Shanmiao Gou

Establishment of clonal colony-forming assay for propagation of pancreatic cancer cells with stem cell properties.

Objective: Pancreatic cancer is among the most aggressive solid malignancies. It is possible that pancreatic cancer contains cancer stem cells responsible for its malignancy. The purposes of this study were (1) to establish an assay in which a subset of pancreatic cancer cell line (PANC-1) cells with stem cell properties can propagate, and (2) to identify the cells obtained from this assay. Methods: The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 supplemented with epidermal growth factor, basic fibroblast growth factor, insulin, transferrin, selenium, and bovine serum albumin at a density of 1000 cells/mL for 10 to 14 days to form spheres. Cells of spheres were cultured in different conditions to evaluate their ability of self-renewal and differentiation. Clone formation assay and tumor formation assay were used to identify the ability of propagation in vitro and in vivo. The cells and spheres were also stained by using Hoechst 33342 dye to evaluate their capacity of excluding Hoechst dye. Real-time polymerase chain reaction was used to detect expressions of LY6E, c-Met, TACSTD1, CD34, and CD44 mRNA. Results: A subpopulation of PANC-1 cells could propagate to form spheres in this assay. Cells of obtained spheres had the hallmark of excluding Hoechst 33342 dye. Cultured in serum-free medium, the dissociated single cells of primary spheres could form filial spheres again; cultured in serum-containing medium, these cells generated both the cells with the ability of excluding Hoechst 33342 dye and the cells without that ability. The propagation capacity of PANC-1 spheres was higher than that of cells cultured in serum-containing medium both in vitro and in vivo. In addition, LY6E, TACSTD1, and CD44 mRNA were overexpressed in PANC-1 spheres. Conclusions: A subpopulation of PANC-1 cells can propagate to form spheres with properties of stem cells in this assay; enough of these cells can be obtained for further study. Considering the overexpression of mRNA, it was tentatively suggested that LY6E, TACSTD1, and CD44 proteins may act as surface markers for sorting pancreatic cancer stem cells with fluorescence-activated cell sorter/magnetic-activated cell sorter.

Cancer Stem-Like Cells Enriched in Panc-1 Spheres Possess Increased Migration Ability and Resistance to Gemcitabine

Pancreatic cancer is one of the most lethal malignancies with poor prognosis. Previously, we found that a subpopulation of cancer stem cells (CSCs) in the Panc-1 pancreatic cancer cell line could propagate to form spheres. Here we characterized the malignant phenotypes of the pancreatic cancer stem CD44+/CD24+ cells, which were enriched under sphere forming conditions as analyzed by flow cytometry. These cells demonstrated increased resistance to gemcitabine and increased migration ability. Moreover, these cells exhibited epithelial to mesenchymal transition characterized by a decreased level of the epithelial marker E-cadherin and an increased level of the mesenchymal marker vimentin. Notably, abnormal expression of Bmi-1, ABCG2, Cyclin D1 and p16 were found in Panc-1 CSCs. Our results suggest that targeted inhibition of CSCs represents a novel therapeutic approach to overcome chemoresistance and metastasis of pancreatic cancer.

Pancreaticoduodenectomy with Vascular Resection for Local Advanced Pancreatic Head Cancer: A Single Center Retrospective Study

IntroductionPancreaticoduodenectomy with vascular resection remains a controversial approach for patients with local advanced pancreatic head cancer for the lack of evidences of survival and quality of life benefits. The aim of this study was to evaluate whether patients of pancreatic head cancer benefit on quality of life, survival, and treatment cost from pancreaticoduodenectomy with vascular resection compared with palliative therapy.Materials and MethodsTwo hundred fourteen patients of pancreatic head cancer whose pancreatic head could not be dissected free from adjacent vascular were involved in this study. Eighty of these patients underwent pancreaticoduodenectomy with vascular resection, whereas other patients underwent palliative therapy.ResultsPancreaticoduodenectomy with artery resection offered worse outcomes on almost all aspects of quality of life and survival compared with palliative therapy. Pancreaticoduodenectomy with vein resection offered better 5-year survival compared with palliative therapy, whereas palliative therapy offered better quality of life after surgery.ConclusionPancreaticoduodenectomy with artery resection is nonsensical on treatment of pancreatic head cancer with artery adhesion/invasion. As for patients with vein adhesion/invasion, pancreaticoduodenectomy with vein resection should be performed cautiously. When actual vein invasion is very possible to have taken place, the choice of treatment strategy should be considered carefully by the pancreatic surgeons.

Use of probiotics in the treatment of severe acute pancreatitis: a systematic review and meta-analysis of randomized controlled trials

IntroductionNecrotic tissue infection can worsen the prognosis of severe acute pancreatitis (SAP), and probiotics have been shown to be beneficial in reducing the infection rate in animal experiments and primary clinical trials. However, the results of multicenter randomized clinical trials have been contradictory. Our aim in this study was to systematically review and quantitatively analyze all randomized controlled trials with regard to important outcomes in patients with predicted SAP who received probiotics.MethodsA systematic literature search of the PubMed, Embase and Cochrane Library databases was conducted using specific search terms. Eligible studies were randomized controlled trials that compared the effects of probiotic with placebo treatment in patients with predicted SAP. Mean difference (MD), risk ratio (RR) and 95% confidence interval (95% CI) were calculated using the Mantel-Haenszel fixed- and random-effects models. A meta-analysis on the use of probiotics in the treatment of critically ill patients was also performed to serve as a reference.ResultsIn this study, 6 trials comprising an aggregate total of 536 patients were analyzed. Significant heterogeneities were observed in the type, dose, treatment duration and clinical effects of probiotics in these trials. Systematic analysis showed that probiotics did not significantly affect the pancreatic infection rate (RR = 1.19, 95% CI = 0.74 to 1.93; P = 0.47), total infections (RR = 1.09, 95% CI = 0.80 to 1.48; P = 0.57), operation rate (RR = 1.42, 95% CI = 0.43 to 3.47; P = 0.71), length of hospital stay (MD = 2.45, 95% CI = −2.71 to 7.60; P = 0.35) or mortality (RR = 0.72, 95% CI = 0.42 to 1.45; P = 0.25).ConclusionsProbiotics showed neither beneficial nor adverse effects on the clinical outcomes of patients with predicted SAP. However, significant heterogeneity was noted between the trials reviewed with regard to the type, dose and treatment duration of probiotics, which may have contributed to the heterogeneity of the clinical outcomes. The current data are not sufficient to draw a conclusion regarding the effects of probiotics on patients with predicted SAP. Carefully designed clinical trials are needed to validate the effects of particular probiotics given at specific dosages and for specific treatment durations.

Bmi-1 Promotes the Chemoresistance, Invasion and Tumorigenesis of Pancreatic Cancer Cells

Background/Aims: The polycomb protein Bmi-1 plays oncogenic roles in various cancers. Here we aimed to investigate the contribution of Bmi-1 on the malignant behaviors of pancreatic cancer such as chemoresistance, invasion and tumorigenesis. Methods and Results: The MTT cell proliferation assay showed that shRNA mediated Bmi-1 knockdown and enhanced the chemosensitivity of pancreatic cancer cells to gemcitabine. The transwell invasion assay showed that Bmi-1 knockdown inhibited the invasion of pancreatic cancer cells in vitro. Notably, the reduced abilities of chemoresistance and invasion were associated with the transition from the mesenchymal phenotype to the epithelial phenotype of pancreatic cancer cells. Moreover, Bmi-1 knockdown led to the inhibition of the PI3K-Akt pathway and disrupted the sphere-forming abilities of pancreatic cancer cells. A nude mouse xenograft experiment demonstrated that pancreatic cancer cells depleted of Bmi-1 showed weak tumorigenicity in vivo. Conclusion: Our data suggest that Bmi-1 plays an important role in the progression of pancreatic cancer and represents a novel target for antitumor therapy of pancreatic cancer.

Antiproliferative effect of resveratrol in pancreatic cancer cells

To investigate resveratrol, one of the food derived polyphenols that might be partially responsible for the beneficial effect on cancer, the in vitro antitumor activity of resveratrol against pancreatic cancer cell lines (PANC‐1, BxPC‐3 and AsPC‐1) was examined, together with the mechanisms involved. The effects of resveratrol on the growth inhibition, apoptosis and cell cycle were assayed. The activity of caspases and the expression of Bcl‐2, Bcl‐xL, XIAP and Bax protein were detected. The results showed that resveratrol inhibited the proliferation of pancreatic cancer cells in a dose‐ and time‐dependent manner. Resveratrol inhibited the cell growth of PANC‐1, BxPC‐3 and AsPC‐1 cells with IC50 values of 78.3 ± 9.6 μmol/L, 76.1 ± 7.8 μmol/L and 123.1 ± 6.5 μmol/L at 48 h, respectively. Incubation of pancreatic cancer cells with resveratrol resulted in cell apoptosis and cell cycle arrests. Resveratrol induced activation of caspases. Simultaneously, resveratrol regulated the expression of the antiapoptotic proteins Bcl‐2, Bcl‐xL and XIAP and the proapoptotic protein Bax. PANC‐1 and BxPC‐3 cells were more chemosensitive to resveratrol than AsPC‐1 cells. In conclusion, resveratrol inhibited the proliferation of pancreatic cancer cells by inducing apoptotic cell death. There was different sensitivity to resveratrol in different pancreatic cancer cell lines. Copyright

ShRNA silencing glycogen synthase kinase-3 beta inhibits tumor growth and angiogenesis in pancreatic cancer.

Glycogen synthase kinase-3 beta (GSK-3β), a serine/threonine protein kinase, plays a vital role in the tumorigenesis of many cancers, but its role in pancreatic cancer remains unknown. In this study, we showed that GSK-3β was aberrantly activated in pancreatic cancer. GSK-3β knockdown resulted in arrested proliferation and increased apoptosis in pancreatic cancer cell lines. Expression of Bcl-2 and vascular endothelial growth factor (VEGF) decreased significantly in a GSK-3β knockdown group. In a xenograft tumor model, GSK-3β knockdown inhibited tumor growth and angiogenesis. Our study showed that GSK-3β may become a promising therapeutic target for human pancreatic cancer.

Pylorus-Preserving Versus Pylorus-Resecting Pancreaticoduodenectomy for Periampullary and Pancreatic Carcinoma: A Meta-Analysis

Background The aim of this meta-analysis was to compare the long-term survival, mortality, morbidity and the operation-related events in patients with periampullary and pancreatic carcinoma undergoing pylorus-preserving pancreaticoduodenectomy (PPPD) and pylorus-resecting pancreaticoduodenectomy (PRPD). Method A systematic search of literature databases (Cochrane Library, PubMed, EMBASE and Web of Science) was performed to identify studies. Outcome measures comparing PPPD versus PRPD for periampullary and pancreatic carcinoma were long-term survival, mortality, morbidity (overall morbidity, delayed gastric emptying [DGE], pancreatic fistula, wound infection, postoperative bleeding, biliary leakage, ascites and gastroenterostomy leakage) and operation related events (hospital stays, operating time, intraoperative blood loss and red blood cell transfusions). Results Eight randomized controlled trials (RCTs) including 622 patients were identified and included in the analysis. Among these patients, it revealed no difference in long-term survival between the PPPD and PRPD groups (HR = 0.23, p = 0.11). There was a lower rate of DGE (RR = 2.35, p = 0.04, 95% CI, 1.06–5.21) with PRPD. Mortality, overall morbidity, pancreatic fistula, wound infection, postoperative bleeding, biliary leakage, ascites and gastroenterostomy leakage were not significantly different between the groups. PPPDs were performed more quickly than PRPDs (WMD = 53.25 minutes, p = 0.01, 95% CI, 12.53–93.97); and there was less estimated intraoperative blood loss (WMD = 365.21 ml, p = 0.006, 95% CI, 102.71–627.71) and fewer red blood cell transfusions (WMD = 0.29 U, p = 0.003, 95% CI, 0.10–0.48) in patients undergoing PPPD. The hospital stays showed no significant difference. Conclusions PPPD had advantages over PRPD in operating time, intraoperative blood loss and red blood cell transfusions, but had a significantly higher rate of DGE for periampullary and pancreatic carcinoma. PPPD and PRPD had comparable mortality and morbidity including pancreatic fistulas, wound infections, postoperative bleeding, biliary leakage, ascites and gastroenterostomy leakage. Our conclusions were limited by the available data. Further evaluations of high-quality RCTs are needed.

Spontaneous Differentiation of Murine Bone Marrow-Derived Mesenchymal Stem Cells into Adipocytes without Malignant Transformation after Long-Term Culture

Some observations have suggested that extensive culture of adult stem cells can lead to malignant transformation. Therefore, it has become commonplace to use stem cells undergoing little or no in vitro culture to circumvent this presumptive limitation. Recently, a detailed study documented that malignant transformation of adult neural stem cells can be avoided under suitable culture conditions. Here, we report the first demonstration that murine bone marrow-derived mesenchymal stem cells (bMSCs) were propagated in vitro for up to 50 passages without any transformation sign under suitable conditions. However, it must be noted that although the long-term cultured bMSCs were comparable with short-term cultured bMSCs in proliferation, migration and invasion, they lost their pluripotent potential. The long-term cultured bMSCs could only differentiate into adipocytes but not into osteocytes or chondrocytes. In conclusion, murine bMSCs can be propagated in vitro for up to 50 passages with no malignant transformation sign under suitable conditions, but how to sustain their pluripotent potential requires further investigation.

Heparin inhibits the inflammatory response induced by LPS and HMGB1 by blocking the binding of HMGB1 to the surface of macrophages.

High mobility group box 1 protein (HMGB1), a nuclear non-histone DNA-binding protein, is secreted extracellularly during inflammation and is a late mediator of inflammatory responses. The pro-inflammatory activity of recombinant HMGB1 proteins is dependent upon the formation of complexes with other mediators, such as lipopolysaccharide (LPS). This study investigated the influence of heparin on LPS+HMGB1-mediated inflammatory responses in cultured macrophages and a murine sepsis model. HMGB1 promoted the phosphorylation of p38 and ERK1/2. HMGB1 enhanced the induction of the pro-inflammatory cytokine, TNF-α, by LPS in macrophages. Heparin blocked the binding of HMGB1 to the surface of macrophages, and suppressed the phosphorylation of p38 and ERK1/2, but not JNK; TNF-α secretion was also decreased. However, heparin alone did not affect LPS-induced production of TNF-α. Heparin reduced lethality in mice exposed to LPS+HMGB1. To conclude, heparin inhibited LPS-induced HMGB1-amplified inflammatory responses by blocking HMGB1 binding to macrophage surfaces. Heparin could be used therapeutically as an effective inhibitor of HMGB1-associated inflammation.