Abstract 894: Sulforaphane as a chemopreventive agent against oral carcinogenesis

Abstract

Abstract Introduction: Epidemiologic studies report an association between reduced head and neck squamous cell carcinoma (HNSCC) risk and a diet rich in the Brassica family of cruciferous vegetables. Broccoli extract induces cytoprotective enzymes that mitigate the effects of environmental carcinogens. The phytochemical sulforaphane (SF) mediates this inducer activity via induction of the NRF2 transcription factor and its target genes. Nrf2−/− mice are more susceptible than WT mice to oral cancer induced by the carcinogen 4NQO, while Keap1−/− mice are less susceptible. We performed pilot preclinical and clinical studies to evaluate the chemopreventive potential of SF against oral carcinogenesis. Methods: First, we evaluated a normal mucosal epithelial (Het-1A) and 2 HPV(-) HNSCC cell lines (UMSCC-22A and UMSCC-1) by immunoblotting for dose and time-dependent induction of NRF2 expression, after varying SF doses and exposure times. Second, we performed qPCR for NRF2 target genes following SF treatment (10 μmol for 6 hrs) to determine if induced NRF2 signaling was functional in these cell lines. Third, we evaluated chemoprevention by SF in the 4NQO model of oral carcinogenesis: 34 WT C57BL/6 mice were treated with 100 μg/ml 4NQO in ad lib drinking water for 16 wks, then randomized to vehicle vs. SF (6 μmol thrice weekly) for 8 wks. Fourth, 10 healthy human volunteers consumed SF-rich broccoli sprout extract (BSE; 100 μmol SF/day) for 3 days (NCT02023931). After a washout, participants were treated for 3 days with topical exposure to the same regimen (swish/spit). We analyzed urine SF metabolites by mass spectroscopy to assess bioavailability, and serial oral mucosa scrapings by PCR to assess NRF2 target gene induction. Results: Basal levels of NRF2 were nearly undetectable, while SF led to dose and time-dependent upregulation of NRF2 expression in all 3 cell lines. SF treatment significantly induced NQO1 and GCLC mRNA in all 3 models. Compared to vehicle, SF reduced both the average incidence of tongue tumors/mouse (1.76 vs. 0.76; p<0.01) and the average tumor volume/mouse (8.97 vs. 2.66 mm3; p<0.01). Seven of 10 subjects showed upregulation of NQO1 mRNA in oral mucosa during oral BSE treatment, compared to baseline. Oral, but not topical, administration of SF-rich BSE resulted in systemic exposure to SF as measured by urinary metabolites. No treatment-related toxicities were observed. Conclusion: SF induced functional NRF2 expression in normal mucosal and HNSCC cell lines, and was protective against oral chemical carcinogenesis in the 4NQO model. Brief oral-systemic exposure to SF-rich BSE was well-tolerated and bioactive in the oral mucosa of healthy human volunteers. These studies provide preliminary evidence that SF may be chemopreventive against HNSCC. Prospective clinical investigation in a high risk population is warranted, including serial assessment of mucosal NRF2 target gene induction as a candidate mechanistic biomarker. Citation Format: Julie E. Bauman, Yan Zang, Malabika Sen, Daniel P. Normolle, Thomas W. Kensler, Sumita Trivedi, Patricia A. Egner, Siddharth H. Sheth, Jennifer R. Grandis, Daniel E. Johnson. Sulforaphane as a chemopreventive agent against oral carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 894. doi:10.1158/1538-7445.AM2015-894