Previously, we reported inhibition of NNK plus BaP-induced lung tumorigenesis in A/J mice by I3C (112 μmol/g diet) given beginning at 50% in the carcinogen treatment phase (21
). In the present study, we further investigated the lung tumor inhibitory effects of I3C by administering the compound at different dose levels and temporal sequences. The results show that I3C given at doses of 1, 10, 30, 71, and 112 μmol/g diet beginning at 50% in the carcinogen treatment phase or at the dose of 112 μmol/g diet during the post-carcinogen treatment phase caused significant reductions (except at the dose level of 1 μmol I3C/g diet) in NNK plus BaP-induced lung tumor multiplicity. The lung tumor inhibitory effects of I3C observed upon administration of the compound during the post-carcinogen treatment phase are likely to be associated with a combination of reduced cell proliferation and induced apoptosis. Our findings are highly relevant for future research on the lung cancer chemopreventive effects of I3C in smokers and ex-smokers.
The mean body weights of mice maintained on I3C-supplemented diets were reduced by 4-19% relative to the group given conventional AIN-93 diet. Moreover, I3C caused a dose-dependent reduction in the amount of perirenal white adipose tissue. Lower body weight gain and perirenal adipose tissue were not due to reduced food intake since mice given I3C-supplemented diet consumed about 2-13% more food than mice maintained on AIN-93 conventional diet. A similar trend in body weight gain was observed in our earlier study in mice (21
) as well as in F344 rats. In the latter study, rats given 0.5% I3C in the diet consumed about 13% more diet but mean body weight was about 7% lower compared to group of rats maintained on a control diet (25
). The lower body weight gain of I3C-treated mice and rats in the face of increased food consumption may be related to induction of increased rate of lipid oxidation, which is typical for phytoestrogens. A recent report (26
) revealed reduced body weight gain (22%), adipose tissue (2-fold), but hyperphagia (41%), in mice maintained on soy-supplemented diet, presumably due to preferential use of lipids as energy source and increased locomotor activity. On the other hand, Maiyoh et al. (27
) showed that I3C significantly reduces cellular lipid synthesis, including tryglycerides, and cholesterol esters and the expression of key lipogenic genes, including diacylglycerol acyltransferase, acyl CoA:cholesterol acyltransferase, fatty acid synthase and sterol regulatory element binding protein, the upstream regulator of fatty acid synthase. We have earlier reported that NNK plus BaP-induced increase in the expression of fatty acid synthase was reversed by I3C (28
). It is not known whether lower body weights in the present study contributed to the chemopreventive activity of I3C. Although calorie restriction is the most potent cancer prevention regimen (29
), effects of calorie restriction and reduced body weight gain on lung tumor development have not been extensively studied. In one study, restriction of food intake by 40 and 60% reduced body weight by about 23 and 34%, respectively, but lung tumor multiplicity was reduced only by 25% in both groups (30
To our knowledge, only four previous reports (21
) are available on the lung tumor inhibitory effects of I3C in the A/J mouse model. When I3C was given at a dose of 10 or 50 μmol/g diet before a single dose of NNK (31
), a moderate 40% reduction in lung tumor multiplicity was observed. Similar results were found upon administration of I3C (13 μmol/g diet) before, during and after a single dose of NNK (32
) or dibenzo[a,l
). We have recently shown that a relatively higher dose of I3C (112 μmol/g diet) given in the diet beginning at one day after the 4th
of eight treatments with a mixture of NNK and BaP reduced lung tumor multiplicity by 86% (21
). In the present study, we not only reproduced the above results but also showed that the lung tumor inhibitory activities of I3C are dose-dependent and the compound is effective when given during the post-carcinogen treatment phase. Administration of I3C beginning 50% through the carcinogen treatment phase or during the post-carcinogen treatment phase was intended to model, to some extent, smokers transitioning to quitting or former smokers, respectively.
The lowest effective dose of I3C (10 μmol I3C /g diet) corresponds, when compared on the basis of body surface area, to the amount of I3C (800 mg/person, orally) given in Phase I clinical trials (19
). This dose of I3C modulated levels of xenobiotic- and steroid-metabolizing enzymes and 2-hydroxyestrone in a manner consistent with chemoprevention without causing toxic effects. It is interesting to note that a dose of I3C that significantly inhibited lung tumor multiplicity in mice without causing toxic effects was well-tolerated by humans and modulated levels of chemopreventive agent efficacy biomarkers. This indicates the great potential of I3C for the chemoprevention of lung tumorigenesis in humans.
I3C inhibits tumorigenesis via different mechanisms (9
). In studies with cell lines in vitro
, I3C caused inhibition of cell proliferation through modulation of various proteins involved in cell cycle regulation, induction of proapoptotic proteins, inhibition of antiapoptotic proteins, and inhibition of signaling pathways involved in cell survival such as PI3K/Akt and NF-κB (9
). In animal models, the main established mechanism by which I3C inhibits tumorigenesis is induction of cytochrome P450 enzymes. I3C inhibited NNK-induced lung tumorigenesis in A/J mice through induction of P450 enzymes resulting in increased hepatic clearance of the carcinogen through α-hydroxylation and N-oxidation (31
). As a result of this, the pulmonary concentration of NNK and its metabolite, NNAL, decreased and pulmonary DNA methylation, a lesion that leads to lung tumors, was reduced. Recent studies using a mouse model with liver-specific deletion of NADPH-cytochrome P450 reductase, the electron donor for the P450 system, further confirmed the role of P450 enzymes in NNK metabolism (34
). Deletion of hepatic cytochrome P450 reductase resulted in decreased P450-mediated hepatic clearance of NNK and a higher multiplicity of NNK-induced tumors in the lung. Smilar results were observed in hepatic cytochrome P450 reductase null mice treated with BaP, in which BaP–DNA adduct levels were higher in the liver (up to 13-fold) and elevated in several extra-hepatic tissues (by 1.7- to 2.6-fold) of mice with the deleted gene relative to mice with the wild type gene (35
). I3C is known to induce P450s involved in the detoxification of BaP (36
In the present study, I3C significantly inhibited lung tumor multiplicity when given during the post-carcinogen treatment phase. These inhibitory activities of I3C could be, at least in part, due to inhibition of cell proliferation and induction of apoptotis. NNK plus BaP-induced increases in the frequency of Ki-67-positive cells and PCNA expression, the most common markers of cell proliferation, were reduced by I3C. Also, I3C reduced NNK plus BaP-induced activation of Akt, a cytosolic signal transduction protein that plays an important role in cell survival pathways through inhibition of apoptosis, and its downstream target phospho BAD. Activated Akt promotes cell survival by phosphorylating and thereby inactivating target proteins such as BAD which results in dissociation of the protein from the Bcl-2/Bcl-X complex and loss of its proapoptotic function (38
). In line with inhibition of Akt and BAD activation, I3C increased cleavage of PARP, a marker of apoptosis. Earlier, I3C was shown to inhibit cell proliferation and induce apoptosis in HPV16-transgenic mice which develop cervical cancer after chronic estradiol exposure (39
), and xenograft models for prostate cancer (41
In conclusion, we showed that I3C given in the diet beginning at 50% in the carcinogen treatment phase inhibited lung tumorigenesis in A/J mice in a dose-dependent manner. Also, upon administration during the post-carcinogen treatment phase, I3C caused a significant reduction in lung tumor multiplicity and modulated the expression of proteins involved in cell proliferation and apoptosis. These results indicate the potential of I3C for lung cancer chemoprevention in smokers transitioning to quitting and in former smokers.