紫草素通过调节PI3K/AKT途径抑制骨肉瘤生长和作用机制研究

Study on the mechanism of shikonin inhibiting osteosarcoma growth by regulating PI3K/AKT pathway

目的研究紫草素(shikonin,SK)的抗骨肉瘤作用及其机制。方法不同浓度的SK处理人骨肉瘤U2OS和MG63细胞及人成骨细胞HFOB1.1924 h或用SK 1μmol/L分别处理24、48和72 h后,CCK-8检测细胞活性;SK 0、0.01、0.1、1μmol/L处理U2OS细胞,克隆形成实验检测细胞增殖;流式检测细胞凋亡,Western blot检测凋亡相关蛋白的表达;U2OS细胞分为对照组,SK1μmol/L组,PI3K激活剂胰岛素样生长因子1(IGF-1)组和SK1μmol/L+IGF-1组,Western blot检测磷脂酰肌醇3-激酶(PI3K)和蛋白激酶B(AKT)磷酸化、胱天蛋白酶(caspase-3,cas3)、cleaved cas3、Ki67及干细胞标记物SOX-2、OCT-4和Nanog的表达;细胞成球实验检测细胞成球能力。右侧腹皮下注射U2OS细胞构建移植瘤裸鼠模型并进行体内验证。结果不同浓度的SK对HFOB1.19细胞无明显毒性作用,而对U2OS和MG63细胞有显著毒性作用(P<0.05),且具有浓度和时间依赖性。紫草素0.1和1μmol/L可显著抑制U2OS细胞增殖和干细胞样特征,诱导细胞凋亡(P<0.05),并抑制PI3K和AKT蛋白的磷酸化(P<0.05)。IGF-1明显逆转紫草素对PI3K/AKT信号通路的抑制作用,以及对U2OS细胞增殖、凋亡,干细胞样特征的影响(P<0.05)。体内实验表明,紫草素能显著抑制肿瘤的生长(P<0.05),并下调瘤组织中p-AKT的表达(P<0.05)。结论紫草素对骨肉瘤U2OS细胞生长和干细胞样特征的抑制作用可能与抑制PI3K/AKT信号通路的激活有关。

Objective To investigate the anti-osteosarcoma effect and mechanism of shikonin(SK).Methods After various concentrations of SK were applied to human U2 OS and MG63 osteosarcoma cells and human osteoblastic cell line HFOB1.19 for 24 h or SK 1μmol/L for 24,48 and 72 h,CCK-8 was used to assay cell viability.U2 OS cells were treated with SK at 0,0.01,0.1 and 1μmol/L and then colony formation assays were employed to assess cell proliferation.Flow cytometry was used to detect apoptosis.Western blot was performed to determine the expression of apoptosis-related proteins.U2 OS cells were divided into control,SK1μmol/L,PI3 K activator insulin-like growth factor-1(IGF-1),and SK1μmol/L+IGF-1 groups.Western blot was used to detect phosphorylation levels of phosphatidylinositol 3-kinase(PI3 K)and protein kinase B(AKT),and expression of caspase-3(cas3),cleaved cas3,Ki67 and stem cell markers SOX-2,OCT-4 and Nanog.Cell spheroid assay was used to detect the ability of cells to form spheres.U2 OS cells were injected subcutaneously into the right abdomen to establish a nude mouse model of tumors and verified in vivo.Results The various concentrations of SK had no obvious toxic effects on HFOB1.19 cells,but had significant toxic effects on U2 OS and MG63 cells(P<0.05)in concentration-and time-dependent manners.SK at 0.1 and 1μmol/L significantly inhibited U2 OS cell proliferation and stem cell-like characteristics,induced apoptosis(P<0.05),and inhibited phosphorylation of PI3 K and AKT proteins(P<0.05).IGF-1 obviously reversed the inhibitory effect of SK on the PI3 K/AKT signaling pathway and its effect on U2 OS cell proliferation,apoptosis,and stem cell-like characteristics(P<0.05).In vivo experiments showed that SK significantly inhibited tumor growth(P<0.05)and downregulated expression of p-AKT in tumor tissues(P<0.05).Conclusions The inhibitory effect of SK on the growth and stem cell-like characteristics of U2 OS osteosarcoma cells may be related to inhibition of PI3 K/AKT signaling pathway activation.