Propolis Has an Anticancer Effect on Early Stage Colorectal Cancer by Affecting Epithelial Differentiation and Gut Immunity in the Tumor Microenvironment

Abstract

Colorectal cancer (CRC) is one of the most common cancers and is the second leading cause of cancer-related death in the world. Due to the westernization of diets, young patients with CRC are often diagnosed at advanced stages with an associated poor prognosis. Improved lifestyle choices are one way to minimize CRC risk. Among diet choices is the inclusion of bee propolis, long recognized as a health supplement with anticancer activities. Understanding the effect of propolis on the gut environment is worth exploring, and especially its associated intratumoral immune changes and its anticancer effect on the occurrence and development of CRC. In this study, early stage CRC was induced with 1,2-dimethylhydrazine (DMH) and dextran sulfate sodium (DSS) for one month in an animal model, without and with propolis administration. The phenotypes of early stage CRC were evaluated by X-ray microcomputed tomography and histologic examination. The gut immunity of the tumor microenvironment was assessed by immunohistochemical staining for tumor-infiltrating lymphocytes (TILs) and further comparative quantification. We found that the characteristics of the CRC mice, including the body weight, tumor loading, and tumor dimensions, were significantly changed due to propolis administration. With further propolis administration, the CRC tissues of DMH/DSS-treated mice showed decreased cytokeratin 20 levels, a marker for intestinal epithelium differentiation. Additionally, the signal intensity and density of CD3⁺ and CD4⁺ TILs were significantly increased and fewer forkhead box protein P3 (FOXP3) lymphocytes were observed in the lamina propria. In conclusion, we found that propolis, a natural supplement, potentially prevented CRC progression by increasing CD3⁺ and CD4⁺ TILs and reducing FOXP3 lymphocytes in the tumor microenvironment of early stage CRC. Our study could suggest a promising role for propolis in complementary medicine as a food supplement to decrease or prevent CRC progression.

Keywords: CD4 protein; cytokeratin 20; early stage colorectal cancer; forkhead box protein P3; gut immunity; propolis; tumor microenvironment.

Figure 1

Change in body weight of CRC-bearing mice following propolis administration. (A) Timing of DMH and DSS induction of CRC and propolis administration. Red line indicated the day for subcutaneous injection of DMH (40 mg/kg body weight) and red boxes were the days for drinking DSS (3%). The days for body weighing and propolis (30 mg/mouse) feeding were indicated in bold. The X-ray microcomputed tomograph (µCT), sacrifice, and colon sampling were performed at day 34. (B) Body weights. Red line, mice with DMH/DSS treatment; green line, mice with DMH/DSS treatment and propolis administration. CRC, colorectal cancer; DMH, 1,2-dimethylhydrazine; DSS, dextran sulfate sodium; DIW, deionized water. *, p < 0.05 and #, 0.05 < p < 0.1 (Mann–Whitney U test).

Figure 2

Detection of CRC in mice by X-ray microcomputed tomograph. Each mouse was imaged 3 h after administration of Baritop LV (30 mg/kg). Control mice, not treated with DMH/DSS and not given propolis; DMH/DSS, mice with DMH (40 mg/kg body weight)/DSS (3%) treatment; DMH/DSS/propolis, mice with DMH/DSS treatment and propolis administration. White arrows indicated the sites with barium accumulation. CRC, colorectal cancer; DMH, 1,2-dimethylhydrazine; DSS, dextran sulfate sodium.

Figure 3

Inhibition of colon shortening in CRC-bearing mice by administration of propolis. Each group was individually framed with a black box including the representative colon image (upper one) and its inner layer (lower one). CRC, colorectal cancer; DMH, 1,2-dimethylhydrazine; DSS, dextran sulfate sodium.

Figure 4

Reduction in DMH/DSS-induced CRC formation following propolis administration. (A) Representative histopathological images of mice colons. Scale bar, 250 µm. Red square indicated an image with a higher magnification (scale bar, 25 µm). (B) The tumor dimensions from the H&E-stained CRC tissue sections. CRC, colorectal cancer; DMH, 1,2-dimethylhydrazine; DSS, dextran sulfate sodium; M, mucosa; LP, lamina propria; MM, muscularis mucosa; H&E, hematoxylin and eosin. *, p < 0.05 (Mann–Whitney U test).

Figure 5

Representative images of immunohistochemical staining for KRT20 in colon of mice. KRT20 staining increased from bottom to the top of the crypt in colonic mucosa of control mice. An intense staining of KRT20 was in the colon sections from the mice with DMH/DSS-induced CRC and a moderate positive reaction was in the mucosal layer of CRC tissues of the mice with propolis administration. KRT20, cytokeratin 20; CRC, colorectal cancer; DMH, 1,2-dimethylhydrazine; DSS, dextran sulfate sodium. Scale bar, 100 µm.

Figure 6

Effects of propolis administration on the density of tumor-infiltrating T lymphocytes in colons of CRC-bearing mice. (A) CD3⁺, CD4⁺, and FOXP3 lymphocytes in the lamina propria with neoplastic lesions in colons of CRC-bearing mice. Scale bar, 250 µm. Red square indicated an image with a higher magnification (scale bar, 25 µm). (B) The signal intensity and density of CD3⁺, CD4⁺, and FOXP3 lymphocytes. CRC, colorectal cancer; DMH, 1,2-dimethylhydrazine; DSS, dextran sulfate sodium. CD3, CD3 protein; CD4, CD4 protein; FOXP3, forkhead box protein 3. *, p < 0.05 and **, p < 0.01 (Mann–Whitney U test).