Human Gallbladder Carcinoma Cell Line Research Articles

Microcystin-leucine-arginine promotes the development of gallbladder carcinoma via regulating ELAC2

Gallbladder carcinoma (GBC) is the most prevalent cancer of the bile tract, with unexpected GBC accounting for almost half of all GBC cases in some tertiary medical centers. Although the involvement of microcystin-leucine-arginine (MC-LR) in the development of intrahepatic cholangiocarcinoma has been established, there is a paucity of data regarding its association with GBC. The present study aims to investigate whether MC-LR level in the gallbladder of patients is associated with GBC development and, if so, to characterize the underlying mechanism in GBC cells. Our clinical data revealed that MC-LR level was significantly increased in GBC patients compared to patients with gallbladder stones only (P = 0.009). Moreover, our findings demonstrated that MC-LR could promote the proliferation and metastasis of human GBC cell lines. Furthermore, ELAC2 was identified as a critical mRNA involved in GBC progression through RNA sequencing. Collectively, our study suggests that MC-LR might be involved in the development of GBC by modulating the expression of ELAC2.

Heterodimerization of cholecystokinin 1 and cholecystokinin 2 receptors in gallbladder cancer: a new mechanism for carcinogenesis.

Cholecystokinin is present in abundance in gallbladder tissue and mediates function through two structurally related receptors, CCK1R and CCK2R. Heterodimerization of these receptors is known to impact cell growth in vitro. However, the significance of these heterodimers in gallbladder carcinogenesis is relatively unknown. Therefore, we evaluated the expression and the dimerization status of CCK1 and CCK2 receptors in human gallbladder carcinoma cell line (GBC-SD) and resected gallbladder tissue from normal (n = 10), cholelithiasis (n = 25) and gallbladder cancer (n = 25) by immunofluorescence/immunohistochemistry and western blot. The dimerization status of CCK1R and CCK2R was evaluated by co-immunoprecipitation. To understand the effect of heterodimerization of these receptors on growth-related signaling pathways, the expression of p-AKT, rictor, raptor and p-ERK was evaluated by western blot. We demonstrated the expression and heterodimerization of CCK1 and CCK2 receptor in GBC-SD gall bladder carcinoma cell line. Knockdown of CCK1R and CCK2R in the cell line led to significant reduction in p-AKT (P = 0.005; P = 0.0001) and rictor (P < 0.001; P < 0.001) levels. In tissue samples, significantly higher expression of CCK1R and CCK2R was observed in gallbladder cancer when compared to other groups both by immunohistochemistry (P = 0.008 and P = 0.013) and western blot (P = 0.009 and P = 0.003). An increase in heterodimer formation of CCK1R with CCK2R was observed in gallbladder cancer when compared to normal and cholelithiasis tissues. No significant difference in the expression of p-AKT and p-ERK was observed between the three groups. Our results provide the first evidence of heterodimerization of CCK1R and CCK2R in gallbladder tissue, and its association with development of gallbladder cancer. This finding has potential clinical and therapeutic significance.

Combination of celecoxib and PD184161 exerts synergistic inhibitory effects on gallbladder cancer cell proliferation.

Cyclooxygenase-2 (COX-2) and extracellular signal-regulated kinase 1/2 (ERK1/2) may serve as potential targets in various types of cancer; however, the roles of these proteins in gallbladder carcinoma (GBC) have not been reported previously. In the present study, the expression levels of COX-2 and phospho (p)-ERK1/2 in GBC were examined and the biological activities of celecoxib and PD184161 (specific inhibitors of COX-2 and p-ERK1/2, respectively) on the proliferation, cell cycle and apoptosis of the GBC-SD and NOZ human GBC cell lines were evaluated by a series of in vitro and in vivo studies. COX-2 and p-ERK1/2 protein expression levels were found to be significantly elevated in GBC tissues as well as in GBC-SD and NOZ cells. Treatments with celecoxib and PD184161 significantly inhibited GBC-SD and NOZ cell growth in a concentration-dependent manner, and their combination produced a synergistic inhibitory effect. In addition, celecoxib and PD184161 significantly inhibited tumor growth in xenograft nude mice. Celecoxib treatment led to G1 arrest via the upregulation of p21 and p27 expression in GBC-SD and NOZ cells, whereas PD184161 did not affect cell cycle distribution. The combination of celecoxib and PD184161 was able to promote cell apoptosis by triggering a collapse of mitochondrial membrane potential and activating caspase-3-mediated apoptosis. In conclusion, COX-2 and p-ERK1/2 protein may serve as potential targets for GBC chemotherapy, and the combination of celecoxib and PD184161 could significantly inhibit GBC cell growth, induce cell G1 arrest and trigger cell apoptosis of GBC cells.

Magnolol inhibits growth of gallbladder cancer cells through the p53 pathway.

Magnolol, the major active compound found in Magnolia officinalis has a wide range of clinical applications due to its anti-inflammation and anti-oxidation effects. This study investigated the effects of magnolol on the growth of human gallbladder carcinoma (GBC) cell lines. The results indicated that magnolol could significantly inhibit the growth of GBC cell lines in a dose- and time-dependent manner. Magnolol also blocked cell cycle progression at G0/G1 phase and induced mitochondrial-related apoptosis by upregulating p53 and p21 protein levels and by downregulating cyclin D1, CDC25A, and Cdk2 protein levels. When cells were pretreated with a p53 inhibitor (pifithrin-a), followed by magnolol treatment, pifithrin-a blocked magnolol-induced apoptosis and G0/G1 arrest. In vivo, magnolol suppressed tumor growth and activated the same mechanisms as were activated in vitro. In conclusion, our study is the first to report that magnolol has an inhibitory effect on the growth of GBC cells and that this compound may have potential as a novel therapeutic agent for the treatment of GBC.

Expression of stem cell marker in side population cells isolated from human gallbladder carcinoma cell line GBC-SD

Objective:To investigate the existence of side population cells with the potency of stem cells in human gallbladder carcinoma cell line GBC-SD and the differences in ABCG2,Oct-4 and CD34 expression among SP cells,non-SP cells and GBC-SD cells.Methods:SP and non-SP cells were sorted from GBC-SD cells by fluorescence-activated cell sorting (FACS).The expression of ABCG2,Oct-4 and CD34 in SP cells, non-SP cells,and GBC-SD cells was detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot,flow cytometry (FCM) and immunofluorescence chemistry.Results:SP cells with stem cell potency were isolated from GBC-SD cells with a proportion of (0.64±0.08)%.The metastatic ability of SP cells was obviously higher than that of non-SP cells and GBC-SD cells (P0.05).The expression of ABCG2 was significantly higher in SP cells than in non-SP cells and GBC-SD cells [(89.56±3.86)% vs.(1.32±0.49)% and (12.37± 1.61)%,P=0.0011.The expression of Oct-4 in these cells was (94.87±1.40)%,(88.16±2.34)% and (90.17± 1.61)%,respectively (P0.05).CD34 was nearly absent in these cells on protein level [(1.78±0.51)% vs. (0.63±0.21)% and (0.96+0.381)%,P0.05)],but it was highly expressed in non-SP cells and GBC-SD cells and absent in SP cells on mRNA level.Conclusion:SP cells,which have the potency of stem cells,exist in hu- man gallbladder carcinoma GBC-SD cell line and have the phenotype of ABCG2+Oct-4+CD34-.

Tetrandrine induces apoptosis in gallbladder carcinoma in vitro.

The aims of this study were to observe the apoptosis effects of tetrandrine on human gallbladder carcinoma cell line (SGC-996), and to explore its related mechanism. First, the anti-proliferative activities of tetrandrine on SGC-996 cells were determined by using the MTT assays. Then, cell cycle changes were detected by flow cytometry analysis. The apoptosis of cells was detected by the annexin V/propidium iodide double-staining assay. Detection of mitochondrial membrane potential was used to validate the ability of tetrandrine on inducing apoptosis. Finally, the expressions of the apoptosis-related proteins (caspase-3, PARP, Bcl-2, and Bax) were analyzed by western blot. Statistical analyses were performed using the Student’s t-test for comparison of the results obtained from cells with or without treatment of tetrandrine. The MTT assay revealed a significant inhibition of cell proliferation in a dose- and time-dependent manner. Cells treated with tetrandrine were arrested at the S phase, according to the flow cytometric analysis. Tetrandrine produced a dose-dependent increase in the apoptotic cell population compared with control cells. Tetrandrine can also affect mitochondrial function by changing the mitochondrial membrane potential. Furthermore, western blot assay demonstrated that the tetrandrine induced apoptosis in SGC-996 cells by regulating the ratio of Bcl-2/Bax and activating the expression of cleaved caspase-3. The results indicate that tetrandrine may be a potential agent for the treatment of gallbladder carcinoma.

Bcl-2 gene silencing by RNA interference inhibits the growth of the human gallbladder carcinoma cell line GBC-SD in vitro and in vivo

Gallbladder carcinoma is the most common malignant tumor in the biliary system; however, the underlying mechanisms of tumor initiation, progression and metastasis are not fully understood to date. The B-cell lymphoma/leukemia-2 (Bcl-2) gene, which is highly expressed in gallbladder carcinoma tissue, is one of the most important regulatory factors in cell apoptosis, and plays an important role in the initiation and progression of gallbladder carcinoma. In the present study, we constructed a eukaryotic expression vector of small interference RNA (siRNA) specific to the Bcl-2 gene and transfected it into GBC-SD human gallbladder carcinoma cells. We demonstrated that the constructed Bcl-2 siRNA vector effectively silenced Bcl-2 gene expression in the GBC-SD human gallbladder carcinoma cells, inhibited cell proliferation, induced cell apoptosis, increased chemotherapeutic sensitivity to 5-fluorouracil and inhibited tumor growth invivo. Collectively, these data reveal an important contribution of Bcl-2 to gallbladder carcinoma. Thus, the use of a synthetic inhibitor of Bcl-2 may be a promising approach for the treatment of gallbladder carcinoma.

Anti-proliferation effect of curcumin on human gallbladder carcinoma cell line

Objective To explore the anti-proliferation effect of curcumin on human gallbladder carcinoma GBC-SD cells and its mechanism.Methods The growth inhibition of GBC-SD cells treated with curcumin of 0,10,30,50,and 70 mg/L was detected by using methyl thiazol tetrazolium (MTT) assay.The growth inhibition of GBC-SD cells treated with curcumin of 30 mg/L at 0,12,24,48 and 72 h was detected.Control group,10 mg/L curcumin group,30 mg/L curcumin group and 50 mg/L curcumin group were set up.The apoptosis rate was tested by using flow cytometry.After curcumin treatment for 24 h,the expression levels of nuclear factor-κB (NF-κB) and Cyclin D1 were detected by using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR),respectively.The mRNA levels of bax and B lymphocytes/leukemia-2 (bcl-2) were examined by using real-time reverse transcription polymerase chain reaction.Results Curcumin could significantly inhibit the viability of GBC-SD cells in a time-and dosedependent manner.After treatment with curcumin (10,30,and 50 mg/L),the survival rate was (95.34 ±9.21)％,(70.63 ± 11.09)％ and (50.58 ± 6.31)％,respectively.There was significant difference between control group and 30 mg/L curcumin group or 50 mg/L curcumin group (P ＜ 0.05) ;The apoptosis rate was (9.83 ± 1.98)％,(37.25 ± 1.21)％ and (57.63 ±7.38)％,respectively.There was significant difference between control group and 30 mg/L curcumin group or 50 mg/L curcumin group (P ＜ 0.05).30 mg/L and 50 mg/L curcumin could down-regulate the expression of NF-κB and CyclinD1,reduce the expression of bcl-2 and enhance the expression of bax.However,10 mg/L curcumin had no evident effect on genes mentioned above.Conclusion Curcumin could not only down-regulate the expression of NF-κB and Cyclin D1,but also reduce the mRNA level of bcl-2 and up-regulate the mRNA level of bax,causing the reduced bcl-2/bax ratio,which could effectively inhibit the proliferation of gallbladder carcinoma GBC-SD cells and induce cell apoptosis. Key words: Curcumin; Gallbladder carcinoma; Apoptosis

Cancer detection by ubiquitin carboxyl-terminal esterase L1 methylation in pancreatobiliary fluids

To evaluate the utility of measuring epigenetic alterations in pancreatic and biliary fluids in determining molecular markers for pancreatobiliary cancers. DNA was extracted from undiluted pancreatic and biliary fluids. As a surrogate for a genome-wide hypomethylation assay, levels of long interspersed nuclear element-1 (LINE-1) methylation were analyzed using bisulfite pyrosequencing. CpG island hypermethylation of 10 tumor-associated genes, aryl-hydrocarbon receptor repressor, adenomatous polyposis coli, calcium channel, voltage dependent, T type α1G subunit, insulin-like growth factor 2, O-6-methyl-guanine-DNA methyltransferase, neurogenin 1, CDKN2A, runt-related transcription factor 3 (RUNX3), secreted frizzled-related protein 1, and ubiquitin carboxyl-terminal esterase L1 (UCHL1), was analyzed using MethyLight. To examine the role of CpG methylation and histone deacetylation in the silencing of UCHL1, human gallbladder carcinoma cell lines and pancreatic carcinoma cell lines were treated with 2 or 5 μmol/L 5-AZA-dC for 72 h or 100 nmol/L Trichostatin A for 24 h. After the treatment, UCHL1 expression was analyzed by real-time reverse transcription-polymerase chain reaction. Pancreatobiliary cancers exhibited significantly lower LINE-1 methylation levels in pancreatic and biliary fluids than did noncancerous pancreatobiliary disease (58.7% ± 4.3% vs 61.7% ± 2.2%, P = 0.027; 53.8% ± 6.6% vs 57.5% ± 1.7%, P = 0.007); however, LINE-1 hypomethylation was more evident in pancreatic cancer tissues than in pancreatic fluids (45.4% ± 5.5% vs 58.7% ± 4.3%, P < 0.001). CpG island hypermethylation of tumor-associated genes was detected at various frequencies, but it was not correlated with LINE-1 hypomethylation. Hypermethylation of the UCHL1 gene was cancer-specific and most frequently detected in pancreatic (67%) or biliary (70%) fluids from patients with pancreatobiliary cancer. As a single marker, hypermethylation of the UCHL1 gene in pancreatic and biliary fluids was most useful for the detection of pancreatic and pancreatobiliary cancers, respectively (100% specificity). Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic (87% sensitivity and 100% specificity) and pancreatobiliary (97% sensitivity and 100% specificity) cancers. Treatment with a demethylating agent, 5-AZA-2'-deoxycytidine, restored UCHL1 expression in pancreatobiliary cancer cell lines. Our results suggest that hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluid might be useful for the diagnosis of pancreatobiliary cancers.

Overexpression of HIF-1α in primary gallbladder carcinoma and its relation to vasculogenic mimicry and unfavourable prognosis

As a novel mode of tumor neovascularization, vasculogenic mimicry (VM) has been reported to increase tumor-related mortality in many different solid tumors. In the present study, two established human gallbladder carcinoma (GBC) cell lines (highly aggressive GBC-SD and poorly aggressive SGC-996) cultured on a three-dimensional matrix were assessed for the ability of VM channel formation under normoxic or hypoxic conditions. In addition, the relationship between HIF-1α gene expression and VM channel formation of GBC cells in vitro was measured using the small interfering RNA (siRNA) technique, western blotting and real-time reverse transcription (RT)-PCR analysis. Furthermore, H&E and CD31/periodic acid-Schiff (PAS) staining were used to observe VM in GBC tissue samples. Additionally, all seventy-one specimens with VM and non-VM were stained for hypoxia inducible factor-1 α (HIF-1α) and its correlation with clinicopathological features and prognosis was analyzed simultaneously. We found that hypoxia could induce more VM channel formation and elevated HIF-1α expression in highly aggressive GBC-SD cells. HIF-1α siRNA efficiently knocked down HIF-1α expression and GBC VM networks under either normoxic or hypoxic conditions. VM was present in human primary GBC and overexpression of HIF-1α was significantly correlated with depth of invasion and perineural involvement in the non-VM group. Moreover, VM and HIF-1α were independent factors for the overall survival of GBC patients and correlated with decreased survival. In conclusion, VM was present in human GBC. As a critical mediator in VM formation, high expression of HIF-1α was associated with VM and tumor progression in GBC patients.

Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substrate Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.

The tumorigenic property of side population cells in a human gallbladder carcinoma cell line GBC-SD

Objective To explore the tumor igenic property of side population cells (SP) from human gallbladder carcinoma cell line GBC-SD. Methods SP and non-SP cells were isolated from GBC-SD staining with Hoechst33342 dye by fluorescence-activated cell sorting (FACS). The soft agar clonal assay and xenograft assay were performed to characterize tumorigenic property of side population cells in vitro and in vivo, respectively. The percentage of SP cells was analyzed by FACS in 5 hu man gallbladder carcinoma specimens. Results The percentage of SP cells accounted for approximately 0.87 ％ of GBC-SD cells. The clone-formed rates of SP was more frequent than that of non-SP cells (14.74％ ± 3.53％ vs 5.17％ ± 1.05％), there was statistically significant difference (t =2.75,P＜0. 05). SP cells could generate tumors with as few as 5 × 103 cells (four of seven animals), whereas at least 1 × 105 non-SP cells were needed to form a tumor (one of seven animals). Re-analysis of SPderived tumors by FACS showed that SP cells under in vivo conditions also have the capacity to regenerate the SP and non-SP fractions. Besides, analysis of Hoechst33342 revealed s small fraction of SP cells, ranging from 0. 27％ to 2.3％ in gallbladder carcinoma specimens. Conclusion SP cells from GBC-SD are highly tumorigenic similar as the cancer stem cells. Key words: Carcinoma of gallbladder; Stem cells; Tumorigenic potential

A pilot histomorphology and hemodynamic of vasculogenic mimicry in gallbladder carcinomas in vivo and in vitro

BackgroundVasculogenic mimicry (VM), as a new blood supply for tumor growth and hematogenous metastases, has been recently described in highly aggressive human melanoma cells, etc. We previously reported VM in human gallbladder carcinomas and its clinical significance. In this study, we further studied histomorphology and hemodynamic of VM in gallbladder carcinomas in vivo and in vitro.MethodsThe invasive potential of human gallbladder carcinoma cell lines GBC-SD and SGC-996 were identified by Transwell membrane. The vasculogenic-like network structures and the signal intensities i.e. hemodynamic in gallbladder carcinomas stimulated via the three-dimensional matrix of GBC-SD or SGC-996 cells in vitro, the nude mouse xenografts of GBC-SD or SGC-996 cells in vivo were observed by immunohistochemistry (H&E staining and CD31-PAS double staining), electron microscopy and micro-MRA with HAS-Gd-DTPA, respectively.ResultsHighly aggressive GBC-SD or poorly aggressive SGC-996 cells preconditioned by highly aggressive GBC-SD cells could form patterned networks containing hollow matrix channels. 85.7% (6/7) of GBC-SD nude mouse xenografts existed the evidence of VM, 5.7% (17/300) channels contained red blood cells among these tumor cell-lined vasculatures. GBC-SD xenografts showed multiple high-intensity spots similar with the intensity observed at tumor marginal, a result consistent with pathological VM.ConclusionsVM existed in gallbladder carcinomas by both three-dimensional matrix of highly aggressive GBC-SD or poorly aggressive SGC-996 cells preconditioned by highly aggressive GBC-SD cells in vitro and GBC-SD nude mouse xenografts in vivo.

Mda-7/IL-24 induces apoptosis in human GBC-SD gallbladder carcinoma cells via mitochondrial apoptotic pathway

mda-7/IL-24 has tumor-suppressor activity in a broad spectrum of human cancer cells. However, the therapeutic effect of the recombinant human IL-24 protein on human gallbladder carcinoma has rarely been explored. In this study, we used a human gallbladder carcinoma cell line (GBC-SD) to explore the effect of adenovirus-mediated IL-24 (Ad-IL24) gene therapy on GBC-SD cells. We show that Ad-IL24 treatment of GBC-SD cells in vitro conspicuously induced apoptosis of GBC-SD cells. We also demonstrate that the in vivo treatment of GBC tumor-bearing athymic nude mice intratumorally injected with Ad-IL24 significantly suppressed GBC growth. To further explore the mechanism that mda-7/IL-24 utilized in tumor cell apoptosis, we examined molecules and pathways involved in apoptotic regulation and found that Ad-IL24 induced the down-regulation of anti-apoptotic gene Bcl-2 and the release of cytochrome c, which subsequently activated caspase-9, caspase-3 and PARP to induce apoptosis. In summary, adenovirus (AdV)-mediated IL-24 overexpression exerted potent antitumor activity via stimulating mitochondrial apoptotic pathway in GBC-SD. Therefore, mda-7/IL-24 has the potential to serve as a tool for targeted gene therapy in the treatment of gallbladder cancer.

TLR4 expression in human gallbladder carcinoma cell Line SGC-996 and affects growth of xenograft tumor in nude mice

Objective To observe the expression of TLR4 in human gallbladder cancer cell line SGC-996,and investigate whether RNA interference targeting TLR4 gene affect growth of the gallbladder carcinoma xenograft in nude mice. Methods The expression of TLR4 in SGC-996 was determined bv im-munohistochemistry and reverse transcription-polymerase chain reaction ( RT-PCR). 12 BALA/C nude mice were randomly divided into two groups. The experimental group was injected 6 x 10 SGC996 cells stable transfected by small interfering RNA for TLR4 per mice. The control group was injected with the same number of untreated SGC996 cells. Aftter continuous observation for 5 weeks, the formation rate, growth rate, weight and volume of the tumors were measured and calculated. Results The results of immunohistochem-istry and RT-PCR indicated the expression of TLR4 in SGC-996. The tumor formation rate of gallbladder carcinoma cell and TLR4-siRNA transfected SGC996 cell was 100%. The average tumor volume in experimental group and control group was (277.94±7.6 ) mm3 and (474.47±9.99) mm3 respectively. The growth rate was 7. 941% and 13. 556% ,and the average tumor weight was (0.76 ±0.16) g and (1.78± 0.23) g,respectively. The tumor growth rate,weight and volume in the experimental group decreased significantly when compared with those in control group (P<0.05). Conclusion Human gallbladder carcinoma cell line SGC-996 expresses TLR4. SGC-996 cells transfected with siRNA can effectively lower the level of TLR4 gene expression. After silence of TLR4 gene,the growth rate,weight and volume of xenograft are decreased significantly. Key words: TLR4; Gallbladder carcinoma; RNA interference; Nude mice

Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to cirsimaritin-induced apoptosis in human gallbladder carcinoma GBC-SD cells

In this study, the anticancer effect of cirsimaritin, a natural flavonoid, against human gallbladder carcinoma cell line GBC-SD and the underlying mechanisms were investigated. Cirsimaritin inhibited the growth of tumor cells and induced mitochondrial apoptosis in GBC-SD cells. In addition, cirsimaritin triggered endoplasmic reticulum (ER) stress and down-regulated the phosphorylation of Akt, while knock-down of CHOP dramatically abrogated the inactivation of Akt and reversed the pro-apoptotic effect of cirsimaritin. Furthermore, cirsimaritin provoked the generation of reactive oxygen species in GBC-SD cells, while the antioxidant N-acetyl cysteine almost completely blocked the activation of ER stress and apoptosis, suggesting cirsimaritin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways in GBC-SD cells.

Establishment of an arsenic-resistant human gallbladder carcinoma cell line and analysis of the differential expressions of genes associated with apoptosis

The aim of the study was to establish an arsenic trioxide (ATO)-resistant cell line of human gallbladder carcinoma, GBC-SD/ATO, and to analyze the differential expressions of its apoptosis-associated genes, so as to investigate a correlation between ATO induced resistance of gallbladder carcinoma and expressions of apoptosis associated genes. The resistant cell line was obtained in vitro by culture of human gallbladder carcinoma cell line GBC-SD with increasingly stepwise concentrations of ATO. The sensitivities of GBC-SD cells and GBC-SD/ATO cells to ATO were determined by MTT assay respectively. cDNA microarray containing 458 apoptosis-related human genes was used to compare the gene expression profiles of GBC-SD/ATO cells and corresponding sensitive cell line GBC-SD. GBC-SD/ATO cell line was established successfully after 8 months of exposure to increasing concentrations of ATO. Compared with the parental cell line, GBC-SD/ATO was 13.6 times more resistant to ATO. Of the 458 apoptosis-related genes, 17 genes were detected having > 2-fold difference of expression between the GBC-SD/ATO and GBC-SD cells, with 6 genes up-regulated and 11 genes down-regulated in GBC-SD/ATO cells. The 17 genes involved in the apoptosis pathway might be relevant to the resistance of GBC-SD/ATO cells to ATO, suggesting that the modulation of expression of apoptosis-related genes may be a main mechanism of acquired resistance in GBC-SD/ATO.

Establishment and characterization of a human gallbladder carcinoma cell line EH-GB1 originated from a metastatic tumor

To establish a human gallbladder carcinoma cell line derived from a metastatic gallbladder carcinoma and identify its biological characteristics. Tissue samples were separated from the surgical specimen obtained from a patient with metastatic carcinoma and single-cell suspension was prepared. Then the cells were cultured in DMEM medium supplemented with 15% fetal bovine serum. The morphology of tumor cells was observed under an electron microscope. The cell growth curve was plotted. The tumorigenicity of the cell line was studied by subcutaneous inoculation in SCID mice. The cells were infected by lentiviral vector carrying fluorescent report genes (lenti-GFP and lenti-Red2) separately for expressions of GFP and Red2, respectively. A novel metastatic gallbladder carcinoma cell line was successfully established and named "EH-GB1". It could be passaged for over 20 generations with typical malignant epithelial morphology and a stable growth cycle of 24 h. Tumors were formed in all of the 10 SCID mice inoculated with EH-GB1 cells subcutaneously, and the tumor cells were tumor marker CA19-9-positive. Continuous expressions of fluorescent report genes were observed in EH-GB1 cells infected by lenti-GFP and lenti-Red2. EH-GB1 cells might be the first stable cell line of human gallbladder carcinoma established from a metastatic focus of gallbladder carcinoma. This cell line with continuous expressions of GFP and Red2 might be a novel and perfect experimental model for clinical and basic research on gallbladder carcinoma.

Histone deacetylase inhibitor (SAHA) and repression of EZH2 synergistically inhibit proliferation of gallbladder carcinoma

Polycomb group protein EZH2, frequently overexpressed in malignant tumors, is the catalytic subunit of polycomb repressive complex 2 (PRC2). PRC2 interacts with HDACs in transcriptional silencing and relates to tumor suppressor loss. We examined the expression of HDAC isoforms (HDAC 1 and 2) and EZH2, and evaluated the possible use of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and EZH2 repressor for gallbladder carcinoma. We used 48 surgically resected gallbladders and cultures of human gallbladder epithelial cells (HGECs), gallbladder carcinoma (TGBC2TKB), and cholangiocarcinoma (HuCCT-1 and TFK-1) cell lines for examination. Immunohistochemically, EZH2 was overexpressed in gallbladder carcinoma, especially poorly differentiated carcinoma, but not in normal epithelium. In contrast, HDAC1/2 were expressed in both carcinoma and normal epithelium in vivo. This pattern was verified in cultured cells; EZH2 was highly expressed only in TGBC2TKB, whereas HDAC1/2 were expressed in HGECs and TGBC2TKB. Interestingly, SAHA treatment caused significant cell number decline in three carcinoma cells, and this effect was synergized with EZH2 siRNA treatment; however, HGECs were resistant to SAHA. In TGBC2TKB cells, the expression of EZH2 and HDAC1/2 were decreased by SAHA treatment, and p16(INK4a), E-cadherin, and p21were simultaneously activated; however, no such findings were obtained in HGECs, suggesting that the effect of SAHA depends on the EZH2-mediated tumor suppressor loss. In conclusion, this study suggests a possible mechanism by which carcinoma cells but not normal cells are sensitive to SAHA and indicates the efficacy of this new anticancer agent in combination with EZH2 repression in gallbladder carcinoma.

Papillary hyperplasia of the gallbladder in pancreaticobiliary maljunction represents a senescence-related lesion induced by lysolecithin

Cellular senescence, an irreversible growth arrest, is considered to play as safeguard against malignant progression, though such a mechanism is speculative in human carcinogenesis. In gallbladder carcinoma, cholecystolithiasis and pancreaticobiliary maljunction (PBM) are major risk factors. Here, by using 113 surgically resected gallbladders and cultures of human gallbladder epithelial cells (HGECs) and gallbladder carcinoma cell line (TGBC2TKB), we examined carcinogenesis with respect to cellular senescence. Among 15 cases of PBM in which carcinoma was found in 4 cases, nonneoplastic gallbladder mucosa showed diffuse papillary hyperplasia (PHP). PHP was not found in gallbladders with cholecystolithiasis. Interestingly, PHP exhibited senescent features such as expression of p16INK4A and low cell proliferative activity. In contrast, EZH2, a polycomb group protein, was overexpressed in intraepithelial neoplasm and carcinoma in gallbladders with cholecystolithiasis. In PBM, EZH2 was expressed only in carcinoma foci but not in PHP. Cultured HGECs treated with lysolecithin, the level of which is elevated in gallbladder bile of PBM, showed increased expression of p16INK4A and senescence-associated β-galactosidase. Conversely, enforced overexpression of EZH2 in senescent HGECs reduced p16INK4A expression. A knockdown of EZH2 in cultured TGBC2TKB cells increased p16INK4a expression. In conclusion, PHP in PBM may act as a barrier to malignant transformation for decades. EZH2 may be responsible for the escape from cellular senescence followed by malignant transformation in the gallbladder of PBM.