Effects Of Dihydromyricetin On Growth Of Human Osteosarcoma MG-63 Cell Xenografts And Carbohydrate Metabolism In Nude Mice

OBJECTIVETo observe effect of dihydromyricetin(DMY)on the human osteosarcoma MG-63 cell xenografts and Warburg effect in nude mice,and further explore its possible mechanism.METHODSHuman osteosarcoma cell line MG-63 was inoculated subcutaneously under the abdomen of nude mice to establish a nude mouse model of human osteosarcoma transplantation.The nude mice with human osteosarcoma xenografts were randomly divided into model group and DMY(50,100and 200 mg/kg/d)treatment group,each one with 6 nude mice.The nude mice in model group and DMY(50,100 and 200mg/kg/d)treatment group were respectively administrated normal saline and DMY(50,100 and 200 mg/kg/d)by gavage for 30 days.The general condition of nude mice and the growth of transplanted tumor were observed.After nude mice were sacrificed,the transplanted tumor was removed,the weight and volume of the transplanted tumor were measured,and the growth inhibition rate was calculated.The content of glucose and lactic acid and lactate dehydrogenase(LDH)activity in the homogenate of transplanted tumor were detected by colorimetry.Immunohistochemistry and Western blot were used to detect the expression of Ki67 in human osteosarcoma xenografts.Expression of hypoxia-inducible factor-1a(HIF-1a),hexokinase-2(HK-2),lactate dehydrogenase A(LDHA),total Akt and p-Akt in human osteosarcoma xenografts in nude mice was detected by Western bloting.RESULTS1.In all nude mice inoculated with human osteosarcoma MG-63 cells,there appeared round rounded mound at the inoculation site.Tumor nodes could be found in the abdomen about 10 days after inoculation.The size of tumor nodules was about72.15±5.42 mm~3.As time goes on,tumor nodules continued to grow.Tumor nodules were irregular in shape,irregular in shape and hard in texture.The tumor formation rate of human osteosarcoma MG-63 cells in nude mice was 100%.Nude mice in the transplanted tumor model group experienced mild food loss,mental depression,slow response to stimulation,and weight loss.But no obvious cachexia was observed in the transplanted tumor model group.All the nude mice did not find other obvious abnormal behaviors.Compared with the transplanted tumor model group,the size of abdominal tumor nodules was significantly reduced,and appetite,reaction and mental state were significantly improved in nude mice in DMY(100 and 200 mg/kg/d)group.All the transplanted tumors were confirmed osteosarcoma by H-E staining.2.Compared with the transplanted tumor model group,the weight and volume of transplanted tumor were significantly decreased in DMY(100 and 200 mg/kg/d)group(all P<0.05).There was no significant difference about the weight and volume of transplanted tumor between transplanted tumor model group and DMY(50mg/kg/d)group(all P>0.05).The tumor inhibition rate of DMY(50,100 and 200mg/kg/d)increased gradually,they were 2.59%,33.26%and 59.61%respectively.3.Compared with the transplanted tumor model group,the percentage of positive cells expressing Ki67 in transplanted tumor of nude mice with osteosarcoma and the protein expression level of Ki67 were significantly decreased in DMY(100 and 200mg/kg/d)group(all P<0.05).There was no significant difference about the percentage of positive cells expressing Ki67 in transplanted tumor of nude mice with osteosarcoma and the protein expression level of Ki67 between transplanted tumor model group and DMY(50 mg/kg/d)group(all P>0.05).4.Compared with the transplanted tumor model group,the level of glucose in transplanted tumor was significantly increased,the level of lactate and the activity level of LDH in transplanted tumor were significantly decreased in DMY(100 and200 mg/kg/d)group(all P<0.05).There was no significant difference about the level of glucose and lactate and the activity level of LDH in transplanted tumor between transplanted tumor model group and DMY(50 mg/kg/d)group(all P>0.05).5.Result of Western blotting showed that the levels of HIF-1?,HK2,LDHA and p-Akt protein expression in transplanted tumor were significantly down-regulated compared with the transplanted tumor model group(all P<0.05).There was no significant difference about total Akt protein expression in transplanted tumor between transplanted tumor model group and DMY(50,100 and 200 mg/kg/d)group(all P>0.05).There was no significant difference about the levels of HIF-1?,HK2,LDHA and p-Akt protein expression in transplanted tumor between transplanted tumor model group and DMY(50 mg/kg/d)group(all P>0.05).CONCLUSIONSDihydromyricetin inhibits the growth of the human osteosarcoma MG-63 cell xenografts in nude mice and the Warburg effect,which the mechanism may be involved to PI3K/Akt signaling pathway.