Effects And Mechanisms Of Creatine Phosphate Sodium For Injection On Invasion Of Human Ovarian Cancer Cell Line SKOV3

Objective: To study the effects of Creatine Phosphate Sodium For Injection(CP)on invasion and migration of human ovarian cancer cell line SKOV3 and its mechanisms. Meanwhile, to investigate the possible mechanisms of CP inhibit invasion,adhesion and migration of SKOV3 cells; and Provide experiment evidence for use CP to treat the anthracycline-induced cardiotoxicity.Methods: SKOV3 cells were incubated in culture medium in vitro, divide into untreated group and treated group(1mmol/l,6mmol/L,12mmol/L). Effect of CP on the adhesion of SKOV3 cells was measured by MTT colorimetric method. Effect of CP on the invasion and migration of SKOV3 cells was measured by transwell method. The expression of nm23-h1,c-myc,MMP-2,MMP-9 protein in SKOV3 cells was analyzed by flow cytometric indirect immunofluorescent technigue. The expression of nm23-h1,c-myc gene in SKOV3 cells was analyzed by RT-PCR .Results: After treated with CP(1,6,12mmol/L )for 60min, measure the ability of adhesion by MTT colorimetric method. The adhesion rate was 0% in untreated group. The adhesion rate was 0.72%±0.92% in CP-treated group (1mmol/l). The adhesion rate was no significant higher, there was no statistically significant difference (P>0.05). The adhesion rate were 36.47%±3.17% in CP-treated group (6mmol/l) and 37.06%±3.30% in CP-treated group (12mmol/l). These two adhesion rates were significant higher than the untreated group, there were statistically significant difference (P<0.05), but there was no statistically significant difference in these two adhesion rates(P>0.05). The results show that CP inhibited adhesion of SKOV3 cells in the range of 6-12mmol/L.After treated with CP(1,6,12mmol/L), test the ability of migration in SKOV3 cells. In migratory experiments, compare with 0mmol/L concentration, the invaded cell numbers in 1mmol/L was no significant lower, there was no statistically significant difference(P>0.05), those numbers in 6 mmol/L and 12mmol/L concentration were statistically significant difference (P<0.05),.but there were no statistically significant difference between 6 mmol/L and 12mmol/L concentration(P>0.05). The Inhibition rates were 0% in untreated group, 3.06%±4.84% in CP-treated group(1mmol/l), 34.87%±3.19% in CP-treated group(6mmol/l), 37.67%±6.06% in CP-treated group (12mmol/l).After treated with CP(1,6,12mmol/L),test the ability of invasion in SKOV3 cells. In invasive experiments,compare with 0mmol/L concentration,the invaded cell numbers in 1mmol/L was no significant lower, there was no statistically significant difference(P>0.05), those numbers in 6 mmol/L and 12mmol/L concentration were statistically significant difference (P<0.05),but there were no statistically significant difference between 6 mmol/L and 12mmol/L concentration(P>0.05).The Inhibition rates were 0% in untreated group, 2.32%±1.47% in CP-treated group(1mmol/l), 31.50%±2.23% in CP-treated group(6mmol/l), 33.51%±2.90% in CP-treated group (12mmol/l).FCM assay results:The expression of nm23-H1,c-myc,MMP-2,MMP-9 protein had been examined in SKOV3 cells being treated with 0,1,6,12mmol/L CP for 72h, the FI-value of nm23-H1 in SKOV3 was 1,1.00±0.07,1.27±0.07,1.28±0.05;the FI-value of c-myc in SKOV3 was 1,1.00±0.04,0.87±0.01,0.85±0.03. To the FI values of nm23-H1 protein, there was statistically significant difference between control group and CP(6,12 mmol/L) group(P<0.05),but there was no statistically significant difference between control group and CP(1 mmol/L) group(P>0.05). To the FI values of c-myc protein, there was statistically significant difference between control group and CP(6,12 mmol/L) group(P<0.05),but there was no statistically significant difference between control group and CP(1 mmol/L) group(P>0.05).But the FI values of MMP-2,MMP-9 protein, there were no statistically significant difference between control group and CPgroup(P>0.05).RT-PCR detection results:After treated with CP(1,6,12mmol/L) for 72h,compared with control group, the expression of c-myc mRNA decreased, and there was statistically significant difference between control group and CP(6,12 mmol/L) group(P<0.05), but there was no statistically significant difference between control group and CP(1 mmol/L) group ( P > 0.05 ) .And compared with control group,the expression of nm23-H1 mRNA increased, and there was statistically significant difference between control group and CP(6,12 mmol/L) group(P<0.05), but there was no statistically significant difference between control group and CP(1 mmol/L) group(P>0.05).Conclusions: 1 CP inhibited the ability of adhesion,invasion and migration in SKOV3 cells.2 CP can increase the expression of nm23-H1 in SKOV3 cells. We infer that CP's effects of inhibiting adhesion,invasion and migration in SKOV3 cells seem to due to up-regulation of the expression of nm23-h1 mRNA and protein.3 CP can reduce the expression of C-myc in SKOV3 cells. We infer that CP's effects of inhibiting adhesion,invasion and migration in SKOV3 cells seem to due to down-regulation of the expression of C-myc mRNA and protein. 4 CP hasn't effect the expression of MMP-2,MMP-9 in SKOV3 cells.5 CP can't promote the adhesion,invasion and migration in SKOV3 cells in vitro,Provide experiment evidence for use CP to treat the anthracycline-induced cardiotoxicity.