Regulation Of Cell Proliferation And Apoptosis By 2-florobenzaldehyde Retinoic Acid Conjugate (RAC) Through Suppression Of STAT3 Activation In Human Glioma Cells

Part Ⅰ EXPRESSION AND SIGNIFICANCE OF STAT3 AND JAK2 IN HUMAN GLIOMASObjective:We detected the expression of STAT3 and JAK2 in human glioma tissues, analyzed and discussed the correlation of them and pathological grade in glioma.Methods:We have collected 50 cases of glioma tissue samples and 10 cases of corresponding normal tissue samples, and then detected the expression of STAT3 JAK2 mRNA by quantitative PCR, we also detected the expression of STAT3 JAK2 protein by western blot, and analysed the relativily between STAT3/JAK2 protein and the clinical pathological grading of glioma patients.Results:(1) The expression of STAT3 and JAK2 mRNA in glioma tissues samples were significantly higher than those in the corresponding normal tissue samples;(2) The expression of STAT3 and JAK2 protein in glioma tissues samples were significantly higher than those in the corresponding normal tissue samples;(3) The expression of JAK2 and STAT3 protein in glioma tissues were positively correlated with pathological grades of glioma.Conclusion:The expression of JAK2 and STAT3 were significantly higher in glioma tissues, suggesting that both of them could be used as a predictive indicator of disease progression in gliomas patients, which could lay the foundation for development of effective treatment programs in clinical treatment.Part II THE INHIBITION OF CELL PROLIFERATION, APOPTOS AND CELL CYCLE OF GLIOMA CELLS BY RACObjective:In this study, we detected the proliferation, apoptosis and cell cycle by treating with different concentrations of RAC in U373 cells, in order to understand the effect of RAC, which was designed to investigate the role of RAC in the above procedure, in order to lay the foundation for the next step.Methods:(1) BrdU cell proliferation assay was used to detect the cell survival by treating with the RAC concentration of 0,10,20,30,40,50 μM in U373 cells;(2) We used flow cytometry (Annexin V-FITC method) to detect the cell apoptosis of U373 cells that were treated with the RAC concentration of 0,10,30,50 μM after 24 h; and we also detected the cell apoptosis of U373, which had treated with 50 μM RAC after 0h,6 h,12 h,18 h;(3) We detected changes of the cell cycle of U373 cells that were treated with different concentration(0,10,30,50μM) of RAC, after 24 h.Results:(1) BrdU cell proliferation assay kit test results show, after treating with 10-50 uL RAC for 48 h, the cell viability of U373 cells decrease gradually (from 98%to 19%), the U373 cell survival rate was the lowest when treated with 50 μM RAC, suggesting that the cell survival rate of U373 cells was decreased gradually with increasing concentrations of RAC; after treating with 50 μM RAC, the cell survival rate of U373 cells in 6,12,24,36,48 h were 96%,81%,76%,43% and 19%, namely U373 cell viability with prolonged duration of action of RAC decreased gradually;(2) Annexin V-FITC test results showed that with increasing concentration of RAC, the number of apoptotic U373 cells also gradually increased. When RAC concentration was 50μM, the number of apoptotic cells was the largest; by treating with 50 μM RAC, the number of apoptotic cells increased gradually as time goes on.(3) Flow cytometry results showed that, RAC could stasis the cell cycle of U373 in G1 phase, and with the increasing concentration of RAC role, the number of cells inG1 phase increased gradually. When the RAC concentration was 50μM, the number of cells in G1 phase was the largest.Conclusion:(1) The inhibition of cell proliferation in U373 cells by RAC was in a time and dose-dependent manner;(2) The apoptosis-promoting of RAC in U373 cells was in a time and dose-dependent manner;(3) RAC enabled the growth of U373 cells at the G1 phase in a dose-dependent manner.Part Ⅲ THE RESEARCH ABOUT THE INHIBITION MECHANISM IN GLIOMA CELL PROLIFERATION BY TREATING WITH RACObjective:In this study, we had discussed the mechanism of action in JAK2-STAT3 signaling pathway by treating with RAC, in order to understand the interaction relations between the two, to provise more accurate experimental evidence for clinical anti-tumor applications by RAC.Methods:(1) We detected the expression of STAT3 and p-STAT3 protein in U373 cells, which were treated with 0,10,20,30,40,50 μM RAC; we detected the expression of STAT3 and p-STAT3 protein in U373 cells, which were treated with 20,30,40,50,100 μM AG490 (JAK2 antagonist) after 3 h; and detected the expression of STAT3 and p-STAT3 after treating with 50 μM RAC in 10,30,60,90,120,150,180 min respectively;(2) U373 cells were treated with IL-6, we detected the expression of p-JAK2 protein by western blot at 0,10,20,30,40,50 and 60 min respectively. The U373 cells previously treated with 50μM RAC, and then treated with IL-6, then we detected the expression of p-JAK2 protein at 0,30,60,90,120,180,340 min respectively by western blot;(3) We had used 0,30,40,50 μM RAC treated U373 cells respectively, and then detected the expression of Bax, Bak, cyclin D1, Bcl-2, Bcl-xL, survivin and VEGF protein after 24 h by western blot.Results:(1) With increasing concentration of RAC, the expression of p-STAT3 protein in U373 cells gradually decreased, while STAT3 protein content had no significant effect; with the increasing of the concentration of AG490, U373 cells, the expression of p-STAT3 protein gradually lower, while STAT3 protein content had no significant effect; with 50 μM RAC role U373 cells, with time, the content of p-STAT3 cells gradually decreased, while there was no significant change in the expression of STAT3;(2) The expression of p-JAK2 protein in U373 cells gradually increased while treated with IL-6, while there was no significant effect on the expression of JAK protein; previously treated with 50μM RAC U373 cells, then these cells were treated with IL-6, with the extension of time, the expression of p-JAK2 protein decreased gradually, but the expression of JAK protein had no significant change;(3) After treated with RAC, the expression of Bak and Bax protein increased significantly; the expression of survivin, Bcl-2, Bcl-xL, VEGF, cyclin D1, Mcl-1 protein decreased gradually.Conclusion:RAC inhibited the proliferation and induce apoptosis by inhibiting JAK2/ STAT3 signaling pathway in U373 cell.