Screening And Studying Of Anti-breast Cancer Lead Drugs Based On MT1-MMP

Breast cancer is a kind of highly heterogenic tumor, whose incidence has been gradually increased, and it has become the second killer of women cancer death. It has become a research hotspot to find therapeutic target and appropriate medicine, for taking individualized comprehensive treatment. Among so many tumor target proteins, membrane type-1matrix metalloproteinase is an ideal drug target, which involved in many processes like tumorigenesis, development, invasion, angiogenesis. Chinese medicine has unique curative effects and is widely used in breast cancer therapy. Hopefully, molecular target and Chinese medicine therapy can bring new perspectives to breast cancer treatment.Firstly, antisense peptides targeting MT1-MMP were screened and researched through computer-aided drug design, then the targeting of antisense peptides to MT1-MMP and breast cancer were determined by molecular docking, molecular dynamics simulation and cell experiments in vitro. Secondly, based on MT1-MMP, the mechanism of the preparation of medical plants (also called Reagent2) against two breast cancers were investigated by MTT, AO-EB, DNA Ladder, Flow Cytometry, Fluorescence Quantitative PCR and Western blot, to determine the target and cell death pathways.Here, we identified the divergent and specific sequence of AYIREGHE (named MT1-loop) located in MTl-MMP loop by multiple sequence alignment and established the antisense peptides library with capacity of1536sequences. After two rounds of virtual screening, five antisense peptides with better scores have been obtained. Then these antisense peptides were docked to other MMP members, and FVTFPYIR was more specific to MT1-MMP. Furthermore, the results from molecular dynamics simulation showed that FVTFPYIR might affect MT1-MMP stability. Based on results above, we had synthesized FVTFPYIR, which was optimal by evaluation, to conduct cell biological experiments, and found that it had better inhibition and specific affinity to MDA-MB-231cell which expressed MT1-MMP.We found that Reagent2significantly inhibited the viabilities of MDA-MB-231and MCF-7cells by MTT assays, meantime their morpholopy changed clearly by light microscopy, indicating that the inhibitory degree is time and dose-dependent. The IC50is3.45%and2.84%after24h, at the same time the IC50is2.36%and2.32%after48h, respectively. On this basis, further study by agarose gel electrophoresis, AO-EB and flow cytometry, it was determined that Reagent2could induce apoptosis and non-apoptotic cell death pathway. Subsequently, FQ-PCR results showed that3%of Reagent2and DOX could up-regulate MMP2, MMP9, MT1-MMP, caspase3and caspase8in MDA-MB-231. MMP2, MMP9, caspase8, caspase9, p53and bcl-2were up-regulated and caspase3was down-regulated in MCF-7. Meanwhile, the proteins (MT1-MMP and p53) increased, and caspase3decreased, however the expression of bcl-2protein had no changed in MDA-MB-231. The caspase3was up-regulated, but bcl-2was down-regulated, at the same time p53decreased at24h, increased at48h in MCF-7. In a word, Reagent2influenced MDA-MB-231and MCF-7cells in different ways.Conclusion: the antisense peptides tagrgeting MT1-MMP could provide an effective way for the development of anti-tumor peptides; the preparation of medical plants (Reagent2) had a strong killing effect on breast cancer cells, which was related to the expression and inhibition of MT1-MMP, p53, caspase3, bcl-2, and other molecular mechanism remains to be further studied.