AIM:To gain molecular insights into the action of the histone deacetylase inhibitor(HDACI) trichostatin-A(TSA) in pancreatic cancer(PC) cells.METHODS:Three PC cell lines,BxPC-3,AsPC-1 and CAPAN-1,were treated with var...AIM:To gain molecular insights into the action of the histone deacetylase inhibitor(HDACI) trichostatin-A(TSA) in pancreatic cancer(PC) cells.METHODS:Three PC cell lines,BxPC-3,AsPC-1 and CAPAN-1,were treated with various concentrations of TSA for def ined periods of time.DNA synthesis was assessed by measuring the incorporation of 5-bromo-2'deoxyuridine.Gene expression at the level of mRNA was quantif ied by real-time polymerase chain reaction.Expression and phosphorylation of proteins was monitored by immunoblotting,applying an infrared imaging technology.To study the role of p38 MAP kinase,the specif ic enzyme inhibitor SB202190 and an inactive control substance,SB202474,were employed.RESULTS:TSA most eff iciently inhibited BrdU incorporation in BxPC-3 cells,while CAPAN-1 cells displayed the lowest and AsPC-1 cells an intermediate sensitivity.The biological response of the cell lines correlated with the increase of histone H3 acetylation after TSA application.In BxPC-3 cells(which are wild-type for KRAS),TSA strongly inhibited phosphorylation of ERK 1/2 and AKT.In contrast,activities of ERK and AKT in AsPC-1 and CAPAN-1 cells(both expressing oncogenic KRAS) were not or were only modestly affected by TSA treatment.In all three cell lines,but most pronounced in BxPC-3 cells,TSA exposure induced an activation of the MAP kinase p38.Inhibition of p38 by SB202190 slightly but signif icantly diminished the antiproliferative effect of TSA in BxPC-3 cells.Interestingly,only BxPC-3 cells responded to TSA treatment by a signif icant increase of the mRNA levels of bax,a pro-apoptotic member of the BCL gene family.Finally,in BxPC-3 and AsPC-1 cells,but not in the cell line CAPAN-1,signif icantly higher levels of the cell cycle inhibitor protein p21Waf1 were observed after TSA application.CONCLUSION:The biological effect of TSA in PC cells correlates with the increase of acetyl-H3,p21Waf1,phospho-p38 and bax levels,and the decrease of phosphoERK 1/2 and phospho-AKT.展开更多
AIM To evaluate the influence of hyperglycemia on the progression of autoimmune pancreatitis.METHODS We induced hyperglycemia by repetitive intraperitoneal(ip) injection of 50 mg/kg streptozotocin in MRL/Mp J mice, wh...AIM To evaluate the influence of hyperglycemia on the progression of autoimmune pancreatitis.METHODS We induced hyperglycemia by repetitive intraperitoneal(ip) injection of 50 mg/kg streptozotocin in MRL/Mp J mice, which develop autoimmune pancreatitis due to a genetic predisposition. We compared the extent of inflammation(histological score, CD3^+ lymphocytes, CD8^+ T-cells, CD4^+ T-cells, Foxp3^+ T-helper cells) in the pancreas of hyperglycemic and normoglycemic mice. We also analyzed the number of leukocytes, lymphocytes, granulocytes and monocytes in the blood. In addition, we determined the percentage of CD3^+ lymphocytes, CD8^+ T-cells, CD4^+ T-cells, Foxp3^+ T-helper cells, Foxp3^+ CD25^+ T-helper and Foxp3^+T-helper cells in the spleen by flow cytometry.RESULTS Treatment with streptozotocin caused a strong induction of hyperglycemia and a reduction in body weight(P < 0.001). Severe hyperglycemia did not, however, lead to an aggravation, but rather to a slight attenuation of autoimmune pancreatitis. In the pancreas, both the histological score of the pancreas as well as the number of CD3+ lymphocytes(P < 0.053) were decreased by hyperglycemia. No major changes in the percentage of CD8^+ T-cells, CD4^+ T-cells, Foxp3^+ T-helper cells were observed between hyperglycemic and normoglycemic mice. Hyperglycemia increased the numbers of leukocytes(P < 0.001), lymphocytes(P = 0.016), granulocytes and monocytes(P = 0.001) in the blood. Hyperglycemia also moderately reduced the percentage of CD3^+ lymphocytes(P = 0.057), significantly increased the percentage of Foxp3^+ T-helper cells(P = 0.018) and Foxp3^+ CD25^+ T-helper cells(P = 0.021) and reduced the percentage of Foxp3^+T-helper cells(P = 0.034) in the spleen. CONCLUSION Hyperglycemia does not aggravate but moderately attenuates autoimmune pancreatitis, possibly by increasing the percentage of regulatory T-cells in the spleen.展开更多
AIM: To study if three clinically available small molecule kinase inhibitors (SMI), erlotinib, sunitinib and sorafenib, exert antifibrogenic effects on pancreatic stellate cells (PSC) and analyze the basis of their ac...AIM: To study if three clinically available small molecule kinase inhibitors (SMI), erlotinib, sunitinib and sorafenib, exert antifibrogenic effects on pancreatic stellate cells (PSC) and analyze the basis of their action.展开更多
AIM： To gain molecular insights into the expression and functions of endothelin-1 （ET-1） in pancreatic stellate cells （PSC）.METHODS： PSCs were isolated from rat pancreas tissue, cultured, and stimulated with ET-...AIM： To gain molecular insights into the expression and functions of endothelin-1 （ET-1） in pancreatic stellate cells （PSC）.METHODS： PSCs were isolated from rat pancreas tissue, cultured, and stimulated with ET-1 or other extracellular mediators. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2＇-deoxyuridine into DNA and cell migration was studied in a transwell chamber assay. Gene expression at the level of mRNA was quantified by real-time Polymerase chain reaction. Expression and phosphorylation of proteins were monitored by immunoblotting, applying an infrared imaging technology. ET-1 levels in cell culture supernatants were determined by an enzyme immunometric assay. To study DNA binding of individual transcription factors, electrophoretic mobility shift assays were performed.RESULTS： Among several mediators tested, transforming growth factor-β1 and tumour necrosis factor-α displayed the strongest stimulatory effects on ET-1 secretion. The cytokines induced binding of Smad3 and NF-κB, respectively, to oligonucleotides derived from the ET-1 promoter, implicating both transcription factors in the induction of ET-1 gene expression. In accordance with previous studies, ET-1 was found to stimulate migration but not proliferation of PSC. Stimulation of ET-1 receptors led to the activation of two distinct rnitogen-activated protein kinases, p38 and extracellular signal-regulated kinases （ERK）1/2, as well as the transcription factor activator protein-1. At the mRNA level, enhanced expression of the PSC activation marker, α-smooth muscle actin and two proinflammatory cytokines, interleukin （IL）-1β and IL-6, was observed. CONCLUSION： This study provides novel lines of evidence for profibrogenic and proinflammatory actions of ET-1 in the pancreas, encouraging further studies with ET-1 inhibitors in chronic pancreatitis.展开更多
文摘AIM:To gain molecular insights into the action of the histone deacetylase inhibitor(HDACI) trichostatin-A(TSA) in pancreatic cancer(PC) cells.METHODS:Three PC cell lines,BxPC-3,AsPC-1 and CAPAN-1,were treated with various concentrations of TSA for def ined periods of time.DNA synthesis was assessed by measuring the incorporation of 5-bromo-2'deoxyuridine.Gene expression at the level of mRNA was quantif ied by real-time polymerase chain reaction.Expression and phosphorylation of proteins was monitored by immunoblotting,applying an infrared imaging technology.To study the role of p38 MAP kinase,the specif ic enzyme inhibitor SB202190 and an inactive control substance,SB202474,were employed.RESULTS:TSA most eff iciently inhibited BrdU incorporation in BxPC-3 cells,while CAPAN-1 cells displayed the lowest and AsPC-1 cells an intermediate sensitivity.The biological response of the cell lines correlated with the increase of histone H3 acetylation after TSA application.In BxPC-3 cells(which are wild-type for KRAS),TSA strongly inhibited phosphorylation of ERK 1/2 and AKT.In contrast,activities of ERK and AKT in AsPC-1 and CAPAN-1 cells(both expressing oncogenic KRAS) were not or were only modestly affected by TSA treatment.In all three cell lines,but most pronounced in BxPC-3 cells,TSA exposure induced an activation of the MAP kinase p38.Inhibition of p38 by SB202190 slightly but signif icantly diminished the antiproliferative effect of TSA in BxPC-3 cells.Interestingly,only BxPC-3 cells responded to TSA treatment by a signif icant increase of the mRNA levels of bax,a pro-apoptotic member of the BCL gene family.Finally,in BxPC-3 and AsPC-1 cells,but not in the cell line CAPAN-1,signif icantly higher levels of the cell cycle inhibitor protein p21Waf1 were observed after TSA application.CONCLUSION:The biological effect of TSA in PC cells correlates with the increase of acetyl-H3,p21Waf1,phospho-p38 and bax levels,and the decrease of phosphoERK 1/2 and phospho-AKT.
基金Supported by the Deutsche Forschungsgemeinschaft(DFG research group FOR 2591),No.321137804,No.ZE 712/1-1 and No.VO 450/15-1
文摘AIM To evaluate the influence of hyperglycemia on the progression of autoimmune pancreatitis.METHODS We induced hyperglycemia by repetitive intraperitoneal(ip) injection of 50 mg/kg streptozotocin in MRL/Mp J mice, which develop autoimmune pancreatitis due to a genetic predisposition. We compared the extent of inflammation(histological score, CD3^+ lymphocytes, CD8^+ T-cells, CD4^+ T-cells, Foxp3^+ T-helper cells) in the pancreas of hyperglycemic and normoglycemic mice. We also analyzed the number of leukocytes, lymphocytes, granulocytes and monocytes in the blood. In addition, we determined the percentage of CD3^+ lymphocytes, CD8^+ T-cells, CD4^+ T-cells, Foxp3^+ T-helper cells, Foxp3^+ CD25^+ T-helper and Foxp3^+T-helper cells in the spleen by flow cytometry.RESULTS Treatment with streptozotocin caused a strong induction of hyperglycemia and a reduction in body weight(P < 0.001). Severe hyperglycemia did not, however, lead to an aggravation, but rather to a slight attenuation of autoimmune pancreatitis. In the pancreas, both the histological score of the pancreas as well as the number of CD3+ lymphocytes(P < 0.053) were decreased by hyperglycemia. No major changes in the percentage of CD8^+ T-cells, CD4^+ T-cells, Foxp3^+ T-helper cells were observed between hyperglycemic and normoglycemic mice. Hyperglycemia increased the numbers of leukocytes(P < 0.001), lymphocytes(P = 0.016), granulocytes and monocytes(P = 0.001) in the blood. Hyperglycemia also moderately reduced the percentage of CD3^+ lymphocytes(P = 0.057), significantly increased the percentage of Foxp3^+ T-helper cells(P = 0.018) and Foxp3^+ CD25^+ T-helper cells(P = 0.021) and reduced the percentage of Foxp3^+T-helper cells(P = 0.034) in the spleen. CONCLUSION Hyperglycemia does not aggravate but moderately attenuates autoimmune pancreatitis, possibly by increasing the percentage of regulatory T-cells in the spleen.
基金Supported by Grant from the Deutsche Forschungsgemeinschaft(to RJ)
文摘AIM: To study if three clinically available small molecule kinase inhibitors (SMI), erlotinib, sunitinib and sorafenib, exert antifibrogenic effects on pancreatic stellate cells (PSC) and analyze the basis of their action.
基金Supported by A grant from the Deutsche Forschungsgemeinschaft (Ja 819/3-2)
文摘AIM： To gain molecular insights into the expression and functions of endothelin-1 （ET-1） in pancreatic stellate cells （PSC）.METHODS： PSCs were isolated from rat pancreas tissue, cultured, and stimulated with ET-1 or other extracellular mediators. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2＇-deoxyuridine into DNA and cell migration was studied in a transwell chamber assay. Gene expression at the level of mRNA was quantified by real-time Polymerase chain reaction. Expression and phosphorylation of proteins were monitored by immunoblotting, applying an infrared imaging technology. ET-1 levels in cell culture supernatants were determined by an enzyme immunometric assay. To study DNA binding of individual transcription factors, electrophoretic mobility shift assays were performed.RESULTS： Among several mediators tested, transforming growth factor-β1 and tumour necrosis factor-α displayed the strongest stimulatory effects on ET-1 secretion. The cytokines induced binding of Smad3 and NF-κB, respectively, to oligonucleotides derived from the ET-1 promoter, implicating both transcription factors in the induction of ET-1 gene expression. In accordance with previous studies, ET-1 was found to stimulate migration but not proliferation of PSC. Stimulation of ET-1 receptors led to the activation of two distinct rnitogen-activated protein kinases, p38 and extracellular signal-regulated kinases （ERK）1/2, as well as the transcription factor activator protein-1. At the mRNA level, enhanced expression of the PSC activation marker, α-smooth muscle actin and two proinflammatory cytokines, interleukin （IL）-1β and IL-6, was observed. CONCLUSION： This study provides novel lines of evidence for profibrogenic and proinflammatory actions of ET-1 in the pancreas, encouraging further studies with ET-1 inhibitors in chronic pancreatitis.
