Progesterone suppresses the progression of colonic carcinoma by increasing the activity of the GADD45α/JNK/c‑Jun signalling pathway

Abstract

Colorectal cancer (CRC) is the third most diagnosed cancer worldwide. Progesterone is associated with a decreased risk of CRC and leads to a favourable prognosis. However, the specific mechanism by which progesterone suppresses malignant progression remains to be elucidated. In the present study, the level of progesterone was first analysed in 77 patients with CRC, and immunohistochemistry was performed to detect the expression of progesterone receptor (PGR) in the paired specimens. The correlations between progesterone, PGR and CRC prognosis were assessed. A Cell Counting Kit‑8 assay was then used to detect proliferation of the CRC cells. Flow cytometry was performed to estimate apoptosis and to evaluate the cycle of the CRC cells. A xenograft tumour model was established in nude mice to assess the role of progesterone in tumour growth. Finally, a PCR microarray was used to screen differentially expressed genes to further interpret the mechanism by which progesterone inhibits the malignant progression of CRC. It was found that low expression of progesterone and PGR were significantly associated with poor prognosis of CRC. In addition, progesterone suppressed CRC cell proliferation by arresting the cell cycle and inducing apoptosis in vitro. Moreover, the inhibitory role of progesterone in tumour growth was verified in vivo. Further investigation showed that the level of growth arrest and DNA damage‑inducible protein α (GADD45α) was up‑regulated by progesterone, and this was followed by the activation of the JNK pathway. Progesterone increased the activity of the JNK pathway via GADD45α to inhibit proliferation by arresting the cell cycle and inducing apoptosis, thereby suppressing the malignant progression of CRC. Therefore, it can be concluded that progesterone and PGR might act as inhibiting factors for poor prognosis of CRC.

Keywords: progesterone; colorectal carcinoma; proliferation; c‑Jun N‑terminal kinases; GADD45α.

Figure 1.

Expression of progesterone and PGR is negatively correlated with prognosis of CRC. (A) Distribution of progesterone levels in 77 patients with colorectal cancer. (B) A representative image showing the expression level of PGR in colorectal cancer tissues. (C) Immunohistochemical staining scores were used to define low and high expression levels of PGR in colorectal cancer tissues. (D) Progesterone was positively correlated with PGR in colorectal cancer tissues. (E) Kaplan-Meier survival analysis of patients with CRC. (F and G) Expression of PGR based on tumour and normal tissue, and individual cancer stages from TCGA database. (H) Levels of PGR in 10 randomly selected paired colorectal cancer tissues were determined via western blot. (I) Semi-quantitative analysis of the western blot results. **P<0.01 and ***P<0.001 vs. low-level or normal groups. CRC, colorectal cancer; PGR, progesterone receptor; TCGA, The Cancer Genome Atlas; P4, progesterone.

Figure 2.

Progesterone inhibits the proliferation of CRC cell lines and arrests the cell cycle. (A) Expression of progesterone receptor in different CRC cell lines (HT29, HCT116, LoVo, SW620 and SW480) and expression of PGR in LoVo (line 1) and SW620 (line 2) cells treated with different concentrations of progesterone (125, 250 and 500 µM) were determined using western blot. Proliferation of (B) LoVo and (C) SW620 cells was inhibited when these cells were treated with varying concentrations of progesterone. Effects of progesterone on (D and E) cell cycle distribution and (F) arrest at the G₂/M phase following treatment with increased concentrations of progesterone in LoVo cell, as well as in (G-I) SW620 cells. Cytokines related to cell cycle in (J) LoVo and (K) SW620 cells were analysed using reverse transcription-quantitative PCR. **P<0.01 and ***P<0.001 vs. control. CRC, colorectal cancer; P4, progesterone.

Figure 3.

Progesterone induces the apoptosis of colorectal cancer cell lines. Apoptosis rates in (A) LoVo and (B) SW620 cells were analysed using flow cytometry. Treatment with increasing concentrations of progesterone significantly promoted the process of apoptosis in (C) LoVo and (D) SW620 cell lines. BCL-2, BAX and cleaved caspase-3 in (E) LoVo and (F) SW620 cell lines were analysed using western blotting. **P<0.01 and ***P<0.001 vs. control. P4, progesterone; PI, propidium iodide; BCL-2, B cell lymphoma-2.

Figure 4.

Progesterone facilitates tumour growth in vivo. (A) Tumour images. (B) Tumour volume and (C) weight were measured in each group. (D) Representative histopathology of xenograft tumours for Ki67 staining. (E) Quantified Ki67 staining. (F) TUNEL assay and (G) quantified results. *P<0.05 and **P<0.01 vs. control. P4, progesterone.

Figure 5.

Progesterone up-regulates the JNK pathway via GADD45α activation to inhibit progression of colonic carcinoma. (A) Heat map depicting progesterone-induced changes in the expression profile of genes that were assessed on the PCR microarray. Blue and red represent low and high gene expression levels in pinnae, respectively. (B) Selection of genes that were altered the most in SW620 cells. Genes are presented alphabetically. RT-PCR was used to investigate the effect of progesterone on proliferation-related genes such as c-Jun, JNK1, JNK2, GADD45α, P15, BAX, BCL2L1 and NCF1 in (C) SW620 and (D) LoVo cells Efficiency of knockdown GADD45α in (E) SW620 and (I) LoVo cells, respectively. Expression of GADD45α, JNK1, JNK2, c-Jun, BCL-2, and Ki67 in (F) SW620 and (J) LoVo cells was analysed using RT-PCR, and GADD45α, phosphorylation of JNK, JNK, c-Jun, BCL-2 and Ki67 in (G) SW620 and (K) LoVo cells were analysed using western blot. Relative expression of phosphorylation of JNK to total protein of JNK in (H) SW620 and (L) LoVo cells. *P<0.05, **P<0.01 and ***P<0.001 vs. control. GADD45α, growth arrest and DNA damage-inducible protein α; JNK, c-Jun N-terminal kinase; BCL-2, B cell lymphoma-2; NCF1, neutrophil cytosol factor 1.