| Background and objective:Lung cancer is the most important cause of tumor death.Due to late discovery,high malignancy and easily metastasis and recurrence,the prognosis of lung cancer is poor with the 5 year survival rate lower than 18%.Traditional treatment include surgery,radiotherapy and chemotherapy,but these treatments are often aimed at lung cancer cells directly.invasion and metastasis is an important cause of treatment failure and poor prognosis,which is closely related to the tumor microenvironment,tumor microenvironment is the internal environment of the tumor during development process,composed of mesenchymal cells,immune cells,microvascular,lymphatic vessels,tissue fluid and their secretion.Tumor angiogenesis is an important symbol of tumor microenvironment.Tumor angiogenesis provides oxygen and nutrients for the continuous growth and development of tumors,carries out metabolic wastes of microenvironment,forms the vascular structure which supports and promotes tumor proliferation and metastasis.PD 0332991 is a cell cycle inhibitor which can phosphorylate CDK4/6 exclusively.in a mouse xenograft model,the tumor angiogenesis of PD 0332991 treated mice was significantly reduced,vascular density was significantly lower than the control group,but the inner mechanism is unknown.At the same time we found that PD 0332991sensitizes lung cancer cells to treatment with epidermal growth factor receptor tyrosine kinase inhibitors,but the sample size was small,(only PC-9),so the purpose of this study is to investigate the effect of PD 0332991 on endothelial cells and the inner molecular mechanism,and further,we intent to use PD combined with gefitinib in several lung cancer cell lines,and discuss its relevant mechanism.Methods:1.EA.hy926,a vascular endothelial cell was used as the research model.MTT method and EdU method was used to detect the effect of PD 0332991 on the activity and proliferation ability of EA.hy926 cell;scratch test and Transwell assay was used to detect the effect of PD 0332991 on the migration ability of endothelial cell;flow cytometry was used to detect the effect of PD 0332991 on the cell cycle of endothelial cells and on the apoptosis;Western blot was used to detect the expression of cell cycle related proteins.2.MTT method was used to detect the sensitivity of 16lung cancer cell lines to gefitinib,PD 0332991,and gefitinib plus PD 0332991.The Combination Index of 16 lung cancer cell lines was determined by Chou-Talalay method,and the expression of cyclin related proteins in 16 lung cancer cell lines was detected by Western blot.Results:1.(1)PD 0332991 significantly inhibited the proliferation and activity of EA.hy926.MTT assay showed that EA.hy926 treated by PD 0332991,the IC50 of24h 48h and 72h were 10.279±2.971 and 4.241±0.292 mol/L mol/L and 2.574±0.692 mol/L.EA.hy926 cells decreased with the increase of drug concentration and time gradient.EdU results showed that the proportion of positive cells in NC group was 77.06+9.19(N=3),and the proportion of positive cells in PD treated group was2.02+1.93(N=3),P<0.0002.(2)PD 0332991 inhibited the migration of endothelial cells.Cell scratch experiments show that after 56 hours the control group cells were covered with scratch clearance,PD treatment groups were not covered,the PD0332991(6μM and 12μM)of different concentrations were treated for 6,18,32hours.The migration rate of EA.hy926 cells in PD 0332991 treatment group was significantly lower than that of the control group(P<0.05),and the cell migration rate decreased with the increase of the concentration of PD 0332991.(3)PD 0332991resulted in Cell cycle arrest in G1 phase and induces apoptosis.after 48h treatment,compared with control group(NC),total apoptosis rate of PD=4μM group increased significantly,20.32±1.23 Vs 2.79±0.1(P<0.001);early and late apoptosis were11.95±0.63,8.37±0.59,compared with the NC group of 1.89±0.07 and 0.89±0.03(P<0.001);PD=2μM,PD=4μM group and NC group,the proportion of cells in G0/G1 phase were 75.8+1.2,75.73+1.7,68.1+1.81(NC vs PD=2μM/PD=4μM group,P<0.05),G2/M cells reduced statistically,(4)PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb,which inhibits endothelial cell cycle progression.2.(1)MTT method showed that the most sensitive cells to PD0332991 was H292 cell line,the most sensitive cell to gefitinib was HCC827 cell line(2)The Chou-Talalay method showed that When inhibiting the growth of lung cancer cell lines at 50%,75%,90%,and 95%,PD 0332991 and gefitinib were synergistic in most lung cancer cell lines.(3)Western blot showed that the lung cancer cell lines which were sensitive to PD 0332991 had higher expression of p-RB~ser780er780 and CDK6as for gefitinib sensitive cell line the expression of E2F1 were higher.Conclusion:1.PD 0332991 inhibits the proliferation and activity of endothelial cell EA.hy926 and induces its apoptosis.2.PD 0332991 inhibits the cell cycle progression by inhibiting the cycle related proteins CDK6,CDK4,and E2F1.3.The most sensitive cell line to PD 0332991 was H292,the most sensitive cell line to gefitinib was HCC827.PD 0332991 showed conjunction effect with gefitinib in most lung cancer cell lines(11/17).4.the PD 0332991 sensitive cell lines had higher expression of p-RB~ser780er780 and CDK6,and the expression of E2F1 was higher in gefitinib sensitive cell lines. |
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