Bruce C. Casto1, Thomas J. Knobloch1, Christopher M. Weghorst1, Laura A. Kresty2, Gary D. Stoner2, Steven M. D’Ambrosio3, Susan R. Mallery4, Dennis K. Pearl5, Herman A.J. Schut6
1Division of Environmental Health Sciences, School of Public health; 2Cancer Chemoprevention and Support Program; 3Division of Radiobiology; 4College of Dentistry; 5Department of Statistics, The Ohio State University; 6Department of Pathology, Medical College of Ohio
Introduction. Cancers of the oral cavity represent approximately 2.5% of the total cancers that occur in the United States and are the sixth most prevalent cancer worldwide. The majority of these cancers (90%) are squamous cell carcinomas (SCC), that have been etiologically linked to the individual's exposure to alcohol and tobacco. The overall prognosis for these patients is poor with a five year survival rate of ~50% that has not changed over the last three decades. Attention has focused on understanding the molecular mechanisms of human oral cancer development and, consequently, alterations in several genes have been directly linked to end‑stage human oral SCC. The studies presented here will test the posit that inhibition of tumor formation by nutritional or dietary chemopreventive agents (black raspberries) and modulation of biomarkers in hamster oral cancer and human oral cancer cells in culture can be used to evaluate efficacy for potentially inhibiting/modulating progression of premalignant lesions in humans. The HCP has a long and reproducible history for identification of chemopreventive agents and human oral cancer cells have been shown to respond to a number of chemopreventive agents of several different classes. The decision to study chemoprevention of oral cancer by lyophilized black raspberries (LBR) was based on the work performed in the laboratories of Dr. Gary Stoner in which LBR, given in the diet, were found to inhibit esophageal and colon cancer. The present investigations evaluated lyophilized black raspberries as inhibitors of carcinogen-induced tumor development in the hamster oral cavity and as inhibitors of cell replication and modifiers of gene expression in human oral tumor cells.
Methods. HCPs of male Syrian Golden hamsters, 3-4 weeks of age, were painted with 0.2% DMBA (7,12-dimethylbenz(a)anthracene) 3H/week for eight weeks or with a 1% mixture of BaP/NNK [benzo(a)pyrene/ 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]3X/week for 12 weeks. Hamsters were given 5% and 10% lyophilized black raspberries (LBR) prior to, during, and after treatment with 0.2% DMBA and for 10 weeks following treatment with BaP/NNK. Animals were sacrificed at 12 or 22 weeks from the beginning of treatment and the number of tumors determined. For inhibition of cell replication, an ethanol extract of LBR was incubated with human tumor cell lines. Cell lines were seeded in 96-well tissue culture plates containing complete growth medium. Fresh medium containing dilutions of the LBR extract [up to 200 µg/ml] dissolved in diluted DMSO or DMSO solvent alone were added after 24 hrs. On days 2 and 4, the medium was removed and replaced with fresh medium containing the LBR extract or solvent. On day 7, the medium was removed, cells were washed in PBS and cell numbers determined using a modified MTT assay.
Results. LBR, in the diet at 5% and 10% concentrations, inhibited tumor formation in the HCP. There was a significant difference (p=0.02) in the number of tumors between the 5% LBR and control groups (27 tumors/14 animals and 48 tumors/15 animals, respectively) and a nonsignificant decrease in the number of tumors in the 10% berry-treated animals (39 tumors/15 animals). All animals had at least one tumor, but only 9 of the 29 berry treated animals (31%) had 3 or more tumors compared with 10 of the 15 (67%) hamsters in the control group (p=0.03).
When hamsters were given 5% LBR in the diet for 48 hr prior to a single DMBA challenge, three major DMBA adducts were reduced by 29% and 55% at 24 and 48 hr, respectively, implying that inhibition of metabolism/initiation was the cause of tumor inhibition. However, when 10% black raspberries were administered topically in substitute saliva, 48 hr after the final DMBA treatment, inhibition of tumor formation was still observed (approximately 37%) in the LBR treated pouches.
In an attempt to duplicate the pathology found in former smokers and to evaluate a treatment strategy that would be amenable for human trials, the HCP were treated for 12 weeks with a mixture of the cigarette carcinogens, BaP and NNK, followed by 10 wk of 5% and 2% LBR in the diet. There was no effect on incidence of microscopic lesions (mild/moderate dysplasia, severe dysplasia, carcinoma-in-situ), but the multiplicity of severe dysplasia and carcinoma-in-situ were reduced by approximately 50% in animals given 5% berries, whereas no effects was seen on multiplicity with 2% berries.
Cultured normal, premalignant, and malignant human oral cells were treated with an ethanol extract of LBR and with selected components of LBR. No significant effect on the proliferation of normal oral cells was observed with any concentration of LBR extract compared to DMSO controls. In contrast, LBR extract inhibited proliferation of SCC cells in a dose dependent manner to a maximum of 40%. To examine the relationship between inhibition of cell proliferation by LBR and the modification of gene expression in cell regulatory pathways, the global expression profiles for over 22,000 transcripts of well-established genes were determined by microarray. Of the approximate 22,000 genes evaluated, roughly 20% were modulated by the LBR extract, with 67% of these being down-regulated and 33% being up-regulated with statistically significant increases and decreases. Genes in several regulatory pathways were modulated by LBR extract. The top 31 down-regulated and 23 up-regulated genes represent molecular pathways that may be affected by LBR extract in human oral SCC cells. LBR down-regulated numerous classes of genes related to tumorigenic processes including: proliferation (c-SKI and c-JUN), cell communication (MUC-1, MUC-5AC, MUC-8), apoptosis (BCL-2, CED-2), tumor invasion (TEB4), proteases (CPA) and up-regulates several genes related to: cell to cell adhesion (PCDHA3, DSC3) and antioxidant processing (CP).
Components of LBR (ellagic and ferulic acids, ß-sitosterol) and berry extract were examined for their ability to selectively inhibit replication of human tumor cells and for their effect on cell cycle progression. Berry extract, ferulic acid, and ß-sitosterol inhibited growth of premalignant and malignant cells, but had no effect on normal oral cells, whereas ellagic acid equally inhibited all cell types. Ferulic acid and ß-sitosterol inhibited progression of the cell cycle at the M/G1 interface, but berry extract appeared not to have any specific effect on stages of the cell cycle.
Future Studies. Clinical trials in human oral cancer, including a pre-surgical model to examine effect of berries on gene expression in oral tumors, a post-surgical model to prevent oral tumor recurrence, and inhibition or modulation of progression of premalignant lesions.
Acknowledgements. Grant support: NIH/NIDCR, PO1DE12704; Elsa U. Pardee Foundation; Cancer Research Foundation of America, NCI P50 5CA16058, OSU Comprehensive Cancer Center, MCC Program. We wish to express our special gratitude to Dale Stokes (Stokes Raspberry Farm, Wilmington, OH) for cultivation and preparation of black raspberries and his continuing interest in our research.
Keywords: black raspberries, chemoprevention, oral cancer, chemical carcinogens, dysplasia