| Part I Inhibitory Effect of Celastrus Orbiculatus Extract on the Proliferation and Stemness of Gastric Cancer Stem CellsObjectives: To construct a gastric cancer stem cell model by serum-free culture to observe the effect of COE on the proliferation and stemness maintenance of gastric cancer stem cells,and make a preliminary investigation on the related mechanism.Methods: Gastric cancer stem cells were isolated from SGC-7901 gastric cancer cell line by in vitro serum-free culture and the protein expressions of stem cell markers（SOX-2,Nanog,Oct-4）,PDCD4 and EIF3 H were detected by Western blot assay;the percentage of CD44+/CD24+ and ALDH+ cell population were examined by flow cytometry assay to further examine the establishment of gastric cancer stem cell model.Then,gastric cancer stem cells were treated with different concentrations（0 20 40 80 μg/m L）of COE.The spheroid-forming ability in each group was observed;proportion of CD24+/CD44+ and ALDH+ cell population was detected by flow cytometry assay;protein expression of Nanog,SOX-2,Oct-4,PDCD4,and EIF3 H was examined by Western blot assay.Results: Compared with parental cells,SGC-7901 cells after in vitro serum-free culture showed significantly enhanced spheroid-forming ability,increased percentage of CD24+/CD44+ and ALDH+ cells（P<0.05）,and upregulated expression of Nanog SOX-2,Oct-4,indicating the successful establishment of gastric cancer stem cell model.In addition,PDCD4 expression was significantly reduced and EIF3 H expression was significantly enhanced in spheroid-forming cells,suggesting that PDCD4 and EIF3 H may be associated with stem cell phenotype acquisition in gastric cancer cells.COE treatment attenuated the spheroid-forming ability,reduced the percentage of CD24+/CD44+ and ALDH+ cells,down-regulated the expression of SOX-2、Oct-4、Nanog and EIF3 H proteins,and up-regulated the expression of PDCD4 in a dosedependent manner.Conclusion: COE could inhibit the proliferation and maintenance of gastric cancer stem cells,and the mechanism may be related to the regulation of PDCD4 and EIF3 H expression by COE.Part II Role of PDCD4 in the Inhibitory Effect of Celastrus Orbiculatus Extract on the Growth of Gastric Cancer and Epithelial-mesenchymal TransitionObjectives: To investigate the impact of COE on the growth of transplanted tumors of gastric cancer in nude mice and the modulation of EMT of transplanted tumor cells,and to investigate the role of PDCD4 in this process.Methods: PDCD4 overexpression SGC-7901 gastric cancer cell line（SGC-7901-pc DNA3.1-PDCD4）and empty plasmid control（SGC-7901-pc DNA3.1）were constructed by plasmid（pc DNA3.1-PDCD4）transfection and G418 screening.Gastric cancer nude mice subcutaneous transplantation tumor model was constructed by injecting SGC-7901-pc DNA3.1-PDCD4 or SGC-7901-pc DNA3.1 gastric cancer cells into the subcutaneous skin of nude mice and were randomly divided into: control group,pc DNA3.1 empty plasmid group,pc DNA3.1-PDCD4 group,COE group,pcDNA3.1+COE group,pcDNA3.1-PDCD4+COE group.Mice in the treatment and control groups were given COE（40 mg/kg）or saline（containing 1% DMSO）by gavage,respectively.During the treatment period,the size of the transplanted tumors was calculated every 2 days for each group and the growth curves of the transplanted tumors were drawn.At the completion of treatment,nude mice were sacrificed,the transplanted tumors were excised,and the tumor suppression rate was calculated.The expression of PDCD4,E-cadherin,N-cadherin,and Vimentin in transplanted tumor tissues was detected by immunohistochemistry.Results: Compared with control group,the growths of transplanted tumors in the COE and pc DNA3.1+COE groups were significantly suppressed,with tumor suppression rates of 57.52% and 58.41%,respectively.（P<0.05）;the expression of E-cadherin and PDCD4 was upregulated and the expression of N-cadherin and Vimentin was downregulated in transplanted tumor tissue.Compared with the control group,tumor growth was not affected in the pc DNA3.1 empty plasmid group,and the expression levels of PDCD4 and EMT-related proteins were not significantly changed（P>0.05）,suggesting that COE could inhibit the growth of transplanted tumors and the development of EMT in gastric cancer cells,and upregulate PDCD4 expression.Compared with the pc DNA3.1 null plasmid group,the growth of transplanted tumors was significantly inhibited in the pc DNA3.1-PDCD4 group（tumor suppression rate: 59.29%）（P<0.05）,and E-cadherin expression was dramatically increased and Ncadherin and Vimentin expression was remarkably reduced（P<0.05）,indicating that PDCD4 could inhibit gastric cancer growth and the EMT of gastric cancer cells.The inhibitory effects of pc DNA3.1-PDCD4+COE group on the growth of transplanted tumors were significantly increased compared with the pc DNA3.1+COE group and pc DNA3.1-PDCD4 group,with a tumor suppression rate of 71.68%.（P<0.05）.The upregulation of E-cadherin and PDCD4,and downregulation of N-cadherin and Vimentin was also more significant（P<0.05）.Conclusion: COE may inhibit the growth of gastric cancer and the occurrence of EMT in gastric cancer cells by upregulating PDCD4 expression. |
PDF Full Text Request