Regulation And Mechanism Of Erianin On Glycolysis In Bladder Cancer Cells

Objective:To investigate the effects of Erianin on the proliferation and glycolysis of bladder cancer cells T24 and 5637,and the reversal effect of Akt overexpression on the Erianin inhibiting human bladder cancer cell line 5637proliferation and glycolysis.Methods:Different concentrations of Erianin（1.95,3.9,7.8,15.6,31.2,62.5,125,250,500,1000 ng/m L）were used to treat bladder cancer cell line T24 and different concentrations of Erianin（3.9,7.8,15.6,31.2,62.5,125,250,500 ng/m L）acted on 5637 of bladder cancer cells,then IC₅₀ was detected respectively;cell proliferation curve was used to detect the effect of different concentrations of Erianin（7.8,15.6,31.2,62.5,125 ng/m L）on the proliferation activity of T24 cell;effect of Erianin（15.6,31.2,62.5,125,250ng/m L）on cell clone of bladder cancer cells was detected by cloning formation experiment;flow cytometry was used to detect the effect of Erianin（125 ng/m L）on the cell cycle distribution of bladder cancer cells;a bladder cancer model was established in nude mice to verify the inhibitory effect of Erianin on proliferation of bladder cancer cell line T24 in vivo;the changes of CDK2,CDK4,p21,p27,p-Akt and Akt in Erianin groups with different concentrations（15.6,31.2,62.5,125 and 250 ng/m L）were detected and analyzed by Western blot;the detection of glucose uptake and lactate production in bladder cancer cell T24 with a final concentration of 125 ng/m L was detected by kits.The 5637cells stably over-expressing Akt were induced using the lentivirus vector.The5637 cells infected with the empty vector were classified into blank group,then the Akt group,empty vector combined with Erianin（62.5 ng/m L）group,and Akt combined with Erianin（62.5 ng/m L）group were set up;the cell viability was detected by cell proliferation curve;the clone formation of 5637 cells in each group was determined in the clone formation experiment;the cell cycle distribution was detected by flow cytometry;Western blot was used to assay the protein expression levels of p-Akt,Akt,p21;the glycolysis of 5637 cells was determined in glucose uptake and lactate secretion assays.Results:The IC₅₀results showed that Erianin inhibited the proliferation of bladder cancer cell line T24 in a dose-dependent manner.The half inhibitory concentration(IC₅₀)of 24h after administration was 217.60±0.08 ng/m L,and the IC₅₀ of 48 h after administration was 160.00±0.07 ng/m L,and the results of follow-up CCK-8showed that Erianin could inhibit the proliferation of bladder cancer cells T24 in a time and dose-dependent manner;clone formation experiment showed that Erianin could inhibit the clone formation of bladder cancer cells T24 in a dose-dependent manner;flow cytometry analysis of cell cycle showed that Erianin induced G0/G1 phase arrest in T24 cells;the results of animal experiments showed that Erianin could inhibit tumorigenesis in nude mice,the tumor weight of the administration group was significantly lower than the control group,and Erianin had no significant effect on the growth of nude mice;Western bolt showed that the effect of Erianin on T24 were promotion of the expression of p21 and p27 and suppression of the expression of p-Akt and Akt;glucose uptake test and lactate secretion test showed that Erianin could inhibit glucose uptake and lactate production in bladder cancer cells T24.In addition,IC₅₀ was 129.10±0.07 ng/m L after 24 h of bladder cancer cells 5637,and the inhibitory effect of Erianin on 5637 cells was reversed after overexpression of Akt with the final concentration 62.5 ng/m L;the results of clone formation experiment showed that Erianin could inhibit the clone formation of 5637 cells,and the inhibitory effect of Erianin on 5637 cells was reversed after overexpression of Akt;flow cytometry showed that Erianin could block 5637cells in G0/G1 phase,and the blocking effect was reversed after overexpression of Akt;Western blot results showed that Erianin could promote the expression of p21 protein and inhibit the expression of p-Akt in bladder cancer 5637 cells,and the promotion effect of the expression of p21 was reversed after overexpression of Akt;kits test showed that Erianin could inhibit glucose uptake and lactate production in bladder cancer cells 5637,and overexpression of Akt could reverse the inhibitory effect of Erianin on glycolysis.Conclusions:Erianin can inhibit the proliferation and colony formation ability of bladder cancer cells T24and 5637,and arrest the cell cycle in G0/G1 phase.In addition,Erianin can up-regulate the expression of p21 and p27 protein,down-regulate the expression of p-Akt,and weaken the glucose uptake and lactate secretion of bladder cancer cells through downregulation of p-Akt;and overexpression of Akt can reverse the inhibitory effect of Erianin on bladder cancer cells.This suggests that the inhibitory effect of Erianin on proliferation and glycolysis of bladder cancer cells is achieved by regulating the expression of Akt.