The Effect Of Luteolin And Its Mechanism On Invasion And Metastasis And Epithelial Mesenchymal Transition Of Breast Cancer Cells

Objective: To investigate the potential effect of luteolin on the invasion, metastasis and epithelial mesenchymal transition of breast cancer cells and to explore its molecular mechanism.Methods:1. In vitro experiment, human breast cancer cell line MDA-MB-231 cells and BT549 cells were treated with different concentration of luteolin. The migration and invasion of breast cancer cells were evaluated by scratch experiment and transwell assay. The mammosphere formation ability was detected by spheroid experiment and the expression of CD24/CD44 on cell surface was analyzed by flow cytometry to evaluate the stem characteristics of breast cancer cells. The expressions and distribution of mesenchymal marker（Vimentin） and cell actin cytoskeleton（F-actin） were tested by immunocytochemistry assay. The expressions of epithelial markers（E-cadherin, Claudin-1） and mesenchymal markers（N-cadherin, Vimentin）and transcription factors（Snail, Slug） were detected by quantitative real-time polymerase chain reaction and western blot experiments to evaluate the effect of luteolin on epithelial mesenchymal transition of breast cancer cells.2. Human breast cancer cell line MDA-MB-231 cells and BT549 cells were treated with different concentration of luteolin. The level of b-catenin protein and m RNA were detected by quantitative real-time polymerase chain reaction and western blot experiments. After transfected Ad-b-catenin into MDA-MB-231 cells, the effect of b-catenin in the influence of luteolin on migration and invasion of breast cancer cells were evaluated by scratch experiment and transwell assay. The effect of b-catenin in the influence of luteolin on epithelial mesenchymal transition of breast cancer was evaluated through detecting the protein and m RNA level of epithelial biomarkers（E-cadherin, Claudin-1） mesenchymal biomarkers（N-cadherin, Vimentin） and transcription factors（Snail, Slug） by quantitative real-time polymerase chain reaction and western blot experiments.3. In vivo experiment, four to five weeks of nude mice were randomly divided into four groups, including blank control group, luteolin intervention group, Ad-GFP + luteolin group, and Ad-b-catenin + luteolin group. Nude mice in blank control group and luteolin intervention group were subcutaneous injected with MDA-MB-231 cells to establish in situ breast cancer mice model. Mice in Ad-GFP + luteolin group were subcutaneous injected with transfected Ad-GFP breast cancer cells. Mice in Ad-b-catenin + luteolin group were subcutaneous injected with transfected Ad-b-catenin breast cancer cells to establish in situ breast cancer mice model over-expression of b-catenin. After the volume of breast cancer tumor growed to 1cm3（about 14 days）, mice in luteolin intervention group, Ad-GFP + luteolin group, and Ad-b-catenin + luteolin group were subcutaneous injected with luteolin（100 mg/kg） every three days, however, mice in blank control group without any intervention. Eight weeks later, nude mice were sacrificed. The tumor nodules, histopathological changes and Ki67 in the lung were counted and detected to evaluate the effect of luteolin on the metastasis of breast cancer cells, and the expression of mesenchymal marker（Vimentin） transcription factor（Slug） and b-catenin in tumor were detected by immunohistochemistry.Results:1. Luteolin dose-dependently inhibited the migration and invasion ability in MDA-MB-231 and BT549 cells.2. Luteolin inhibited the epithelial mesenchymal transition of breast cancer cells dramatically. Luteolin inhibited breast cancer cells from epithelial type to mesenchymal type. Luteolin inhibited the expression of F-actin and changed its distribution in breast cancer cells. Luteolin up-regulated the expression of epithelial markers（E-cadherin, Claudin-1）, but down-regulated the expression of mesenchymal markers（N-cadherin, Vimentin） and transcription factors（Snail, Slug） dose-dependently in breast cancer cells. Among them, Slug changed obviously than Snail.3. Luteolin apparently inhibited the mammosphere formation ability and the expression of CD24^low/CD44^high on cell surface of breast cancer cells. 4. Luteolin apparently inhibited the expression of b-catenin at both protein and m RNA level in breast cancer cells.5. Over-expression of b-catenin in breast cancer cells reversed the inhibition effect of luteolin on the migration, invasion and epithelial mesenchymal transition of breast cancer cells.6. Luteolin significantly alleviated lung metastasis of breast cancer and inhibited the process of epithelial mesenchymal transition in nude mice. Hover, over-expression of b-catenin could reverse this protective effect. Results of lung tumor nodules number counting, HE staining and Ki67 indicated that luteolin inhibited the lung metastasis of breast cancer cells dramatically in nude mice. Immunohistochemistry results showed that, after mice treated with luteolin, the expression of mesenchymal marker（Vimentin） and transcription factor（Slug） and b-catenin were down-regulated. However, lung metastasis of breast cancer was enhanced and the expression of mesenchymal marker（Vimentin）, transcription factor（Slug） and b-catenin were significantly up-regulated when b-catenin was over-expressed in breast cancer cells.Conclusion: Luteolin can restrain the invasion and metastasis in breast cancer cells. The inhibition mechanism of luteolin may be through down-regulation of the expression of b-catenin, inhibiting the activation of the transcription factor Slug in the process of epithelial mesenchymal transition, and then down-regulation the expression of mesenchymal markers（Vimentin, N-cadherin） but up-regulation the expression of epithelial markers（E-cadherin, Claudin-1）, inhibition the epithelial mesenchymal transition of breast cancer cells to complete.