Kleiner-Hancock et al. BMC Cancer 2010, 10:394 http://www.biomedcentral.com/1471-2407/10/394 ATRA + ACA/AUR, starting 1 week prior to tumor cell inoculation and for the duration of the study. Test diets were administered prior to the injection of tumor cells to be a chemoprevention protocol. In humans, this might be applicable to individuals who have been trea- ted for a primary tumor, but are susceptible to the development of subsequent tumors. This could be a window of opportunity for chemoprevention. Mice were shaved (2 days prior to injection), then injected with 1 × 106 SRB12-p9 cells suspended in sterile PBS, s.c., pal- pated and weighed twice a week for 25-28 days follow- ing injection. Tumors were measured using digital 3 calipers and tumor volume (mm ) was calculated using the following formula, based on the assumption of a near-spherical tumor shape: V = ((l + w)/4)3 * 1.33 * PI, where l = length, w = width. The studies were con- ducted at different times, so the timing was kept to a 25-28 day window for consistency. All mice in a particu- lar study were euthanized on the same day so that com- parisons could be made. microPET imaging Tumor bearing mice were investigated by non-invasive imaging using a R4 microPET scanner (Siemens Medical Solutions USA, Inc., Knoxville, TN). In brief, the system operates in three dimensions and is composed of 6144 lutetium oxyorthosilicate crystal (LSO) detector ele- ments, with a 7.8 cm axial and a 10 cm transaxial field- of-view. The [18F]-3’-fluoro-3’-deoxy-L-thymidine ([18F] FLT) radiotracer, a pyrimidine nucleoside analogue, has been validated as a promising PET radiopharmaceutical for monitoring tumor proliferation as well as response to therapy [24]. The [18F]FLT was synthesized using a 3-N-Boc-5’-O-dimethoxytrityl-3’-O-nosyl-thymidine pre- cursor according to published method of Eisenhut et al. [25]. No-carrier-added [18F]FLT was administered intra- venously by tail vein injection into experimental animals at a dose of 300 mCi/mouse. Sixty minutes after injec- tion, the animals were anesthetized, positioned in the field of view of the scanner, and imaged for 15 minutes. Subsequently, the microPET images were reconstructed using an iterative reconstruction technique. Glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase (NQO) activities Liver cytosolic GST activities were assayed spectropho- tometrically (Shimadzu, Columbia, MD, kinetic mode) using two different substrates [26]. First, liver cytosol enriched fractions were prepared by differential centrifu- gation as previously described [27]. Protein concentra- tions were estimated using the Bradford method with BSA as a standard [28]. Aliquots of cytosolic samples were incubated with either 1 mM 1,2-dichloro-4- nitrobenzene (DCNB), 5 mM GSH in 0.1 M Page 3 of 10 KH2PO4 buffer, pH 7.5; or with 1 mM 1-chloro- 2,4-dinitrobenzene (CDNB), 1 mM GSH, in 0.1 M KH2PO4 buffer, pH 6.5. Changes in absor- bances were assessed at 340 nm (CDNB) or 345 nm (DCNB), and enzyme activities were calculated using extinction coefficients of 9.6 nM-1/cm-1 and 8.5 nM-1/ cm-1, respectively [29]. Liver cytosolic NQO activity was assayed spectrophotometrically (kinetic mode) at 600 nm using 2,6-dichloroindophenol (1.25 mM) as a sub- strate and an extinction coefficient of 21 nM-1/cm-1 as previously described [30]. Statistical analysis For NF- B and GST data, differences between groups were compared using ANOVA followed by post-hoc analysis with Fisher’s PLSD test and/or Tukey’s test. Group comparisons are shown in the figure and table legends. Descriptive statistics of body weight and tumor volume were presented as means and least squared means along with standard deviation and sample size for a particular sub-group. Analysis of variance method was used to compare the body weight and tumor volume among groups (ACA, AUR and ATRA). Subse- quently, the multiple comparison using Dunnett method was made among the group (all other group compares to control group). To perform the non-parametric data analysis, the body weight and tumor volume were ranked first and then similar analysis were performed as described above. A linear trend test was performed to assess the effect of ATRA dosage of 0, 5, 10, and 30 with/without ACA on the body weight and tumor volume at particular days (13, 15, 20, and 22) after treat- ment. Only these days have the same measurement for the dosage of ATRA, the number of mice in the control dosage level (0) were combination of two separated experiment in this study (n = 13), the number of mice are 8, 8, and 5 for dose level of 5, 10 and 30. All p- values, ≤ 0.05 were considered statistically significant. All statistical data analysis was performed by using SAS system 9.2 (SAS Inc, Gary, NC). Results ACA and AUR suppressed LPS-induced NF- B activation in NF- B-RE-luc (Oslo) mice Many phytochemicals suppress NF- B activation, so we used a simple in vivo method using NF- B-RE-luc (Oslo) to first determine whether ACA and AUR also pos- sessed this inhibitory activity. 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. On day 3, mice were imaged as a pre-screen to ensure that ACA and AUR did not activate the NF- B reporter. On day 4, 30 min after the last pretreatment, mice were injected