The pro-adhesive and pro-survival effects of glucocorticoid in human ovarian cancer cells

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   Figure 1

   DEX upregulates the expression of FN, but does not affect the expression of collagens and secretion of laminin in ovarian cancer cells. HO-8910 cells were treated with 10⁻⁷mol L⁻¹ DEX for the indicated times. The protein levels of collagen I, III, and IV were assessed by Western blot (A) and secreted protein of laminin was determined by ELISA (B) as described in Materials and methods. The expression of FN1 mRNA in HO-8910 cells treated with 10⁻⁷mol L⁻¹ DEX for the indicated times was assessed by Real-time PCR (C) normalized to GAPDH. Data shown are representative of at least three separate experiments. FN expression in HO-8910 (D) and SKOV-3 cells (E) treated with 10⁻⁷mol L⁻¹ DEX for the indicated times were assessed by Western blot and β-actin was used as normalization control. Relative density for Western blot was analyzed by ImageJ 1.40g software and the results were expressed as fold of control and representative of at least three independent experiments. The secreted levels of FN in SKOV-3 cells treated with or without 10⁻⁷mol L⁻¹ DEX for 72h were determined by ELISA. The concentration of FN was calibrated from a dose–response curve based on reference standards. The experiments were performed in triplicate (F). *P<0.05, **P<0.01 vs control.
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   Figure 2

   Upregulated expression of FN contributes to the pro-adhesive effect of DEX in ovarian cancer cells. HO-8910 or SKOV-3 cells were transfected with 10−8mol L−1 FN1 siRNA or Con siRNA for 24h and then treated with or without 10−7mol L−1 DEX for another 24h. FN knockdown was monitored at the protein level by Western blot and β-actin was used as a loading control (A and B). Single-cell suspension was prepared and 8×104 cells were seeded into a noncoated 96-well plate. 45 min later, cells were washed thrice with PBS, and the number of the remaining cells attached was determined by MTT assay (C and D). Data are summarized from three independent sets of experiments. *P<0.05, **P<0.01 vs Con siRNA without DEX, #P<0.05, ##P<0.01 vs Con siRNA with DEX.
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   Figure 3

   CD44 is involved in the pro-adhesive effect of DEX through interaction with the increased FN. HO-8910 cells were cultured with or without 10−7mol L−1 DEX for the indicated times, and the protein level of CD44 (A) and the secreted level of HA (B) were assessed by Western blot and ELISA, respectively. HO-8910 (C) or SKOV-3 (D) cells were treated with or without 40μg/mL CD44-blocking antibody for 45 min, and then seeded into 96-well plates coated with or without human fibronectin (50μg/mL). Cell adhesion was assayed as described in Materials and Methods. (E and F) Cells were treated with or without 10−7mol L−1 DEX for 24h, and then cultured continuously in the presence or absence of 20 or 40μg/mL CD44-blocking antibody for another 45 min. Cell adhesion was assayed with noncoated 96-well plates. Data are summarized from three independent sets of experiments. **P<0.01 vs control, #P<0.05, ##P<0.01 vs FN-coated (C and D), #P<0.05 vs DEX (E and F).
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   Figure 4

   Activation of Akt pathway is involved in pro-survival effect of DEX in ovarian cancer cells. The protein levels of p-AKT and total AKT were assessed in HO-8910 (A) and SKOV-3 (B) cells treated with 10−7mol L−1 DEX for the indicated times. Total AKT was detected as an internal control. The viable cellswere analyzed by CCK-8 kit after treatment of HO-8910 (C) and SKOV-3 (D) cells treated with paclitaxel (PTX), DEX, wortmannin, PTX and DEX, DEX and wortmannin, or the combination of PTX, DEX, and wortmannin, for 24h. Figure 4E was operated under the same condition besides replacing wortmannin by MK-2206. *P<0.05, **P<0.01 vs control, ##P<0.01 vs PTX, &P<0.05, &&P<0.01 vs PTX with DEX.
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   Figure 5

   DEX upregulates the expression of MUC1 in ovarian cancer cells. HO-8910 (A and C) and SKOV-3 (B and D) cells were treated with 10−7mol L−1 DEX for the indicated times, and the levels of MUC1 mRNA and protein were assessed by real-time PCR and western blot analysis using GAPDH or β-actin as normalization controls, respectively. The results were expressed as fold over control and represented at least three independent experiments. *P< 0.05, **P<0.01 vs 0.
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   Figure 6

   MUC1 partially mediates DEX-induced pro-adhesion, AKT activation, and enhancement of chemotherapeutic resistance in ovarian cancer cells. HO-8910 (A) or SKOV-3 (B) cells were transfected with 5×10−9mol L−1 MUC1 siRNA or Con siRNA for 24h and then cultured with or without 10−7mol L−1 DEX for another 24h followed by cell adhesion assessment. Knockdown of MUC1 was confirmed by Western blot. The protein levels of p-AKT and total AKT in HO-8910 cells in each group were examined by western blot, and total AKT was used as a normalization control (C). HO-8910 (D) or SKOV-3 (E) cells were seeded in 96-well culture plates in triplicate and were transfected with 5×10−9mol L−1 MUC1 siRNA or Con siRNA for 24h. Cells were pretreated with or without 10−7mol L−1 DEX for 24h, and then cultured continuously in the presence or absence of 5×10−7mol L−1 PTX for another 48h. Cell survival was analyzed by CCK-8 kit. Data are summarized from three independent sets of experiments. *P<0.05, **P<0.01 vs Con siRNA, or Con siRNA combined with PTX.#P<0.05 vs Con siRNA combined with PTX and DEX.

• © 2016 Society for Endocrinology