The Effects And Mechanisms Of The IGF-1R Inhibitor PPP On Human Osteosarcoma Cells U-2OS

[Objective]: To observe the effects of the IGF-1R inhibitor PPP on human osteosarcoma cells U-2OS, and preliminary explore its possible mechanism of resistance-transfer of osteosarcoma. [Methods]: 1. The human osteosarcoma cell line U-2OS were cultured in vitro, determined by MTT method to detect the influence of different drug concentration and action time on the proliferation of U-2OS cell. In addition, cell morphology was observed under an inverted phase contrast microscope, and pictures were taken with an Canon SX270 HS camera. 2. Using medication cells for 24 h, flow cytometry detection of cell cycle of osteosarcoma cell U-2OS. 3. Scratch test and Transwell method for determining the influence of the migration and invasion ability,after the PPP on osteosarcoma cell U-2OS. 4. Western blotting analysis was used to evaluate the change of expression level of E-cadherin and Vimentin, which are EMT markers of tumor cells. [Results]: 1. Under the inverted phase contrast microscope observation of the cells U-2OS, which were be treated by the IGF-1R inhibitor PPP, showed with the increase of drug concentration or the extension of time, the slower growth of osteosarcoma cell U-2OS, cells decreased significantly, arranged loosely, cells in different volume sizes, shape changed irregularly, losed their original characteristics, the cell-body shriveled, intercellular space increased, and some cells losed adherent abilities and floated, refraction sex is reduced, the cell vitality become poor. 2. Determined by method MTT to detect the influence of different drug concentration and action time on the proliferation of cell U-2OS. The concentration of PPP is set to 0.5μM,1.0μM,2.0μM,4.0μM,8.0μM, and be respectively dealed with 24 h, 48 h, 72 h. Results show that PPP can significantly inhibit the proliferation of the human osteosarcoma cell U-2OS, and with the increase of drug concentration and the extension of action time, the proliferation inhibition rate of the human osteosarcoma cell U-2OS increased significantly, the difference is statistically significant(P < 0.05). Under the condition of same action time, the comparison between any two in treated-groups indicates that:F24h=208.580, F48h=395.674, F72h=412.220, P24 h 、 48h、72h=0.000;and then same the condition of drug concentrations, 24 h, 48 h and 72 h compared between the groups showed that: F0.5=40.825, F1.0=22.038, F2.0=91.701, F4.0=69.781, F8.0=22.999, P values < 0.01; at the same time, there are interaction between drug concentration and action time, F=13.471, P=0.000. All in all, our experimental results show that PPP can obviously inhibit the proliferation of the human osteosarcoma cell U-2OS in a time-and concentration-dependent manner in vitro. 3. Flow cytometry analysis: The concentration of PPP is set to 0.5μM,1.0μM,2.0μM,4.0μM,6.0μM, and be processed for 24 h. Compared with the control group, significant changes in the cell cycle, the cell number of G2/M phase increased obviously, S phase cells decreased, and the blocking effect in a obvious concentration dependent manner, the difference was statistically significant(P < 0.01). The result shows that PPP blocked S phase and G2�'M period of transition, namely under the effect of PPP, the DNA synthesis, at the same time are copied and mitotic barriers, by influencing cell cycle so as to achieve the purpose of inhibiting cell proliferation. 4. In order to define the PPP whether can inhibit the metastasis of osteosarcoma, Scratch test and Transwell method are used to evaluate the migration and invasion ability of PPP on osteosarcoma cells. Result shows,(1) Control,0.5μM,1.0μM,2.0μM each group treated the cells U-2OS for 24 h, the migration distance were 179.71±16.91,191.86±16.31,218.14±10.10,266.14±19.54 respectively, the migration distance of 48 h were 102.71±16.22, 154.28±22.71, 216.85±18.82, 282.03±52.61 respectively. ANOVA: Time factor: F=154.66 P=0.00; Concentration factor: F=81.46 P=0.00; the interaction of the time factors and processing factors: F=21.27 P=0.00.(2) Control,0.5μM,1.0μM,2.0μM groups through the microporous membrane cell number were 67.66±1.52, 54.66±2.08, 38.00±2.00, 23.66±2.51; the concentration and the control group had significant difference between groups(P<0.05), the invasion ability of the human osteosarcoma cell U-2OS between each treated groups there were significant differences(F=260.54,P<0.001), suggesting the PPP inhibition of infiltrating invasion ability of osteosarcoma cells shows obvious concentration dependence, consistent with scratches healing experimental results. Therefore draw the conclusion: PPP can inhibit the migration and invasion of osteosarcoma cell U-2OS, and presenting the concentration dependence. This is consistent with PPP reported in other tumor. 5. Western blot analysis to examine the expression changes for E-cadherin and Vimentin protein of the osteosarcoma cells U-2OS. Results show that with the increase of concentration of PPP, E-cadherin expression increased significantly(p<0.05), while Vimentin expression level gradually reduced(p<0.05), the PPP might inhibit the occurrence of EMT in human osteosarcoma cell line U-2OS, thereby inhibiting tumor cell metastasis. [Conclusion]: 1. The PPP can obviously inhibit the proliferation of human osteosarcoma U-2OS cells in vitro, presenting the dependency on time and concentration, its mechanism may be related to cell cycle S phase and G2/M phase retardation. 2. The PPP can induce osteosarcoma cells S phase and G2/M phase of the block, and the cycle retardation with dose Dependencies. 3. The PPP induced time-and dose-dependent inhibition of migration in human osteosarcoma U-2OS cells. 4. The PPP can inhibit the invasion of human osteosarcoma U-2OS cells, presenting the concentration dependence. 5. The PPP may inhibit the occurrence of EMT in human osteosarcoma cell line U-2OS, thereby inhibiting tumor cell metastasis.