Interleukin-37 mediates the antitumor activity in colon cancer through β-catenin suppression

Abstract

The occurrence and development of colon cancer is closely related to inflammation. Thus, we conducted the present retrospective study to investigate the effects of IL-37 (Interleukin 37), a newly identified anti-inflammatory factor, on colon cancer development. We first evaluated the IL-37 expression in 186 pairs of colon cancer samples and their adjacent normal mucosa by real-time PCR, ELISA (Enzyme-linked immunoassay) and tissue microarrays. Then the role of IL-37 on patient survival rates, colon cancer progression and their sensitivity to chemotherapy drugs were assessed. IL-37 was barely expressed in the colon cancer tissue but highly expressed in the adjacent normal tissue. The down-regulation of IL-37 was significantly correlated with the results of American Joint Committee on Cancer stage, nodal involvement, invasion depth, distant metastasis, differentiation and it was also shown to be an independent prognostic indicator of disease-free survival and overall survival for patients with colon cancer. Overexpression of IL-37 in colon cancer cell suppressed cell migration, invasion, proliferation, colony formation and cancer stem cells through suppressing β-catenin. IL-37 inhibited colon tumor formation in the mice model and sensitize the cancer cell to chemotherapy drugs. Our results showed that IL-37 plays an inhibitory role in colon cancer development and function as a novel prognostic indicator and a potential therapeutic target.

Keywords: IL-37; colon cancer; interleukin-37; β-catenin.

Figure 1. Lack of IL-37 expression correlated with the poor prognosis of colon cancer

(A) IL-37 mRNA levels in cancer tissues and adjacent normal tissues were determined by real-time PCR (n = 186). *P < 0.05. (B) IL-37 protein levels in cancer tissues (95% CI: 11.5–109.6 pg/mg) and adjacent normal tissues (95% CI: 119.1–216.9 pg/mg) were determined by ELISA (n = 186). Result is depicted as box plots; middle line indicates median; bottom of box, 25th percentile; and top of box, 75th percentile. *P < 0.05. (C) Representative figure for IL-37 protein levels in cancer tissues and adjacent normal tissues were determined by western blot. N: adjacent normal tissues; C: cancer tissues. (D) Kaplan-Meier survival curve of patients with negative, weak or strong expression of IL-37.

Figure 2. IL-37 suppresses colon cancer in a dose-dependent manner

(A) Wound healing assay of DLD1 and HT-29 cells with different concentrations of rhIL-37 protein (0, 1, 10, 100 ng/mL). n = 3. *P < 0.05. (B) Cell invasion assay of DLD1 and HT-29 cells with different concentrations of rhIL-37 protein (0, 1, 10, 100 ng/mL). n = 3. *P < 0.05. (C) Analysis of colon cancer cell apoptosis following treatment of rhIL-37. DLD1 and HT-29 cells were treated at the indicated doses, harvested, and stained with Annexin V-FITC and 7-AAD. Annexin V-FITC-positive apoptotic cells were determined by flow cytometry. n = 3. *P < 0.05. (D) The survival rate of DLD1 and HT-29 cells treated with different concentrations of rhIL-37 (0, 1, 10, 100 ng/mL) were analyzed. n = 3. *P < 0.05. (E) The clone formation number of DLD1 and HT-29 cells treated with different concentrations of rhIL-37 (0, 1, 10, 100 ng/mL) were analyzed. n = 3. *P < 0.05. (F) The percentage of CD44⁺CD133⁺ cancer stem cells of DLD1 and HT-29 cells treated with different concentrations of rhIL-37 (0, 1, 10, 100 ng/mL) were analyzed. n = 3. *P < 0.05.

Figure 3. IL-37 inhibits β-catenin expression in colon cancer cells

(A) The mRNA level of β-catenin was measured by qPCR. DLD1 and HT-29 cells were treated with 100 ng/mL rhIL-37. n = 3. *P < 0.05. (B) The protein level of β-catenin was measured by western blot. DLD1 and HT-29 cells were treated with 100 ng/mL rhIL-37. n = 3. *P < 0.05. (C–H) The wound healing, cell invasion, apoptosis, survival rate, clone formation capability and the percentage of cancer stem cells of cells overexpressing IL-37, β-catenin, both IL-37 and β-catenin or empty vector were analyzed. n = 3. *P < 0.05.

Figure 4. IL-37 suppresses colon tumorigenesis in vivo

The adeno-associated virus expressing IL-37 or GFP was administrated via tail-vein injection. The average tumor number per mice (A) and the tumor size (B) were analyzed. The nuclear translocation of β-catenin in the tumors was assessed by western blot (C). Tumor cells were isolated from the mice and then subjected to chemotherapeutic drugs treatment (D). n = 12. *P < 0.05.