Fig. 4. SC66 regulates cell sensitivity to anticancer drugs and cell invasiveness through inhibition of COL11A1.

a A2780CP70 and ES-2/CP cells were treated with different concentrations of SC66 for 24 h and then COL11A1 expression was evaluated by real-time RT-PCR. All experiments were performed in triplicate. *P < 0.05 and **P < 0.005, SC66 vs. control. b A2780CP70 and ES-2/CP cells transfected with the COL11A1 promoters were treated with different concentrations of SC66 for 24 h. Luciferase activity was measured and normalized to Renilla luciferase activity. All experiments were performed in triplicate. *P < 0.05 and **P < 0.005, SC66 vs. control. c ChIP assays were performed to evaluate c/EBPβ and NF-YA binding to the COL11A1 promoter in A2780CP70 and ES-2/CP cells after treatment with different concentrations of SC66 for 24 h. d Left panel: Protein expression levels of COL11A1, p-Akt, and Akt in A2780 cells transfected with the COL11A1 plasmid and in A2780CP70 cells transfected with the shCOL11A1 plasmid were evaluated by western blotting. β-Actin protein was used as an internal loading control. Right panel: Ovarian cancer cells were treated with different concentrations of cisplatin (CDDP, 0–32 μM) or paclitaxel (PAC, 0–64 μM), or combined with 2 μM SC66 for 48 h. Each combination was tested with n = 5 replicates. After 48 h, cell viability was assessed by MTT assays. All experiments were performed in triplicate. The IC₅₀ values of each agent in single or combination treatments and CI values of the SC66 + CDDP or SC66 + PAC combinations. P-value between the IC₅₀ values of single vs. combination treatment. e A2780/V and A2780/COL11A1 cells were treated for 24 h with 2 μM SC66 alone or with the addition of anticancer drugs (10 μM) indicated. The percentage of apoptotic cells was determined by Annexin V and PI staining. Mean ± SD for three independent experiments are shown. **P < 0.005, SC66 + CDDP vs. CDDP or SC66 + PAC vs. PAC. f Colony formation assay. A2780/V and A2780/COL11A1 cells were treated with 2 μM SC66 alone or with the addition anticancer drugs (10 μM) as indicated for 14 d. After treatment, cells were stained with crystal violet. Mean ± SD for three independent experiments are shown. **P < 0.005, SC66 + CDDP vs. CDDP or SC66 + PAC vs. PAC. g Invasion activity in vitro of A2780/V and A2780/COL11A1 cells after treatment with different concentrations of SC66 for 24 h. All data represent the mean ± SD of three separate experiments. *P < 0.05 and **P < 0.005, SC66 vs. control. h Protein expression levels of COL11A1, p-Akt, Akt, p-p70S6K, p70S6K, p-4EBP1, 4EBP1, and MMP3 in A2780/V and A2780/COL11A1 cells treated with different concentrations of SC66 for 24 h were evaluated by western blotting. β-Actin was used as an internal loading control. All experiments were performed in triplicate. i MMP3 activity was evaluated by casein zymography in A2780/V and A2780/COL11A1 cells treated with different concentrations of SC66 for 24 h. All experiments were performed in triplicate. j A model illustrating the hypothetical role of SC66 in controlling COL11A1-mediated chemoresistance and invasiveness in ovarian cancer cells