细菌氧化还原蛋白azurin选择性诱导人骨肉瘤U2OS细胞凋亡

Selective Inducement Effect of Bacterial Redox Protein Azurin on Apoptosis of Human Osteosarcoma Cell Line U2OS

背景与目的:已有研究表明细菌氧化还原蛋白azurin,在体外和荷瘤裸鼠体内可选择性的抑制某些黑色素瘤细胞和人乳腺癌细胞增殖并诱导和激发肿瘤细胞的凋亡,同时发现azurin在荷瘤裸鼠体内诱导肿瘤凋亡使肿瘤体积明显减小,但未发现毒副作用。azurin如何诱导肿瘤细胞凋亡从而达到治疗肿瘤的机制目前仍不明确。本研究旨在探讨azurin对人骨肉瘤U2OS细胞诱导凋亡作用及其可能的分子机制。方法:以不同浓度的azurin培养U2OS细胞。通过MTT法测细胞活性,用Bliss法计算IC50值,荧光显微镜、电镜观察亚细胞结构和凋亡小体形成,琼脂糖凝胶电泳观察DNA“ladder”形成,流式细胞仪检测细胞凋亡发生率,Westernblot检测细胞Bax、Bcl-2和caspase-3表达情况。结果:细菌氧化还原蛋白azurin能选择性抑制人骨肉瘤U2OS细胞生长且呈浓度依赖关系并能诱导细胞凋亡,IC50值为(114.54±7.65)mg/L,均高于相同浓度组的人骨肉瘤细胞MG63及正常人肝细胞L-02细胞组(P<0.05)。100mg/L和200mg/Lazurin作用24h后,AO、Hoechst33258和透射电镜见较多的凋亡细胞,出现凋亡小体,部分细胞坏死。琼脂糖凝胶电泳见典型的“ladder”,Annexin-V/PI的FCM检测azurin作用3h就出现凋亡现象,200mg/L的azurin作用48h时凋亡指数达到峰值35.8%,细胞停滞于G1期,而对?

BACKGROUND & OBJECTIVE: Bacterial redox protein azurin can selectively inhibit proliferation, and induce apoptosis in various human cancer cells. These in vitro findings have been confirmed in vivo in nude mice bearing tumor xenograft. Furthermore, azurin could diminish tumor volume in nude mice bearing tumor xenograft with no evidence of toxicity. The mechanism of its action is unknown. This study was designed to explore the effects of azurin on apoptosis of osteosarcoma cell line U2OS, and its molecular mechanism. METHODS: U2OS cells were cultured with different concentrations of azurin. Cell viability was detected by MTT assay. The 50% inhibitory concentration (IC50) of azurin was calculated by Bliss method. Apoptotic morphology and apoptotic body of U2OS cells were observed under fluorescent microscope, and transmission electron microscope. DNA ladder was observed through agarose gel electrophoresis. Cell apoptosis was determined by flow cytometry (FCM). Protein levels of Bax, Bcl-2, and Caspase-3 were quantified by Western blot. RESULTS: Azurin inhibited growth and induced apoptosis of U2OS cells in a dose-dependent manner 48 h after treatment, with the IC50 of(114.54±7.65) mg/L. Moreover, its inhibitory effect was significantly higher on U2OS cells than on MG63 cells or L-02 cells under the same concentrations (P<0.05). When treated with 100 or 200 mg/L of azurin for 24 h, U2OS cells showed typical apoptotic morphology, typical DNA ladder bands were observed. No apoptotic feature was observed in control cells. Sub-G1 peak and apoptotic peak of U2OS cells were observed when exposed to 200 mg/L of azurin for 48 h with the apoptosis index of 35.8%, cell cycle was arrested in G1 phase. Bcl-2 was down-regulated when U2OS cells were treated with azurin for 24 h, while Bax and caspase-3 were up-regulated. CONCLUSIONS: Azurin could selectively induce apoptosis of human osteosarcoma U2OS cells. The induction of apoptosis by azurin may be closely associated with down-regulation of Bcl-2, up-regulation of Bax, and activation of Caspase-3.