Upregulation of α-ENaC induces pancreatic β-cell dysfunction,ER stress,and SIRT2 degradation

Islet beta cells(β-cells)produce insulin in response to high blood glucose levels,which is essential for preserving glucose homeostasis.Voltage-gated ion channels inβ-cells,including Na+,K+,and Ca2+channels,aid in the release of insulin.The epithelial sodium channel alpha subunit(α-ENaC),a voltage-independent sodium ion channel,is also expressed in human pancreatic endocrine cells.However,there is no reported study on the function of ENaC in theβ-cells.In the current study,we found thatα-ENaC was expressed in human pancreatic glandule and pancreatic isletβ-cells.In the pancreas of db/db mice and high-fat diet-induced mice,and in mouse isletβ-cells(MIN6 cells)treated with palmitate,α-ENaC expression was increased.Whenα-ENaC was overexpressed in MIN6 cells,insulin content and glucose-induced insulin secretion were significantly reduced.On the other hand,palmitate injured isletβ-cells and suppressed insulin synthesis and secretion,but increasedα-ENaC expression in MIN6 cells.However,α-ENaC knockout(Scnn1a−/−)in MIN6 cells attenuatedβ-cell disorder induced by palmitate.Furthermore,α-ENaC regulated the ubiquitylation and degradation of sirtuin 2 inβ-cells.α-ENaC also modulatedβ-cell function in correlation with the inositol-requiring enzyme 1 alpha/X-box binding protein 1(IRE1α/XBP1)and protein kinase RNA-like endoplasmic reticulum kinase/C/EBP homologous protein(PERK/CHOP)endoplasmic reticulum stress pathways.These results suggest thatα-ENaC may play a novel role in insulin synthesis and secretion in theβ-cells,and the upregulation ofα-ENaC promotes isletβ-cell dysfunction.In conclusion,α-ENaC may be a key regulator involved in isletβ-cell damage and a potential therapeutic target for type 2 diabetes mellitus.

The Effect of Tuberculosis Infection on Pancreatic Beta-Cell Function in Patients with Type 2 Diabetes Mellitus

Objective: The aim of this study is to investigate how individuals with type 2 diabetes mellitus’ pancreatic β-cell function index and insulin resistance index are affected by tuberculosis infection. Methods: The study group consisted of 89 patients with type 2 diabetes mellitus and tuberculosis infection who were admitted to Jingzhou Chest Hospital between March 2019 and March 2021. Gender and duration of diabetes were matching conditions. The control group was made up of 89 patients with type 2 diabetes who were admitted to Jingzhou Central Hospital’s endocrinology department during the same period. The two patient groups provided general information such as gender, age, length of diabetes, and blood biochemical indexes such as glycosylated hemoglobin (HbA1c), fasting glucose (FPG), and fasting C-peptide (FC-P). The HOMA calculator was used to calculate the HOMA-β and the HOMA-IR, and intergroup comparisons and correlation analyses were carried out. Results: Regarding gender, age, disease duration, FC-P, and HbA1c, the differences between the two groups were not statistically significant (P > 0.05). However, BMI, FPG, HOMA-β, and HOMA-IR showed statistically significant differences (P < 0.05). In comparison to the control group, the study group’s HOMA-β was lower and its HOMA-IR was greater. According to Spearman’s correlation analysis, HOMA-β had a negative association (P th FPG, HbA1c, and the length of the disease, and a positive correlation with BMI and FC-P. A positive correlation was found between HOMA-IR and BMI, FPG, and FC-P (P < 0.01), as well as a correlation with the length of the disease (P > 0.05) and HbA1c. Conclusions: In type 2 diabetes mellitus combined with tuberculosis infection, the patients had higher FPG levels and lower FC-P levels, the secretory function of pancreatic β-cells was more severely impaired, and insulin resistance was more obvious.

Apolipoprotein E2 inhibits mitochondrial apoptosis in pancreatic cancer cells through ERK1/2/CREB/BCL-2 signaling

Background: Apolipoprotein E2(ApoE2) is a pleiotropic protein that influences several aspects of cancer metabolism and development. Evading apoptosis is a vital factor for facilitating cancer cell growth. However, the role and mechanism of ApoE2 in regulating cell apoptosis of pancreatic cancer remain unclear. Methods: In this study, we firstly detected the m RNA and protein expressions of ApoE2 in PANC-1 and Capan-2 cells by real-time polymerase chain reaction and Western blotting. We then performed TUNEL and flow cytometric analyses to explore the role of recombinant human ApoE2, p CMV6-ApoE2 and si ApoE2 in the apoptosis of PANC-1 and Capan-2 cells. Furthermore, we investigated the molecular mechanism through which ApoE2 affected apoptosis in PANC-1 cells using immunofluorescence, immunoprecipitation, Western blotting and co-immunoprecipitation analysis. Results: ApoE2 phosphorylated ERK1/2 and inhibited pancreatic cancer cell apoptosis. In addition, our data showed that ApoE2/ERK1/2 altered the expression and mitochondrial localization of BCL-2 via activating CREB. ApoE2/ERK1/2/CREB also increased the total BCL-2/BAX ratio, inhibited the opening of the mitochondrial permeability transition pore and the depolarization of mitochondrial transmembrane potential, blocked the leakage of cytochrome-c and the formation of the apoptosome, and consequently, suppressed mitochondrial apoptosis. Conclusions: ApoE2 regulates the mitochondrial localization and expression of BCL-2 through the activation of the ERK1/2/CREB signaling cascade to evade the mitochondrial apoptosis of pancreatic cancer cells. ApoE2 may be a distinct prognostic marker and a potential therapeutic target for pancreatic cancer.

The effect of miR-129-5p in pancreatic cancer cells on apoptosis through targeted of HMGB1

Objective:To investigate the role of miR-129-5p in regulating HMGB1 expression in pancreatic cancer cell apoptosis.Methods:The untreated pancreatic cancer SW1990 cells were used as the control group.Mimics-NC(empty vector),miR-129-5p mimics,inhibitor-NC(empty vector)and miR-129-5p inhibitor were transfected into SW1990 cells by liposome transfection method as the mimics-NC group,miR-129-5p overexpression group(miR-129-5p mimics group),inhibitor-NC group and miR-129-5p low expression group(miR-129-5p inhibitor group).The binding site of miR-129-5p and HMGB1 was predicted by online target gene prediction website Target genes,and the targeting relationship between miR-129-5p and HMGB1 was verified by dual luciferase gene report experiment.The expression of miR-129-5p in each group was detected by qRT-PCR,and the expression of HMGB1 protein and apoptosis-related proteins Caspase 3 and Bcl-2 by Western blot.Hoechst staining was used to observe the changes of apoptosis.Results:Compared with the mimics-NC group and control group,miR-129-5p mimics transfection significantly up-regulated miR-129-5p level(P<0.01),inhibited HMGB1(P<0.01)and Bcl-2(P<0.05)protein expression,pro-moted Caspase 3 protein expression(P<0.05),and promoted apoptosis;compared with the inhibitor-NC group and control group,miR-129-5p inhibitor transfection significantly down-regulated miR-129-5p level(P<0.05),promoted HMGB1 and Bcl-2 protein expression(all P<0.05),inhibited Caspase 3protein expression(P<0.01),and inhibited apoptosis.The results of dual luciferase reporter gene assay showed that miR-129-5p could inhibit the fluorescence activity of wildtype HMGB1 cells and target the expression of HMGB1.Conclusion:miR-129-5p promotes the apoptosis of pancreatic cancer SW1990 cells by targeting inhibition of HMGB1 expression.

Visfatin Protects Rat Pancreatic β-cells against IFN-γ-Induced Apoptosis through AMPK and ERK1/2 Signaling Pathways

Objective Interferon-γ （IFN-γ） plays an important role in apoptosis and was shown to increase the riskof diabetes. Visfatin, an adipokine, has anti-diabetic, anti-tumor, and regulating inflammatoryproperties. In this study we investigated the effect of visfatin on IFN-γ-induced apoptosis in ratpancreatic β-cells.Methods The RINm5F （rat insulinoma cell line） cells exposed to IFN-γ were treated with or withoutvisfatin. The viability and apoptosis of the cells were assessed by using MTT and flow cytometry. Theexpressions of mRNA and protein were detected by using real-time PCR and western blot analysis.Results The exposure of RINm5F cells to IFN-γ for 48 h led to increased apoptosis percentage of thecells. Visfatin pretreatment significantly increased the cell viability and reduced the cell apoptosisinduced by IFN-γ. IFN-γ-induced increase in expression of p53 mRNA and cytochrome c protein,decrease in mRNA and protein levels of anti-apoptotic protein Bcl-2 were attenuated by visfatinpretreatment. Visfatin also increased AMPK and ERK1/2 phosphorylation and the anti-apoptotic actionof visfatin was attenuated by the AMPK and ERK1/2 inhibitor.Conclusion These results suggested that visfatin protected pancreatic islet cells against IFN-γ-inducedapoptosis via mitochondria-dependent apoptotic pathway. The anti-apoptotic action of visfatin ismediated by activation of AMPK and ERK1/2 signaling molecules.

Propofol induces apoptosis and increases gemcitabine sensitivity in pancreatic cancer cells in vitro by inhibition of nuclear factor-κ B activity

AIM:To investigate the effect of propofol on human pancreatic cells and the molecular mechanism of propofol action.METHODS:We used the human pancreatic cancer cell line MIAPaCa-2 for in vitro studies measuring growth inhibition and degree of apoptotic cell death induced by propofol alone,gemcitabine alone,or propofol followed by gemcitabine.All experiments were conducted in triplicate and carried out on three or more separate occasions.Data were means of the three or more independent experiments±SE.Statistically significant differences were determined by two-tailed unpaired Student’s t test and defined as P<0.05.RESULTS:Pretreatment of cells with propofol for 24 h followed by gemcitabine resulted in 24%-75% growth inhibition compared with 6%-18%when gemcitabine was used alone.Overall growth inhibition was directly correlated with apoptotic cell death.We also showed that propofol potentiated gemcitabine-induced killing by downregulation of nuclear factor-κB(NF-κB).In contrast,NF-κB was upregulated when pancreatic cancer cells were exposed to gemcitabine alone,suggesting a potential mechanism of acquired chemoresistance.CONCLUSION:Inactivation of the NF-κB signaling pathway by propofol might abrogate gemcitabineinduced activation of NF-κB,resulting in chemosensitization of pancreatic tumors to gemcitabine.

Growth inhibition and apoptosis induction by alternol in pancreatic carcinoma cells

AIM: To investigate the effect of alternol on pancreatic cancer cells.METHODS: Pancreatic cancer cells PANC-1 and Bx PC3 were treated with various concentrations of alternol for 24, 48 and 72 h. Cell proliferation was measured by cell counting. Cell cycle distribution and mitochondrial membrane potential were determined by flow cytometry. Apoptosis was determined by a Td T-mediated d UTP nick end labeling assay and Hoechst staining. Expression of caspase 3, Bcl-2, p53 and p21 was measured by western blotting.RESULTS: Alternol showed dose- and time-dependent inhibition of the proliferation of PANC-1 and Bx PC3 cells in vitro. Alternol induced apoptosis and cell cycle arrest at S phase and decreased mitochondrial membrane potential. Alternol activated caspase 3, upregulated p53 and p21 expression, and downregulated Bcl-2 expression in a dose-dependent manner.CONCLUSION: Our results suggested that alternol is a candidate for treatment of pancreatic cancer.

microRNA-218 suppresses the proliferation, invasion and promotes apoptosis of pancreatic cancer cells by targeting HMGB1

Objective： To detect the expression profiles of micro RNA-218（mi R-218） in human pancreatic cancer tissue（PCT） and cells and their effects on the biological features of human pancreatic cancer cell line PANC-1 and observe the effect of mi R-218 on the expression of the target gene high mobility group box 1（HMGB1）, with an attempt to provide new treatment methods and strategies for pancreatic cancer.Methods： The expressions of mi R-218 in PCT and normal pancreas tissue as well as in various pancreatic cancer cell lines including As PC-1, Bx PC-3, and PANC-1 were determined with quantitative real-time reverse transcription polymerase chain reaction（q RT-PCR）. The change of mi R-218 expression in PANC-1 cells was detected using qR T-PCT after the transfection of miR-218 mimic for 48 h. Cell Counting Kit-8（CCK-8） was applied for detecting the effect of mi R-218 on the activity of PANC-1 cells. The effects of mi R-218 on the proliferation and apoptosis of PANC-1 cells were analyzed using the flow cytometry. The effect of mi R-218 on the migration of PANC-1 cells was detected using the Trans-well migration assay. The HMGB1 was found to be a target gene of mi R-218 by luciferase reporter assay, and the effect of mi R-218 on the expression of HMGB1 protein in cells were determined using Western blotting.Results： As shown by q RT-PCR, the expressions of mi R-218 in PCT and in pancreatic cancer cell line significantly decreased when compared with the normal pancreatic tissue（NPT）（P〈0.01）. Compared with the control group, the miR-218 expression significantly increased in the PANC-1 group after the transfection of mi R-218 mimic for 48 h（P〈0.01）. Growth curve showed that the cell viability significantly dropped after the overexpression of mi R-218 in the PANC-1 cells for two days（P〈0.05）. Flow cytometry showed that the S-phase fraction significantly dropped after the overexpression of mi R-218（P〈0.01） and the percentage of apoptotic cells significantly increased（P〈0.01）. As shown by the Trans-well migration assay, the enhanced mi R-218 expression was associated with a significantly lower number of cells that passed through a Transwell chamber（P〈0.01）. Luciferase reporter assay showed that, compared with the control group, the relative luciferase activity significantly decreased in the mi R-218 mimic group（P〈0.01）. As shown by the Western blotting, compared with the control group, the HMGB1 protein expression significantly decreased in the PANC-1 group after the transfection of mi R-218 mimic for 48 h（P〈0.01）.Conclusions： The mi R-218 expression decreases in human PCT and cell lines. mi R-218 can negatively regulate the HMGB1 protein expression and inhibit the proliferation and invasion of pancreatic cancer cells. A treatment strategy by enhancing the mi R-218 expression may benefit the patients with pancreatic cancer.

Apoptosis of human pancreatic carcinoma cell-1 cells induced by Yin Chen Hao Decoction

AIM: To evaluate human pancreatic carcinoma cell line(PANC-1) cells apoptosis and Bcl-2 and Bax expression induced by Yin Chen Hao Decoction(YCHD).METHODS: The cell growth inhibitory rate was determined by MTT assay. Apoptosis of PANC-1 cells before and after treatment with YCHD was determined by TUNEL staining. Expression of the apoptosisassociated genes, Bcl-2 and Bax, was detected by immunohistochemical staining and reverse transcription-PCR.RESULTS: YCHD inhibited the growth of PANC-1 cells. Following treatment with YCHD for 24-96 h, the apoptotic rate of PANC-1 cells increased with time. In addition, the positive rate of Bcl-2 protein expression decreased in a time-dependent manner, whereas the positive rate of Bax protein expression increased in a time-dependent manner. Following treatment of with YCHD for 24-96 h, expression of BAX m RNA increased gradually and BCL-2 m RNA reduced gradually with time.CONCLUSION: YCHD induces apoptosis of PANC-1 cells mediated in part via up-regulation of BAX and down-regulation of BCL-2.

Tanshinone IIA Could Inhibit Pancreatic Cancer BxPC-3 Cells through Increasing PERK, ATF6, Caspase-12 and CHOP Expression to Induce Apoptosis

Tanshinone IIA (Tan-IIA) is extracted from Dan-Shen. Tan-IIA could inhibit human pancreatic cancer BxPC-3 cells through decreasing TCTP, Mcl-1 and Bcl-xl expression in vitro. Our previous study showed that Tan-IIA can inhibit hepatocellular carcinoma hep-J5 cells and human breast cancer BT-20 cells through inducing endoplasmic reticulum (ER) stress. In the present study, we investigated the ER stress related protein expressions in human pancreatic cancer BxPC3 cells were treated with Tan-IIA. The ER stress related protein expressions in human pancreatic cancer BxPC-3 cells were evaluated by western blotting. The results showed that Tan-IIA can increase the protein expressions of PERK, ATF6, Caspase-12 and CHOP, but decrease Bip, PDI, Calnexin, Calreticulin and Bcl-2 expression. These findings indicated that Tan-IIA can inhibit human pancreatic cancer BxPC-3 cells by inducing ER stress to induce apoptosis.

Human antigen R mediated post-transcriptional regulation of inhibitors of apoptosis proteins in pancreatic cancer

AIM To determine the association of human antigen R(HuR) and inhibitors of apoptosis proteins(IAP1, IAP2) and prognosis in pancreatic cancer.METHODS Protein and mRNA expression levels of IAP1, IAP2 and HuR in pancreatic ductal adenocarcinoma(PDAC) were compared with normal pancreatic tissue. The correlations among IAP1/IAP2 and HuR as well as their respective correlations with clinicopathological parameters were analyzed. The Kaplan-Meier method and log-rank tests were used for survival analysis. Immunoprecipitation assay was performed to demonstrate HuR binding to IAP1, IAP2 mRNA. PANC1 cells were transfected with either anti-HuR siRNA or control siRNA for 72 h and quantitative reverse transcription polymerase chain reaction(RT-PCR), western blot analysis was carried out.RESULTS RT-PCR analysis revealed that HuR, IAP1, IAP2 mRNA expression were accordingly 3.3-fold, 5.5-fold and 8.4 higher in the PDAC when compared to normal pancreas(P < 0.05). Expression of IAP1 was positively strongly correlated with HuR expression(P < 0.05, r = 0.783). Western blot analysis confirmed RTPCR results. High IAP1 expression, tumor resection status, T stage, lymph-node metastases, tumor differentiation grade, perineural and lymphatic invasion were identified as significant factors for shorter survival in PDAC patients(P < 0.05).Immunohistological analysis showed that HuR was mainly expressed in the ductal cancer cell's nucleus and less so in cytoplasm. RNA immunoprecipitation analysis confirmed IAP1 and IAP2 post-transcriptional regulation by HuR protein. Following siHuR transfection, IAP1 mRNA and protein levels were decreased, however IAP2 expression levels were increased.CONCLUSION HuR mediated overexpression of IAP1 significantly correlates with poor outcomes and early progression of pancreatic cancer. Further studies are needed to assess the underlying mechanisms.

Glucocorticoid receptor regulates expression of microRNA-22 and downstream signaling pathway in apoptosis of pancreatic acinar cells

AIM To elucidate the underlying mechanism that microRNA-22(miR-22) promotes the apoptosis of rat pancreatic acinar cells(AR42 J) and the elements that regulate the expression of miR-22.METHODS One hundred nanomoles per liter of caerulein(Cae)was administrated to induce the apoptosis of AR42 J cells and the apoptosis rate was detected by flow cytometry analysis. An amylase assay kit was used to measure the amylase expression level in the supernatant. Quantitative real-time PCR(qRT-PCR)was adopted to measure miR-22 expression. We used online tools to predict the potential transcription promoter of miR-22 and the binding sites, which was further identified by using luciferase reporter analysis,chromatin immunoprecipitation(ChIP) and ChIPqP CR assays. Then, a mimic of miR-22, Nr3 c1 plasmid encoding the glucocorticoid receptor(GR), and siNr3 c1 were used to transfect AR42 J cells, respectively.The mRNA expression of miR-22, Nr3 c1, and Erb-b2 receptor tyrosine kinase 3(ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42 J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3 k, PI3 kp85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3.RESULTS After inducing apoptosis of AR42 J cells in vitro, the expression of miR-22 was significantly increased by2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at3 h and 6 h in comparison with the control group.As revealed by qRT-PCR assay, the expression of miR-22 was 78.25 ± 6.61 times higher in the miR-22 mimic group relative to the miRNA control group,accompanied with an obviously increased acinar cell apoptosis rate(32.53 ± 1.15 vs 18.07 ± 0.89, P =0.0006). The upregulation of miR-22 could suppress its target gene, ErbB3, and the phosphorylation of PI3 k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and sitedirected mutagenesis indicated that the binding site(GACAGCCATGTACA) of the GR, which is encoded by the Nr3 c1 gene. Downregulation of the expression of GR could upregulate the expression of miR-22, which further promoted the apoptosis of AR42 J cells.CONCLUSION GR transcriptionally represses the expression of miR-22,which further promotes the apoptosis of pancreatic acinar cells by downregulating the downstream signaling pathway.

Metformin induces apoptosis of pancreatic cancer cells

AIM:To assess the role and mechanism of metformin in inducing apoptosis of pancreatic cancer cells.METHODS:The human pancreatic cancer cell lines ASPC-1,BxPc-3,PANC-1 and SW1990 were exposed to metformin.The inhibition of cell proliferation and colony formation via apoptosis induction and S phase arrest in pancreatic cancer cell lines of metformin was tested.RESULTS:In each pancreatic cancer cell line tested,metformin inhibited cell proliferation in a dose dependent manner in MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays).Flow cytometric analysis showed that metformin reduced the number of cells in G1 and increased the percentage of cells in S phase as well as the apoptotic fraction.Enzymelinked immunosorbent assay(ELISA) showed that metformin induced apoptosis in all pancreatic cancer cell lines.In Western blot studies,metformin induced poly-ADP-ribose polymerase(PARP) cleavage(an indicator of caspase activation) in all pancreatic cancer cell lines.The general caspase inhibitor(VAD-fmk) completely abolished metformin-induced PARP cleavage and apoptosis in ASPC-1 BxPc-3 and PANC-1,the caspase-8 specific inhibitor(IETD-fmk) and the caspase-9 specific inhibitor(LEHD-fmk) only partially abrogated metformin-induced apoptosis and PARP cleavage in BxPc-3 and PANC-1 cells.We also observed that metformin treatment dramatically reduced epidermal growth factor receptor(EGFR) and phosphorylated mitogen activated protein kinase(P-MAPK) in both a time-and dose-dependent manner in all cell lines tested.CONCLUSION:Metformin significantly inhibits cell proliferation and apoptosis in all pancreatic cell lines.And the metformin-induced apoptosis is associated with PARP cleavage,activation of caspase-3,-8,and-9 in a time-and dose-dependent manner.Hence,both caspase-8 and-9-initiated apoptotic signaling pathways contribute to metformin-induced apoptosis in pancreatic cell lines.

Ellagic acid induces apoptosis through inhibition of nuclear factor κB in pancreatic cancer cells

AIM:To determine the effect of ellagic acid on apop-tosis and proliferation in pancreatic cancer cells and to determine the mechanism of the pro-survival effects of ellagic acid. METHODS:The effect of ellagic acid on apoptosis was assessed by measuring Phosphatidylserine externalization,caspase activity,mitochondrial membrane potential and DNA fragmentation;and proliferation by measuring DNA thymidine incorporation. Mitochondrial membrane potential was measured in permeabilized cells,and in isolated mitochondria. Nuclear factor kB(NF-kB) activity was measured by electromobility shift assay(EMSA) . RESULTS:We show that ellagic acid,a polyphenolic compound in fruits and berries,at concentrations 10 to 50 mmol/L stimulates apoptosis in human pancreatic adenocarcinoma cells. Further,ellagic acid decreases proliferation by up to 20-fold at 50 mmol/L. Ellagic acid stimulates the mitochondrial pathway of apoptosis associated with mitochondrial depolarization,cytochrome C release,and the downstream caspaseactivation. Ellagic acid does not directly affect mitochondria. Ellagic acid dose-dependently decreased NF-kB binding activity. Furthermore,inhibition of NF-kB activity using IkB wild type plasmid prevented the effect of ellagic acid on apoptosis. CONCLUSION:Our data indicate that ellagic acid stimulates apoptosis through inhibition of the prosu-rvival transcription factor NF-kB.

Effects of large dose of dexamethasone on inflammatory mediators and pancreatic cell apoptosis of rats with severe acute pancreatitis

AIM: To investigate the influence of high dose of dexamethasone on inflammatory mediators and apoptosis of rats with severe acute pancreatitis (SAP). METHODS: SAP rats were randomly assigned to the model group and treatment group while the normal rats were assigned to the sham operation group. The mortality,ascite volumes,ascites/body weight ratio and pancreas pathological changes of all rats were observed at 3,6 and 12 h after operation. Their contents of amylase and endotoxin in plasma and contents of tumor necrosis factor (TNF-α),phospholipase A2 (PLA2) and IL-6 in serum were also determined. The microarray sections of their pancreatic tissues were prepared,terminal transferase dUTP nick end labeling (TUNEL) staining was performed and apoptotic indexes were calculated. RESULTS: There was no marked difference between treatment group and model group in survival. The contents of amylase and endotoxin in plasma and contents of TNF-α,PLA2 and IL-6 in serum,ascite volumes,ascites/body weight ratio and pancreas pathological scores were all lower in treatment group than in model group to different extents at different time points P < 0.05,58.3 (26.4) ng/L vs 77.535 (42.157) ng/L in TNF-α content,8.00 (2.00) points vs 9.00 (2.00) points in pathological score of pancreas respectively; P < 0.01,0.042 (0.018) EU/mL vs 0.056 (0.0195) EU/mL in endotoxin content,7791 (1863) U/L vs 9195 (1298) U/L in plasma amylase content,1.53 (0.79) vs 2.38 (1.10) in ascites/body weight ratio,8.00 (1.00) points vs 11.00 (1.50) points in pathological score of pancreas; P < 0.001,3.36 (1.56) ng/L vs 5.65 (1.08) ng/L in IL-6 content,4.50 (2.00) vs 7.20 (2.00),4.20 (1.60) vs 6.40 (2.30),3.40 (2.70) vs 7.90 (1.70) in ascite volumes,respectively. The apoptotic indexes of pancreas head and pancreas tail were all higher in treatment group than in model group at 6 h P < 0.01,0.00 (2.00)% vs 0.00 (0.00)%,0.20 (1.80) vs 0.00 (0.00) in apoptosis indexes,respectively. CONCLUSION: The mechanism of dexamethasone treatment in acute pancreatitis is related to its inhibition of inflammatory mediator generation and induction of pancreatic acinar cell apoptosis.

Apoptosis of human pancreatic cancer cells induced by Triptolide

AIM: To investigate apoptosis in human pancreatic cancer cells induced by Triptolide (TL), and the relationship between this apoptosis and expression of caspase-3' bcl-2 and bax. METHODS: Human pancreatic cancer cell line SW1990 was cultured in DMEM media for this study. MTT assay was used to determine the cell growth inhibitory rate in vitro. Flow cytometry and TUNEL assay were used to detect the apoptosis of human pancreatic cancer cells before and after TL treatment. RT-PCR was used to detect the expression of apoptosis-associated gene caspase-3' bcl-2 and bax. RESULTS: TL inhibited the growth of human pancreatic cancer cells in a dose-and time-dependent manner. TL induced human pancreatic cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of human pancreatic cancer cells with 40 ng/mL TL for 12 h and 24 h, the apoptotic rates of human pancreatic cancer cells increased significantly. RT-PCR demonstrated that caspase-3 and bax were significantly up-regulated in SW1990 cells treated with TL while bcl-2 mRNA was not. CONCLUSION: TL is able to induce the apoptosis in human pancreatic cancer cells. This apoptosis may be mediated by up-regulating the expression of apoptosis- associated caspase-3 and bax gene.

Induction of Apoptosis and Anoikis by Bit1 in Pancreatic Cancer Cells

Pancreatic cancer is a highly aggressive disease with a very high mortality rate among all human cancers. The poor prognosis is in part due to intrinsic resistance to the apoptosis-inducing effects of radio- and chemotherapy. To find alternative cell death pathways that can bypass the apoptotic resistance of pancreatic cancer cells, we examined the role of the novel anoikis effector Bit1 (Bcl-2 inhibitor of transcription) in the survival and apoptotic resistance of pancreatic cancer cells. Bit1 is a mitochondrial protein that induces a caspase-independent apoptosis upon its release into the cytosol following loss of integrin-mediated attachment to extracellular matrix (anoikis). In this report, we observed that ectopic expression of Bit1 in the cytosol reduced viability and induced caspase-independent apoptosis in human pancreatic cancer cell lines, Miapaca-2 and PANC-1. While increased expression of mitochondrial Bit1 in these cells did not induce apoptosis under attached conditions, detachment significantly induced higher level of apoptosis in mitochondrial Bit1-transfected cells than in control transfected cells. Conversely, downregulation of endogenous Bit1 in PANC-1 cells further enhanced their anoikis resistance. Furthermore, exogenous expression of mitochondrial Bit1 in Miapaca-2 cells inhibited their anchorage-independent growth and enhanced their sensitivity to etoposide-mediated apoptosis. Mechanistically, we found that the Bit1 apoptosis function is in part dependent on the groucho related Amino-terminal Enhancer of Split (AES) expression and is abrogated by the transcriptional corepressor TLE1 protein. Consistent with our in vitro findings that Bit1 is an effector of apoptosis in pancreatic tumor cells, we find that Bit1 is significantly downregulated in a fraction of advanced stages of human pancreatic carcinoma tissues. Taken together, these findings indicate that the Bit1-apoptotic pathway can be targeted to trigger cell death in pancreatic cancer cells and implicate Bit1 as a novel therapeutic agent in attenuating pancreatic chemoresistance.

THE EFFECT OF ISCHEMIC RE-PERFUSION INJURY PLUS PARTICLE INFUSION EMBOLISM ON THE APOPTOSIS OF RATS WITH PANCREATIC CANCER

Objectives. In an attempt to develop new method of treating the end ormid stage pancreatic cancer, we examined the effect of ischemic re perfusion injury plus particle embolism on the pathology and cell apoptosis of pancreatic cancer in Sprague Dawely rats. Methods. 9 mg dimethylbeneanthracine (DMBA) were implanted directly into the parenchyma of pancreatic tail of Sprague Dawely rats. After establishment of tumor, the inferior splenic artery, a main supplying vessel to pancreatic tail was subjected to blockade and re opening for 30 min separately, then embolism particles were infused via the artery. Afterwards, artery was ligated. Pathological changes and cell apoptosis indicators (AI) of pancreatic cancer were observed by light microscopy and ISEL respectively 14 days after the operation. Results. The prevalence of pancreatic cancer among DMBA implanted rats evaluated 3 months to 4 months after implantation was 59%. The volumes of the tumor in positive control group (B), pancreatic ischemic group (C), pancreatic ischemic re perfusion injury group (D) were significantly larger than pancreatic ischemic re perfusion injury plus particle thrombus group (E) (P< 0.01). Thevolumes of the tumor in groups D, E were significantly smaller than that in group C (P< 0.01). There was a significant difference in tumor size between group Band group C (P< 0.01), but the difference was not significant between group D and group E (P >0.05). There was a significant infiltration of tumor tissue in group B rats, but strong inflammatory reaction was not noted. In groups C, D, E, alocalized tumor growth was observed; infiltration of inflammatory cells and proliferation of fibroblasts and connective fiber were obvious, and some of these fibers grew into cancer nests and separate the tumor. The above findings were most conspicuous in group E. There was a significant difference in AI between group E (13.7±1.5)and other groups (P< 0.01), with the difference being also significant between group C(4.3±2.4), D (8.5±1.1)and group B (1.2±0.8)(P< 0.01), and between group C and group D (P< 0.01) or between group D and group E (P< 0.01). In the samples of group A, the apoptotic cells were not found. Conclusions. Pancreatic ischemic re perfusion injury plus particle thrombus can cause significant infiltration of inflammatory cells in tumor tissues thereby limiting its growth, and inducing cell apoptosis of pancreatic cancer. This effect is superior to either pancreatic ischemia alone or pancreatic ischemia plus re perfusion injury.

Targeting X-linked inhibitor of apoptosis protein inhibits pancreatic cancer cell growth through p-Akt depletion

AIM: To determine whether lentivirus-mediated shRNA targeting the X-linked inhibitor of apoptosis protein (XIAP) gene could be exploited in the treatment of pancreatic cancer. METHODS: Human pancreatic cancer cells Panc-1, Mia-paca2, Bxpc-3 and SW1990, infected with lentivirus, were analyzed by real-time polymerase chain reaction (PCR). Western blotting was used to examine XIAP protein levels, survivin and p-Akt to confirm the result of real-time PCR and determine the possible mechanism. The 3-(4,5-cimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay was used to measure IC50 to determine chemosensitivity to the chemotherapeutic drugs 5-fluorouracil (5-FU) and gemcitabine. A colony assay, MTT assay and a tumorigenicity experiment were used to study cell proliferation in vitro and in vivo . Caspase-3/7 activity, 4',6-diamidino-2-phenylindole-staining and flow cytometric measurements were used to study apoptosis in SW1990 cells. RESULTS: XIAP proteins were found to be differen- tially expressed among pancreatic cancer cell lines Panc-1, Mia-paca2, Bxpc-3 and SW1990. Data of real-time PCR and Western blotting showed that XIAP was reduced persistently and markedly by lentivirus-mediated shRNA. Downregulation of XIAP by transfection with XIAP shRNA resulted in decreased p-Akt expression. XIAP shRNA also inhibited the growth of pancreatic cancer cells in vitro and in vivo , enhanced drug-induced apoptosis and increased chemosensitivity to 5-FU and gemcitabine. Results also suggest that inhibition of XIAP and subsequent p-Akt depletion may have an anti-tumor effect through attenuating the ability of cancer cells to survive. CONCLUSION: Lentivirus-mediated gene therapy is an attractive strategy in the treatment of pancreatic cancer and justifies the use of lentivirus in pancreatic cancer gene therapy studies.

Apoptosis of Human Pancreatic Carcinoma Cells Induced By All-Trans Retinoic Acid and Interferon

Objective： To investigate the apoptosis of human pancreatic carcinoma PC3 cells induced by the combination of all-trans retinoic acid （ATRA） with interferon alpha （IFN-α）. Methods： PC3 cells were treated with ATRA and IFN-α. The inhibitory rate of PC3 cell proliferation was detected using MTT method. Cellular apoptosis was determined with flow cytometry. The percentage of PC3 cell apoptosis was assayed using TUNEL methods. Results： ATRA and IFN-α could inhibit cellular proliferation and induces cellular apoptosis of PC3 cells. The inhibitory effect was stronger when the ATRA and IFN-α were combined as a therapy. Conclusion： ATRA inhibits the proliferation of PC3 cells and induce the apoptosis of PC3 cells. The combination of IFN-α with ATRA may enhance these effects on PC3 cells.