Apoptosis Of Gallbladder Carcinoma Cells Research Articles

Expression and inhibitory effects of p53-upregulated modulator of apoptosis in gallbladder carcinoma.

The p53-upregulated modulator of apoptosis (PUMA) has been reported to be involved in various types of cancer. However, its potential biological role in gallbladder carcinoma (GBC) has not been fully elucidated. The present study aimed to determine the expression levels of PUMA and its biological effects on GBC. The mRNA and protein expression levels of PUMA in GBC tissues and cell lines were measured using reverse transcription-quantitative PCR and western blotting, respectively. The effects of PUMA overexpression on cell viability, proliferation and invasive ability were determined in vitro using the MTT, colony formation and Transwell invasion assays, respectively. The apoptotic rates were detected using the Annexin V-FITC apoptosis detection kit. Furthermore, follow-up of patients with GBC was performed to identify the association between PUMA expression levels and GBC prognosis. The results of the present study demonstrated that the expression levels of PUMA were significantly lower in the GBC tissues and cell lines compared with those in adjacent normal gallbladder tissues and normal gallbladder cells, respectively. Further experiments indicated that overexpression of PUMA inhibited the viability, proliferation and invasive ability of GBC cells compared with those in the control-transfected GBC cells. In addition, overexpression of PUMA significantly promoted apoptosis in GBC cells. Furthermore, overexpression of PUMA inhibited epithelial-mesenchymal transition, and promoted Bax upregulation and Bcl-2 downregulation compared with those in the control group. Low PUMA expression levels were associated with a short overall survival time in patients with GBC. In conclusions, PUMA may act as a tumor suppressor in GBC and may serve as a potential novel treatment target for human GBC.

MiR-1231 enhances docetaxel sensitivity to gallbladder carcinoma cells by downregulating FOXC2.

To illustrate the role of microRNA-1231 (miR-1231) in regulating malignant proliferative potential and DTX sensitivity to gallbladder carcinoma (GBC) by regulating FOXC2 level. Expression levels of miR-1231 in GBC tissues and paracancerous ones were detected. The relationship between miR-1231 level and clinical parameters of GBC patients was analyzed. After overexpression of miR-1231, changes in proliferative and apoptotic potentials in GBC-SD and NOZ cells were examined by Cell Counting Kit-8 (CCK-8), colony formation assay and flow cytometry, respectively. Regulatory effects of miR-1231 on its downstream gene FOXC2 were determined by Luciferase assay. Finally, the role of miR-1231 in regulating DTX sensitivity to GBC cells was assessed. MiR-1231 was downregulated in GBC tissues compared to paracancerous ones. GBC patients expressing lower level of miR-1231 had worse tumor staging and larger tumor size. Overexpression of miR-1231 attenuated proliferative potential, and induced apoptosis in GBC cells. FOXC2 was upregulated in GBC and negatively linked to miR-1231. Luciferase activity confirmed that FOXC2 was the target gene binding miR-1231. DTX treatment dose-dependently suppressed viability in GBC cells and overexpression of miR-1231 could enhance DTX sensitivity in GBC. Notably, overexpression of FOXC2 abolished regulatory effects of overexpressed miR-1231 on proliferative and apoptotic potentials in GBC cells. MiR-1231 is downregulated in GBC species. Its level is closely linked to tumor staging and tumor size in GBC patients. By downregulating FOXC2, miR-1231 enhances DTX sensitivity to GBC cells and thus alleviates the malignant development of GBC.

Smac mimetic promotes TNF-α to induce apoptosis of gallbladder carcinoma cells

Gallbladder carcinoma has a high degree of malignancy. No effective treatment exists for patients with advanced tumors. The second mitochondria-derived activator of caspases (Smac) is the antagonist of the inhibitors of apoptosis protein. Smac mimetics are a class of effective tumor-targeted drugs undergoing clinical trials. However, studies on the effect of Smac mimetics on gallbladder cancer are unavailable. In this study, Smac mimetics can promote tumor necrosis factor-α (TNF-α) to inhibit the proliferation of gallbladder cancer cells and activate the apoptotic pathway, thereby promoting the ubiquitination of Lys48 on Receptor interacting protein kinase-1 (RIPK1) and leading to proteasomal degradation that causes damage to RIPK1 protein integrity. The formation of complex I (RIPK1, tumor necrosis factor 1-associated death domain protein, and TNF receptor-associated factor 2) is inhibited. Then, nonubiquitinated RIPK1 binds with the Fas-associated death domain and caspase-8 to form complex II and promotes the death receptor pathway of apoptosis. Animal experiments further verify that TNF-α combined with Smac mimetics can inhibit the growth of transplanted tumors and induce the apoptosis of transplanted tumor cells. This research provides a new direction for the targeted therapy of gallbladder cancer.

MiRNA-145 Induces Apoptosis in a Gallbladder Carcinoma Cell Line by Targeting DFF45.

BakcgroundWe measured expression of miRNA-145 in gallbladder carcinoma and its influence on propagation, invasion, and apoptosis of gallbladder carcinoma cells in vitro.MethodsmiRNA-145 expression was compared between normal gallbladder epithelial cells and GBS-SD (gallbladder series) cells using miRNA chip technology. Propagation, apoptosis, and invasion properties of each cell group were tested using MTT, a clone-formation assay, flow cytometry, Western blot, and Transwell assays.ResultsExpression of miRNA-145 was observed to be down-regulated and GBC-SD cell clones transiently transfected with hsa-miRNA-145 were substantially reduced compared with controls (p<0.01). We observed that GBC-SD cells transfected with hsa-miRNA-145 and double-positive (Annexin V and PI) for apoptosis were more numerous than controls. Moreover, GBC-SD cells over-expressing miRNA-145 had significantly greater expression of apoptosis-related protein, caspase-3. A Transwell assay confirmed that GBC-SD cells over-expressing miRNA-145 that migrated to the lower chamber were fewer compared with controls. Post-transcriptional regulation of gene expression was measured using dualluciferase reporter assays and data show that miRNA-145 facilitates the inhibition of GBC-SD cell growth and invasion while inducing apoptosis by targeting DFF45.ConclusionThus, we speculate that miRNA-145 facilitates inhibition of GBC-SD cell growth and invasion while inducing apoptosis by targeting DFF45; however, miRNA-145 does not directly affect the GBC-SD cell cycle.

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.

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.

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.

Baicalin Induces Apoptosis of Gallbladder Carcinoma Cells in vitro via a Mitochondrial-Mediated Pathway and Suppresses Tumor Growth in vivo

Baicalin, the main active ingredient in the Scutellaria baicalensis (SB), is prescribed for the treatment of various inflammatory diseases and tumors in clinics in China. In the present study, we evaluated the antitumor activity of baicalin for gallbladder carcinoma and the underlying mechanisms both in vitro and in vivo. Our results indicate that baicalin induced potent growth inhibition, cell cycle arrest, apoptosis and colony-formation inhibition in a dose-dependent manner in vitro. We observed inhibition of NF-κB nuclear translocation, up-regulation of Bax and down-regulation of Bcl-2, as well as increased caspase-3 and caspase-9 expression after baicalin treatment in vitro and in vivo, which indicates that the mitochondrial pathway was involved in baicalin-induced apoptosis. In addition, daily intraperitoneally injection of baicalin (15, 30 and 60 mg/kg) for 21 days significantly inhibited the growth of NOZ cells xenografts in nude mice, which improved the survival of baicalin-treated mice. In summary, baicalin exhibited a significant anti-tumor effect by suppressing cell proliferation, promoting apoptosis, and inducing cell cycle arrest in vitro, and by suppressing tumor growth and improving survival in vivo, which suggested that baicalin represents a novel therapeutic option for gallbladder carcinoma.

Effects of Matrine on Proliferation and Apoptosis in Gallbladder Carcinoma Cells (GBC‐SD)

Although matrine, a primary active component of dried Sophora flavescens root (ku shen), is known to induce apoptosis in a variety of tumor cells in vitro, the molecular mechanism of such apoptosis remains elusive. This analysis of the cell cycle and apoptosis in matrine-treated human gallbladder carcinoma cells (GBC-SD) showed that matrine can indeed inhibit cell proliferation and induce G1 cell cycle arrest and apoptosis in a dose- and time-dependent manner. An additional western blot analysis of matrine-treated cells also showed caspase-3 and Bcl-2 activation, as well as cyclinE down-regulation. Overall, the results indicate that matrine perturbs gallbladder cancer cell progression during the G1 phase by down-regulating cyclinE and induces apoptosis by decreasing the expression of the antiapoptotic protein Bcl-2 and increasing expression of the proapoptotic protein Bax.

Over-Expression of c-FLIP Confers the Resistance to TRAIL-Induced Apoptosis on Gallbladder Carcinoma

Gallbladder carcinoma (GBC) is an aggressive malignancy with high mortality, mainly due to the reduced chance of curative resection and the resistance to chemotherapeutic drugs. Here, we showed that cellular Fas-associated death domain-like interleukin-1 converting enzyme inhibitory protein (c-FLIP), an anti-apoptotic protein, was over-expressed in the most of gallbladder carcinoma tissues, as judged by immunohistochemistry. Semi-quantitation was performed by determining the percentage of c-FLIP-positive cells: no positive cells (-), approximately 1% positive cells (+), approximately 30% positive cells (++), and >70% positive cells (+++). Out of the 35 tissue specimens of gallbladder carcinoma, positive c-FLIP expression was found in 26 samples (6/positive+++, 13/++, 7/+), whereas negative or weak c-FLIP staining was detected in normal (1/+, 9/-) and adenomatous (2/+, 8/-) gallbladder tissues. Then, we used a small interference RNA (siRNA), which can substantially down-regulate the expression levels of c-FLIP mRNA and protein in GBC-SD and SGC-996 human gallbladder carcinoma cells, as confirmed by real-time PCR and western blot analyses. Furthermore, the combined treatment with the c-FLIP siRNA and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) significantly induced apoptosis in gallbladder carcinoma cells, as judged by the increases in pyknosis, caspase-3/7 activities, and Annexin V-propidium iodide labeling, a marker for chromatin condensation. Thus, the siRNA-mediated down-regulation of c-FLIP profoundly enhances the sensitivity to TRAIL-induced apoptosis. In conclusion, c-FLIP expression is up-regulated in gallbladder carcinoma and the down-regulation of c-FLIP sensitizes TRAIL-induced apoptosis. The present study provides a potent strategy for the treatment of gallbladder carcinoma by targeting the c-FLIP.