Gene expression analysis of oral cancer chemoprevention by lyophilized strawberries in DMBA-induced hamster cheek pouch

B9

Oral cancers represent 2.5-3.0% of all cancers worldwide. Prognosis for oral cancers is poor with 5- and 10-year survival rates of 59% and 44%, respectively. Even with advances in treatment, these survival rates have not changed dramatically in three decades. Since current long-term treatments are relatively ineffective, it is important to develop novel strategies for the prevention of oral cancer.Chemoprevention by natural food products, e.g., black raspberries, has been demonstrated to be a viable approach for aerodigestive cancer prevention.The hamster cheek pouch (HCP) model was used to evaluate administration of lyophilized strawberries (LS) during the initiation and progression of oral cancer induced by 7,12-dimethylbenz(a)anthracene (DMBA). LS were incorporated into AIN-76A pellets at 5% and 10%, and fed to hamsters before, during, and after carcinogen treatment (Complete Chemoprevention Assay; CC), or after establishment of premalignant lesions (Post-initiation Assay; PI). Animals were sacrificed at 12 weeks and examined for total lesions, and tumor incidence and multiplicity. HCP tissues were excised and quick frozen for RNA isolation. Using Real-Time RT-PCR, we analyzed gene expression in apoptosis (Bcl2, Bax), angiogenesis (Vegfa), and proliferation (Myc, Ccnb2, Cdk2, Psmd10) pathways, as well as in select tumor suppressor genes (Cdkn2ap16, Cdkn2ap13-ARF, Trp53) in HCP tissues.LS at 5% and 10% inhibited tumor formation by 28% (p<0.05) and 41% (p<0.01) in the CC Assay, and 54% (p<0.01) and 36% (p<0.01) in the PI Assay, respectively. Total lesions (leukoplakias, papillomas, tumors) were inhibited in both assays. Histological analysis of HCPs demonstrated that 10% LS in the CC Assay inhibited moderate and severe dysplasias by 31% (p<0.01) and 34% (p<0.01), respectively. With respect to untreated HCP, DMBA-initiated HCP significantly increased Cdkn2ap16 (34-fold), Cdkn2ap13-ARF (23-fold), Bcl2 (3-fold), Ccnb2 (3-fold), Trp53 (3-fold), and Myc (1.4-fold) expression. In contrast, only Psmd10 (2-fold) demonstrated significantly decreased gene expression (p<0.000 for all genes). Conversely, with respect to DMBA-initiated HCP, DMBA + 5% LS-treatment in the PI Assay resulted in decreased Cdkn2ap16 (2-fold) and Cdkn2ap13-ARF (2-fold) expression, and increased Psmd10 (1.3-fold) expression (p<0.003 for all genes). These findings demonstrate that 5% LS in the PI Assay significantly attenuate cancer gene expression towards a more “normal” expression pattern.These experiments provide strong evidence that LS prevent oral tumorigenesis under conditions mimicking those in former tobacco users. Furthermore, this study extends our data supporting the ability of natural food products to modulate oral carcinogenesis when delivered in a mode directly amenable to the human oral cavity. Translation of preclinical oral cancer chemoprevention data into patient intervention strategies is essential. Ongoing human clinical trials targeting oral cancer prevention and recurrence will further define the utility of LS chemoprevention.