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.

Ischemia/reperfusion in clamped lobes facilitates liver regeneration of non-clamped lobes after selective portal vein ligation.

BACKGROUND: Hypertrophy of non-clamped liver lobes and the atrophy of clamped lobes have been shown to be interactive. Here, a rat model of selective lobe occlusion was established to study the effect of contralateral ischemia/reperfusion (I/R) on regeneration of non-clamped lobes. METHODS: Left lateral and middle liver lobes were pretreated with I/R. In the experimental (IR + PVL) group, portal veins of the left and middle lobes were ligated. A group given similar portal vein ligation but no I/R (PVL) was the positive control. RESULTS: Compared with the PVL group, the IR + PVL had higher, but not remarkable, levels of serum transaminases; weights of non-clamped lobes in the IR + PVL group comparatively increased much more notably. At 24-h post-surgery, the IR + PVL group's PCNA mRNA was up-regulated compared with the PVL group. At 72-h post-surgery, PCNA protein was up-regulated significantly, while TGF-ß1 was down-regulated in the IR + PVL group notably, compared with the PVL group. CONCLUSION: The I/R pretreatment given to the clamped lobes facilitates liver regeneration of non-clamped lobes after selective portal vein ligation, which may result from down-regulated TGF-ß1 expression in non-clamped lobes.

Impact of pancreatic margin status and lymph node metastases on recurrence after resection for invasive and noninvasive intraductal papillary mucinous neoplasms of the pancreas: a meta-analysis.

BACKGROUND: Accurate information is currently lacking regarding the values of positive margins (M(+)) and lymph node (LN) metastases as independent predictors of postoperative recurrence in invasive and noninvasive intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. METHODS: A comprehensive online literature search identified all types of primary studies that included M(+) and LN metastases as risk factors and defined recurrence as an outcome in patients with IPMNs. Suitable articles were also identified by manually researching references in qualifying articles. A meta-analysis of the result was performed using a random effects model. RESULTS: The recurrence rate in noninvasive IPMNs was 3.72% in patients with negative margin (M(-)) versus 9.56% in those with M(+) (odds ratio, OR = 0.37, 95% confidence interval, 95% CI: 0.17-0.78, p = 0.010). The recurrence rate in invasive M(-) IPMNs in was 33.85% compared to 53.66% in M(+) IPMNs (OR = 0.47, 95% CI: 0.25-0.88, p = 0.020). The recurrence rate in invasive IPMNs with positive LN was 76.92% compared to 30.86% with negative LN; OR = 0.15, 95% CI: 0.06-0.37, p < 0.0001). CONCLUSIONS: M(+) were associated with disease recurrence in all patients with IPMN, and nodal metastases were significantly associated with recurrence in invasive IPMN.

Prognostic significance of regional lymphadenectomy in T1b gallbladder cancer: Results from 24 hospitals in China.

BACKGROUND: Whether regional lymphadenectomy (RL) should be routinely performed in patients with T1b gallbladder cancer (GBC) remains a subject of debate. AIM: To investigate whether RL can improve the prognosis of patients with T1b GBC. METHODS: We studied a multicenter cohort of patients with T1b GBC who underwent surgery between 2008 and 2016 at 24 hospitals in 13 provinces in China. The log-rank test and Cox proportional hazards model were used to compare the overall survival (OS) of patients who underwent cholecystectomy (Ch) + RL and those who underwent Ch only. To investigate whether combined hepatectomy (Hep) improved OS in T1b patients, we studied patients who underwent Ch + RL to compare the OS of patients who underwent combined Hep and patients who did not. RESULTS: Of the 121 patients (aged 61.9 ± 10.1 years), 77 (63.6%) underwent Ch + RL, and 44 (36.4%) underwent Ch only. Seven (9.1%) patients in the Ch + RL group had lymph node metastasis. The 5-year OS rate was significantly higher in the Ch + RL group than in the Ch group (76.3% vs 56.8%, P = 0.036). Multivariate analysis showed that Ch + RL was significantly associated with improved OS (hazard ratio: 0.51; 95% confidence interval: 0.26-0.99). Among the 77 patients who underwent Ch + RL, no survival improvement was found in patients who underwent combined Hep (5-year OS rate: 79.5% for combined Hep and 76.1% for no Hep; P = 0.50). CONCLUSION: T1b GBC patients who underwent Ch + RL had a better prognosis than those who underwent Ch. Hep + Ch showed no improvement in prognosis in T1b GBC patients. Although recommended by both the National Comprehensive Cancer Network and Chinese Medical Association guidelines, RL was only performed in 63.6% of T1b GBC patients. Routine Ch + RL should be advised in T1b GBC.

IL-6/STAT3/TFF3 signaling regulates human biliary epithelial cell migration and wound healing in vitro.

Interleukin-6 (IL-6), through activation of the signal transducer and activator of transcription 3 (STAT3) and trefoil factor family 3 (TFF3), has been implicated in the promotion of mouse biliary epithelial cell (BEC) proliferation and migration. However, it is still unclear whether the IL-6/STAT3/TFF3 signaling had similar effects on human BECs. Here, we showed that exposure of human BECs to recombinant IL-6 resulted in STAT3 phosphorylation and increased the expression of TFF3 at both mRNA and protein levels. Moreover, inhibition of STAT3 using RNA interference significantly abrogated IL-6-induced TFF3 expression. In an in-vitro wound healing model, IL-6 facilitated human BEC migration. This promotion of cell migration by IL-6 was blocked when STAT3 was knocked down. Interestingly, the addition of exogenous TFF3 could rescue the cell migration defects caused by STAT3 silencing. In conclusion, our data indicate that STAT3 plays a critical role in IL-6-induced TFF3 expression in human BECs and the IL-6/STAT3/TFF3 signaling is involved in human BEC migration and wound healing.

Reconstitution of secreted frizzled-related protein 1 suppresses tumor growth and lung metastasis in an orthotopic model of hepatocellular carcinoma.

BACKGROUND: Secreted Frizzled-related protein 1 (sFRP1) is frequently silenced in many types of cancer, including hepatocellular carcinoma (HCC), leading to aberrant activation of Wnt signaling and thereby facilitating tumor development. In this study, we aimed to investigate whether restoration of sFRP1 affected HCC growth and metastasis. METHODS: We generated stable cell lines overexpressing sFRP1 in MHCC97-H cells, which naturally do not express detectable sFRP1 messenger RNA (mRNA) and have high metastatic properties. The effects of sFRP1 reexpression on tumor growth and metastasis were assessed in vitro and in vivo. It was also tested whether ß-catenin signaling mediated the function of sFRP1 in tumor progression. RESULTS: Overexpression of sFRP1 substantially diminished the proliferation and invasion potentials of MHCC97-H cells. Furthermore, sFRP1 expression significantly inhibited MHCC97-H xenograft growth and metastasis in vivo, which was accompanied by decreased angiogenesis and increased tumor cell apoptosis. Moreover, sFRP1 overexpression caused less expression of ß-catenin and its downstream effector genes cyclin D1 and matrix metalloproteinase (MMP)-2. CONCLUSION: Together these findings demonstrate that sFRP1 reconstitution suppresses tumor growth, angiogenesis, and metastasis in MHCC97-H xenografts, which may be associated with inactivation of ß-catenin signaling, thus providing a possible therapeutic strategy against HCC.

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.

[Study on combined therapy of hyperlipidemic severe acute pancreatitis].

OBJECTIVE: To investigate the principle and measures of combined treatment of the patients with hyperlipidemic severe acute pancreatitis (HL-SAP). METHODS: The clinical data of 54 patients with HL-SAP including two phases from January 1996 to December 2000 and from January 2001 to August 2006 were analyzed retrospectively. In the first phase, 25 patients were performed by routine methods to decrease triglyceride, or additional operative treatments. In the second phase, 29 cases were treated by multiple ways of non-operative combined therapy, or additional operative treatments mainly by minimally invasive procedures. RESULTS: Among 54 cases with HL-SAP, 33 cases (61.1%) received non-operative therapy and 21 cases (38.9%) received surgical intervention. Overall mortality was 18.5% (10/54). In the first phase of 25 cases, the mortality in non-operative group and surgical intervention group was 21.4% (3/14) and 36.3% (4/11), respectively. In the second phase of 29 cases, the mortality in non-operative group and surgical intervention group was 10.5% (2/19) and 10.0% (1/10), respectively. The overall curative rate, morbidity, overall mortality, content of triglyceride at the fourth day after onset, APACHE II score at the fourth day after onset and average stay were obviously improved in the second phase compared with the first phase (P < 0.05). CONCLUSIONS: According to individualized therapy principles, treatment for HL-SAP should emphasis on multiple ways of non-operative combined therapy and appropriate choices of the timing, indication in surgical intervention. And the choice of operative procedure should follow the principle of minimally invasive surgery. Meanwhile, pay more attention to monitoring and controlling the level of triglyceride post-discharge for the patients with the history of HL-SAP.

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.

Prevention of core cell damage in isolated islets of Langerhans by low temperature preconditioning.

AIM: To study the core cell damage in isolated islets of Langerhans and its prevention by low temperature preconditioning (26 degrees). METHODS: Islets were cultured at 37 degrees for 7-14 d after isolation, and then at 26 degrees for 2, 4 and 7 d before additional culture at 37 degrees for another 7 d. Core cell damage in the isolated islets was monitored by video-microscopy and analyzed quantitatively by use of a computer-assisted image analysis system. The analysis included daily measurement of the diameter and the area of the isolated islets and the area of the core cell damage that developed in those islets over time during culture. Histology and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to characterize the cell damage and to monitor islet function. RESULTS: Microscopic analysis showed that during the 7 to 14 d of culture at 37 degrees, core cell damage occurred in the larger islets with diameters >200 microm, which included both necrotic and apoptotic cell death. Low temperature (26 degrees) culture could prevent core cell damage of isolated islets. The 7-d culture procedure at 26 degrees could inhibit most of the core cell (excluding diameters >300 microm) damages when the islets were re-warmed at 37 degrees. CONCLUSION: Our results indicate that core cell damage within isolated islets of Langerhans correlates with the size of islets. Low temperature (26 degrees) culture can prevent core cell damage in isolated islets, and successfully precondition these islets for incubation at 37 degrees. These novel findings may help to understand the pathophysiology of early loss of islet tissue after transplantation, and may provide a new strategy to improve graft function in the clinical setting of islet transplantation.

Prevention of central cell damage to isolated islets of Langerhans in hamsters by low temperature preconditioning.

BACKGROUND: The efficacy of clinical islet transplantation has been demonstrated with autografts, and although islet allografts have established insulin independence in a small number of IDDM patients, the treatment is confounded by the necessity of central cell damage immunosuppression, the lack of donor tissue, and recurring islet immunogenicity. These limitations underscore a need to develop therapies to serve the large population of diabetic patients. This study was designed to document central cell damage to isolated islets of Langerhans in hamsters and its prevention. METHODS: Islets were cultured at 37 degree centigrade for 7-14 days after isolation, and then at 26 degree centigrade for 2,4 and 7 days before additional culture at 37 degree centigrade for an additional 7 days. Central cell damage in the isolated islets was monitored by video-microscopy and analyzed quantitatively by a computer-assisted image analysis system. The analysis included daily measurement of the diameter and the area of the isolated islets and the area of the central cell damage that developed in those islets over time during culture. Histological examination and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to characterize cell damage and to monitor islet function. RESULTS: Microscopic analysis showed that during the 7 to 14 days of culture at 37 degree centigrade, central cell damage appeared in the larger islets with diameters greater than 200 microm, which included both necrotic and apoptotic cell death. Low temperature (26 degree centigrade) culture prevented central cell damage of isolated islets. The 7-day culture procedure at 26 degree centigrade could inhibit most of the central cell (excluding diameters greater than 300 microm) damage when the islets were rewarmed to 37 degree centigrade. CONCLUSIONS: Our results indicate that central cell damage to isolated islets of Langerhans correlates with the size of the islets. Low temperature (26 degree centigrade) culture can prevent central cell damage to the isolated islets, and is capable to successfully precondition these islets for 37 degree centigrade culture. These novel findings may help to understand the pathophysiology of early loss of islet tissue after transplantation, and may provide a new strategy to improve graft function in the clinical setting of islet transplantation.

Enhanced expression of suppresser of cytokine signaling 3 inhibits the IL-6-induced epithelial-to-mesenchymal transition and cholangiocarcinoma cell metastasis.

It was recently demonstrated that interleukin-6 (IL-6) induces the epithelial-to-mesenchymal transition (EMT) in cholangiocarcinoma (CCA), but the underlying molecular mechanism remains to be explored. In this study, we studied the role of suppresser of cytokine signaling 3 (SOCS3), a negative feedback regulator of IL-6/STAT3, in the IL-6-induced EMT in CCA. Treatment with IL-6 induced the EMT by decreasing the E-cadherin expression and increasing the expression of N-cadherin and vimentin. Using wound healing and invasion assays, we found that IL-6 promoted cell motility. Further, a stably transfected cell line overexpressing SOCS3 was constructed. Enhanced SOCS3 expression decreased IL-6-induced cell invasion and EMT in parallel with downregulating the IL-6/STAT3 pathway. In contrast, SOCS3 silencing using siRNA exhibited no effect on the cell invasive ability and EMT. Finally, an in vivo study indicated that the enhancement of SOCS3 expression decreased metastasis compared with the control, and this effect was achieved by the repression of p-STAT3, N-cadherin and vimentin, and the induction of E-cadherin assessed by Western blot analysis. Our results suggest that enhanced expression of SOCS3 can antagonize IL-6-induced EMT and cell metastasis by abrogating the IL-6/STAT3 pathway. These data establish that SOCS3 plays a role in the EMT in CCA and may provide novel therapeutic strategies for CCA.

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.

Synergism of cytotoxicity effects of triptolide and artesunate combination treatment in pancreatic cancer cell lines.

BACKGROUND: Triptolide, extracted from the herb Tripteryglum wilfordii Hook.f that has long been used as a natural medicine in China, has attracted much interest for its anti-cancer effects against some kinds of tumours in recent years. Artesunate, extracted from the Chinese herb Artemisia annua, has proven to be effective and safe as an anti-malarial drug that possesses anticancer potential. The present study attempted to clarify if triptolide enhances artesunate-induced cytotoxicity in pancreatic cancer cell lines in vitro and in vivo. METHODS: In vitro, to test synergic actions, cell viability and apoptosis were analyzed after treatment of pancreatic cancer cell lines with the two agents singly or in combination. The molecular mechanisms of apoptotic effects were also explored using qRT-PCR and Western blotting. In vivo, a tumor xenograft model was established in nude mice, for assessment of inhibitory effects of triptolide and artesunate. RESULTS: We could show that the combination of triptolide and artesunate could inhibit pancreatic cancer cell line growth, and induce apoptosis, accompanied by expression of HSP 20 and HSP 27, indicating important roles in the synergic effects. Moreover, tumor growth was decreased with triptolide and artesunate synergy. CONCLUSION: Our result indicated that triptolide and artesunate in combination at low concentrations can exert synergistic anti-tumor effects in pancreatic cancer cells with potential clinical applications.

PI3K/AKT/mTOR signaling is involved in (-)-epigallocatechin-3-gallate-induced apoptosis of human pancreatic carcinoma cells.

PI3K/AKT/mTOR signaling promotes cell survival, proliferation and progression in cancer cells. Targeting this pathway may lead to the development of novel therapeutic approaches for human cancers. Here, we examined the effects of (-)-epigallocatechin-3-gallate (EGCG) on the PI3K/AKT/mTOR pathway in pancreatic cancer cells, and assessed its therapeutic potential. In this study, the proliferation and apoptosis of PANC-1 cells were examined by MTT assay and flow cytometry, respectively. The expression of genes and proteins involved in the PI3K/AKT/mTOR pathway were measured by RT-PCR and western blotting, respectively. Our results revealed that EGCG dramatically inhibited the proliferation of PANC-1 cells and induced apoptosis simultaneously. Furthermore, it upregulated PTEN mRNA and protein expression levels, as well as downregulating the expression of phospho-AKT and phospho-mTOR. In conclusion, these results suggest that EGCG can suppress proliferation and induce apoptosis of PANC-1 cells in a time- and dose-dependent manner; moreover, EGCG also can upregulate PTEN expression and downregulate the expression of pAKT and p-mTOR to modulate the PI3K/AKT/mTOR signaling pathway.

MicroRNA-421 functions as an oncogenic miRNA in biliary tract cancer through down-regulating farnesoid X receptor expression.

MicroRNAs (miRNAs) are involved in the development of most cancers. However, few studies have been conducted to determine their relationship to biliary tract cancer (BTC). Farnesoid X receptor (FXR) has been reported to be a tumor suppressor for hepatocellular carcinoma and breast cancer; but few studies have focused on its correlation with BTC. In this study, we identified miR-421 as a potential regulator of FXR expression. We found that their expression amount was inversely correlated as FXR was aberrantly down-regulated in both primary tumor specimens and cell lines; while miR-421 was significantly up-regulated. Ectopic expression of miR-421 significantly decreased FXR protein concentration in BTC cells and promoted cell proliferation, colony formation and migration in vitro. Furthermore, a decrease in miR-421 expression induced G(0)/G(1) cell cycle arrest. In conclusion, our study identified microRNA-421 functions as an oncomiR in BTC by targeting FXR. This finding may provide a novel therapeutic strategy for treatment of biliary tract cancer.

CDK10 functions as a tumor suppressor gene and regulates survivability of biliary tract cancer cells.

Cyclin-dependent kinase 10 (CDK10) is a member of the Cdc2 family of kinases, and has been demonstrated to be an important determinant of resistance to endocrine therapy for breast cancer. To investigate the expression and possible function of CDK10 in biliary tract cancer (BTC), we systematically examined CDK10 in tissues and cell lines. We found that expression of CDK10 was downregulated in both biliary tract tumors and cell lines. Remarkably, the expression of CDK10 correlated with clinical characteristics. Overexpression or knockdown of CDK10, respectively, inhibited or promoted cell proliferation, colony formation and migration. This suggests that CDK10 functions as a tumor suppressor gene in BTC. Overexpression of CDK10 caused malignant cells to become sensitive to chemotherapy and other hostile environments, suggesting that CDK10 functions to regulate survivability of BTC cells. We investigated the expression of six genes to resolve the mechanism. c-RAF was negatively regulated by CDK10 in both cells and specimens. Our results indicate that CDK10 plays a crucial role in the growth and survivability of biliary tract cancer, and offers a potential therapeutic target for this fatal disease.