Metformin Prevents the Development of Oral Squamous Cell Carcinomas from Carcinogen-Induced Premalignant Lesions

Optimization scheme of the 4NQO oral-specific chemical carcinogenesis model shows time-dependent malignant progression of oral epithelial dysplastic lesions. A, the indicated groups of mice (n = 20) were randomly divided and administered with 4NQO in the drinking water (50 μg/mL) for the indicated time (8, 10, 12, 14, and 16 weeks). Mice were then sacrificed or switched to regular water and sacrificed at week 22 from the initiation of the 4NQO administration. The tongue of each mouse was processed for detailed histologic examination as described in Materials and Methods. The average of the number of lesions per mouse, classified as low-grade dysplasia, high-grade dyplasia, or carcinoma based on histopathologic examination was recorded at the end of the 4NQO administration as well as on week 22 from the initiation of carcinogen exposure. B, representative photographs of lesions observed in mice at the indicated time since the initiation of carcinogen exposure in mice treated with 4NQO for 14 weeks. No evident gross alteration were observed at week 4; small elevated white lesions and discoloration are seen at week 8; the lesions grow to small berry-like elevations at week 16 and to ill-defined indurated white or congestive areas, often ulcerated, at week 22. Microscopically, the series of photograph depict the sequence of histologic changes on the dorsal tongue from a normal epithelium at week 4, through mild to moderate atypia at week 8, several dysplastic cellular alterations at week 16 (note the increasing thickness of the epithelium), to a fully infiltrating well-differentiated SCC at week 22.

Metformin decreases the number and size of oral tumors induced by orally administered 4NQO carcinogen. A, C57Bl/6 mice were administered 4NQO diluted in the drinking water for 14 weeks, as described in Materials and Methods, and remained on regular water until week 22. At week 14, the mice were divided into 2 groups and given either daily i.p. injections of metformin at a dose of 50 mg/kg/d or an equal volume of sterile saline for a period of 8 weeks. Grossly evident lesions started to appear by week 16, and by week 18 the difference between metformin-treated and -untreated groups was significant. The cumulative incidence of the number of macroscopic lesions per animal is shown; the different colors indicate their respective size: 2 to 4 mm (blue), 4 to 6 mm (orange), 6 to 8 mm (green), and 8 to 10 mm (red). B, gross morphology of the lesions developed in the animals treated with 4NQO in the metformin-treated and control untreated groups. At week 14, when 4NQO treatment was stopped, the alterations are more evident in the dorsal tongue. A rather extensive white and elevated tumor composed of 3 different outgrowths is evident in this control animal (arrow, left topmost photograph). Smaller lesions are seen in the ventral aspect of the tongue (arrow, bottom rightmost photograph). C, at week 22, an untreated control animal has developed a significant papillomatous tumor in the middle of the tongue as well as another similar lesion in the tip of the organ (arrows). Only small discolorations are observed in an animal treated with metformin. In the ventral tongue the control animal has developed 2 evident berry-like outgrowths, whereas only one small white papillomatous lesion is seen in the metformin-treated animal (arrows). D, at the conclusion of the experimental period (week 22), the difference in the incidence of tumor lesions per animal was evident between metformin- and control-treated mice and is significant.

Metformin decreases the number and size of oral tumors and prevents the malignant conversion of preneoplastic lesions. A, a sagittal section of a tongue in a control animal at week 22 after the initiation of the exposure to 4NQO in the drinking water for 14 weeks, where a total of 3 tumors are observed. The lesion closest to the tip of the tongue has a papillary (1) as well as a clearly infiltrative aspect (2); closer to the middle of the dorsal tongue another infiltrative SCC is evident (3). The third tumor, a papilloma, is observed in the ventral tongue (4). B, a higher magnification of lesion 2; atypical epithelial cells showing squamous differentiation (white arrowhead) infiltrate the skeletal muscle of the tongue (black arrowhead). They are surrounded by a dense inflammatory infiltrate (white star). C, in this metformin-treated animal a single large benign papillomatous lesion is seen in the posterior dorsal tongue (1). D, at a higher magnification, the squamous epithelium (white arrowhead) lines thin projections of the stroma (black arrowhead), it is evidently hyperplastic, although the cells display no evident atypia; mitotic figures are not evident in this photograph. E and F, tumor multiplicity, including benign and malignant tumors (E) as well as SCC multiplicity (F) are significantly different between metformin-treated and untreated control groups. G and F, the number of preneoplastic lesions per tongue, both low- and high-grade dysplasias was also significantly reduced in the metformin-treated animals.