Study On The Anti-bladder Cancer Mechanism Of Resveratrol-Amino Acid-NO Donor Conjugate

Bladder cancer is a disease with high morbidity,mortality,and treatment costs.As one of the top ten most common cancers worldwide,bladder cancer has a significant gender difference,with almost four times more common in men than in women.The combination of cisplatin and gemcitabine（GC）is the standard regimen for the clinical treatment of muscleinvasive bladder cancer.However,the emergence of cell resistance has brought new challenges to the treatment and prognosis of patients with bladder cancer.In the search for bladder cancer treatment drugs with significant efficacy and no obvious toxic side effects,resveratrol has attracted wide attention due to its low toxic side effects,strong autoimmune activity and anti-tumor activity.At the same time,as an important gas messenger molecule in vivo,the effect of NO on tumor proliferation,apoptosis,metastasis and other stages has gradually attracted people’s attention.In addition,current studies have found that NO donors can improve the drug resistance of tumor cells by regulating the expression of multidrug resistance（MDR）proteins and other ways.A number of NO donor anti-tumor drugs with different release mechanisms are emerging,which provide new ideas and methods for the research of anti-tumor drugs.The traditional concept of "one drug,one target,one disease" was the main paradigm of drug discovery in the past.However,studies in systems biology have shown that complex diseases may not be effectively treated by interventions against a single target.As a classical bioinformatics method,network pharmacology can save cost and time compared with traditional experiments.More importantly,network pharmacology can systematically reflect the association between drugs and disease targets from a holistic perspective,and can reveal the mechanism of drug action on complex diseases from multiple directions.Therefore,in this study,we combined network pharmacological analysis and experimental validation to evaluate the possible therapeutic effects and underlying mechanisms of resveratrol-amino acid-NO donor conjugates in bladder cancer,with the aim of providing a promising therapeutic option for bladder cancer.The specific research content of this work is as follows:1.Based on six resveratrol-amino acid-NO donor derivatives synthesized by our research group,the antiproliferative activities of two kinds of bladder cancer cells and human normal liver cells were detected by MTT method.The results showed that compound E16/20 exhibited good cytotoxicity on both bladder cancer cell lines,and had no significant effect on normal liver cells.Therefore,compounds E16 and E20 were selected as potential agents for the treatment of bladder cancer for more in-depth activity studies.The results of wound healing assay,crystal violet staining and live/dead cell staining showed that E20 could significantly inhibit the growth and proliferation of two kinds of bladder cancer cells.Flow cytometry showed that E16 and E20 induced apoptosis in a concentration-dependent manner,and promoted the generation of reactive oxygen species and the influx of Ca²⁺ in the cells.Western blot results showed that E16 and E20 could induce apoptosis of bladder cancer cells by regulating the expression of endogenous apoptosis pathway proteins such as P53,Bcl-XL,Bax,Bcl-2,Cytochrom-c,and Caspase3/8.In addition,immunofluorescence results showed that both compounds could induce the production of autophagosomes in bladder cancer T24 cells under half inhibitory concentration.At the same time,the results of Western blot also verified that E16 and E20 could induce the accumulation of LC3-II in a certain concentration range.The results of Griess assay indicated that the NO produced by compound E20 may be involved in the inhibition of drug resistance and sensitive proliferation of bladder cancer cells in vitro.In SCaBER cells,E20 significantly reduced the relative expression levels of drug resistanceassociated proteins such as MDRI,MRP1 and BCRP.2.Further explore the mechanism of the compound on bladder cancer cells based on network pharmacology.First of all,the known targets of parent compound resveratrol were summarized through Pubchem,FAFdrugs4 and other databases,and the potential targets of resveratrol were predicted using STITCH,Pharm Mapper and other databases.DisGeNET disease gene database was used to summarize bladder cancer disease genes.The summarized potential targets of resveratrol and bladder cancer disease genes were intercrossed by Veen map,and 224 common affected gene targets were obtained.Then the pathway analysis of the disease-compound interaction targets was conducted through functional annotation databases such as DAVID and KEGG,and the pathway with high enrichment scores was obtained: PD-1/PD-L1 immune checkpoint pathway and PI3KAKT-m TOR signaling pathway.Auto dock molecular docking results showed that the key targets of PD-1/PD-L1 immune checkpoint pathway,such as SHP2,PD-1 and PD-L1,can bind to the parent compound resveratrol through hydrophobic interaction,hydrogen bond and other ways by docking.In addition,key proteins in PI3K-AKT-m TOR signaling pathway and PD-1/PD-L1 immune checkpoint pathway were verified by Western blot.The results showed that compounds E16 and E20 could significantly down-regulate the expression of EGFR,PI3 K,AKT,m TOR,P70/S6,STAT-3,NF-κ B and other proteins in a concentration-dependent manner.These results suggested that the compound could inhibit the PI3K-AKT-m TOR signaling pathway and PD-1/PD-L1 immunoassay site pathway in tumor cells.