Inhibition of Pro-inflammatory and Anti-apoptotic Biomarkers during Experimental Oral Cancer Chemoprevention by Dietary Black Raspberries

Abstract

Oral cancer continues to be a significant public health problem worldwide. Recently conducted clinical trials demonstrate the ability of black raspberries (BRBs) to modulate biomarkers of molecular efficacy that supports a chemopreventive strategy against oral cancer. However, it is essential that a preclinical animal model of black raspberry (BRB) chemoprevention which recapitulates human oral carcinogenesis be developed, so that we can validate biomarkers and evaluate potential mechanisms of action. We therefore established the ability of BRBs to inhibit oral lesion formation in a carcinogen-induced rat oral cancer model and examined potential mechanisms. F344 rats were administered 4-nitroquinoline 1-oxide (4NQO) (20 µg/ml) in drinking water for 14 weeks followed by regular drinking water for 6 weeks. At week 14, rats were fed a diet containing either 5 or 10% BRB, or 0.4% ellagic acid (EA), a BRB phytochemical. Dietary administration of 5 and 10% BRB reduced oral lesion incidence and multiplicity by 39.3 and 28.6%, respectively. Histopathological analyses demonstrate the ability of BRBs and, to a lesser extent EA, to inhibit the progression of oral cancer. Oral lesion inhibition by BRBs was associated with a reduction in the mRNA expression of pro-inflammatory biomarkers Cxcl1, Mif, and Nfe2l2 as well as the anti-apoptotic and cell cycle associated markers Birc5, Aurka, Ccna1, and Ccna2. Cellular proliferation (Ki-67 staining) in tongue lesions was inhibited by BRBs and EA. Our study demonstrates that, in the rat 4NQO oral cancer model, dietary administration of BRBs inhibits oral carcinogenesis via inhibition of pro-inflammatory and anti-apoptotic pathways.

Keywords: biomarker; black raspberry; chemoprevention; oral cancer; pro-inflammatory.

Figure 1

(A) Scheme of rat oral carcinogenesis and chemoprevention. Rats were exposed to oral carcinogen 4-nitroquinoline 1-oxide (4NQO) (20 μg/ml in drinking water) for 14 weeks. Administration of dietary chemopreventive agent began at week 14 and continued for 6 weeks. Animals were sacrificed at week 20 for gross and histological analysis of tongue lesions and examination of cellular and molecular markers of carcinogenesis. N represents number of rats per group. (B) Average food consumption for the control and experimental rat groups during oral carcinogenesis and dietary intervention phases of the experimental protocol. Data are expressed as mean food consumption (grams/cage/day) ± SE in each rat cage belonging to an experimental group (three rats per cage). *p-value < 0.05 for food intake comparison between 4NQO-only rat group and 4NQO + ellagic acid (EA) rat group using independent Student’s t-test. (C) Average body weight measurements for control and experimental rat groups during the carcinogenesis and intervention phases of the experimental protocol. With the exception of the sentinel animals, all experimental animals were administered 4NQO and were on AIN-76A defined diet prior to intervention with dietary black raspberries (BRBs) or EA. Data are expressed as mean ± SE of all rats in the group.

Figure 2

(A–C) Average number of (A) premalignant, (B) malignant, and (C) total lesions in 4-nitroquinoline 1-oxide (4NQO)-induced rat tongues fed control diet (4NQO group) or diet supplemented with 5% black raspberry (BRB) (4NQO + 5% BRB group), 10% BRB (4NQO + 10% BRB group), or 0.4% ellagic acid (EA) (4NQO + 0.4% EA group). Data are expressed as mean ± SE of all rats in the group. *p-value < 0.05 for group comparisons using independent Student’s t-test.

Figure 3

(A–E) Histopathological analysis of tongue tissues from untreated and treated carcinogen-induced rats. Distribution of the histopathological grades (normal, low-grade dysplasia, high-grade dysplasia, and SCC) in tongue epithelia of (A) sentinel rats, (B) 4-nitroquinoline 1-oxide (4NQO)-induced rats fed control diet, (C) 4NQO-induced rats fed diet supplemented with 5% black raspberry (BRB), (D) 4NQO-induced rats fed diet supplemented with 10% BRB, and (E) 4NQO-induced rats fed diet supplemented with 0.4% ellagic acid (EA). Note the significantly reduced percentage of SCCs in 4NQO exposed rats treated with 5% BRB. *p-value < 0.05 for SCC incidence comparison between 4NQO-only exposed rats and 4NQO + 5% BRB rats, using independent Student’s t-test.

Figure 4

(A) Gene expression profiles of the pro-inflammatory markers Cxcl1, Mif, Nfe212, Il-1β, Nfkb1, Arg1, Ptgs1, and Ptgs2 during 4-nitroquinoline 1-oxide (4NQO)-induced oral carcinogenesis determined by reverse transcription quantitative PCR. Data are presented as mean fold induction over sentinels ± SE. *p-value < 0.05 for each group (N ≥ 20) comparison using analysis of variance analysis. (B) Serum concentrations of cyclooxygenase 1 (Cox-1) and Cox-2 in randomly selected rats from each experimental group (N = 15) during 4NQO-induced oral carcinogenesis as determined by enzyme-linked immunosorbent assay. Data are presented as mean ± SE. *p-value < 0.05 for each respective group comparison using independent Student’s t-test.

Figure 5

(A) Representative microscopic images (×200) of tongue tissues from sentinel rats and 4-nitroquinoline 1-oxide (4NQO) exposed rats fed control diet, 5% black raspberry (BRB), 10% BRB, or 0.4% ellagic acid (EA). Paraffin sections were stained with anti-Ki-67 antibody and counterstained with hematoxylin. (B) Quantitation of Ki-67 positive cells in tongue epithelial tissue from sentinel rats as well as 4NQO exposed rats fed with control diet, 5% BRB, 10% BRB, or 0.4% EA. Graphic representation of percent Ki-67 positive nuclei relative to total nucleated cells in six random fields of tongue epithelium from selected rats from each experimental group. Data are expressed as percent cells ± SE. *p-value < 0.05 for each group comparison using independent Student’s t-test. (C) Gene expression profiles of the cell cycle associated biomarkers Aurka, Ccna1, and Ccna2 during 4NQO-induced oral carcinogenesis as determined by reverse transcription quantitative PCR. Data are presented as mean fold induction over sentinels ± SE. *p-value < 0.05 for each group (N ≥ 20) comparison using analysis of variance analysis.

Figure 6

(A) Gene expression profiles of the apoptotic biomarkers Birc5 and Casp14 during 4-nitroquinoline 1-oxide (4NQO)-induced oral carcinogenesis as determined by reverse transcription quantitative PCR. Data are presented as mean fold induction over sentinels ± SE. *p-value < 0.05 for each group (N ≥ 20) comparison using analysis of variance analysis. (B) Representative immunohistochemistry stained microscopic images (×100) of tongue tissues from sentinel rats and 4NQO exposed rats fed control diet, black raspberry (BRB) supplemented diet, or ellagic acid (EA) supplemented diet. Paraffin sections were stained with antibodies against cleaved caspase 3 and counterstained with hematoxylin. In 4NQO-exposed sections, regions of dysplasia or squamous cell carcinomas are shown. Arrow points to positively stained (apoptotic) cells.