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

Effects of sfltuximab on the interleukin-6/Stat3 signaling pathway in ovarian cancer

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.