Gallbladder Carcinoma Cells Research Articles

Desulfation of cell surface HSPG is an effective strategy for the treatment of gallbladder carcinoma

Cell surface heparan sulfate proteoglycan (HSPG) is a group of critical glycoproteins that mediates signal transduction. Sulfated HSPG can mediate the activation of a variety of cell growth factor signal pathway to promote the progression of gallbladder carcinoma (GBC). This study analyzed 527 clinical GBC specimens and confirmed that the HSPG sulfation level was significantly higher in GBC tissues than in gallbladder mucosa (GBM) tissues. The high HSPG sulfation level was closely associated with poor differentiation, local metastasis, and advanced clinical stage of GBC; it was also associated with the shortening of disease-free survival (DFS) and overall survival (OS) and influenced the outcome of chemotherapy or radio-chemotherapy in patients with GBC recurrence. Inhibition of HSPG sulfation on the GBC cell surface using human sulfatase 1 (hSulf-1) significantly reduced the phosphorylation levels of growth factor receptors and signaling protein kinases in GBC cells, decreased cell responses to growth factors, and inhibited cell proliferation and migration abilities. In a nude mouse model with GBC xenografts, we observed that the xenograft tumor growth was suppressed and the phosphorylation levels of signaling proteins were downregulated, together with decreased expression of Ki67 and reduced sensitivity to bFGF (basic fibroblast growth factor) induction after inhibition of HSPG sulfation. Our study demonstrated that a high HSPG sulfation endows GBC with high malignant biological behaviors and a poor prognosis. Desulfation of cell surface HSPG can inhibit the kinase activities of a variety of signaling proteins, hinder the cell response to growth factors, and effectively inhibit the malignant biological behaviors of GBC cells.

The lncRNA MALAT1 functions as a competing endogenous RNA to regulate MCL-1 expression by sponging miR-363-3p in gallbladder cancer.

Gallbladder carcinoma (GBC) is an aggressive neoplasm, and the treatment options for advanced GBC are limited. Recently, long non‐coding RNAs (lncRNAs) have emerged as new gene regulators and prognostic markers in several cancers. In this study, we found that metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) expression was up‐regulated in GBC tissues (P < 0.05). Luciferase reporter assays and RNA pull down assays showed that MALAT1 is a target of miR‐363‐3p. Real‐time quantitative PCR and Western blot analysis indicated that MALAT1 regulated Myeloid cell leukaemia‐1 (MCL‐1) expression as a competing endogenous RNA (ceRNA) for miR‐363‐3p in GBC cells. Furthermore, MALAT1 silencing decreased GBC cell proliferation and the S phase cell population and induced apoptosis in vitro. In vivo, tumour volumes were significantly decreased in the MALAT1 silencing group compared with those in the control group. These data demonstrated that the MALAT1/miR‐363‐3p/MCL‐1 regulatory pathway controls the progression of GBC. Inhibition of MALAT1 expression may be to a novel therapeutic strategy for gallbladder cancer.

Downregulation of stathmin 1 in human gallbladder carcinoma inhibits tumor growth in vitro and in vivo.

Gallbladder carcinoma (GBC) is a highly lethal malignancy of the gastrointestinal tract. Despite extensive research, the underlying molecular mechanism of GBC remains largely unclear. Stathmin 1 (STMN1) is an important cytosolic protein associated with microtubule stability that was reported to be involved in tumorigenesis. Up to our knowledge, its role in gallbladder carcinoma has not been analyzed. In this study, we found that STMN1 was significantly highly expressed in GBC by immunohistochemistry (IHC). Further research demonstrated that silencing of STMN1 inhibited cell growth in vitro. Moreover, knockdown of STMN1 induced apoptosis and delayed G2/M phase transformation in GBC cells. Our data support a rationale for further studies that the silencing of STMN1 may regulate the activity of p38 MAPK kinase and p53/p21 signal pathway. Besides, xenografted gallbladder carcinoma cells growth were significantly impaired after STMN1 was silenced in vivo. These results suggested that STMN1 played an important role in cell proliferation and migration. This provided a potential clue for investigating the therapeutic target in GBC.

CD133 promotes gallbladder carcinoma cell migration through activating Akt phosphorylation.

Gallbladder carcinoma (GBC) is the fifth most common malignancy of gastrointestinal tract. The prognosis of gallbladder carcinoma is extremely terrible partially due to metastasis. However, the mechanisms underlying gallbladder carcinoma metastasis remain largely unknown. CD133 is a widely used cancer stem cell marker including in gallbladder carcinoma. Here, we found that CD133 was highly expressed in gallbladder carcinoma as compared to normal tissues. CD133 was located in the invasive areas in gallbladder carcinoma. Down-regulation expression of CD133 inhibited migration and invasion of gallbladder carcinoma cell without obviously reducing cell proliferation. Mechanism analysis revealed that down-regulation expression of CD133 inhibited Akt phosphorylation and increased PTEN protein level. The inhibitory effect of CD133 down-regulation on gallbladder carcinoma cell migration could be rescued by Akt activation. Consistent with this, addition of Akt inhibitor Wortmannin markedly inhibited the migration ability of CD133-overexpressing cells. Thus, down-regulation of CD133 inhibits migration of gallbladder carcinoma cells through reducing Akt phosphorylation. These findings explore the fundamental biological aspect of CD133 in gallbladder carcinoma progression, providing insights into gallbladder carcinoma cell migration.

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

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

Histone Deacetylase Inhibitors Inhibit the Proliferation of Gallbladder Carcinoma Cells by Suppressing AKT/mTOR Signaling.

Gallbladder carcinoma is an aggressive malignancy with high mortality mainly due to the limited potential for curative resection and its resistance to chemotherapeutic agents. Here, we show that the histone deacetylase inhibitors (HDACIs) trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA) reduce the proliferation and induce apoptosis of gallbladder carcinoma cells by suppressing the AKT/mammalian target of rapamycin (mTOR) signaling. Gallbladder carcinoma SGC-996 cells were treated with different concentrations of TSA and SAHA for different lengths of time. Cell proliferation and morphology were assessed with MTT assay and microscopy, respectively. Cell cycle distribution and cell apoptosis were analyzed with flow cytometry. Western blotting was used to detect the proteins related to apoptosis, cell cycle, and the AKT/mTOR signaling pathway. Our data showed that TSA and SAHA reduced SGC-996 cell viability and arrested cell cycle at the G1 phase in a dose- and time-dependent manner. TSA and SAHA promoted apoptosis of SGC-996 cells, down-regulated the expression of cyclin D1, c-Myc and Bmi1, and decreased the phosphorylation of AKT, mTOR p70S6K1, S6 and 4E-BP1. Additionally, the mTOR inhibitor rapamycin further reduced the cell viability of TSA- and SAHA-treated SGC-996 cells and the phosphorylation of mTOR, whereas the mTOR activator 1,2-dioctanoyl-sn-glycero-3-phosphate (C8-PA) exerted the opposite influence. Our results demonstrate that histone deacetylase inhibitors (HDACIs) suppress the proliferation of gallbladder carcinoma cell via inhibition of AKT/mTOR signaling. These findings offer a mechanistic rationale for the application of HDACIs in gallbladder carcinoma treatment.

Loss of NDRG2 promotes epithelial-mesenchymal transition of gallbladder carcinoma cells through MMP-19-mediated Slug expression

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract and one of the most lethal forms of human cancer. However, there is limited information about the molecular pathogenesis of GBC. Here, we examined the functional role of the tumor suppressor N-myc downstream-regulated gene 2 (NDRG2) and the underlying molecular mechanisms of disease progression in GBC. Clinical correlations between NDRG2 expression and clinicopathological factors were determined by immunohistochemical analysis of tumor tissues from 86 GBC patients. Biological functions of NDRG2 and NDRG2-mediated signaling pathways were determined in GBC cell lines with NDRG2 knockdown or overexpression. Loss of NDRG2 expression was an independent predictor of decreased survival and was significantly associated with a more advanced T stage, higher cellular grade, and lymphatic invasion in patients with GBC. GBC cells with loss of NDRG2 expression showed significantly enhanced proliferation, migration, and invasiveness in vitro, and tumor growth and metastasis in vivo. Loss of NDRG2 induced the expression of matrix metalloproteinase-19 (MMP-19), which regulated the expression of Slug at the transcriptional level. In addition, MMP-19-induced Slug, increased the expression of a receptor tyrosine kinase, Axl, which maintained Slug expression through a positive feedback loop, and stabilized epithelial-mesenchymal transition of GBC cells. The results of our study help to explain why the loss of NDRG2 expression is closely correlated with malignancy of GBC. These results strongly suggest that NDRG2 could be a favorable prognostic indicator and promising target for therapeutic agents against GBC.

Abstract 3404: NDRG2 suppresses growth and invasion of gallbladder carcinoma via regulation of matrix metalloproteinase 19/slug interaction

Abstract Background: Gallbladder cancer is a relatively uncommon tumor worldwide but it has a feature of highly invasive and malignant tumor with poor prognosis. However, relevant molecules and/or genes associated with growth and metastasis of gallbladder carcinoma are poorly understood. Although NDRG2 is known as a tumor suppressor gene, its exact role in gallbladder carcinoma remains unknown. Materials and method: For deciphering biological roles of NDRG2 in gallbladder carcinoma, first, we examined the expression of NDRG2 in tumor specimens from 86 gallbladder carcinoma patients by immunostaining and analyzed the correlation between NDRG2 expression and clinicopathologic parameters as well as patient survival. Second, we investigated the functional significance of NDRG2 in gallbladder carcinoma progression by stable knock-down or overexpression of NDRG2 in in vitro and in vivo studies. Results: NDRG2 was differentially expressed in gallbladder carcinoma tissues. The negative expression of NDRG2 was significantly associated with a pathological T stage (P = 0.015), high histological grade (P = 0.020), and perineural invasion (P = 0.016). Multivariate analyses clearly showed that the loss of NDRG2 expression was an independent poor prognostic factor for cancer-specific survival. The stable expression of NDRG2 shRNA in gallbladder carcinoma cell lines significantly increased cell proliferation, anchorage-independent growth, migration and invasion. In addition, NDRG2 shRNA remarkably increased metastasis from the spleen to the liver in mice. Furthermore, loss of NDRG2 expression in cells caused an epithelial-mesenchymal transition through Slug induction by MMP-19 interaction with Slug promoter. Conclusions: In the present study, we showed that loss of NDRG2 expression leads to poor prognosis and enhanced metastasis in gallbladder carcinoma. NDRG2 suppresses MMP-19 which causes an epithelial-mesenchymal transition through Slug induction. These data indicate that NDRG2 suppresses tumor progression via regulating MMP19/Slug axis in gallbladder carcinoma. The loss of NDRG2 is a novel prognostic factor suggesting it could be a useful biomarker in patients with gallbladder carcinoma and a potential therapeutic target. Citation Format: Nayoung Kim, Jeong-Ki Min, Jin-Man Kim, Hyo Jin Lee. NDRG2 suppresses growth and invasion of gallbladder carcinoma via regulation of matrix metalloproteinase 19/slug interaction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3404. doi:10.1158/1538-7445.AM2015-3404

Adeno-associated virus mediated gene transfer of Shepherdin inhibits gallbladder carcinoma growth in vitro and in vivo

Gene therapy, a significantly crucial strategy for treatment of malignancies, has been gradually accepted in recent years. However, this therapeutic approach has being facing great challenges concerning problems which include complicated development of cancer with multiple gene control, effective target shortage, low efficiency of gene transferring and safety of the vector delivery system. Shepherdin, a novel peptidomimetic molecule designed from Lys-79 to Leu-87 of survivin, has been identified as a tumor suppressor with the function that can not only competitively interfere with the interaction between survivin and Hsp90 (heat shock protein-90) leading to the degradation of survivin to anti-tumor, but also competitively target the ATP-dependent binding pocket of Hsp90 resulting in the dysfunction of Hsp90 chaperone to cell apoptosis via a mitochondrial dependent or independent pathway. In the present study, we designed and constructed a recombinant Adeno-associated virus (rAAV) loading fusion gene NT4–TAT–6His–Shepherdin. The expression of Shepherdin in gallbladder carcinoma (GBC) cells was detected and its strong inhibitory effects against GBC growth were evaluated after AAV mediated gene transfer of Shepherdin into GBC cells and xenograft tumors. MTT assay and flow cytometric analysis demonstrated that rAAV containing Shepherdin gene could significantly inhibit the growth of GBC and this effect was closely associated with apoptosis. These results indicated that rAAV–NT4–TAT–6His–Shepherdin may be considered a novel therapeutic strategy in the gene therapy for gallbladder carcinoma.

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-.

MiR-138 Suppresses Cell Proliferation by Targeting Bag-1 in Gallbladder Carcinoma.

BackgroundMiR-138 is frequently downregulated in different cancer types and is thought to be involved in the progression of tumorigenesis. However, the molecular mechanism of miR-138 involvement in gallbladder carcinoma still remains unknown.MethodsThe expression of miR-138 in 49 gallbladder carcinoma samples and paired normal gallbladder samples was analyzed using quantitative reverse transcription–polymerase chain reaction. The biological functions of miR-138 and Bag-1 (Bcl-2-associated athanogene-1) on cell proliferation were examined using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and apoptosis assays. Targets of miR-138 were predicted using bioinformatics and validated using luciferase reporter and Western blot analyses. The in vivo effects of miR-138 were examined using subcutaneous inoculation of gallbladder carcinoma cells in Balb/c nude mice.ResultsCompared with their paired normal gallbladder samples, the gallbladder carcinoma samples had decreased expression of miR-138 and increased expression of Bag-1. Overexpression of miR-138 inhibited the proliferation of gallbladder carcinoma cells. Bag-1 was defined as a novel target of miR-138. Both the inhibition of Bag-1 by miR-138 and the silencing of Bag-1 by siRNA led to alterations of apoptosis-related proteins such as Bcl-2 and Bax. Restoring expression of Bag-1 eliminates the effects of miR-138 on cell proliferation and apoptosis. Furthermore, overexpression of miR-138 markedly inhibited the growth of tumors in the gallbladder carcinoma xenograft model in nude mice.ConclusionsExpression of miR-138 is frequently reduced in gallbladder carcinoma when compared to normal cells. Overexpression of miR-138 inhibited cell proliferation by directly suppressing the expression of Bag-1. These results suggest that miR-138 plays an important role in inhibiting the growth of gallbladder carcinoma.

Protein phosphatase PHLPP induces cell apoptosis and exerts anticancer activity by inhibiting Survivin phosphorylation and nuclear export in gallbladder cancer.

Many factors regulate cancer cell apoptosis, among which Survivin has a strong anti-apoptotic effect and PHLPP is a tumor suppressor gene that can induce significant apoptosis. However, the relationship between PHLPP and Survivin in gallbladder carcinoma (GBC) has not been reported. This study found that PHLPP expression is decreased and Survivin expression is increased in GBC tissues and cell lines. Their expression levels showed an inverse relationship and were associated with poor prognosis of GBC patients. Loss of PHLPP can increase the level of phosphorylated Survivin and induce the nuclear export of Survivin, which thus inhibit cell apoptosis and promote cell proliferation in GBC cells. The process that PHLPP regulates Survivin phosphorylation and intracellular localization is involved in AKT activity. Re-overexpression of PHLPP in GBC cells can decrease AKT phosphorylation level. Reduced expression of PHLPP in GBC is associated with high expression of miR-495. Increasing PHLPP expression or inhibiting miR-495 expression can induce apoptosis and suppress tumor growth in GBC xenograft model in nude mice. The results revealed the role and mechanism of PHLPP and Survivin in GBC cells and proposed strategies for gene therapies targeting the miR-495 / PHLPP / AKT / Survivin regulatory pathway.

Establishment and biological characterization of gallbladder carcinoma cell sublines with higher metastatic potential ability

Objective To establish high transfer potential,high attack ability gallbladder carcinoma cell line (GBC-SD) subline and study its biological characteristics.Methods using Transwell chamber from gallbladder carcinoma cell line (GBC-SD) screening high transfer invasion ability gallbladder carcinoma subline GBC-SD-I-2,then compare cells form; Clone formation test and four methyl azo azole blue (MTT) method to detect the change of cell proliferation ability; Real-time polymerase chain reaction (Real-time PCR) and Western blotting method detection and epithelial mesenchymal transition (EMT) related N-cadherin,β-catenin protein expression change.Results The use of small room Transwell invasion from gallbladder carcinoma cell line were high transfer potential cell subsets and maternal form there are significant differences.Clone formation assay (0.711 ± 0.021 vs.0.445 ± 0.017,P ＜ 0.01),MTT method shows subline proliferation ability noticeably (P ＜ 0.05).Real-time PCR test showed that the group of cells N-cadherin expression level increased significantly (0.881 ± 0.018 vs.0.102 ± 0.020,P ＜ 0.01),β-catenin protein expression decreased obviously (0.625 ± 0.041 vs.0.275 ±0.052,P ＜ 0.05).Western blotting method showed N-cadherin expression level increased significantly (0.758 ± 0.012 vs.0.156 ± 0.011,P ＜ 0.01) ; β-catenin protein expression decreased obviously (0.243 ± 0.014,0.720 ± 0.011,P ＜ 0.01).Conclusion Screening gallbladder carcinoma cell subsets GBC-SD-I-2 attacks,transfer ability enhanced obviously,to explore the gallbladder carcinoma transfer mechanism provides the ideal experimental tools. Key words: Gallbladder neoplasms; Cell line; Neoplasm metastasis

Ursolic acid induces cell cycle arrest and apoptosis of gallbladder carcinoma cells.

BackgroundUrsolic acid (UA), a plant extract used in traditional Chinese medicine, exhibits potential anticancer effects in various human cancer cell lines in vitro. In the present study, we evaluated the anti-tumoral properties of UA against gallbladder carcinoma and investigated the potential mechanisms responsible for its effects on proliferation, cell cycle arrest and apoptosis in vitro.MethodsThe anti-tumor activity of UA against GBC-SD and SGC-996 cells was assessed using MTT and colony formation assays. An annexin V/PI double-staining assay was used to detect cell apoptosis. Cell cycle changes were detected using flow cytometry. Rhodamine 123 staining was used to assess the mitochondrial membrane potential (ΔΨm) and validate UA’s ability to induce apoptosis in both cell lines. The effectiveness of UA in gallbladder cancer was further verified in vivo by establishing a xenograft GBC model in nude mice. Finally, the expression levels of cell cycle- and apoptosis-related proteins were analyzed by western blotting.ResultsOur results suggest that UA can significantly inhibit the growth of gallbladder cancer cells. MTT and colony formation assays indicated dose-dependent decreases in cell proliferation. S-phase arrest was observed in both cell lines after treatment with UA. Annexin V/PI staining suggested that UA induced both early and late phases of apoptosis. UA also decreased ΔΨm and altered the expression of molecules regulating the cell cycle and apoptosis. In vivo study showed intraperitoneally injection of UA can significantly inhibited the growth of xenograft tumor in nude mice and the inhibition efficiency is dose related. Activation of caspase-3,-9 and PARP indicated that mitochondrial pathways may be involved in UA-induced apoptosis.ConclusionsTaken together, these results suggest that UA exhibits significant anti-tumor effects by suppressing cell proliferation, promoting apoptosis and inducing 7cell cycle arrest both in vitro and in vivo. It may be a potential agent for treating gallbladder cancer.

Tetrandrine induces apoptosis in gallbladder carcinoma in vitro.

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

Bufalin induces cell cycle arrest and apoptosis in gallbladder carcinoma cells.

Bufalin, a major digoxin-like immunoreactive component of the Chinese medicine Chan Su, has been shown to exert a potential for anticancer activity against various human cancer cell lines in vitro. However, no detailed studies have so far been reported on its action on human gallbladder carcinoma cells. In this study, bufalin remarkably inhibited growth in human gallbladder cancer cells by decreasing cell proliferation, inducing cell cycle arrest and apoptosis in a dose-dependent manner. Bufalin also disrupted the mitochondrial membrane potential (ΔΨm) and regulated the expression of cell cycle and apoptosis regulatory molecules. Activation of caspase-9 and the subsequent activation of caspase-3 indicated that bufalin may be inducing mitochondria apoptosis pathways. Intraperitoneal injection of bufalin for 3 weeks significantly inhibited the growth of gallbladder carcinoma (GBC-SD) xenografts in athymic nude mice. Taken together, the results indicate that bufalin may be a potential agent for the treatment of gallbladder cancer.

Down-regulation of FoxM1 inhibits viability and invasion of gallbladder carcinoma cells, partially dependent on inducement of cellular senescence.

To investigate the effect of knockdown of Forkhead box M1 (FoxM1) on the proliferation and invasion capacities of human gallbladder carcinoma (GBC)-SD cells. Four FoxM1 shRNAs were transfected into GBC-SD cells with Lipofectamine 2000 to select the appropriate shRNA for down-regulation of FoxM1. A recombinant lentivirus for shFoxM1 (Lv-shFoxM1), which expresses FoxM1-specific shRNA, and a negative control carrying green fluorescent protein, which expresses a scrambled RNA, were constructed. After transfection with the recombinant adenovirus and screened with puromycin, RT-PCR and Western blot were utilized to evaluate the inhibition efficiency. Cell viability was evaluated by MTT assay, and cell migration and invasion were assessed using the Transwell system. Cells were suspended in serum-free medium and seeded into Transwell inserts either uncoated (for migration assay) or coated (for invasion assay) with growth factor-reduced Matrigel. To verify the involvement of FoxM1 in the senescence of tumor cells, staining of senescence β-galactosidase (SA β-gal), the widely used biomarker of cellular senescence, was also performed. After successful transfection of four FoxM1 small interfering RNAs (shRNAs) with Lipofectamine 2000, the shF1822 was selected as the most appropriate shRNA according to its obvious inhibitory effect. The recombinant adenovirus was then constructed with the shF1822 and successfully transfected into the GBC-SD cells, resulting in the significant inhibition of FoxM1 expression at both the mRNA and protein levels, compared with the negative control (P < 0.05). After transfection, down-regulation of FoxM1 significantly inhibited cell viability according to the MTT assay (P < 0.05). In addition, Transwell migration and invasion assays also suggested the suppression of invasion ability of the transfected cells. SA β-gal staining showed that down-regulation of FoxM1 could induce more senescent GBC cells (P < 0.05), suggesting the possible involvement of the senescence process of the FoxM1-deficient cells in GBC. FoxM1 is functionally involved in viability of GBC cells, partially dependent on the inducement of cellular senescence, and is a potential target for GBC therapy.

Targeting gallbladder cancer: oncolytic virotherapy with myxoma virus is enhanced by rapamycin in vitro and further improved by hyaluronan in vivo.

BackgroundGallbladder carcinoma (GBC) is highly lethal, and effective treatment will require synergistic anti-tumor management. The study is aimed at investigating the oncolytic value of myxoma virus (MYXV) infection against GBC and optimizing MYXV oncolytic efficiency.MethodsWe examined the permissiveness of GBC cell lines to MYXV infection and compared the effects of MYXV on cell viability among GBC and control permissive glioma cells in vitro and in vivo after MYXV + rapamycin (Rap) treatment, which is known to enhance cell permissiveness to MYXV by upregulating p-Akt levels. We also assessed MYXV + hyaluronan (HA) therapy efficiency by examinating Akt activation status, MMP-9 expression, cell viability, and collagen distribution. We further compared hydraulic conductivity, tumor area, and survival of tumor-bearing mice between the MYXV + Rap and MYXV + HA therapeutic regimens.ResultsMYXV + Rap treatment could considerably increase the oncolytic ability of MYXV against GBC cell lines in vitro but not against GBC xenografts in vivo. We found higher levels of collagen IV in GBC tumors than in glioma tumors. Diffusion analysis demonstrated that collagen IV could physically hinder MYXV intratumoral distribution. HA–CD44 interplay was found to activate the Akt signaling pathway, which increases oncolytic rates. HA was also found to enhance the MMP-9 secretion, which contributes to collagen IV degradation.ConclusionsUnlike MYXV + Rap, MYXV + HA therapy significantly enhanced the anti-tumor effects of MYXV in vivo and prolonged survival of GBC tumor-bearing mice. HA may optimize the oncolytic effects of MYXV on GBC via the HA–CD44 interaction which can promote viral infection and diffusion.

MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA (lncRNA), is associated with metastasis and is an independent prognostic factor for lung cancer. Recent studies have demonstrated that MALAT1 plays an important role in other malignancies. However, little is known about the role of MALAT1 in gallbladder carcinoma (GBC), which is the most common cancer of the biliary tract and has an extremely poor prognosis. In this study, we focused on the expression, biological functions and mechanism of MALAT1 in GBC and found that MALAT1 was significantly upregulated in GBC tissues compared with corresponding non-cancerous tissues. Knockdown of MALAT1 in GBC cell lines using lentivirus-mediated RNA interference significantly inhibited the proliferation and metastasis of the GBC cells both in vitro and in vivo. Furthermore, ERK/MAPK pathway was found to be inactivated in the GBC cell lines after MALAT1 knockdown. These results indicated that MALAT1 might serve as an oncogenic lncRNA that promotes proliferation and metastasis of GBC and activates the ERK/MAPK pathway

Suppression of autophagy by chloroquine sensitizes 5-fluorouracil-mediated cell death in gallbladder carcinoma cells

Autophagy1 is a complex of adaptive cellular response that enhances cancer cell survival in the face of cellular stresses such as chemothery. Here we show that in human gallbladder carcinoma (GBC) cells lines, SGC-996 and GBC-SD, autophagy is induced by the DNA damaging agent 5-fluorouracil (5-FU). While in combination with the pre-treatment of chloroquine (CQ), a inhibitor of autophagy, the inhibition of 5-FU to the proliferation and viability of GBC cells was potentiated. Furthermore, 5-FU treatment resulted in a general increase of the apoptotic rate and G0/G1 arrest of GBC cells, and the effect was potentiated by CQ pre-treatment. Since 5-FU induced autophagy in GBC cells, and CQ inhibited autophagy, our findings suggest a possible mechanism that CQ inhibited 5-FU-induced autophagy, which modified the cytotoxicity of 5-FU. The combination therapy of CQ and 5-FU should be considered as an effective strategy for the treatment of gallbladder carcinoma.