Abstract A49: Chemoprevention of esophageal cancer using PCA, a metabolite of black raspberry-derived anthocyanins
Authors: Peiffer Dan , Siddiqui Jibran H. , Kuo Chieh-Ti , Huang Yi-Wen , Zimmerman Noah P. , Wang Li-Shu , Stoner Gary D. , Carmella Steven G. , Ransom Ben , Hecht Stephen S. 
Published: January 2014

Background: We have shown that black raspberries (BRB) as well as their component anthocyanins (AC) inhibit N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in rats. Like many polyphenolic compounds, the bioavailability of BRB anthocyanins is low. The majority of BRB anthocyanins are metabolized by gut bacteria to produce protocatechuic acid (PCA). PCA has been shown to be bioavailable and also to be chemopreventive. Furthermore, we have shown that BRB and their anthocyanins decrease methylation and increase tumor suppressor gene (TSG) expression; e.g., SFRP and WIF1, Wnt pathway antagonists, as well as p16 and PAX6a, in human colon tumors. The current study was designed to determine if PCA is effective in preventing NMBA-induced rat esophageal tumorigenesis by reducing DNA methylation and increasing expression of TSG(s).

Materials and Methods: Male Fischer 344 rats aged 3-5 weeks were randomized into five groups and injected s.c. with either the vehicle (20% DMSO/water) or NMBA (0.35 mg/kg b.w.) three times a week for five weeks. During this time all rats were fed control diet (AIN-76A). At the beginning of week 6, all rats were started on their study diets (Group 1: vehicle + AIN-76A, Group 2: NMBA + AIN-76A, Group 3: NMBA + 3.8 umoles AC/g of AIN-76A, Group 4: NMBA+ 6% BRB in AIN-76A, Group 5: NMBA+ 500 ppm PCA in AIN-76A). The concentration of 3.8 umole AC/g of diet for Group 3 was chosen to compare with past studies, 6% BRB were used to match the anthocyanin content of Group 3, and 500 ppm PCA is the estimated amount of anthocyanins converted to PCA from 3.8 umole AC/g diet in the gut. A total of 9 animals per group were harvested at weeks 15 and 25, and 30 rats per group will be harvested at week 35. Esophagi were harvested and esophageal tumors counted and sized. After counting the tumors, one half of the esophagus was used for histological analysis and the other half for DNA/RNA extraction to investigate relevant TSG expression and methylation.

Results: The study is still on going. H&E slide scoring revealed significantly reduced hyperplasia in all test diet groups (BRB, AC, PCA) when compared to the NMBA control group at week 15. At week 25, the test diets had no effect on dysplasia frequency, however, all test diets reduced tumor incidence. NMBA treatment led to an increase in dimethyltransferase 3b (DNMT3b) mRNA expression at week 15 and week 25, and 500 ppm PCA decreased DNMT3b expression at both time points. NMBA treatment caused increased methylation and down-regulated mRNA expression of SFRP4 when compared to the vehicle control group at week 25 but not at week 15. None of the test diets influenced DNA methylation or mRNA expression of SFRP4 in NMBA-treated rats at either week 15 or 25. No differences in methylation status of PTX3 (Pentraxin 3), a cytokine released by phagocytic and dendritic cells and shown to be methylated in human esophageal cancer, was observed across all groups at either time point.

Conclusion: These results suggest for the first time that PCA may be protective against NMBA-induced esophageal tumorigenesis in rats. PCA significantly reduced tumor incidence, similar to that of BRB and their anthocyanins, by preventing the conversion of dysplastic lesions into papillomas. Preliminary evidence suggests that PCA influences the expression of a key methyltransferase (DNMT3b) in NMBA-treated rat esophagus.

Citation Format: Dan Peiffer, Jibran H. Siddiqui, Chieh-Ti Kuo, Yi-Wen Huang, Noah P. Zimmerman, Li-Shu Wang, Gary D. Stoner, Steven G. Carmella, Ben Ransom, Stephen S. Hecht. Chemoprevention of esophageal cancer using PCA, a metabolite of black raspberry-derived anthocyanins. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A49.