Anticancer Mechanisms Of A Small Molecular Inhibitor BHX Targeting Wnt Signaling Pathway

Objective:Wnt signaling pathway plays a clear role in the tumor. Gene mutations of?-catenin and abnormal expression of ?-catenin were found in a variety of tumor,which is closely related to the development of tumors. For the current toxic chemotherapy drugs have the feature of more side effects, discovering the novel targeted inhibitor with determined targets, determine efficacy is of great significance.BHX is a novel small molecule inhibitors aimed at Wnt signaling pathway, the main purpose of our task is to research the efficacy and mechanism of BHX from the following several aspects around the small molecule inhibitor:1. To investigate the effects of Wnt pathway inhibitors BHX on lung tumor cells and mammary gland tumor cell in vitro and vivo.2. To further explore the inhibition mechanism of BHX from the Wnt pathway perspective.3. To explore the possible mechanisms of BHX inhibition mechanism to Wnt signaling pathway.Methods:1. To detect inhibition on tumor of BHX in vitro, we choose two kinds of tumor cell lines A549(lung adenocarcinoma cells) and MCF-7(breast cancer cells) which are Wnt signaling activated, and normal lung epithelial cells Beas-2b, Then used the MTT assay and colony formation assay to anlyze the proliferation inhibition of BHX; A549 cells were grown subcutaneously in nude mice model was prepared, then grouped into blank control group, cisplatin positive control group, BHX low dose group, high dose group BHX randomly for consecutive 21 days in vivo to detect the efficacy of BHX;2. For the further research of BHX on the Wnt signaling pathway,Immunofluorescence assay were carried out to detected the Wnt key protein?-catenin localization; Western Blot assay to analyze the expression of?-catenin in nuclear and the expression of downstream proteins c-myc,cyclin D1; Flow cytometry analysis the effect of BHX to cell cycle regulated by Wnt pathway downstream genes;3. After BHX were shown to inhibit Wnt signaling pathway, we then preliminary analysis possible mechanism for Wnt signaling pathway.Western blot were carried out to detect the expression of total key protein?-catenin and its phosphorylated form p-?-catenin expression level, PCR were carried out to analysis the gene transcriptional level of ?-catenin;Results:1. MTT result showed that BHX effectively inhibited the proliferation of tumor cells A549 and MCF-7, but less effect on normal lung cells Beas-2b by MTT assay; clonogenic assay proved the long-term inhibitory effect of BHX is obvious on A549 and MCF-7; xenograft tumor experiment showed BHX suppressed the xenograft model established by A549 obviously, but barely toxicity.2. BHX reduced the nucleus distribution of Wnt signaling key protein, ?-catenin;Western blot results showed that BHX reduced the expression of ?-catenin protein in nucleus, downstream protein c-myc and cyclin D1; The results of FCM illustrated that BHX arrest cell in G1 phase;3. Western blot results indicated that BHX reduced the expression of the total intracellular protein ?-catenin, however, the expression of p-?-catenin did not increase but decrease a little; RT-PCR declared that BHX decreased the gene transcriptional level of ?-catenin;Conclusions:1) BHX effectively inhibited the proliferation of tumor in vitro and vivo.Simultaneously, BHX has barely toxicity.2) BHX suppress the Wnt signaling pathway, inhibits the expression of cell nucleus protein ?-catenin and downstream protein c-myc, cyclin D1; cell cycle arrest in G1 phase;3) BHX inhibit Wnt signaling pathway not by the Wnt signaling cascade, but directly interaction with ?-catenin then reduced the transcriptional level of it.