Effective chemoprevention of lung cancer, the principal cause of cancer deaths in the United States, has not been achieved. Characterization and use of effective chemopreventive agents have become important issues in the prevention and control of this deadly disease, particularly in the case of former smokers who are known to remain at high risk. Aiming to identify novel chemopreventive agents for lung cancer, we have determined the efficacy of several agents, including polyphenon E, ginseng, and rapamycin, in preventing lung tumorigenesis in mice and found that these three agents can significantly inhibit lung tumorigenesis in A/J mice.
Green tea has been shown to be chemopreventive in several animal models [32–36
]. However, the effect of the administration of polyphenon E in the diet on lung tumorigenesis has not yet been determined. Polyphenon E is a well-standardized decaffeinated green tea catechin mixture containing five different catechins: epicatechin, gallocatechin gallate, epigallocatechin, epicatechin gallate, and EGCG [12
]; it is the recommended form of green tea for clinical chemoprevention trials [13,14
]. In this study, we treated the mice with different concentrations of polyphenon E (0.5%, 1.0%, 1.5%, and 2%) in AIN-76A diet. We initiated the polyphenon E treatment after BP treatment because green tea in drinking water was already known to be effective, and because this post-initiation schedule made the most sense in modeling clinical trials in former smokers. We found that polyphenon E (2% in diet) reduced tumor multiplicity by 46% () and tumor load by 94% (). The latter result implies that, cumulatively, polyphenon E has a profound effect on tumor growth. Because we have shown that the development of adenocarcinomas in this model is highly dependent on tumor size [32
], one would readily hypothesize that this treatment should profoundly decrease adenocarcinoma formation. We believe that this is the first report of the efficacy of polyphenon E given in the diet against lung tumorigenesis in A/J mice, and this lends strong support to the planned clinical phase II chemoprevention study of lung cancer.
The results of administering polyphenon E in the diet are consistent with many previously reported studies that used green tea in the drinking water, which showed the inhibition of carcinogenesis in many rodent models, including the skin, lung, forestomach, esophagus, liver, colon, and mammary glands [15,16
]. Green tea and one of its components (EGCG), when given in drinking water, have been shown to inhibit 4-(N
-methylnitrosamino)-1-(3-pyridyl)-butanone (NNK)-induced mouse lung tumorigenesis by 63% and 28%, respectively [37
]. Green tea has also been found to inhibit the growth—and to cause the regression—of established benign skin tumors, suggesting that it may be preventive at all stages of carcinogenesis [16
]. Tea polyphenols have various biologic activities, including antioxidation, modulation of enzyme systems for metabolizing chemical carcinogens, inhibition of nitrosation reactions, scavenging of activated metabolites of chemical carcinogens, and inhibition of tumor promotion [11,38,39
]. Recently, several lines of evidence have suggested that green tea is a potent inducer of apoptosis in tumor cell lines [38,39
]. It is likely that some degree of apoptosis and inhibition of cell proliferation may contribute to achieving striking decreases in tumor load, which we have observed.
Ginseng has been used for thousands of years in Asia. It has antioxidant, antitumor promotion, and anti-inflammatory properties [40
]. Ginseng contains more than 30 different ginsenosides of protopanaxadiol, protopanaxatriol, or oleanane [22
]. Ginseng is a potent inhibitor of carcinogenesis in many rodent models, including the lung, skin, liver, mammary gland, uterine cervix, brain, and colon [23–27,41
]. The chemopreventive efficacy of ginseng has been demonstrated in a mouse lung tumor model using urethane, DMBA, or aflatoxin B1
as a carcinogen [23–27
]—a result that we have readily confirmed in this standard BP model of lung carcinogenesis. Furthermore, three specific ginsenosides Rg3, Rg5, and Rh2 showed a reduction of lung tumor incidence in mice [17,41
]. In a two-stage mouse skin model with DMBA, ginseng exhibited inhibitory effects against the development of skin papillomas in a dose-dependent manner [42
]. When ginseng was given in the diet, it suppressed preneoplastic lesions [aberrant crypt foci (ACF)] induced by 1,2-dimethylhydrazine or azoxymethane [43
]. More significantly, ginseng appears to be effective against ACF development at the postinitiation stage of colon carcinogenesis [44
]. There are some reports on the mechanisms of the chemopreventive activity of ginseng. These studies suggest that the possible actions of ginseng are antioxidation, antitumor promotion, induction of p21 or p27, activation of NF-κB, activation of ERK, inhibition of cell proliferation, and induction of apoptosis [45,46
]. In this study, red ginseng treatment in drinking water did not cause any gross toxicity and did not affect body weight. Treatment with red ginseng significantly inhibited both lung tumor multiplicity and tumor load. Our results demonstrated that red ginseng was a potent chemopreventive agent for the prevention of lung tumorigenesis in A/J mice.
Rapamycin is an antifungal agent originally purified from S. hygroscopicus
]. It is a drug clinically used to suppress immune response after organ transplantation [28
]. Rapamycin has also been used to inhibit restenosis due to smooth muscle cell overgrowth after angioplasty. The cellular target of rapamycin has been well characterized [31
]. Rapamycin forms a tight complex with FKBP12, and the rapamycin-FKBP12 complex binds to and inhibits mTOR, which is a protein kinase [31
]. mTOR plays a central role in the regulation of cell growth and has been shown to directly phosphorylate S6K and 4EBP1 [31
]. S6K stimulates cell growth by phosphorylating the ribosomal protein S6 and by stimulating translation [47
]. S6K activity is stimulated by mitogens, nutrients, and energy sufficiency [48
]. The immunosuppressant drug rapamycin inhibits S6K activation, which demonstrates that mTOR plays an important role in S6K regulation [49
]. mTOR is a member of the phosphoinositide kinase-related family of protein kinases. It has been reported that mTOR phosphorylates S6K on T389 and activates S6K in vitro
]. T389 is therefore the primary rapamycin-sensitive phosphorylation site in vivo
]. The rationale for testing the chemopreventive efficacy of rapamycin in the mouse lung tumor model is the observation that the PI3K/Akt/mTOR pathway was activated in mouse lung tumors [51
]. A recent study has performed an immunohistochemical analysis of paraffin-embedded lung tissues using phosphospecific antibodies against serine 473 of Akt, serine 2448 of mTOR, and serine 9 of GSK3β [51
]. It was shown that Akt and mTOR (but not GSK3β) were activated in lung tumors induced by NNK in A/J mice [51
]. Another line of evidence is the finding that blocking an enzyme (mTOR) that acts further down the PI3K/Akt/mTOR pathway sensitizes tumors to killing by conventional chemotherapy agents [52
]. In this study, we showed that rapamycin is chemopreventive in the mouse lung tumor carcinogenesis model of A/J mice induced by BP. Administration of rapamycin reduced both lung tumor multiplicity and tumor load. This is the first attempt, based on our knowledge, to test rapamycin in animal models of lung carcinogenesis to establish the efficacy of this compound as a chemopreventive agent of lung cancer. Thus, the activation of the PI3K/Akt/mTOR pathway in lung tumors is a new target for chemoprevention, especially by agents that act on this pathway, namely rapamycin.
In summary, the results of this study show that polyphenon E, red ginseng, and rapamycin are novel lung chemopreventive agents that are effective in A/J mice. Interestingly, the effects were substantially greater on tumor volume than on multiplicity in mice treated with polyphenon E, ginseng, or rapamycin, suggesting that the major effect of these agents is on later stages of the carcinogenic process. To follow up on these observations, we are actively working on potential mechanisms by which these agents may prevent lung tumorigenesis by conducting microarray studies and real-time polymerase chain reactions of genes that are relevant to their mechanisms of action, which are reported to be separate once they are completed. Finding new and effective agents that can prevent lung cancer is urgently needed because cancer of the lungs remains the principal cause of cancer deaths in the United States and because effective chemoprevention of this cancer type remains elusive. For example, carotene, retinol, and vitamin E/C have been shown to have little—or a negative—effect on human lung cancer development in smokers [11
]. Thus, polyphenon E, red ginseng, and rapamycin are among the more promising new preventive agents for lung cancer and should be considered for further studies in animal models and clinical trials.