Purpose: Epigenetic alteration which leads to the development of various cancer hallmarks is induced by chromatin instability stimulated by a mix of genetic and non-genetic factors and Inflammation is one of the important non-genetic stimuli. During UV- and chemical-induced skin carcinogenesis, Nuclear factor E2- related factor 2 (Nrf2) is a well-recognized transcription factor in activating antioxidant responsive element (ARE) to exert anti-inflammatory activity against skin cancer transformation. Flavonoids from fruits and vegetables, which possess anti-inflammatory activities can be used as potential epigenetic modulators for cancer prevention. Anthocyanidin such as pelargonidin and delphinidin is one of the major subtypes of flavonoid and has been shown to have anti-inflammatory potency. The purpose of this study is to examine whether delphinidin and pelargonidin can prevent skin carcinogenesis by demethylating Nrf2 promoter, which leads to the activation of Nrf2-ARE signaling pathway.
Methods: A phytochemical model was established combining epigenetic modification, transcriptional/translational gene expression of anti-inflammatory pathway and cancer prevention effect. First, chemoprophylaxis effect of delphinidin and pelargonidin was examined through the soft agar colony formation assay with 12-O-tetradecanoylphorbol-13-acetate (TPA) as a tumor promoter in murine skin epidermal JB6 P+ cells. Second, the inhibitory mechanism was then elucidated by the induction of Nrf2-ARE promoter activity on ARE-luciferase reporter assay. The induction of Nrf2-mediated antioxidant/anti-inflammatory enzymes was confirmed at the mRNA and protein levels by qPCR and Western blot. Finally, to examine the epigenetic modulation, the methylation status of the Nrf2 promoter was analyzed using bisulﬁte genomic DNA sequencing and then the protein expression of DNA methyltransferases and the histone deacetylase was analyzed.
Results: Delphinidin and pelargonidin have shown to demethylate the first 15 CpG sites in the mouse Nrf2 promoter region between −1226 and −863, which corresponds to the increased Nrf2 promoter activity and elevated mRNA and protein expression of Nrf2 downstream genes in murine skin epidermal JB6 P+ cells. The reduced CpG methylation ratio in the Nrf2 promoter region was exhibited through the decreased protein expression of DNA methyltransferases and the histone deacetylase. Next, the upregulation of Nrf2 signaling pathway by delphinidin and pelargonidin correlated to the chemoprophylaxis effect exerted through inhibiting anchorage-independent cellular transformation induced by tumor promoter, TPA, in JB6 P+ cells.
Conclusion: Overall, our results suggest that delphinidin and pelargonidin as epigenetic activators of Nrf2-ARE pathway could be used as skin cancer chemopreventive agents. Most importantly, the experimental design integrating phytochemical, inflammation and the epigenetic modification, can serve as a useful model to study epigenetic modulation in cancer prevention. (Supported in part by NIH grant R01CA200129 to A.N.K)
Shanyi Li– Postdoc, Rutgers University Ernest Mario School of Pharmacy
Ah-ng Kong– Distinguished Professor, Glaxo Endowed Chair in Pharmaceutics; Graduate Director, Rutgers University Ernest Mario School of Pharmacy