siltuximab单克隆抗体对卵巢上皮性癌中白细胞介素6/Stat3信号传导通路的影响

摘要

目的 探讨白细胞介素6(IL-6)单克隆抗体--siltuximab对卵巢上皮性癌(卵巢癌)中IL-6/信号传导及转录活化因子3(Stat3)信号传导通路的影响.方法 (1)选取美国哈佛大学麻省总医院近20年来确诊为卵巢癌的26例患者的癌组织标本,每例患者的标本均包含原发性、转移性和复发性癌组织,免疫组化法检测26例卵巢癌患者癌组织中IL-6蛋白的表达;(2)蛋白印迹法检测IL-6、siltuximab联合IL-6处理后卵巢癌细胞株SKOV3细胞中磷酸化Stat3(pStat3)蛋白的表达,以及siltuximab处理后卵巢癌紫杉醇耐药细胞株SKOV3/TR和CAOV3/TR细胞中Stat3介导的抗凋亡蛋白--bcl-XL、MCL-1、survivin蛋白的表达;(3)实时细胞技术检测siltuximab联合IL-6处理后SKOV3-pEGFP-Stat3细胞中pEGFP-Stat3融合蛋白的核转移情况;(4)四甲基偶氮唑蓝比色法检测siltuximab处理后SKOV3/TR和CAOV3/TR细胞对紫杉醇的敏感性,以50%抑制浓度(IC50)表示.结果 (1)卵巢癌患者转移性和复发性癌组织中IL-6蛋白的染色强度明显高于原发性癌组织;且转移性和复发性癌组织中IL-6蛋白的阳性表达率[分别为69%(18/26)和77%(20/26)]明显高于原发性癌组织[23%(6/26),P＜0.05].(2)IL-6处理的SKOV3细胞中pStat3蛋白的表达强度明显高于未经IL-6处理者;siltuximab(浓度分别为0.01、0.1、1和10μg/ml)联合IL-6处理后,SKOV3细胞中pStat3蛋白的表达强度随siltuximab浓度的增加明显减弱;经不同浓度(0.001、0.01、0.1、1、10μg/ml)的siltuximab处理后,SKOV3/TR和CAOV3/TR细胞中bcl-XL、MCL-1、survivin蛋白的表达强度均明显低于未经siltuximab处理者.(3)IL-6处理后,pEGFP-Stat3融合蛋白迅速转移到SKOV3-pEGFP-Stat3细胞的细胞核中;siltuximab(浓度分别为0.001、0.01、0.1、1、10μg/ml)联合IL-6处理后,pEGFP-Stat3融合蛋白的核转移随siltuximab浓度的增加逐渐减少.(4)不同浓度的siltuximab(分别为1、10 μg/ml)处理后,SKOV3/TR细胞对紫杉醇的IC50(分别为0.49和0.19μg/ml)明显低于未经siltuximab处理者(0.71μg/ml;P＜0.05);CAOV3/TR细胞对紫杉醇的IC50(分别为0.0010和0.0008μg/ml)明显低于未经siltuximab处理者(0.0021 μg/ml;P＜0.01).结论 siltuximab能有效阻断卵巢癌中IL-6/Stat3信号传导通路,可能对卵巢癌的治疗有重要作用.%Objective To study the effects of siltuximab on the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (Stat3) signaling pathway in ovarian epithelial carcinoma.Methods (1) Expressions of IL-6 in ovarian cancer patient specimens were assessed by immunohistochemistry.(2) Expression of phosphorylation Stat3 (pStat3) protein in siltuximab and IL-6 treated SKOV3 cell lines was determined by western blot, and expression levels of Stat3-induced bcl-XL,MCL-1, survivin, in siltuximab treated SKOV3/TR and CAOV3/TR cells lines were also determined by western blot.(3) Real-time image analysis was used to study the nuclear translocation of pEGFP-Stat3 fusion protein in ovarian cancer cell line SKOV3-pEGFP-Stat3 treated with siltuximab and IL-6.(4)Paclitaxel sensitivity in siltuximab treated SKOV3/TR and CAOV3/TR cell lines were assessed using the methyl thiazolyl tetrazolium (MTT).The 50% inhibiting concentration ( IC50 ) was defined as the paclitaxel concentration required to decrease the A490 value to 50%.Results ( 1 ) There were significantly difference in IL-6 staining density and the positive rate of IL-6 protein stained among the metastatic, and drug-resistant recurrent tumors,and matched primary tumors [69%(18/26)] vs.77% (20/26)vs.23% (6/26), P＜0.05].(2)A clear increase in Stat3 phosphorylation levels was observed in the IL-6-treated SKOV3 cell lines as compared to the SKOV3 cell lines.When the IL-6-treated SKOV3 cells were incubated with siltuximab with a range of concentrations of 0.001,0.01,0.1, 1.0 and 10 μg/ml, there were trends toward reduced pStat3 expression in the treated cell lines.Compared without treatment with siltuximab, the expression of the anti-apoptotic proteins MCL-1, bcl-XL and survivin in SKOV3/TR and CAOV3/TR cell lines were significantly decreased after treated with siltuximab.(3) In resting cells, the majority of pEGFPStat3 was cytoplasmic until the addition of human IL-6, which promptly induced the translocation of fluorescent Stat3 molecules to the nucleus.Exposure of cells to siltuximab with a range of concentrations of 0.001, 0.01,0.1, 1.0 and 10 μg/ml, followed by an incubation in IL-6 significantly reduced pEGFP-Stat3 nucleocytoplasmic translocation.(4) MTT cytotoxicity assay demonstrated that siltuximab increased paclitaxel-induced cell death and partially overcame paclitaxel resistance.Treated with siltuximab ( 1 and 10 μg/ml) ,the paclitaxel IC50 value of siltuximab in SKOV3/TR (0.49, 0.19 μg/ml) and CAOV3/TR (0.0010, 0.0008 μg/ml) cells were significantly lower than those in untreated cells (0.71,0.0021 μg/ml;all P ＜ 0.05).Conclusions These results demonstrated that siltuximab effectively block the IL-6 signaling pathways, which .Blockage of IL-6 signaling may provide benefits for the treatment of ovarian cancer.