Timp-1 By Inhibiting Cyclin B1 To Reduce The Breast Cancer Cell Line Mcf-7 Paclitaxel Sensitivity

Paclitaxel is a very effective drug in treating tumors.It disturbs microtubule dynamics and impairs the transition of cells from metaphase to anaphase in mitosis,leading to cell death by apoptosis.However,effectiveness of paclitaxel in cancer chemotherapy is hampered by drug resistance in portion of patients.Tissue inhibitor of metalloproteinase-1(TIMP-1)is well known to be capable of inhibiting apoptosis.Elevated tumor tissue TIMP-1 levels were significantly associated with a poor response to chemotherapy.We hypothesized that TIMP-1 could reduce the sensitivity of breast cancer cells to paclitaxel by inhibiting apoptosis.To test this hypothesis,Firstly,The TIMP-1 expressing plasmid and control plasmid were transfected into MCF-7 cells, respectively.Then the TIMP-1 protein was identified by Western Blot and ELISA.The results showed that the T1MP-1 protein expression level of TIMP-1-transfected clones(T10 and T13)was significantly higher than that of control clone.Then the effects of paclitaxel on different clones were detected by cytometry and immunofluorescence staining.The results showed that stable expression of TIMP-1 significantly decreased sensitivity of MCF-7 cells to paclitaxel-induced apoptosis and decreased the percentage of G₂/M in cell cycle. We further investigated the effect of TIMP-1 on the protein level and the stability of Cyclin B1 that critically regulates metaphase to anaphase transition in mitosis and found TIMP-1 down-regulated the protein level of Cyclin B1 by decreasing the stability of Cyclin B1.To further investigate whether TIMP-1 decreases paclitaxel-induced apoptosis is mediated by adjusting Cyclin B1,the Cyclin B1-expression plasmid was transfected into T13 and the apoptosis-induced effects of paclitaxel were analyzed by cytometry.The data showed that the effect of TIMP-1 decreasing paclitaxel-induced apoptosis was reversed by Cyclin B1.Our data indicated TIMP-1 could protect breast cancer cells from paclitaxel-induced apoptosis by decreasing the stability of Cyclin B1, suggesting the expression levels of TIMP-1 in primary breast cancer tissues may be a potential marker for predicting the sensitivity of cancer cells to paxlitaxel.