Expression of PCNA, cytokeratin, Bcl-2 and p53 during chemoprevention of hamster buccal pouch carcinogenesis by ethanolic neem (Azadirachta indica) leaf extract

Objectives: To evaluate the effect of ethanolic neem leaf extract (ENLE) on cell proliferation, differentiation and apoptosis associated proteins during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Design and methods: Hamsters were divided into four groups. The right buccal pouches of animals in group 1 were painted with 0.5% DMBA three times a week. Animals in group 2 painted with DMBA as in group 1, received in addition, intragastric administration of ENLE (200 mg/kg bw) on days alternate to DMBA application. Group 3 animals were given ENLE (200 mg/kg bw) alone. Animals in group 4 served as control. All the animals were sacrificed after an experimental period of 14 weeks. The expression of proliferating cell nuclear antigen (PCNA), cytokeratin, Bcl-2 and p53 in the buccal pouch tissues were investigated using immunohistochemical staining. In addition, the expression of p53 was confirmed by Western blot analysis. Results: Topical application of DMBA for 14 weeks induced buccal pouch carcinomas associated with increased expression of PCNA, mutant p53 and Bcl-2 and decreased expression of cytokeratin. Administration of ENLE significantly inhibited the development of HBP carcinomas as revealed by decreased expression of PCNA, mutant p53 and Bcl-2 and overexpression of cytokeratin. Conclusion: These findings suggest that ENLE exerts its anticancer properties by inhibiting cell proliferation and inducing differentiation and apoptosis.