Ubagai et al.
) described a protocol for the efficient induction of large intestine tumors in the rat by intermittent administration of PhIP and a HF diet. Specifically, 2 weeks of PhIP treatment at 400 p.p.m. in the diet were followed by 4 weeks of HF diet, which was repeated three times, ending with continuous feeding of HF diet for 42 weeks—this regimen produced a final incidence of large intestine tumors similar to that seen with continuous dietary PhIP treatment for an entire year (7
). Although it reduced dramatically the amount of PhIP needed to produce colon tumors in the rat, we modified the protocol in order to further optimize carcinogen use. We also sought to eliminate HF exposure in the latter part of the study to facilitate post-initiation experiments with phytochemicals. Thus, PhIP was given by oral gavage at a dose (50 mg/kg body wt/day) that was matched to the daily carcinogen intake from 400 p.p.m. PhIP in the diet, and after three such cycles of PhIP/HF treatment, standard AIN-93M diet rather than HF diet was given until the study was terminated at 1 year. Using this modified protocol, the colon tumor incidence was 41.6%, comparable with the 45% incidence reported by Ubagai et al.
). Animals in the PhIP/HF control group developed a wide spectrum of tumors () and less than one-third survived tumor free for 1 year (). Rats were euthanized early due to the presence of Zymbal’s gland tumors and/or skin lesions or after the appearance of blood in the stools, which was usually indicative of bleeding into the gastrointestinal tract from one or more tumors in the large or small intestine.
We reported previously (12
) that white tea, EGCG and caffeine protected in the rat against PhIP-induced ACF, which are putative preneoplastic lesions and used as biomarkers of final colon tumor outcome (16
). However, in the present investigation, EGCG had no inhibitory effect, and white tea and caffeine promoted rather than suppressed colon tumors. It is unclear whether this reflects a basic limitation of ACF as biomarkers of tumor outcome, as reported by others for genistein and cholic acid (19
), or alternatively that caffeine and white tea exert a true biphasic response in the rat colon, being beneficial (protective) during short-term treatment but deleterious (promotional) after prolonged exposure. Importantly, however, we observed that caffeine offered significant protection in other target organs of PhIP-induced tumorigenesis (). The cumulative results include animals that died early, but the overall trends were similar in each of the treatment groups for rats that survived to 1 year (data not shown). We interpret this as evidence that protection by caffeine in other target organs was real, and not due to fewer rats surviving to 1 year. Previously (22
), caffeine was reported to protect against PhIP-induced mammary tumors in female rats but also promoted tumor formation in the colon.
Consistent with their tumor-promoting activities, caffeine and white tea altered colonic crypt homeostasis by enhancing cell proliferation versus apoptosis (). Similar findings were obtained before in rats given chlorophyllin or indole-3-carbinol, and colon tumor promotion was associated with a shift in the β-catenin mutation spectrum (3
). Thus, we rationalized that a likely mechanism for tumor promotion by white tea and caffeine might be via the dysregulation of β-catenin/Tcf signaling since β-catenin mutation and increased β-catenin expression are clearly associated with tumor progression (1
). An increase in β-catenin mutations thus would be consistent with enhanced tumor growth by caffeine and white tea.
An increase in β-catenin mutation frequency coupled with a shift in the β-catenin mutation spectrum indeed was observed in colon tumors from rats post-treated with caffeine, but not white tea, indicating that β-catenin mutation status alone could not explain the promotional activities seen here. The colon tumors from rats given caffeine harbored codon 34 β-catenin mutants at a higher frequency than codon 32 mutants, suggesting that one or more promotional mechanisms ‘selected’ certain oncogenic forms of β-catenin to progress in preference to others. We do not know the reason for this apparent selection pressure, given that oncogenic β-catenin mutants with substitutions at either Asp32 or Gly34 were equally effective in activating a β-catenin/Tcf-responsive reporter construct (3
Downstream targets of β-catenin/Tcf were highly expressed in colon tumors, namely c-myc
and cyclin D1
and so too was Ctnnb1
mRNA itself. Overexpression of β-catenin protein therefore might arise due to mutational events that stabilize the β-catenin protein (1
) or via dysregulation of Ctnnb1
expression at the transcriptional level, as reported for 1,2-dimethylhydrazine-induced colon tumors (13
). Importantly, among the three β-catenin/Tcf target genes examined, only c-myc
showed any concordance with tumor promotion by white tea and caffeine (). Thus, rather than β-catenin, c-Myc overexpression may be important in the promotional mechanism of white tea and caffeine. In its capacity as an ataxia telangiectasia mutated/ATM and Rad3-related kinase inhibitor, caffeine was reported to prevent p53 accumulation upon activation of c-Myc or E2F1 (24
). We recently described a pathway in which overexpression of c-Myc elevated E2F1 and enhanced Bcl-2 levels, resulting in suppression of apoptosis (25
). Suppression of apoptosis in the present study was evident for caffeine and white tea using cleaved caspase-3 as a biomarker, in animals treated with PhIP/HF diet and also in the negative controls given three cycles of vehicle/HF diet. However, we did not examine the possible role of E2F1, Bcl-2 or p53 in the promotional mechanism of caffeine and white tea in the rat colon, and this is worthy of future study.
Finally, it is important to note that a substantial literature exists on tea and coffee consumption in humans, which does not indicate an adverse effect on tumor outcome—indeed, some studies suggest a protective role for these caffeinated beverages. For example, Tavani et al.
) reviewed epidemiological studies for the period 1990–2003 on coffee, decaffeinated coffee and tea and cancer of the colon and rectum. For coffee consumption, most case–control studies found risk estimates below unity for colon cancer, but no relation for rectal cancer. A meta-analysis of 5 cohort and 12 case–control studies found a pooled relative risk of 0.75 for colon cancer, which was significant. No such relationship was seen for tea or decaffeinated coffee. A more recent meta-analysis (27
) concluded that ‘despite the strong evidence from in vitro
and non-human in vivo
studies in support of green and black tea as potential chemopreventive agents against colorectal cancer, available epidemiologic data are insufficient to conclude that either tea type may protect against colorectal cancer in humans’. In light of the findings from the present investigation and the increased risk for colorectal adenoma associated with meat cooked at high temperature (28
), it would be interesting to reassess the epidemiological evidence for possible increased risk of colon cancer in a subset of individuals consuming tea (or coffee) plus large amounts of cooked meat and a HF diet.
In summary, we report here that in rats treated with three short cycles of PhIP/HF diet, post-initiation exposure to white tea and caffeine promoted rather than suppressed colon tumorigenesis, whereas EGCG had no effect. Caffeine increased the frequency of β-catenin mutations, and the colon tumors harbored almost exclusively β-catenin mutants with direct substitutions of Gly34. One important conclusion from these studies is that the final spectrum of β-catenin mutants in colon tumors is clearly influenced by exposure to dietary phytochemicals, as reported before for chlorophyllin and indole-3-carbinol (3
). There was no direct concordance between the changes in tumor outcome and the expression of β-catenin or its downstream target genes. However, c-Myc was identified as a possible key player in the promotional mechanism of white tea and caffeine. Caffeine protected in other target organs of PhIP-induced tumorigenesis, suggesting a complex pattern of tumor modulation, in line with the general heterogeneity noted in human epidemiology studies of caffeinated beverages.