| BackgroundsEsophageal carcinoma is one of the most common solid tumors in China,with high incidence and poor prognosis.Henan province is the characteristic high-risk area of esophageal cancer.Despite efforts for many years,the incidence of esophageal cancer has declined to some extent,but the recurrence and metastasis rate of esophageal cancer is still high?34%to 79%?,and the five-year survival rate is low?20-30%?.At present,comprehensive treatment such as radiotherapy combined with chemotherapy is used to treat the disease,but its recurrence rate and metastasis rate remain high,which is one of the important factors that threaten the survival of patients with esophageal cancer.Recurrence and metastasis are the most important biological characteristics of malignant tumors,which is the important cause of patients'treatment failure and death.Studies have shown that the invasion and metastasis of malignant tumors are mainly related to the formation of angiogenesis and lymphatic vessels.Notch signaling pathway is a highly conservative signaling pathway,which plays a role in stem cell differentiation and self-renewal in embryos and somatic cells and regulates multiple aspects of angiogenesis.In recent years,Dll4-Notch signaling pathway and multiple signaling pathways have been found to interact with each other to regulate the neovascularization,which is a new target of tumor anti-angiogenesis.Vascular endothelial growth factor-C?VEGF-C?is the first discovered lymphangiogenic factor and stimulates the renewal of lymphatic vessels by binding to the corresponding receptors,thereby promoting the diffusion and metastasis of tumor cells throughout the body.Studies have shown that VEGF-C play an important role in lymphangiogenesis.All-trans retinoic acid?ATRA?is a natural vitamin derivative,which is involved in embryonic development,cell growth and differentiation;it also plays a role in tumor cell proliferation apoptosis and angiogenesis,especially in the treatment of acute promyelocytic leukemia.Previous studies have confirmed that ATRA has a role in promoting differentiation and inducing apoptosis in a variety of solid tumor cells,including esophageal cancer,and may inhibit tumor angiogenesis by regulating multiple signaling pathways.Studies have also shown that retinoic acid interacts with the Notch pathway in skin and neural stem cells,but there is little research on the effect of ATRA on Notch signaling and lymphangiogenesis-related factors in esophageal cancer.In this experiment,the effect of ATRA on the expression of Notch1,Dll4,and VEGF-C in esophageal cancer cell line KYSE70 through retinoic acid receptor??RAR-??was observed by in vitro experiments,laying a theoretical foundation for ATRA's treatment of angiogenesis and lymphangiogenesis of esophageal cancer.ObjectiveTo investigate the effect of all-trans retinoic acid alone or/and combined with RAR-?agonists or inhibitors on the proliferation and migration of esophageal cancer KYSE70 cell lines,and on the regulation to the expression of Notch1,Dll4,and VEGF-C proteins.To explore the possible mechanism of ATRA on anti-microvascular and lymphangiogenesis in esophageal cancer.MethodsThe KYSE70 cells in logarithmic growth phase were randomly divided into 4groups:ATRA group,ATRA+CD2314 group,ATRA+LE135 group and control group.CCK-8 assay was used to determine the proliferation inhibition and cell activity changes in cells treated with different drugs.The migration ability of cells in each group was determined by Wound healing assay.Western blot was used to detect the expression of RAR-?,Notch1,Dll4 and VEGF-C proteins in each group.Results1.Western blot results:KYSE70 cells were cultured in 10?M ATRA plus 1?M LE135,10?M ATRA,10?M ATRA plus 1?M CD2314,and media alone for 48 h,respectively.The relative expression of RAR-?protein in each group was?11.33±0.99?×10-2,?28.32±0.93?×10-2 and?33.70±0.77?×10-2,and the control group was?20.00±0.99?×10-2.After statistical analysis,compared with the control group,the expression of RAR-?protein in each experimental group were changed,and all had statistical significance?p<0.05?;the differences were also has statistical significance between the experimental groups?p<0.05?.2.CCK-8 results:To investigate the effect of ATRA and its receptor RAR-?on the proliferation of KYSE70 cells,we employed the CCK-8 to detect the cell proliferation of KYSE70 cells.The cells were treated with 10?M ATRA,10?M ATRA combined with 1?M CD2314,10?M ATRA in combination with 1?M LE135or untreated medium,and cultured for 24 h,48 h and 72 h,respectively.Results display cells incubated in 10?M ATRA decreased by 29.04%,40.29%and 51.5%at24 h,48 h and 72 h,relative to untreated cells,respectively?p<0.05;Fig.2B?.Cells incubated at 10?M ATRA combined with 1?M CD2314 showed a decrease of 36.7%,50.1%and 62.1%at 24 h,48 h and 72 h,relative to untreated cells,respectively?p<0.05;Fig.2B?.Cells cultured in 10?M ATRA in combination with 1?M LE135showed 24.39%,30.83%,40.46%reduction compared to untreated cells at 24 h,48 h,72 h?p<0.05;Fig.2B?.Compared with incubated in ATRA,the proliferation of cells incubated in ATRA and CD2314 were significantly suppressed?p<0.05;Fig.2A?.The proliferation of cells incubated in ATRA plus LE135 was obviously better than that of cells incubated with ATRA?p<0.05;Fig.2A?.3.Wound healing assay:The effect of ATRA and RAR-?on the migration of KYSE70 cells was determined by the wound healing assay.The photographs of the cultures were taken immediately at 0 h and 24 h after wound formation,respectively?Fig.3A?.Cells migration behavior was compared by selecting the corresponding 10points at the edge of the cells,measuring the distance and calculating the mobility?p<0.05;Fig.3B?.Compared with untreated,the scratch distance of cells in 10?M ATRA was decreased to 47.1%,the scratch distance of cells in 10?M ATRA plus 1?M CD2314 was decreased to 32.5%,and in cells treated with 10?m ATRA plus1?M LE135,the scratch distances were reduced to 60.1%?p<0.0001;Fig.3C?.Activation of RAR-?widens the gap between cells scratches wounds.4.Western blot results:To investigate the effects of ATRA and RAR-?on the Notch signaling pathway in esophageal cancer KYSE70 cells,the expression of Notch1,Dll4,and VEGF-C proteins were evaluated by western blot?Fig 4A?.After48 h of KYSE70 cells treated with ATRA+LE135,ATRA and ATRA+CD2314 in each experimental group,the relative expression levels of Notch1 were?52.89±1.24?×10-2,?108.31±1.77?×10-2 and?117.60±3.02?×10-2,respectively,and the control group was?81.36±0.77?×10-2.The relative expression levels of Dll4 protein were:?47.04±1.44?×10-2,?30.59±0.52?×10-2 and?26.51±2.37?×10-2,and the control group was?50.37±1.32?×10-2.The relative expression levels of VEGF-C were?93.12±2.32?×10-2,?53.97±2.09?×10-2 and?26.90±2.06?×10-2,respectively,and the control group was?117.28±3.17?×10-2.Statistically,there was a significant difference between the experimental group and the control group?p<0.05?,there were also significant differences between experimental groups?p<0.05?.Conclusion1.The increase of RAR-?expression not only enhanced the inhibitory effect of ATRA on the proliferation and migration of esophageal cancer KYSE70 cells,but also enhanced the regulation of Notch1 protein up-regulation and down-regulation of Dll4 and VEGF-C protein expression by ATRA.2.ATRA enhances the expression of Notch1 in esophageal cancer cells,inhibits the expression of Dll4 and VEGF-C,which may affect the Dll4-Notch1 signaling pathway and interfere with lymphangiogenesis,thereby inhibiting the formation of tumor microvessels and lymphatic vessels. |
PDF Full Text Request