| Breast cancer is a highly heterogeneous cancer, more and more interests have been focused on its prevention and treatment, therefore, finding a new and more effective therapeutic drug with low toxicity is urgently needed. Recently, the function of trypsin inhibitor extracted from plant to prevent and resist cancer has become special attention topic. The primary investigation indicated that rBTI can inhibit the proliferation of the human leukemia cells (K562, HL60) and human solid tumor cells (HepG2, EC9706), and induce cell apoptosis. However, the mechanism of rBTI-inhibit cell proliferation, induced apoptosis and molecular target are poorly understood. Considerable evidence has indicated that NF-κB activation possesses an important role in the initiation and progression of breast cancer. To investigate the mechanism of rBTI on MCF-7 cells, our study working as follows:1. MTT assay results indicated that rBTI could inhibit the proliferation of MCF-7 cells in a dose- and time-dependent manner. The wound-healing assay indicated that rBTI could inhibit the cell migration. Then MCF- 7 cells were treated with different concentration of rBTI for 24 h; apoptosis and cell cycle in human MCF- 7 cells were analyzed by flow cytometry. It showed that the cell cycle progression was blocked and obvious cell apoptosis. qRT-PCR was used to detect the expression of cycle factors p53、CDK1、CDK2 and cell apoptosis factors Fas、Bax、Bcl-2 at mRNA level, found that pro-apoptosis genes decreased and anti-apoptosis genes increased notably.2. When MCF-7 cells were treated with different concentration of rBTI, Western Blot and qRT-PCR were used to analysis the expression of p65、 IκBα IKKα/β at both transcriptional and translational levels; An immunofluorescence assay using an anti p65 monoclonal antibody was performed; The result showed that the expression of IKKα/β notably increased, the phosphorylation of IκBa increased obviously. Meanwhile, p65 protein gradually increased in the nucleus and decreased in the cytoplasm. By the flow cytometry detecting, it indicated that rBTI could induce ROS generation. When ROS was inhibited by NAC, the NF-κB/p65 nuclear translocation was reduced, meanwhile the phosphorylation of IκBα noticeably decreased. It showed that the NFκB/p65 signal pathway activation is associated with the increase of ROS.In conclusion, rBTI blocked cell cycle progression and promoted cell apoptosis which may be dependent on the activation of NF-κB/p65 signaling pathway.3. rBTI and paclitaxel could both inhibit proliferation and induce apoptosis of tumor cells. However, the effects of the combination of rBTI with paclitaxel on tumor cells are less understood. In this study, the effects of rBTI combined with paclitaxel on the proliferation of MCF-7 cells were tested by using MTT assays. Flow cytometry was used to determine cell apoptosis and ROS level, as well as the expression level of apoptosis factors by qRT-PCR and western blot. As the result, MTT assay showed that rBTI (2.5 μM) combined with paclitaxel (0.05～0.5 μM) could significantly inhibit the viability of MCF-7 cells in vitro. Flow cytometry showed that rBTI combined with paclitaxel was synergistic and induced the MCF-7 cells apoptosis and the generation of ROS. Meanwhile, rBTI combined with paclitaxel could significantly up-regulate the expression of p53 and Bax, promoted IκBα phosphorylation and nuclear localization of NF-κB/p65 compared with rBTI only. The combination of rBTI with paclitaxel down-regulate the expression of Bcl-2 and CyclinD1 compared with rBTI and paclitaxel used respectively. Taken together, combination of rBTI and paclitaxel could promote the ROS generation, activate NFκB/p65, thereby synergetic induced MCF-7 cell apoptosis.The results of this work will lay the foundation for rBTI of antitumor mechanism, and provides a new strategy for the cancer treatment and combination drug therapy. |
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