| Objective:We investigated the influence of isothiocyanate combined with adriamycin on the apoptosis of human osteosarcoma U2-OS cell line in vitro. It was found that isothiocyanate could facilitate the adriamycin-induced apoptosis of U2-OS cells. The possible action mechanism was proposed.Method:The cryopreserved US-OS cells were resuscitated and observed and photographed under the microscope after culture. The cell viability was detected. The second-generation US-OS cells in good growth status were harvested. The cells reaching the logarithmic growth phase were treated by adriamycin with the concentration of 1ug/ml,2.5ug/ml,5ug/ml, 10ug/ml and 25ug/ml and isothiocyanate with the concentration of 2uM/ml,4uM/ml,8uM/ml,16uM/ml, 32uM/ml and 64uM/ml, respectively. The effect of the drugs on the inhibition rate was determined, and the IC50 was calculated. Next the cells were treated by adriamycin (ug/ml,3ug/ml, 10ug/ml) in combination with isothiocyanate (2uM/ml,2.5uM/ml and 4uM/ml) for 24h, respectively. The inhibition rate was estimated by MTT assay. Moreover, the histogram showing the relationship between the inhibition rate and the concentration was plotted. The combined effect of the two drugs was characterized by Q value, based on which the optimal effective concentration was determined. Then the cells were divided into blank group, adriamycin group, isothiocyanate group and adriamycin+isothiocyanate group based on the optimal effective concentration. After treatment for 24h, the cell apoptosis rate was detected by flow cytometry, and the variations of the cell apoptosis rate were observed. The same grouping method was applied to detect the activity of Caspase3 under the optimal effective concentration. Cell apoptosis was characterized by using TUNEL assay kit. The variations of cell apoptosis rate were measured in each group. The results were compared against those by flow cytometry to check for consistency. To discover the action mechanism, the AAH-APO-1 array (cell apoptosis antibody array) was used to detect the apoptosis-related factors. The cultured cells were divided into 4 groups, namely, blank group, adriamycin group, isothiocyanate group and adriamycin+isothiocyanate group. After treatment for 24h, about 40 common apoptosis-related factors were identified using the array. The factors with the largest variation were chosen for subsequent experiments. The mRNA expressions of Caspase3, Caspase9, Bcl-2 and Bax were detected in each group using real-time PCR. The protein expressions of Caspase3, Caspase 9, Bcl-2 and Bax were determined by Western Blotting. The results were compared between the groups. The consistency in variation was checked by comparing the Western Blotting results with the detection using cell apoptosis antibody array and real-time PCR.Results:The resuscitated cells were cultured conventionally. The second-generation cells adhered to the walls within 48h. The cells grew vigorously with compact arrangement and good viability. MTT assay indicated that the higher the concentrations of adriamycin and the higher the inhibition rate was. The IC50 of adriamycin was 10.322ug/ml, and that of isothiocyanate was 5.33uM/L. In the combination of 3ug/mladriamycin and 2.5uM/ml isothiocyanate, the inhibition rate of U2-OS cells was 70.482%, and the Q value 1.886. This suggested the good synergistic effect of the two drugs. These concentrations were selected for further study. The cultured U2-OS cells were randomly divided into four groups, namely, blank group, adriamycin group (3ug/ml), isothiocyanate group (2.5uM/ml) and adriamycin (3ug/ml)+isothiocyanate group (2.5uM/ml). The cell apoptosis rate was detected by flow cytometry after treatment for 24h. Compared with the earlier apoptosis rate, the apoptosis rate (%) of blank group, adriamycin group (3ug/ml), isothiocyanate group (2.5uM/ml) and adriamycin (3ug/ml)+isothiocyanate (2.5uM/ml) group was 3.2±0.6,8.5±2.3,9.2±3.1 and 35.2±7.9, respectively. Compared with the blank group and the single drug groups (SPSS 16.0 software, n=10), the combined treatment group showed a significant increase of cell apoptosis rate (p<0.05). The activity of Caspase3 was 42.86±10.376 in the combined treatment group, which was much higher than that of blank group (5.48±2.153), adriamycin group (13.62±4.874), isothiocyanate group (10.56±4.162) (p<0.05, n=3). Caspase 3 is the main executor of apoptosis, and its expression was positively correlated with cell apoptosis, as was confirmed by the results of flow cytometry. The apoptosis of U2-OS cells was detected by TUNEL staining. There were very few early apoptotic cells in the blank group. The apoptosis rate (%) of adriamycin group, isothiocyanate group and combined treatment group was 6.5±2.16,7.32.47 and 38.6±11.35, respectively. An increase of statistical significance was observed in the combined treatment group compared with the blank group and the single drug groups (SPSS 16.0 software, n=5) (p<0.01). The detection using AAH-APO-1 array indicated that the expressions of Caspase3, Caspase9 and Bax were significantly upregulated in the combined treatment group compared with the blank group and the single drug groups. However, the expression of Bcl-2 was downregulated considerably compared with the other three groups. In addition, XIAP-associated factor closely related to Bcl-2 family was also highly expressed, though the difference was not so marked in the combined treatment group. RT-PCR suggested that the expressions of Caspase3, Caspase9 and Bax were lower in the blank group, and an obvious increase was noted in the combined treatment group. The variation of Bcl-2 expression showed the opposite trend. The expression was higher in the blank group and lower in the combined treatment group. Consistent results were obtained by Western Blotting, with a significant increase observed in the expressions of Caspase 3, Caspase 9 and Bax. The largest variation amplitude was found with Caspase 3, and the expression of Bcl-2 was much decreased in the combined treatment group compared with the blank group.Conclusion1. Isothiocyanate and adriamycin had a synergistic effect in inhibiting the U2-OS cells.2. Isothiocyanate with a low concentration could promote the adriamycin-induced apoptosis of U2-OS cells in vitro.3. Isothiocyanate combined with adriamycin may promote cell apoptosis via the regulatory effect on the Bcl-2 family proteins.
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