Epithelial-mesenchymal transition inhibition by metformin reduces melanoma lung metastasis in a murine model

Abstract

Melanoma is an aggressive cancer with fast metastatic spread and reduced survival time. One common event during the neoplastic progression is the epithelial-mesenchymal transition (EMT), which enhances invasiveness, cell migration, and metastasis. In this study, we investigated the effects of metformin at EMT in melanoma cell lines B16-F10 and A-375, in vitro, and the impact of EMT downregulation on melanoma progression in vivo. The metformin cells treatment reduces the migration potential in vitro and reduced the development of pulmonary metastases and the expressions of N-cadherin, vimentin, ZEB1, and ZEB2 at the metastases site, in vivo. These results indicate that metformin can promote EMT downregulation impairing the metastatic potential of melanoma cells.

Figure 1

Effects of metformin on cell viability. Cells viability assessment by MTT assay of A-375 and B16-F10 cells lines treated for 24- and 48-h with metformin at 0.5 mM and 5 mM. *p < 0.05; **p < 0.01; ****p < 0.0001 (One-way ANOVA).

Figure 2

Effects of metformin on cell migration and invasion. (a) Number of A-375 invasion cells treated for 24-h with 0.5 mM or 5 mM metformin. (b,c) Wound closure percentual of A-375 (b) and B16-F10 (c) cells treated with 0.5 mM or 5 mM metformin. *p < 0.05 (One-way ANOVA).

Figure 3

A-375 mRNA expression. A-375 cell line mRNA expression of EMT-related genes after 24 h of treatment with metformin at 0.5 mM and 5 mM. Data are plotted as mean ± SD of 2^−ΔΔCT, directly proportional to the relative gene expression.

Figure 4

B16-F10 mRNA expression. B16-F10 cell line mRNA expression of EMT-related genes after 24 h of treatment with metformin at 0.5 mM and 5 mM. Data are plotted as mean ± SD of 2^−ΔΔCT, directly proportional to the relative gene expression.

Figure 5

Lung metastases development in C57BL/6 mice inoculated with metformin-treated or untreated B16-F10 cells. Lung tissue, stained with hematoxylin–eosin, 21 days after inoculation of B16-F10 cells: (a) untreated with metformin; (b) treated, in vitro, with 0.5 mM of metformin; (c) treated, in vitro, with 5 mM of metformin. ×4. Scale bar: 100 µM.

Figure 6

Histopathological and immunohistochemical parameters of pulmonary metastases from metformin-treated or untreated B16-F10 cells in C57BL/6 mice. (a) Frequency of pulmonary metastases. Fisher’s exact test. (b) Measurement of the pulmonary area affected by metastases. One-sample t-test. (c) Proliferative index of pulmonary metastases. One-sample t-test. **p < 0.01; ***p < 0.001; **** p < 0.0001. (d–f) Lung tissues, collected 21 days after inoculation of B16-F10 cells, were submitted to immunohistochemistry technique for CDC47 identification. Nuclear immunoexpression was evaluated in 500 neoplastic cells, allowing the determination of the mean proliferative index. (d) Cells untreated with metformin; (e) Cells treated, in vitro, with 0.5 mM of metformin; (f) Cells treated, in vitro, with 5 mM of metformin. ×60. Scale bar: 50 µM.

Figure 7

Immunohistochemical expression of N-cadherin and Vimentin in lung metastases from C57BL/6 mice inoculated with metformin-treated or untreated B16-F10 cells. (a,b) Lung tissues showing membrane and cytoplasmic imunoexpression of N-cadherin 21 days after inoculation of B16-F10 cells: (a) untreated with metformin; (b) treated, in vitro, with 5 mM of metformin. (c,d) Lung tissues showing cytoplasmic imunoexpression of vimentin 21 days after inoculation of B16-F10 cells: (c) untreated with metformin; (d) treated, in vitro, with 5 mM of metformin. ×40. Scale Bar: 50 µM. Arrowhead: Neoplastic cells with positive immunoexpression.

Figure 8

Immunohistochemical expression of Twist, ZEB1, and ZEB2 in lung metastases from C57BL/6 mice inoculated with metformin-treated or untreated B16-F10 cells. (a,b) Lung tissues showing cytoplasmic imunoexpression of Twist, 21 days after inoculation of B16-F10 cells: (a) untreated with metformin; (b) treated, in vitro, with 0.5 mM of metformin. (c,d) Lung tissues showing membrane imunoexpression of ZEB1 21 days after inoculation of B16-F10 cells: (c) untreated with metformin; (d) treated, in vitro, with 0.5 mM of metformin. (e,f) Lung tissues showing membrane and nuclear imunoexpression of ZEB2, 21 days after inoculation of B16-F10 cells: (e) untreated with metformin; (f) treated, in vitro, with 0.5 mM of metformin. ×40. Scale Bar: 50 µM. Arrowhead: Neoplastic cells with positive immunoexpression.

Figure 9

Immunohistochemical expression of Nanog and Sox10 in lung metastases from C57BL/6 mice inoculated with metformin-treated B16-F10 cells. (a,b) Lung tissues showing nuclear imunoexpression of Nanog 21 days after inoculation of B16-F10 cells: (a) untreated with metformin; (b) treated, in vitro, with 5 mM of metformin. (c,d) Lung tissues showing cytoplasmatic imunoexpression of Sox10 21 days after inoculation of B16-F10 cells: (c) untreated with metformin; (d) treated, in vitro, with 0.5 mM of metformin. ×40. Scale Bar: 50 µM. Arrowhead: Neoplastic cells with positive immunoexpression.