Sildenafil Inhibits the Growth and Epithelial-to-mesenchymal Transition of Cervical Cancer via the TGF-β1/Smad2/3 Pathway

Abstract

Aims: The study aims to explore new potential treatments for cervical cancer.

Background: Cervical cancer is the second most common cancer in women, causing >250,000 deaths worldwide. Patients with cervical cancer are mainly treated with platinum compounds, which often cause severe toxic reactions. Furthermore, the long-term use of platinum compounds can reduce the sensitivity of cancer cells to chemotherapy and increase the drug resistance of cervical cancer. Therefore, exploring new treatment options is meaningful for cervical cancer.

Objective: The present study was to investigate the effect of sildenafil on the growth and epithelial-tomesenchymal transition (EMT) of cervical cancer.

Methods: HeLa and SiHa cells were treated with sildenafil for different durations. Cell viability, clonogenicity, wound healing, and Transwell assays were performed. The levels of transforming growth factor-β1 (TGF-β1), transforming growth factor-β type I receptor (TβRI), phosphorylated (p-) Smad2 and p-Smad3 in cervical cancer samples were measured. TGF-β1, Smad2 or Smad3 were overexpressed in HeLa cells, and we measured the expression of EMT marker proteins and the changes in cell viability, colony formation, etc. Finally, HeLa cells were used to establish a nude mouse xenograft model with sildenafil treatment. The survival rate of mice and the tumor size were recorded.

Results: High concentrations of sildenafil (1.0-2.0 μM) reduced cell viability, the number of HeLa and SiHa colonies, and the invasion/migration ability of HeLa and SiHa cells in a dose- and time-dependent manner. The expression of TGF-β1, TβRI, p-Smad2 and p-Smad3 was significantly enhanced in cervical cancer samples and cervical cancer cell lines. Sildenafil inhibited the expression of TGF-β1-induced EMT marker proteins (Snail, vimentin, Twist, E-cadherin and N-cadherin) and p-Smad2/3 in HeLa cells. Overexpression of TGF-β1, Smad2, and Smad3 reversed the effect of sildenafil on EMT, viability, colony formation, migration, and invasion ability of HeLa cells. In the in vivo study, sildenafil significantly increased mouse survival rates and suppressed xenograft growth.

Conclusion: Sildenafil inhibits the proliferation, invasion ability, and EMT of human cervical cancer cells by regulating the TGF-β1/Smad2/3 pathway.

Keywords: Sildenafil; cervical cancer; epithelial-to-mesenchymal transition; invasion; migration; platinum.

Fig. (1)

The effects of sildenafil on the proliferation and clonogenicity of HeLa and SiHa cells. The effects of sildenafil on the proliferation of (A) HeLa and (B) SiHa cells. The effects of sildenafil on the clonogenicity of (C) HeLa and (D) SiHa cells. The cells in the control group were cultured in normal DMEM with 10% fetal bovine serum. The data are expressed as the mean ± standard error (n=10). *: p<0.05 compared with the control group.

Fig. (2)

The effects of sildenafil on the migratory and invasive activities of HeLa and SiHa cells. The effect of sildenafil on the wound closure rate of (A) HeLa and (B) SiHa cells. The effects of sildenafil on the invasive activities of (C) HeLa and (D) SiHa cells. The cells in the control group were cultured in normal DMEM without fetal bovine serum. The data are expressed as the mean ± standard error (n=10). *: p<0.05 compared with control cells. **: p<0.01 compared with the control group. Scar bar=20 μM.

Fig. (3)

The expression levels of TGF-β1, TβRI, p-SMAD2 and p-SMAD3 in cervical cancer tissues. (A) The expression levels of TGF-β1 in cervical cancer and normal cervical tissues measured by Western blot. (B) The expression levels of TβRI in cervical cancer and normal cervical tissues measured by Western blot. (C) The expression levels of p-SMAD2 and SMAD2 in cervical cancer and normal cervical tissues measured by Western blot. (D) The expression levels of p-SMAD3 and SMAD3 in cervical cancer and normal cervical tissues measured by Western blot. The data are expressed as the mean ± standard error (n=10). **: p<0.01 compared with control cells. TGF-β1, transforming growth factor-β1; TβRI, transforming growth factor β type I receptor; p-, phosphorylated. **: p<0.01 compared with the control group.

Fig. (4)

The expression levels of TGF-β1, TβRI, p-SMAD2 and p-SMAD3 in cervical cancer cells. (A) The mRNA expression levels of TGF-β1 in cervical cancer cells measured by qPCR method. (B) The mRNA expression levels of TβRI in cervical cancer cells measured by qPCR method. (C) The expression levels of p-SMAD2 and SMAD2 in HeLa and SiHa cells measured by Western blot. (D) The expression levels of p-SMAD3 and SMAD3 in HeLa and SiHa cells measured by Western blot. The data are expressed as the mean ± standard error (n=10). **: p<0.01 compared with control cells. TGF-β1, transforming growth factor-β1; TβRI, transforming growth factor β type I receptor; p-, phosphorylated. **: p<0.01 compared with the control group.

Fig. (5)

The effects of sildenafil on TGF-β1-induced EMT in HeLa cells. (A) Representative images of the western blots corresponding to the expression of the EMT-associated marker proteins (Snail, vimentin, Twist, E-cadherin and N-cadherin). (B) The results of the expression analysis of Snail, vimentin proteins in HeLa cells. (C) The results of the expression analysis of Twist, E-cadherin and N-cadherin proteins in HeLa cells. The cells in the control group were cultured in normal DMEM with 10% fetal bovine serum. The data are expressed as mean ± standard error (n=10). *: p<0.05 compared with control; **: p<0.01 compared with the control group. EMT, epithelial-mesenchymal transition; Snail, snail family transcriptional repressor 1; p-, phosphorylated; Twist, twist family bHLH transcription factor 1.

Fig. (6)

The effects of sildenafil on the TGF-β1/SMAD2/3 signaling pathway in HeLa cells. (A) Representative images of western blots corresponding to TGF-β1 and TβRI expression. (B) Semi-quantification of the expression levels of TGF-β1 and TβRI protein in HeLa cells. (C) Representative images of the western blots for p-SMAD2, SMAD2, p-SMAD3 and SMAD3. (D) Semi-quantification of SMAD2/3 phosphorylation levels in HeLa cells. The cells in the control group were cultured in normal DMEM with 10% fetal bovine serum. The data are expressed as mean ± standard error (n=10). *: p<0.05 compared with control; **: p<0.01 compared with the control group. TGF-β1, transforming growth factor-β1; TβRI, transforming growth factor β type I receptor; p-, phosphorylated.

Fig. (7)

The effects of TGF-β1, SMAD2 and SMAD3 overexpression and sildenafil treatment on EMT in HeLa cells. (A) Representative images corresponding to the western blot experiments. (B-C) The results of the expression analysis of the EMT-associated marker proteins (Snail, vimentin, Twist, E-cadherin, N-cadherin) in HeLa cells. The cells in the control group were cultured in normal DMEM with 10% fetal bovine serum. The data are expressed as mean ± standard error (n=10). **: p<0.01 compared with the control group. TGF-β1, transforming growth factor-β1; EMT, epithelial-mesenchymal transition; Snail, snail family transcriptional repressor 1; Twist, twist family bHLH transcription factor 1.

Fig. (8)

Overexpression of TGF-β1, SMAD2 and SMAD3 reverses the effects of sildenafil on viability, clonogenicity and migratory and invasive activities of HeLa cells. (A) Estimation of the viability of HeLa cells. (B) Estimation of the number of HeLa cell colonies. (C) Determination of the wound healing rate of SiHa cells. (D) Determination of the invasive activity of HeLa cells. The cells in the control groups were cultured in normal DMEM with 10% FBS for determination of cell viability and clonogenicity. The cells in the control groups were cultured in normal DMEM without FBS for the determination of migratory and invasive activities. The data are expressed as mean ± standard error (n=10). **: p<0.01 compared with control cells. TGF-β1, transforming growth factor-β1, FBS, fetal bovine serum.

Fig. (9)

Effects of sildenafil on mouse survival rate and xenograft tumor growth. Nude mice were subcutaneously injected with HeLa cells to establish xenograft tumors. The mice were subsequently treated with sildenafil. The survival rates (A) and tumor size (B, C) were measured. The mice in the control group received no treatment. The data are expressed as mean ± standard error (n=12 per group). **, p<0.01 compared with the control group.