Effects of overexpression of LncRNA MEG3 on autophagy, apoptosis and mTOR pathway in pancreatic cancer cells PANC1 / 中华内分泌外科杂志

Objective:To investigate the effects of overexpression of long non-coding RNA maternally expressed gene 3 (LncRNA MEG3) on autophagy, apoptosis and mammalian rapamycin target protein (mTOR) pathway in pancreatic cancer cells (PANC1) .Methods:The pCMV-N-Flag-MEG3 expression plasmid was constructed and transfected into PANC1 cells. The expression of LncRNA MEG3 in hpde6c7 (normal pancreatic cells) group, PANC1 (blank control) , Vector (PANC1 cell transfected empty vector) group and MEG3 (PANC1 cell transfected with pCMV-N-Flag-MEG3 recombinant plasmid) group was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) ; methyl thiazolyl tetrazolium (MTT) , flow cytometry and monodansylcadaverin (MDC) staining were used to detect the effects of overexpression of LncRNA MEG3 on the proliferation, apoptosis and autophagy of PANC1 cells; Western blot was used to detect the effects of overexpression of LncRNA MEG3 on the expression levels of Bcl-2, Bax and Beclin-1 in PANC1 cells, and the phosphorylation levels of mTOR, ribosomal p70S6 kinase protein (SK61) and uclear initiation factor 4E binding protein 1 (4E-BP1) in mTOR pathway.Results:Compared with those in PANC1 group and Vector group, the expression level of LncRNA MEG3 (0.36±0.08 vs 0.35±0.11 vs 0.69±0.09) , proliferation inhibition rate (3.35%±0.12 vs 3.23%±0.09 vs 36.77%±0.13) , autophagy rate (29.32%±1.03 vs 26.73%±1.32 vs 57.76%±1.09) , apoptosis rate (9.85%±1.58 vs 9.73%±1.12 vs 35.89%±1.05) , expression levels of Bax (0.26±0.08 vs 0.29±0.05 vs 0.83±0.08) and Beclin 1 (0.15±0.06 vs 0.17±0.02 vs 0.61±0.03) of PANC1 cells in MEG3 group were significantly higher (all P<0.05) , and the expression level of Bcl-2 (0.79±0.12 vs 0.81±0.09 vs 0.30±0.03) and phosphorylation levels of mTOR (1.08±0.05 and 1.06±0.08 vs 0.37±0.10) , SK61 (1.12±0.06 and 1.11±0.09 vs 0.41±0.03) and 4E-BP1 (0.97±0.07 and 0.95±0.03 vs 0.39±0.05) in mTOR pathway were significantly lower (all P<0.05) . Conclusion:Overexpression of LncRNA MEG3 can inhibit the proliferation of PANC1 cells, promote apoptosis and formation of autophagic vesicles, which may be related to the blocking of mTOR pathway.

Effect of high voltage electric field on cell growth, apoptosis of human pancreatic cancer cell line PANC-1 / 中华肝胆外科杂志

Objective To investigate the effects of high intensity electric field on cell growth,apoptosis and microstructure of human pancreatic cancer cell line PANC-1.Methods The PANC-1 cells in the logarithmic growth period were selected,and cells in the high voltage electrical treatment group were treated with high voltage electric field 250,500,750,1000 V/cm,respectively.The effects of different high voltage electric fields on cell growth and microstructure of PANC-1 cells were determined by cell viability,cell death staining,apoptosis detection,transmission electron microscopy and scanning electron microscopy.Results Compared with control group,the high voltage electric pulse significantly inhibited the growth of PANC-1 cells in the field dependent manner.Moreover,when the field was more than 500 V/cm,the cell viability was significantly decreased (P＜0.05).High voltage electric pulse could induce cell apoptosis.When the field was higher than 750 V/cm,serious necrosis was noticed.In the 1000 V/cm group,the integrity of cell membrane and the structure of organelles was seriously damaged.Conclusion High voltage electric pulse could significantly inhibit the growth of PANC-1 cells and would be a promising method in cancer treatment.

Silencing ANO9 increases the radiosensitivity of pancreatic cancer cell AsPC-1 / 中华放射肿瘤学杂志

Objective To evaluate the effect of ANO9 on the radiosensitivity of pancreatic cancer cell AsPC-1,aiming to provide new targets for clinical radiotherapy of pancreatic cancer.Methods Western blot was performed to detect the expression of ANO9 in pancreatic cancer cell lines (BxPC-3,PANC-1,AsPC-1)and normal pancreatic cell line (HPNE).The AsPC-1 cell line with stable silencing ANO9 was constructed by using lentivirus and validated by Western blot.MTT assay was adopted to detect the cell viability of AsPC-1 with stable silencing ANO9 after irradiation.Colony formation assay was conducted to evaluate the effect of silencing ANO9 upon the radiosensitivity of AsPC-1 cells.Western blot was performed to assess the effect of ANO9 silencing on the expression of EGFR/ERK signaling protein.Results The expression levels of ANO9 were significantly up-regulated in three pancreatic cancer cell lines compared with that in the normal pancreatic cell line HPNE (t =7.426,5.543,11.850,all P＜0.05).After silencing ANO9,the expression level of ANO9 protein was significantly down-regulated than that in the control group (t =9.670,P＜0.05).The AsPC-1 cells with stable silencing ANO9 were successfully constructed.The sensitivity of AsPC-1 cells to irradiation was significantly increased after silencing ANO9,and the sensitivity enhancement ratio was 1.566.The expression levels of EGFR/ERK signaling proteins (EGFR and p-ERK 1/2) were significantly downregulated after silencing ANO9 (t =7.949,13.160,both P＜ 0.05).Conclusions Silencing ANO9 can significantly increase the sensitivity of AsPC-1 cells to radiotherapy,which is probably associated with the inhibition of EGFR/ERK signaling transduction.ANO9 might be a new therapeutic target for preventing the progression of pancreatic cancer.

The "target + activity" method for rapid discovery of active compounds targeting Hsp90 in pancreatic cancer cells from Physalis angulata L / 药学学报

The purpose of this study was to select the active compounds targeting Hsp90 protein in pancreatic cancer cells through a new dual "target + activity" rapid discovery technique. We combined an in vitro anti-cancer activity screening method with a dual-luciferase reporter gene and multi-chromatography separation technology, for rapid discovery of potential Hsp90 inhibitors from the Chinese herbal medicine Physalis angulata L. The anti-proliferation activity of those compounds was assessed in pancreatic cancer cell line BxPC-3 by MTT assays. The molecular mechanisms of Hsp90 inhibition were explored by Western blot and shRNA knockdown assays. As a result, two withanolides, withanolide E (WE) and 4β-hydroxywithanolide E (HWE), were identified from Physalis angulata L. The half maximal inhibitory concentration (IC50) of WE and HWE were 0.71±0.03 and 1.23±0.10 μmol·L-1 for the growth of BxPC-3 cells in 48 h. Luciferase reporter assay demonstrated that WE and HWE significantly induced heat shock element (HSE) activity in a dose- and time-dependent manner. The molecular mechanism study showed that after exposing to 5 μmol·L-1 WE or HWE for 48 h, the aggregation of Hsp90 dimer was upregulated to 6.5±1.3 and 11.8±2.0 fold, while the expression of Hsp90 client protein Akt was downregulated to 21.7%±2.8% and 9.8%±1.4% of the control group. Moreover, the Hsp90 inhibitory activity of WE or HWE was canceled by shRNA mediated Hsp90 knockdown. Overall, based on the dual "target + active" rapid discovery technique, two new Hsp90 inhibitors WE and HWE were found from Physalis angulata L. The Hsp90 inhibitory mechanism of WE and HWE may be mediated by induction of Hsp90 aggregate dimer and inhibition of Hsp90 client protein Akt expression.

The role of Tif1γ on apoptosis of pancreatic cancer cells / 复旦学报（医学版）

Objective To investigate the effect of transcriptional internediary factor 1 gamma (Tif1γ)and its signal pathway related proteinson apoptosis of human pancreatic cancer cell line Capan-1 treated with either gemcitabine (GEM) or raltitrexed (RTX).Methods Capan-1 cells were treated with GEM of 559 μmol/L or RTX of 0.86 μmol/L for 36 h.The cell apoptosis of Capan-1 was determined using flow cytometry.Protein levels of Tif1γ,TGF-β1,Smad3,p-Smad3,Smad4,Bcl2,BAX and Caspase3 in Capan-1 cells were determined by Western blot.Results The late apoptosis and death ratio after RTX treatment were 28.7％ ± 5.1％ and 3.7％ ± 0.5％,respectively,showing significant difference from that after GEM treatment or untreated control group (P＜0.01).The results of Western blot showed that the relative protein levels of Tif1 γ,TGF-β1,p-Smad3,BAX and Caspase3 in Capan-1 cells were increased in RTX group compared with those in GEM group or control group (P＜0.05).The relative protein levels of Smad4 and the Bcl2/Bax ratio were decreased in RTX group compared with those in GEM group or control group (P＜0.05).Conclusions Increased level of Til1γ by RTX treatment resulted in decreased level of Smad4 to regulate the balance of Bcl2/Bax,increased Caspase3 and increased apoptosis in Capan-1 cells.

Effects of chemokine Fractalkine on proliferation and invasion of human pancreatic cancer cell lines by regulating IL-6/STAT3 signal pathway / 重庆医学

Objective To explore the effects of chemokine Fractalkine(FKN) on the proliferation and invasion of human pancreatic cancer cell lines PANC-1 and SW-1990 by regulating IL-6/STAT3 signal pathway.Methods Adenovirus served as the vector to construct and synthesizing FKN-small interfering RNA(siRNA),then which was transfected into PANC-1 and SW-1990.The proliferation and invasion ability of cells was determined by CCK-8 assay and Transwell assay.Expression of FKN,IL-6 and STAT3 protein and mRNA was detected by Western blot and RT-qPCR.Results After transfecting FKN-siRNA for 24 h,the absorbance values(A value) in the PANC-1 and SW-1990 groups had no significant changes,the A value at 48,72 h in the FKN-siRNA group was significantly higher than that in the control group and FKN-siRNA negative group (P＜0.05).After transfecting FKN-siRNA,the cellular invasive ability in the PANC-1 and SW-1990 FKN-siRNA group was significantly stronger than that in the control group and FKN-siRNA negative group(P＜0.05).After transfecting FKN-siRNA in cell lines PANC-1 and SW-1990,compared with the control group and FKN-siRNA negative group,the FKN protein and mRNA expression in the FKN-siRNA group was significantly decreased(P＜0.05),while IL-6 and STAT3 protein and mRNA expression was significantly increased(P＜0.05).Conclusion Chemokine FKN might play the inhibiting effect on the biological activity of pancreatic cancer cells by regulating IL-6/ STAT3 signal pathway.

Effects of chemokine Fractalkine on proliferation and invasion of human pancreatic cancer cell lines by regulating IL-6/STAT3 signal pathway / 重庆医学

Objective To explore the effects of chemokine Fractalkine(FKN) on the proliferation and invasion of human pancreatic cancer cell lines PANC-1 and SW-1990 by regulating IL-6/STAT3 signal pathway.Methods Adenovirus served as the vector to construct and synthesizing FKN-small interfering RNA(siRNA),then which was transfected into PANC-1 and SW-1990.The proliferation and invasion ability of cells was determined by CCK-8 assay and Transwell assay.Expression of FKN,IL-6 and STAT3 protein and mRNA was detected by Western blot and RT-qPCR.Results After transfecting FKN-siRNA for 24 h,the absorbance values(A value) in the PANC-1 and SW-1990 groups had no significant changes,the A value at 48,72 h in the FKN-siRNA group was significantly higher than that in the control group and FKN-siRNA negative group (P＜0.05).After transfecting FKN-siRNA,the cellular invasive ability in the PANC-1 and SW-1990 FKN-siRNA group was significantly stronger than that in the control group and FKN-siRNA negative group(P＜0.05).After transfecting FKN-siRNA in cell lines PANC-1 and SW-1990,compared with the control group and FKN-siRNA negative group,the FKN protein and mRNA expression in the FKN-siRNA group was significantly decreased(P＜0.05),while IL-6 and STAT3 protein and mRNA expression was significantly increased(P＜0.05).Conclusion Chemokine FKN might play the inhibiting effect on the biological activity of pancreatic cancer cells by regulating IL-6/ STAT3 signal pathway.

Silencing pancreatic adenocarcinoma up-regulated factor increases the sensitivity of pancreatic cancer cell line to gemcitabine / 中华肝胆外科杂志

Objective To observe the influence on the sensitivity of pancreatic cancer cell line BxPC-3 to gemcitabine of silencing PAUF gene.Methods BxPC-3 cells,which overexpress PAUF,was stably transfected with PAUF-shCtrl and PAUF-shRNA to establish BxPC-3_shCtrl and BxPC-3_shPAUF cells as control and experiment group.Then the mRNA and protein expression level of PAUF in these two cell lines were detected by RT-PCR and western blot,respectively.The growth inhibition rates of these two cell lines treated with different concentrations of gemcitabine (0,3.1,6.25,12.5,25,50,100,200 nmol/L) were detected by MTT.Apoptosis rates in the cells treated with different concentrations of gemcitabine (0,75,100 nmol/L) were then observed by flow cytometry.Results The relative PAUF mRNA expression level in BxPC-3_shCtrl and BxPC-3 cells were 1.00 ± 0.06 and 0.83 ± 0.07,which were significantly high er than that in BxPC-3_shPAUF cells (0.25 ± 0.02;both P ＜ 0.05).The relative PAUF protein expression level in BxPC-3_shCtrl and BxPC-3 cells were 0.89 ± 0.07 and 0.95 ± 0.04,which were significantly high er than that in BxPC-3_shPAUF cells (0.31 ± 0.03;both P ＜ 0.05).The IC50 value of gemcitabine to BxPC-3_shCtrl cell was (22.88 ± 2.43) nmol/L,which was significantly higher than that of BxPC-3_shPAUF cells [(1.06 ± 0.02) nmol/L;P ＜ 0.05];apoptosis rate of BxPC-3_shPAUF cells treated by gemcitabine increased faster than that of BxPC-3_shCtrl cells.Conclusion PAUF silencing could greatly enhance the sensitivity of BxPC-3 cells to gemcitabine.

Insulin Promotes Proliferative Vitality and Invasive Capability of Pancreatic Cancer Cells via Hypoxia-inducible Factor 1α Pathway / 华中科技大学学报（医学）（英德文版）

This study examined whether insulin-stimulated hypoxia-inducible factor 1α(HIF-1α)expression plays a crucial role in promoting the proliferative vitality and invasive capability in human pancreatic cancer cells.PANC-1 cells were divided into three groups: Control group,insulin group and insulin+YC-1(a pharmacological inhibitor of HIF-1α)group in terms of different treatments.Cells in the insulin group or insulin+YC-1 group were treated with insulin(0.1,1,10 and 100 nmol/L)alone or combined with 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole(YC-1,0.1,1,10 and 100μmol/L).HIF-1α mRNA and protein expression in PANC-1 cells was determined by real-time RT-PCR and Western blotting respectively.Cell proliferation and invasion were measured by using growth curve and invasion assay,respectively.Western blot analysis demonstrated that insulin dose-dependently increased the HIF-1α protein expression,and YC-1 could dose-dependently block this effect.However,neither insulin nor YC-1 altered HIF-1α mRNA levels in PANC-1 cells.Moreover,insulin could enhance the proliferation and invasion of PANC-1 cells,while YC-1 could weaken this effect.It was concluded that the malignant proliferation and local invasion of pancreatic cancer cells may be related to high-insulin microenvironment.The tumor biological behavior change resulting from high-insulin microenvironment may be associated with the increased expression of HIF-1αprotein.

Inhibitory Effect of Phenolicalkaloids of Menispermum Dauricum on the BXPC-3 Tumor and Its Effect on iNOS Content in Tumor-bearing Mice / 中国药房

OBJECTIVE:To observe the inhibitory effect of phenolicalkaloids of menispermum dauricum(PAMD)on the human pancreatic cell line(BXPC-3)tumor and study the effect of PAMD on serum content of inducible nitric oxide synthase(iNOS)in tumor-bearing mice.METHODS:The BXPC-3 tumor-bearing mice model was established,and the model mice were randomly assigned to 6 groups:model control group,blank control group,cyclophosphamide group,3 PAMD groups(high,medium and low dosages).The blank control group was not inoculated with tumor strain but given same volume of normal saline.The tumor-inhibition ratio of PAMD was detected and the content of iNOS of in peripheral blood of tumor-bearing mice was determined by fluorospectrophotometry.RESULTS:PAMD(high,medium and low dosages)showed marked inhibitory effect on tumor growth of BXPC-3 cell line in tumor-bearing mice,with the inhibition ratios at 34.91%,52.83% and 41.51%,respectively.As compared with model control group,PAMD-treated groups showed significantly lower content of iNOS in peripheral blood of tumor-bearing mice.CONCLUSION:PAMD showed marked inhibitory effect on tumor growth of BXPC-3 cell line in tumor-bearing mice,which might be attributed to the lowered content of iNOS.

Construction of eukaryotic expression vector expressing double shRNA sections targeting Survivin gene in Bx-PC3 cells / 中国临床药理学与治疗学

AIM:To construct eukaryotic expression vector expressing double shRNA sections targeting Survivin gene.METHODS: Eukaryotic expression vector expressing double shRNA sections targeting Survivin gene were designed and chemically synthesized.They were directionally inserted into plasmid pGenesil-1 with respectively U6 promoter and termination code,the common green fluorescence protein(EGFP) gene and Neo gene. In this way,the vector of pGenesil-1 shRNA containing 2 sections of Survivin shRNA were constructed and they were transfected into the pancreatic cancer cell Bx-PC3.Transfection was detected by fluorescence microscope.The inhibition expression of Survivin mRNA was measured by RT-PCR.RESULTS: HE1 and HE2 plasmids were identified by the biocatalyst cut which confirmed the exactitude and were analyzed by the sequence analysis which verified the perfect clone plasmid inserted by them.CONCLUSION: A eukaryotic expression vector of double short hairpin RNA for Survivin gene is successfully constructed.The pancreatic cancer cells Bx-PC3 succeed to be transfected and expression of Survivin mRNA is inhibited obviously.

Effect of CD95 system in the pancreatic cancer cells sensitivity to chemotherapy / 中华消化杂志

Objective To explore the role of CD95 system in chemotherapeutic sensitivity of pancreatic cancer cells, in an attempt to enhance the efficacy of chemotherapy by transfection CD95 gene, and to provide the evidence for the immunological treatment of pancreatic cancer. Methods CD95 gene was transfected into the pancreatic cancer cell line SW1990 by lipofectamine. The transfected cells were selected by G418. CD95 expressions of the transfected cells were detected by Northern blot and Western blot. MTT assay was used to analyze the response of the transfected cells to 5 fluorouracil, adriamycin (ADM), gemcitabine and in combination with anti CD95 monoclonal antibody (mAb). The drug induced apoptosis of transfected cells was measured by flow cytometry. Results The transfected pancreatic cancer cell SW1990 could overexpress CD95 stably. The CD95 mRNA and protein expressions were significantly increased in the transfected cells than in the controls. Anti CD95 mAb could inhibit the growth of the transfected cells. In addition, transfected cells were more sensitive to clinically relevant concentrations of chemotherapeutic drugs than non transfected cells. Anti CD95 mAb addition could enhance the cytotoxic effect of chemotherapeutic drugs. Drug induced apoptosis in ADM treated transfected cells more pronounced than in non transfected cells. Conclusions CD95 transfection could increase the sensitivity of pancreatic cancer cell SW1990 to, and partly reverse the resistance to, chemotherapeutic drugs. The combination of chemotherapeutic drugs with anti CD95 mAb showed a synergistic cytotoxicity to pancreatic cancer cells.

The role of pituitary adenylate cyclase activating polypeptide in the growth modulation of human pancreas carcinoma / 中华消化杂志

Objective To investigate the role pituitary adenylate cyclase activating polypeptide (PACAP) in the growth modulation of PACAP of human pancreas carcinoma cells and determine whether sphingomyolin (SM) may act as a second messenger involved in the postreceptor signal transduction. Methods Human pancreas carcinoma cell strains, JF305, HS766T and ASPC 1 cells were cultivated, reproduced and then treated with PACAP 1 38 (10 -12 -10 -6 M). The amounts of proliferated carcinoma cells were estiimated with Mosmann's method (MTT). The concentrations of intracellular SM in cells were determined with thin layer chromotograph. Intracellular adenosine monophosphate and Ca 2+ levels were detected by radioimmunoassay and Fura 2/AM respectively. Results It was found that three kind of human pancreatic cancer cells were proliferated and the intracellular levels of SM, cAMP and cytosolic Ca 2+ were increased by treating PACAP 1 38 . The effect of PACAP 1 38 in JF305, HS766T and ASPC 1 could be inhibited by Somatostatin. Conclusion PACAP 1 38 may play a role in the proliferation of human pancreatic cancer cells. The postreceptorsignal transduction of PACAP may be mediated by both adenosine cyclinase and Calcium calmodin pathways. SM may be a second messenger involved in this process.

THE CYTOTOXIC EFFECTS OF CRUDE BILE ON HUMAN PANCREATIC CANCER CELL LINES / 药物分析学报

Objective To identify effects of bile acids on pancreatic cancer, The ultrastructure and growth of PANC-1 and MIA PaCa-2 cell lines in crude bile modified medium were studied. Methods The growth of PANC-1 and MIA PaCa-2 cells in RPMI 1640 with or without 1％, 2％ and 4％ of the purified crude bile (containing total bile acids 10.17mmol/L) was assessed for 2, 4, 6, 8d by using MTT assay to determine inhibitory rate. The cell surface and intracellular ultrastructure of PANC-1 cells was investigated by SEM and TEM at 24h and 48h, respectively. Re sults The proliferation of both cell lines in bile treated medium were greatly retarded (P ＜0.001). The inhibitory rate of 1％, 2％ and 4％ bile on Panc-1 cells in 4d were 38％, 60％ and 66％, respectively (P ＜0. 05), on MIA PaCa-2 cells at 4d were 28％, 39％ and 52％, respectively (P ＜0. 05). The cells grown in bile for 48h lost their mi crovilli, their mitochondria and other organelles became vacuolated. Conclusion The bile acids in bile has cytotoxicity on PANC-1 and MIAPACA-2 cells, which may inhibit pancreatic cancer progress in patients clinically.