Kleiner-Hancock et al. BMC Cancer 2010, 10:394 http://www.biomedcentral.com/1471-2407/10/394 1’-acetoxychavicol acetate (ACA) is a natural compo- nent of traditional Thai condiments, present in the seeds (15000 ppm) and rhizomes (27300 ppm) of the ethnomedicinal plant Languas galanga Stuntz (Zingiber- aceae) [5]. In the skin model, pre-treatment of mice with ACA prior to TPA treatment in 7,12-dimethylbenz [a]anthracene (DMBA)-initiated mice suppressed skin tumor promotion [6]. Recently it was found that ACA blocked TNF-a induced activation of NF- B indirectly through I B [7]. In this regard, ACA inhibited TNF-a- induced phosphorylation and degradation of I Ba, sug- gesting that it inhibits the I Ba kinase, IKK. Auraptene (AUR) is a geranyloxy-coumarin obtained from citrus fruits [8], the aromatic plant Zanthoxylum schinifolium [9] and the fruits of Paliurus ramosissimus [10]. AUR is also known to suppress skin tumor promotion [8], in addition to its many other chemopreventive effects. AUR suppresses superoxide generation indirectly, most likely through inhibition of the multi-component NADPH oxidase system [8]. AUR, possessing anti- inflammatory activities [11] also suppresses ulcerative colitis and ulcerative colitis induced colon cancer [12,13]. Consistent with these observations, AUR also activated PPARg in adipocytes, which led to the increased production of adiponection, which is known to suppress the inflammatory process [14]. These varied mechanisms and protective effects in rodent models of cancer, combined with the low risk of toxicity [15], sug- gest that AUR might be a good candidate for skin SCC chemoprevention. Retinoids are a class of chemical compounds, which include active metabolites of vitamin A (retinol), as well as a diverse array of synthetic derivatives. Retinoids have been shown to modulate multiple cellular processes, including proliferation, differentiation, homeostasis, malignant transformation and apoptosis [16-18]. ATRA has long been known as one of the most effective sup- pressors of tumor formation in the 2-stage mouse skin chemical carcinogenesis model [19,20]. We have recently used this model to further explore the mechan- ism of ATRA action and have demonstrated that ATRA targets the B-Raf/Mek/Erk signaling pathway and this activity coincides with its chemopreventive activity [21]. We have also shown recently that ATRA can suppress Stat3 signaling in skin carcinogenesis, and that Stat3 activity lies downstream of the B-Raf/Mek/Erk pathway [22]. It is interesting to note that many downstream sig- naling effectors involved in malignancy are regulated by both Stat3 and NF- B We hypothesized that suppression of both B-Raf/ Mek/Erk/Stat3 and NF- B signaling through combined administration of ATRA and ACA/AUR, may exert a stronger anti-tumor effect than either agent alone. Page 2 of 10 Methods Cell culture The human skin SCC cell line SRB12-p9 was derived by single cell cloning from SRB12 cells (a gift from Dr. Reuben Lotan, Department of Thoracic Head and Neck Medical Oncology, University of Texas M.D. Ander- son Cancer Center). Cells were cultured in a humidified atmosphere at 5% CO2, in Dulbecco’s Modified Eagle’s Media-F12 supplemented with 10% fetal bovine serum. Severe combined immunodeficient (SCID) beige mice SCID/bg mice were housed sterile cages in a tempera- ture and humidity controlled AAALAC facility under a normal 12 hour light/dark cycle. All procedures were approved by LSUHSC Institutional Animal Care and Use Committee in accordance with NIH guidelines. The mice were allowed access to autoclaved food and water ad libitum. NF- B-RE-luc (Oslo) mice All mice were allowed food and water ad libitum and were housed according to institutional and NIH guide- lines. Mice were purchased from the Xenogen Corpora- tion (Alameda, California) and are bred and genotyped in our breeding colony. NF- B-RE-luc is a light produ- cing animal model that carries a transgene containing 3 NF- B response elements from the Ig light chain pro- moter and modified firefly luciferase cDNA (Promega pGL-3) [23]. The reporter is expressed under basal con- ditions in the lymph node (neck), thymus (thoracic region), and Peyer’s patches (abdominal region). Various inflammatory stimuli induce the reporter, such as LPS, TNF-a, and arthritis, which makes it suitable for the study of transcriptional regulation of NF- B. Male and female mice (n = 3-4) were pre-treated with ACA (100 mg/kg bw), AUR (200 mg/kg bw) or vehicle (0.2 mL corn oil/25 g bw) once a day for 4 consecutive days. At 30 min after the last pretreatment, mice were injected with LPS (2.7 mg/kg bw). Mice were imaged on an IVIS 100 Imaging System at 30 min after the 3rd pre-treat- ment dose as a pre-screen to ensure that the pre-treat- ments themselves did not activate NF- B. Mice were imaged again at 3 h and 24 h after LPS treatment. Mice were injected with 2.5 mg D-luciferin substrate, along with ketamine/xylazine anesthetic, 15 minutes prior to each imaging. Luminescence intensity was quantified using Living Image (R) 3.0 software (Caliper Life Sciences, Inc., Hopkinton, MA). Xenograft Model Groups of 5-8 female SCID/bg mice (6-12 weeks of age) each were fed control diets or diets containing ATRA (5, 10, 30 ppm), ACA (500 ppm) or AUR (1000 ppm) or