In the present study, we examined inhibition by I3C of NNK-induced lung adenocarcinoma in mice given the chemopreventive agent either during the post-initiation phase or tumor progression phase. In the post-initiation protocol, chemopreventive agents are given 2–3 weeks post-carcinogen initiation and continued until tumors are formed, whereas the tumor progression protocol is designed to determine effects on tumor progression to carcinoma by administering preventive agents beginning around the time tumors are formed. Testing the efficacy of chemopreventive agents during the tumor progression phase is more relevant than the post-initiation protocol since this phase of lung tumorigenesis is frequently targeted in human clinical chemoprevention trials. Although our earlier studies showed that I3C administered to mice during the post-initiation phase inhibited vinyl carbamate-induced [16
] or NNK-induced [17
] lung adenocarcinoma, to our knowledge, this is the first report on the efficacy of I3C to delay malignant tumor progression to adenocarcinoma when given in the progression protocol. Moreover, we assessed, for the first time, modulation by I3C of cancer-related kinome and pro-apoptotic and anti-apoptotic proteins in A549 cells on a global level.
Administration of I3C during week 4–27 (post-initiation protocol) reduced the multiplicity all tumor size classes, albeit at different efficacy levels, and all forms of histological lesions (hyperplastic foci, adenoma, adenoma with dysplasia, and adenocarcinoma). On the other hand, when I3C was given during week 28–52 (tumor progression phase), multiplicities of smaller tumors (≤ 2 mm) increased, whereas that of bigger tumors (> 2 mm) decreased. Similarly, multiplicities of early stage histological lesions increased, whereas advanced lesions (adenoma with dysplasia and adenocarcinoma) decreased. Despite these differences, I3C effectively inhibited the development of pulmonary adenocarcinoma when administered during the progression phase, the most critical stage in lung cancer development.
In an attempt to identify potential mechanisms for the lung cancer preventive activities of I3C, we examined modulation by the compound of cell proliferation- and apoptosis-related proteins in lung tissues and receptor tyrosine kinases, non-receptor kinases and pro-and anti-apoptotic proteins in A549 cells. Since Akt is frequently activated during lung tumorigenesis [20
] and our earlier reports showed that Akt was activated in carcinogen-induced mouse lung tumors and I3C suppressed its activation [16
], our focus was on Akt and related proteins. AKT, a major downstream target of growth factor receptor tyrosine kinases that signals via phosphatidylinositol-3 kinase (PI3K), plays a central role in tumor cell survival, growth, migration, and angiogenesis [23
]. Immunoblotting studies in mouse lung tissues showed that I3C reduced expression of phospho-Akt and its downstream target, survivin, but enhanced PARP cleavage, a marker for apoptosis. In subsequent studies using A549 cells, we showed that I3C inhibited activation of receptor tyrosine kinases such as ErbB2, ErbB3, c-Met, VEGF receptors and IGFR-1, all of which are known to be over-expressed in human lung cancer and enhance activation of Akt via a phosphatidyliositoal-3 kinase (PI3K)-dependent pathway [24
]. Although our results on inactivation of EGFR by I3C were inconsistent, EGF-induced over-activation of Akt was inhibited by I3C (data not shown), which indicated that I3C blocks the EGFR/PI3K/Akt signaling pathway. In a previous report, I3C abrogated EGFR expression and activation and EGF-induced activation of PI3K and Akt in prostate cancer cells [28
Intracellular serine/threonine/tyrosine kinases that were modulated by I3C include Akt itself, Src, Hck, FAK, STAT3, CREB, mTOR, beta-catenin, p53, Ch2, and the MAPKs ERK, p38 and JNK. Src and Hck are members of the Src family of non-receptor tyrosine kinases and increased expression of Src has been reported in 60%–80% of lung adenocarcinomas and bronchioloalveolar cancers and in 50% of squamous cell carcinomas [29
]. The main pathways through which Src increases tumor cell survival is via its down-stream effectors, STAT3, and FAK, both of which are transcription factors that mediate expression of genes involved in cell cycle progression and apoptosis. STAT3 and FAK are down-stream effectors of the PI3K/Akt pathway [30
]. Similarly, CREB, a transcription factor down-stream of the PI3K/Akt pathway, activates the expression of genes related to cell survival, inflammation and proliferation such as Bcl-2, Bcl-xl, cyclooxygenase-2, and tumor necrosis factor-α [32
]. The most widely studied down-stream effector of Akt is mTOR, which has been shown to be over-expressed in 74% of NSCLC [33
]. Although some mTOR inhibitors such as rapamycin are known to induce upstream receptor tyrosine kinase signaling and thereby activate Akt in both cancer cells and in patient tumors [34
], I3C inhibited activation of both proteins. Another protein whose function is intimately associated with Akt is p53. Akt and p53 are major but opposing players in signaling pathways that regulate cell survival. p53 counteracts the effects of Akt via promotion of caspase-mediated cleavage and subsequent degradation of the AKT protein itself and induction of PTEN expression [35
]. However, comparison the apoptotic effects of I3C in p53 wild type and p53 mutant lung cancer cells and in A549 cells pretreated with pifithrin-α, a small molecule inhibitor of p53, did not show any differences (data not shown), indicating that p53 is probably not necessary for the chemopreventive effects of I3C in these cells.
Activation of the MAPK proteins JNK and p38 by I3C indicates involvement of cellular stress responses in the antiproliferative and apoptotic effects of the compound in A549 cells. However, activation by I3C of ERK in both A549 cells and mouse lung tissues is intriguing since this protein is often associated with mitogenic effects. Indeed, similar effects were observed in prostate cancer cells treated with I3C or phenethyl isothiocyanate [36
] and colon cancer cells treated with selenomethionine [38
]. Phenethyl isothiocyanate-induced apoptosis in prostate cancer cells was even dependent on ERK activation since pharmacological inhibition of ERK activation abolished the apoptotic effect of the chemopreventive agent [37
]. Activation of ERK was also shown to inhibit CDK activity and induce cell cycle arrest and these effects were blocked by the pharmacological inhibitor of ERK [39
]. All these studies indicate that ERK activation, in addition to its well-known mitogenic effects, could also be a negative effector of cell growth and survival. The differential effect of ERK on cell proliferation and survival was found to be correlated with the intensity and duration of ERK activation, i.e., persistent activation of ERK leads to cell cycle arrest, whereas transient activation of the protein increases cell proliferation [36
Studies in mice and human volunteers showed that I3C was metabolized quickly and completely into more than 18 products [9
]. The parent compound was not detected in the plasma or tissues 1 h after administration [40
]. On the other hand, under cell culture conditions, only 0.3% of I3C enters the cells and the compound was surprisingly inert to metabolism with a half-life in medium of approximately 40 h [42
]. In this study, only a fraction of the intracellular I3C was converted into diindolylmethane. Thus, the high concentration of I3C required in the present studies could be related to the poor intracellular accumulation and metabolism of I3C in cell culture models. I3C was also found to be stable when left in the open in the mouse feeders [43
In conclusion, our results showed that I3C is a potent inhibitor of lung adenocarcinoma in mice when given during the early phase of lung tumorigenesis or after tumors had developed, indicating the potential of the compound not only to prevent lung cancer but for therapy as well. The major mechanism through which I3C inhibited lung adenocarinoma is modulation of activation of proteins in the RTK/PI3K/Akt/ pathway. The exact role of each protein in the pathway remains to be determined.