| Objective:Twist, a transcription factor, plays an important role in epithelial-mesenchymal transition (EMT) of malignant tumor cells. To explore if silybinin can inhibit EMT of 4T1 and MDA-MB-231 breast cancer cell lines by decreasing Twist gene expression and evaluate the effect of silybinin on invasion and metastasis of breast cancer cells, we evaluated the expression of Twist gene and EMT markers E-cadherin and Vimentin proteins in cultured 4T1 and MDA-MB-231 breast cancer cells in the presence or absence of silybinin treatment. In addition we evaluated the expression of E-cadherin and Vimentin proteins in tumor tissue of tumor-bearing mice after silybinin treatment.Methods:(1) In vitro experiments:4T1 and MDA-MB-231 cell lines were used as EMT models due to their high metastasis potential. MTT assay was used to determine the effect of silybinin on cell proliferation and viability of 4T1 and MDA-MB-231 breast cells. Cell scratch experiment was used to determine the effect of silybinin on migration of breast cancer cells. Migration and invasive analysis was done by transwell assay for the above breast cancer cells. mRNA transcription level of Twist gene was detected by real-time quanti-tative PCR and E-cadherin and vimentin protein expressions levels were detected by Western Blot.(2) In vivo experiments:4T1 cells were implanted into the 4th mammary gland of BALB/c mices to establish breast cancer models. After stratified by tumor volume, BALB/c mices were randomly assigned to either control group, low-dose group (50mg/kg silybinin), intermediate-dose group (100mg/kg) and high-dose group (200mg/kg) (n=8 per group). Silybinin was injected intraperitoneally once daily for 14 days. In the control group, the same volume of saline was administered intraperitoneally. Liver and kidney as well as tumor tissues were harvested at the end of the experiments. Comparison of tumor weight and volume were used to assess the effect of silybinin on tumor growth. Tumor metastasis was validated by HE staining. Vimentin and E-cadherin protein expression levels were detected by Western Blot.Result:(1) In vitro experiments:MTT assay showed that silybinin (50μg/ml, 100μg/ml and 200μg/ml) treatment could significantly decreased the cell viability of 4T1 and MDA-MB-231 cells in dose-and time-dependent manners. According to the results of MTT, the rest of cell experiments were performed with 50-200μg/ml silybinin and a culture time of 24 h. The results of scratch and transwell assays indicated that silybinin treatment reduced the ability of migration and invasion, of MDA-MB-231 breast cancer cell in a dose-dependent manner. Scratch experiment indicated that silybinin treatment inhibited migration of 4T1 cells. Gene expression level of Twist was prominently inhibited in 4T1 and MDA-MB-231 cells (p<0.05). Protein expression level of Vimentin was down-regulated in 4T1 and MDA-MB-231 cells, while expression level of E-cadherin was significant up-regulated after silybinin treatment.(2) In vivo experiments:Silybinin was injected intraperitoneally in mices at three different dosages (50mg/kg, 100mg/kg and 200mg/kg). At the end of the experiment, the mean tumor weight of each groups were 2.11±0.37g,1.37±0.21g, and 1.45±0.15g respectively, while mean tumor weight was 2.18±0.31g in the control group. The difference between low-dose silybinin group and the control group was not significant, but the difference between the control and the other two silybinin groups were significant (p<0.05). These results demonstrated that silybinin can inhibit 4T1 tumor in vivo. After silybinin treatment, the mean numbers of tumor nodules on liver of each silybinin group were 7.09±0.11,4.76±0.37, and 1.03±0.23 respectively. The mean numbers of tumor nodules on lung were 9.24±0.28,6.58±0.19, and 2.21±0.13, respectively. Compared with the control group (10.81±0.15 for liver and 13.72±0.26 for lung respectively), the differences were significant (p<0.05). HE staining also showed that metastasis of transplanted 4T1 cells to liver and lungs could be inhibited by administration of Silybinin. These results demonstrated the silybinin can inhibit 4T1 tumor metastasis. Western blot analysis showed that silybinin administration significantly inhibited the expression of vimentin, while promoted E-cadherin expression.Conclusion:Silybinin could inhibit cell viability and growth of 4T1 and MDA-MB-231 cells. Silybinin treatment significantly inhibited tumor weight and volume in breast cancer model. Silybinin can effectively down-regulated Twist gene expression in 4T1 and MDA-MB-231 cells, inhibit vimentin expression, while promote E-cadherin expression, reduce epitheial-mesenchymal transition, and effectively inhbit breast cancer migration and invasion. |
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