Cancer chemoprevention has become an important area of cancer research, which, in addition to providing a practical approach to identifying potentially useful inhibitors of cancer development, also affords excellent opportunities to study the mechanisms of carcinogenesis (26
). One excitement of chemoprevention is that agents can be targeted for intervention either at the initiation, promotion or progression stage of multistage carcinogenesis. The intervention of cancer at the promotion stage appears to be the most appropriate and practical. The major reason for this relates to the fact that tumor promotion is a reversible event at least in early stages and requires repeated and prolonged exposure of a promoting agent (28
). For this reason, it is important to identify mechanism-based effective novel antitumor-promoting agents. It is appreciated that those agents, which have the ability to intervene at more than one critical pathway in the carcinogenic process, will have greater advantage over other single-target agents. This study was designed to show the chemopreventive potential of GUG by using carcinogenesis-associated biochemical endpoints in a mouse skin tumorigenesis model. The topical application of TPA to mouse skin or its treatment in certain epidermal cells is known to result in a number of biochemical alterations, changes in cellular functions and histologic changes leading to skin tumor promotion (13
). Our data clearly demonstrate that topical application of GUG prior to TPA application affords significant inhibition of TPA-induced skin edema and hyperplasia ( and ).
ODC, the first and the rate-limiting enzyme in the biosynthesis of polyamines, plays an important role in the regulation of cell proliferation and development of cancer (30
). Studies with the mouse skin model have shown an excellent correlation between the induction of ODC activity and the tumor-promoting ability of a variety of substances (31
). Several lines of evidence indicate that aberrations in ODC regulation and subsequent polyamine accumulation are intimately associated with neoplastic transformation (33
). Elevated levels of ODC gene products are consistently detected in transformed cell lines, virtually all animal tumors and in certain tissues predisposed to tumorigenesis (33
). Agents that block induction of ODC can prevent tumor formation; therefore, its inhibition was shown to be a promising tool for screening inhibitors of tumorigenesis (35
). In the present study, topical application of GUG prior to that of TPA resulted in a significant inhibition of TPA-mediated induction of epidermal ODC activity (). It is reasonable to believe that GUG application inhibited the action of the tumor promoter and/or the enzymatic pathways that regulates the ODC induction rather than interacting directly with the enzyme. In addition, our data obtained from western blot analysis demonstrate that prior application of GUG to that of TPA showed an inhibitory effect of GUG against TPA-induced increases in the levels of epidermal ODC protein in the mouse skin (). The magnitude of the inhibitory effect of topical application of GUG on TPA-induced increases in ODC protein expression seems to be similar to that for inhibition of TPA-induced increases in ODC enzyme activity.
Tumor promotion is closely linked to inflammation and oxidative stress, and it is probable that compounds that have anti-inflammatory and antioxidative properties act as antitumor promoters as well (37
). COX-2 isoform and iNOS are important enzymes involved in mediating the inflammatory process (38
). COX-2 and iNOS have been reported to play an important role in cutaneous inflammation, cell proliferation and skin tumor promotion (40
). There is considerable body of compelling evidence that inhibition of COX-2 and iNOS expression or activity is important for not only alleviating inflammation but also for the prevention of cancer (41
). Previous studies have demonstrated that GUG inhibits cytokine-induced COX-2 expression and NF-κB activation (42
). In this study, we showed the inhibitory effects of GUG against TPA-caused induction of epidermal COX-2 and iNOS protein expression in SENCAR mice (). These inhibitory effects also correlate with the inhibitory effect of GUG against TPA-caused induction of skin edema () and hyperplasia (). These inhibitory effects of GUG against TPA-mediated responses in the mouse skin suggest that the primary effect of GUG may be against inflammatory responses, which may then result in the inhibition of tumor promotion.
MAPKs constitute a superfamily of proteins that include ERK1/2, JNK1/2 and p38 kinase (15
). The involvement of MAPKs pathway in tumor proliferation is well documented. Activation of the MAPKs pathway occurs in response to integrin-mediated cellular adhesion to the extracellular matrix, which plays a critical role in both tumor metastasis and angiogenesis (43
). In the present study, employing western blot analysis, we found that topical application of TPA resulted in a marked increase in the phosphorylated form of ERK1/2, JNK1/2 and p38 protein expression. Importantly, topical application of GUG prior to TPA application was found to inhibit TPA-mediated phosphorylation of MAPKs (). Several studies have shown that JNK pathway plays a major role in cellular function, such as cell proliferation and transformation (45
), whereas the ERK pathway suppresses apoptosis and enhances cell survival or tumorigenesis (46
Studies have shown that ERK1/2 and p38 are involved in the transcriptional activation of NF-κB (47
). NF-κB has emerged as one of the most promising molecular targets in the prevention of cancer. We next investigated the effect of GUG on the pattern of NF-κB activation and its nuclear translocation by TPA in SENCAR mice skin. NF-κB resides in the inactive state in the cytoplasm as a heterotrimer consisting of p50, p65 and IκBα subunits. An IκBα kinase, IKKα, phosphorylates serine residues in IκBα at position 32 and 36 (49
). Upon phosphorylation and subsequent degradation of IκBα, NF-κB activates and translocates to the nucleus, where it binds to DNA and activates the transcription of various genes (49
). Several lines of evidence suggest that proteins from the NF-κB and IκB families are involved in carcinogenesis. Studies have also shown that NF-κB activity affects cell survival and determines the sensitivity of cancer cells to cytotoxic agents as well as ionizing radiation (51
). Shishodia et al.
) have shown that GUG inhibits tumor necrosis factor-induced IKK activity and also suppresses inducible and constitutive NF-κB activation without directly affecting binding of NF-κB to DNA. In the present study, we have demonstrated that topical application of TPA to mouse skin resulted in activation and nuclear translocation of NF-κB/p65 (). We also found that TPA application to mouse skin resulted in an increased expression of IKKα and phosphorylation and degradation of IκBα protein (). Interestingly, we observed that topical application of GUG prior to TPA application to mouse skin inhibited TPA-induced NF-κB/p65 and IKKα activation and phosphorylation and degradation of IκBα protein (). Because GUG inhibited IκBα phosphorylation and degradation, this study suggests that the effect of GUG on NF-κB/p65 is through inhibition of phosphorylation and subsequent proteolysis of IκBα. Since GUG contains two α,β-unsaturated carbonyl moiety, it seems that the compound may cause cysteine thiol modification in key molecule such as IKK of the NF-κB-signaling pathway (52
The results in show the protective effects of skin application of GUG on TPA-caused tumor promotion in DMBA-initiated SENCAR mouse skin. The preapplication of GUG to that of TPA showed substantially reduced tumor incidence and lower tumor body burden when assessed as total number of tumors per group, percent of mice with tumors and number of tumors per animal, as compared with animals that did not receive GUG (). These chemopreventive and antitumor promotion observations in murine skin by GUG can be explained by the biochemical mechanisms examined in the present study.
In summary, our results suggest that topical application of GUG prior to TPA application to SENCAR mice resulted in a significant decrease in skin edema, hyperplasia, epidermal ODC activity and protein expression of ODC, COX-2 and iNOS classical markers of inflammation and tumor promotion. In addition, our results also suggest that topical application of GUG prior to TPA application also resulted in inhibition of phosphorylation of MAPKs, activation of NF-κB/p65 and IKKα/β and degradation and phosphorylation of IκBα. Our data clearly demonstrate that GUG could be a potent antitumor-promoting agent because it inhibits several biomarkers of TPA-induced tumor promotion in an in vivo animal model. One might envision the use of chemopreventive agents such as GUG in an emollient or patch for chemoprevention or treatment of skin cancer.