Studies On The Anti-angiogenesis Effect Of Muscone Derivatives In Tumor Targeting On HuR Protein

Objective:Traditional Chinese medicine（TCM）has been practiced for thousands years and at present is widely accepted as an effective treatment for cancer with its wide range of pharmacological properties and low side effects.In order to better develop the resources of traditional Chinese medicine,our group has been performing the structure-modification of a variety of traditional Chinese medicine monomers,providing good resources for screening of new active compounds.Human antigen R（HuR）,an RNA-binding protein,regulates m RNAs of multiple angiogenic factors by binding to the adenylate-uridylate-rich element in their 3′untranslated region and promotes tumor angiogenesis.HuR overexpression has been commonly observed in human cancers,thus HuR may serve as a valuable drug target for anti-tumor angiogenesis therapy.This Ph D thesis presents the studies of HuR inhibitors derived from traditional Chinese medicine aiming to inrich anti-tumor angiogenesis treatment.Methods:1.Establishment of HuR inhibitor screening model based on the interaction of HuR protein and ARE^Vegf-a,and for screening of the compound library:HuR protein was expressed and purified.5’-FAM-labeled ARE sequence was synthesized.The binding activity of HuR and 5’-FAM-ARE^Vegf-a was verified by EMSA assay.The high-throughput screening（HTS）system based on HuR and ARE^Vegf-a interaction was established with fluorescence polarization（FP）technique.A library of 4200 compounds was screened.2.Feasibility study of the HTS system based on the HuR-ARE^Vegf-a interaction for screening anti-tumor angiogenesis drugs:At molecular level,EMSA assay and FP technique were used to verify the inhibitory activity of the positive compound ELB against binding of HuR or HuR RRM1/2 to ARE^Vegf-a,and IC₅₀ was calculated.The interaction mode between ELB and HuR RRM1/2 was simulated by molecular docking.The binding activity of ELB to HuR RRM1/2 protein was detected by surface plasmon resonance（SPR）.At cellular level,HuR knockout and HuR overexpressed 4T1 cell lines were constructed to detect the effects of ELB on the m RNA and protein levels of Vegf-a and Mmp9 genes in 4T1,4T1 HuROE and 4T1ΔHuR cells.m RNA stability assay and immunoprecipitation assay were used to detect the effect of ELB on HuR function in 4T1 cells.The HuR-dependent angiogenic genes regulation mechanism of ZM-32 in Raw264.7 cells were studied by q RT-PCR assays,RNA stability assays and RNA immunoprecipitation.3.Screening and validation of muscone derivatives:FP assay was used to established a screening model on the basis of the interaction between HuR RRM1/2 and ARE^Vegf-a.All the muscone derivatives were screened.The IC₅₀ value of positive compounds disrupting the interaction between HuR RRM1/2 and ARE^Vegf-awere determined by FP assays.The binding activity of positive compounds to HuR RRM1/2 protein was detected by SPR.The interaction mode between positive compounds and HuR RRM1/2 was simulated by molecular docking.4.Muscone derivative ZM-32 inhibited angiogenesis in vitro:CCK-8 assay,wound healing assay and matrigel angiogenesis assay in HUVECs were performed to detect the effect of ZM-32 on the proliferation,migration and tubule formation of HUVECs.The effect of ZM-32-treated macrophage supernatants on HUVECs was also detected.q RT-PCR,Western blot and ELISA assays were used to detect the effects of ZM-32 on the expression of VEGF-A and MMP9 in macrophages.Western blot assay was used to detect the effect of ZM-32-treated macrophage supernatants on the activation of VEGFR signaling pathways in HUVECs.5.Muscone derivative ZM-32 inhibits tumor growth and angiogenesis in vivo:CCK-8assay was used to detect the toxicity of ZM-32 on cancer cell lines.Transwell assay was used to detect the effect of ZM-32 on the migration of MDA-MB-231 cells.The in vivo inhibitory activity of ZM-32 on tumor growth and tumor angiogenesis was verified in MDA-MB-231 xenograft models.The safety of ZM-32 was evaluated by routine blood tests,biochemical analyses and H&E staining of tissues and organs.6.Muscone derivative ZM-32 inhibits tumor angiogenesis in HuR-dependent manner:The HuR-dependent angiogenic genes regulation mechanism of ZM-32 in macrophages were studied by RNA stability assays,RNA immunoprecipitation and RNA pull down assays.q RT-PCR and Western blot assays were used to detect the effects of ZM-32 on the expression of VEGF-A and MMP9 in MDA-MB-231 cells.The HuR-dependent angiogenic genes regulation mechanism of ZM-32 in MDA-MB-231 cells were studied by RNA stability assays,RNA immunoprecipitation and luciferase assays.Results:1.Screening model based on the HuR-ARE^Vegf-a interaction was successfully established.At present,4200 compounds have been screened,of which two are positive compounds,which are ZM-32 and ELB.Our previous study found that ELB had the ability of inhibiting the growth of 4T1 breast cancer and inhibiting macrophage-mediated angiogenesis.In this study,we verified the HuR-dependent angiogenic genes regulations mechanism of ELB at molecular and cellular levels.While verifying the new target of ELB,we also confirmed the feasibility of the established HTS system for screening anti-tumor angiogenesis drugs.2.Forty-eight muscone derivatives were tested for their inhibitory activity on HuR RRM1/2-ARE^Vegf-a interaction using FP experiments.ZM-1,ZM-12,and ZM-32 were found to be positive in this assay.ZM-32 was the most effective in preventing HuR RRM1/2-Vegf-a m RNA complex formation with an IC₅₀ of 204.20±1.38 nmol/L.ZM-32 bound to HuR RRM1/2 protein with a K_D value of 521.7 nmol/L.3.When HUVECs were incubated directly with ZM-32,the proliferation,migration and tube formation capacity of HUVECs remain unaffected.However,when HUVECs were incubated with ZM-32-treated macrophage supernatants,the proliferation,migration and tube formation capacity of HUVECs were all inhibited.Furthermore,ZM-32 inhibited the expression and release of VEGF-A and MMP9 in Raw264.7 cells and thus inhibit the activation of VEGFR-2 signaling pathway in HUVECs.4.Muscone derivative ZM-32 effectively prevented the proliferation and migration of breast cancer cells and inhibited the growth and angiogenesis of MDA-MB-231 xenograft tumors without any obvious toxicity in vivo.5.Muscone derivative ZM-32 regulates the expression of angiogenesis-related genes（Vegf-a and Mmp9）via disrupting HuR-m RNA interactions which inhibited m RNA stability both in Raw264.7 and MDA-MB-231 cells.Conclusion:The HuR inhibitor screening model built in this study could be used to screen anti-tumor angiogenesis drugs.The positive compound muscone derivative ZM-32 could disrupt the interaction between HuR and m RNA,which reduce m RNA stability and inhibit the expression of angiogenesis-related genes.It has shown better anti-tumor angiogenic activity.In summary,this study has established a good screening model for the rapid development and application of traditional Chinese medicine,and provided theoretical basis for development of anti-tumor angiogenesis drugs targeting HuR protein.