| Background:Gastric cancer is a common general digestive tumor in our country. Most of the patients are diagnosed in advanced stages and lose the opportunity of surgery treatment. Thus, gastric cancer chemotherapy has attracted more attention than in previous years. However, the result of chemotherapy is not satisfactory. It is realized that the failure of chemotherapy could be attributed to chemoresistance. Chemoresistance of gastric cancer cells may involve a series of complex mechanism. But in recent years a growing number of researches have shown that the singnaling pathway activation (inactivation), which is responsible for the growth and apoptosis of cells is an important aspect of chemotherapy resistance in tumor cells. These signaling pathways through post-transcriptional regulation mechanisms are widely involved in the cell’s life activities, which relates to the resistance of tumor cells. Professor Yu preliminary study confirmed that the phosphoinositide3-kinase/AKT (PI3K/AKT) pathway played an important role in chemoresistance of gastric cancer cells. PTEN gene can weaken the chemoresistance behavior of gastric cancer cells, but the specific mechanism remains to be further studied. Gastric cancer as a malignant tumor is relatively sensitive to chemotherapy, and reverse its resistance to chemotherapeutic drugs has great significance. Thus, in-depth study of the regulation mechanisms of intracellular signaling pathways has great significance on definitude the resistant molecular mechanisms of gastric cancer, and also provide a theoretical basis for the new method of treatment for cancer.Part one:The role of AKT1gene in doxorubicin resistance of gastric cancer cells and relationships to Notch-1and PTEN geneObjectives:To investigate the expression and function of AKT1, Notch-1,and PTEN in gastric cancer cells induced by doxorubicin, and explore AKT1in gastric cancer cells resistance to doxorubicin.Methods:1.We first observed the AKT1, Notch-1and PTEN expression changes induced by doxorubicin at different times in MKN28, MKN45BGC823, and SGC7901cell lines by immunoblotting (Western blot) and real-time fluorescence quantitative polymerase chain reaction (Real-time PCR).2.AKT1RNAi lentiviral vector (AKT1 shRNA) and active AKT1vector were constructed, and the recombinant vectors were transfected into gastric cancer cells (MKN28). Next, the effects of AKT1on the apoptosis of MKN28cells in the presence or absence of doxorubicin (3μM) were investigated. And the Notch-1and PTEN expression were also detected.3. Electrophoretic mobility shift assay (EMSA) was used to detect general NFκB and CBF-1DNA binding activity.Results:1. Doxorubicin was time gradient increased mRNA and protein expression levels of AKT1, Notch-1and PTEN.2. The chemosensitivity of MKN28cells to doxorubicin increased significantly following the downregulation of AKT1expression (P<0.01), which associated with the inactivation of the PI3K/AKT1signaling pathway, followed by the reduced expression of Notch-1and PTEN expression. Overexpression of active AKT1increased Notch-1and PTEN expression, while PDTC (pyrrolidine dithiocarbam-ate, a specific inhibitor of NFκB activity,10μM) could inhibit the increase.3. Knockdown of AKT1expression inhibited general NFκB DNA binding activity, which were increased by doxorubicin.Conclusion:1. AKT1, Notch1, and PTEN were obviously increased in parallel after doxorubicin treatment.2. Downregulation of AKT1reduced chemotherapy tolerance of gastric cancer cells to doxorubicin treatment, with Notch-1and PTEN expression decreased.3. AKT1may be the upstream of Notch-1and PTEN gene, AKT1may affect Notch-1and PTEN gene expression through NFκB pathway.Part two:The possible mechanism of AKT1regulation of Notch-1gene transcription induced by doxorubicinObjectives:The aim of the study was to investigate the specific mechanisms of Notch-1gene transcription.Methods:1.Different length Notch-1promoter luciferase reporter constructs were generated. MKN28cells were infected with the recombinant reporter constructs using Lipofectamine2000, and Dual Luciferase Reporter Gene Assay Kit was used to assess Notch-1promoter activity in different condition.2. Specificity of NFκB (containing the sequence of the binding sites) and mutant NFκB probe activity were detected in MKN28cells by EMSA.3. Further application of Chromatin Immunoprecipitation (ChIP) to observe the combination between NFκB (p65) protein and Notch-1promoter sequence.Results:1. Knockdown of AKT1expression and PDTC1(10μM) inhibited Notch-1luciferase reporter gene activity induced by doxorubicin.2. NFκB effectively bound to oligonucleotides containing wild type NFκB binding sites but not to those with mutations in Notch-1promoter region of MKN28cells.3. NFκB subunit p65may be a transcription factor for the transcription of Notch-1.Conclusion:Doxorubicin increased AKT1and Notch-1expression, and doxorubicin-induced AKT1activation promoted the transcription and expression of Notchl through binding of NFκB on Notchl promoter region.Part three:The role and mechanism of Notch-1and PTEN in the doxorubicin chemoresistance of gastric cancer cellsObjectives:To investigate expression and function of Notch-1and PTEN in gastric cancer tissues and cells, study the regulatory mechanism of PTEN expression and explore the specific role in the resistance to doxorubicin in gastric cancer.Methods:1.Forty four adult patients with gastric cancer were collected for the study. Observe the expression of Notch-1and PTEN in gastric cancer tissues.2. PTEN luciferase reporter gene construction was used to detected PTEN promoter activity under different conditions.3. RNAi interference technology was uesd to downregulation Notch-1expression, after transfection, the effects of Notch-1downregulation on transcription factor CBF-1activity in the presence or absence of doxorubicin were investigated by EMSA, on PTEN expression were investigated by Western blot, on apoptosis ratio were by Annexin V-FITC/PI staining.4. After transfection PTEN plasmid vector and PTEN siRNA vector, the effects of PTEN on the apoptosis and protein levels of MKN28cells in the presence or absence of doxorubicin were detected by flow cytometric analysis of annexin V-FITC/PI staining, and Western blot.Results:1. Strong expression of Notchl and PTEN were visible in the cytoplasm nornal gastric mucosal cells, whereas no or very weak expression of Notchl and PTEN could be observed in foci of gastric cancer. Statistical analysis demonstrated the Notchl expression correlated with that of PTEN in gastric cancer tissues and the paired normal mucosa. The correlation between the Notch-1expression and PTEN expression was strongly and positively (r=0.866).2. The PTEN reporter vector containing the sequence of CBF-1binding sites, Lentiviral AKT1shRNA, PDTC (10μM) and Notch-1siRNA reduced the PTEN promoter activity (P<0.05), but increased PTEN promoter activity induced by doxorubicin.3. Knockdown of Notch-1expression decreased CBF-1binding activity, inhibitted PTEN expression, and reduce doxorubicin-induced apoptosis rate (P<0.05).4. Overexpression of PTEN decreased AKT phosphorylation levels, but improve doxorubicin-induced apoptosis rate (P<0.05). however, the effects of knockdown of PTEN expression was just opposite. There were no significant effect on AKT1gene expression in two conditions.Conclusion:1. Notch-1and PTEN expression was significantly decreased in the gastric tissues.2.Notch-1gene played an important role in gastric cancer cell resistance to doxorubicin, through mediation of Notch1downstream target CBF-1on suppressor gene PTEN promoter. |
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