| Objective: To study the anti-tumor effect of arsenic trioxide combining with cisplatin on the human osteosarcoma cells line MG-63, and explore its mechanism of interaction. Methods: MG-63 cells were cultured in the environment of 37℃, 5% CO2 with MEM medium, which was supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100μg/ml streptomycin. 1 After cultured for several generations, the cells were digested and transferred into 96-well culture plates. The MTT method was used to obtain the IC50 of the drugs on MG-63 cells. The IC50 of each drug and half the IC50 of CDP added half the IC50 of arsenic trioxide was used to treat MG-63 cells for 48h in order to obtain the inhibition rate. 2 For detection of the apoptosis affected by the drugs, the cells were harvested and stained by Acridine Orange and Propidium Iodide Fluorescence to determine the morpological alteration of apoptotic cells and the apoptotic rate. 3 The cells DNA were extracted using saturation phenol/chloroform and stained by Ethidium Bromide to evaluate DNA strands breaks using agarose gel electrophoresis and observed with UV lamp. 4 After harvested, cells were stained according to the reagent descriptions, then cell cycle alteration and apoptotic rate of the cells were determined by flow cytometry. 5 In order to measure the alteration of the content of glutathione and the activity of glutathione S-transferases in tumor cells, the IC50 of each drug and half the IC50 of CDP added half the IC50 of arsenic trioxide was used to treat MG-63 cells for 48h, then the MG-63 cells were harvested and broken by ultrasonic wave. Glutathione reductase recycling assay and common ultraviolet spectrophotometer assay were used to analyze the content of glutathione and the activity of glutathione S-transferases. 6 After the MG-63 cells were treated with drugs for 48h, the cells were collected, then marked by anti-Bcl-2-FITC or by anti-Bax-FITC. The positive rates of MG-63 cells with Bcl-2 or Bax protein were examined with flow cytometry. Results: It was revealed by MTT assay that IC50 of cisplatin was 1.13μg/ml, while arsenic trioxide was 2.15μmol/ml. The MTT assay also showed that the inhibition rate of As2O3,CDP and combinatorial treatment group was (52.20±0.19)%,(57.11±0.64)%,(71.10±0.25)%, respectively. The apoptotic rates of the cells observed through AO and PI staining were (3.20±0.90)%,(21.33±1.58)%,(25.53±1.83)% and (37.80±1.73)% for the control group,As2O3,CDP and combinatorial treatment group, respectively. When tested byflow cytometry, the apoptotic rates were (8.24±0.14)% for control group, (22.46±0.22)% for As2O3, (24.84±0.320% for CDP and (35.76±0.24)% for combinatorial treatment group. The cellular DNA strand breskages were found obviously in every drug treatment group by agarose gel electrophoresis, but combinatorial treatment group were more obviously. It was found that CDP had no influence on the content of GSH and the activity of GST. As2O3 could reduce them obviously, while in the combinatorial treatment group, they were reduced more obviously. There was no chang of Bcl-2/Bax in the CDP treatment group or control group. As2O3 could raise the expression of Bax and reduce the expression of Bcl-2 obviously. Compared with As2O3 treatment group, the combinatorial treatment group's Bcl-2/Bax had no chang. Conclusion: 1 Arsenic trioxide of low concentration combining with cisplatin of low concentration may obviously increase the anti-osteosarcoma effect in compare to using arsenic trioxide or cisplatin in high concentration respectively, either in inhibiting MG-63 cells proliferation or inducing apoptosis. 2 The mechanisms of interaction are mainly that arsenic trioxide may reduce the content of glutathione and inhibit the the activity of glutathione S-transferases or reduce the... |
