Effects of ginkgetin on proliferation and apoptosis of gastric cancer cells by regulating cell cycle and action mechanism
Objective To investigate the effect of ginkgetin on proliferation and apoptosis of gastric cancer cells by regulating cell cycle and its action mechanism.Methods Human gastric cancer MKN-45 cells werein vitro cultured.Ginkgetin was dissolved in dimethyl sulfoxide(DMSO)and used to treat gastric cancer MKN-45 cells with a series of concentrations(0,12.5,25.0,50.0,100.0 µmol/L)of Ginkgetin solution prepared by MKN-45 special culture medium for 48 h respectively.The cell counting kit(CCK-8)was used to verify the proliferation ability of cells and determine the action concentration and treatment time.Flow cytometry was used to verify the effects of Ginkgetin on apoptosis and cell cycle of each group.The cultured gastric cancer cell MKN-45 was treated with 1‰ DMSO,50.0 µmol/L Ginkgetin and c-myc inhibitor for 48 h and divided into blank control group(negative control,NC),Ginkgetin group and Ginkgetin+c-myc inhibitor group.The expression levels of apoptosis-related markers[B cell lymphoma/leukemia-2 associated X protein(bax),B cell lymphoma/leukemia-2(bcl-2)]and cell cycle-related mark-ers(CDK2,Cyclin B1)of the key gene c-myc downstream of Wnt pathway were detected by Western blot-ting.Comparison between groups was conducted using t-test.Results CCK-8 assay showed that the cell survival rate in 12.5 µmol/L Ginkgetin group was lower than in 0 μmol/L Ginkgetin group[(83.75± 1.16)％vs.(102.26±3.08)％t=10.83,P＜0.01].The cell survival rate in 25.0 μmol/L group was lower than in 0 μmol/L Ginkgetin group[(75.60±1.64)％vs.(102.26±3.08)％t=12.68,P＜0.01].The cell survival rate in 50.0 μmol/L group was significantly lower than in 0 µmol/L group[(53.29±1.33)％vs.(102.26±3.08)％t=30.36,P＜0.01].The cell survival rate in 100.0 µmol/L group was significantly lower than in 0 µmol/L group[(25.41±1.02)％vs.(102.26±3.08)％t=47.66,P＜0.01].The half maximal inhibitory concentration(IC50)value of Ginkgetin for gastric cancer MKN-45 cells was 51.39 at 48 h.The results of flow cytometry showed that Ginkgetin could induce apopto-sis of gastric cancer MKN-45 cells(the apoptosis rate increased from 12.36％to 49.60％)and lead to G2 phase arrest of gastric cancer cells(the number of cells in G2/M phase increased from 1.85％to 27.00％).The results of Western blotting showed that the expression of bax protein in Ginkgetin group was higher than in NC group(571 241±7 627 vs.364 248±351,t=71.72,P＜0.01).The level of bcl-2 protein in Ginkgetin group was lower than in NC group(6 040±7 597 vs.681 315±7 017,t=19.76,P＜0.01).The expression of CDK2 protein in Ginkgetin group was lower than in NC group(627 653± 9 488 vs.742 450±5 791,t=27.32,P＜0.01).The expression of cyclinb1 protein in Ginkgetin group was lower than in NC group(558 302±11 687 vs.708 767±20 598,t=16.81,P＜0.01).The expres-sion of c-myc protein in Ginkgetin group was lower than in NC group(643 589±6 304 vs.836 895± 6 428,t=56.80,P＜0.01).The expression of c-myc protein in Ginkgetin+c-myc inhibitor group was lower than in Ginkgetin group(504 992±6 779 vs.643 589±6 304,t=39.61,P＜0.01).Conclusion Ginkgetin regulates the cell cycle by regulating the Wnt pathway and the expression of c-myc in gastric cancer cells,thus affecting the proliferation and apoptosis of gastric cancer cells.
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