Preparation of Nexrutine® diet
Nexrutine® was provided by Next Pharmaceuticals, Irvine, CA. For in vivo experiments, pelleted diet containing different doses of Nexrutine (300 and 600 mg/kg) was prepared at Dyets, Inc. (Bethlehem, PA). Stability of Nexrutine® in the diet pellets was evaluated every month by thin-layer chromatographic (TLC) analysis. Briefly, 5 g of pelleted diet was extracted with 70% methanol and dried under vacuum. Dried powder was resuspended in water and TLC was performed using dichloromethane and methanol (24:1 w/v) as the solvent systems. The chromatographic profile was observed under UV light. Nexrutine® was used as a positive control. The chromatograms of pure Nexrutine® and the extracted Nexrutine® from the pelleted diet were identical (data not shown).
Transgenic mouse experiments
TRAMP model was developed by prostate specific expression of SV40 large T antigen using the rat probasin promoter (22
). TRAMP mice develop prostate tumors with 100% frequency, in progressive stages that facilitates preclinical studies in the prevention, intervention and regression setting as demonstrated by various groups, including our own (24
). TRAMP mice, with a pure C57BL/6 background, were obtained from Jackson Laboratories (The Jackson Laboratories, Bar Harbor, Maine, USA). All mice were maintained in a climate-controlled environment with a 12-h light/dark cycle. Diet and water were supplied ad libitum
. For imaging, animals were anesthetized with ketamine (80mg/kg i.m.) and scanned in a Siemens 3T TRIO MRI scanner using a receive-only surface coil that was custom-built for this experiment to provide a full volumetric coverage of the prostate region. A 3-D Fast Low Angle Shot (FLASH) sequence with fat suppression was optimized (TR=27ms, TE=5ms, flip angle=22 degrees) to provide an SNR of 25 at isotropic spatial resolution of 400 microns and total scan time of about 10 min. PSVC volume was measured using MNI Display program1
. Following the final MRI scan, animals were euthanized and all organs were collected for further analysis. At necropsy, animals were examined to determine if there were any gross organ abnormalities. Animal care and handling was conducted in accordance with established humane guidelines and protocols approved by the University of Texas Health Science Center at San Antonio’s Institutional Animal Care and Use Committee.
Tumors were weighed, harvested and fixed in 10% neutral buffered formalin. Tumors were paraffin embedded, sectioned, placed on poly-lysine slides and stained with H&E to visualize cell nuclei and cytoplasm. Prostate lesions were scored using an established grading system for TRAMP mice (21
). Non-cancerous lesions were graded as 1, 2 or 3, indicating normal tissue, low PIN and high PIN, respectively. Grades 4, 5 and 6 indicated well-differentiated, moderately differentiated and poorly differentiated cancerous lesions, respectively. Images were recorded using a light microscope.
Preparation of extracts and Western blot analysis
50 mg of prostate tumor or tissue (dorso-lateral) was homogenized in liquid nitrogen and lysed in buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP40, 50 mM NaF, 1 mM NaVO4
, 1 mM phenylmethylsufonyl fluoride, 25 μg/ml leupeptin, 25 μg/ml aprotinin, 25 μg/ml pepstatin and 1 mM DTT; pH 7.4). After passing the lysate through a 25G needle, cell debris was removed by centrifugation at 12,000 rpm for 30 min. Nuclear extracts were prepared according to the method of Dignam and protein content of the extracts was determined by the method of Bradford as described earlier (32
). Equal amounts of extracts were fractionated on a 10% SDS-polyacrylamide gel. Following electrophoresis, proteins were transferred to a nitrocellulose membrane. The blotted membrane was blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween 20, and incubated with indicated antibodies (Santa Cruz Biotechnology, CA; Cell Signaling Technology, Inc. Beverly, MA) followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Sigma) in blocking solution. Bound antibody was detected by enhanced chemiluminescence using Supersignal West Pico Chemiluminescent Substrate, following the manufacturer’s directions (Pierce, Rockford, IL). All the blots were stripped and re-probed with GAPDH to ensure equal loading of protein.
Sections from formalin fixed, paraffin embedded tissue blocks of prostate were cut and stained with pCREB (Cell Signaling Danvers, MA), pAkt (Ser473, rabbit monoclonal, 1:50, Cell Signaling, Danvers, MA); CREB (Cell Signaling Danvers, MA). Proliferation was assessed using the Ki-67 (SP6) antibody (Lab vision, Fremont, CA). The secondary and tertiary antibodies were biotinylated link and streptavidin HRT (Biocare 4 plus Kit, Biocare Medical, Concord, CA or Vector Labs).
Proliferation and TUNEL staining in tumors
Proliferation was assessed using the Ki-67 (SP6) antibody (Lab Vision, Fremont, CA). The secondary and tertiary antibodies were a biotinylated link and streptavidin HRP (Biocare 4 plus Kit, Biocare Medical, Concord, CA). Apoptosis was assessed using in situ the terminal transferase dUTP nick end-labeling (TUNEL) assay, with biotin-16-dUTP (Roche Applied Science, Indianapolis, IN) and terminal deoxynucleotidyl transferase (TdT) according to vendor recommendations (Invitrogen, Carlsbad, CA).
CREB DNA binding activity
CREB DNA binding activity was measured in TRAMP nuclear extracts using TransAM™ CREB as described earlier (21; Active Motif, Carlsbad, CA). The sequence of the wild type CREB was 5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′ (Mutated nucleotides shown in bold). Extracts were incubated with CREB consensus oligonucleotide that was immobilized in a 96-well plate. A primary antibody specific for an epitope on the bound and active form of CREB was added, followed by subsequent incubation with secondary antibody and developing solution. Following incubation, CREB activity was measured, colorimetrically, at 450 nm. Nuclear extracts prepared from human fibroblast WI-38 cells stimulated with Forskolin (CREB activator) was used as a positive control. For competition experiments, the wells containing immobilized oligo were pre-incubated with a 100-fold molar excess of wild type and mutant oligonucleotide for 30 min before adding the nuclear extract.
Prostate cancer tissue array
Immunohistochemical staining for CREB, pCREB and pAkt was conducted on a human prostate cancer tissue array containing 15 specimens of different Gleason grades with paired normal prostate. The array was prepared and provided by Dr Dean Troyer (Department of Pathology. University of Texas Health Science Center, San Antonio, TX). Each specimen was analyzed for immunoreactivity using a1-4+ scoring system for stain intensity and percentage of positive cells. Grading scale for intensity ranged from undetectable signal (1+) to strong signal (4+). The % of staining was scored by counting the positive stained cells and total number of cells in four random microscopic fields.
Dichotomous measures, such as the presence/absence of (1
) prostate tumors, (2
) cancerous lesions with grade of 4 or higher were evaluated for treatment group differences using Fisher’s exact tests. Owing to the small sample size and similarity of outcome frequencies for the low- and high-dose Nexrutine® treatment groups, the groups were then combined and compared vs. controls using Fisher’s exact tests. Since the reduction in tumor frequency was a primary hypothesis of the study, a power analysis was performed to determine the likelihood of observing a clinically significant response with the proposed sample size of 15 control and 20 experimental mice. In the population of TRAMP mice, if 50% or more mice receiving the control diet develop prostate tumors, while 10% or fewer mice receiving the experimental diet develop prostate tumors, then this difference in tumor rates can be considered clinically significant. This can be detected with the proposed sample size by Fisher’s Exact Test at the 0.05 level with power of 80%. One-way ANOVA was performed to determine if there were any significant treatment group mean differences for prostate weights. Post-hoc tests adjusting for the number of comparisons were performed to identify group differences if the F-test was significant. The weekly mouse weight data were analyzed using mixed-model ANOVA, assuming a first-order autoregressive covariance structure for the repeated effect by week. The mixed-model ANOVA tested for interaction between treatment group and week, as well as the individual main effects and post-hoc comparisons, were performed when the estimated treatment group means for a given week were significantly different (determined by F-test). Because weight changes were substantial at monthly intervals relative to weekly intervals, the mixed-model ANOVA was performed by restricting the data to the baseline, 4, 8, 12 and 16-week measures. For all statistical tests, p-values < 0.05 were considered significant, with the exception that the test for interaction in the ANOVA model was considered significant if p<0.10. The power analysis was performed using PASS 6.0 software. Statistical analyses and graphics were performed using SPSS and Stata software.