Ethanol-induced mast cell-mediated inflammation leads to increased susceptibility of intestinal tumorigenesis in the APC Δ468 min mouse model of colon cancer - PubMed (original) (raw)

Ethanol-induced mast cell-mediated inflammation leads to increased susceptibility of intestinal tumorigenesis in the APC Δ468 min mouse model of colon cancer

Andre L Wimberly et al. Alcohol Clin Exp Res. 2013 Jan.

Abstract

Background: Chronic and frequent alcohol (ethanol [EtOH]) intake has been associated with an increased incidence of several types of cancers including breast, mouth, throat, esophageal, stomach, and colorectal (CRC). The underlying mechanism of this deleterious carcinogenic effect of alcohol has not been clearly established but inflammation may be 1 unifying feature of these cancers. We have recently shown that intestinal mast cells play a central role in intestinal carcinogenesis. In this study, we tested our hypothesis that mast cell-mediated inflammation is 1 underlying mechanism by which chronic alcohol promotes intestinal tumorigenesis.

Methods: APC(Δ468) mice were fed either an alcohol-containing Nanji liquid diet or isocaloric dextrose-containing Nanji diet for 10 weeks and then sacrificed to collect small and large intestine samples. Assessments of tumor number and size as well as mast cell number and mast cell activity and histology score for invasion were compared between Control (dextrose-fed) and alcohol-fed APC(∆468) mice. The effect of alcohol on mast cell-mediated tumor migration was also assessed using an in vitro migration assay.

Results: Alcohol feeding increased both polyp number and size within both the small and the large intestines of APC(∆468) mice. Only alcohol-fed mice showed evidence of tumor invasion. Chronic alcohol feeding also resulted in an increased mast cell number and activity in tumor stroma and invading borders. In vitro migration assay showed that alcohol significantly increases mast cell-mediated tumor migration in vitro.

Conclusions: Our data show that chronic alcohol intake promotes: (i) intestinal tumorigenesis and tumor invasion in genetically susceptible mice; (ii) increases in polyp-associated mast cells; and (iii) mast cell-mediated tumor migration in vitro. Both our in vivo and in vitro studies suggest that mast cell-mediated inflammation could be 1 mechanism by which alcohol promotes carcinogenesis.

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Conflict of interest statement

CONFLICT OF INTERES

All authors declare no conflict of interest.

Figures

Figure 1

Figure 1a. Chronic alcohol feeding results in increased numbers of intestinal polyp tumors in APC_Δ_468 mice. After 8 weeks of the Nanji alcohol diet treatment, mice were sacrificed and the intestines were removed. Polyps were quantified via macroscopic exam as well as microscopy (Materials and Methods). Intestines were fixed and processed for paraffin embedding. H&E staining was performed after obtaining 5 μm paraffin sections. There was a significant increase in the number of intestinal polyps in the Alcohol fed (EtOH) APC Δ468 mice versus Control treated. EtOH (N=5) and Control (N=6). All results are in Means ± SE: EtOH = *95±2.7 vs. Control = 68±3.0, **p≤ 0.01. Figure 1b. Chronic alcohol feeding results in increased size of intestinal polyp tumors in APC_Δ_468 mice. After sacrifice and quantification of the number of polyps, the sizes of polyps were measured for comparison. Polyps (mm) of 0.5, 1.0, 2.0, and 3.0 were measured in the EtOH (dark grey) and Control (light grey) groups. The size of polyps in alcohol fed APC Δ468 mice increased significantly as compared to the Control treated group. There was an increase of average number of polyps of the same size (2.0 mm) as well as larger (3.0 mm) polyp formation in alcohol fed mice that were not present in the control. Sizes in mm- Alcohol: 0.5 mm = 22.8±2.5, 1.0 mm = 25.1±1.9, **2.0 mm = 38.8±1.9 versus Control, p≤0.01 and **3.0 mm = 3.0±0.7 versus Control, p≤0.01 compared to dextrose Control treated: 0.5 mm = 20±2.6, 1.0 mm = 25.6±2.1, *2.0 mm = 18.2±1.4, **3.0 mm = 0.00. Data are means ± SE. ** p ≤ .01 vs. the respective Control value.

Figure 1

Figure 2

Figure 2a. Chloroacetate Esterase (CAE) staining demonstrates increased intestinal polyp mastocytosis in alcohol fed APC_Δ_468 mice. Paraffin embedded intestinal sections were obtained as described in Methods after chronic alcohol feeding for 8 weeks and stained for presence of mast cells with CAE. Micrographs of intestinal polyps were obtained to quantify mast cells. The circled areas are of polyps in representative Control (A) and EtOH treated (B) mice at 20x. CAE positive stained MC are red in color and indicated by arrows: Control intraepithelial MC (C) were significantly less than EtOH MC (D) at 40x. Overall, a significant increase of mast cell numbers occurred in alcohol fed APCΔ468 mice versus Controls in the intestinal polyps. Bar line = 30μm Figure 2b. Mast cell numbers are increased in polyps with alcohol treatment in APC_Δ_468 mice. Mast cells were quantified as mast cells/field at 20x magnification as described in Methods. A significant increase of intestinal mast cell number was found in intestinal polyps of alcohol treated APC Δ468 mice versus dextrose Control mice polyps. Control (dextrose fed) polyp average mast cell number per polyp was significantly lower (39±1.0) as compared to alcohol fed mice polyps (**50.4±1.0; p≤0.01).

Figure 2

Figure 3

Figure 3a. Chronic alcohol feeding results in increased numbers of degranulating MC in stromal but not epithelial zones of intestinal polyps of APC_Δ_468 mice. MC Toluidine Blue staining revealed MC releasing contents of granules within the microenvironment of the intestinal polyps, with both intraepithelial and stromal staining. Paraffin embedded tissue slides (5 μm) were obtained and stained as described in Methods. Representative polyps (A, C, E, G) have circles with area of interest at 10x and (B, D, F, H) are the areas at 20x. Intraepithelial MC (D) in alcohol treated mice did not exhibit a significant difference in number vs. Control (B). However, in the stroma, there was a significant increase in degranulating MC in the alcohol fed mice (H) as compared to Controls (F). Representative mast cell degranulation is shown in I and J, at magnification of 40x and 100x respectively. Bar line = 30μm Figure 3b. Chronic alcohol feeding results in increased average numbers of degranulating mast cells as revealed by Toluidine Blue staining of MC in stromal but not epithelial zones of intestinal polyps of APC_Δ_468 mice. Intraepithelial MC showed no significant difference in staining between Control (1± 1) and Alcohol fed (2± 2) groups. However, Stromal MC showed a significant increase in staining in alcohol fed mice: Control (9.5±1.0) vs. Alcohol fed mice 21±1.0, **p≤0.01.

Figure 3

Figure 4

Figure 4a. Tryptase and Chymase staining for MC was increased in intestinal polyps of Alcohol fed compared to Control fed APC_Δ_468 mice. Mast cells were identified via staining with Tryptase and Chymase antibodies of paraffin embedded intestinal tissue sections as described in Methods. Representative images on the left (Control: A, C, I, K, and Alcohol: E, G, M, O; at magnification of 10x) are of polyps of respective mice and have circles of an area of concentration. Images on the right of them (Control: B, D, F, H, J, L, N, and P at magnification of 20x) are highlighted with arrows showing MC positively stained for tryptase or chymase respectively. Bar line = 30μm Figure 4b. Numbers of intestinal polyp MC staining for Tryptase and Chymase were increased in Alcohol fed compared to Control fed APC_Δ_468 mice. Numbers of intestinal tryptase and chymase positive mast cells (MC) in intestinal polyps increased with chronic alcohol feeding. Intraepithelial Tryptase positive MC: Control (26.5±1.2) was significantly lower than in the Alcohol fed (dark grey) APCΔ468 mice (38.0±1.2; *p≤0.01). Intraepithelial Chymase positive MC: Control (light grey) (20±1.0) was significantly lower than the Alcohol fed APCΔ468 mice (42±1.8; **p≤0.01). Stromal Tryptase positive MC: Control (2.0±1.0) was significantly lower than the Alcohol fed APCΔ468 mice (25.4±1.0; ***p≤0.01). Stromal Chymase positive MC: There was no significant difference in stromal chymase positive mast cells in the Alcohol fed mice (1.2±0.5) compared to the dextrose Control fed mice (1.8±0.5). Figure 4c. Intestinal polyps in alcohol fed APCΔ468 mice exhibit a dramatic increase in stromal tryptase (mMCP6) positive mast cells compared to Control fed mice. In order to elucidate relative contributions for alcohol promotion of tumorigenesis by subsets of mouse mast cells we used data from Fig. 4b above to determine the ratio of alcohol/control mean mast cell number for each mast cell type (chymase/mMCP2 or tryptase/mMCP6) in either the intraepithelial or stromal tissue locations. Ratios from left to right are Intraepithelial: 1.5 (tryptase), 2.1(chymase); Stromal: 12.5 (tryptase), 1.0 (chymase).

Figure 4

Figure 5

Figure 5a. Intestinal polyps of alcohol fed but not Control APC_Δ_468 mice exhibit evidence of tumor invasion. Polyps stained as described above for H&E and CAE (mast cell marker) were examined for signs of tumor invasion of the basement membrane. Numerous polyps from Alcohol fed mice exhibited signs of invasion while no such invasion was found in Control fed mice polyps. Representative images show: (A) Control fed mouse polyp with no invasion, 10x. (B) Alcohol fed mouse polyp with invasion (arrow) and dysplasia, 10x. (C) CAE staining for MC at same invasive polyp shown in B, 20x. Note MC are intraepithelial (right) as well as stromal (arrows). Bar line = 30 μm Figure 5b. Alcohol stimulates KU-812 mast cell promotion of DLD-1 colon cancer cell migration in vitro. The human basophilic leukemia cell line KU-812 was differentiated to become mast cell like in vitro as described in Materials and Methods. These cells were tested in a cell migration assay (modified Boyden chamber) using 8μM Falcon inserts in 12-well plates (lower well, 2 × 105 cells/well) for their ability to stimulate migration of DLD-1 human colon cancer cells (upper well, 1 × 105 cells/well) ± 0.2% alcohol for 48h. Control wells had no KU-812 in lower well. Data are for migrated cells per high power field (HPF, 200x) visualized with crystal violet staining and are means ± SE for 3 random fields in triplicate wells (N=9) by a blinded counter.

Figure 5

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Ethanol-induced mast cell-mediated inflammation leads to increased susceptibility of intestinal tumorigenesis in the APC Δ468 min mouse model of colon cancer - PubMed (original) (raw)