In the present study, we showed that low doses of PEITC-NAC and/or I3C/DIM, products of commonly consumed cruciferous vegetables, when combined with MI, effectively reduced the multiplicity of tobacco smoke carcinogen-induced mouse lung tumors, proliferation of A549 cells and CSC-treated HBEC and levels of p-Akt, p-ERK and p-NF-κB in lung tumor tissues, A549 cells and HBEC. We reported in earlier studies that higher doses of PEITC-NAC (9 and 15 μmol/g diet) and I3C (30, 71 and 120 μmol/g diet) significantly reduced the multiplicity of carcinogen-induced lung tumors in mice (17
). However, these effects were accompanied by reductions in body weight (~10%). In the present study, body weights of the mice treated with the individual chemopreventive agents or their combinations were similar to those of the control animals. Also, in both in vitro
and in vivo
studies, the agents were used at concentrations that could be achieved physiologically in humans. For instance, plasma PEITC concentrations of 1.15 μmol/l could be achieved upon consumption of 100 g of watercress (25
), which is equivalent to the concentration of PEITC-NAC (1 μmol/l) used in cell culture studies with HBEC. Similarly, the concentration of DIM used for cell proliferation and western assays in HBEC was similar to that achieved in the plasma of women who participated in a phase I clinical trial with I3C (2.46 μmol/l, 26
). For the in vitro
studies, we used DIM, a major in vivo
condensation product of I3C, instead of the parent compound because I3C could not be optimally converted to DIM in cell culture conditions. MI is a promising chemopreventive agent with negligible toxic effects. In earlier studies, it has been administered to mice at a dose level of 168 μmol/g diet, which is three times the amount used in the present study (56 μmol/g diet), without causing any toxic effects (27
). In phase I clinical trials of MI in smokers, a dose level of 18 g/day was well tolerated (13
). Although tissue or plasma levels of MI were not determined in this study, in another investigation in which 100 mg/kg MI was given, plasma levels of 100 μmol/l MI were achieved (28
). Given the fact that the dose level of MI used in phase I trials (18 g/day, 300 mg/kg for a person with a 60 kg body wt) was 3-fold higher than that used in the study by Groenen et al.
, it is plausible to speculate that the dose level of MI used in our in vitro
studies (300 μmol/l) was relevant to the human situation.
Two important criteria for the development of chemopreventive agents are safety and efficacy. Potential approaches to reduce the toxicity of chemopreventive agents are the use of combinations of low doses of promising chemopreventive agents such as PEITC and I3C or mixtures of low doses of promising agents and high doses of relatively safe agents such as MI. Also, combinatorial treatment could increase the efficacy of cancer chemoprevention as it enables one to target multiple carcinogens or cancer-related signaling pathways. This is particularly justified in view of the fact that cigarette smoke, the main cause of lung cancer, is a complex mixture of >5000 compounds including >70 carcinogens (29
) that contribute to lung carcinogenesis via different mechanisms. Moreover, the chronic and multi-step nature of carcinogenesis and the fact that lung cancer is a heterogenous group of diseases with alterations in several signaling proteins provide further support for a multi-agent approach to the chemoprevention of lung cancer (19
In the present study, we showed that NNK plus BaP-treated mice given combinations of PEITC-NAC plus I3C plus MI had significantly lower tumor multiplicities than those mice given the compounds individually or a combination of PEITC-NAC plus I3C (). However, there was no significant difference between the chemopreventive activity of mixtures of all three compounds and PEITC-NAC plus MI or I3C plus MI (). Therefore, for chemoprevention trials in current-smokers/former smokers, the use of mixtures of PEITC-NAC plus MI or I3C plus MI would be more advisable than the combination of all three agents. Possible explanations for the greater efficacy of combinatorial treatment, especially when given beginning in the mid-phase of carcinogen treatment, could be that the chemopreventive agents cooperatively inhibit carcinogen metabolism and other aspects of carcinogenesis that lead to initiation in addition to their post-initiation effects. Indeed, PEITC and I3C modulate the metabolic activation of tobacco carcinogens via different mechanisms and also target separate cancer-related signaling pathways although cooperation in modulating a single pathway is also possible (7
). Similar effects were observed in our previous study (24
) in which a combination of PEITC-NAC plus MI showed the greatest lung tumor inhibitory activities when given during the entire experiment although additive effects were observed in all temporal sequences (carcinogen treatment phase, post-carcinogen treatment phase and 50 or 75% in the carcinogen treatment phase). summarizes our present and earlier findings on inhibition of mouse lung tumorigenesis by combinations of PEITC-NAC plus MI/PEITC-NAC plus I3C plus MI.
Of the different signal transduction pathways that are deregulated during lung carcinogenesis, Akt, ERK and NF-κB pathways have been proposed to play a central role (31
). Akt and ERK are involved in cell survival and proliferation, respectively, whereas NF-κB regulates the expression of inflammation-related genes. All three pathways were found to be activated in normal bronchial and alveolar cells and non-small cell lung cancer cells treated with tobacco smoke constituents (18
), human lung cancer lesions (36
) and lung tumors from tobacco carcinogen-treated mice or transgenic mice (17
). There is also a close interaction between Akt, ERK and NF-κB. For instance, the function of both phosphatidylinositol 3-kinase–Akt and RAS–ERK pathways was required to transform human melanocytes by v-SEA and neither of the genes was sufficient to transform the cells (42
). Also, a cross talk was shown between the RAF–MAPK/ERK kinase–ERK and phosphatidylinositol 3-kinase–Akt pathways, mediated by the direct interaction of Akt with and its phosphorlyation of RAF that switched the biological response of breast cancer cells from growth arrest to proliferation (43
). One of the mechanisms by which Akt promotes cell survival is via induction of IkappaB degradation that results in the release of NF-κB from the cytoplasm to the nucleus (44
). The expression of p-Akt, p-ERK and p-NF-κB in transformed and malignant lung cells as well as the close interaction among the proteins makes them attractive targets for lung cancer chemoprevention. Indeed, in the present study, levels of p-Akt, p-ERK and p-NF-κB were reduced by PEITC-NAC, I3C/DIM and MI or their combinations, combinatorial treatment being more effective than treatment with a single agent, and the trend in p-Akt, p-ERK and p-NF-κB modulation is similar to the effects observed in the tumor bioassay and MTT assay. Thus, the higher chemopreventive activities of the mixtures may be explained, at least partly, by their additive effects in abrogating Akt, ERK and NF-κB activation. We and others have previously reported that PEITC-NAC, I3C and MI, when given individually to mice at relatively higher doses in the diet, inhibited carcinogen-induced lung tumorigenesis through modulation of activities of cell proliferation- and apoptosis-related proteins (17
). In the present study, the individual effects of PEITC-NAC and I3C on lung tumor multiplicity and activation of Akt, ERK and NF-κB were weaker than those observed in the above studies. This discrepancy could be due to differences in the dose levels of the chemopreventive agents. Our findings with MI are in line with the reports of Han et al.
) in which the compound significantly decreased Akt activation in dysplastic lesions from heavy smokers and endogenous and tobacco carcinogen-induced treated immortalized HBEC but not in lung cancer cell lines. Therefore, for MI to be effective against lung tumorigenesis, it should be administered during the relatively early phase of the carcinogenic process.
Although data on the effect of combinatorial chemopreventive treatments on lung tumor multiplicity and the associated lung cancer-related proteins are lacking, studies in other models are in line with our results. For instance, combination treatment with PEITC and curcumin caused additive apoptotic effects in PC-3 human prostate cancer cells through simultaneous targeting of epiderma growth factor receptor, Akt and NF-κB signaling pathways (48
). The same group showed that a combination of curcumin and PEITC significantly reduced the growth of tumor xenografts through inhibition of Akt and NF-κB signaling pathways, whereas PEITC or curcumin alone had little effect (49
In conclusion, we showed that the efficacy of combinations of PEITC-NAC, I3C/DIM and MI was higher than the individual compounds in inhibiting NNK plus BaP-induced lung tumor multiplicity in mice and proliferation of A549 cells and CSC-treated HBEC. Also, combinatorial treatment with the chemopreventive agents led to a stronger abrogation of Akt, ERK and NF-κB activation in lung tumor tissues, A549 cells and HBEC, indicating that the higher chemopreventive activities of the mixtures could be mediated, at least partly, via the combined inhibitory effects of the agents on activation of these crucial mitogenic and anti-apoptotic proteins. Moreover, combinatorial treatment exerted differential toxicity toward CSC-treated HBEC as compared with DMSO-treated cells and did not affect body weight gains in mice. The higher chemopreventive efficacy and safety of combinations of PEITC-NAC, I3C/DIM and MI indicates the promise of mixtures for prevention of lung cancer in current and former smokers.