Objective Cytotoxin-associated protein （CagA） of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we...Objective Cytotoxin-associated protein （CagA） of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA＋ strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21WAF1/CIP1 and p27KIP1, and also in releasing IL-8 in host cells. Methods Akt1 phosphorylation and IL-8 levels of CagA＋ and CagAˉ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21WAF1/CIP1 and p27KIP1 mRNA levels in the CagA＋ and CagAˉ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release. Results CagA＋ strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagAˉ strain failed to do so. The CagA＋ H. pylori strain infected AGS cells showed significant drops both in p21WAF1/CIP1 and p27KIP1 mRNA levels, whereas the CagAˉ H. pylori strain caused a remarkable increase in p21WAF1/CIP1 mRNA without affecting p27KIP1 gene transcription in the AGS cells. Both the CagA＋ and CagAˉ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels. Conclusion CagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21^WAF1/CIP1 and p27^KIP1 expression might be involved in CagA-associated carcinogenesis.展开更多
AIM:To investigate the effects of macrophage migration inhibitory factor (MIF) on proliferation of human gastric cancer MGC-803 cells and expression of cyclin D1 and p27Kip1 in them,and further determine whether the e...AIM:To investigate the effects of macrophage migration inhibitory factor (MIF) on proliferation of human gastric cancer MGC-803 cells and expression of cyclin D1 and p27Kip1 in them,and further determine whether the effects are related to the PI3K/Akt signal transduction pathway. METHODS:Gastric cancer MGC-803 cells were cultured and then treated with 50 μg/L recombinant human MIF (rhMIF) with and without a PI3K inhibitor,LY294002 (25 μmol/L). MTT assay was used to detect the prolifer-ation of MGC-803 cells. Cell cycle was detected by flow cytometry. Expression of cyclin D1 and p27Kip1 mRNA was by reverse transcription-polymerase chain reaction. Protein expression of phosphorylated Akt (p-Akt),Akt,cyclin D1 and p27Kip1 was examined by immunocyto-chemistry and Western blotting. RESULTS:rhMIF signifi cantly stimulated the prolifera-tion of MGC-803 cells and cell cycle progression from G1 phase to S phase in a concentration-and time-de-pendent manner. After the MGC-803 cells were treated with rhMIF for 24 h,the expression of cyclin D1 was signifi cantly up-regulated compared with the cells not treated with rhMIF at both mRNA and protein levels(0.97 ± 0.02 vs 0.74 ± 0.01,P = 0.002; 0.98 ± 0.05 vs 0.69 ± 0.04,P = 0.003). The p27Kip1 was down-regulated but only statistically significant at the protein level. rhMIF significantly increased the expression of p-Akt,which reached the peak at 30 min,but did not affect the expression of Akt. However,LY294002 inhibited all the effects of rhMIF.CONCLUSION:Macrophage MIF increases the proliferation of gastric cancer cells,induces the expression of cyclin D1 at the transcriptional level and inhibits the expression of p27Kip1 at the post-transcriptional level via the PI3K/Akt pathway.展开更多
AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27...AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27kip1in patients with extrahepatic cholangiocarcinoma.METHODS:Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December1994 to March 2008 were enrolled.Immunohistochemical staining for Skp2,Cks1,p27kip1,and Ki67,along with other relevant molecular biologic experiments,were performed.RESULTS:By Cox regression analyses,advanced age(>65 years),advanced AJCC tumor stage,poorly differentiated histology,and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma.Exogenous epidermal growth factor(EGF,especially 0.1-10 ng/mL)significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1in SNU-1196,SNU-1079,and SNU-245 cells.The protein levels of Skp2/Cks1(from nuclear lysates)and p27kip1(from cytosolic lysate)were also significantly increased in these cells.There were significant reductions in the protein levels of Skp2/Cks1and p27kip1(from nuclear lysate)after the treatment of LY294002.By chromatin immunoprecipitation assay,we found that E2F1 transcription factor directly binds to the promoter site of Skp2.CONCLUSION:Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma.EGF upregulates the mRNA and protein levels of Skp2/Cks1and p27kip1via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.展开更多
基金supported by a grant (2008ZZ06) from the National Key Laboratory for Diagnosis and Treatment of Infectious Diseases of China
文摘Objective Cytotoxin-associated protein （CagA） of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA＋ strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21WAF1/CIP1 and p27KIP1, and also in releasing IL-8 in host cells. Methods Akt1 phosphorylation and IL-8 levels of CagA＋ and CagAˉ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21WAF1/CIP1 and p27KIP1 mRNA levels in the CagA＋ and CagAˉ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release. Results CagA＋ strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagAˉ strain failed to do so. The CagA＋ H. pylori strain infected AGS cells showed significant drops both in p21WAF1/CIP1 and p27KIP1 mRNA levels, whereas the CagAˉ H. pylori strain caused a remarkable increase in p21WAF1/CIP1 mRNA without affecting p27KIP1 gene transcription in the AGS cells. Both the CagA＋ and CagAˉ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels. Conclusion CagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21^WAF1/CIP1 and p27^KIP1 expression might be involved in CagA-associated carcinogenesis.
基金Supported by Grant from Hunan Provincial Science and Technology Department (2008 FJ 3088), China
文摘AIM:To investigate the effects of macrophage migration inhibitory factor (MIF) on proliferation of human gastric cancer MGC-803 cells and expression of cyclin D1 and p27Kip1 in them,and further determine whether the effects are related to the PI3K/Akt signal transduction pathway. METHODS:Gastric cancer MGC-803 cells were cultured and then treated with 50 μg/L recombinant human MIF (rhMIF) with and without a PI3K inhibitor,LY294002 (25 μmol/L). MTT assay was used to detect the prolifer-ation of MGC-803 cells. Cell cycle was detected by flow cytometry. Expression of cyclin D1 and p27Kip1 mRNA was by reverse transcription-polymerase chain reaction. Protein expression of phosphorylated Akt (p-Akt),Akt,cyclin D1 and p27Kip1 was examined by immunocyto-chemistry and Western blotting. RESULTS:rhMIF signifi cantly stimulated the prolifera-tion of MGC-803 cells and cell cycle progression from G1 phase to S phase in a concentration-and time-de-pendent manner. After the MGC-803 cells were treated with rhMIF for 24 h,the expression of cyclin D1 was signifi cantly up-regulated compared with the cells not treated with rhMIF at both mRNA and protein levels(0.97 ± 0.02 vs 0.74 ± 0.01,P = 0.002; 0.98 ± 0.05 vs 0.69 ± 0.04,P = 0.003). The p27Kip1 was down-regulated but only statistically significant at the protein level. rhMIF significantly increased the expression of p-Akt,which reached the peak at 30 min,but did not affect the expression of Akt. However,LY294002 inhibited all the effects of rhMIF.CONCLUSION:Macrophage MIF increases the proliferation of gastric cancer cells,induces the expression of cyclin D1 at the transcriptional level and inhibits the expression of p27Kip1 at the post-transcriptional level via the PI3K/Akt pathway.
基金Supported by A grant from Samsung Biomedical Research Institute,No.C-A9-210-1
文摘AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27kip1in patients with extrahepatic cholangiocarcinoma.METHODS:Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December1994 to March 2008 were enrolled.Immunohistochemical staining for Skp2,Cks1,p27kip1,and Ki67,along with other relevant molecular biologic experiments,were performed.RESULTS:By Cox regression analyses,advanced age(>65 years),advanced AJCC tumor stage,poorly differentiated histology,and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma.Exogenous epidermal growth factor(EGF,especially 0.1-10 ng/mL)significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1in SNU-1196,SNU-1079,and SNU-245 cells.The protein levels of Skp2/Cks1(from nuclear lysates)and p27kip1(from cytosolic lysate)were also significantly increased in these cells.There were significant reductions in the protein levels of Skp2/Cks1and p27kip1(from nuclear lysate)after the treatment of LY294002.By chromatin immunoprecipitation assay,we found that E2F1 transcription factor directly binds to the promoter site of Skp2.CONCLUSION:Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma.EGF upregulates the mRNA and protein levels of Skp2/Cks1and p27kip1via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.
