Annexin A1 peptide and endothelial cell-conditioned medium modulate cervical tumorigenesis

Abstract

Cervical cancer is one of the leading causes of cancer death in women worldwide, and its tumorigenesis can be influenced by the microenvironment. The anti-inflammatory protein annexin A1 (ANXA1) has been reported to be associated with cancer progression and metastasis, suggesting that it plays a role in regulating tumour cell proliferation. Here, we examined the effect of the N-terminal peptide Ac2-26 of ANXA1 on the HaCaT cell line (normal) and HeLa cell line (cervical cancer) co-cultured with endothelium cell-conditioned medium (HMC). Treatment with Ac2-26 decreased proliferation and increased motility of cervical cancer cells, but did not affect cellular morphology or viability. Combined HMC stimulus and Ac2-26 treatment resulted in an increase in apoptotic HeLa cells, upregulated expression of MMP2, and downregulated expression of COX2,EP3 and EP4. In conclusion, Ac2-26 treatment may modulate cellular and molecular mechanisms underlying cervical carcinogenesis.

Keywords: ANXA1; carcinogenesis; cervical cancer; inflammation; peptide treatment.

Figure 1

Response to Ac2‐26 peptide treatment of HaCaT and HeLa cell lines in proliferation, motility and cytotoxicity assays. The cells were cultured in complete minimum essential medium (MEM) and treated with Ac2‐26 (10 μg·mL⁻¹). (A) Proliferation; (B) motility; (C) images illustrating motility; (D) cytotoxicity. P < 0.05 was considered significant; one symbol, P < 0.05; two symbols, P < 0.01; three symbols, P < 0.001: * vs HaCaT, # vs HeLa; ANOVA followed by Bonferroni's test. Assays were performed with three independent experiments. Error bars indicate SD. Scale bars: 500 μm.

Figure 2

Response to Ac2‐26 peptide treatment by HaCaT and HeLa cell lines in an apoptosis assay and gene expression. The cells were cultured in complete MEM and treated with Ac2‐26 (10 μg·mL⁻¹). (A) Densitometry and DotBlot apoptosis; y = cell percentage in 10 000 events. (B) Gene expression. P < 0.05 was considered significant; one symbol, P < 0.05; two symbols, P < 0.01; three symbols, P < 0.001: * vs HaCaT; # vs HeLa; ANOVA followed by Bonferroni's test. Assays were performed with three independent experiments. Error bars indicate SD.

Figure 3

Response of the HaCaT cell line to conditioned medium induction and Ac2‐26 peptide treatment. The cells were cultured in complete MEM and stimulated with conditioned HUVEC cell medium (HMC) (at a ratio of 1 : 1) that was untreated (HMCS; A,C,E) or treated (HMCT; B,D,F) with Ac2‐26 (at 10 μg·mL⁻¹). (A,B) HaCaT proliferation; (C,D) HaCaT motility; (E,F) HaCaT cytotoxicity. P < 0.05 was considered significant; *P < 0.05; **P < 0.01; ***P < 0.001: (A,C,E) vs HaCaT; (B,D,F) vs HaCaT + Ac2‐26; ANOVA followed by Bonferroni's test. Assays were performed with three independent experiments. Error bars indicate SD.

Figure 4

Response of the HeLa cell line to conditioned medium induction and Ac2‐26 peptide treatment. The cells were cultured in complete MEM and stimulated with conditioned HUVEC cell medium (HMC) (at a ratio of 1 : 1), untreated (HMCS; A,C,E) or treated (HMCT; B,D,F) with Ac2‐26 (at 10 μg·mL⁻¹). (A,B) HeLa proliferation; (C,D) HeLa Motility; (E,F) HeLa cytotoxicity. P < 0.05 was considered significant; *P < 0.05; **P < 0.01; ***P < 0.001: (A,C,E) vs HeLa (B,D,F) vs HeLa + Ac2‐26; ANOVA followed by Bonferroni's test. Assays were performed with three independent experiments. Error bars indicate SD.

Figure 5

Response of HaCaT and HeLa cell lines to conditioned medium induction and Ac2‐26 peptide treatment. The cells were cultured in complete MEM and stimulated with conditioned HUVEC cell medium (HMC) (at a ratio of 1 : 1), untreated (HMCS; A–C) or treated (HMCT; B–D) with Ac2‐26 (at 10 μg·mL⁻¹). (A,B) HaCat densitometry and DotBlot apoptosis; (C,D) HeLa densitometry and DotBlot apoptosis; y = cell percentage in 10 000 events. P < 0.05 was considered significant; *P < 0.05; **P < 0.01; ***P < 0.001: (A–C) vs Control of each group; (B–D) vs Control + Ac2‐26 of each group; ANOVA followed by Bonferroni's test. Assays were performed with three independent experiments. Error bars indicate SD.

Figure 6

Response of HaCaT and HeLa cell lines to conditioned medium induction and Ac2‐26 peptide treatment. The cells were cultured in complete MEM and stimulated with conditioned HUVEC cell medium (HMC) (at a ratio of 1 : 1) untreated (HMCS; A) or treated (HMCT; B) with Ac2‐26 (at 10 μg·mL⁻¹). (A) Gene expression after HMCS induction. (B) Gene expression after Ac2‐26 treatment and HMCT induction. P < 0.05 was considered significant; one symbol, P < 0.05; two symbols, P < 0.01; three symbols, P < 0.001: (A) * vs HaCaT; # vs HeLa; (B) * vs HaCaT; $ vs HaCaT + Ac2‐26; # vs HeLa; £ vs HeLa + Ac2‐26; ANOVA followed by Bonferroni's test. Assays were performed with three independent experiments. Error bars indicate SD.