[ 摘要] 目的 探讨白细胞介素6（IL -6）单克隆抗体-siltuximab（CNTO328）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；（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介导的抗调往蛋白——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（IC 50 ）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 河南省人民医院妇科郭玉琪
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和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和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、10µg/ml）处理后，SKOV3/TR细胞对紫杉醇的IC 50 （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IC 50 （分别为0.0010和0.0008µg/ml）明显低于未经siltuximab处理者（0.0021µg/ml； P <0.01）。 结论 siltuximab能有效阻断卵巢癌中IL-6/Stat3信号通路，可能对卵巢癌的治疗有重要作用。
[ 关键词] 抗体，单克隆；卵巢 肿瘤 ；白细胞介素6；STAT3转录因子；信号传导
Effects of Siltuximab on the interleukin-6/Stat3 Signaling Pathway in Ovarian Cancer
[Abstract ] 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) Expressions 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-p EGFP-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 thiazolium(MTT). The 50%inhibiting concentration(IC 50 ) was defined as the paclitaxel concentration required to decrease the A 490 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 10µg/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 pEGFP-Stat3 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 concentration of 0.001, 0.01, 0.1, 1.0, and 10µg/ml, followed by an incubation in IL-6 significantly reduced pEGFP-Stat3 nucleocytoplasmic ntranslocation. (4) MTT cytotoxicity assay demonstrated that siltuximab increased paclitaxel-induced cell death and partially overcame paclitaxel resistance. Treated with siltuximab (1 and 10µg/ml), the paclitaxel IC 50 value of siltuximab in SKOV3/TR (0.49, 0.19µg/ml) and CAOV3/TR (0.0010, 0.008µg/ml) cells were significantly lower than those in untreated cells (0.71, 0.0021µg/ml; all P <0.05). Conclusions These results demonstrated that siltuximab effectively block the IL-6 signaling pathway s. Blockage of IL-6 signaling may provide benefits for the treatment of ovarian cancer.
[Key words] Antibodies, monoclonal; Ovarian neoplasms Interleukin-6; STAT3 transcription factor; Signal transduction
