INTRODUCTION

1. Pre-clinical Evaluation of Strawberries and Strawberries with Selenium on the Chemoprevention of Oral Cancer Blake M. Warner1,2, Bruce C. Casto1, Thomas J. Knobloch1, Richard C. Funt3, Brent Accurso2, Brett Daly1, Rebecca Galioto4, and Christopher M. Weghorst1 The Ohio State University, College of Public Health, Division of Environmental Health and the Comprehensive Cancer Center 1, College of Dentistry, Comprehensive Training in Oral and Craniofacial Sciences Program2, College of Agriculture and Horticulture3, College of Veterinary Medicine4, Columbus, OH METHODS STUDY DESIGN AND RESULTS ABSTRACT Animals. Male Syrian hamsters (Mesocricetus auratus), 5 weeks of age were obtained from Harlan Laboratories (Indianapolis, IL). Animals acclimated for 7 days before carcinogen treatment. Food (AIN-76A, Dyets Inc., Bethlehem, PA) and water were given ad libitum. Berry and Diet Preparation (Table 1). Strawberries (Fragraria annassa) used in this study were grown at Dr. Richard C. Funt’s berry farm in Bellville, OH. Strawberries with bioincorporated selenium were prepared by spraying 0.1% sodium selenate solution (98%, Sigma) three times on the leaves of the strawberry plants just before and during blossom. At this stage, plants contain elevated levels of methionine resulting in maximum selenium (Se) uptake. In preliminary lots of berries, the Se content of the freeze-dried berries from four different harvests ranged from 12.9-14.4ppm after just two applications of Se solution. It is theorized that Se is transported to chloroplasts and rapidly reduced [using sulfur metabolic pathways] to organic selenium amino acids (Se-Methionine, Se-Cysteine) that are essential for selenium accumulation in plants and for the synthesis of antioxidant selenoproteins such as glutathione peroxidase and superoxide dismutase. After harvest, strawberries were washed, flash frozen, and sent to Van Drunen Farms (Momence, IL) where they were lyophilized and ground into a fine powder. Three concentration levels of bioincorporated Se (<0.25ppm, 4.53ppm, and 10.9ppm) were established in the lots to be used for chemoprevention studies. Powdered strawberries were then sent to Dyets Inc., and incorporated at 10% w/w into pellets of AIN-76A diet. Caloric equivalence was achieved by reserving 10% starch from berry-laden diets. Complete Chemoprevention (Figure 1). 5 week-old male hamsters were started on 10% LS+Se (0.025ppm, 0.453ppm, and 1.09ppm) modified AIN-76A pellets and unmodified AIN-76A pellets (chemoprevention controls). After acclimation period, bilateral HCPs were painted with 0.2% DMBA in DMSO 3x/week for 6 weeks. Animal tissues were harvested after 12 weeks, tumors were enumerated and cryopreserved in liquid nitrogen. Bilateral HCPs were collected, split, and separated for cryopreservation and FFPE. Histologic Grading of Lesions (Figure 2). Each HCP was embedded in paraffin, cut on edge and prepared for histology. Serial 5µm sections were mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). A hematoxylin and eosin stained slide from each of the left and right HCPs was characterized at 100X magnification. Each field of view was categorized into one of four histologic categories: normal epithelium, low-grade dysplasia, high-grade dysplasia or carcinoma in situ(Table 2). Statistical Analysis of Lesion Counts. All statistical analyses were done using STATA IC V-10.2. Based on the unit of analysis being the individual contralateral cheek pouches from each animal in the study, Poisson regression and linear regression, with the dependent variable being lesion number and the independent variables being group and cheek pouch were used to evaluate the differences between mean lesion numbers. Both types of regression gave similar results, but Poisson regression was a more appropriate statistical analysis method based on the assumptions. Logistic regression was used to predict the theoretical bivariate outcome low-vs-high tumor number in each cheek pouch. INTRODUCTION In the US, prognosis for oral cancer is poor with low 5- and 10-year survival rates. These rates have remained relatively constant over the last three decades, with 20%-30% of oral cancer survivors experiencing a recurrence within two years. Since current treatments are relatively ineffective at inhibiting recurrence, it is important that novel preventive strategies be developed. The hamster cheek pouch (HCP) model has shown that biochemical, molecular and histological changes between humans and hamsters are maintained during oral carcinogenesis. We have used the HCP to demonstrate the chemopreventive efficacy of black raspberries and translated these findings into human clinical trials. Recently, we have investigated strawberries as a food-based chemopreventive agent for oral cancer. Epidemiologic and chemoprevention studies have demonstrated that selenium (Se) can decrease the risk and incidence of some human cancers through pathways such as apoptosis, detoxification, and inhibition of proliferation. We have demonstrated that dietary lyophilized strawberries (LS) significantly inhibited tumor formation in Complete Chemoprevention (CC) and Post-initiation (PI) assays. The current study illustrates ongoing pre-clinical efforts aimed at evaluating the chemopreventive efficacy of selenium (Se)-enriched lyophilized strawberries (LS+Se). Previous studies have shown that elemental and organoselenium compounds inhibited tongue carcinogenesis in animal models. Therefore, we hypothesize that the combinatorial administration of LS+Se will inhibit oral tumorigenesis to a greater extent than LS-alone. METHODS Strawberries with bioincorporated selenium (0.5 and 1.0 ppm) were prepared by spraying sodium selenate solution three times on the leaves of the plants just before and during blossom. The HCP model was used to evaluate the ability of LS and LS+Se to inhibit 7,12-dimethylbenz(a)anthracene (DMBA)-induced oral tumors. LS and LS+Se were incorporated into AIN-76A pellets at a rate of 10% and given to hamsters before, during, and after carcinogen treatment (CC). At 12 weeks, lesions in each cheek pouch were enumerated. HCP were excised and stored appropriately for subsequent studies. RESULTS Poisson regression with the dependent variable being tumor number and the independent variable being treatment group demonstrated that 10% LS and 10% LS+Se [0.5 ppm] significantly inhibited tumor formation by 43% and 59%, respectively, over DMBA-only treated controls. Using logistic regression with the dependent variable being presence or absence of tumors, it was found that LS+Se were 3x as likely to have no tumors (95% CI: 1.31–8.33) as DMBA-only treated HCP. LS-alone was 2.2x as likely to have no tumors, but this difference was not significantly different than DMBA-only treated groups. The presence of leukoplakias was significantly increased with LS+Se and LS, suggesting that progression from premaligant lesions to overt tumors was inhibited by LS and to an even greater effect with LS+Se. CONCLUSIONS While trends were evident for the combinatorial effects of LS+Se, the current study could not demonstrate statistical significances between the chemopreventive role of LS-alone versus LS+Se. Overall, these experiments provide strong preclinical evidence that the strawberries and strawberries with selenium can prevent or delay the development of oral cavity cancer. Figure 1 –Experimental Design Table 1 –Bioincorporated Selenium Figure 2 –Representative H&E Slides Table 1 – Two lots of strawberry plants were sprayed with 0.1% sodium selenate (Na2SeO4) solution 3x before and during blossom. A third lot of untreated plants served as control to test combinatorial effects of bioincorporated Se. Concentrations of bioincorporated Se were determined by Graphite Furnace Atomic Absorption Spectroscopy (GFAA). The treated LS contained 10.9ppm and 4.53ppm Se, respectively, and <0.25ppm in the untreated LS. A high Se, low Se, and CC control LS would be used in the animal study. Figure 1 – A complete chemoprevention (CC) protocol (chemopreventive agents given before, during and after carcinogen treatment) was used to evaluate the additive or synergistic chemopreventive effects of strawberries with bioincorporated selenium (Se) in preventing DMBA-induced oral cancers. Animals were given experimental diets one week prior to carcinogen treatment. 0.2% DMBA in DMSO was painted to contralateral cheek pouches 3x/wk for 6 weeks. Groups 1 (High) received AIN-76A laden with 10% lyophilized strawberries (LS) with ~1.0 PPM Se; 2 (Low) received diet laden with 10% LS with ~0.5 PPM Se; 3 (CC Ctl) received diet laden with 10% LS w/o Se, 4 received control AIN-76A diet only. Group 5 would serve as sentinel controls and would receive no carcinogen treatment. Table 2 –Histological Analysis of FFPE HCP Figure 2 – Representative H&E slides demonstrating the histology of sequential carcinogenesis in the DMBA-induced hamster cheek pouch model of oral cancer. Table 2 – It was found that there were no significant differences in the numbers of low-grade, high-grade and CIS per cheek pouch across the experimental treatment groups. Figure 3-6 –Strawberries and Strawberries + Selenium Inhibit the Formation of Tumors in the HCP Model of Oral Cancer Table 3 – Summary of Gross Lesion Counts Figure 3 Figure 4 Table 3 – At twelve weeks after the commencement of carcinogen treatment, animals were sacrificed, HCP were expressed, and gross lesions (tumors, leukoplakias, and papillomas) were enumerated. In total it was found that Group 4 had more tumors but less leukoplakias than the LS and LS+Se groups (Groups 1-3). A very limited number of papillomas were enumerated ( less than 1 per animal). Figure 5 Figure 6 Table 4 – Logistic Regression Analysis RESULTS ✔ Se is readily taken up by strawberry plants leading to as high as 40x higher concentrations of bioincorporated Se than in untreated berries. ✔ No overt toxic effects associated with bioincorporated Se at rates as high as 1.09ppm in the diet. ✔ LS and LS with bioincorporated Se inhibit the initiation and progression of oral cavity cancer. ✔ Although trends were evident for the combinatorial effects of strawberries with bioincorporated selenium, there were no statistically significant additive effects. ACKNOWLEDGEMENTS • Research Support • The Ohio State University Comprehensive Cancer Center - Molecular Carcinogenesis and Chemoprevention (OSU - CCCMC) • The Ohio State University College of Dentistry • Comprehensive Training in Oral and Craniofacial Sciences Program (CTOC) NIDCR T32 DE14320 Table 4 – Using logistic regression with the dependent variable low or high tumor number and the independent variables being group and HCP (R or L), it was demonstrated that LS and LS+Se are significantly more likely to have less tumors per HCP than DMBA-only treated controls. This relationship holds true across all LS+Se treated groups with respect to low-vs-high tumor number at three levels ( 0 vs >0, 0,1 vs >1, and 0-2 vs >2). Figures 3-4. In Figure 3, DMBA-only treated controls have a right-shifted frequency distribution relative to LS and LS+Se treated groups. This difference is indicative of the highly significant results achieved through logistic regression on low-vs-high tumor number at tumor counts less than 3 tumors per HCP. Figure 4, opposed to Figure 3, demonstrates that frequency distribution of leukoplakias in LS and LS+Se treated cheek pouches is shifted to right relative to DMBA-only treated controls. Figure 5-6. Using Poisson regression and plotting the mean number of lesions per cheek pouch, Figures 5-6 demonstrate that there are significantly less tumors in LS and LS+Se (p<0.005) relative to DMBA-only treated controls. There were not significant differences in tumors per cheek pouch between LS and LS+Se treated groups (p>0.05), but a trend toward significance was observed with an estimated increased sample size. With respect to leukoplakias, only Group 1 (p<0.01) had significantly more leukoplakias than DMBA-only treated controls. However, all LS+Se treated groups had high numbers of tumors per cheek pouch and may be indicative of the sustained arrest of premalignant lesions (leukoplakias) relative to DMBA-only treated controls.