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OBJECTIVE： To study the effects of curcumin on gemcitabine （GEM）-resistant pancreatic cancer SW1990 cells and its mechanism. METHODS： CCK8 assay was used to detect the effects of different concentrations of GEM （50， 100， 150， 200， 250 μmol/L） on the survival rate of SW1990 cells and GEM-resistant SW1990 cells （SW1991/GEM resistant cells）； half inhibitory concentration （IC50） and drug resistance multiple were calculated. CCK8 assay was performed to detect the effects of different concentrations of curcumin （1， 5， 10， 20， 40 μmol/L） on survival rate of SW1990/GEM resistant cells， and IC50 was calculated. CCK8 assay was used to detect the effects of curcumin 2.41 μmol/L combined with different concentrations of GEM （25， 50， 75， 100， 125 μmol/L） on the survival rate of SW1990 cells and SW1990/GEM resistant cells， and IC50 and drug resistance reversal fold of GEM were calculated. Flow cytometry was carried out to detect the cell cycle distribution and apoptosis rate of SW1990 after treated with GEM alone or curcumin （2.41 μmol/L） combined with GEM using IC50 of GEM as drug concentration. Western blot assay was used to the protein expression of FAS， AKT， p-AKT， PI3K， p-PI3K， Caspase-3， Bcl-2 and related X protein （Bax）. RT-PCR was used to detect mRNA expression. RESULTS： IC50 of GEM to SW1990 cells was 92 μmol/L. IC50 of GEM to SW1990/GEM resistant cells was 216 μmol/L， and drug resistance multiple SW1990 cells to GEM was 2.35. IC50 of curcumin to SW1990/GEM resistant cells was 9.2 μmol/L. Under 2.41 μmol/L curcumin， IC50 of GEM to SW1990 cells was 75 μmol/L， and IC50 of GEM to SW1990/GEM resistant cells was 98 μmol/L； drug resistance reversal multiple of SW1990/GEM resistant cells to GEM was 2.2. Compared with GEM alone， the apoptosis rate of SW1990 cells and SW1990/GEM resistant cells were increased significantly after curcumin combined with GEM （P＜0.05）， blocking at G0/G1 phase； the protein expression of FAS， p-AKT， p-PI3K and Bcl-2 and mRNA expression of FAS and Bcl-2 were decreased significantly （P＜0.05）； the protein and mRNA expression of Bax and Caspase-3 were increased significantly （P＜0.05）. CONCLUSIONS： Curcumin can reverse drug resistance of SW1990 cells to GEM， the mechanism of which may be associated with PI3K/AKT pathway.
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Objective:Study on the inhibitory effect of celecoxib on pancreatic cancer SW1990 cells growth.Methods:The experiment was divided into two parts.(1) SW1990 cells were divided into fluorouracil group (50 μ g/mL),celecoxib group(50 μ mol/L),the combined group (celecoxib 50 μ mol/L+fluorouracil 50 μ g/mL),blank group,after 24 h culture of each group using the MTT assay SW1990 cell growth inhibition rate was detected by flow cytometry cell cycle distribution ratio;(2) were used at different concentrations (12.5,25.0,50.0 and 100.0 μ mol/L) of celecoxib,while using different concentrations above celecoxib were combined with 50 μ g/mL of fluorouracil treated SW1990 cells were treated 24 h,using real-time fluorescence polymerase chain reaction(RT-PCR) to detect differential expression Survivn RNA and detect the cyclooxygenase-2 (COX-2) enzyme-linked immunosorbent assay(ELISA),matrix metalloproteinase-14(MMP-14) expression differences.Results:Celecoxib group,fluorouracil group was significantly higher than the rate of the combination group cytostatic blank group (P＜0.05),the inhibition rate of the combination group was significantly higher than the celecoxib group and fluorouracil group (P＜0.05),celecoxib cloth inhibition rate was significantly lower than the fluorouracil group(P＜0.05);significantly higher than the proportion of cells celecoxib group,fluorouracil group,joint group of G0/G1 phase control group (P＜0.05),celecoxib group was significantly higher than the proportion of cells fluorouracil group G0/G1 phase combination group(P＜0.05),significantly higher than the proportion of cells fluorouracil group G0/G1 phase celecoxib group (P＜0.05);with the increasing concentration of celecoxib,celecoxib group,celecoxib+Survivn RNA fluorouracil group,COX-2 and MMP-14 protein levels were decreased and group differences were between statistically significant(P＜0.05),Under the condition of the same concentration of celecoxib and fluorouracil plus celecoxib group RNA survivin,COX-2 and MMP-14 protein expression levels were significantly lower than that of celecoxib group,the difference has statistical significance (P＜0.05).Conclusion:Celecoxib on the growth of pancreatic cancer SW1990 cells was significantly inhibited in a dose dependent manner,and its mechanism may be related to reduced Survivn RNA,COX-2 and MMP-14 protein expression.
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Objective The purpose of this study is to investigate the effect of miR-449a on pancreatic cancer cells and the molecular mechanism. Methods The expression levels of miR-449a in pancreatic cancer cells treated or untreated with radiation was detected by qRT-PCR.High expression of miR-449a was achieved by transfecting miR-449a mimics into SW1990 cells. The cell growth,apoptosis and colony formation ability was assessed by MTT assay,flow cytometry and colony formation assay,respectively. The relationship of miR-449a and Cyclin D1 was determined by the TargetScan and dual luciferase reporter. Immunohistochemistry was used to examine protein levels of Cyclin D1 in pancreatic cancer and normal pancreas tissues. Si-Cyclin D1 was used to detecte the effect of Cyclin D1 on radiosensitivity of pancreatic cancer cells. Results The expression levels of miR-449a in pancreatic cancer cells with radiation treatment were decreased significantly. Mir-449a mimics increased the cell proliferation rates and apoptosis rates obviously,and decreased the colony formation ability in SW1990 cells treated with radiation. Results from the TargetScan and dual luciferase reporter showed that Cyclin D1 was the target of miR-449a. The positive staining rates of Cyclin D1 in pancreatic cancer tissue(85.7%,30/35)was higher than those in normal pancreas tissue(20%,2/10).Knockdown of Cyclin D1 enhanced the radiosensitivity of pancreatic cancer cells.Conclusion MiR-449a enhances the radiosensitivity of pancreatic cancer cells by targeting Cyclin D1.
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Objective To study the effect of small interfering RNA targeted on Apollon for proliferation on pancreatic cancer cells and its possible acting mechanism.Methods The small interfering RNA targeted on apollon in our previous study was transfected to the cells using LipofectamineTM 2000,after 48 hours transtection.The inhibitory effects of small interfering RNA targeted on Apollon (Apollon siRNA)on cell proliferation were detected by WST-8.Their inhibition rate and IC50 were calculated.The percentage of apoptosis cells were determined by flow cytometry.The expression of Apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction.The Apollon protein ex-pression levels were detected by western blotting.Results Apollon siRNA could effectively inhibit the proliferation of pan-creatic cancer cell.The amount of apoptotic cells increased significantly.The early apoptotic rate was 37.1%,and the RT-PCR results showed that the relative expression levels of Apollon mRNA were down-regulate,and shows a dose-effective-ness relations.The protein expression levels were decreased by Apollon siRNA.Conclusion Apollon siRNA can effectively inhibit the proliferation of pancreatic cancer cell.The mechanism may be work together to promote pancreative cancer cell early apoptosis and decreased the expression levels of gene and protein,which provides a novel potential approach for treat-ment of target therapy of pancreatic cancer.
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Objective To construct pEGFP-C1-HSP27 recombinant eukaryotic expression vector and establish human pancreatic cancer SW1990 cell line stably expressing HSP27.Methods RT-PCR was applied to amplify human HSP27 cDNA from human pancreatic cancer SW1990 cells with a pair of specific primers carrying a restriction enzyme site BamH Ⅰ or Hind Ⅲ on each 5' end.HSP27 cDNA was inserted into pEGFP-C1 vector and then identified by restriction enzyme digestion and sequencing.Successful constructed pEGFP-C1-HSP27 or empty vector was transfected into SW1990 cells by lipofectamine 2000,respectively.The location of HSP72 was determined by fluoroscopy,RT-PCR and Western blot was used to detect the expression of HSP27 in transfected cell.Results The DNA sequence of pEGFP-C1-HSP27 recombinant plasmid was completely correct,and it was successfully transfected into SW1990 cell lines and stably transfected SW1990 cell lines were obtained,which were confirmed by restriction enzyme and sequencing.The expression of EGFP was distributed in cytoplasm,the HSP27mRNA expression was significantly increased (1.458 ± 0.160vs0.897 ±0.051,P ＜0.05).In addition,it was showed that EGFP-HSP27 fusion protein was expressed.Conclusions The eukaryotic expression vector pEGFP-C1-HSP27 was constructed successfully and stably transfected SW1990 cell line expressing HSP27 was obtained.
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Objective To study the influence of the small interfering RNA (siRNA) interference TMPRSS4 expression on human pancreatic cancer SW1990 cell's proliferation and invasion. Methods The four eukaryotic expression vector of TMPRSS4 gene were synthesized in vitro and were transfected transiently into human pancreatic cancer SW1990 cells. TMPRSS4 mRNA expression of transfected cells was detected by real-time RT-PCR. The most efficient eukaryotic expression vector was used to be transfected into SW1990 cells. By using G418, cell strain that can silence TMPRSS4 gene stably was screened. The TMPRSS4 mRNA expression of the stable cell strain was detected by real time PCR TMPRSS4 protein expression was detected by western blot. The proliferation ability of transfected SW1990 cells was detected by CCK-8 method. By Transwell, the invasion change of SW1990 cell was detected. Results A stable cell strain, SW1990/psi TMPRSS4, was successfully constructed, in which the expression level of TMPRSS4 could be reduced stably by RNA interference. Cell transfection efficiency was 82.9%. Compared with the control group, the TMPRSS4 mRNA and protein levels were reduced by 80.1% and 60% ,and number of penetrating cells was 118.6 ±13.4 in SW1990/psi TMPRSS4 group, which was significantly lower than those in the negative control group (157.4 ± 12.9) and control group (157.0±9.5, P <0.01). Cells invasion inhibitory rate was 24.5% in SW1990/psi TMPRSS4 group. The cell proliferation was not significantly different among all the groups. Conclusions A stable cell strain is screened successfully in which the expression level of TMPRSS4 can be reduced stably. The down-regulation of TMPRSS4 gene expression level can inhibit the invasion of SW1990 cells, but has no effect on cell proliferation.
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Objective: In order to investigate the effects of activating and blocking Stat3 signaling pathway on invasion ability of human pancreatic cancer cells and explore its action mechanism.Methods:Human pancreatic cancer Capan-2 cells were treated with IL-6. SW1990 human pancreatic cancer cells were treated with AG490. Cell proliferation was measured by MTT assay. Western blotting and immunocytochemistry were performed to detect expression of phosphorylated Stat3 (p-Stat3) protein. Real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blotting were used to detect the mRNA and protein expression of VEGF and MMP-2 mRNA, respectively. The invasion abilities of SW1990 and Capan-2 cells were determined by cell invasion assay in vitro. Results:IL-6 stimulated the proliferation of Capan-2 cells (P<0.05), elevated the expression of p-Stat3, increased the mRNA and protein expressions of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 (MMP-2) (P<0.05), and enhanced the invasion ability of Capan-2 cells. AG490 inhibited the proliferation of SW1990 cells (P<0.05), down-regulated the expression of p-Stat3, markedly decreased the mRNA and protein expression of VEGF and MMP-2 (P<0.05), and weakened the invasion ability of SW1990 cells. Conclusion:Stat 3 signaling pathway plays an important role in the invasion and metastasis of pancreatic cancer. Stat 3 signaling transduction pathway may provide a novel therapeutic target for the treatment of pancreatic cancer.
