Inhibition of protease-activated receptor-2 induces apoptosis in cervical cancer by inhibiting signal transducer and activator of transcription-3 signaling

Abstract

Objective: The present study explored how the inhibition of protease-activated receptor-2 (PAR-2) induced proliferation and apoptosis in cervical cancer in vitro and in vivo.

Methods: mRNA and protein expression of PAR2 and signal transducer and activator of transcription-3 (STAT-3) was determined by quantitative real-time PCR and western blotting. The proliferation and apoptosis of cervical cancer cells were assayed by the cell counting kit-8 kit, flow cytometry, and western blotting. The effects of PAR2 inhibition on cervical cancer were also examined in BALB/c nude mice in vivo.

Results: SLIGRL-NH2 (SL), a selective PAR-2 agonist, promoted proliferation and inhibited apoptosis of healthy cervical cancer cells and HeLa cells, while the PAR-2 antagonist FSLLRY-NH2 (FS) inhibited proliferation and led to apoptosis. SL also promoted the activation of STAT-3, while FS inhibited it and inhibited cancer growth in vivo.

Conclusion: FS inhibited cervical cancer by reducing proliferation and inducing apoptosis by interfering with STAT-3 signaling.

Keywords: Cervical cancer; FSLLRY-NH2; apoptosis; proliferation; protease-activated receptor-2; signal transducer and activator of transcription-3.

Figure 1.

PAR-2 promoted the proliferation of HeLa cells and primary cervical cells (n = 6). A: Proliferation was assayed by CCK-8 after treatment with different concentrations of SL or FS for 48 hours. B: PAR-2 mRNA expression after treatment with different concentrations of FS or SL for 48 hours was assayed by real-time PCR (n = 5). C: PAR-2 protein expression after treatment with different concentrations of FS or SL for 48 hours was assayed by western blotting. *P < 0.05 and ** P < 0.01 vs. 0 hours.

Figure 2.

PAR-2 inhibited the apoptosis of HeLa cells and primary cervical cells (n = 5). A: Apoptosis was assayed by flow cytometry after treatment with 100 µM of FS or SL for 48 hours. B: Caspase-3 expression was assayed by western blotting after treatment with 100 µM of FS or SL for 48 hours. *P < 0.05 and **P < 0.01 vs. control.

Figure 3.

PAR-2 promoted the expression of STAT-3 in HeLa cells and primary cervical cells (n = 5). A: STAT-3 mRNA expression was assayed by real-time PCR after treatment with 100 µM of FS or SL for 48 hours. B: p-STAT-3 and STAT-3 were assayed by western blotting after treatment with 100 µM of FS or SL for 48 hours. c: p-STAT-3 immunofluorescence staining. *P<0.05 and **P < 0.01 vs. control.

Figure 4.

FS inhibited cervical cancer growth in vivo (n=12). Nude mice were subcutaneously injected with 1 × 10⁷ HeLa cells, and half were simultaneously injected with 20 mg/kg FS. A: Tumor growth (**P = 0.0153). B: Ki67 immunohistochemical staining (magnification ×400). C: PAR-2 protein expression was assayed by western blotting.