| Background Osteosarcoma is the most common primary malignant bone neoplasms,it frequently occurs at teenagers,which is characterized by high malignant degree,poor prognosis and high mortality.In the research of nearly 30 years,it found that patients with osteosarcoma after neoadjuvant chemotherapy combined limb-salvage surgery treatment,the 5-year survival rate is about 70%,and there was no significant change,which made the treatment of osteosarcoma has entered a period of stagnation.Therefore,it is necessary to explore new drugs or new methods of treatment of osteosarcoma.Thalidomide,which was mainly used in the treatment of pregnancy vomiting reaction in the earliest stage,was almost prohibited to use around the world due to its severe fetal teratogenic effect.But in recent years,with the deepening of its pharmacological mechanism study,it has been found that thalidomide had the obvious antitumor effect.In the field of tumor treatment,thalidomide had become a part of the standard treatment of relapse and refractory multiple myeloma,on other tumors such as prostate cancer,colorectal cancer,non-small cell lung cancer,breast cancer and renal cell carcinoma and other solid tumors,thalidomide had also been proved to have certain curative effect.But what the influence did the thalidomide have on proliferation,invasion and metastasis of osteosarcoma cell,was rarely reported.this experiment aimed to investigate the antitumor effect of thalidomide,and to explore the possible pathways of apoptosis,after treatment with different concentration thalidomide to osteosarcoma cells in vitro,which would provide experimental foundation and theoretical basis of thalidomide may be applied to the clinical treatment of osteosarcoma.Objetive : This experiment aimed to evaluate the effect of thalidomide on proliferation and apoptosis in human osteosarcoma cell line MG-63 in vitro,to further investigate possible mechanism of its anti-tumor effect.Methods: 1.Using CCK-8 method to detect the influence of thalidomide,to the cell activity of MG-63 and U2 OS cells.2.Using Hoechst33258 fluorescent dye to detect effects of different concentrations thalidomide on MG-63 cell nuclei.3.Detecting the apoptosis of MG-63 cells after treatment with different concentrations of thalidomide by flow cytometry.4.Detecting the cell cycle changes of MG-63 cells after treatment with different concentrations of thalidomide by flow cytometry.5.Detecting the mitochondrial membrane potential changes of MG-63 cells after treatment with different concentrations of thalidomide by JC-1 fluorescent probe.6.Using DCFH-DA fluorescent probe to detect the level of reactive oxygen species changes of MG-63 cell after treatment with different concentrations of thalidomide.7.Using Western Blotting analysis to detect the expression of apoptosis related proteins BCL2 and BAX,Nf-?b and Caspase3 of MG-63 cells after treatment with different concentrations of thalidomide.Results 1.In vitro cytotoxicity experimental results showed that thalidomide could significantly inhibit the cell activity of human osteosarcoma cell line MG-63 and U2 OS with dose and time-dependent effect;The IC50 of U2 OS cells for 24 h,48 h and 72 h: >500?g/ml ?476.13±93.3?g/ml and 156.61±40.65?g/ml,respectively;The IC50 of MG-63 cells for 24 h,48 h and 72 h: >500?g/ml?151.05±8.09?g/ml and 94.76±10.52?g/ml,respectively.2.Results of hoechst-33258 dyeing observation under the fluorescence microscope: Compared with the experimental group,the cells nucleus of control group is complete,and have uniform color and dispersive fluorescence;After treatment with different concentrations of thalidomide,the significant nuclear condensation and morphological changes,such as nuclear shrinkage,chromatin condensation and the color is bright,were observed,and with the increasing of thalidomide dose the changes became more obvious.3.Annexin V FITC /PI double dye apoptosis detection showed that thalidomide can induce apoptosis and necrosis in MG-63 cells,and dose-dependently.The apoptosis rate after different concentrations of thalidomide(0,50,100 and 200?g/ml)dealed with MG-63 cells were(7.98±1.26)%,(10.58±1.18)%,(28.74±6.08)% and(38.00±6.40)%,respectively.4.The results of Flow cytometry to detect the cell cycle changes showed that,after treatment 48 h with different concentrations of thalidomide(0,50,100 and 200?g/ml),MG-63 were induced G0/G1 phase retardation in dose-dependent manner,(63.68±1.76)%,(71.9±0.83)%,(73.87±1.72)% and(76.37±1.12)%,respectively;And S phase cells to be decreasing in dose-dependent manner —ratio of S phase of 0,50,100 and 200?g/ml to be(19.95±3.11)%,(15.08±3.35)%,(13.53±2.96)% and(12.38±2)%,respectively;G2 / M phase change is also presented decreasing trend.5.The results of JC-1 probe method to detect mitochondrial membrane potential: thalidomide can significantly reduce mitochondrial membrane potential in osteosarcoma MG-63 cells by flow cytometry.After treatment 24 h with different concentrations of thalidomide(0,50,100 and 200?g/ml),ratio of low mitochondrial membrane potential cells to be(4.84 ± 0.31)%,(7.63 ± 0.94)%,(9.57 ± 7.63)% and(13.62 ± 5.92)%,respectively;And not damaged mitochondria membrane potential cell proportion to be(95.17 + 95.17)%,(92.37 + 0.97)%,(90.4 + 2.62)%,(85.53 + 4.70)%,respectively.6.The results of DCFH-DA fluorescent probe to detect intracellular reactive oxygen species level: The range of drug concentrations from 100 ug/ml to 400 ug/ml dealed with MG63 cells,thalidomide can increase the intracellular ros level,and presents the dose-dependent effect(P<0.05);the ratio between experimental group(100?g/ml,200?g/ml,400?g/ml)and normal control group of the fluorescence intensity of DCF was(1.26 ± 0.16),(1.40 ± 1.40)and(1.88 ± 0.32),respectively;while the ratio between positive control group(ROSUP)and normal control group was(1.34 ± 0.07).The reactive oxygen species levels between high concentrations of thalidomide group in which 100 ug/ml to 400 ug/ml)and low concentration group(12.5 ug/ml to 50 mu g/ml)has also significant statistical significance(P<0.05).7.Western Blot method was used to detect the expression change of apoptosis related proteins BAX,BCL2,Caspase3 and Nf-?b,the results showed that after treatment of different concentrations of thalidomide(0?50?g/ml?100?g/ml?200?g/ml)to osteosarcoma MG-63 cells 48 h,the expression levels of antiapoptotic BCL2 protein and protein Nf-?b dropt,and the expression levels of apoptosis protein BAX and Caspase3 rise,compared with negative control group.Conclusion thalidomide has effect of restraining proliferation and promoting apoptosis to osteosarcoma MG-63 cell in vitro,the possible mechanism may be through ascending intracellular ros level,reducing the mitochondrial membrane potential,promoting the expression levels of apoptosis protein BAX and Caspase3,and inhibiting the expression levels of antiapoptotic BCL2 protein and protein Nf-?b,which suggests its possible mechanism may be through the intrinsic mitochondrial apoptotic pathways to induced apoptosis. |
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