Induction of cell-cycle arrest and apoptosis in human cholangiocarcinoma cells by pristimerin.

Pristimerin, a triterpenoid isolated from Celastraceae and Hippocrateaceae, is known to induce cytotoxicity in several cancer cell lines. However, whether pristimerin can induce apoptosis in cholangiocarcinoma cells and the underlying mechanism remain unexplored. We assessed the function of human cholangiocarcinoma QBC and RBE cell lines using various experimental methods such as the cell viability assay to elucidate the viability of cells, flow cytometry to detect the death rate of cells, and Western blot analysis to evaluate the expression of cell cycle-related proteins and autophagy-related proteins. Human cholangiocarcinoma QBC cells were transplanted to nude mice to establish an animal model, and the effect of pristimerin on tumor growth in this model was observed. QBC and RBE cell lines treated with pristimerin (0, 5, 10, and 20 µmol/L) demonstrated the induction of apoptosis in a dose-dependent manner. The cell viability assay revealed a reduction in the cell viability with an increase in the pristimerin concentration. Similarly, flow cytometry revealed a gradual increase in the cell death rate with an increase in the pristimerin concentration. In addition, pristimerin significantly lowered the expression of apoptosis-related proteins (Bcl-2, Bcl-xL, and procaspase-3), but increased the Bax expression. Furthermore, pristimerin resulted in the G0/G1 cell-cycle arrest, reducing the expression of cell cycle-related proteins (cyclin E, CDK2, and CDK4), and increased the expression of autophagy-related proteins (LC3) in QBC cell line. Treatment with pristimerin could inhibit tumor growth in the nude mouse model. Overall, this study suggests the potential effect of pristimerin on the cell-cycle arrest and apoptosis in human cholangiocarcinoma cells.

miR-191 Inhibition Induces Apoptosis Through Reactivating Secreted Frizzled-Related Protein-1 in Cholangiocarcinoma.

BACKGROUND/AIMS: Cholangiocarcinoma (CCA) is one of the most common malignant tumors of the biliary tract originating from biliary epithelial cells. Although many therapeutic strategies have been developed to treat CCA, the survival rate for CCA patients is still quite low. Thus it is urgent to elucidate the pathogenesis of CCA and to explore novel therapeutic targets. miR-191 has been shown to be associated with many human solid cancers, but the function of miR-191 in CCA is still poorly understood. METHODS: We first investigated the expression level of miR-191 in human CCA tissues and cell lines with quantitative real-time PCR (qRT-PCR). The effects of miR-191 on CCA cells were determined by Cell Counting Kit-8 assay, colony formation assay and acridine orange/ethidium bromide staining. Finally, we utilized qRT-PCR, western blot and luciferase reporter assays to verify the miR-191 target gene. RESULTS: We showed that miR-191 was up-regulated in CCA cell lines and patients. Knockdown of miR-191 by transfection of its inhibitor sequence blocked RBE cells viability and induced apoptosis of RBE cells. Both qRT-PCR and western blot analysis showed that the secreted frizzled-related protein-1 (sFRP1) level was negatively correlated with that of miR-191. Luciferase assay validated that sFRP1 was a direct target of miR-191. Moreover, knockdown of miR-191 led to suppression of Wnt/ß-catenin signaling activation. Co-transfection of sFRP1 small interfering RNA (siRNA) and miR-191 inhibitor re-activated the Wnt/ß-catenin signaling pathway as detected by an increased level of ß-catenin and phosphorylation of GSK-3ß, and restored the expression of survivin and c-myc in RBE cells. Co-transfection of sFRP1 siRNA with miR-191 inhibitor restored the colony formation ability and viability of RBE cells. CONCLUSION: Taken together, our results demonstrate a novel insight into miR-191 biological function in CCA. Our findings suggest that miR-191 is a potential therapeutic target of CCA treatment.

Silenced LASP1 interacts with DNMT1 to promote TJP2 expression and attenuate articular cartilage injury in mice by suppressing TJP2 methylation.

To investigate the regulatory mechanisms and effects of LIM and SH3 protein 1 (LASP1) on osteoarthritis (OA). IL-1ß was used to induce OA in cell models. Viability and apoptosis of chondrocytes were assessed. The expressions of tumor necrsis factor-α (TNF-α) and IL-6 were measured by ELISA kit, and Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were performed to test the expression of related proteins. The STRING database was used to predict the relationship between LASP1 and DNA methyltransferase 1 (DNMT1). The tight junction protein 2 (TJP2) and Gene Expression Omnibus data were analyzed for differential OA genes. Methylation-specific PCR detected methylation of the TJP2 promoter region, and chromatin immunoprecipitation detected the enrichment of DNMT1 in the TJP2 promoter region. Safranin O-Fast Green staining and hematoxylin and eosin staining were used to determine the OARSI score and evaluate the pathological conditions of the joint tissues. LASP1 was highly expressed in IL-1ß-induced cell models. Silencing of LASP1 promoted chondrocyte proliferation and expression of Collagen II and Aggrecan and inhibited chondrocyte apoptosis, inflammatory factors, and matrix metalloprotein expression. TJP2 is weakly expressed in OA models, and LASP1 promotes methylation of the TJP2 promoter region by interacting with DNMT1. Silencing of LASP1 attenuated IL-1ß-induced chondrocyte degeneration by promoting TJP2 expression. Similarly, silencing LASP1 promotes TJP2 expression to alleviate articular cartilage injury in mice with OA. Silencing of LASP1 inhibited the methylation of the TJP2 promoter region by interacting with DNMT1, thereby alleviating articular cartilage damage in OA mice.

Radical modular pancreatoduodenectomy for pancreatic head cancer using a combination of multiple artery-first approaches technique.

The aim of this study was to describe and assess the efficacy of a combination of multiple artery-first approaches (CMAFA) in pancreatoduodenectomy (PD) depending on the tumor location from an embryonic point of view.Between January 2011 and December 2016, seventy-nine consecutive patients with pancreatic head cancer (PHC) underwent PD with curative intent. Patients were classified into two groups according to the surgical procedure: CMAFA-PD group (n = 38) and conventional PD (Co-PD) group (n = 41). Clinicopathlogical variables and clinical outcomes were compared among the two groups.The CMAFA technique demonstrated an improved rate of R0 resection (89.5% vs. 70.7%, P = .038) and a higher median lymph node yield (24 vs.20, P = .034). The CMAFA-PD group was associated with reduced blood loss (450 vs. 600 ml, P = .049), lower rate of blood transfusion (23.7% vs. 46.3%, P = .035), and shorter length of hospital stay (19 vs. 26 days, P < .001). The rates of 90-day mortality, major morbidity, and readmission were comparable among the two groups.This study demonstrates that CMAFA is a feasible and efficient technique with acceptable perioperative and oncological outcomes in treating patients with PHC.

Results of adjuvant radiation therapy for locoregional perihilar cholangiocarcinoma after curative intent resection.

PURPOSE: This study sought to define the role of adjuvant radiation therapy (RT) for patients with curative intent resection of perihilar cholangiocarcinoma (pCCA). PATIENTS AND METHODS: By using the Surveillance, Epidemiology and End Results (SEER) registry, 1,917 patients with non-metastatic pCCA who underwent surgical resection from 1988 to 2009 were included in this study. Propensity score methods were used to compare the survival outcomes of patients treated with and without adjuvant RT after controlling for selection bias. RESULTS: Of the 1,917 patients, 762 (39.7%) received adjuvant RT. In the unmatched population, median overall survival (OS) for patients receiving adjuvant RT compared with those undergoing surgery alone was 23 versus 22 months (P=0.651). Patients who received adjuvant RT were younger (65 vs 68 years, P<0.001), had more regional diseases (86.0% vs 76.7%, P<0.001), and had more positive lymph nodes (43.8% vs 32.2%, P<0.001). In the matched population, adjuvant RT did not show better OS (22 vs 23 months, P=0.978) or cancer-specific survival (CSS) (17 vs 18 months, P=0.554). CONCLUSION: Adjuvant RT is not associated with improved survival of patients with resected pCCA. These data suggest that adjuvant RT should not be routinely used to treat patients with pCCA outside research trials. Ideally, prospective randomized trials should be performed to verify the conclusion of this study.

Interleukin­6 induces epithelial­mesenchymal transition in human intrahepatic biliary epithelial cells.

The aim of the present study was to determine the role of interleukin-6 (IL-6) in the epithelial-mesenchymal transition (EMT) of human intrahepatic biliary epithelial cell (HIBEC) lines in vitro. HIBECs were stimulated with IL-6 at concentrations of 0, 10, 20, 50 and 100 µg/l for 24 h. A wound healing and Transwell assay were performed to determine the migratory and invasive capacity of HIBECs, respectively. Following 24 h of incubation, IL-6 at 10 and 20 µg/l significantly increased the number of migrated and invaded cells (P<0.05), while stimulation with 50 and 100 µg/l IL-6 resulted in a further increase of the migratory and invasive capacity compared to that in all other groups (P<0.05). Furthermore, reverse-transcription quantitative polymerase chain reaction and western blot analyses were used to detect the mRNA and protein expression of EMT markers E-cadherin and vimentin in HIBECs. Decreased mRNA levels of E-cadherin accompanied by higher mRNA levels of vimentin were observed in the 10, 20, 50, 100 µg/l IL-6 groups compared to those in the 0 µg/l group (all P<0.05). Furthermore, the protein expression of E-cadherin was decreased, while that of vimentin was increased in the 50 and 100 µg/l IL-6 groups compared to those in the 0, 10 and 20 µg/l IL-6 groups (all P<0.05). The present study therefore indicated that IL-6 promoted the process of EMT in HIBECs as characterized by increased migration and invasion of HIBECs and the typical changes in mRNA and protein expression of the EMT markers E-cadherin and vimentin.

Identifying survival-associated ceRNA clusters in cholangiocarcinoma.

Competing endogenous RNAs (ceRNAs) represent a novel layer regulations of long non-coding RNAs (lncRNAs) and genes that play important roles in cancer pathogenesis by binding microRNAs (miRNAs). However, the competition mechanism of ceRNAs in cholangiocarcinoma (CHOL) is not fully understood. In this study, we constructed a dysregulated ceRNA competitive network (CCEN) to globally characterize the competing difference between CHOL and normal tissues. Then, we integrated affinity propagation and Kaplan­Meier (K-M) methods to identify functional clusters associated with survival. A total of 7 key ceRNA clusters were identified. Further functional annotation analyses found that Cluster23 and Cluster32 involved cell based functions, and the loss of ceRNA competitive relations in clusters may contribute to CHOL, by disturbing important biological processes, such as 'Pathway in cancer', MAPK and Neurotrophin signaling pathway. This study provides further insights into understanding the competitive mechanism of ceRNAs in CHOL.

The Roles of MicroRNA-122 Overexpression in Inhibiting Proliferation and Invasion and Stimulating Apoptosis of Human Cholangiocarcinoma Cells.

Our study investigated whether microRNA-122 (miR-122) played important roles in the proliferation, invasion and apoptosis of human cholangiocarcinoma (CC) cells. QBC939 and RBE cells lines were chosen and divided into five groups: miR-122 mimic group, anti-miR-122 group, negative control (NC) group, mock group and blank group. MiR-122 expression was measured by qRT-PCR. Roles of miR-122 in cell proliferation, apoptosis and invasion were investigated using MTT assay, flow cytometer and Transwell invasion assay, respectively. MiR-122 expression was lower in CC tissues and QBC939 cell than that in normal bile duct tissues, HCCC-9810 and RBE cells. In both QBC939 and RBE cells lines, miR-122 expression was higher in miR-122 mimic group than that in NC group, mock group and blank group; opposite results were found in anti-miR-122 group. Cell proliferation and invasion were remarkably inhibited in miR-122 mimic group after 48 h/72 h transfection, while apoptotic cells numbers were much greater in miR-122 mimic group; the opposite results were obtained from anti-miR-122 group (all P < 0.05). MiR-122 expression was significantly weaker in CC tissues, and miR-122 overexpression might play pivotal roles in inhibiting proliferation, stimulating apoptosis and suppressing invasion of CC cells, suggesting a new target for CC diagnosis and treatment.