Topical application of a bioadhesive black raspberry gel modulates gene expression and reduces cyclooxygenase 2 protein in human premalignant oral lesions

Abstract

Reduced expression of proapoptotic and terminal differentiation genes in conjunction with increased levels of the proinflammatory and angiogenesis-inducing enzymes, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), correlate with malignant transformation of oral intraepithelial neoplasia (IEN). Accordingly, this study investigated the effects of a 10% (w/w) freeze-dried black raspberry gel on oral IEN histopathology, gene expression profiles, intraepithelial COX-2 and iNOS proteins, and microvascular densities. Our laboratories have shown that freeze-dried black raspberries possess antioxidant properties and also induce keratinocyte apoptosis and terminal differentiation. Oral IEN tissues were hemisected to provide samples for pretreatment diagnoses and establish baseline biochemical and molecular variables. Treatment of the remaining lesional tissue (0.5 g gel applied four times daily for 6 weeks) began 1 week after the initial biopsy. RNA was isolated from snap-frozen IEN lesions for microarray analyses, followed by quantitative reverse transcription-PCR validation. Additional epithelial gene-specific quantitative reverse transcription-PCR analyses facilitated the assessment of target tissue treatment effects. Surface epithelial COX-2 and iNOS protein levels and microvascular densities were determined by image analysis quantified immunohistochemistry. Topical berry gel application uniformly suppressed genes associated with RNA processing, growth factor recycling, and inhibition of apoptosis. Although the majority of participants showed posttreatment decreases in epithelial iNOS and COX-2 proteins, only COX-2 reductions were statistically significant. These data show that berry gel application modulated oral IEN gene expression profiles, ultimately reducing epithelial COX-2 protein. In a patient subset, berry gel application also reduced vascular densities in the superficial connective tissues and induced genes associated with keratinocyte terminal differentiation.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

Dr. Russell Mumper is a co-Founder and Director of NanoMed Pharmaceuticals, and Dr. Susan Mallery is a member of NanoMed’s Scientific Advisory Board. At the present time, NanoMed is negotiating with Ohio State University and The University of Kentucky to obtain an exclusive license agreement to the gel technology described in this report. NanoMed also has an awarded STTR grant in which Dr. Mallery is the PI. The subject of the awarded STTR is related to the topic of the gel technology; however, no data of any kind funded via the STTR is contained in the current article. Importantly, all of the data and studies described in the current report were planned, completed, and funded independently of NanoMed. The other authors disclosed no potential conflicts of interest.

Figures

Figure 1

Although the majority of patients with oral IEN showed a reduction in epithelial levels of COX-2 and iNOS at the conclusion of the trial, the extent of protein decreases varied among patients. The pretreatment COX-2 photomicrograph of patient no. 20 shows moderate to intense positivity in the spinous and granular layers, which was markedly reduced following treatment (88.9% decrease for patient no. 20). In contrast, iNOS showed a more uniform distribution throughout the epithelium, inclusive of basal cell staining, as is apparent in the pretreatment iNOS photomicrograph for patient no. 9. This patient showed an overall high therapeutic response rate (47.4%), inclusive of a 24.1% decrease in epithelial iNOS following berry gel application (all photomicrographs taken at × 100 image scale, using an Olympus BX51 microscope equipped with a Nikon DS-Fi1 digital camera).

Figure 2

Eleven clinical trial participants retained sufficient pretreatment and posttreatment tissues for the evaluation of the effects of berry gel on MVD. A, seven of the 11 participants showed posttreatment decreases, with four participants showing MVD reductions that ranged from 19.8% to 67.3%. B, pretreatment and posttreatment photomicrographs of patient no. 2. This individual showed the highest overall therapeutic response rate of 60%, including a decrease in lesional histologic grade apparent in the H&E-stained sections (pretreatment diagnosis—mild dysplasia; posttreatment diagnosis—atypia) and a 67.3% reduction in MVD, as noted in the pretreatment and posttreatment CD34 sections (all photomicrographs taken at ×100 image scale using an Olympus BX51 microscope equipped with a Nikon DS-Fi1 digital camera).