Effects Of SM-164 Combined With Doxorubicin(ADM)on Proliferation And Apoptosis In Human Osteosarcoma U-2 Cell Line

Background and Objective:As a new kind of mitochondrial protein, Smac has been widely studied since it was first revealed by Du et al. [16] As a novel anti-cancer drug in July 2000,SM-164,as a subtype of Smac protein, has a more significant effect to anticancer. The team confirmed that SM-164 can significantly enhance the sensitivity and anti-cancer effects on breast cancer, colon and prostate cancer by further exploration[21,25].The paper aims to explore the inhibition of growth and apoptosis inducing effect of U-2-OS by doxorubicin(ADM) which is enhanced by SM-164. Method:(1) MTT assay the growth inhibition for SM-164 alone and combined with doxorubicin(ADM) in U-2-OS cells. 1) Detecting the proliferation inhibition of U-2-OS and drawing the growth curve after different concentrations of SM-164 treating U-2-OS cells separately for 24 hours;2) According to preliminary concentration gradient experiments,the density of the SM-164 was set to 100 nM and 200 nM,and the the concentration of ADM were set to clinical drug concentration 0.5μg / mL and half-dose clinical concentration 0.25μg / mL.There are night groups to be divided: SM-164-100 nM group, SM-164-200 nM group, ADM(0.25μg/ml)group,ADM(0.5μg/ml)group,SM-164-100nM+ADM(0.25μg/ml)group,SM-164-100nM+ADM(0.5μg/ml)group,SM-164-200nM+ADM(0.25μg/ml)group,SM-164-200nM+ ADM(0.5μg/ml) group and control group at last.(2) Hoechst staining tests the apoptosis of U-2 cell. The control group, the experimental group 1(ADM0.5μg /ml) and the experimental group 2(ADM0.5μg / ml + SM164100 nM) will be set up.(3) FCM( flow cytometry) detects the apoptosis of U-2 cells impacted by ADM(0.25μg / mL, 0.5μg / mL) and/or SM-164(100nM). Results:(1) MTT assay showed that:1) When different concentrations(1nM, 10 nM, 50 nmol / L, 100 nM, 200 nM, 500 nM, 1μmol / L) of SM-164 treating U-2-OS cells for 24 hours respectively, the proliferation of U-2-OS is suppressed, and the degree of inhibition with increasing doses of SM-164 gradually increased, but the inhibition effect is not obvioue,because the U-2 cell viability has no significant change(seeing Table 1, Figure 1);2)Cell growth inhibition rate of SM-164 combined with ADM group was significantly enhanced than that of the corresponding ADM group, displaying that they have a cooperative effect.(2) Hoechst staining test displayed that there are more U-2 cells in the control group vision, but only appears a faint blue fluorescence; The number of cells within ADM(0.5μg / mL) group vision decreased,just a small quantity of blue fluorescence could be caught sight; A large amount of darker nuclei and strong blue fluorescence cound be found in the SM-164 combined with ADM group.(3) FCM displayed that the apoptosis rate of ADM(0.25μg/ml, 0.5μg/ml) groups were(8.53±2.41)% and(12.2±3.11)%, SM-164(100n M) group was(6.03±2.01)%, and the SM-164(100n M) combined with ADM(0.25μg/ml, 0.5μg/ml) groups were(36.4±3.68)% and(50.2±5.19)%, Q values were1.97±0.292、and 2.24±0.226., bringing out an explicitly cooperative effect.Conclusion: 1)SM-164 monotherapy for the U-2-OS proliferation is limited, but the SM-164 combined with ADM on the U-2-OS has a significant proliferation resistance, and the synergistic effect is very obvious; 2)SM-164 can significantly strengthen the ADM apoptosis to U-2-OS, the synergistic induction of apoptosis is also significant.