Inhibition of 4NQO-Induced Oral Carcinogenesis by Dietary Oyster Shell Calcium

Abstract

Oyster has gained much attention recently for its anticancer activity but it is unclear whether calcium, the major antitumor ingredient in oyster shell, is responsible for the anticarcinogenic role of the oyster. To address this issue, C57BL/6 mice were fed with the carcinogen 4-nitroquinoline-1-oxide (4NQO, 50 µg/mL) and normal diet or a diet containing oyster powder, oyster calcium, or calcium depleted oyster powder. The tongue tissue specimens isolated from these mice were histologically evaluated for hyperplasia, dysplasia, and papillary lesions, and then analyzed for proliferation and differentiation markers by immunohistochemistry. The results showed that mice on the diet containing oyster calcium significantly reduced rates of tumors in the tongue and proliferation and enhanced differentiation in the oral epithelium compared with the diet containing calcium depleted oyster powder. These results suggest that calcium in oyster plays a critical role in suppressing formation of oral squamous cell carcinoma and proliferation and promoting differentiation of the oral epithelium.

Keywords: calcium; differentiation; oral carcinogenesis; oyster; proliferation.

Figure 1.

(A) The experimental timelines and effects of the diet containing oyster powder (OPow), oyster extract (OEx), or oyster calcium (OCa) on the tumor rates in mice treated by 4NQO. Mice were randomly divided into 4 groups including the group on the normal diet (the control group), the group on OPow diet, the group on OCa diet, and the group on OEx diet. All the groups were given 50 mg/mL of 4NQO in drinking water for 16 consecutive weeks. The diets include the normal diet containing 1.3% calcium, OPow containing 2.0% calcium, OEx containing 1.3% calcium, and OCa containing 2.0% calcium. The concentration of calcium in each diet was determined as described in Materials and Methods. The body weight in male mice (B) and female mice (C) were recorded and plotted. At the end of study, the mice were sacrificed and examined for oral tumors. The number of tumors is shown the bar graph (D). The data are expressed as mean ± SD. *P < .05 (compared with the control group). The results showed that the mice on OPow, OCa, or OEx did not display any difference in body weight when compared with mice on the normal diet. The rates of oral tumors larger than 1.0 mm in diameter were almost 93% in all the groups and there is no difference between each group. The rate of tumors larger than 2.5 mm in the OCa group is significantly lower than that in the normal diet group. However, there was no significant difference in the rate of tumors larger than 2.5 mm between the OEx group or the OPow group and the normal diet group.

Figure 2.

Effects of OPow, OCa, or OEx diet on the proliferation and differentiation of oral epithelia. (A) The tongue was removed from the mouse on the normal diet (the control group) or the diet containing OPow, OCa, or OEx and the tongue tissue was fixed in formalin solution and embedded in paraffin blocks for pathological analysis and immunohistochemistry using the antibodies against the proliferating cell nuclear antigen (PCNA), keratin 1, involucrin, filaggrin, or loricrin. Positive expression is shown in brown and the counterstaining is shown in blue. (B) The bar graph shows quantitation of the levels of differentiation markers in the cells. The quantitation for each section was obtained by counting the number of positive cells and total number of cells in the corresponding region in 5 representative regions in each section. The data are expressed as mean ± SD, *P < .05 (compared with the control group). The results showed that levels of PCNA in the normal oral epithelium was significantly lower and levels of keratin 1, involucrin, filaggrin, and loricrin in mice on OPow or OCa were higher compared with that in mice on OEx or on the normal diet (P < .05).