE and human extrahepatic cholangiocarcinoma (ECC) cell line QBC-939 to observe its effects on the biological behavior of cholangiocarcinoma (CC) cells, including proliferation, apoptosis and migration. In vivo xenograft model was constructed to explore the role of TGR5 in CC growth. Proteins that interacted with TGR5 were screened using an immunoprecipitation spectrometry approach, and the identified protein was down-regulated to investigate its contribution to CC growth. The present study demonstrated that TGR5 is highly expressed in CC tissues, and strong TGR5 expression may indicate high malignancy in CC. Furthermore, TGR5 promotes CC cell proliferation, migration, and apoptosis resistance. TGR5 boosts CC growth in vivo. In addition, TGR5 combines with mortalin and regulates mortalin expression in the CC cell line. Mortalin participates in the TGR5-induced increase in CC cell proliferation. In conclusion, TGR5 is of clinical significance based on its implications for the degree of malignancy in patients with CC. Mortalin may be a downstream component regulated by TGR5, and TGR5 promotes cholangiocarcinoma at least partially by interacting with mortalin and upregulating its expression. Both TGR5 and mortalin are positive regulators, and may serve as potential therapeutic targets for CC.

Autophagy Suppression Accelerates Apoptosis Induced by Norcantharidin in Cholangiocarcinoma

Norcantharidin is a cantharidin demethylated analog with antitumor effects in many tumors, including cholangiocarcinoma. Autophagy suppression is known to increase chemosensitivity in cholangiocarcinoma. This study aimed to determine whether autophagy suppression accelerates apoptosis induced by norcantharidin in human cholangiocarcinoma cells. The human cholangiocarcinoma cell line QBC939 was incubated in RPMI 1640 medium with or without norcantharidin. Autophagy was induced using HBSS media with Ca2+ and Mg2+ supported by 10mM HEPES or suppressed by treatment with 3-MA or transfection with siRNA against Atg5. The comparison was drawn between these conditions in mitochondrial membrane potential disturbance, the levels of reactive oxygen species (ROS), apoptotic proteins, and apoptosis. Cholangiocarcinoma cell apoptosis was accelerated by norcantharidin. Autophagy suppression up-regulated norcantharidin's pro-apoptotic effect, but autophagy induction weakened it. As apoptosis was accelerated, ROS production was up-regulated. Bax protein expression, cytochrome c levels and localization, mitochondrial membrane disturbance, and the levels of caspase-9, caspase-3, and cleaved PARP were higher when autophagy was suppressed, and all of those were down-regulated when autophagy was induced. To sum up, it was found that norcantharidin induced cholangiocarcinoma cell death, and autophagy suppression enhanced the pro-apoptotic action of norcantharidin, which appears to involve the mitochondrial apoptosis pathway activation and ROS generation.

Defensive Mechanism in Human Cholangiocarcinoma Cells Against Hypoxia by Cyanidin-3-glucoside Treatment

Abstract: Background and Objective: Cyanidin-3-glucoside (C3G) is one of the major bioactive compounds in anthocyanins, which have been shown to prevent a large spectrum of diseases. It has been reported that hypoxia is a common characteristic of many types of solid tumor and that the hypoxia-inducible factor 1α (HIF-1α) plays an important role in the process of angiogenesis and metastasis in cancer cells. The objective of this study was to explore the effects of C3G on HIF-1α activation in cholangiocarcinoma (CCA) cells. Materials and Methods: The human cholangiocarcinoma cell line QBC939 was used in this study. Hypoxic conditions were achieved by using the chemical hypoxia agent cobalt chloride or incubation at 1% O2. and MTT assay, Luciferase reporter assay, flow cytometric apoptosis assay, western blot, RT-PCR and immuno fluorescence methods were used to determine the C3G efficacy. Results: C3G markedly decreased the hypoxia-induced accumulation of HIF-1α reporter activity and protein expression in a dose-dependent manner and exerted a large apoptotic effect in CCA cells. Furthermore, C3G reduced expression of several genes downstream of HIF-1α, such as vascular endothelial growth factor and erythropoietin, without affecting the expression level of HIF-1α mRNA, thus indicating that C3G acts as an inhibitor of HIF-1α mRNA translation. Conclusion: This study provides a novel strategy to develop a low toxicity, high efficiency and multi-target natural active substance for the treatment of cancers.

Enhanced chemosensitivity of cholangiocarcinoma cell line QBC939 to gemcitabine induced by curcumin

Objective To investigate the effect of curcumin in enhancing chemosensitivity of cholangiocarcinoma cell line QBC939 to gemcitabine and its mechanism. Methods QBC939 cells were divided into control group, 5 μmol/L curcumin group, 0.625 μg/ml gemcitabine group and 5 μmol/L curcumin combined 0.625 μg/ml gemcitabine group. Methyl thiazol tetrazolium (MTT) assay was used to detect the proliferation inhibition effect, flow cytometry was used to detected the change of cell cycle and apoptosis rate. Expressions of nuclear factor-κB (NF-κB), Cyclin B1 and inhibitor of apoptosis proteins (IAP)-1 in different groups were detected by Western blotting. Results Compared with control group, curcumin group and gemcitabine group, the inhibitory rates of cells in curcumin combined gemcitabine group at 12, 24, 48 and 72 h were (10.00±0.02)%, (23.10±0.03)%, (33.23±0.01)%, (48.30±0.05)% respectively, which were significantly higher than those of control group and single medicaments treatment group (P=0.001, 0.000, 0.000, 0.000); besides, curcumin combined with gemcitabine blocked (28.75±3.23)% human cholangiocarcinoma cell line QBC939 in G2/M phase, which was significantly higher than those of curcumin group and gemcitabine group [(14.49±2.20)%, (11.82±2.10)%; P=0.000], the apoptosis rate in combined group was (13.64±3.40)%, significantly higher than those of control group, curcumin group and gemcitabine group [(4.53±0.56)%, (6.47±0.98)%, (9.87±1.12)%; P=0.000]; moreover, the relative expression levels of NF-κB and its downstream moleculor Cyclin B1 and IAP-1 proteins in curcumin combined with gemcitabine group were 0.300±0.001, 0.407±0.036, 0.475±0.032 respectively, significantly lower than those of control group, curcumin group and gemcitabine group (P=0.000, 0.001, 0.003). Conclusion Curcumin can enhance the sensitivity of human cholangiocarcinoma cell line QBC939 to gemcitabine through the inactivation of NF-κB signaling pathway. Key words: Cholangiocarcinoma cell; Curcumin; Gemcitabine; Cholangiocarcinoma cell line QBC939; Nuclear factor-κB

Mir-637 inhibits the proliferation of cholangiocarcinoma cell QBC939 through interfering CTSB expression.

To investigate the role of mir-637 on the proliferation, migration and apoptosis in human cholangiocarcinoma (CCA) cell line QBC939, and the impact of mir-637 on Cathepsin B (CTSB) expression. Expression of mir-637 and CTSB in CCA tissues and adjacent non-tumor tissues were measured by Real-time PCR. CTSB expression in human CCA cell lines was detected with or without mir-637 exogenous overexpression through fluorescent quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method. The efficiency of lentivirus-mir-637 vector infection, and the effect of mir-637 on the proliferation, migration ability, apoptosis and cell cycle of CCA cell were detected, respectively. The possibility of mir-637 targeting CTSB was also tested by bioinformatics method, correlation analysis and molecular biology method. Decreased mir-637 and increased CTSB expression were observed in CCA tissue compared with adjacent non-tumor tissues. CTSB expression in mir-637 exogenously overexpressed QBC939 cells decreased compared with negative control. Mir-637 overexpression caused significant decrease of proliferation and migration ability of QBC939 cell. In addition, mir-637 overexpression induced that the cell cycle was blocked in G0/G1 phase, and cell apoptosis rate increased significantly. mir-637 and CTSB play an important role in the proliferation and migration of CCA cells. Mir-637 could inhibit CTSB expression significantly, which in-turn down-regulates the proliferation, migration and invasion ability of QBC939 cell, and promotes apoptosis in QBC939 cell line.

The in vivo study of dihydroartemisinin inhibits the growth of human cholangiocarcinoma

Objective To investigate the effect and the potential mechanisms of dihydroartemisinin on the treatment of human cholangiocarcinoma cell line QBC939 xenograft in nude mouse.Methods A total of 18 animal models of nude mouse xenograft by human cholangiocarcinoma cell line QBC939 were established and randomly divided into three groups:the dihydroartemisinin group (DHA),the control group (saline),and the solvent control group (vegetable oil).The weight of tumor,the tumor inhibition rate,the expression of hypoxia-inducible factor (HIF)-1α,the microvessel density (MVD) and the apoptotic index (AI) in each group were compared and analyzed.Results The volume and the weight of xenograft tumor in DHA group [(1 979.63 ± 357.29) mm3 and (1.77 ± 0.23) g] were significantly lower than those in the control group [(2 827.69 ± 549.00) mm3 and (2.50 ± 0.65) g,P ＜ 0.05].The expression of HIF-1α in the experimental group (0.228 0 ± 0.043 9)was less than that in the control group (0.336 7 ± 0.089 6,P ＜ 0.05).MVD in the experimental group (11.06 ± 1.44) was less than that in the control group (17.44 ± 2.24,P ＜ 0.05).The AI in the experimental group [(13.18 ± 2.42) ％] was significantly greater than that in the control group [(3.28 ± 1.04) ％,P ＜ 0.05].Conclusion Dihydroartemisinin has an inhibitory effect on the tumor growth of human cholangiocarcinoma in nude mice,and the mechanism may be related to the induction of the apoptosis of tumor cells,and the inhibition of tumor angiogenesis. Key words: Cholangiocarcinoma; Dihydroartemisinin ; Hypoxia-inducible factor-1 α ; In vivo study

Expression of carcinoembryonic antigen-related cell adhesion molecule 6 in human hilar cholangiocarcinoma cell line QBC939 and its role in invasion and metastasis

Objective To explore the expression of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) in human hilar cholangiocarcinoma cell line QBC939 and its roles in invasion and metastasis.Methods Real-time polymerase chain reaction (real-time PCR) was used to detect the expression of CEACAM6 in QBC939 cells.Lentiviral-mediated CEACAM6 small interfering RNA (siRNA) was established to infect QBC939 cells.The silencing effect was assayed by real-time PCR.The invasive activity of infected QBC939 cells was analyzed by Transwell invasion assay.Results There was high expression of CEACAM6 in QBC939 cells.Recombinant lentiviral vector expressing siRNA targeting CEACAM6 gene was established successfully and confirmed by PCR and DNA sequencing.The expression of CEACAM6 mRNA in QBC939 cells infected with LV-CEACAM6-siRNA was decreased by 93.1％ as compared with control group (P ＜ 0.05).After CEACAM6 gene was knocked down,Transwell test revealed that the invasion potency of QBC939 cells was significantly suppressed by 69.2％ as compared with control group (P ＜ 0.05).Conclusion There was high expression of CEACAM6 in QBC939 cells.There is a positive correlation between the expression level of CEACAM6 and the invasion potency of hilar cholangiocarcinoma. Key words: RNA interference; Carcinoembryonic antigen-related cell adhesion molecule 6; Hilar cholangiocarcinoma

Experimental study of norcantharidins effects on proliferative growth and apoptosis of human cholangiocarcinoma cell line QBC939

Objective To study the growth effect of human cholangiocarcinoma cell line QBC939 treated by norcantharidin (NCTD) and preliminary illustrate the potential mechanism.Methods The human cholangiocarcinoma cell line QBC939 was detected by MTT assay,flow cytometry,immunocytochemistry after the treatment of NCTD in vitro.Results NCTD displayed inhibitory effect on growth of QBC939 from different doses of 0.125,0.75,2.5,10,120 μg/ml after 48 h (P ＜0.05).It was in a dose and time dependent manner.Dose-effect curve was drawn and IC50 value was (3.66±1.14) μg/ml.The flow cytometric profiles showed that the rate of cell apoptosis enhanced following increasing the concentration of NCTD[(8.6±0.4) ％,(17.6±0.3) ％,(22.9±0.4) ％,(25.5±0.9) ％ and (31.1±1.5) ％,respectively]and cells blocked in the G2/M phase after treatment with 2.5 μg/ml NCTD[(14.1±1.0) ％ and (5.7±0.3) ％].The expression of the protein caspase-3 elevated after different concentrations of NCTD co-cultured with QBC939 compare with contrast group.Conclusion NCTD has an inhibitory effect on proliferation of QBC939 cell line,and the mechanism might be related to the induction of cell apoptosis and blockade of cell cycle. Key words: Norcantharidin; Bile duct neoplasms; Cell proliferation; Apoptosis

Immunomodulation of inducible co‐stimulator (ICOS) in human cytokine‐induced killer cells against cholangiocarcinoma through ICOS/ICOS ligand interaction

To evaluate the immunomodulation of inducible co-stimulator (ICOS) in cytokine-induced killer (CIK) cells against cholangiocarcinoma. CIK cells were generated from normal peripheral blood mononuclear cells. Methyl thiazolyl tetrazolium assay was performed to assess proliferation of CIK-ICOS and controlled CIK cells; ELISA was used to analyze the expression of cytokines. Reverse transcription-polymerase chain reaction and immunohistochemistry were performed to evaluate the expression of ICOS ligand (ICOSL) in CIK cells and human cholangiocarcinoma cell line QBC939 cells. The cytotoxicity of CIK cells was determined either by lactate dehydrogenase-releasing assay in vivo or alteration of tumor size prior to and after the treatment of CIK cells in vivo. CIK-ICOS cells proliferated more and expressed higher secretion a level of interferon-γ than the controlled CIK. These cells exhibited higher cytotoxicity against cholangiocarcinoma cell lines at all efficacy: toxicity (E:T) ratios tested than the controlled CIK cells. More importantly, the anti-ICOSL antibody was able to attenuate the elevated cytotoxicity mediated by ICOS overexpression. When injected into cholangiocarcinoma xenografts in severe combined immunodeficiency mice, CIK-ICOS cells survived better than the controlled CIK cells around xenografts and significantly reduced the growth rate of cholangiocarcinoma, with least volume increase and more severe necrosis of the xenografts than controlled mice treated with saline, CIK or CIK-enhanced green fluorescent protein. ICOS can enhance the cytotoxic effect of CIK cells against cholangiocarcinoma both in vitro and in vivo. This effect is mediated by ICOS-augmented cytokine secretion and cell proliferation, and in part through ICOS-ICOSL interaction.

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

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.

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 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。

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。

IFN-γ regulates Fas/FasL expression in cholangio carcinoma cells

Objective: To study the regulation of Fas receptor (Fas)/Fas ligand (FasL) expression by IFN-γ in cholangiocarcinoma cell lines. Methods: We studied the expressions of Fas and FasL gene in the human cholangiocarcinoma cell line QBC939 by RT-PCR, Western blot, and immunohistochemistry and their apoptosis effects on human T cell line Jurkat cell. At the same time, we investigated the regulative effect of IFN-γ on them. Results: Fas, FasL mRNA and protein were expressed by cholangiocarcinoma cells. We also found IFN-γ could up-regulate the expressions of these two genes. However, IFN-γ could also down-regulate the ability of them to make Jurkat cells apoptotic. With the increasing of dosage and time, the effect was enhanced. Conclusion: IFN-γ could regulate the expression of Fas and FasL in cholangiocarcinoma cells, and might influence the ability of cholangiocarcinoma to regulate immune escape. This study provides new theoretical basis for immunological therapy of cholangiocarcinoma.

Antitumor effects of soluble TRAIL in human hepatocellular carcinoma.

The therapeutic potential of soluble TRAIL (sTRAIL) in hepatocellular carcinoma (HCC) was studied. The expression of TRAIL receptors was detected in 60 HCC tissues, 20 normal liver samples and 2 HCC cell lines (HepG2 and SMMC-7721) by in situ hybridization. Before and after HepG2 and SMMC-7721 were treated with sTRAIL protein with various concentrations, the apoptosis rate was observed by using flow cytometry and in situ terminal deoxynucleotidyl tranferase (TdT) labeling. The results showed death receptor 4 (DR4) and DR5 were expressed in 60 HCC tissues and 20 normal liver samples, while the expression intensity of DR in HCC tissues was stronger than in normal liver samples. DcR1 and DcR2 were not detectable in 54 (90%) and 25 (41.%) HCC tissues, while in 20 normal liver samples, DcR was detectable. The high expression of DR and low expression of DcR in HCC tissues were significantly differed from the low expression and high expression in normal liver samples. The expression of DR5, DR4 and DcR2 in both HCC cell lines was detectable, but the expression of DcR1 was not detectable. The expression of DR in HCC tissues was related to the differentiation and grades of HCC. In the poor differentiated HCC, the expression of DR was decreased (P < 0.01). The expression of DR in Il/IV grades was significantly lower than that in I / 11 grades (P < 0.05). The expression of DR was not related to gender, age, HBsAg, AFP, tumor size and metastasis. The expression of DR in the HCC drugresistant lines was decreased. After treatment with TRAIL (100 ng/ml) for 24 h, the apoptosis rate of HCC cells, Jurkat cells and human cholangiocarcinoma cell line QBC939 was 10%, 70%, 50% respectively. It was suggested that the TRAILR expression is prevalent in HCC with different expression patterns of different receptor types. HCC is resistant to TRAIL-mediated apoptosis. The treatment of TRAIL alone has a limited effect on inducing apoptosis of HepG2 and SMMC7721.

Induction of cell-cycle arrest and apoptosis by 4HPR in human cholangiocarcinoma cells and its possible mechanism

目的 探讨N-hydroxyphenyl-retinamide(4HPR)对人胆管癌细胞QBC939细胞增殖和凋亡的影响及其可能机制.方法采用四甲基氮唑蓝实验(MTT)、免疫组织化学染色、流式细胞术等技术,检测经5×10-6 mol/L 4HPR处理后,QBC939细胞生长增殖、细胞周期和凋亡的变化,以及对P21waf1、P27kip1、P53蛋白表达的影响.结果 4HPR显著抑制人胆管癌细胞QBC939细胞的生长增殖,并呈时间、剂量依赖性.用5×10-6 mol/L 4HPR处理细胞后,细胞周期停滞于G1期.流式细胞术检测发现,4HPR处理12 h细胞凋亡率达9.7%,至48 h达57.2%.而P21waf1、P27kip1蛋白表达呈上升趋势,P53蛋白表达则无明显变化.结论 4HPR具有抗增殖和诱导QBC939细胞发生凋亡的作用,并可导致细胞周期停滞于G1期.此种机制与其上调P21waf1、P27kip1蛋白表达有关,而与P53蛋白表达无关。