[Effects of siltuximab on the interleukin-6/Stat3 signaling pathway in ovarian cancer].

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 (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 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).

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.