Roflumilast enhances cisplatin-sensitivity and reverses cisplatin-resistance of ovarian cancer cells via cAMP/PKA/CREB-FtMt signalling axis

Abstract

Objective: We previously demonstrated the roflumilast inhibited cell proliferation and increased cell apoptosis in ovarian cancer. In this study, we aimed to investigate the roles of roflumilast in development of cisplatin (DDP)-sensitive and -resistant ovarian cancer.

Methods: OVCAR3 and SKOV3 were selected and the corresponding DDP-resistant cells were constructed. Cell viability, proliferation, apoptosis, cycle were performed. Expression cAMP, PKA, CREB, phosphorylation of CREB and FtMt were detected. The roles of roflumilast in development of DDP-sensitive and -resistant ovarian cancer were confirmed by xenograft model.

Results: Roflumilast + DDP inhibited cell proliferation, and induced cell apoptosis and G0/G1 arrest in OVCAR3 and SKOV3 cells, roflumilast induced expression of FtMt, the activity of cAMP and PKA and phosphorylation of CREB in ovarian cancer cells and the above-effect were inhibited by H89. Downregulation of CREB inhibited the roflumilast-increased DDP sensitivity of ovarian cancer cells, and the roflumilast-induced FtMt expression and phosphorylation of CREB. Also, roflumilast reversed cisplatin-resistance, and induced expression of FtMt and activation of cAMP/PKA/CREB in DDP-resistant ovarian cancer cells. Similarly, treated with H89 or downregulation of CREB inhibited the changes induced by roflumilast. In vivo, roflumilast inhibited the development of SKOV3 or SKOV3-DDP-R xenograft models.

Conclusions: Roflumilast enhanced DDP sensitivity and reversed the DDP resistance of ovarian cancer cells via activation of cAMP/PKA/CREB pathway and upregulation of the downstream FtMt expression, which has great promise in clinical treatment.

Figure 1

Effect of roflumilast on DDP sensitivity in ovarian cancer cells. OVCAR3 and SKOV3 cells were exposed to different concentrations of roflumilast. Inhibition rates in A, OVCAR3 and B, SKOV3 cells were determined. Then, after administration of DDP (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 μg/mL) or DDP (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 μg/mL) + roflumilast (15 μM), flow cytometry and Hoechst assays were performed. Apoptosis in C, OVCAR3 and D, SKOV3 cells was evaluated. Hoechst staining was performed in E, OVCAR3 and F, SKOV3 cells. Cell cycle distribution was determined in G, OVCAR3 and H, SKOV3 cells

Figure 2

Effect of roflumilast on expression of FtMt. OVCAR3 and SKggOV3 cells were treated with DDP (1 μg/mL) or DDP (1 μg/mL) + roflumilast (15 μM) for 24 h. The expression of FtMt in mRNA and protein levels in OVCAR3 and SKOV3 cells was assessed by A–B, qRT‐PCR, C–D, Western blotting, and E–F, immunofluorescence, respectively. **P < .01, ***P < .001 vs control; ##P < .01 vs DDP

Figure 3

Effect of roflumilast on activity of cAMP/PKA/CREB. After administration with DDP (1 μg/mL) or DDP (1 μg/mL) + roflumilast (15 μM) for 24 h, the activity of A, cAMP and B, PKA were detected by ELISA in OVCAR3 cells. The expression of CREB and phosphorylation of CREB were detected by Western blotting in both C, OVCAR3 and D, SKOV3 cells. **P < .01, ***P < .001 vs control; ##P < .01 vs DDP

Figure 4

Effect of PKA on DDP sensitivity of ovarian cancer cells. Cells were treated with H89 before administration of DDP and roflumilast. A, Viability of OVCAR3 cells. B, Viability of SKOV3 cells. C, Edu staining in OVCAR3 cells. D, Edu staining in SKOV3 cells. E, Apoptosis in OVCAR3 cells. F, Apoptosis in SKOV3 cells. G, Hoechst staining in OVCAR3 cells. H, Hoechst staining in SKOV3 cells. I, Cell cycle in OVCAR3 cells. J, Cell cycle in SKOV3 cells

Figure 5

Expression of FtMt and p‐CERB, and PKA activity in cells treated with H89. Cells were treated with H89 before administration of DDP and roflumilast. A, FtMt mRNA in OVCAR3 cells. B, FtMt mRNA in SKOV3 cells. C, Staining of FtMt in OVCAR3 cells. D, Staining of FtMt in SKOV3 cells. E, Western blotting of FtMt and p‐CERB in OVCAR3 cells. F, Western blotting of FtMt and p‐CERB in SKOV3 cells. G, PKA activity in SKOV3 cells. **P < .01, ***P < .001 vs control; ##P < .01 vs DDP. %%P < .01 vs DDP + roflumilast

Figure 6

Effect of CREB shRNA on cell proliferation, cell apoptosis, and cell cycle. Cells were transfected with CREB shRNA and then treated with DDP and roflumilast. Viability of A, OVCAR3 and B, SKOV3 cells. Edu staining in C, OVCAR3 and D, SKOV3 cells. Apoptosis in E, OVCAR3 and F, SKOV3 cells. Hoechst staining in G, OVCAR3 and H, SKOV3 cells. Cell cycle in I, OVCAR3 and J, SKOV3 cells

Figure 7

Effect of CREB shRNA on expression of FtMt and p‐CERB in cells treated with DDP and roflumilast. Cells were transfected with CREB shRNA and then treated with DDP and roflumilast. FtMt mRNA in A, OVCAR3 and B, SKOV3 cells. Staining of FtMt in C, OVCAR3 and D, SKOV3 cells. Western blotting of FtMt and p‐CERB in E, OVCAR3 and F, SKOV3 cells. **P < .01, ***P < .001 vs control; ##P < .01 vs DDP. %%P < .01 vs DDP + roflumilast

Figure 8

The anti‐tumour effects of roflumilast in vivo. A total of 2 × 10⁶ SKOV3 cells were subcutaneously injected in rear flank of nude mice (4 per group) and 5 mg/kg roflumilast or PBS was administered (i.p.) in mice. A, Tumour. B, The mean tumour size was measured in mm³. C, HE and Tunel staining, and IHC staining of Ki‐67, FtMt, CREB, and p‐CREB were determined. D, Western blots of FtMt, CREB, and p‐CREB

Figure 9

Effect of roflumilast on cisplatin resistance of DDP‐resistant ovarian cancer cells. OVCAR3‐DDP‐R and SKOV3‐DDP‐R were constructed. After treatment with DDP, the inhibitor rate of A, OVCAR3‐DDP‐R and B, SKOV3‐DDP‐R cells was detected. C and D, Viability of OVCAR3‐DDP‐R and SKOV3‐DDP‐R after treatment with indicated concentration of roflumilast. E–F, Cell viabilities of OVCAR3‐DDP‐R and SKOV3‐DDP‐R after treatment with roflumilast for the indicated time. G–H, Inhibitor rate in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells after treatment with roflumilast at the indicated concentration. The cells were treated with roflumilast (15 μM) for 24 h. I–J, Edu staining in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. K, Colon formation in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. L, Apoptosis in OVCAR3‐DDP‐R and SKOV3‐DDP‐R. cells. M, Hoechst staining in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. N, Cell cycle in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells

Figure 10

Effect of roflumilast on expression of FtMt and activity of cAMP/PKA/CREB in DDP‐resistant ovarian cancer cells. OVCAR3‐DDP‐R and SKOV3‐DDP‐R were treated with roflumilast. Then, A and B, FtMt mRNA expression in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells was detected by RT‐qPCR. C, FtMt protein expression in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells was detected by Western blotting. D, Staining of FtMt in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells was detected. E and F, Activities of cAMP and PKA in SKOV3‐DDP‐R cells were detected. G, Phosphorylation of CREB in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells was detected

Figure 11

Effect of PKA on roflumilast‐mediated reversal of cisplatin‐resistance in DDP‐resistant ovarian cancer cells. OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells were treated with H89 before administration of roflumilast. A, Viability of OVCAR3‐DDP‐R cells. B, Viability of SKOV3‐DDP‐R cells. C, Edu staining in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. D, Apoptosis in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. E, Hoechst staining in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. F, Colon formation of OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. G, Cell cycle in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. H, Staining of FtMt in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. I, Western blotting of FtMt and p‐CERB in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. J, PKA activity in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells. **P < .01, ***P < .001 vs control; ##P < .01 vs roflumilast

Figure 12

Effect of CREB shRNA on cell proliferation, cell apoptosis, cell cycle, expression of FtMt and p‐CERB in DDP‐resistant ovarian cancer cells. A, Viability of OVCAR3‐DDP‐R and B, SKOV3‐DDP‐R cells. C, Edu staining. D, Apoptosis was detected by flow cytometry. E, Hoechst staining. F, Colon formation. G, Cell cycle. H, Staining of FtMt. I, Western blotting of FtMt and p‐CERB in OVCAR3‐DDP‐R and SKOV3‐DDP‐R cells

Figure 13

The effects of roflumilast on DDP resistance in ovarian cancer in vivo. A total of 2 × 10⁶ SKOV3‐DDP‐R cells were subcutaneously injected in the rear flank of nude mice (4 per group) and 5 mg/kg roflumilast or PBS was administered (i.p.) to mice. A, Tumour. B, The mean tumour size measured in mm³. C and D, Activities of cAMP and PKA in SKOV3‐DDP‐R cells were detected. E, HE and Tunel staining, and IHC staining of Ki‐67, FtMt, CREB, and p‐CREB were determined. F, WB results of FtMt, CREB, and p‐CREB