Kleiner-Hancock et al. BMC Cancer 2010, 10:394 http://www.biomedcentral.com/1471-2407/10/394 10 ppm in the diet, the inhibitory effect can be main- tained to the end of the study when combined with either ACA or AUR. Although neither ACA nor AUR possessed strong inhibitory effects on their own, these data suggest that they may be useful in combination with ATRA. This is particularly important because ATRA is known to produce hypervitaminosis A-related side effects, and if its dose can be limited and efficacy improved by combining with naturally occurring dietary agents, these combinations could be clinically useful. However, diets containing ATRA led to a dose-related decrease in body weights, which was not different when combined with ACA or AUR. Many other phytochemicals are known to suppress NF- B activation [38]. Among these, several are related to the Zingiberaceae family, including gingerol and cur- cumin. These agents have been known to suppress many types of cancer, including that of skin. Taken together, these data lend to an intriguing suggestion that suppression of NF- B activation in combination with suppression of the B-raf/Mek/Erk/Stat3 pathway may be a useful strategy in the suppression of cancer progression. Many downstream signaling effectors involved in malignancy are regulated by both Stat3 and NF- B, such that targeting both of these signaling path- ways at once by combining ATRA + AUR, may produce a synergistic tumor suppressive effect. New evidence is also helping to decipher the connections between NF- B and STAT3. In 2007, Yang and associates discovered that unphosphorylated STAT3 competes with IKB for binding to unphosphorylated NF- B. This is turn results in nuclear localization of this complex, leading to activa- tion of NF- B regulated genes [39]. Most recently, Lee and co-workers demonstrated that STAT3 impairs nuclear export of NF- B via activation of RelA acetyla- tion [40]. Thus it appears that STAT3 may enhance the effects of NF- B through multiple mechanisms. The combinatorial effects of ATRA and AUR were encouraging. AUR is known to activate Nrf2/ARE and induces carcinogen and oxidant-detoxifying enzymes. However, recently, a “dark side” of Nrf2 has been sug- gested: that Nrf2 is upregulated in cancer (particularly lung cancer) and provides cancer cells with protection against the hostile tumor microenvironment [41]. In fact, shRNA against Nrf2 in lung cancer cells attenuated their growth in mouse xenografts compared to vector control treated cells [42]. It is argued that chemopreven- tive agents that increase Nrf2 activity may produce che- moresistance to therapy [43]. However, Wang and colleagues demonstrated that ATRA reduced binding of Nrf2 to the ARE enhancer [42]. ATRA has been reputed to possess pro-oxidant activities at therapeutic concen- trations. If it suppresses Nrf2 activation it may render cells more susceptible to oxidative stress. In fact, Tan Page 8 of 10 and colleagues confirmed that ATRA activates the Nrf2/ ARE transcription pathway [44]. Although beyond the scope of the present study, it appears that perhaps ATRA behaves in combination with AUR as a partial agonist: that is, ATRA has a mild effect on activating Nrf2 by itself, but is milder than AUR, and in combina- tion, may mitigate the effects of AUR, leading to an intermediate effect on Nrf2/ARE. Considering the potentially controversial role of Nrf2/ARE in carcino- genesis, we believe that the combination of ATRA with AUR for cancer therapy is even more enticing, because both agents were shown to be more effective at suppres- sing tumor growth in combination, despite the appar- ently intermediate effect on Nrf2/ARE. Conclusions In conclusion, we report that the combination of two different phytochemicals, ACA and AUR in the diet, enhanced the chemoprotective effect of ATRA against human skin cancer cell growth in a xenograft model. In particular, AUR appeared to be synergistic with the effects of ATRA, whereas ACA appeared to prolong the tumor suppressive effects of ATRA. We believe these mechanisms to be related to the suppression of at least two pathways, Stat3 and NF- B. Future studies may be warranted to capitalize on using these combinations to target multiple oncogenic pathways to enhance efficacy and lower potential off-target effects. Additional material Additional file 1: Supplemental Table S1. 2 × 2 Factorial design for statistical analyses. Supplemental Table S2. Tumor volume (TV) statistics for ACA ± ATRA xenograft study. Supplemental Table S3. Body weight (BW) statistics for ACA ± ATRA xenograft study. Supplemental Table S4: Tumor volume (TV) as a function of ATRA dose: statistics for ACA ± ATRA xenograft study. Supplemental Table S5: Body weight (BW) statistics for ACA ± ATRA xenograft study as a function of ATRA dose. Supplemental Table S6: Trend effect of ATRA dose in presence or absence of ACA on tumor volume (TV). Supplemental Table S7: Tumor volume (TV) statistics for AUR ± ATRA study. Supplemental Table S8: Body weight (BW) data for AUR ± ATRA study. Abbreviations ACA: 1’-acetoxychavicol acetate; ARE: antioxidant response element; ATRA: all-trans retinoic acid; AUR: auraptene; CDNB: 1-chloro-2,4- dinitrobenzene; DCNB: 1,2-dichloro-4-nitrobenzene; GST: glutathione S-transferase; LPS: lipopolysaccharide; NF- B: nuclear factor kappa-B; NMSC: nonmelanoma skin cancer; NQO: NAD(P)H quinone oxidoreductase; SCC: Squamous cell carcinoma; SCID: severe combined immunodeficiency; TPA: 12-O-tetradecanoylphorbol-13-acetate. Acknowledgements The authors gratefully acknowledge the expert technical assistance of Tracee Terry, in the Small Animal Imaging Center. This research was supported in part by grants from the Feist-Weiller Cancer Center and the Department of Pharmacology, Toxicology & Neuroscience. This research was also supported, in part, by a National Cancer Institute grant 1K22CA102005-01A2 (HEK) and