GBC-SD Cells Research Articles

Effects of matrix metalloproteinase inhibitor doxycycline and CD147 antagonist peptide-9 on gallbladder carcinoma cell lines.

Gallbladder carcinoma is the most common and aggressive malignancy of the biliary tree and highly expresses CD147, which is closely related to disease prognosis in a variety of human cancers. Doxycycline exhibited anti-tumor properties in many cancer cells. CD147 antagonist peptide-9 is a polypeptide and can specifically bind to CD147. The effect of these two drugs on gallbladder cancer cells has not been studied. The aim of this study is to investigate the effect of doxycycline and antagonist peptide-9 on gallbladder carcinoma cells and the possible mechanism of inhibition on cancer cell of doxycycline. To investigate the effects of doxycycline and antagonist peptide-9 on gallbladder carcinoma cells (GBC-SD and SGC-996), cell proliferation, CD147 expression, and early-stage apoptosis rate were measured after treated with doxycycline. Matrix metalloproteinase-2 and matrix metalloproteinase-9 activities were measured after treated with different concentrations of doxycycline, antagonist peptide-9, and their combination. The results demonstrated that doxycycline inhibited cell proliferation, reduced CD147 expression level, and induced an early-stage apoptosis response in GBC-SD and SGC-996 cells. The matrix metalloproteinase-2 and matrix metalloproteinase-9 activities were inhibited by antagonist peptide-9 and doxycycline, and the inhibitory effects were enhanced by combined drugs in gallbladder carcinoma cell lines. Taken together, doxycycline showed inhibitory effects on gallbladder carcinoma cell lines and reduced the expression of CD147, and this may be the mechanism by which doxycycline inhibits cancer cells. This study provides new information and tries to implement the design of adjuvant therapy method for gallbladder carcinoma.

The expression and clinical significance of hypoxia-induced factor-1α in gallbladder carcinoma tissues and its role on metformin-suppressed metastasis in GBC-SD cells

Objective To study the expression and the clinical significance of hypoxia-induced factor-1α (HIF-1α ) in gallbladder cancer tissues, and the role and mechanism of HIF-1α in metformin-suppressed metastasis in gallbladder carcinoma GBC-SD cells. Methods 24 specimens of gallbladder cancer tissues and 5 specimens of chronic cholecystitis were collected from the First Affiliated Hospital of Zhengzhou University between June 2016 and February 2017. Immunohistochemistry and qPCR were used to detect the expression of HIF-1α in gallbladder cancer tissues, in adjacent non-cancer tissues and in chronic cholecystitis, and the clinical significance was analyzed. The model of metastasis was induced by hypoxia; the wound healing assay and the Transwell assay were used to detect the ability of cell metastasis; the expressions of HIF-1α and VEGF in gallbladder carcinoma GBC-SD cells were detected by western blotting assay and immunofluorescence. Results The expression of HIF-1α in gallbladder cancer tissues was higher than the adjacent non-cancer tissues and in chronic cholecystitis. The expression of HIF-1α was correlated with lymph node metastasis and TNM staging in gallbladder cancer tissues (P<0.05). The wound healing rate after 48 h in the negative control group and in the treatment with hypoxia group (1%O2) in GBC-SD cells were (46.5±4.8)% and (67.3±4.0)%, respectively. The Transwell data showed that the numbers of metastasis after 24 h in the negative control group and in the treatment with hypoxia group GBC-SD cells were (147.4±11.7) and (234.4±17.7), respectively. When compared with the negative control group, treatment with hypoxia significantly increased the ability of metastasis and up-regulated the expression of HIF-1α and VEGF in GBC-SD cells (P<0.05). The wound healing rate after 48 h in the negative control group, the metformin group, the hypoxia group and the metformin and hypoxia group in GBC-SD cells were (40.6±7.1)%, (16.4±9.4)%, (69.5±4.0)% and (22.4±7.4)%, respectively. The Transwell data showed that the numbers of metastasis after 24 h in the negative control group, the metformin group, the hypoxia group and the metformin and hypoxia group in GBC-SD cells were (148.4±6.9), (90.0±8.4), (185.8±10.2) and (113.4±8.6), respectively. When compared with the hypoxia group, treatment with metformin and hypoxia significantly decreased the ability of metastasis and down-regulated the expression of HIF-1α and VEGF in GBC-SD cells (P<0.05). The wound healing rate after 48 h in the negative control group, the 2MeoE2 group, the hypoxia group, the 2MeoE2 and hypoxia group in GBC-SD cells were (43.4±4.4)%, (25.9±9.0)%, (63.3±2.2)%, (46.2±4.5)%, respectively. The Transwell data showed that the numbers of metastasis after 24 h in the negative control group, the 2MeoE2 group, the hypoxia group, the 2MeoE2 and hypoxia group in GBC-SD cells were (144.2±12.6), (80.2±7.7), (203.8±7.0), (124.0±5.2), respectively. When compared with the hypoxia group, treatment with HIF-1α inhibitor 2MeoE2 and hypoxia significantly decreased the ability of metastasis and down-regulated the expression of HIF-1α and VEGF in GBC-SD cells (P<0.05). Conclusions The expression of HIF-1α was correlated with lymph node metastasis and TNM staging in gallbladder cancer tissues. Treatment with hypoxia significantly increased the expression of HIF-1α and VEGF and promoted metastasis of GBC-SD cells, while treatment with metformin decreased the ability of metastasis induced by hypoxia via inhibiting the HIF-1α/VEGF pathway in GBC-SD cells. Key words: Gallbladder carcinoma; Hypoxia-induced factor-1α; Metformin; Hypoxia; Metastasis

CCK1 receptor is involved in the regulation of protein lysine acetylation in GBC-SD cells and gallbladder carcinoma.

CCK1 (cholecystokinin receptor 1) and protein lysine acetylation were associated with several cancers, respectively. However, whether they are involved in the alternation of gallbladder carcinoma is unknown. This study investigated the characteristics of CCK1 and protein lysine acetylation in GBC-SD cells and carcinoma of gallbladder. The expression and localization of CCK1 were detected by western blot analysis and indirect immunofluorescence. GBC-SD cells were treated with CCK-8 and CCK-8+CCK1 inhibitor. The protein lysine acetylation from cells, as well as tissues of gallbladder carcinoma, was examined by western blotting. CCK1 receptor was expressed and localized in the GBC-SD cells. The synthetic octapeptide of CCK (CCK-8) could accelerate the lysine acetylation of a subset of proteins in dose-dependent manners in GBC-SD cells. Further investigation demonstrated that the specific inhibitor (CR1409) of CCK1 receptor could attenuate the CCK8-induced increase of protein lysine acetylation. In addition, we revealed that the rise of CCK1 receptor expression is associated with the increase of protein lysine acetylation in tissues from carcinoma of gallbladder. CCK might regulate protein lysine acetylation via CCK1 receptor.

Somatostatin enhances growth inhibition by cisplatin in gallbladder cancer cells through inducing PTEN expression

Objective To investigate the combined effects of somatostatin (SST) and cisplatin (DDP) on proliferation and apoptosis in gallbladder cancer cells, and to investigate the mechanism of the combined effects. Methods We performed immunohistochemistry to detect the PTEN expression in gallbladder cancer. We then investigated the combined effects of SST and DDP on cell proliferation in vitro with CCK-8 assay and analyzed the interaction between these two drugs using isobologram analysis. We also investigated the combined effects on cell proliferation in vivo using a xenograft nude mouse model. FITC-Annexin V/PI assay and TUNEL staining assay were performed to detect the proportion of apoptosis after combined treatment in vitro and in vivo. Reactive oxygen species and mitochondrial membrane potential were detected with DCFH-DA assay and JC-1 staining assay after the combined treatment. We finally detected the PTEN and p-AKT associated proteins using western blotting after the combined treatment. Results PTEN was abnormally decreased in gallbladder cancer tissues. PTEN expression was negatively correlated with cancer differentiation and was positively correlated with patients' survival time. DDP treatment decreased while combined treatment with SST induced PTEN expression and inhibited AKT activation by reversing resistance to DDP. Isolated SST or DDP treatment inhibited gallbladder cancer GBC-SD and SGC996 cell proliferation which was dose-dependence. These two drugs synergistically inhibited gallbladder cancer cell growth in vivo and in vitro. Isolated SST or DDP treatment induced cell apoptosis and combined treatment induced cell apoptosis the most. SST inhibited AKT activation but did not induce ROS. DDP induced ROS resulting in increased cell apoptosis. Either SST or DDP alone increased the levels of cytoplasmic cytochrome C protein and activated caspase-3. Conclusions SST enhanced growth inhibition by cisplatin in gallbladder cancer cells through inducing PTEN expression. This study provides the theoretical basis for further combined clinical chemotherapeutic applications. Key words: Somatostatin; Cisplatin; PTEN protein; Gallbladder cancer

Heparanase overexpression down-regulates syndecan-1 expression in a gallbladder carcinoma cell line.

ObjectiveTo discuss the relevance of heparanase and syndecan-1 and regulation of the heparanase-syndecan1 axis in the invasiveness of gallbladder carcinoma cells.Methods1. Generation of a gallbladder cancer cell line overexpressing a heparanase (GBD-SD) transgene. 2. Western blot analysis of syndecan-1 levels of GBD-SD and control gallbladder carcinoma (GBC-SD) cells. 3. RT-PCR analysis of syndecan-1 mRNA levels of GBD-SD and GBC-SD. 4. Evaluation of invasion and migration of GBD-SD and GBC-SD cells.Results1. Heparanase expression in GBD-SD cells was significantly increased. 2. The syndecan-1 mRNA level of GBD-SD cells was significantly lower compared with that of GBC-SD cells. 3. The syndecan-1 DNA copy number in GBD-SD cells was significantly lower compared with that of GBC-SD. 4. The invasiveness and migration of GBD-SD cells were significantly higher compared with GBC-SD cells.Conclusions1. The expression of heparanase negatively correlated with that of syndecan-1 in a gallbladder carcinoma cell line. 2. The expression of heparanase and syndecan-1 in gallbladder carcinomas negatively correlated, similar to other tumours. 3. The heparanase/syndecan1 axis in gallbladder carcinoma plays an important role in the invasion and metastasis, thus providing a new therapeutic target. 4. Further research is required to identify the detailed mechanisms.

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.

AICAR activates ER stress-dependent apoptosis in gallbladder cancer cells

AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine) is an AMP-activated protein kinase (AMPK) agonist, its activity in human gallbladder cancer cells was evaluated here. We show that AICAR provoked significant apoptosis in human gallbladder cancer cell lines (Mz-ChA-1, QBC939 and GBC-SD) and primary gallbladder cancer cells. AICAR-induced cytotoxicity in gallbladder cancer cells appears independent of AMPK activation. Inhibition of AMPK, via AMPKα shRNA knockdown or dominant negative mutation (T172A), failed to rescue GBC-SD cells from AICAR. Further, forced-activation of AMPK, by adding two other AMPK activators (A769662 and Compound 13), or expressing a constitutively-active mutant AMPKα (T172D), didn't induce GBC-SD cell death. Remarkably, AICAR treatment in gallbladder cancer cells induced endoplasmic reticulum (ER) stress activation, the latter was tested by caspase-12 activation, C/EBP homologous protein (CHOP) expression and IRE1/PERK phosphorylation. Contrarily, salubrinal (the ER stress inhibitor), z-ATAD-fmk (the caspase-12 inhibitor) or CHOP shRNAs significantly attenuated AICAR-induced gallbladder cancer cell apoptosis. Together, we conclude that AICAR-induced gallbladder cancer cell apoptosis requires ER stress activation, but is independent of AMPK.

Overexpression of LncRNA AFAP1-AS1 predicts poor prognosis and promotes cells proliferation and invasion in gallbladder cancer.

Long non-coding RNA actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) has been elucidated to be associated with some kinds of human cancers. However, whether lncRNA AFAP1-AS1 implicates in tumor development of gallbladder cancer (GBC) remains largely unknown. This study aims to elucidate the tumorigenic role and regulatory function of lncRNA AFAP1-AS1 in gallbladder cancer. We analyzed lncRNA AFAP1-AS1 expression by quantitative real time PCR (qRT-PCR) in 40 gallbladder cancer tissue and adjacent normal tissues, survival plots were generated by Kaplan-Meier analysis and the log-rank test. The expression levels of transcription factor Twist1 and epithelial-to mesenchymal transition (EMT) makers (E-cadherin and Vimentin) were detected by quantitative real time PCR and western blotting analysis after knockdown of lncRNA AFAP1-AS1. The expression levels of lncRNA AFAP1-AS1 were significantly elevated in GBC tissues and GBC cell lines. In addition, the expression level of lncRNA AFAP1-AS1 was significantly associated with tumor sizes and the higher expression of lncRNA AFAP1-AS1 was correlated with poor prognosis in GBC patients. Knockdown of LncRNA AFAP1-AS1 suppressed cell growth and invasion in NOZ and GBC-SD cells. Furthermore, we found that knockdown of LncRNA AFAP1-AS1 in GBC cells inhibited EMT by down-regulating the transcription factor Twist1 and Vimentin and up-regulated the E-cadherin. Our results suggested lncRNA AFAP1-AS1 was correlated with poor prognosis in GBC patients and lncRNA AFAP1-AS1 might be novel therapeutic target in gallbladder cancer.

IL-1β promotes proliferation and migration of gallbladder cancer cells via Twist activation

Increasing evidence has revealed a correlation between chronic inflammation and gallbladder cancer (GBC). However, the underlying molecular mechanisms remain to be elucidated. In the present study, secretion of interleukin (IL)-1β was examined in tissues of GBC, chronic cholecystitis and normal gallbladder, as well as in the supernatant of GBC-SD, SGC996 and HIBEpiC cells. The effect of IL-1β on the proliferation and migration of GBC cell lines was also evaluated. In addition, the role of Twist in IL-1β-induced proliferation of GBC cells was also studied. It was observed that the level of IL-1β protein in normal gallbladder tissue was low, while it was significantly increased in GBC and chronic cholecystitis tissues. The level of IL-1β protein and mRNA in GBC-SD and SGC996 cells was markedly higher than those in HIBEpiC cells. Exogenous IL-1β promoted the proliferation of GBC-SD and SGC996 cells in vitro and in vivo, and also promoted migration in vitro. The level of Twist protein was significantly increased following treatment with exogenous IL-1β. In addition, gene silencing of Twist blocked IL-1β-induced proliferation and migration of GBC-SD and SGC996 cells. Taken together, these results indicate that IL-1β promotes proliferation and migration of GBC cells via Twist activation.

The effects of miR-1207-5p expression in peripheral blood on cisplatin-based chemosensitivity of primary gallbladder carcinoma.

ObjectiveThe aim of this study was to investigate the association between miR-1207-5p expression in peripheral blood and the chemosensitivity of primary gallbladder carcinoma (PGBC).MethodsA total of 85 patients with PGBC undergoing preoperative chemotherapy were divided into effective (n=18) and ineffective (n=67) groups. Another 70 healthy individuals were selected as the control group. An miR-1207-5p mimic (mimic group), an inhibitor (inhibitor group), and a negative control (NC group) sequence were transfected into human gallbladder carcinoma GBC-SD cells. Real-time quantitative polymerase chain reaction was used to determine miR-1207-5p expression. After 48 hours of cisplatin treatment, CCK-8 method was used to detect cell proliferation and flow cytometry were performed to examine cell apoptosis.ResultsmiR-1207-5p expression in peripheral blood was significantly associated with tumor node metastasis staging of PGBC (P<0.05). Before chemotherapy, miR-1207-5p expression in patients was higher than in healthy individuals (P<0.05). After chemotherapy, the effective group had lower miR-1207-5p expression than the ineffective group (P<0.05). The rates of positive expression of Ki67 protein in the effective group were significantly lower than those in the ineffective group (P<0.05). Receiver operating characteristic curves showed that the area under curve, sensitivity, and specificity of miR-1207-5p used to diagnose PGBC were 0.898, 77.6%, and 97.1% at a cutoff of 1.470, respectively. After 48 hours of cisplatin treatment, compared with the NC group and nontransfected (non-T) group, the mimic group had decreased rates of cell inhibition and apoptosis, but the inhibitor group had increased rates (all P<0.05). The expression levels of caspase3 protein were increased in the mimic group and decreased in the inhibitor group. Cell survival rates in the mimic group at different time points after cisplatin treatment were significantly higher than the corresponding rates in the NC and non-T groups, whereas the cell survival rates in the inhibitor group were significantly lower than the rates in the NC and non-T groups (all P<0.05). The concentration and action time of cisplatin were negatively associated with the cell survival rate in each group (all P<0.05).ConclusionCisplatin-based chemosensitivity of PGBC increased as expression of miR-1207-5p in peripheral blood declined. Thus, miR-1207-5p appears to be a promising and novel chemosensitizer for the treatment of PGBC.

Proteasome inhibitor MG132 potentiates TRAIL-induced apoptosis in gallbladder carcinoma GBC-SD cells via DR5-dependent pathway.

TRAIL is a tumor-selective apoptosis-inducing cytokine playing a vital role in the surveillance and elimination of some tumor cells. However, some tumors are resistant to TRAIL treatment. Proteasome inhibitor MG132 exhibits anti-proliferative and pro-apoptotic properties in many tumors. In this study, we demonstrated that proteasome inhibitor MG132 invitro and invivo potentiates TRAIL-induced apoptosis in gallbladder carcinoma GBC-SD cells. MG132 was able to inhibit the proliferation of GBC-SD cells and induce apoptosis in a dose-dependent manner. The induction of apoptosis by proteasome inhibitor MG132 was mainly through the extrinsic apoptotic pathways of caspase activation such as caspase-8, caspase-3 and PARP cleavage. In addition, this process was also dependent on the upregulation of death receptor 5 (DR5), which promoted TRAIL-induced apoptosis in GBC-SD cells. Taken together, these findings indicate that MG132 possesses anti-gallbladder cancer potential that correlate with regulation of DR5-dependent pathway, and suggest that MG132 may be a promising agent for sensitizing GBC-SD cells to TRAIL-induced apoptosis.

TNF-alpha promotes lymphangiogenesis and lymphatic metastasis of gallbladder cancer through the ERK1/2/AP-1/VEGF-D pathway.

BackgroundTumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gallbladder cancer (GBC). Vascular endothelial growth factor D (VEGF-D) is a key lymphangiogenic factor that is associated with lymphangiogenesis and lymph node metastasis in GBC. However, whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC remains undetermined.MethodsThe expression of VEGF-D in patient specimens was detected by immunohistochemistry and the relationship between VEGF-D in the tissue and TNF-α in the bile of the matching patients was analyzed. The VEGF-D mRNA and protein levels after treatment with exogenous TNF-α in NOZ, GBC-SD and SGC-996 cell lines were measured by real-time PCR and ELISA. The promoter activity and transcriptional regulation of VEGF-D were analyzed with the relative luciferase reporter assay, mutant constructs, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, RNA interference and Western blotting. Inhibitors of JNK, p38 MAPK and ERK1/2 were used to explore the upstream signaling effector of AP-1. We used lentiviral vector expressing a VEGF-D shRNA construct to knockdown VEGF-D gene in NOZ and GBC-SD cells. The role of the TNF-α-VEGF-D axis in the tube formation of human dermal lymphatic endothelial cells (HDLECs) was determined using a three-dimensional coculture system. The role of the TNF-α - VEGF-D axis in lymphangiogenesis and lymph node metastasis was studied via animal experiment.ResultsTNF-α levels in the bile of GBC patients were positively correlated with VEGF-D expression in the clinical specimens. TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo.ConclusionTaken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway.

Artemisinin inhibits proliferation of gallbladder cancer cell lines through triggering cell cycle arrest and apoptosis

To evaluate the effects of artemisinin on proliferation, cell cycle and apoptosis of gallbladder cancer cells. Gallbladder carcinoma cell lines(GBC-SD and NOZ)were cultured in vitro. The effects of artemisinin in different concentration on proliferation of the two cell lines in vitro were examined using MTT assay. The cell cycle distribution of GBC-SD and NOZ cells 24 h after treatments with artemisinin(20 μmol/L) were examined using flow cytometry. The apoptosis of GBC-SD and NOZ cells 24 h after treatments with artemisinin (20 μmol/L) were examined using Annexin V/PI staining.The expressions of p-ERK1/2, CDK4, cyclin D1, p16, cytochrome C and caspase-3 were examined by Western blot assay. t-test and one way ANOVA were used to evaluate the differences between two groups and more than two groups, respectively. The cell proliferation was significantly inhibited by artemisinin, the IC50 of artemisinin against GBC-SD and NOZ cells were 14.05 μmol/L and 12.42 μmol/L, respectively.Artemisinin induced cycle arrest, and G1 population of GBC-SD and NOZ cells increased to 74.60% and 78.86%. Cell apoptosis and apoptotic population of GBC-SD and NOZ cells were increased to 15.67% and 16.51% after dealt with artemisinin, respectively. In addition, expression of p16 was increased, and expressions of p-ERK1/2, CDK4 and cyclin D1 were down-regulated by artemisinin(all P<0.05). Cytochrome C was released from mitochondria to cytoplasm leading to the activation of caspase-3 and PARP after dealt with artemisinin(P<0.05). The inhibition effect of artemisinin on the proliferation gallbladder cancer cells is accompanied by down-regulation of ERK1/2 signaling pathway, G1 phase arrest and triggering caspase-3-mediate apoptosis.

NOX1 mediates chemoresistance via HIF1α/MDR1 pathway in gallbladder cancer

NADPH oxidase 1 (NOX1) plays a key role in tumorigenesis and metastasis through generating reactive oxygen species (ROS), an important intracellular signaling molecule. However, how it is expressed in gallbladder cancer (GBC) tissue sample and whether it associates with GBC chemoresistance have never been investigated. Our study analyzed the relationship between NOX1 expression and cisplatin-sensitivity both in vivo and in vitro. We found that reduced NOX1 expression promoted cisplatin efficiency in GBC-SD cells, whereas overexpression of which potentially inhibited the sensitivity of cisplatin in SGC-996 cells. Further study into the mechanism we found that increased NOX1 expression elevated intracellular ROS levels, which then activated HIF-1α/MDR1 pathway. These findings established NOX1 a novel accelerant of chemoresistance in GBC, and NOX1-targeted therapeutics might be exploited as a strategy for increasing the efficacy of cisplatin treatment.

20(S)-ginsenoside Rg3 promotes senescence and apoptosis in gallbladder cancer cells via the p53 pathway.

Gallbladder cancer (GBC), the most frequent malignancy of the biliary tract, is associated with high mortality and extremely poor prognosis. 20(S)-ginsenoside Rg3 (20(S)-Rg3) is a steroidal saponin with high pharmacological activity. However, the anticancer effect of 20(S)-Rg3 in human GBC has not yet been determined. In this study, we primarily found that 20(S)-Rg3 exposure suppressed the survival of both NOZ and GBC-SD cell lines in a concentration-dependent manner. Moreover, induction of cellular senescence and G0/G1 arrest by 20(S)-Rg3 were accompanied by a large accumulation of p53 and p21 as a result of murine double minute 2 (MDM2) inhibition. 20(S)-Rg3 also caused a remarkable increase in apoptosis via the activation of the mitochondrial-mediated intrinsic caspase pathway. Furthermore, intraperitoneal injection of 20(S)-Rg3 (20 or 40 mg/kg) for 3 weeks markedly inhibited the growth of xenografts in nude mice. Our results demonstrated that 20(S)-Rg3 potently inhibited growth and survival of GBC cells both in vitro and in vivo. 20(S)-Rg3 attenuated GBC growth probably via activation of the p53 pathway, and subsequent induction of cellular senescence and mitochondrial-dependent apoptosis. Therefore, 20(S)-Rg3 may be a potential chemotherapeutic agent for GBC therapy.

Zinc finger X-chromosomal protein (ZFX) is a significant prognostic indicator and promotes cellular malignant potential in gallbladder cancer

The Zinc finger X-chromosomal protein (ZFX), a novel member of the Krueppel C2H2-type zinc finger protein family, has been implicated in multiple human cancers. However, the clinical significance of ZFX expression in gallbladder cancer (GBC) remains largely unknown. In this study, we focused on the clinical significance, biological function and mechanism of ZFX in GBC, and found that ZFX protein overexpression was frequently detected in GBC tissues. The expression of ZFX was significantly correlated with histological grade, perineural invasion, and margin status and lead to a significantly poorer prognosis in GBC patients(P <0.001). Furthermore, knockdown of ZFX result in significant inhibition of proliferation, migration, invasion and cause cell cycle arrest in GBC-SD cells, while over-expression of ZFX in NOZ shows the opposite results. Activation of PI3K/AKT pathway maybe the potential mechanism behind these effects. In conclusion, ZFX may serve as a oncogene and could be used as a potential prognostic marker and genetic treatment target for GBC patients.

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-.

Oleanolic acid induces mitochondrial-dependent apoptosis and G0/G1 phase arrest in gallbladder cancer cells.

Oleanolic acid (OA), a naturally occurring triterpenoid, exhibits potential antitumor activity in many tumor cell lines. Gallbladder carcinoma is the most common malignancy of the biliary tract, and is a highly aggressive tumor with an extremely poor prognosis. Unfortunately, the effects of OA on gallbladder carcinoma are unknown. In this study, we investigated the effects of OA on gallbladder cancer cells and the underlying mechanism. The results showed that OA inhibits proliferation of gallbladder cancer cells in a dose-dependent and time-dependent manner on MTT and colony formation assay. A flow cytometry assay revealed apoptosis and G0/G1 phase arrest in GBC-SD and NOZ cells. Western blot analysis and a mitochondrial membrane potential assay demonstrated that OA functions through the mitochondrial apoptosis pathway. Moreover, this drug inhibited tumor growth in nude mice carrying subcutaneous NOZ tumor xenografts. These data suggest that OA inhibits proliferation of gallbladder cancer cells by regulating apoptosis and the cell cycle process. Thus, OA may be a promising drug for adjuvant chemotherapy in gallbladder carcinoma.

Effects of CXC chemokine receptor 4 on proliferation of primary gallbladder cancer cells

Objective To investigate the effect of CXC chemokine receptor 4(CXCR4)on the growth of the gallbladder cancer cell line GBC- SD. Methods Using real- time quantitative polymerase chain reaction(Real- time PCR), Western blotting and flow cytometry, the expression of CXCR4 was detected in GBC- SD cells.After GBC- SD cells were transfected with CXCR4 shRNA, the proliferation was measured by MTT. Results The level of mRNA and protein of CXCR4 in GBCSD cells, as well as the surface CXCR4 molecule on GBC- SD cells were all highly expressed than that in ECV-304 alls[(70.30±3.50)% vs.(0.76±0.11)%].The proliferation of GBC- SD cells was inhibited when CXCR4 was knocked down(3.19±0.16 vs.1.63±0.23). Conclusion The CXCR4 was highly expressed in GBC- SD cells, and the targeting of stromal cell derived factor- 1(SDF- 1)/CXCR4 axis may be a novel strategy to control the progress of gallbladder cancer. Key words: CXC chemokine receptor 4; Cell proliferation; Gallbladder cancer

Fibronectin promotes cell proliferation and invasion through mTOR signaling pathway activation in gallbladder cancer

Fibronectin (FN), a heterodimeric glycoprotein overexpressed in several types of tumors, has been implicated in cancer progression via the activation of integrin-mediated pro-oncogenic pathways. The FN level in human bile fluid is dramatically increased in malignant biliary diseases; however, FN expression and its biological functions in gallbladder cancer (GBC) remain unknown. In this study, we found that FN was overexpressed in GBC tissues and was associated with a worse prognosis in GBC patients. In vitro experimental studies indicated that exogenous FN significantly enhanced cell proliferation, invasion and active MMP-9 secretion in human GBC cell lines (GBC-SD and NOZ). Moreover, the key kinases of the mTOR signaling pathway, including FAK, Akt, mTOR and 4E-BP1, were markedly activated in a time-dependent manner in FN-treated GBC-SD and NOZ cells. The IHC statistical analyses validated that FN expression was positively correlated with the phosphorylation levels of the 4E-BP1 protein in GBC tissues. Furthermore, rapamycin, a specific inhibitor of mTOR, almost completely blocked FN-induced phosphorylation of 4E-BP1 and also partially abrogated the stimulatory effects of FN on GBC cell proliferation and invasion. In vivo, FN treatment significantly promoted the proliferation and metastasis of GBC cells and markedly activated Akt/mTOR/4E-BP1 signaling cascade. These findings demonstrate that FN may play a critical role in the modulation of cell proliferation and invasion via mTOR signaling pathway activation during GBC progression.