Inhibitory Effect Of Galangin On Bladder Cancer And Its Molecular Mechanism

Objective: Bladder cancer is the tenth most common cancer worldwide,accounting for2.1% of all cancer deaths worldwide.It has the fourth and tenth highest incidence rates in men and women,respectively.According to the results of Global Cancer Statistics 2020,the estimated number of new cases of bladder cancer is about 573,000,and the number of deaths is about 212,000,with the incidence and mortality rate in men being about four times higher than that in women.In China,the incidence of bladder cancer ranks the 13 th in the incidence of malignant tumors in general and the seventh in the incidence of malignant tumors in men,with an incidence rate 3.8 times higher than that of women and a mortality rate 4 times higher than that of women.According to the 2022 US-China cancer statistics,the new cases and deaths of bladder cancer in China in 2022 are much higher than those in the US.The current clinical treatments for bladder cancer are mainly surgery,chemotherapy,radiotherapy,immunotherapy,and targeted therapy.However,due to the different pathological characteristics of bladder cancer,there are still many problems after the above treatments,such as the high recurrence rate after surgery for non-muscle-infiltrating bladder cancer and the poor prognosis and low 5-year survival rate for muscle-infiltrating bladder cancer.Therefore,it is urgent to find new anti-bladder cancer drugs with low toxicity and good efficacy.Chinese medicine has been reported for the treatment of bladder cancer,which has attracted widespread attention both at home and abroad,such as extracts of traditional Chinese medicine or traditional Chinese herbal medicines like tannisol,artesunate and bitter ginseng are able to exert anti-bladder cancer effects.There have been more reports on the anti-tumor studies of galangin,but there are almost no studies on the anti-bladder cancer of galangin,so this study was intended to explore the anti-bladder cancer effect of galangin through network pharmacology and in vitro experiments.Methods:(A)Network pharmacology techniques were used to analyze the mechanism of action of galangin exerting anti-bladder cancer effects.The TCMSP,Swiss Target Prediction and targetnet databases were used to predict the targets of galangin action.Bladder cancer-related targets were obtained from the Gene Cards database.The intersection of the two was taken as the target of galangin’s action against bladder cancer.The intersecting targets were screened for core targets using STRING database and Cytoscape 3.9.0 software to build a protein-protein interaction(PPI)network of targets.The core targets were subjected to gene ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis using the online annotation and visual integration analysis tool DAVIDBioinformatics Resources(2021Update).The drug-disease-target-pathway network was constructed using Cytoscape 3.9.0 software.(B)Human bladder cancer cells 5637,HT-1376 and normal bladder epithelial SV-HUC-1 cell lines were used to detect the effect of different concentrations(0μM,40μM,80μM,120μM,160μM,200μM)of galangin on the proliferation of bladder cancer cells after treatment of the above cells for 24 hours using CCK-8 kit.(C)To investigate the role of p53 signaling pathway in the anti-bladder cancer of galangin on the basis of network pharmacology.Bladder cancer cells 5637 and HT-1376 were used for the study,and the experimental validation was performed by setting the drug concentration of galangin at 0μM,120μM and 160μM in combination with CCK-8 assay.The effect of galangin on cell proliferation was examined by plate cloning assay;the apoptosis of bladder cancer cells induced by galangin was examined by Hoechst 33258 staining and flow cytometry;the effect of galangin on apoptosis-related proteins bax,Bcl-2,Cleaved-PARP and Cleaved-PARP and p53 signaling was examined by Western blot.Caspase3 and the expression of p53 signaling pathway-related proteins MDM2,p53,p-p53 and cytc.si-p53silencing was followed by further validation of the inhibition of bladder cancer by galangin.Results:(A)A total of 204 galangin-acting targets and 2912 bladder cancer targets were obtained after de-duplication by different databases.The GO analysis yielded 101 GO items for biological process(BP),16 GO items for cellular component(CC),53 GO items for molecular function(MF),and 53 GO items for KEGG pathway enrichment analysis,including cancer signaling pathway,cellular senescence,viral oncogenesis,chemo-oncogenic-receptor activation,and PI3K-Akt signaling pathway.PI3K-Akt signaling pathway,p53 signaling pathway,non-small cell lung cancer,prostate cancer,progesterone-mediated oocyte maturation,cell cycle and 53 other signaling pathways.(b)The value-added rate of bladder cancer cells 5637 and HT-1376 as well as normal bladder epithelial cells SV-HUC-1 gradually decreased with the increase of galangin concentration,and the effect of galangin on the proliferation rate of normal bladder epithelial cells was less than that of bladder cancer cells at the same concentration.(iii)Plate cloning assay showed that galangin inhibited the proliferation of bladder cancer cells in a concentration-dependent manner;Hoechst 33258 staining and flow cytometry results suggested that galangin induced apoptosis of bladder cancer cells in a concentration-dependent manner;protein immunoblot(westernblot)assay suggested that galangin could inhibit the proliferation of apoptosis-related proteins bax,Cleaved-PARP,and bax in a dose-dependent manner.Cleaved-PARP and Cleaved-Caspase3 expression was up-regulated and Bcl-2 expression was down-regulated;p53 signaling pathway-related protein MDM2 expression was down-regulated and p53,p-p53 and cytc expression was up-regulated.After using si RNA to silence p53 protein,bladder cancer cells were treated with galangin,and it was found that galangin was able to upregulate p53 protein expression in cells after p53 silencing,and also to affect the proliferation of cells with p53 protein silencing.Conclusions: 1.The results of network pharmacology suggest that galangin can exert anti-bladder cancer effects through multiple targets and pathways,among which the p53 signaling pathway may play an important role in galangin’s anti-bladder cancer effects.2.The results of CCK-8 and plate cloning experiments suggest that galangin can inhibit bladder cancer cell proliferation in a dose-dependent manner.3.In vitro experiments suggest that galangin can induce apoptosis of bladder cancer cells by activating the p53 signaling pathway.