Guo Yu-qi, Lu Ping, Duan Zhen-feng, Zhang Zhan
Department of Obstetrics and Gynecology, Zhengzhou University, Zhengzhou 450052, China.
Zhonghua Fu Chan Ke Za Zhi. 2010 Nov;45(11):854-9.
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.
(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 (IC(50)) was defined as the paclitaxel concentration required to decrease the A(490) value to 50%.
(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 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 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 IC(50) 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).
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.
研究西妥昔单抗对卵巢上皮癌白细胞介素-6（IL-6）/信号转导及转录激活因子3（Stat3）信号通路的影响。
（1）采用免疫组织化学法评估卵巢癌患者标本中IL-6的表达。（2）通过蛋白质免疫印迹法检测西妥昔单抗和IL-6处理的SKOV3细胞系中磷酸化Stat3（pStat3）蛋白的表达，并用蛋白质免疫印迹法检测西妥昔单抗处理的SKOV3/TR和CAOV3/TR细胞系中Stat3诱导的bcl-XL、MCL-1、生存素的表达水平。（3）采用实时图像分析研究西妥昔单抗和IL-6处理的卵巢癌细胞系SKOV3-pEGFP-Stat3中pEGFP-Stat3融合蛋白的核转位。（4）用噻唑蓝（MTT）法评估西妥昔单抗处理的SKOV3/TR和CAOV3/TR细胞系对紫杉醇的敏感性。将50%抑制浓度（IC50）定义为使A490值降低至50%所需的紫杉醇浓度。
（1）转移瘤、耐药复发性肿瘤及配对的原发性肿瘤中IL-6染色密度及IL-6蛋白染色阳性率存在显著差异[69%（18/26）对77%（20/26）对23%（6/26），P<0.05]。（2）与SKOV3细胞系相比，IL-6处理的SKOV3细胞系中Stat3磷酸化水平明显升高。当IL-6处理的SKOV3细胞与浓度为0.001、0.01、0.1、1.0和10μg/ml的西妥昔单抗孵育时，处理后的细胞系中pStat3表达有降低趋势。与未用西妥昔单抗处理相比，西妥昔单抗处理后SKOV3/TR和CAOV3/TR细胞系中抗凋亡蛋白MCL-1、bcl-XL和生存素的表达明显降低。（3）在静息细胞中，大多数pEGFP-Stat3位于细胞质中，直到加入人IL-6，其迅速诱导荧光Stat3分子转位至细胞核。将细胞暴露于浓度为0.001、0.01、0.1、1.0和10μg/ml的西妥昔单抗，然后在IL-6中孵育，显著降低了pEGFP-Stat3的核质转位。（4）MTT细胞毒性试验表明，西妥昔单抗增加了紫杉醇诱导的细胞死亡并部分克服了紫杉醇耐药性。用西妥昔单抗（1和10μg/ml）处理后，西妥昔单抗在SKOV3/TR（0.49，0.19μg/ml）和CAOV3/TR（0.0010，0.0008μg/ml）细胞中的紫杉醇IC50值显著低于未处理细胞（0.71，0.0021μg/ml；均P<0.05）。
这些结果表明西妥昔单抗有效阻断IL-6信号通路，阻断IL-6信号通路可能对卵巢癌治疗有益。
