目的: 研究真菌次级代谢产物rasfonin对骨肉瘤143B细胞增殖与迁移能力的影响。方法: 以骨肉瘤细胞系143B为模型,利用3-(4,5-二甲基) -5-(3-羧甲基苯环)-2-(4-硫基苯)-2H-四唑盐复合物检测法[3-(4,5-dime-thylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, MTS]观察rasfonin对细胞活性的影响,以二甲基亚砜(dimethyl sulfoxide,DMSO)组为对照,采用rasfonin (3 μmol/L和6 μmol/L)处理12或24 h,观察143B细胞活性的变化;采用克隆形成实验检测rasfonin对细胞集落形成能力的影响,以DMSO组为对照,采用rasfonin (3 μmol/L)处理1周,对比两组克隆形成数目。划痕实验及侵袭实验检测rasfonin对细胞侵袭迁移能力的影响,以DMSO组为对照,采用rasfonin (3 μmol/L)处理24 h,对比两组的伤痕愈合率及侵袭细胞数。以DMSO组为对照,透射电子显微镜观察rasfonin(3 μmol/L)处理4 h后细胞内自噬体含量的变化。蛋白免疫印迹的方法检测rasfonin对p62蛋白(sequestosome 1)、微管相关蛋白1轻链3融合蛋白(microtubule associated protein 1 light chain 3 fusion protein, LC3)及聚腺苷酸二磷酸核糖转移酶[poly (ADP-ribose) polymerase-1, PARP-1]表达的影响。结果: 在12和24 h,随着rasfonin浓度的升高,骨肉瘤143B细胞的细胞活性均逐渐降低,3组间差异有统计学意义(12 h: F=31.36, P<0.01; 24 h: F=67.07, P<0.01),任意两组间比较差异也均有统计学意义(P<0.01)。3 μmol/L的rasfonin可以显著抑制143B细胞的集落形成能力(P<0.01)。Rasfonin处理后,143B细胞在24 h内的伤痕愈合比率明显低于DMSO对照组(33.91%±0.83% vs. 65.11%±0.94%, P<0.01);同样24 h内穿过覆盖有Matrigel的基底膜的细胞数明显低于DMSO对照组[(21.33±1.45)个vs.(49.33±2.40)个, P<0.01]。Rasfonin处理4 h后骨肉瘤143B细胞中自噬体增多,p62水平降低,LC3-Ⅱ累积,自噬抑制剂氯喹存在条件下LC3-Ⅱ累积更为明显,并且随着rasfonin浓度的提高PARP-1的切割增多。结论: Rasfonin既可以抑制骨肉瘤143B细胞的增殖迁移,还可以引起细胞的自噬与凋亡,这些结果为rasfonin作为骨肉瘤新的治疗药物提供了初步的实验基础。

Objective: To investigate the effects of rasfonin,a fungal secondary metabolite, on the proliferation and migration of osteosarcoma 143B cells.Methods: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay was performed to exa-mine 143B cell viability following treatment of rasfonin. Using dimethyl sulfoxide (DMSO) group as control, cell viability was detected when 143B cells were treated with rasfonin (3 μmol/L and 6 μmol/L) for 12 or 24 hours. The effect of rasfonin on colony forming ability was detected by clone formation assay.143B cells treated with DMSO or rasfonin (3 μmol/L) for one week, and the number of clones formed in the two groups was counted. Wound healing and transwell assay were employed to analyze cell invasion and migration upon rasfonin challenge. The DMSO group was used as control while rasfonin (3 μmol/L) was used for 24 hours. The wound healing rate and the number of invasive cells were compared between the two groups. The intracellular autophagosomes were monitored by transmission electron microscopy when 143B cells were treated with DMSO or rasfonin (3 μmol/L) for 4 hours. The expression of p62, microtubule-associated protein 1 light chain 3 fusion protein (LC3) and poly (ADP-ribose) polymerase-1(PARP-1) in response to rasfonin were detected by immunoblotting assay.Results: Rasfonin reduced the viability of 143B cells in a dose-dependent manner (12 h: F=31.36, P<0.01; 24 h: F=67.07, P<0.01). Rasfonin (3 μmol/L) completely inhibited the clonal formation of 143B cells (P<0.01). The wound healing result revealed that rasfonin significantly decreased migratory ability of 143B cells (33.91%±0.83% vs. 65.11%±0.94%, P<0.01), whereas its treatment significantly reduced the number of 143B cells penetrating through Matrigel-containing basement membrane (21.33±1.45 vs. 49.33±2.40, P<0.01). Compared with the control group, rasfonin markedly increased the number of autophagic vacuoles. The immunoblotting results revealed that rasfonin increased LC3-Ⅱ accumulation and decreased p62 levels. Choloroquine (CQ), an often used autophagic inhibitor, further accumulated rasfonin-induced LC3-Ⅱ. In addition, rasfonin appeared to cause the cleavage of PARP-1.Conclusion: Rasfonin induced autophagy and activated caspase-dependent apoptosis in 143B cells concurring with suppressing the proliferation and migration of the cells; these results provide an experimental basis for rasfonin as a potential therapeutic agent for osteosarcoma.