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 the molecular effects of three different D-vitamins, vitamin D2, vitamin D3 and calcipotriol, in pancreatic stellate cells(PSCs).METHODS Quiescent PSCs were isolated from mouse pancreas and activated in v...AIM To study the molecular effects of three different D-vitamins, vitamin D2, vitamin D3 and calcipotriol, in pancreatic stellate cells(PSCs).METHODS Quiescent PSCs were isolated from mouse pancreas and activated in vitro by seeding on plastic surfaces. The cells were exposed to D-vitamins as primary cultures(early-activated PSCs) and upon re-culturing(fullyactivated cells). Exhibition of vitamin A-containing lipid droplets was visualized by oil-red staining. Expression of α-smooth muscle actin(α-SMA), a marker of PSC activation, was monitored by immunofluorescence and immunoblot analysis. The rate of DNA synthesis was quantified by 5-bromo-2'-deoxyuridine(Brd U) incorporation assays. Real-time PCR was employed to monitor gene expression, and protein levels of interleukin-6(IL-6) were measured by ELISA. Uptake of proline was determined using 18 F-proline.RESULTS Sustained culture of originally quiescent PSCs inducedcell proliferation, loss of lipid droplets and exhibition of stress fibers, indicating cell activation. When added to PSCs in primary culture, all three D-vitamins diminished expression of α-SMA(to 32%-39% of the level of control cells; P < 0.05) and increased the storage of lipids(scores from 1.97-2.15 on a scale from 0-3; controls: 1.49; P < 0.05). No such effects were observed when D-vitamins were added to fully-activated cells, while incorporation of Brd U remained unaffected under both experimental conditions. Treatment of re-cultured PSCs with D-vitamins was associated with lower expression of IL-6(-42% to-49%; P < 0.05; also confirmed at the protein level) and increased expression of the vitamin D receptor gene(209%-321% vs controls; P < 0.05). There was no effect of D-vitamins on the expression of transforming growth factor-β1 and collagen type 1(chain α1). The lowest uptake of proline, a main component of collagen, was observed in calcipotriol-treated PSCs.CONCLUSION The three D-vitamins inhibit, with similar efficiencies, activation of PSCs in vitro, but cannot reverse the phenotype once the cells are fully activated.展开更多
Origin and fate of pancreatic stellate cells （PSCs） before, during and after pancreatic injury are a matter of de- bate. The crucial role of PSCs in the pathogenesis of pancreatic fibrosis is generally accepted. How...Origin and fate of pancreatic stellate cells （PSCs） before, during and after pancreatic injury are a matter of de- bate. The crucial role of PSCs in the pathogenesis of pancreatic fibrosis is generally accepted. However, the turnover of the cells remains obscure. The present study addressed the issue of a potential bone marrow （BM） origin of PSCs. We used a model of stable hematopoietic chimerism by grafting enhanced green fluorescence protein （eGFP）-express- ing BM cells after irradiation of acceptor rats. Chimerism was detected by FACS analysis of eGFP-positive cells in the peripheral blood. Dibutyltin dichloride （DBTC） was used to induce acute pancreatic inflammation with subse- quent recovery over 4 weeks. Investigations have been focused on isolated cells to detect the resting PSC population. The incidence of eGFP-positive PSC obtained from the pancreas of chimeric rats was approximately 7% in healthy pancreatic tissue and increased significantly to a mean of 18% in the restored pancreas 4 weeks after DBTC-induced acute inflammation. Our results suggest that BM-derived progenitor cells represent a source of renewable stellate cells in the pancreas. Increased numbers of resting PSCs after regeneration point toward enhanced recruitment of BM-derived cells to the pancreas and/or re-acquisition of a quiescent state after inflammation-induced activation.展开更多
AIM： To investigate the biological effects of cishydroxyproline （CHP） on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms. METHODS： The effect of CHP on DSL6A cell p...AIM： To investigate the biological effects of cishydroxyproline （CHP） on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms. METHODS： The effect of CHP on DSL6A cell proliferation was assessed by using BrdU incorporation. The expression of focal adhesion kinase （FAK） was characterized by Western blotting and immunofluorescence. Induction of endoplasmic reticulum （ER） stress was investigated by using RT-PCR and Western blotting for the glucose-related protein-78 （GRP78） and growth arrest and DNA inducible gene （GADD153）. Cell viability was determined through measuring the metabolic activity based on the reduction potential of DSL6A cells. Apoptosis was analyzed by detection of caspase-3 activation and cleavage of poly（ADP-ribose） polymerase （PARP） as well as DNA laddering. RESULTS： In addition to inhibition of proliferation, incubation with CHP induced proteolytic cleavage of FAK and a delocalisation of the enzyme from focal adhesions, followed by a loss of cell adherence. Simultaneously, we could show an increased expression of GRP78 and GADD153, indicating a CHP-mediated activation of the ER stress cascade in the DSL6A cell line. Prolonged incubation of DSL6A cells with CHP finally resulted in apoptotic cell death. Beside L-proline, the inhibition of intracellular proteolysis by addition of a broad spectrum protease inhibitor could abolish the effects of CHP on cellular functions and the molecular processes. In contrast, impeding the activity of apoptosis-executing caspases had no influence on CHP-mediated cell damage.CONCLUSION： Our data suggest that the initiation of ER stress machinery by CliP leads to an activation of intracellular proteolytic processes, including caspaseindependent FAK degradation, resulting in damaging pancreatic carcinoma cells.展开更多
Background:Pancreatic stellate cells(PSCs)foster the progression of pancreatic adenocarcinoma and chronic pancreatitis(CP)by producing a dense fibrotic stroma.However,the incomplete knowledge of PSCs biology hampers t...Background:Pancreatic stellate cells(PSCs)foster the progression of pancreatic adenocarcinoma and chronic pancreatitis(CP)by producing a dense fibrotic stroma.However,the incomplete knowledge of PSCs biology hampers the exploration of antifibrotic therapies.Here,we explored the role of the Hippo pathway in the context of PSCs activation and experimental CP.Methods:CP model was created in rats with the tail vein injection of dibutyltin dichloride(DBTC).The expression of Yes-associated protein(YAP)in CP tissue was assessed.Primary and immortalized rats PSCs were treated with the YAP-inhibitor verteporfin.Furthermore,YAP siRNA was employed.Subsequently,DNA synthesis,cell survival,levels ofα-smooth muscle actin(α-SMA)protein,presence of lipid droplets and PSCs gene expression were evaluated.Upstream regulators of YAP signaling were studied by reporter gene assays.Results:In DBTC-induced CP,pronounced expression of YAP in areas of tubular structures and periduc-tal fibrosis was observed.Verteporfin diminished DNA replication in PSCs in a dose-dependent fash-ion.Knockdown of YAP reduced cell proliferation.Primary cultures of PSCs were characterized by a de-crease of lipid droplets and increased synthesis ofα-SMA protein.Both processes were not affected by verteporfin.At the non-cytotoxic concentration of 100 nmol/L,verteporfin significantly reduced mRNA levels of transforming growth factor-β1(Tgf-β1)and Ccn family member 1(Ccn1).YAP signaling was acti-vated by TGF-β1,but repressed by interferon-γ.Conclusions:Activated YAP enhanced PSCs proliferation.The antifibrotic potential of Hippo pathway in-hibitors warrants further investigation.展开更多
BACKGROUND： Modulation of the stroma response is considered a promising approach for the treatment of chronic pancreatitis and pancreatic cancer. The aim of this study was to evaluate the effects of three clinically ...BACKGROUND： Modulation of the stroma response is considered a promising approach for the treatment of chronic pancreatitis and pancreatic cancer. The aim of this study was to evaluate the effects of three clinically available small molecule kinase inhibitors, regorafenib, trametinib and dactolisib, on effector functions of activated pancreatic stellate cells （PSCs）, which play a key role in pancreatic fibrosis.展开更多
Background:Current animal models of chronic pancreatitis(CP)often provide only limited pathophysio-logical insights since they incompletely reflect the human disease.CP induced by injection of dibutyltin dichloride(DB...Background:Current animal models of chronic pancreatitis(CP)often provide only limited pathophysio-logical insights since they incompletely reflect the human disease.CP induced by injection of dibutyltin dichloride(DBTC-pancreatitis)shares with human CP the important feature of extended fibrosis and would be an even more attractive model if it could be transferred from rats to mice,as recently sug-gested in the context of combined ethanol and DBTC application.This study aimed to evaluate the effects of DBTC in pancreas and liver of C57BL/6 mice,a strain commonly used to engineer genetic mouse mod-els.Methods:C57BL/6 mice and Lewis rats were exposed to variable doses of DBTC.After an investigation period of up to 4 weeks,laboratory findings and histopathological changes of pancreas and liver were evaluated.Results:Chronic DBTC-pancreatitis in rats was characterized by acinar cell damage,ductal changes,fi-brosis,and inflammatory cell infiltrates.Mice treated with DBTC at 6–8 mg/kg body weight,the standard doses in rats,showed transient increases of lipase activities but no morphological signs of chronic DBTC-pancreatitis 4 weeks after injection of the drug.Increased doses of 10–12 mg/kg DBTC were intolerable due to their high toxicity.In contrast,mice and rats presented with a similar histopathology of the liver that can be characterized as a chronic-proliferative DBTC-cholangitis with predominating damage and proliferation of the small bile ducts as well as secondary portal inflammatory cell infiltrates and a begin-ning portal fibrosis.Conclusions:The DBTC-model cannot be transferred from rats to C57BL/6 mice with respect to chronic DBTC-pancreatitis,but might be of interest to study DBTC-cholangitis in both species.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
BACKGROUND Crohn’s disease(CD)is characterized by a multifactorial etiology and a significant impact of genetic traits.While NOD2 mutations represent well established risk factors of CD,the role of other genes is inc...BACKGROUND Crohn’s disease(CD)is characterized by a multifactorial etiology and a significant impact of genetic traits.While NOD2 mutations represent well established risk factors of CD,the role of other genes is incompletely understood.AIM To challenge the hypothesis that single nucleotide polymorphisms(SNPs)in the genes CLEC5 A and CLEC7 A,two members of the C-type lectin domain family of pattern recognition receptors,may be associated with CD.METHODS SNPs in CLEC5 A,CLEC7 A and the known CD risk gene NOD2 were studied using real time PCR-based SNP assays.Therefore,DNA samples from 175 patients and 157 healthy donors were employed.Genotyping data were correlated with clinical characteristics of the patients and the results of gene expression data analyses.RESULTS In accordance with previous studies,rs2066844 and rs2066847 in NOD2 were found to be significantly associated with CD(allelic P values=0.0368 and 0.0474,respectively).Intriguingly,for genotype AA of rs1285933 in CLEC5 A,a potential association with CD(recessive P=0.0523;odds ratio=1.90)was observed.There were no associations between CD and SNPs rs2078178 and rs16910631 in CLEC7 A.Variants of rs1285933 had no impact on CLEC5 A gene expression.In contrast,genotype-dependent differences of CXCL5 expression in peripheral blood mononuclear cells were observed.There is no statistical interactionbetween the tested SNPs of NOD2 and CLEC5 A,suggesting of a novel pathway contributing to the disease.CONCLUSION Our data encourage enlarged follow-up studies to further address an association of SNP rs1285933 in CLEC5 A with CD.The C-type lectin domain family member also deserves attention regarding a potential role in the pathophysiology of CD.展开更多
AIM:To gain insights into the molecular action of erlotinib in pancreatic cancer (PC) cells. METHODS:Two PC cell lines, BxPC-3 and Capan-1, were treated with various concentrations of erlotinib, the specific mitogen-a...AIM:To gain insights into the molecular action of erlotinib in pancreatic cancer (PC) cells. METHODS:Two PC cell lines, BxPC-3 and Capan-1, were treated with various concentrations of erlotinib, the specific mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and protein kinase B (AKT) inhibitor XIV. DNA synthesis was measured by 5-bromo-2'-deoxyuridine (BrdU) assays. Expression and phosphorylation of the epidermal growth factor receptor (EGFR) and downstream signaling molecules were quantified by Western blot analysis. The data were processed to calibrate a mathematical model, based on ordinary differential equations, describing the EGFRmediated signal transduction. RESULTS:Erlotinib significantly inhibited BrdU incorporation in BxPC-3 cells at a concentration of 1 mol/L, whereas Capan-1 cells were much more resistant. In both cell lines, MEK inhibitor U0126 and erlotinib attenuated DNA synthesis in a cumulative manner, whereas the AKT pathway-specific inhibitor did not enhance the effects of erlotinib. While basal phosphorylation of EGFR and extracellular signal-regulated kinase (ERK) did not differ much between the two cell lines, BxPC-3 cells displayed a more than five-times higher basal phospho-AKT level than Capan-1 cells. Epidermal growth factor (EGF) at 10 ng/mL induced the phosphorylation of EGFR, AKT and ERK in both cell lines with similar kinetics. In BxPC-3 cells, higher levels of phospho-AKT and phospho-ERK (normalized to the total protein levels) were observed. Independent of the cell line, erlotinib efficiently inhibited phosphorylation of EGFR, AKT and ERK. The mathematical model successfully simulated the experimental findings and provided predictions regarding phosphoprotein levels that could be verified experimentally. CONCLUSION:Our data suggest basal AKT phosphorylation and the degree of EGF-induced activation of AKT and ERK as molecular determinants of erlotinib efficiency in PC cells.展开更多
AIM To investigate disease-specific gene expression profiles of peripheral blood mononuclear cells(PBMCs) from Crohn's disease(CD) patients in clinical remission.METHODS Patients with CD in clinical remission or w...AIM To investigate disease-specific gene expression profiles of peripheral blood mononuclear cells(PBMCs) from Crohn's disease(CD) patients in clinical remission.METHODS Patients with CD in clinical remission or with very low disease activity according to the Crohn's disease activity index were genotyped regarding nucleotidebinding oligomerization domain 2(NOD2),and PBMCs from wild-type(WT)-NOD2 patients,patients with homozygous or heterozygous NOD2 mutations and healthy donors were isolated for further analysis.The cells were cultured with vitamin D,peptidoglycan(PGN) and lipopolysaccharide(LPS) for defined periods of time before RNA was isolated and subjected to microarray analysis using Clariom S assays and quantitative realtime PCR.NOD2-and disease-specific gene expression profiles were evaluated with repeated measure ANOVA by a general linear model.RESULTS Employing microarray assays,a total of 267 genes were identified that were significantly up-or downregulated in PBMCs of WT-NOD2 patients,compared to healthy donors after challenge with vitamin D and/or a combination of LPS and PGN(P < 0.05;threshold:≥ 2-fold change).For further analysis by real-time PCR,genes with known impact on inflammation and immunity were selected that fulfilled predefined expression criteria.In a larger cohort of patients and controls,a disease-associated expression pattern,with higher transcript levels in vitamin D-treated PBMCs from patients,was observed for three of these genes,CLEC5 A(P < 0.030),lysozyme(LYZ;P < 0.047) and TREM1(P < 0.023).Six genes were found to be expressed in a NOD2-dependent manner(CD101,P < 0.002;CLEC5 A,P < 0.020;CXCL5,P < 0.009;IL-24,P < 0.044;ITGB2,P < 0.041;LYZ,P < 0.042).Interestingly,the highest transcript levels were observed in patients with heterozygous NOD2 mutations.CONCLUSION Our data identify CLEC5 A and LYZ as CD-and NOD2-associated genes of PBMCs and encourage further studies on their pathomechanistic roles.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
Background: Pancreatic ductal adenocarcinoma(PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over ti...Background: Pancreatic ductal adenocarcinoma(PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. Methods: We took advantage of a model of accelerated aging, uncoupling protein 2-deficient( Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically transplanted Ucp2 wild-type(WT) PDAC cells(cell lines Panc02 and 6606PDA) in vivo and to study strain-dependent differences of the PDAC microenvironment. Results: Measurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to Ucp2 KO mice. In tumors in the knockout strain, higher levels of interferon-γ m RNA despite similar numbers of tumor-infiltrating T cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 KO mice than in WT animals. Accordingly, pancreatic stellate cells from Ucp2 KO mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. Conclusions: Ucp2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms.展开更多
Background:The combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy.Here,we studied the efficacy of the genotoxic agent etoposide(Eto)and irradiation in indu...Background:The combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy.Here,we studied the efficacy of the genotoxic agent etoposide(Eto)and irradiation in inducing senescence of Panc02 pancreatic cancer cells,and the capability of the Bcl-2 inhibitor navitoclax(ABT-263;Nav)to trigger senolysis.Methods:Panc02 cells were treated with Eto or irradiated with 5–20 Gy before exposure to Nav.Cell survival,proliferation,and senescence were assessed by trypan blue staining,quantification of DNA synthesis,and staining of senescence-associatedβ-galactosidase(SA-β-Gal)-positive cells,respectively.Levels of mRNA were determined by real-time polymerase chain reaction,and protein expression was analyzed by immunoblotting.Panc02 cells were also grown as pancreatic tumors in mice,which were subsequently treated with Eto and Nav.Results:Eto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav.In vivo,Eto and Nav together,but not Eto alone,significantly reduced the proportion of proliferating cells.The expression of the senescence markerγH2AX and tumor infiltration with T-cells were not affected by the treatment.In vitro,almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-β-Gal-positive.Application of Nav reduced the percentage of SA-β-Gal-positive cells after irradiation but not after pretreatment with Eto.In response to triggers of senescence,cultured Panc02 cells showed increased protein levels ofγH2AX and the autophagy marker LC3B-II,and higher mRNA levels of Cdkn1a,Mdm2,and PAI-1,while the effects of Nav were variable.Conclusions:In vitro and in vivo,the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone.Our data also provide some evidence for senolytic effects of Nav in vitro.展开更多
BACKGROUND： Uncoupling protein 2 （UCP2） has been suggested to inhibit mitochondrial production of reactive oxygen species （ROS） by decreasing the mitochondrial membrane potential. Experimental acute pancreatitis ...BACKGROUND： Uncoupling protein 2 （UCP2） has been suggested to inhibit mitochondrial production of reactive oxygen species （ROS） by decreasing the mitochondrial membrane potential. Experimental acute pancreatitis is associated with increased UCP2 expression, whereas UCP2 deficiency retards regeneration of aged mice from acute pancreatitis. Here, we have addressed biological and molecular functions of UCP2 in pancreatic stellate cells （PSCs）, which are involved in pancreatic wound repair and fibrogenesis. METHODS： PSCs were isolated from 12 months old （aged） UCP2^-/- mice and animals of the wild-type （WT） strain C57BL/6. Proliferation and cell death were assessed by em- ploying trypan blue staining and a 5-bromo-2＇-deoxyuridine incorporation assay. Intracellular fat droplets were visualized by oil red O staining. Levels of mRNA were determined by RT-PCR, while protein expression was analyzed by immunoblotting and immunofluorescence analysis. Intracellular ROS levels were measured with 2＇,7＇-dichlorofluorescin diacetate. Expression of senescence-associated β-galactosidase （SA β-Gal） was used as a surrogate marker of cellular senescence. RESULTS： PSCs derived from UCP2^-/- mice proliferated at a lower rate than cells from WT mice. In agreement with this observation, the UCP2 inhibitor genipin displayed dose- dependent inhibitory effects on WT PSC growth. Interestingly, ROS levels in PSCs did not differ between the two strains, and PSCs derived from UCP2^-/- mice did not senesce faster than those from corresponding WT cells. PSCs from UCP2^-/- mice and WT animals were also indistinguishable with respect to the activation-dependent loss of intracellular fat droplets, expression of the activation marker α-smooth muscle actin, type I collagen and the autocrine/paracrine mediators interleukin-6 and transforming growth factor-I~ 1. CONCLUSIONS： A reduced proliferative capacity of PSC from aged UCP2^-/- mice may contribute to the retarded regeneration after acute pancreatitis. Apart from their slower growth, PSC of UCP2^-/- mice displayed no functional abnormalities. The antifibrotic potential of UCP2 inhibitors deserves further attention.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 FORUN program of the Rostock University Medical Center
文摘AIM To study the molecular effects of three different D-vitamins, vitamin D2, vitamin D3 and calcipotriol, in pancreatic stellate cells(PSCs).METHODS Quiescent PSCs were isolated from mouse pancreas and activated in vitro by seeding on plastic surfaces. The cells were exposed to D-vitamins as primary cultures(early-activated PSCs) and upon re-culturing(fullyactivated cells). Exhibition of vitamin A-containing lipid droplets was visualized by oil-red staining. Expression of α-smooth muscle actin(α-SMA), a marker of PSC activation, was monitored by immunofluorescence and immunoblot analysis. The rate of DNA synthesis was quantified by 5-bromo-2'-deoxyuridine(Brd U) incorporation assays. Real-time PCR was employed to monitor gene expression, and protein levels of interleukin-6(IL-6) were measured by ELISA. Uptake of proline was determined using 18 F-proline.RESULTS Sustained culture of originally quiescent PSCs inducedcell proliferation, loss of lipid droplets and exhibition of stress fibers, indicating cell activation. When added to PSCs in primary culture, all three D-vitamins diminished expression of α-SMA(to 32%-39% of the level of control cells; P < 0.05) and increased the storage of lipids(scores from 1.97-2.15 on a scale from 0-3; controls: 1.49; P < 0.05). No such effects were observed when D-vitamins were added to fully-activated cells, while incorporation of Brd U remained unaffected under both experimental conditions. Treatment of re-cultured PSCs with D-vitamins was associated with lower expression of IL-6(-42% to-49%; P < 0.05; also confirmed at the protein level) and increased expression of the vitamin D receptor gene(209%-321% vs controls; P < 0.05). There was no effect of D-vitamins on the expression of transforming growth factor-β1 and collagen type 1(chain α1). The lowest uptake of proline, a main component of collagen, was observed in calcipotriol-treated PSCs.CONCLUSION The three D-vitamins inhibit, with similar efficiencies, activation of PSCs in vitro, but cannot reverse the phenotype once the cells are fully activated.
文摘Origin and fate of pancreatic stellate cells （PSCs） before, during and after pancreatic injury are a matter of de- bate. The crucial role of PSCs in the pathogenesis of pancreatic fibrosis is generally accepted. However, the turnover of the cells remains obscure. The present study addressed the issue of a potential bone marrow （BM） origin of PSCs. We used a model of stable hematopoietic chimerism by grafting enhanced green fluorescence protein （eGFP）-express- ing BM cells after irradiation of acceptor rats. Chimerism was detected by FACS analysis of eGFP-positive cells in the peripheral blood. Dibutyltin dichloride （DBTC） was used to induce acute pancreatic inflammation with subse- quent recovery over 4 weeks. Investigations have been focused on isolated cells to detect the resting PSC population. The incidence of eGFP-positive PSC obtained from the pancreas of chimeric rats was approximately 7% in healthy pancreatic tissue and increased significantly to a mean of 18% in the restored pancreas 4 weeks after DBTC-induced acute inflammation. Our results suggest that BM-derived progenitor cells represent a source of renewable stellate cells in the pancreas. Increased numbers of resting PSCs after regeneration point toward enhanced recruitment of BM-derived cells to the pancreas and/or re-acquisition of a quiescent state after inflammation-induced activation.
文摘AIM： To investigate the biological effects of cishydroxyproline （CHP） on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms. METHODS： The effect of CHP on DSL6A cell proliferation was assessed by using BrdU incorporation. The expression of focal adhesion kinase （FAK） was characterized by Western blotting and immunofluorescence. Induction of endoplasmic reticulum （ER） stress was investigated by using RT-PCR and Western blotting for the glucose-related protein-78 （GRP78） and growth arrest and DNA inducible gene （GADD153）. Cell viability was determined through measuring the metabolic activity based on the reduction potential of DSL6A cells. Apoptosis was analyzed by detection of caspase-3 activation and cleavage of poly（ADP-ribose） polymerase （PARP） as well as DNA laddering. RESULTS： In addition to inhibition of proliferation, incubation with CHP induced proteolytic cleavage of FAK and a delocalisation of the enzyme from focal adhesions, followed by a loss of cell adherence. Simultaneously, we could show an increased expression of GRP78 and GADD153, indicating a CHP-mediated activation of the ER stress cascade in the DSL6A cell line. Prolonged incubation of DSL6A cells with CHP finally resulted in apoptotic cell death. Beside L-proline, the inhibition of intracellular proteolysis by addition of a broad spectrum protease inhibitor could abolish the effects of CHP on cellular functions and the molecular processes. In contrast, impeding the activity of apoptosis-executing caspases had no influence on CHP-mediated cell damage.CONCLUSION： Our data suggest that the initiation of ER stress machinery by CliP leads to an activation of intracellular proteolytic processes, including caspaseindependent FAK degradation, resulting in damaging pancreatic carcinoma cells.
文摘Background:Pancreatic stellate cells(PSCs)foster the progression of pancreatic adenocarcinoma and chronic pancreatitis(CP)by producing a dense fibrotic stroma.However,the incomplete knowledge of PSCs biology hampers the exploration of antifibrotic therapies.Here,we explored the role of the Hippo pathway in the context of PSCs activation and experimental CP.Methods:CP model was created in rats with the tail vein injection of dibutyltin dichloride(DBTC).The expression of Yes-associated protein(YAP)in CP tissue was assessed.Primary and immortalized rats PSCs were treated with the YAP-inhibitor verteporfin.Furthermore,YAP siRNA was employed.Subsequently,DNA synthesis,cell survival,levels ofα-smooth muscle actin(α-SMA)protein,presence of lipid droplets and PSCs gene expression were evaluated.Upstream regulators of YAP signaling were studied by reporter gene assays.Results:In DBTC-induced CP,pronounced expression of YAP in areas of tubular structures and periduc-tal fibrosis was observed.Verteporfin diminished DNA replication in PSCs in a dose-dependent fash-ion.Knockdown of YAP reduced cell proliferation.Primary cultures of PSCs were characterized by a de-crease of lipid droplets and increased synthesis ofα-SMA protein.Both processes were not affected by verteporfin.At the non-cytotoxic concentration of 100 nmol/L,verteporfin significantly reduced mRNA levels of transforming growth factor-β1(Tgf-β1)and Ccn family member 1(Ccn1).YAP signaling was acti-vated by TGF-β1,but repressed by interferon-γ.Conclusions:Activated YAP enhanced PSCs proliferation.The antifibrotic potential of Hippo pathway in-hibitors warrants further investigation.
文摘BACKGROUND： Modulation of the stroma response is considered a promising approach for the treatment of chronic pancreatitis and pancreatic cancer. The aim of this study was to evaluate the effects of three clinically available small molecule kinase inhibitors, regorafenib, trametinib and dactolisib, on effector functions of activated pancreatic stellate cells （PSCs）, which play a key role in pancreatic fibrosis.
文摘Background:Current animal models of chronic pancreatitis(CP)often provide only limited pathophysio-logical insights since they incompletely reflect the human disease.CP induced by injection of dibutyltin dichloride(DBTC-pancreatitis)shares with human CP the important feature of extended fibrosis and would be an even more attractive model if it could be transferred from rats to mice,as recently sug-gested in the context of combined ethanol and DBTC application.This study aimed to evaluate the effects of DBTC in pancreas and liver of C57BL/6 mice,a strain commonly used to engineer genetic mouse mod-els.Methods:C57BL/6 mice and Lewis rats were exposed to variable doses of DBTC.After an investigation period of up to 4 weeks,laboratory findings and histopathological changes of pancreas and liver were evaluated.Results:Chronic DBTC-pancreatitis in rats was characterized by acinar cell damage,ductal changes,fi-brosis,and inflammatory cell infiltrates.Mice treated with DBTC at 6–8 mg/kg body weight,the standard doses in rats,showed transient increases of lipase activities but no morphological signs of chronic DBTC-pancreatitis 4 weeks after injection of the drug.Increased doses of 10–12 mg/kg DBTC were intolerable due to their high toxicity.In contrast,mice and rats presented with a similar histopathology of the liver that can be characterized as a chronic-proliferative DBTC-cholangitis with predominating damage and proliferation of the small bile ducts as well as secondary portal inflammatory cell infiltrates and a begin-ning portal fibrosis.Conclusions:The DBTC-model cannot be transferred from rats to C57BL/6 mice with respect to chronic DBTC-pancreatitis,but might be of interest to study DBTC-cholangitis in both species.
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.
文摘BACKGROUND Crohn’s disease(CD)is characterized by a multifactorial etiology and a significant impact of genetic traits.While NOD2 mutations represent well established risk factors of CD,the role of other genes is incompletely understood.AIM To challenge the hypothesis that single nucleotide polymorphisms(SNPs)in the genes CLEC5 A and CLEC7 A,two members of the C-type lectin domain family of pattern recognition receptors,may be associated with CD.METHODS SNPs in CLEC5 A,CLEC7 A and the known CD risk gene NOD2 were studied using real time PCR-based SNP assays.Therefore,DNA samples from 175 patients and 157 healthy donors were employed.Genotyping data were correlated with clinical characteristics of the patients and the results of gene expression data analyses.RESULTS In accordance with previous studies,rs2066844 and rs2066847 in NOD2 were found to be significantly associated with CD(allelic P values=0.0368 and 0.0474,respectively).Intriguingly,for genotype AA of rs1285933 in CLEC5 A,a potential association with CD(recessive P=0.0523;odds ratio=1.90)was observed.There were no associations between CD and SNPs rs2078178 and rs16910631 in CLEC7 A.Variants of rs1285933 had no impact on CLEC5 A gene expression.In contrast,genotype-dependent differences of CXCL5 expression in peripheral blood mononuclear cells were observed.There is no statistical interactionbetween the tested SNPs of NOD2 and CLEC5 A,suggesting of a novel pathway contributing to the disease.CONCLUSION Our data encourage enlarged follow-up studies to further address an association of SNP rs1285933 in CLEC5 A with CD.The C-type lectin domain family member also deserves attention regarding a potential role in the pathophysiology of CD.
基金Supported by A grant of the Bundesministerium für Bildung und Forschung through the FORSYS partner program, No.0315255the Helmholtz Society as part of the Systems Biology Network
文摘AIM:To gain insights into the molecular action of erlotinib in pancreatic cancer (PC) cells. METHODS:Two PC cell lines, BxPC-3 and Capan-1, were treated with various concentrations of erlotinib, the specific mitogen-activated protein kinase kinase (MEK) inhibitor U0126, and protein kinase B (AKT) inhibitor XIV. DNA synthesis was measured by 5-bromo-2'-deoxyuridine (BrdU) assays. Expression and phosphorylation of the epidermal growth factor receptor (EGFR) and downstream signaling molecules were quantified by Western blot analysis. The data were processed to calibrate a mathematical model, based on ordinary differential equations, describing the EGFRmediated signal transduction. RESULTS:Erlotinib significantly inhibited BrdU incorporation in BxPC-3 cells at a concentration of 1 mol/L, whereas Capan-1 cells were much more resistant. In both cell lines, MEK inhibitor U0126 and erlotinib attenuated DNA synthesis in a cumulative manner, whereas the AKT pathway-specific inhibitor did not enhance the effects of erlotinib. While basal phosphorylation of EGFR and extracellular signal-regulated kinase (ERK) did not differ much between the two cell lines, BxPC-3 cells displayed a more than five-times higher basal phospho-AKT level than Capan-1 cells. Epidermal growth factor (EGF) at 10 ng/mL induced the phosphorylation of EGFR, AKT and ERK in both cell lines with similar kinetics. In BxPC-3 cells, higher levels of phospho-AKT and phospho-ERK (normalized to the total protein levels) were observed. Independent of the cell line, erlotinib efficiently inhibited phosphorylation of EGFR, AKT and ERK. The mathematical model successfully simulated the experimental findings and provided predictions regarding phosphoprotein levels that could be verified experimentally. CONCLUSION:Our data suggest basal AKT phosphorylation and the degree of EGF-induced activation of AKT and ERK as molecular determinants of erlotinib efficiency in PC cells.
基金Supported by a grant from the Damp-Foundation(2016-04) to Schaffler H and Rohde S
文摘AIM To investigate disease-specific gene expression profiles of peripheral blood mononuclear cells(PBMCs) from Crohn's disease(CD) patients in clinical remission.METHODS Patients with CD in clinical remission or with very low disease activity according to the Crohn's disease activity index were genotyped regarding nucleotidebinding oligomerization domain 2(NOD2),and PBMCs from wild-type(WT)-NOD2 patients,patients with homozygous or heterozygous NOD2 mutations and healthy donors were isolated for further analysis.The cells were cultured with vitamin D,peptidoglycan(PGN) and lipopolysaccharide(LPS) for defined periods of time before RNA was isolated and subjected to microarray analysis using Clariom S assays and quantitative realtime PCR.NOD2-and disease-specific gene expression profiles were evaluated with repeated measure ANOVA by a general linear model.RESULTS Employing microarray assays,a total of 267 genes were identified that were significantly up-or downregulated in PBMCs of WT-NOD2 patients,compared to healthy donors after challenge with vitamin D and/or a combination of LPS and PGN(P < 0.05;threshold:≥ 2-fold change).For further analysis by real-time PCR,genes with known impact on inflammation and immunity were selected that fulfilled predefined expression criteria.In a larger cohort of patients and controls,a disease-associated expression pattern,with higher transcript levels in vitamin D-treated PBMCs from patients,was observed for three of these genes,CLEC5 A(P < 0.030),lysozyme(LYZ;P < 0.047) and TREM1(P < 0.023).Six genes were found to be expressed in a NOD2-dependent manner(CD101,P < 0.002;CLEC5 A,P < 0.020;CXCL5,P < 0.009;IL-24,P < 0.044;ITGB2,P < 0.041;LYZ,P < 0.042).Interestingly,the highest transcript levels were observed in patients with heterozygous NOD2 mutations.CONCLUSION Our data identify CLEC5 A and LYZ as CD-and NOD2-associated genes of PBMCs and encourage further studies on their pathomechanistic roles.
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.
基金supported by a grant from the Bundesminis-terium für Bildung und Forschung (01ZX1903A)。
文摘Background: Pancreatic ductal adenocarcinoma(PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. Methods: We took advantage of a model of accelerated aging, uncoupling protein 2-deficient( Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically transplanted Ucp2 wild-type(WT) PDAC cells(cell lines Panc02 and 6606PDA) in vivo and to study strain-dependent differences of the PDAC microenvironment. Results: Measurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to Ucp2 KO mice. In tumors in the knockout strain, higher levels of interferon-γ m RNA despite similar numbers of tumor-infiltrating T cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 KO mice than in WT animals. Accordingly, pancreatic stellate cells from Ucp2 KO mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. Conclusions: Ucp2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms.
基金supported by a grant from the Bundesministerium fur Bildung und Forschung(01ZX1903A).
文摘Background:The combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy.Here,we studied the efficacy of the genotoxic agent etoposide(Eto)and irradiation in inducing senescence of Panc02 pancreatic cancer cells,and the capability of the Bcl-2 inhibitor navitoclax(ABT-263;Nav)to trigger senolysis.Methods:Panc02 cells were treated with Eto or irradiated with 5–20 Gy before exposure to Nav.Cell survival,proliferation,and senescence were assessed by trypan blue staining,quantification of DNA synthesis,and staining of senescence-associatedβ-galactosidase(SA-β-Gal)-positive cells,respectively.Levels of mRNA were determined by real-time polymerase chain reaction,and protein expression was analyzed by immunoblotting.Panc02 cells were also grown as pancreatic tumors in mice,which were subsequently treated with Eto and Nav.Results:Eto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav.In vivo,Eto and Nav together,but not Eto alone,significantly reduced the proportion of proliferating cells.The expression of the senescence markerγH2AX and tumor infiltration with T-cells were not affected by the treatment.In vitro,almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-β-Gal-positive.Application of Nav reduced the percentage of SA-β-Gal-positive cells after irradiation but not after pretreatment with Eto.In response to triggers of senescence,cultured Panc02 cells showed increased protein levels ofγH2AX and the autophagy marker LC3B-II,and higher mRNA levels of Cdkn1a,Mdm2,and PAI-1,while the effects of Nav were variable.Conclusions:In vitro and in vivo,the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone.Our data also provide some evidence for senolytic effects of Nav in vitro.
基金supported by a grant from the Bundesministerium für Bildung und Forschung(0315892A,GERONTOSYS program)
文摘BACKGROUND： Uncoupling protein 2 （UCP2） has been suggested to inhibit mitochondrial production of reactive oxygen species （ROS） by decreasing the mitochondrial membrane potential. Experimental acute pancreatitis is associated with increased UCP2 expression, whereas UCP2 deficiency retards regeneration of aged mice from acute pancreatitis. Here, we have addressed biological and molecular functions of UCP2 in pancreatic stellate cells （PSCs）, which are involved in pancreatic wound repair and fibrogenesis. METHODS： PSCs were isolated from 12 months old （aged） UCP2^-/- mice and animals of the wild-type （WT） strain C57BL/6. Proliferation and cell death were assessed by em- ploying trypan blue staining and a 5-bromo-2＇-deoxyuridine incorporation assay. Intracellular fat droplets were visualized by oil red O staining. Levels of mRNA were determined by RT-PCR, while protein expression was analyzed by immunoblotting and immunofluorescence analysis. Intracellular ROS levels were measured with 2＇,7＇-dichlorofluorescin diacetate. Expression of senescence-associated β-galactosidase （SA β-Gal） was used as a surrogate marker of cellular senescence. RESULTS： PSCs derived from UCP2^-/- mice proliferated at a lower rate than cells from WT mice. In agreement with this observation, the UCP2 inhibitor genipin displayed dose- dependent inhibitory effects on WT PSC growth. Interestingly, ROS levels in PSCs did not differ between the two strains, and PSCs derived from UCP2^-/- mice did not senesce faster than those from corresponding WT cells. PSCs from UCP2^-/- mice and WT animals were also indistinguishable with respect to the activation-dependent loss of intracellular fat droplets, expression of the activation marker α-smooth muscle actin, type I collagen and the autocrine/paracrine mediators interleukin-6 and transforming growth factor-I~ 1. CONCLUSIONS： A reduced proliferative capacity of PSC from aged UCP2^-/- mice may contribute to the retarded regeneration after acute pancreatitis. Apart from their slower growth, PSC of UCP2^-/- mice displayed no functional abnormalities. The antifibrotic potential of UCP2 inhibitors deserves further attention.
