Serum levels of IL-6, IL-10, IL-5, IL-4, IFNγ, and GM-CSF were increased during preneoplastic stages of colon carcinogenesis
We first wanted to determine the stagewise changes in serum because, ultimately, we are interested in intervening to attenuate early stages of carcinogenesis. To identify possible indicators of colon carcinogenesis risk, a multiplex-based electrochemiluminescence assay to measure serum levels of 14 proteins was used. Serum samples from AOM-induced ob/ob mice were analyzed in mice that were tumor-bearing (i.e., adenomas, adenocarcinomas), bore only preneoplastic lesions (i.e., hyperplasia, dysplasia), or bore no lesions at the end of the study. Because the number of lesion-bearing mice on bean diets was not sufficient to compare stages within diet groups, we initially compared stagewise changes in the colon carcinogenesis process (i.e., normal versus hyperplastic/dysplastic versus tumor) without regard to intervention. Serum levels of IL-6 (P = 0.004), IL-10 (P = 0.003), IL-5 (P = 0.005), and IFNγ (P = 0.006) were statistically significantly increased or had a statistical trend (i.e., IL-4: P = 0.026, GM-CSF: P = 0.021) during preneoplastic stages of colon carcinogenesis compared with serum from AOM-induced ob/ob mice with normal colons (; data not shown for IL-4 and GM-CSF). Serum levels of IL-10 (P = 0.001), IL-5 (P = 0.00008), IFNγ (P = 0.0004), and GM-CSF (P = 0.009) were significantly reduced or had a significant trend in reduction (i.e., IL-4: P = 0.011) in tumor-bearing, AOM-induced ob/ob mice compared with serum from mice with hyperplastic or dysplastic colons (; data not shown for IL-4 and GM-CSF), to levels similar to or less than those seen in mice with normal colons. Similar changes were observed when comparing exclusively stagewise changes in mice on control diet (data not shown).
Serum levels of IL-6 were not significantly reduced in tumor-bearing mice compared with mice with hyperplastic or dysplastic colons (). IL-5 serum levels had a statistical trend toward lower levels in tumor-bearing mice than in mice with normal colons (P = 0.037; ). No significant differences in serum levels of IL-1β, IL-2, KC, IL-12, insulin, resistin, or MCP-1 were seen among mice in normal versus hyperplastic/dysplastic versus tumor groups (data not shown). TNFα levels were not sufficiently detectable to permit analysis. Thus, serum levels of IL-10, IL-5, IL-4, IFNγ, and GM-CSF were increased during preneoplastic stages of colon carcinogenesis relative to tumor-bearing stages.
Bean-based diets produce changes in serum levels of IL-6 and MCP-1 in AOM-induced ob/ob mice
We next wanted to know which serum protein levels would be attenuated by the bean diet. Although the number of mice per group was sufficient to compare serum levels by stage, the number of lesion-bearing mice was not sufficient for subdividing the stages into those that did or did not receive the bean diets.
Thus, we ascertained changes in inflammation-associated serum proteins that occurred in response to bean diet interventions (). Serum levels of the inflammation-associated protein IL-6 were significantly decreased in AOM-induced ob/ob mice on bean-based diets overall (P = 0.0035) and in mice on residue (P = 0.009) and had a statistical trend in decreasing IL-6 in mice on extract fraction (P = 0.018) bean diets compared with controls (). Serum levels of IL-6 were less detectable in AOM-induced ob/ob mice on bean-based diets (27% and 37% for residue and extract fraction, respectively) compared with mice on control diet (68%; ). Serum levels of the chemokine MCP-1 were significantly increased in AOM-induced ob/ob mice on bean-based diets overall (P = 0.002) and in mice on whole bean diet (P = 0.004) and had a statistical trend in increase in mice on residue and extract diets (P = 0.02 and P = 0.02, respectively; ). Thus, bean-based diets seem to target serum IL-6 and MCP-1.
Bean diets change gene expression of a subset of inflammation-related cytokines in colon mucosa of AOM-induced ob/ob mice
To identify possible indicators of response to bean diets in colon mucosa, the expression of 84 inflammation-associated genes was queried using real-time quantitative PCR array analysis. Cd4 expression changes were observed in both whole bean (4.97-fold) and bean residue (5.25-fold) fractions, suggesting that this residue-associated gene may contribute to the cancer-preventive effect of the whole beans (). Sftpd transcript changes were observed with both whole bean (−6.37-fold) and bean extract (−5.04-fold), suggesting that extract components provided the source of the whole bean effect on this gene. We also observed Tnfrsf8 transcript changes in both residue (−1.81-fold) and extract (−2.16-fold) bean fractions. Fold differences that were statistically significant at the P < 0.01 level included Tnfrsf8 (−2.16-fold for bean extract; P = 0.005) and Stat4 (−5.56-fold for bean extract; P = 0.007). The remaining fold differences had a significant trend where P values ranged from P = 0.012 (Tnfrsf8; bean residue) to P = 0.047 (Icos).
Fig. 3 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 (more ...)
The expression of 10 genes was changed overall in AOM-induced ob/ob
mice consuming bean-based diets (). Five of these genes were up-regulated (Cxcr3, Icos, Irf4, Cd4,
) and five were down-regulated (IL-2, Sftpd, Tnfrsf8, IL-6,
). Fold differences for the up-regulated genes ranged from 1.86 to 5.25, whereas fold difference for down-regulated genes ranged from −1.81 to −6.37. A trend emerged in the direction of gene change. Four of five up-regulations occurred in mice consuming whole bean diet. These up-regulated genes are largely proinflammatory. Two of three up-regulated genes were seen in mice consuming bean residue diet with one of the two gene changes (Cd4
) in common with whole bean. The third gene expression change (i.e., down-regulation of Tnfrsf8
) occurred in common with mice consuming bean extract diet. Four of five gene down-regulations occurred in mice consuming bean extract diet, the most efficacious of the three bean diets (4
). Because these down-regulated genes are involved principally in the proinflammatory response, this down-regulation may contribute to the greater efficacy of the bean extract compared with the other bean diets.
There were n = 6 mice per comparison group unless otherwise noted. This sample size was less than the total number of samples used for the serum analysis because colon tissues were not collected from every mouse. Colon samples were selected from mice that had conclusive pathology and serum analysis data. Every effort was made to ensure that the colon samples collected were representative of the animals in each group.
In addition to evaluating gene expression changes in each respective bean group, we wanted to determine if there were gene expression differences in tumor-bearing versus non–tumor-bearing mice. To carry this out, all three bean groups (i.e., All Bean) were combined and compared with the No Bean group. The results indicate that there was a distinct set of genes whose expressions changed based on tumor status, with only one gene in common between the two groups (Maf; ). The up-regulation of Maf in both groups is consistent with the possibility that Maf contributes to but is not sufficient to account for the efficacy of the beans. The set of gene changes occurring in non–tumor-bearing mice may or may not be sufficient to protect against colon tumorigenesis. In contrast, the set of gene changes occurring in tumor-bearing mice is not a sufficient set to protect against colon tumorigenesis.
Fig. 4 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 (more ...)
Among non–tumor-bearing mice, the overall bean diets (n = 24 mice: whole bean, residue, and extract groups combined) significantly changed expression of Cd4 (P = 0.003) and had a statistical trend in the remaining genes (Ptprc, Ccl5, IL-6, Cd40, and Maf) compared with mice on control diet (n = 6) showing −1.86- to 4.85-fold differences (). Statistical trend ranged from P = 0.014 (Ccl5) to P = 0.046 (Cd40). Among tumor-bearing mice, the bean diets overall (n = 3 mice) significantly changed gene expression of IL-4ra (P = 0.005) and had a statistical trend in the remaining genes (Jak3, Mapk8, IL-2, IL-5, Stat4, and Maf) compared with tumor-bearing mice on control diet (n = 4 mice) showing −15.23-to 3.54-fold differences (). Statistical trend ranged from P = 0.010 (IL-5) to P = 0.045 (Mapk8).
The false discovery rate was estimated for the gene expression changes observed. For genes that were statistically significant at the 0.01 significance level (P < 0.01), false discovery rate = 0.01(84)/2 = 42%. Therefore, 58% of our significant findings are expected to be correct. For genes that showed a statistical trend (P < 0.05), false discovery rate = 0.05(84)/8 = 53%. Therefore, 47% of the significant findings are expected to be correct.
Taken together, the results of gene expression analysis suggest several bean diet-induced changes in inflammation-associated genes. Those expression changes shared in mice on bean extract and whole beans (i.e., Sftpd) and those shared in mice on bean residue and whole beans (i.e., Cd4) implicate extract or residue, respectively, as the source of the changes seen with whole beans.
Bean diets attenuate the effects of AOM on a subset of inflammation-related genes in AOM-induced ob/ob mice
To ascertain whether bean diets counteract gene expression changes induced by the carcinogen, colon mucosa RNAs from no-carcinogen controls (i.e., No AOM No Bean) were compared with those treated with AOM. No AOM group was used as the reference group to which all other samples were compared. That is, both AOM-induced and bean attenuated values are compared with a constant denominator to determine a ratio. This calculation was carried out to determine which of the AOM-induced gene expression events were counteracted by bean diet (). The AOM-induced change was then compared with the AOM plus bean diet change. shows genes whose expression was altered by AOM in the absence of bean diet and genes whose expression was altered by bean diets in AOM-induced mice. All fold changes are relative to expression levels in the group of mice that did not receive AOM or bean diets [i.e., AOM (−), bean (−)]. The total number of genes found to change significantly or show a significant trend in response to AOM was 55. The expression of only five of the AOM-altered genes was counteracted by bean diets. IL-6,Tnfrsf8, Stat4, and Sftpd showed expression levels that were induced by AOM, but attenuated (i.e., less induced) in ob/ob mice on overall bean, whole bean, residue, or extract diet. Expression of Ccl5 was repressed by AOM, but attenuated by bean diets. The changes in Stat4 and Tnfrsf8 were statistically significant at the P < 0.001 level, whereas changes in IL-6, Ccl5, and Sftpd showed a significant trend (). Furthermore, the false discovery rate of the genes that were significant at the 0.01 significance level (P < 0.01) in the assessment of AOM-treated mice versus the reference No AOM group was estimated as 0.01(84)/39 = 2%. Therefore, 98% of the significant findings are expected to be correct. Thus, several inflammation-associated gene expression changes induced during AOM-induced carcinogenesis seem to be counteracted by bean diets.
Fig. 5 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, (more ...)