Human Gallbladder Cancer Cell Lines Research Articles

Establishment and characterization of a novel human gallbladder cancer cell line, GBC-X1

In this study, we successfully established a novel gallbladder cancer cell line, designated as GBC-X1, derived from a primary tumor of a gallbladder cancer patient. By comprehensively analyzing the cell line’s phenotype, molecular characteristics, biomarkers, and histological characteristics, we confirmed that GBC-X1 serves as a valuable model for investigating the pathogenesis of gallbladder cancer and developing therapeutic agents. GBC-X1 has been continuously cultured for one year, with over 60 stable passages. Morphologically, GBC-X1 exhibits typical features of epithelial tumors. The population doubling time of GBC-X1 is 32 h. STR analysis validated a high consistency between GBC-X1 and the patient’s primary tumor. Karyotype analysis revealed an abnormal hypertetraploid karyotype for GBC-X1, characterized by representative karyotypes of 98, XXXX del (4) p (12) del (5) p (21) der (10). Under suspension culture conditions, GBC-X1 efficiently forms tumor balls, while subcutaneous inoculation of GBC-X1 cells into NXG mice leads to xenograft formation with a rate of 80%. Drug sensitivity testing demonstrated that GBC-X1 is resistant to oxaliplatin and sensitive to 5-FU, gemcitabine, and paclitaxel. Immunohistochemistry revealed positive expression of CK7, CK19, E-cadherin, MMP-2, CD44, SOX2, and TP53 in GBC-X1 cells, weak positive expression of Vimentin, and a Ki67 positive rate of 35%. Our research highlights GBC-X1 as a novel gallbladder cancer cell line and emphasizes its potential as an effective experimental model for investigating the pathogenesis of gallbladder cancer and drug development.

Establishment and characterization of the gemcitabine-resistant human gallbladder cancer cell line NOZ GemR.

Patients with gallbladder cancer (GBC) generally receive gemcitabine as the standard treatment; however, its efficacy is often limited owing to the development of resistance. To identify the mechanisms underlying gemcitabine resistance in GBC, a gemcitabine-resistant GBC cell line (NOZ GemR) was established by exposing the parental NOZ cell line to increasing concentrations of gemcitabine. Morphological changes, growth rates, and migratory and invasive capabilities were evaluated. Protein expression was detected using western blotting. The results demonstrated that the IC50 of NOZ and NOZ GemR was 0.011 and 4.464μM, respectively, and that the resistance index ratio was 405.8. In comparison, NOZ GemR cells grew slower and had significantly lower migration and invasion abilities than NOZ cells. There were altered levels of epithelial-mesenchymal transformation markers in NOZ GemR cells, as well as increased levels of the Akt/mTOR pathway protein. The NOZ GemR cell line could be used as an effective in vitro model to improve our understanding of gemcitabine resistance in GBC.

MicroRNA-34a-5p: A pivotal therapeutic target in gallbladder cancer

Gallbladder cancer incidence has been increasing globally, andit remains challenging to expect long prognosis with the current systemic chemotherapy. We identified a novel nucleic acid-mediated therapeutic target against gallbladder cancer by using innovative organoid-based gallbladder cancer models generated from KrasLSL-G12D/+; Trp53f/f mice. Using comprehensive microRNA expression analyses and a bioinformatics approach, we identified significant microRNA-34a-5p downregulation in both murine gallbladder cancer organoids and resected human gallbladder cancer specimens. In three different human gallbladder cancer cell lines, forced microRNA-34a-5p expression inhibited cell proliferation and induced cell-cycle arrest at the G1 phase by suppressing direct target (CDK6) expression. Furthermore, comprehensive RNA sequencing revealed the significant enrichment of gene sets related to the cell-cycle regulators after microRNA-34a-5p expression in gallbladder cancer cells. In a murine xenograft model, locally injected microRNA-34a-5p mimics significantly inhibited gallbladder cancer progression and downregulated CDK6 expression. These results provide a rationale for promising therapeutics against gallbladder cancer by microRNA-34a-5p injection, as well as a strategy to explore therapeutic targets against cancers using organoid-based models, especially for those lacking useful genetically engineered murine models, such as gallbladder cancer.

Taurodeoxycholic acid-YAP1 upregulates OTX1 in promoting gallbladder cancer malignancy through IFITM3-dependent AKT activation.

Orthodenticle homeobox (OTX1) is reported to be involved in numerous cancers, but the expression level and molecular function of OTX1 in gallbladder cancer (GBC) remain unknown. Here, we found the elevated level of OTX1 associated with poor prognosis in human gallbladder cancer. In vitro and in vivo studies of human gallbladder cancer cell lines demonstrated that overexpression of OTX1 promoted cell proliferation, whereas the downregulation inhibited it. Additionally, we found a tight correlation between the serum level of taurodeoxycholic acid (TDCA) and OTX1 expression. TDCA-induced activation of YAP1 by phosphorylation inhibition contributed to the transcriptional activation of OTX1. Mechanistically, we identified that OTX1 activated AKT signaling pathway by transactivating the expression of IFITM3 and thus promoted the proliferation of GBC cells. Taken together, our results showed that TDCA-YAP1-dependent expression of OTX1 regulated IFITM3 and affected GBC proliferation via the AKT signaling pathway. Our experiments also suggested that OTX1 is a novel therapeutic target for GBC.

Anthocyanin-Rich Extracts of Calafate (Berberis microphylla G. Forst.) Fruits Decrease In Vitro Viability and Migration of Human Gastric and Gallbladder Cancer Cell Lines

Currently, gastric cancer (GC) and gallbladder cancer (GBC) constitute important causes of human deaths related to cancer worldwide. In the last years, several researches are focused on the role of dietary compounds in preventing cancers. The consumption of fruits with high antioxidants, mainly anthocyanins, represents a good option to reduce the risk of chronic human diseases. Calafate (Berberis microphylla G. Forst.) berries, recognized by their remarkable antioxidant properties and high content of anthocyanins, appear as a new alternative to treat degenerative diseases of public interest. The present work was aimed to evaluate the impact of crude and anthocyanin-rich extracts from Calafate fruits on in vitro cell viability and migration capacity of gastric (AGC) and gallbladder (G415) human cancer cell lines, as related with their antioxidant properties. Crude and anthocyanin-rich extracts were obtained from fruits of Calafate grown under field conditions in the south of Chile. Antioxidants, phenols, anthocyanins, and anthocyanidins were determined. In vitro cell viability and migration of AGS and G415 human cancer cell lines at different concentrations of extracts (25–800 μg mL−1) were determined. Anthocyanin-rich extracts of Calafate berries showed comparable antioxidant activity (up to 1200 μg Trolox eq. g−1DW), slightly lower total phenolic content (12%), but higher total anthocyanin content (25%) compared to the crude extract. The major anthocyanidin molecule detected in both extracts was delphinidin, followed by malvidin, and low concentrations of petunidin, cyanidin, and peonidin. As expected, all of these compounds were detected in higher levels in anthocyanin-rich extracts (up to 2-fold). Noteworthy, our study revealed that Calafate fruit extracts strongly decrease in vitro viability and migration capacity of gastric carcinoma (AGC model) and gallbladder carcinoma (G415 model) human cell lines; however, the anthocyanin-rich extract displayed higher inhibitory effects (up to 70%) compared to crude extracts. These findings allow suggesting that the in vitro antiproliferative potential of Calafate fruit extract is strongly related to the anthocyanin concentration, especially delphinidin.

Osthole inhibits the progression of human gallbladder cancer cells through JAK/STAT3 signal pathway both in vitro and in vivo.

Osthole is an antitumor compound, which effect on Gallbladder cancer (GBC) has been not elucidated. This study focused on its anti-GBC effect and mechanism both in vitro and in vivo. The antiproliferation effect on cell lines NOZ and SGC-996 were measured by cell counting kit-8 (CCK-8) and colony formation assay. The effects on cell apoptosis and cell cycle were investigated by flow cytometry assay. The migration effect was checked by transwell assay and the expressions of proteins were examined by Western Blots. Also, we did an in-vivo experiment by intraperitoneal injection of osthole in nude mice. The results showed that cell proliferation and viability were inhibited in a dose- and time-dependent manner. The similar phenomenon was also found in vivo. Flow cytometric assay confirmed that osthole inhibited cells proliferation via inducing apoptosis and G2/M arrest. Transwell assay indicated that osthole inhibited the migration in a dose-dependent manner. Expression of key proteins related with apoptosis and cell cycle were testified after osthole treatment. Also, we found the key proteins involved in the JAK/STAT3 signal way decreased after osthole treatment. This study suggested that osthole can inhibit the progression of human GBC cell lines, thus maybe a potential drug for GBC treatment.

Near-infrared fluorescence imaging and photodynamic therapy with indocyanine green lactosomes has antineoplastic effects for gallbladder cancer.

BACKGROUND: The diagnostic use and therapeutic effect of near infrared fluorescence (NIF) imaging and photodynamic therapy (PDT) was investigated for gallbladder cancer using indocyanine green (ICG)-lactosomes. RESULTS: PDT was toxic for NOZ cells treated with ICG-lactosomes. Fluorescence intensity in the tumor region of mice administered ICG-lactosomes, but not ICG alone, was higher than the healthy contralateral region ≥24 hours after injection. PDT exerted immediate and continuous phototoxic effects in NOZ implanted mice injected with ICG-lactosomes. Enhanced antitumor effects were observed in the twice irradiated group compared with the once irradiated group. METHOD: ICG or ICG-lactosomes were added to the human gallbladder cancer cell line NOZ followed by PDT and cell viability was measured. Mass spectrometry of ICG and ICG-lactosomes was performed after PDT. ICG or ICG-lactosomes were intravenously administered to BALB/c nude mice implanted subcutaneously with NOZ cells and fluorescence was evaluated by NIF imaging. Implanted tumors underwent PDT and antitumor effects were analyzed after performing irradiation once or twice in ICG-lactosome groups. CONCLUSIONS: ICG-lactosomes accumulated in xenograft tumors and PDT had an antitumor effect on these malignant tumors. NIF imaging with ICG-lactosomes and PDT may be useful diagnostic and/or therapeutic agents for gallbladder cancer.

Establishment of Cas9 stably expressed human hepatocellular carcinoma and cholangiocarcinoma cell lines

Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry(-)EGFP(+) cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry(-)EGFP(+) cells accounted from 0.3% to 93.6%. Conclusion: We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.

Transglutaminase 2 Mediates the Cytotoxicity of Resveratrol in a Human Cholangiocarcinoma and Gallbladder Cancer Cell Lines

ABSTRACTResveratrol is a polyphenolic compound extracted from plants and is also a constituent of red wine. Our aim was to evaluate if the cytotoxic effect of resveratrol (RES) on cholangiocarcinoma (CC) and gallbladder cancer (GBC) cell lines could be abolished by TG2 inhibition. Human CC and GBC cell lines (SK-ChA-1 and MZ-ChA-1), grown in a three-dimensional cell culture system (MCTS, multicellular tumor spheroids), were treated for 72 h with RES (32, 64 µM) alone or combined with different TG2 inhibitors (Cystamine, B003, T101). We investigated: cells viability; cell morphology with light microscopy (LM) and transmission electron microscopy (TEM); immunoreactivity with immunohistochemistry; Q-Banding karyotype analysis; TG2 activity; Western blotting. RES treatment induced a significant inhibition of cell growth, ranging from 24% to 76% in both cell lines. The inhibitors successfully reduced TG2 activity without any variation of protein quantity as demonstrated by immunohistochemistry and Western blot. TG2 inhibition resulted in cell growth normalization. In addition, morphologic analysis by light and transmission electron microscopy confirmed the cytotoxic effect of RES and its reduction consequent to TG2 inhibition. Our data demonstrated a connection between the cytotoxic effect of RES in SK-ChA-1 and MZ-ChA-1 and TG2 activity.

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.

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.

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.

Maslinic acid potentiates the antitumor activities of gemcitabine invitro and invivo by inhibiting NF-κB-mediated survivalsignaling pathways in human gallbladder cancer cells.

Gemcitabine (GEM) is one of the first-line drugs in the treatment of gallbladder cancer (GBC), although the therapeutic effect is not sustained due to resistance to the drug over time. Maslinic acid (MA) has been shown to inhibit transcription factor nuclear factor-κB (NF-κB), resulting in the suppression of survival signaling. The authors of the present study investigated whether MA enhanced the antitumor activity of GEM in GBC. Anti-proliferative effects of MA, GEM and MA+GEM were assessed using the MTT assay. Apoptosis was assessed using Annexin V and by western blot analysis of various mediators of apoptosis. Xenograft tumors of EH-GB2 GBC cells were established in athymic nude mice and were monitored following treatment with MA, GEM and MA+GEM. Immunohistochemistry of the tumors was used to examine various survival proteins. MA inhibited the in vitro proliferation of various GBC cell lines and potentiated the apoptosis and cell invasion inhibition induced by GEM. Western blot analysis showed that the combination of MA and GEM inhibited constitutive NF-κB activation and NF-κB-regulated gene products, including cyclin D1, Bcl-2, Bax, MMP-2 and MMP-9, to a greater extent. In vivo, the group that was treated with MA+GEM showed significant reductions in tumor volume and a decreased expression of NF-κB-regulated gene products. In conclusion, the results suggest that MA potentiates the antitumor effects of GEM in human GBC cell lines by suppressing the activation of NF-κB and its dowstream gene products, which are involved in survival signaling.

Cordycepin induces S phase arrest and apoptosis in human gallbladder cancer cells.

Gallbladder cancer is the most common malignant tumor of the biliary tract, and this condition has a rather dismal prognosis, with an extremely low five-year survival rate. To improve the outcome of unresectable and recurrent gallbladder cancer, it is necessary to develop new effective treatments and drugs. The purpose of the present study was to evaluate the effects of cordycepin on human gallbladder cells and uncover the molecular mechanisms responsible for these effects. The Cell Counting Kit-8 (CCK-8) and colony formation assays revealed that cordycepin affected the viability and proliferation of human gallbladder cancer cells in a dose- and time-dependent manner. Flow cytometric analysis showed that cordycepin induced S phase arrest in human gallbladder cancer cell lines(NOZ and GBC-SD cells). Cordycepin-induced apoptosis was observed using an Annexin V/propidium iodide (PI) double-staining assay, and the mitochondrial membrane potential (ΔΨm) decreased in a dose-dependent manner. Additionally, western blot analysis revealed the upregulation of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP and Bax and the downregulation of Bcl-2, cyclin A and Cdk-2 in cordycepin-treated cells. Moreover, cordycepin inhibited tumor growth in nude mice bearing NOZ tumors. Our results indicate that this drug may represent an effective treatment for gallbladder carcinoma.

Inhibition of tumor angiogenesis by interferon-γ by suppression of tumor-associated macrophage differentiation.

Tumor-associated macrophages (TAMs) differentiate from monocytes and are the M2-polarized macrophages in most human tumors, secreting generous vascular endothelial growth factor (VEGF) to promote angiogenesis. Although it has been shown in vitro that interferon-γ (IFN-γ) can inhibit monocytes differentiating to M2 macrophages in the tumor microenvironment and switch TAMs from M2 into M1, suppressing the ability of secreting VEGF, its effects on TAMs in vivo remains unknown. Here we tried to examine the effects of IFN-γ on the recruitment of monocyte/macrophage differentiation of TAMs and tumor angiogenesis in vivo. We built a gallbladder cancer model by inoculating subcutaneously the human gallbladder cancer cell line (GBC-SD) into BALB/C nude mice and injected the recombinant mouse IFN-γ intratumorally. We found that in the IFN-γ group, the number of monocytes/macrophages was significantly higher than that in the control group (p < 0.01), and TAM differentiation rate, which we defined as the number of TAMs / the number of monocytes/macrophages × 100%, mice-VEGF concentration, and microvessels density (MVD) were significantly lower than those in the control group (p < 0.01, p < 0.05, and p < 0.01). Our results suggest that IFN-γ can induce monocytes/macrophages recruiting into the tumor microenvironment, but inhibit them, differentiating to TAMs in vivo, which may reduce the concentration of VEGF and angiogenesis in tumor.

Hydroxytyrosol inhibits cholangiocarcinoma tumor growth: An in vivo and in vitro study

Cholangiocarcinoma (CCA) is a type of digestive tumor that is associated with a high rate of mortality due to the difficulty of early diagnosis and the resistance of this tumor type to chemotherapy. Hydroxytyrosol (HT), which is derived from virgin olive oil (VOO), has recently been reported to inhibit the proliferation of various types of human cancer cells. In the present study, we investigated the effect of HT on CCA. The antiproliferative and proapoptotic effects of HT on CCA were evaluated in the human CCA cell lines TFK-1 and KMBC and the human gallbladder cancer cell line GBS-SD. We also assessed this effect in vivo. We found that 75 µM HT inhibited the proliferation of the TFK-1, KMBC and GBS-SD cell lines. However, 200 µM HT treatment did not affect the proliferation of the human bile duct cell line HIBEpiC. More importantly, HT (250 and 500 mg/kg/day) markedly inhibited the growth of CCA xenografts in mice. G2/M phase cell cycle arrest and apoptosis were observed using flow cytometry and western blotting, and we also noted a time- and dose-dependent inhibition of phospho-ERK, with no changes in total-ERK, during treatment with HT. The present study showed that HT induces cell cycle arrest and apoptosis in vitro and in vivo. These data suggest that HT, which possesses excellent biocompatibility and few side-effects, could be developed as a novel agent against CCA.

High expression of microRNA-155 is associated with the aggressive malignant behavior of gallbladder carcinoma

The prognosis of gallbladder cancer (GBC) remains poor despite recent advances in diagnostics and therapeutic strategies. Although the role of microRNAs (miRs) in GBC have not been well documented, miR-155 is known to be associated with inflammation-associated carcinogenesis in various types of cancers. The aim of this study was to investigate the clinical significance of miR-155 expression and the biological functions of miR-155 in GBC. The expression levels of miR-155 in surgically resected GBCs and gallbladders with pancreaticobiliary maljunction (PBM) were assessed by quantitative reverse transcription-polymerase chain reaction. The relationship between the expression levels of miR-155 and clinicopathological features of GBCs was analyzed. Human GBC cell lines were transfected with miR-155 inhibitors or mimics, and the effects on proliferation and invasion were assessed. miR-155 was significantly overexpressed in GBCs when compared with that in gallbladders with PBM (P=0.007) and normal gallbladders (P=0.04). The high expression level of miR-155 in GBCs was significantly associated with the presence of lymph node metastasis (P=0.01) and a poor prognosis (P=0.02). In vitro assays showed that aberrant expression of miR-155 significantly enhanced GBC cell proliferation and invasion. In conclusion, high miR-155 expression correlates with the aggressive behavior of GBCs, and miR-155 may become a prognostic marker and therapeutic target for GBC.

Ethyl Pyruvate Administration Suppresses Growth and Invasion of Gallbladder Cancer Cells via Downregulation of Hmgb1-Rage Axis

High mobility group box B1 (HMGB1)-receptor for advanced glycation end products (RAGE) axis has been previously known to be involved in carcinogenesis and development of multiple malignancies. Some studies have confirmed that Ethyl pyruvate (EP), a potent inhibitor of HMGB1, exerts the therapeutic effects on metastatic live tumor from gastric cancer. However, the effects and possible molecular mechanisms of EP on gallbladder cancer (GBC) need to be further explored. In the present study, human GBC cell lines (GBC-SD and SGC-996) were treated with different concentrations of EP. Then, the expression levels of HMGB1, RAGE and some transcription factors were identified by Real-time PCR and Western blot assays. Cell proliferative activities indicated by MTT assay, invasive potential by Transwell assay and cell apoptosis and cycle distribution were performed for functional analysis of GBC cell lines in vitro. As a result, EP decreased the expression of HMGB11, RAGE, PCNA and matrix metallopeptidase-9 (MMP-9), while it increased the expression of p53. Moreover, EP administration decreased GBC cell proliferation, inhibited the invasive potential, and induced apoptosis and cycle arrest in S phase in GBC cells. In conclusion, EP administration inhibits growth and invasion of gallbladder cancer cells possibly via down-regulation of the HMGB1-RAGE axis, suggesting that EP may play a critical role in the treatment of cancer in conjunction with other therapeutic agents.

Side population cells in human gallbladder cancer cell line GBC-SD regulated by TGF-β-induced epithelial-mesenchymal transition

Mounting evidence has shown that side population (SP) cells are enriched for cancer stem cells (CSCs) responsible for cancer malignancy. In this study, SP technology was used to isolate a small subpopulation of SP cells in human gallbladder cancer cell line GBC-SD, and SP cells which had superior potential for proliferation in vitro and tumorigenesis in vivo were identified. Importantly, the abundance of GBC-SD SP cells was increased by a transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT), and this effect was accompanied with a strong up-regulation of ABCG2 mRNA expression, and a decreased sensitivity to mitoxantrone. SP cells were restored upon the removal of TGF-β and the reversion of the cells to an epithelial phenotype, and smad3-specific siRNA reduced SP abundance in response to TGF-β. In conclusion, TGF-β-induced EMT by smad-dependent signaling pathway promotes cancer development and anti-cancer drug resistant phenotype by augmenting the abundance of GBC-SD SP cells, and a better understanding of mechanisms involved in TGF-β-induced EMT may provide a novel strategy for preventing cancer progression.

Vascular endothelial growth factor-D promotes growth, lymphangiogenesis and lymphatic metastasis in gallbladder cancer

Lymph node metastasis is a major prognostic factor for patients with gallbladder cancer (GBC), and greater understanding of the molecule mechanism of lymph node metastasis in GBC is needed to improve prognosis. VEGF-D has been implicated in the control of lymphangiogenesis in many carcinomas, but the biological function of VEGF-D in human GBC remains unclear. In this study, we analyzed the role of the VEGF-D in human GBC cells and addressed the functional role of VEGF-D using a xenograft mouse model. We examined the expression of VEGF-D in three human gallbladder cancer cell lines. A lentivirus-based effective VEGF-D siRNA vector was infected into GBC NOZ cells. The effect of VEGF-D siRNA on GBC NOZ cells was investigated by cell proliferation assay and invasion assay. Furthermore, we examined the role of VEGF-D-SiRNA on GBC NOZ cells in the mice of subcutaneous and orthotopic xenograft tumor. Our results are as follows: VEGF-D mRNA and protein were expressed in all three GBC cell lines (GBC-SD, NOZ, and SGC-996). We successfully selected D-3/siRNA as the most effective siRNA to silence VEGF-D expression after four VEGF-D siRNA plasmid transfection in NOZ cells. VEGF-D mRNA and protein expression were suppressed by lentivirus-mediated D-3/siRNA. D-3-RNAi-LV inhibited NOZ cells proliferation and invasion ability in vitro. D-3-RNAi-LV inhibited tumor growth and lymphangiogenesis in the NOZ cell subcutaneous xenograft model. D-3-RNAi-LV inhibited lymphangiogenesis and lymphatic metastasis in the NOZ cell orthotopic xenograft model. Furthermore, D-3-RNAi-LV inhibited tumor ascites and hepatic invasion in the NOZ cell orthotopic xenograft model. In conclusion, VEGF-D is involved and plays an important role in GBC progression, suggesting that VEGF-D may be a potential molecular target in the treatment of GBC.