The Research On Multi-target Mechanism Of Novel Flavonoid Spiro-skeleton Derivatives Based On P53 Pathway

Tumor has become one of the major diseases that pose a serious threat to human health.Its formation and development is a complex process involving multi-stage,multi-factor,multi-gene and multi-cell signaling pathways.Due to the heterogeneity and complexity of cancer cells,multi-target drugs based on system biology may have better advantages in cancer treatment than single-target drugs and drug combination.Therefore,designing a lead compound that synergistically regulates multiple pathological targets and is safe and effective has become a hot topic in the development of novel anti-tumor drugs.The transcriptional regulator p53 is an important tumor suppressor gene in the human body,which has functions such as inhibiting cell proliferation,inducing cell cycle arrest and inducing apoptosis.5-Lox is overexpressed in a variety of malignant tumors,and its role in promoting cell proliferation,inhibiting apoptosis and differentiation is closely related to signaling pathways such as PI3K/Akt and p53.Studies have shown that Akt can delay p53-mediated apoptosis and stabilize p53expression by inhibiting MDM2 autophosphorylation.There is a synergistic dialogue between p53-MDM2 signaling pathway and PI3K/Akt.Inhibition of Akt activity by acting on the p53 pathway in tumor cells may enhance the therapeutic effect of p53activators.Therefore,in this paper,a series of novel flavonoid spiro-skeleton derivatives were designed and synthesized by introducing pharmacophore indole oxide of p53-MDM2 binding inhibitor reasonably on the flavone skeleton targeting5-Lox and Akt.Two compounds 3d and 3e with the highest cytotoxicity towards tumor cells were obtained by in vitro anticancer activity and their anti-tumor mechanisms were explored.On this basis,the in vivo anti-tumor mechanism of compound 3e was studied by establishing a mouse model of Lewis lung cancer xenografts.The purposes of this study are to obtain anti-tumor lead compounds with better curative effect and lower toxic side effects,and to provide a theoretical reference for the development of multi-target anti-tumor drugs.The main work of this paper is as follows:1.Synthesis of target compounds and study of antitumor activity in vitroWith p53 signaling pathway as the research object,26 novel flavonoid spiro-skeleton derivatives were rationally designed and synthesized by pharmacophore combination method.The structures of all compounds were confirmed by¹H NMR,¹³C NMR,HR-ESI-MS and single-crystal X-ray analysis.MTT assay showed that some compounds significantly inhibited the proliferation of A549,K562,NCI-H1299 and PC-3 cells.In particular,compounds 3d(IC₅₀=4.72+0.35?M)and 3e(IC₅₀=3.15+0.51?M)were the most sensitive to the inhibition of wild-type p53-expressing A549 cells,showing better inhibition than the positive control cisplatin(IC₅₀=17.58+1.12?M)and parent compound chrysin(IC₅₀>50?M).Furthermore,compounds 3d and 3e have good selectivity between A549 cells and normal cells.On this basis,through the molecular docking of computer simulation,the mode of action and binding ability of compound 3e and receptors Akt,5-Lox and MDM2 were preliminarily investigated.The results demonstrate that the anti-tumor target of the compound 3e is related to the Akt,5-Lox and MDM2 targets of the design concept.Biological experiments showed that target compounds 3d and3e significantly activated the expression of p53 protein and m RNA in A549 cells in a dose-or time-dependent manner,which proved that target compounds could indeed affect the p53 pathway.2.Inhibitory effects of compounds 3d and 3e on proliferation of A549 cellsHuman lung cancer A549 cells were used as the research object to study the effects of compounds 3d and 3e on cell proliferation from cell proliferation,apoptosis,cell cycle,cell migration and invasion.Soft agar colony formation and plate cloning experiments showed that compounds 3d and 3e significantly inhibited the colony formation of A549 cells,and confirmed the anti-proliferative effect of target compounds on A549 cells.The wound-healing and transwell experiments showed that compounds 3d and 3e significantly inhibited the migration and invasion of A549cells.Morphological observation,PI and AO/EB fluorescence staining showed that compounds 3d and 3e induced apoptosis of A549 cells in a dose-dependent manner.Subsequently,agarose gel electrophoresis showed that 20?M of compound 3d and3e treated A549 cells for 24 h,respectively,and the formation of DNA ladder was observed,indicating that it induced fragmentation of genomic DNA in A549 cells.Flow cytometry showed that the percentage of apoptosis in A549 cells in a dose-or time-dependent manner,and the cells tend to be late apoptosis.Treatment of cells with compounds 3d and 3e for 24 h caused G1 arrest in A549 cells.The above results indicate that compounds 3d and 3e have an inhibitory effect on the proliferation of A549 cells,and this effect is associated with induction of apoptosis and cell cycle arrest.3.Mechanism of apoptosis of A549 cells induced by compounds 3d and 3eBy detecting intracellular ROS levels,it was found that compounds 3d and 3e increased ROS levels in A549 cells in a dose-dependent manner.The results of Caspase activity assay showed that compounds 3d and 3e significantly activated the apoptosis-related Caspase-3,Caspase-8 and Caspase-9 enzyme activities.These results suggest that activated p53 increases mitochondrial membrane permeability by regulating related gene expression,and apoptosis of A549 cells may be related to Caspase cascade.In order to further explore its mechanism,related proteins and genes in p53 signaling pathways such as Bcl-2,Bax,Cyt c,p53,p21,MDM2,Akt,and5-Lox were selected for study.Western blot and Real-time PCR showed that compounds 3d and 3e significantly up-regulated the expression of Bax,Cyt c,p21and p53 protein in A549 cells,and down-regulated the expression of Bcl-2,MDM2,Akt and 5-Lox protein.In addition,compounds 3d and 3e up-regulated p53 m RNA levels in A549 cells and down-regulated Akt m RNA levels.Based on the above results,the mechanism of apoptosis of A549 cells was predicted:compounds 3d and 3e inhibited MDM2 activity,released p53,and activated p53 signaling pathway;simultaneously,inhibited the expression of Akt and 5-Lox protein,and synergistically increased the anti-tumor effect of p53 pathway.Activated p53 induces G1 arrest in A549 cells by regulating p21 protein expression;at the same time,changing the ratio of Bax/Bcl-2,increasing intracellular ROS levels,leading to intracellular redox imbalance,DNA damage,increased mitochondrial membrane permeability,and release of pro-apoptotic factors Cyt c,thereby activating Caspase-9,to initiate the endogenous mitochondrial apoptotic pathway.In addition,the activation of the exogenous death receptor apoptosis pathway is initiated by activating Caspase-8enzyme activity.4.Acute toxicity of compounds 3d and 3e and anti-tumor mechanism in vivoThe toxicity of compounds 3d and 3e is low toxicity according to the criteria for acute toxicity.On this basis,the mechanism of anti-tumor effect of compound 3e in vivo was studied by constructing a Lewis lung cancer mouse xenograft model.The results showed that compound 3e effectively inhibited the growth of transplanted tumors in Lewis lung cancer mouse,and had a good dose-dependent relationship.Among them,high dose(400 mg/kg)had the highest tumor inhibition rate(56.87±1.47%).Western blot analysis showed that the anti-tumor mechanism in vivo was:compound 3e activates and synergizes with p53 signaling pathway by up-regulating the expression of p53 protein and inhibiting the expression of MDM2,Akt and 5-Lox proteins in tumor cells.On the one hand,up-regulated of Bax expression of proapoptotic protein,down-regulated of anti-apoptotic protein Bcl-2expression,increase of Bax/Bcl-2 ratio,and release of Cyt c,thereby inducing apoptosis of tumor cells.On the other hand,down-regulated of VEGF,cell adhesion factor ICAM-1 and matrix metalloproteinase MMP-2 protein,block tumor cell angiogenesis and inhibit tumor metastasis in Lewis lung cancer mouse.In summary,26 novel flavonoid spiro-skeleton derivatives were designed and synthesized in this study.Screening by in vitro anti-tumor activity,then studying the effects of high-activity compounds 3d and 3e on the proliferation of A549 cells,and compound 3e inhibited the growth of transplanted tumors in Lewis lung cancer mouse,revealing the anti-tumor mechanism of target compounds in vitro and in vivo.It provides theoretical basis and technical support for the further development of multi-target flavonoid anti-tumor drugs,which has certain theoretical value and guiding significance.