Breast cancer and obesity: in vitro interferences between adipokines and proangiogenic features and/or antitumor therapies?

Abstract

Obesity is now considered as a risk factor for breast cancer in postmenopausal women. Adipokine levels are modulated in obesity, and may play a role in carcinogenesis. Moreover, obesity increases risk of cancer mortality. Here, we hypothesized that this increase could be due to a modification in angiogenesis, capital event in the development of metastases, and/or in effectiveness of cancer treatments. To test these assumptions, following a same experimental design and simultaneously the effects of leptin and adiponectin on angiogenesis were investigated, and the impact of hyperleptinemia on anticancer drug effectiveness was measured in physiological and obesity situations. Focusing on angiogenesis, the proliferation of endothelial cells (HUVEC), which expressed leptin and adiponectin receptors, was stimulated by leptin and inhibited by adiponectin. Both adipokines globally reduced apoptosis and caspase activity. Leptin increased migration whereas adiponectin decreased migration, and leptin enhanced the area of the tubes formed by HUVEC cells while adiponectin inhibited their formation. MCF7 and MDA-MB-231 cells treated with leptin secreted more VEGF than untreated cells, whereas adiponectin treatment inhibited VEGF secretion. Finally, MCF7 cells pre-treated with leptin were more invasive than untreated cells. This effect was not reproduced in MDA-MB-231 cells. In the MCF7 breast cancer cell line, leptin could induce cell proliferation and reduced the efficacy of all breast cancer therapies (tamoxifen, 5-fluorouracil, taxol and vinblastin). These results suggest that, in obesity situation, leptin- in contrast to adiponectin - may promote tumor invasion and angiogenesis, leading to metastases 'apparition, and reduce treatment efficacy, which could explain the increased risk of cancer mortality in cases of overweight. The evidence suggests adipokines influence breast cancer issue and could play a significant role, especially in obese patients for which hyperleptinemia, hypoadiponectinemia and increased metastatic potential are described.

Figure 1. Protein expression of leptin and adiponectin receptors in untreated HUVEC.

The protein expression was analysed by immunochemistry (magnification x 400).

Figure 2. Effect of leptin or adiponectin on the proliferation of HUVEC cells.

A. HUVECs were cultured in the presence of increasing concentrations of human recombinant leptin (10 to 1,000 ng/mL) for 72 and 96 hours in a complete medium containing VEGF and bFGF (n = 6). B. HUVECs were cultured in the presence of increasing concentrations of human recombinant leptin (10 to 1,000 ng/mL) for 72 and 96 hours in a medium without VEGF and bFGF (n = 6). C. HUVECs were cultured in the presence of increasing concentrations of human recombinant adiponectin (100 to 10,000 ng/mL) for 72 and 96 hours in a complete medium with VEGF and bFGF (n = 4). Data are presented as mean ± SEM *p<0.05 vs control.

Figure 3. Effect of leptin and adiponectin on global apoptosis and on caspase activation in HUVEC cells.

A. HUVECs were cultured in the presence of increasing concentrations of human recombinant leptin (10 to 1,000 ng/mL) for 72 in a medium without VEGF and bFGF (n = 3). B. HUVECs were cultured in the presence of increasing concentrations of human recombinant adiponectin (100 to 10,000 ng/mL) for 72 hours in a complete medium (n = 3). Data are presented as mean ± SEM *p<0.05 vs control. C. HUVECs were cultured in the presence of increasing concentrations of human recombinant leptin (10 to 1,000 ng/mL) for 72 in a medium without VEGF and bFGF (n = 1) D. HUVECs were cultured in the presence of increasing concentrations of human recombinant adiponectin (100 to 10,000 ng/mL) for 72 hours in a complete medium (n = 1).

Figure 4. Migration assay for HUVECs treated by leptin or adiponectin.

Migration pattern of HUVECs with (A) or without (B) leptin treatment (100 ng/mL) and with (C) or without (D) adiponectin traitement (10,000 ng/mL). The percentage of migration ability (E) was analyzed by averaging the scraped area of each well under each condition (n = 6). Original magnification ×40 (A, B, C and D). Data were expressed as mean ± SEM (E and F). *p<0.05 vs control.

Figure 5. Endothelial tube formation for HUVECS treated by leptin or adiponectin.

Differentiation of HUVEC in three-dimensional Matrigel cultured in treatment with (A) or without (B) leptin (100 ng/ml). Differentiation of HUVEC in three-dimensional Matrigel cultured in treatment with (C) or without (D) adiponectin (10,000 ng/ml). Average of number of tube (E) and mean area (F) in different concentrations of leptin or adiponectin-treated HUVEC was calculated using computer software (ImageJ software) (n = 6). Original magnification ×40 (A, B, C and D). Data were expressed as mean ± SEM (E and F). *p<0.05 vs control.

Figure 6. Effects of leptin on VEGF secretion in MCF7 (A) and MDA-MB-231 (B) (n = 1).

Figure 7. Effect of leptin on invasion of MCF7 cells.

Invasion of MCF7 with (A) or without (B) leptin treatment (1,000 ng/mL). The number of cells (C) was analyzed under each condition (n = 3). Data expressed as mean ± SEM (C). *p<0.05 vs control.

Figure 8. Effects of leptin and Tx (A), 5-FU (B), Taxol (C) and Vinblastin (D) on MCF7 cells proliferation at 72 h (n = 6).

Data are presented as means ± SEM *p<0.05 vs control.