华蟾素诱导骨肉瘤细胞凋亡及其机制

Effect and Mechanism of Cinobufotalin Induces Apoptosis in Osteosarcoma Cells

目的:探讨华蟾素诱导骨肉瘤U2OS细胞凋亡的效果及其可能机制。方法:体外培养人骨肉瘤细胞系U2OS,将其分为第1,2,3,4,5组,于其培养基内分别加入0,0.5,1.0,1.5,2.0μg/mL华蟾素,每组分别于培养的第24,48,72h进行检测。MTT法检测各组骨肉瘤细胞生长的抑制率,TUNEL染色观察第4组细胞凋亡程度并计算其凋亡率,免疫荧光染色检测第4组凋亡相关蛋白Bax和Bcl-2的含量并计算其比值,RT-PCR检测Bax和Bcl-2的表达。结果:MTT试验显示,随着各组华蟾素浓度的增加,细胞生长抑制率明显增加,其中第5组48h生长抑制率增加至(59.56±1.95)%,相当于第2组同期(22.85±1.34)%的2.5倍(P<0.01)。随着培养时间的延长,各组细胞生长抑制均有不同程度的增加,以第5组72h最为显著。Tunel染色显示,随着华蟾素干预时间的延长,U20S细胞凋亡率显著上升,与华蟾素作用于细胞24,48时相比,作用72h后凋亡率达到(32.35±1.57)%,凋亡率明显增加(P<0.01)。免疫荧光和RT-PCR提示,随着华蟾素作用时间的延长,第4组Bax的表达逐渐增加,而Bcl-2的表达逐渐减少,同时Bax/Bcl-2含量的比值也逐渐增加。结论:华蟾素具有抑制骨肉瘤U2OS细胞增殖和诱导其凋亡的作用,其疗效呈剂量和时间依赖性,而其机制可能与华蟾素影响Bax和Bcl-2的表达有关。华蟾素具有治疗骨肉瘤的潜力,值得进一步研究探讨。

Objective：To investigate the effect and mechanism of cinobufotalin induced apoptosis in osteosarcoma cell line U2OS. Methods： Human osteosareoma cell line U2OS were cultured in vitro and divided into 1st, 2nd, 3rd, 4th, and 5th group. Then, different concentration of cinobufotalin （0,0.5,1.0,1.5,and 2.0 vg/mL） were added to their respective medium. The 3-[4,5-dimethyhhiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT）assay was used to evaluate the inhibition rate of osteosarcoma cell growth. The TUNEL staining methods was used to detect the change in cell morphology of apoptosis in the 4th group, and the apoptosis rate was calculated. The immunofluorescence and reverse transcription-polymerase chain reaction （RT-PCR）were used to detect the expression of apoptosis-related proteins,and then the Bax/Bcl-2 ratio was calculated. Results：The MTT assay showed that with the increasing of the concentration of cinobufotalin, the inhibition of cell growth was significantly increased. The growth inhibition rate of 4th group was increased to （59.56 ± 1.95）%, equiv alent to 2.5 times of 1st group （22.85±1.34）% 48 h after treated with cinobufotalin （P〈0. 01）. With the extension of incubation time,the cell growth of each group was all inhibited, especially in the 4th group 72 h after treatment. Tunel staining showed that with the extension of incubation time, the U2OS cell apoptosis rate was significantly increased. The cell apoptosis rate was significantly increased to （32.35 ±1.57）% at 72 h, which was significantly higher than that at 24 h and 28 h after treated with cinobufotalin （P〈0.01）. The results of immunofluorescence and RT-PCR showed that with the extension time of incubation with cinobufotalin in the 4th group, the expression of Bax was increased, while Bcl-2 was decreased,moreover,the ratio of Bax/Bcl-2 was also increased. Conclusion. Cinobufotalin can induce apoptosis in U2OS cells,and its affects are in a dose-and time-dependent manner. The possible mechanisms are related to the expression of Bax and Bel-2. Cinobufotalin is potentially effective to treat osteosarcoma, so it is worthy for further investigation.