Serum levels of inflammation-associated proteins were increased during preneoplastic stages of colon carcinogenesis in AOM-induced ob/ob mice. Serum levels of IL-6 (A), IL-10 (B), IL-5 (C), and IFNγ (D) were increased in hyperplastic and dysplastic stages of colon carcinogenesis. Columns, geometric mean (pg/mL); bars, 95% confidence interval. Total mice per group: normal, n = 111; hyperplasia/dysplasia (hyp/dys), n = 12; tumor-bearing (tumor), n = 14. Normal is classified as any ob/ob mouse that was free of tumors or preneoplastic lesions. Tumor-bearing includes mice with adenomas or adenocarcinomas. Comparisons are shown for hyperplasia/dysplasia versus normal and hyperplasia/dysplasia versus tumor. Similar changes were observed in stages of colon carcinogenesis whether or not bean-fed mice were excluded from the comparison.

Bean diets produced changes in serum levels of inflammation-associated proteins in AOM-induced ob/ob mice. A, mice on bean-based diets overall (P < 0.0035) showed significant decrease in serum levels of IL-6. Significant reductions in IL-6 were seen in mice on residue (P = 0.009) and bean extract (P = 0.018) diets. Columns, percent detectable IL-6; bars, SEM. B, mice on bean-based diets overall (P = 0.002) and on whole bean (P = 0.004) showed significant increase in serum levels of MCP-1. A significant trend in MCP-1 increase was seen in mice on residue (P = 0.02) and bean extract (P = 0.015) diets. Columns, geometric mean (pg/mL); bars, 95% confidence interval. For all figures, P values spanning bar graphs represent overall bean diets (i.e., whole bean, residue, and extract groups combined). All comparisons are to mice on control (no bean) diet. Total mice per group: control, n = 37; whole bean, n = 32; residue, n = 33; and extract, n = 35. The above decreases and increases were observed with bean diets whether or not tumor-bearing mice were excluded from the comparison.

Bean diets change gene expression of a subset of inflammation-related cytokines in colon mucosa of AOM-induced ob/ob mice. RNA isolated as described in Materials and Methods. The expression of 84 Th1 Th2 Th3 inflammation-associated genes was analyzed using Superarray real-time quantitative PCR arrays. Shown are Venn diagrams depicting bean-induced gene expression changes that were overlapping or not overlapping between diet groups. Arrows, direction of fold change depicted in parentheses. Criteria for gene selection were P < 0.05 for the difference between bean fed and control, and fold change −1.7. Fold change range for all genes was −2.0 to +6.0. n = 6 mice per group. Analysis was done on non–tumor-bearing mice. Comparison groups included whole bean versus no bean, bean residue versus no bean, and bean extract versus no bean, respectively. Statistical significance: Tnfrsf8 (Bean Extract; P = 0.005) and Stat4 (Bean Extract; P = 0.007). All other genes in the figure had a significant trend where P values ranged from P = 0.012 (Tnfrsf8; Bean Residue) to P = 0.047 (Icos). Genes include CD4 antigen (Cd4), surfactant-associated protein D (Sftpd), TNF receptor superfamily member 8 (Tnfrsf8), chemokine C-X-C motif receptor 3 (Cxcr3), inducible T-cell costimulator (Icos), IFN regulatory factor 4 (Irf4), IL-2, chemokine C-C motif receptor 10 (Ccr10), IL-6, and signal transducer and activator of transcription 4 (Stat4).

Bean diets as a group change the expression of a subset of inflammation-related cytokine genes in colon mucosa of AOM-induced ob/ob mice. The fold change range for all genes was −15.00 to +5.0. Cd4 (P = 0.003) and IL-4ra (P = 0.005) were statistically significant by t test analysis. All other genes in the figure had a statistical trend with P values ranging from P = 0.010 (IL-5) to P = 0.046 (Cd40). Total mice per group: All Bean, n = 18; No Bean, n = 6. Comparison groups include All Bean versus No Bean among non–tumor-bearing mice and All Bean versus No Bean among tumor-bearing mice. All Bean group is combination of mice in whole, residue, and extract bean fed groups. Genes include CD4antigen (Cd4), protein tyrosine phosphatase receptor type C (Ptprc), chemokine C-C motif ligand 5 (Ccl5), IL-6, CD40 antigen (Cd40), IL-4 receptor α (IL-4ra), Janus kinase 3 (Jak3), mitogen-activated protein kinase 8 (Mapk8), IL-2, IL-5, signal transducer and activator of transcription 4 (Stat4), and avian musculoaponeurotic fibrosarcoma v-maf AS42 oncogene homologue (Maf).

Bean diet attenuates some of the gene expression changes induced by AOM in ob/ob mice. Graph represents fold changes relative to non–AOM induced (i.e., −AOM) ob/ob mice, which was the overall comparison group for this analysis. Right, clustergram of fold changes depicted in graph (red, high-expressing genes; green, low-expressing genes). All mice from The Jackson Laboratory (No AOM) were free of colon lesions and tumors, showing normal colon on histopathologic examination. As expected in ob/ob mice, some fat infiltration was observed. In contrast, the AOM-treated mice included some with preneoplastic lesions or tumors as indicated in Fig. 1 (4). Blue columns, ratio of +AOM to −AOM in the absence of beans. Red columns, ratio of +beans to −beans following AOM exposure. The determined (blue columns) and calculated (red columns) fold change values have been ascertained as follows: To identify genes whose expression is induced by AOM (A) and attenuated/counteracted by the bean diets (or repressed by AOM and attenuated by the bean diets) (B), all expression levels were normalized to the level found without AOM or bean diet. The following calculations were done using Superarray results. Expression values were measured on Superarray Th1 Th2 Th3 array plates with one sample run per plate. Calculations were determined using the following x, y, and z groups: x = AOM (+) Beans (−), y = AOM (+) Beans (+), and z = AOM(−) Beans (−). Two ratios were determined: (a) y/x or AOM (+) Beans (+)/AOM (+) Beans (−) and (b) x/z or AOM (+) Beans (−)/AOM (−) Beans (−). To calculate the ratio of expression with AOM with beans to expression without AOM without beans (y/z), the following equation was used: y/z = y/x × x/z. Letter(s) in parentheses following the gene indicates the bean group: A.B., all bean; W, whole bean; R, bean residue; E, bean extract. Total mice per group: No AOM No Bean, n = 5; ob/ob mice, +AOM No Bean, n = 6; +AOM All Bean, n = 18; +AOM whole bean, n = 6; +AOM residue, n = 6; +AOM extract, n = 6. Values for AOM + beans (red columns) were significantly different or had a statistical trend from those for AOM alone (blue columns). Stat4 and Tnfrsf8 genes were significant at the P < 0.001 level. *, genes statistically significant or having a statistical trend. Genes include chemokine C-C motif ligand 5 (Ccl5), IL-6, chemokine C-X-C motif receptor 3 (Cxcr3), TNF receptor superfamily member 8 (Tnfrsf8), chemokine C-C motif receptor 10 (Ccr10), signal transducer and activator of transcription 4 (Stat4), and surfactant associated protein D (Sftpd).