Publicação
Study of the mechanisms underlying the cytotoxic effects of bovine lactoferrin on breast cancer cells
| Resumo: | Lactoferrin (LF) is an iron-binding protein predominantly found in mammalian secretions. This protein and its variants have been proposed for cancer therapy for many years owing to their tumor-targeting properties. Previous studies showed that LF and its derived peptides inhibit the proliferation of cancer cells. However, the detailed mechanisms by which LF exerts its effect are still fairly unknown. Moreover, there are few reports concerning LF effect on breast cancer cells, which is one of the most common malignant tumors in the World. In this sense, the present thesis aimed to investigate the cytotoxicity of bovine lactoferrin (bLF) and its variants against several breast cancer cells, namely T-47D, MDA-MB-231, Hs578T and MCF-7 cell lines. The results showed that bLF at concentrations of 1.875 μM, 3.75 μM, 7.5 μM, 15 μM and 30 μM could efficiently inhibit the growth of cancer cells but showed a very low effect on normal breast cells (MCF-10-2A). Moreover, its variants (apo-bLF, holo-bLF and LfcinB17-41) were also able to inhibit cancer cells’ growth, except for LfcinB26-36. Additionally, bLF, apo-bLF and holo-bLF did not promote the proliferation of breast cancer cells at low concentrations (0.25 μM, 0.5 μM and 1 μM) as reported for other cancer cell lines. Simultaneously, the degradation assay excluded the possibility that bLF anticancer effects could be due to its degraded peptides under cell culture conditions. On the other hand, it was found that most of the bLF was blocked outside the cells, despite that a few amount was able to be internalized to the cytoplasm. Its peptide LfcinB17-41 also succeeded in penetrating the cell membrane but could not enter the nucleus. Subsequently, we found that the inhibitory effects of bLF on the breast cancer cells resulted from the cell cycle arrest without effects in cell death by apoptosis. Depending on the cell lines, this prevention of cell cycle progression induced by bLF occurred at different phases. Nevertheless, the MAPK/ERK and PI3K/AKT signaling pathways were not implicated in the cell cycle arrest observed. bLF anticancer effect was associated, however, with an increase of AMPKα phosphorylation and a decrease in the levels of mTOR and its phosphorylation. To our knowledge this is the first time this pathway has been implicated in the mechanisms underlying bLF cytotoxicity against cancer. These findings suggest that bLF could be a new mTOR-targeting drug in cancer therapy. However, it is important to notice that no apoptotic cells could be found in bLF-treated cancer cells. The use of higher bLF concentrations (12.5 μM, 50 μM, 125 μM and 175 μM) was expected to exhibit different effects on the breast cancer cells as compared with the low concentrations range. In fact, in the high range of concentrations bLF selectively induced cell death by apoptosis in MCF-7 cells. The mechanisms of bLF-induced apoptosis included the intrinsic pathway since it was observed the mitochondrial membrane depolarization and a decrease in Bcl-2 levels. In addition, bLF also induced significantly the cell cycle arrest of these cells at the G1 phase, while the same concentration of another protein source (bovine serum albumin - BSA) did not affected significantly the cells. This suggests that bLF cytotoxicity is not due to the addition of great amounts of exogenous proteins in the cell microenvironment. The western bolt analysis confirmed that bLF blocked the cell cycle progression by adjusting cell cycle related regulators, such as CDC25c. Additionally, bLF showed a clear inhibitory effect on the MCF-7 cells ability to form colonies, which is one of the favorite features of anticancer drugs for preventing metastasis. We also found that the promoting effect on the migration of MCF-7 cells may be due to the fact that bLF changes cell microenvironment positively for cell migration similarly to BSA. The results gathered in this thesis demonstrated the potential of bLF as an anticancer agent and provided some new insights on its mechanisms of action. However, further work is still required before bLF can be considered for clinical applications. Being a food-derived protein, bLF is commonly consumed in the daily life, as well as in supplements for health care. Nevertheless, the relation between its consumption and cancer prevention remains to be elucidated. |
|---|---|
| Autores principais: | Zhang, Yunlei |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Lactoferrin (LF) is an iron-binding protein predominantly found in mammalian secretions. This protein and its variants have been proposed for cancer therapy for many years owing to their tumor-targeting properties. Previous studies showed that LF and its derived peptides inhibit the proliferation of cancer cells. However, the detailed mechanisms by which LF exerts its effect are still fairly unknown. Moreover, there are few reports concerning LF effect on breast cancer cells, which is one of the most common malignant tumors in the World. In this sense, the present thesis aimed to investigate the cytotoxicity of bovine lactoferrin (bLF) and its variants against several breast cancer cells, namely T-47D, MDA-MB-231, Hs578T and MCF-7 cell lines. The results showed that bLF at concentrations of 1.875 μM, 3.75 μM, 7.5 μM, 15 μM and 30 μM could efficiently inhibit the growth of cancer cells but showed a very low effect on normal breast cells (MCF-10-2A). Moreover, its variants (apo-bLF, holo-bLF and LfcinB17-41) were also able to inhibit cancer cells’ growth, except for LfcinB26-36. Additionally, bLF, apo-bLF and holo-bLF did not promote the proliferation of breast cancer cells at low concentrations (0.25 μM, 0.5 μM and 1 μM) as reported for other cancer cell lines. Simultaneously, the degradation assay excluded the possibility that bLF anticancer effects could be due to its degraded peptides under cell culture conditions. On the other hand, it was found that most of the bLF was blocked outside the cells, despite that a few amount was able to be internalized to the cytoplasm. Its peptide LfcinB17-41 also succeeded in penetrating the cell membrane but could not enter the nucleus. Subsequently, we found that the inhibitory effects of bLF on the breast cancer cells resulted from the cell cycle arrest without effects in cell death by apoptosis. Depending on the cell lines, this prevention of cell cycle progression induced by bLF occurred at different phases. Nevertheless, the MAPK/ERK and PI3K/AKT signaling pathways were not implicated in the cell cycle arrest observed. bLF anticancer effect was associated, however, with an increase of AMPKα phosphorylation and a decrease in the levels of mTOR and its phosphorylation. To our knowledge this is the first time this pathway has been implicated in the mechanisms underlying bLF cytotoxicity against cancer. These findings suggest that bLF could be a new mTOR-targeting drug in cancer therapy. However, it is important to notice that no apoptotic cells could be found in bLF-treated cancer cells. The use of higher bLF concentrations (12.5 μM, 50 μM, 125 μM and 175 μM) was expected to exhibit different effects on the breast cancer cells as compared with the low concentrations range. In fact, in the high range of concentrations bLF selectively induced cell death by apoptosis in MCF-7 cells. The mechanisms of bLF-induced apoptosis included the intrinsic pathway since it was observed the mitochondrial membrane depolarization and a decrease in Bcl-2 levels. In addition, bLF also induced significantly the cell cycle arrest of these cells at the G1 phase, while the same concentration of another protein source (bovine serum albumin - BSA) did not affected significantly the cells. This suggests that bLF cytotoxicity is not due to the addition of great amounts of exogenous proteins in the cell microenvironment. The western bolt analysis confirmed that bLF blocked the cell cycle progression by adjusting cell cycle related regulators, such as CDC25c. Additionally, bLF showed a clear inhibitory effect on the MCF-7 cells ability to form colonies, which is one of the favorite features of anticancer drugs for preventing metastasis. We also found that the promoting effect on the migration of MCF-7 cells may be due to the fact that bLF changes cell microenvironment positively for cell migration similarly to BSA. The results gathered in this thesis demonstrated the potential of bLF as an anticancer agent and provided some new insights on its mechanisms of action. However, further work is still required before bLF can be considered for clinical applications. Being a food-derived protein, bLF is commonly consumed in the daily life, as well as in supplements for health care. Nevertheless, the relation between its consumption and cancer prevention remains to be elucidated. |
|---|