Research On The Anti-tumor Effects Of Cinobufacini In Osteosarcoma Cells And Its Mechanisms

Part1Research of Cinobufacini induces apoptosis in osteosarcoma cells and its possible mechanism[Aim]:To investigate the effects of Cinobufacini induces apoptosis in osteosarcoma cell line, U2OS and the possible mechanism:[Methods]:MTT assay was used to evaluate the effects of Cinobufacini on cell proliferation in U2OS cells. Changes in cell apoptosis was detected by TUNEL staining. The expression of apoptosis-related and Wnt/β-catenin pathway proteins were detected by immunofluorescence, RT-PCR, and western blot analysis.[Results]:The result of MTT indicated that Cinobufacini significantly inhibited cell proliferation in a dose-and time-dependent manner. Marked changes in cell apoptosis was clearly observed after cinobufacini treatment. Compared with cells were treated after24,48h, When cells were treated after72h, the expression of Bax was gradually increased, while Bcl-2was decreased, the ratio of bax/bcl-2was also increased (P<0.05). The Wnt/β-catenin pathway-related protein, the expression of β-catenin was positive in cells after Cinobufacini treatment. [Conclusion]:Cinobufacini can induce apoptosis in U2OS cells, the affects are in a dose-and time-dependent manner. The possible mechanism is related to the activation of Wnt/p-catenin pathway. Part2Research on the anti-tumor effects of Cinobufacini combined with chemotherapy in osteosarcoma cells[Aim]:To investigate the anti-tumor effects of Cinobufacini combined with chemotherapy in osteosarcoma cells.[Methods]:The experiment was divided into four groups, namely the control group, Cinobufotalin group, paclitaxel group, and combination group (cinobufotalin+paclitaxel). MTT assay was used to evaluate the effects of each group on cell proliferation in U2OS cells. Change in cell apoptosis was detected by TUNEL staining. The expression of apoptosis-related and Wnt/β-catenin pathway proteins were detected by immunofluo-rescence, RT-PCR, and Western Blot analysis.[Results]:The result of MTT indicated that the cell inhibition rate in cinobufacini group was lower than in paclitaxel group, while the rate was highest in combination group. Marked change in cell apoptosis rate was clearly observed after Cinobufacini combined with chemotherapy treatment on cells for48h when compared with monotherapy (P<0.05). Immunofluorescence staining showed:after the cells were treated by Cinobufotalin, paclitaxel, and the combination of both, the fluorescence intensity of bax, P-catenin were all increased when compared with the control group, while the fluorescence intensity of bcl-2was weakened, but the changes of their fluorescence intensity in combined group were more obvious. Immunofluorescence staining and RT-PCR results showed that: Cinobufotalin, paclitaxel, and their combination could increase the expression of both bax、 β-catenin and reduce the expression of bcl-2, but the combination therapy on up-regulating the expression of bax、β-catenin and lowering bcl-2expression was stronger than monotherapy(P<0.05). [Conclusion]:the anti-tumor effects of Cinobufacini combined with chemotherapy were significant in osteosarcoma cells, the expression of bax was increased, while bcl-2was reduced, which may be related to activation of Wnt/(3-catenin pathway.