Arctigenin hinders the invasion and metastasis of cervical cancer cells via the FAK/paxillin pathway

Abstract

Context: Cervical cancer is the most common gynecological pernicious tumor with high morbidity and mortality worldwide, especially in developing countries. Arctigenin (ARG), a nature-derived component, has exhibited anti-tumor activity in various tumors.

Objective: To explore the effect of ARG on cervical cancer.

Materials and methods: The effect and mechanism of ARG on cervical cancer cells were explored by cell counting kit-8 (CCK-8), flow cytometry, transwell and Western blot assays. Additionally, in vivo experiment was conducted in xenografted mice by immunohistochemistry (IHC), terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) and Western blot assays.

Results: ARG treatment induced both concentration-dependent and time-dependent reductions in the cell viability of SiHa and HeLa cells with a IC50 value of 9.34 μM and 14.45 μM, respectively. ARG increased the apoptosis rate and the protein levels of cleaved-caspase 3 and E-cadherin, but decreased the invaded cell numbers and the protein levels of Vimentin and N-cadherin in vitro. Mechanically, ARG inhibited the expression of focal adhesion kinase (FAK)/paxillin pathway, which was confirmed by the overexpression of FAK in SiHa cells. The inhibitory role of overexpression of FAK in proliferation and invasion, as well as its promoted role in apoptosis were reversed with ARG treatment. Meanwhile, ARG suppressed growth and metastasis, and enhanced apoptosis in vivo. Consistently, ARG administration reduced the relative protein level of p-FAK/FAK and p-paxillin/paxillin in tumor tissues of xenografted mice.

Conclusion: ARG inhibited proliferation, invasion and metastasis, but enhanced apoptosis of cervical cancer via the FAK/paxillin axis.

Keywords: Arctigenin; Cervical cancer; FAK/Paxillin; Invasion; Metastasis.

Fig. 1

ARG inhibited the proliferation of SiHa and HeLa cells. (A) The 2D and 3D structure of ARG downloaded from https://pubchem.ncbi.nlm.nih.gov/compound/64981. (B–D) The cell viability of End1/E6E7, SiHa or HeLa cells was examined by CCK-8 assays after cells were incubated with 0, 1.25, 2.5, 5, 10, 20, 40 μM ARG for 24 h *P˂0.05 and **P˂0.01 vs. 0 μM ARG. (E) The cell viability of SiHa or HeLa cells was detected by CCK-8 assays after cells were hatched with 20 μM ARG for 24, 48 and 72 h, respectively. **P˂0.01 and ***P˂0.001 vs. 0 h.

Fig. 2

ARG accelerated apoptosis of SiHa and HeLa cells. (A and B) The apoptosis rate of SiHa and HeLa cells was assessed by flow cytometry after both cells were treated with 5, 10 and 20 μM ARG for 24 h. (C) The relative protein expression of cleaved-caspase 3 was detected by Western blot. Data were present after normalized with GAPDH. *P˂0.05, **P˂0.01 and ***P˂0.001 vs. 0 μM ARG.

Fig. 3

ARG inhibited the invasion and EMT of SiHa and HeLa cells. (A) The invaded cell numbers of SiHa and HeLa cells were counted by transwell assays after cell were administrated with 5, 10 and 20 μM ARG for 24 h. (B) The relative protein expressions of E-cadherin, Vimentin and N-cadherin in SiHa and HeLa cells were determined by Western blot after cell were incubated with 5, 10 and 20 μM ARG for 24 h. Data were exhibited after normalized with GAPDH. *P˂0.05 and **P˂0.01 vs. 0 μM ARG.

Fig. 4

ARG restrained cell proliferation and invasion with enhanced apoptosis of SiHa cells through suppressing the FAK/paxillin axis. (A) The relative protein expression of FAK, p-FAK, paxillin and p-paxillin was examined by Western blot after SiHa and HeLa cells were treated with ARG (5, 10 and 20 μM) for 24 h. Data were displayed after normalized with GAPDH. *P˂0.05 and **P˂0.01 vs. 0 μM ARG. (B) The relative protein expression of FAK, p-FAK, paxillin and p-paxillin was detected by Western blot after SiHa cells transfected with FAK overexpression plasmid before the treatment of 20 μM ARG for 24 h. Data were displayed after normalized with GAPDH. **P˂0.01 vs. con; #P˂0.05 vs. FAK. (C) The SiHa cell viability was measured by CCK-8 assays. *P˂0.05 vs. con; ##P˂0.01 vs. FAK. (D) The apoptosis of SiHa cells was analyzed by flow cytometry. (E) The invaded cell numbers of SiHa and HeLa cells were determined by transwell assays. **P˂0.01 vs. con; #P˂0.05 vs. FAK.

Fig. 5

ARG restrained cervical tumor cell growth and enhanced cell apoptosis in vivo. (A) Representative images of tumors from nude mice in two groups, and the supervision of tumor volumes for successive seven weeks. **P < 0.01 (B) The monitor of mice body weight for sequential thirty-five days. (C) Tumor weight after nude mice were administrated with PBS or 50 mg/kg ARG for five weeks. **P < 0.01 vs. sham. (D) Representative images of IHC staining for Vimentin (Scale bar = 200 μm) and TUNEL staining (Scale bar = 50 μm). (E) The quantitative analysis of IHC staining for Vimentin and TUNEL staining with the histograms. **P < 0.01 vs. sham. (F) The relative protein expressions of p-FAK, FAK, p-paxillin and paxillin were detected by the Western blot. The results were normalized to GAPDH. **P < 0.01 vs. sham.