Gallbladder Cancer Cell Lines Research Articles

Tea polyphenols induce S phase arrest and apoptosis in gallbladder cancer cells.

Gallbladder cancer (GBC) is the most common malignancy in the biliary tract. Without effective treatment, its prognosis is notoriously poor. Tea polyphenols (TPs) have many pharmacological and health benefits, including antioxidant, anti-inflammatory, anti-tumor, anti-thrombotic, antibacterial, and vasodilatory properties. However, the anti-cancer effect of TPs in human gallbladder cancer has not yet been determined. Cell viability and colony formation assay were used to investigate the cell growth. Cell cycle and apoptosis were evaluated by flow cytometry analysis. Western blot assay was used to detect the expression of proteins related to cell cycle and apoptosis. Human tumor xenografts were used to examine the effect of TPs on gallbladder cancer cells in vivo. TPs significantly inhibited cell growth of gallbladder cancer cell lines in a dose- and time-dependent manner. Cell cycle progression in GBC cells was blocked at the S phase by TPs. TPs also induced mitochondrial-related apoptosis in GBC cells by upregulating Bax, cleaved caspase-3, and cleaved PARP expressions and downregulating Bcl-2, cyclin A, and Cdk2 expressions. The effects of TPs on GBC were further proven in vivo in a mouse xenograft model. Our study is the first to report that TPs inhibit GBC cell growth and these compounds may have potential as novel therapeutic agents for treating gallbladder cancer.

Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133.

The present study aimed to isolate and identify the properties of the cluster of differentiation (CD)133+ subset in human gallbladder cancer cells. The CD133+ and CD133− subpopulations of the GBC-SD cell line were separated using immunomagnetic separation, and the biological features of the two subpopulations were analyzed in vitro and in vivo. In particular, the present study aimed to determine whether the two subpopulations were resistant to anti-tumor reagents and to identify the underlying molecular mechanisms involved. Following cell sorting of GBC-SD cells using immunomagnetic beads, 90.2±2% of cells were identified as CD133+. Immunofluorescence confirmed that CD133 was expressed at higher levels in the Cd133+ group compared with the CD133− group. The proliferation of the CD133+ group was significantly increased compared with the CD133− group in vitro and in vivo. Following treatment with fluorouracil or gemcitabine, cells in the CD133+ group exhibited a decreased sensitivity to these drugs. The number of transmembrane cells was significantly increased in the CD133+ group compared with the CD133− group. In addition, the expression levels of ATP binding cassette subfamily G member 2, CD44, C-X-C motif chemokine receptor 4 (CXCR4), phosphorylated-protein kinase B (Akt) and CD133 in the CD133+ group were significantly increased, compared with those in the CD133− group. In CD133+ GBC-SD cells, stromal cell-derived factor 1α (SDF-1α) or treatment with AMD3100, an inhibitor of CXCR4, promotes or suppresses the SDF-1α/CXCR4 axis, respectively, resulting in increased or decreased CD133 expression through the Akt signaling pathway. Inhibition of the Akt signaling pathway resulted in decreased CD133 expression in GBC-SD cells. Immunomagnetic beads were successfully used for isolation of the CD133+ subset from GBC-SD cells. Furthermore, the CD133+ subset revealed an increased potential for tumor formation, cell proliferation, invasion and resistance to chemotherapeutic agents with expression of stem cell-associated genes. Therefore, in GBC-SD cells, the CXCR4/Akt/CD133 signaling pathways may be activated.

Downregulation of BRD4 inhibits gallbladder cancer proliferation and metastasis and induces apoptosis via PI3K/AKT pathway.

Bromodomain containing protein 4 (BRD4) has been demonstrated to play a critical role in tumor progression. However, the expression and function of BRD4 in gallbladder cancer (GBC) are still unknown. In this study, we report that BRD4 expression level was significantly upregulated in GBC tissues and GBC cell lines. We explored the correlation between BRD4 levels and clinicopathological data of GBC patients. The high expression level of BRD4 was notably correlated with the poor prognosis of GBC patients. Knockdown of BRD4 suppressed proliferation and migration in NOZ and EH-GB1 cells. The depletion of BRD4 in GBC cell lines resulted in obvious cell apoptosis and downregulated the expression levels of Bcl-2, p-PI3K and p-AKT. In vivo, tumor volumes of nude mice were significantly decreased in BRD4 silenced group. Our data suggested that downregulation of BRD4 in GBC cells induced apoptosis by PI3K/AKT pathway. Inhibition of BRD4 expression may be a novel therapeutic strategy for patients with GBC.

Isoform switch of CD44 induces different chemotactic and tumorigenic ability in gallbladder cancer.

Gallbladder cancer (GBC) is one of the most unfavorable prognostic tumor, and immediate growth and distant metastasis are important factors associated with the poor prognosis of patients with this disease. Standard and variant isoforms of CD44 are associated with tumor growth, metastasis, and epithelial-mesenchymal transition (EMT), although their roles in GBC are unclear. We investigated the relationship between the CD44 isoforms with EMT, chemotaxis, and tumorigenicity. We analyzed CD44 expression in the GBC cell line NOZ and found that it comprises a major population that expressed CD44std+/CD44v9− (CD44s) and the minor population that expressed CD44std−/CD44v9+ (CD44v). CD44s cells exhibited increased chemotaxis and invasiveness compared with CD44v cells in in vitro cell migration and invasion assays. CD44s cells expressed higher and lower levels of mRNAs that encode vimentin and E-cadherin, respectively, compared with those of CD44v cells. CD44s cells expressed high levels of the transcription factors ZEB1 and ZEB2 that mediate EMT, and low levels of a splicing factor ESRP1 that controls the CD44 isoform switch. We performed in vivo mouse xenotransplantation analyses of CD44s and CD44v cells and found that CD44v cells exhibited relatively increased tumorigenicity. Immunohistochemical analysis of tissue microarrays revealed that high levels of CD44v9 and CD44std were associated with poorer prognosis. The expression of CD44std was also associated with poorly differentiated tumors and distant metastasis. In conclusion, CD44s was associated with a mesenchymal phenotype, increased chemotaxis and invasiveness, and decreased tumorigenicity. In contrast, CD44v cells exhibited an epithelial phenotype, decreased chemotaxis, decreased invasiveness, and increased tumorigenicity. These findings suggest that CD44v and CD44s cells play differently important roles in the progression and metastasis of GBC and the isoform switch triggers EMT.

Abstract 5794: TrkB-mediated signaling contributes to malignant phenotypes of gallbladder cancer

Abstract Background: Brain derived neurotrophic factor (BDNF)/ Tropomyosin-related kinase B (TrkB) signaling has been shown to be associated with aggressive phenotype in some cancers. However, the contribution of BDNF/TrkB signaling to gallbladder cancer (GBC), one of refractory malignancies, still remains unclear. This study aims to analyze the biological significance of BDNF/TrkB signaling in GBC. Methods: 1) Clinical experiment: 69 patients with primary GBC who underwent curative surgical resection were enrolled in this study. We investigated TrkB expression by immunohistochemistry and analyzed the correlation between TrkB expression and clinicopathological findings. 2) In vitro experiment: BDNF/TrkB signaling was inhibited using k252a or siRNA, and was activated by recombinant human BDNF (rhBDNF). Then, whether BDNF/TrkB signaling contributes to the biological function was estimated by proliferation assay and Matrigel invasion assay, using TrkB-expressing 5 GBC cell lines (NOZ, TGBC2TKB, GBd15, TYGBK-1, TYGBK-8). Furthermore, we determined the mechanisms of invasion in terms of epithelial mesenchymal transition (EMT) and matrix metalloproteinases (MMPs). We also examined the relationship between TrkB expression and the level of vascular endothelial growth factors (VEGFs) and hypoxia-inducible factor-1α (HIF-1α) in GBC. 3) In vivo experiment: Tumorigenesis and tumor growth of TrkB siRNA-transfected GBC cells were analyzed, using xenograft mice model. Results: 1) Clinical results; TrkB expression was detected in 63 (91.3%) GBC specimens. TrkB expression in the invasive front correlated with T factor (p=0.0391) and clinical staging (p=0.0391). Overall survival was lower in patients with high TrkB expression in the invasive front than in those with low TrkB expression (p=0.0363). 2) In vitro results; Proliferation was unaffected by rhBDNF treatment; however, K252a treatment and TrkB siRNA transfection decreased proliferation. rhBDNF treatment increased invasiveness by inducing EMT and activating MMP-2/MMP-9, whereas K252a treatment abrogated these effects. TrkB or BDNF siRNA transfection suppressed invasiveness. TrkB siRNA transfection decreased HIF-1α, VEGF-A, and VEGF-C/-D expressions. 3) In vivo results; TrkB siRNA transfection decreased tumorigenicity and tumor growth in NOZ and TYGBK-1. Conclusion: These findings demonstrate that TrkB-mediated signaling contributes to the induction of malignant phenotypes (proliferation, invasiveness, angiogenesis, lymphangiogenesis, and tumorigenesis) in GBC. Citation Format: Makoto Kawamoto, Hideya Onishi, Keigo Ozono, Akio Yamasaki, Akira Imaizumi, Masafumi Nakamura. TrkB-mediated signaling contributes to malignant phenotypes of gallbladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5794. doi:10.1158/1538-7445.AM2017-5794

Abstract 589: Hedgehog signaling augments PDL-1 expression in cancer cells under hypoxic condition to inhibit antitumor effects by activated lymphocytes

Abstract [Background] Hedgehog (Hh) signaling is activated under hypoxic condition in cancerous tissue. This is thought to be one of the mechanisms of the induction of malignant phenotype of cancers. We have shown that Hh inhibitor decreases proliferation, invasiveness, and tumorigenesis. We have also revealed that Hh signaling plays pivotal roles for the maintenance of function in activated lymphocytes and dendritic cells under hypoxia. Recently, immune checkpoint inhibitor takes much attention, however, response rate is still limited. The mechanisms regulating PDL-1 expression also remains unclear. In this study, to increase reponse rate of immune checkpoint inhibitor, we investigated whether hypoxia-induced activation of Hh signaling contributes to PDL-1 expression in cancer and whether it affects the antitumor function of activated lymphocytes. [Materials and Methods] Pancreatic cancer cell line, Panc-1, gallbladder cancer cell line, NOZ and GBd, and lung small cell cancer cell line, SBC-5 were used as target cancer cells. Human lymphocytes derived from PBMC were activated by using anti CD3 mAb and IL-2, and were used as effector cells. For normoxic conditions, cells were cultured in 5% CO2 and 95% air. For hypoxic conditions, cells were cultured in 1% O2, 5% CO2, and 94% N2 using a multigas incubator. For Hh signaling inhibition, cyclopamine, polysaccharide-K (PSK), and small interfering RNA targeting Gli1, Smo, MAML3, and RBPJ were used. Cell numbers were counted by light microscope. Expression of cell surface molecules was estimated by FACS. [Results] 1) Hypoxia augmented PDL-1 expression in all 4 cancer cell lines. 2) Inhibition of Hh signaling using MAML3 siRNA, cyclopamine and PSK reduced PDL-1 expression under hypoxia in all 4 cancer cell lines. 3) When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor, activated lymphocyte cell numbers significantly increased under hypoxia. 4) In contrast, this increase was abrogated when cancer cells were treated with a PDL-1 neutralizing antibody. 5) When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor and/or anti-PDL-1 Ab, the percent of CD8+ lymphocytes decreased in both the cyclopamine- and anti-PDL-1-treated groups under hypoxic conditions, while there was no significant change in CD3 expression. 6) NKG2D expression increased on activated lymphocytes in anti PDL-1Ab-treated group. [Conclusion] These results suggest that Hh signaling is one of regulatory pathways of PDL-1 expression under hypoxia and that inhibiting Hh signaling may induce lymphocyte antitumor activity. Therefore, Hh inhibition could be a promising drug target, not only as a direct tumor suppressor agent, but also as a PDL-1 inhibitor. Citation Format: Hideya Onishi, Akiko Fujimura, Yasuhiro Oyama, Makoto Kawamoto, Akio Yamasaki, Takashi Morisaki. Hedgehog signaling augments PDL-1 expression in cancer cells under hypoxic condition to inhibit antitumor effects by activated lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 589. doi:10.1158/1538-7445.AM2017-589

EIF3D promotes gallbladder cancer development by stabilizing GRK2 kinase and activating PI3K-AKT signaling pathway

Recent evidence suggests that dysregulated eIF3d expression may be critical in various genetic disorders as well as cancer. In this study, we observed that EIF3d levels increased in gallbladder cancer (GBC) samples compared with non-tumor tissue. High eIF3d levels were associated with advanced tumor stage and metastasis and were correlated with poor prognosis in 92 patients with GBC. Depletion of EIF3d in GBC cell lines inhibited cell proliferation, colony formation and metastasis and induced apoptosis and cell cycle arrest in vitro and in vivo. In contrast, ectopic expression of eIF3d had the opposite effects. Moreover, in this study, we revealed that a novel non-translational factor function of eIF3d mediated its protumoral effects. In details, eIF3d stabilizes GRK2 protein by blocking ubiquitin-mediated degradation, consequently activates PI3K/Akt signaling, and promotes GBC cell proliferation and migration. In conclusion, eIF3d promotes GBC progression mainly via eIF3d–GRK2–AKT axis and it may be used as a prognostic factor. The therapeutic targeting of eIF3d–GRK2 axis may be a potential treatment approach for GBC.

Ring finger protein 125, as a potential highly aggressive and unfavorable prognostic biomarker, promotes the invasion and metastasis of human gallbladder cancers via activating the TGF- β1-SMAD3-ID1 signaling pathway.

Human gallbladder cancer (GBC) is a lethal aggressive malignant neoplasm. Identification of potential molecular biomarkers and development of targeted therapeutics for GBC patients is very necessary. In this study, we firstly investigated the correlation between ring finger protein 125 (RNF125) expression and the metastasis and prognosis of GBC, and the underlying molecular mechanism. RNF125 expression in a cohort of GBC tissues was examined; its correlation with clinicopathological and prognostic factors of GBC patients was analyzed. Moreover, the metastasis-related difference expressed genes in highly and lowly aggressive GBC cell lines were identified; and the influence of RNF125 knockdown on the metastatic phenotypes and characteristic EMT markers in highly aggressive GBC NOZ cells was detected. Furthermore, the underlying molecular mechanism of RNF125 effect was explored. The results showed that RNF125 was highly expressed in GBC tissues and related with aggressive characteristics such as Nevin stage (P = 0.041) etc. and unfavorable prognosis of GBC patients (P = 0.023, log-rank test). And, RNF125 was proved to a positive metastasis-related gene in vitro. RNF125 knockdown inhibited the invasion and migration, enhanced the adhesion, upregulated E-cadherin and β-catenin expression, and downregulated vimentin and N-cadherin expression (all P < 0.001) of NOZ cells in vitro. RNF125 promoting effect on GBC tumor progression was identified to relate with the activation of TGF-β1-SMAD3-ID1 signaling pathway. These findings firstly confirm that high RNF125 expression is related with aggressive characteristics and unfavorable prognosis of GBC patients; RNF125 promotes the invasion and metastasis of human GBCs via activating the TGF-β1-SMAD3-ID1 signaling pathway.

Tumor Lysate Loaded Dendritic Cells Induce a T cell Specific Antitumor Response Against Gallbladder Cancer

Abstract Introduction Although infrequent, Gallbladder cancer (GBC) has high incidence rates in South America. In fact, Chile has the highest GBC incidence and mortality worldwide. Currently, surgery is the only effective treatment, and the five-year survival rate is less than 10%. Previously, we demonstrated that allogeneic melanoma cell lysates-loaded dendritic cells (DCs) provide a standardized, applicable tumor-specific vaccine; capable to induce a cell mediated immune response in melanoma patients, improving survival. Here, we investigated the effect of tumor lysates derived from gallbladder cancer cell (GCC) lines on immature DCs and their capacity to induce a T cell mediated anti GBC responses in vitro. Material and Methods DCs were stimulated with GCC lysates and analyzed by flow cytometry for a panel of surface markers. Autologous CD3+ T cells were co-cultured with these DCs for 14 days. CD4+ and CD8+ T cells were analyzed by flow cytometry for activation and chemokine receptor expression. CD8+ T cells were isolated and co-cultured with GCC lines GBd1, TGBC-2TKB and melanoma cell line Mel1 for 16hrs. IFN-γ secretion was measured by ELISPOT. Results All GCC lysates induced DCs maturation, increasing surface expression of MHC-I, MHC-II, CD80, CD83, CD86, CCR7. DCs maturated with GCC lysate induced CD3+ T cells activation, increasing expression of CD25, CD69, CXCR3, CXCR4 in both CD4+ and CD8+ T cells compared with non-stimulated lymphocytes. Additionally, CD8+ T cells co-cultured with GBC-loaded DCs, released higher amounts of IFN-γ than controls. Discussion Based on these results, DCs maturated with GCC lysates are capable to induce specific T cells activation against GBC and can be considered for future immunotherapy approaches.

The NmrA-like family domain containing 1 pseudogene Loc344887 is amplified in gallbladder cancer and promotes epithelial-mesenchymal transition.

The expression pattern and biological role of long non-coding RNA (lncRNA) in cancer has been reported to be involved in many cancers. Here, we report the expression and biological role of a newly discovered lncRNA NmrA-like family domain containing 1 pseudogene (Loc344887) in gallbladder cancer (GBC). In this study, we found that the expression of Loc344887 was significantly elevated in GBC tissues and cell lines when compared with matched normal tissues and normal epithelial bile duct cell line, respectively. High Loc344887 was associated with larger tumor size. Loc344887 was upregulated significantly after ectopic expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in GBC cells. Downregulation of Loc344887 in GBC cells suppressed cell proliferation, blocked cells in G0/S phase, and decreased the migration and invasion cell numbers. In addition, downregulation of Loc344887 decreased the expression of transcription factor Twist, mesenchymal marker Vimentin, and N-cadherin and increased the expression of epithelial maker E-cadherin, which could prompt a mesenchymal-to-epithelial transition phenotype. These results demonstrated that Loc344887 might contribute to cell proliferation and epithelial-to-mesenchymal transition process in GBC, which might be a potential therapeutic target.

TROP2 promotes proliferation, migration and metastasis of gallbladder cancer cells by regulating PI3K/AKT pathway and inducing EMT.

The human trophoblast cell surface antigen 2 (TROP2) is overexpressed in many cancers. However, its effect on proliferation, migration and metastasis of gallbladder cancer remains unclear. In this study, we found that TROP2 was highly expressed in gallbladder cancer. Overexpression of TROP2 was associated with poor prognosis. Knockdown of TROP2 in gallbladder cancer cell lines strongly inhibited the cell proliferation, clone formation, invasion and migration in vitro, while TROP2 overexpression had opposite effects. In addition, knockdown of TROP2 increased the expression of total PTEN, p-PTEN and PDK-1 but reduced p-AKT via PI3K/AKT pathway. TROP2 downregulation also inhibited vimentin and increased E-cadherin expression during epithelial-mesenchymal transition (EMT). Moreover, gallbladder cancer cells with TROP2 knockdown formed smaller xenografted tumors in vivo. In consistent with in vitro results, TROP2 inhibition decreased Akt phosphorylation, increased PTEN expression and postponed EMT of gallbladder cancer cells in vivo. In conclusion, we revealed that TROP2 promoted the proliferation, migration and metastasis of gallbladder cancer cells by regulating PI3K/AKT pathway and inducing EMT. TROP2 could serve as a potential prognostic biomarker and therapeutic target for the clinical management of 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.

Tropomyosin-related kinase B mediated signaling contributes to the induction of malignant phenotype of gallbladder cancer.

This study aims to demonstrate the clinical and biological significance of Brain derived neurotrophic factor (BDNF)/Tropomyosin-related kinase B (TrkB) signaling in gallbladder cancer (GBC) through a series of in vitro and in vivo experiments. TrkB expression was detected in 63 (91.3%) out of 69 surgically resected primary GBC specimens by immunohistochemistry. TrkB expression in the invasive front correlated with T factor (p=0.0391) and clinical staging (p=0.0391). Overall survival was lower in patients with high TrkB expression in the invasive front than in those with low TrkB expression (p=0.0363). In vitro experiment, we used five TrkB-expressing GBC cell lines with or without K-ras mutation. TrkB-mediated signaling increased proliferation and the invasiveness by inducing epithelial mesenchymal transition, and activating matrix metalloproteinases-2 (MMP-2) and MMP-9. Inhibition of TrkB-mediated signaling also decreased hypoxia-inducible factor-1α, vascular endothelial growth factor A (VEGF-A), VEGF-C, and VEGF-D expression. In vivo experiment, inhibition of TrkB-mediated signaling suppressed tumorigenicity and tumor growth in GBC. These findings demonstrate that TrkB-mediated signaling contributes to the induction of malignant phenotypes (proliferation, invasiveness, angiogenesis, lymphangiogenesis, and tumorigenesis) in GBC, and could be a promising therapeutic target regardless of K-ras mutation status.

MicroRNA-125b predicts clinical outcome and suppressed tumor proliferation and migration in human gallbladder cancer.

We intended to investigate the functional role and clinical relevance of microRNA-125b in human gallbladder cancer. Quantitative real-time polymerase chain reaction was used to examine microRNA-125b expression in gallbladder cancer cell lines, and 79 pairs of gallbladder cancer and normal gallbladder clinical tissues. Clinical correlations between tumorous microRNA-125b expression and gallbladder cancer patients' clinicopathological variances or overall survivals were statistically analyzed. In gallbladder cancer cell lines, TYGBK-8 and G-415 cells, microRNA-125b was upregulated to examine its regulatory effect on gallbladder cancer proliferation and migration in vitro. MicroRNA-125b was significantly downregulated in gallbladder cancer cell lines and human gallbladder cancer tumors. MicroRNA-125b in gallbladder cancer was significantly correlated with patients' clinical stage, tumor differentiation, lymph metastasis, and tumor invasion. Low tumorous microRNA-125b expression was also found to be associated with poor overall survivals among gallbladder cancer patients. In vitro studies demonstrated that microRNA-125b upregulation significantly suppressed proliferation and migration in TYGBK-8 and G-415 cells. Tumorous microRNA-125b is an independent prognostic biomarker for patients with gallbladder cancer and possibly acts as a tumor suppressor in gallbladder cancer.

The ERK/MAPK pathway is overexpressed and activated in gallbladder cancer

Gallbladder cancer (GBC) is a highly fatal disease with poor prognosis and few therapeutic alternatives. Molecular profiling has revealed that the deregulation in the ERK/MAPK signaling pathway plays a crucial role in many disease and malignancies, including GBC. The aim of this study was to measure the expression of ERK1/2 and p-ERK1/2 in a population with high GBC-related mortality, such as the Chilean population, and characterize the protein expression of this ERK/MAPK pathway in seven GBC cell lines. Immunohistochemistry (IHC) for ERK1/2 and p-ERK1/2 was performed in 123 GBC tissues and 37 chronic cholecystitis (CC) tissues. In addition, protein expression analysis by western blot for ERK1/2, p-ERK1/2, EGFR, ERBB2 and ERBB3 were performed in seven GBC cell lines (GB-d1, G415, NOZ, OCUG-1, TGBC-1, TGBC-2 and TGBC-24). A higher ERK1/2 and p-ERK1/2 expression was found in GBC tissues compared to chronic cholecystitis (CC) tissues (P<0.001). However, neither significant differences in overall survival nor significant associations with any of the clinicopathological features were found by comparing low and high expression of both ERK1/2 and p-ERK1/2. Western blot analysis of seven GBC cell lines showed that, in general, GB-d1, G415 and NOZ cells evidenced a strong expression of ERK1/2, p-ERK1/2, EGFR, ERBB2 and ERBB3. Therefore, ERK1/2 and p-ERK1/2 seem to be important in the development of GBC and GB-d1, G415 and NOZ cell lines may be used as experimental models for further in vitro and in vivo studies that help to decipher the role of MAPK/ERK pathway in gallbladder carcinogenesis.

Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer.

Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors.In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies.In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05).The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%).Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.

Establishment and characterization of a novel highly aggressive gallbladder cancer cell line, TJ-GBC2

BackgroundHuman gallbladder cancer (GBC) is an aggressive malignant neoplasm with a poor prognosis. The development of ideal tools for example tumor cell lines for investigating biological behavior, metastatic mechanism and potential treatment in GBCs is essential. In present study, we established and characterized a GBC cell line derived from primary tumor.MethodsPrimary culture method was used to establish this cell line from a primary GBC. Light and electron microscopes, flow cytometry, chromosome analysis, heterotransplantation and immunohistochemistry were used to characterize the epidemic tumor characteristics and phenotypes of this cell line.ResultsA novel GBC cell line, named TJ-GBC2, was successfully established from primary GBC. This cell line had characteristic epithelial tumor morphology and phenotypes in consistent with primary GBC, such as polygon and irregular cell shape, increased CA19-9 and AFP levels, and positive expression of CK7, CK8, CK19 and E-cadherin with negative vimentin. Moreover, about 25% of the cells were in the S-G2/M phase; abnormity in structure and number of chromosome with a peak number of 90–105 and 80% hypertetraploid were observed. Furthermore, this cell line had higher invasion and highest migration abilities compared to other GBC cell lines; and metastatic-related marker MMP9 and nm23 were positively expressed.ConclusionsA novel highly aggressive GBC cell line TJ-GBC2 was successfully established from primary GBC. TJ-GBC2 cell line may be efficient tool for further investigating the biological behaviors, metastatic mechanism and potential targeted therapy of human GBC.

DUSP1 inhibits cell proliferation, metastasis and invasion and angiogenesis in gallbladder cancer.

DUSP1/MKP1 is a dual-specific phosphatase that regulates MAPK activity and is known to play a key role in tumor biology. Its function in gallbladder cancer (GBC) remains largely unknown, however. By exploring its activities in two GBC cell lines (SGC996 and GBC-SD), DUSP1 was found to inhibit GBC cell proliferation, migration and invasion. Moreover, DUSP1 inhibited GBC growth and metastasis in nude mice subcutaneously xenografted with SGC996 cells. The tumor suppression appeared to be mediated via the DUSP1-pERK/MAPK-MMP2 signal pathway. Angiogenesis was associated with the tumor metastasis in the mouse model and was impaired by DUSP1, which suppressed VEGF expression. These results suggest that DUSP1 suppresses GBC growth and metastasis by targeting the DUSP1-pERK-MMP2/VEGF axis. Identification of the DUSP1-pERK-MMP2/VEGF signals may provide new biomarkers and/or therapeutic targets to better suppress GBC metastasis in the future.

Long non-coding RNA LINC00152 promotes gallbladder cancer metastasis and epithelial-mesenchymal transition by regulating HIF-1α via miR-138.

Long non-coding RNA LINC00152 had been reported as an oncogene in gastric and hepatocellular cancer. In this study, we show that LINC00152 is overexpressed in gallbladder cancer (GBC) tissue samples and cell lines. The high LINC00152 levels correlated negatively with the overall survival time in GBC patients. Functionally, LINC00152 dramatically promoted cell migration, invasion and epithelial–mesenchymal transition (EMT) progression in vitro. In vivo, LINC00152 overexpression significantly promoted tumour peritoneal spreading and metastasis. Mechanistic analyses indicated that LINC00152 functions as a molecular sponge for miR-138, which directly suppresses the expression of hypoxia inducible factor-1α (HIF-1α). We revealed that miR-138 is a suppressor of GBC cell metastasis and EMT progression, and a similar phenomenon was observed in HIF-1α knockdown NOZ cells. Through binding to miR-138, LINC00152 has an oncogenic effect on GBC. Overall, our study suggested that the LINC00152/miR-138/HIF-1α pathway potentiates the progression of GBC, and LINC00152 may be a novel therapeutic target.

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.