| Objective:The present study was designed to investigate the in vitro effect of LY294002( LY) and pirarubicin(THP、PI) combination on proliferation, apoptosis, invasion and migration of OS cell lines, in addition to providing new insights into the treatment of OS.Methods:①The effect of LY294002 and pirarubicin, administered separately and in combination for 24 h on OS cell growth was examined by MTT. The half maximal inhibitory concentration(IC50) values was computed respectively and the interaction between pirarubicin and LY294002 was assessed by the combination index(CI).②The effect of LY294002 and pirarubicin, administered separately and in combination for 24 h on OS cell apoptosis was detected by nuclear staining with4,6-diamidino-2-phenylindole(DAPI). The apoptotic rate was calculated and the adjunctive therapy was assessed applyling Jin’s Q-value.DAPI③The effect of LY294002 and pirarubicin, administered separately and in combination for 24 h on OS cell migration and invasion was measured by wound healing and transwell invasion assay Results:①The growth inhibition of MG-63 and U2-OS cells, which treated with LY294002, pirarubicin and combination treatment, occurred in a dose-dependent manner. For U2-OS cells, the pirarubicin and LY294002 IC50 values were 0.47±0.05 and 19.71 ±1.87μg/ml, respectively. When these two reagents were administered to U2-OS cells simultaneously at 1:25, 1:50 and 1:100 proportions, the IC50 values were6.54±0.62、9.15 ±1.21、11.28±1.32ug/ml, respectively. Thus the CI values were0.74±0.05,0.81±0.07 and 0.87±0.06 at the relevant proportions above respectively.For MG-63 cells, the pirarubicin and LY294002 IC50 values were 3.61±0.35 and17.36±2.07ug/ml, respectively. When these two reagents were administered toMG-63 cells simultaneously at 1:5, 1:10 and 1:20 proportions, the IC50 values were5.56±0.65, 5.04 ±0.55 and 5.36±0.62μg/ml, respectively. So the CI values were0.54±0.08, 0.37±0.06 and 0.39±0.07, respectively accroding to the relevant proportions. All the CI value were <0.95.②The promotion of cell apoptosis also turned out to be dose-dependent regardless of whether cells were treated with LY294002, pirarubicin or the combination of the two. The apoptotic rate of MG63 cells were 7.5±1.9 %,14.5±3.2%, 24±5.4% and 10.5±2.1.%, 8.9±3.5%, 42.4±5.1%, respectively when applying 0.25, 0.5 and 1μg/ml of pirarubicin and 5、10、20μg/ml of LY294002. When pirarubicin plus LY294002 were administered to MG63 cells at 0.25+5, 1+5 and1+20 μg/ml, the apoptotic rate were 30.8±4.2%, 39.5±5.4% and 74.5±8.9%,respectively, futher figured out the Jin’s Q-value being 1.5±0.28, 1.23±0.25 and1.49±0.32 respectively. Whlie the combined apoptotic rate of U2-OS cells were25.6±3.2%, 33.5±4.5% and 79.4±9.6% following 1.5±0.28, 1.23±0.25 and 1.49±0.32 of Jin’s Q-value respectively, when pirarubicin plus LY294002 were administered at0.1+5 、0.2+5 and 0.2+20 ug/ml. All the Q value were >1.15.③The migration rates of MG63 cells were 82±12%, 35±7%, 41±8% and 12±3%in control, pirarubicin, LY294002 and combination groups respectively, aslo the U2-OS cells did it to 0.75±14%, 33±8%, 26±6% and 10±3% correspondingly,revealing that the migration rate of cells treated with LY294002 and pirarubicin together was significantly lower than those treated by either LY294002 or pirarubicin alone(p<0.05).④The number of transmenbranous MG63 cells were 165±22, 72±8, 63±7 and28±3 in control, pirarubicin, LY294002 and combination group respectively, and the number of U2-OS cells reached to 155±20, 68±8, 52±6 and 25±4 correspondingly,demonstrating that the number of transmenbranous cells following combined LY294002 and pirarubicin treatment was significantly lower than those treated by LY294002 and pirarubicin separately(p<0.01).Conclusion :The results indicated that LY294002 enhanced the suppressive effect on the malignant phenotype of osteosarcoma cells to pirarubicin chemotherapy in asynergistic manner, leading to more effctive antitumor effects which induced apoptosis, and inhibited the growth, migration and invasion of OS cells. The study provided theoretical support for the following in vivo experiment, as well as novel chemotherapy solutions for OS treatment. |
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