Proinflammatory Cytokines IL-6 and TNF-α Increased Telomerase Activity through NF-κB/STAT1/STAT3 Activation, and Withaferin A Inhibited the Signaling in Colorectal Cancer Cells

Figure 3

STAT3 binds hTERT promoter region. There are two STAT3 binding sites located in the promoter region of hTERT. Primer sets were designed flanking the putative STAT3-binding sites 1 and 2. Chip assay was performed with the primers. (a) Diagram of hTERT promoter with location of consensus STAT3-binding sites 1 and 2 indicated. (b) Chip assay was performed with the DLD1 lysates using anti-STAT3 antibody. Cells were pretreated with IL-6 and TNF-α alone or in combination. (c) Chip assay was performed with DLD1 cells stimulated with IL-6 and TNF-α, and DLD1 cells cotreated with IL-6, TNF-α, and withaferin A. (d) Western blot of hTERT proteins. DLD1 cells were treated with IL-6 and TNF-α and cotreated with IL-6, TNF-α, and withaferin A and monitored for the hTERT expression levels. (e) TRAP-PCR-ELISA assay for telomerase activity. DLD1 cells of 2 × 105 were treated with IL-6 (10 ng/ml) for 24 h, TNF-α (25 ng/ml) for 24 h alone, and in combination. Withaferin A (10 μM) was treated with the cytokines and applied to TRAP-PCR-ELISA assay to monitor telomerase assay. Telomerase activities were measured three times independently. All of the data are presented as mean ± SD ( in each group). , versus untreated control. (f) Less amount of cells, 1 × 104, was treated with IL-6 and TNF-α alone and in combination. We decreased the cell amounts to discern cytokine and withaferin A effects on telomerase more clearly. , versus untreated control.

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