The Role And Mechanisms Of Brucea Javanica And Its Monomer In Colon Cancer

Objective: Colon cancer is one of the top killers of human health in the world.It has high morbidity,mortality,recurrence,and poor prognosis.It is important to explore new drugs or treatment strategies to effectively inhibit the progression of colon cancer.Brucea javanica is a traditional Chinese herb.It exerts strong anti-cancer activity in various types of cancers.Brucea javanica oil emulsion injection(BJOEI)is a proprietary Chinese medicine extracted from crow gall.Brucea javanica has been widely used to treat gastrointestinal and lung cancer.However,the effects of brucea javanica on colon cancer have been reported very few.Therefore,this study first analyzes the literature related to brucea javanica with the help of bibliometrics,explores the researchers’ hot spots for brucea javanica,and plays a guiding role in the selection of topics.At the same time,the network pharmacological analysis of brucea javanica was carried out,and the monomer components of the natural products of brucea javanica were analyzed.Next,we explored the effect of the natural product monomer of brucea javanica on colon cancer cells through a series of basic experiments,which provided a strong theoretical basis for the pharmacological value of Brucea javanica.Methods:Part 1.The bibliometrics of Brucea javanica related literature.1.1.Literature retrieval: Chinese Literature: Chinese literatures that published between January 1,2001 and December 31,2021 were searched in CNKI database with brucea javanica as the main inscription by using the correlation analysis module of the database,we had a view of the brucea javanica in Chinese literature.English Literature: Web of Science Core Collection database of all relevant literatures about brucea javanica was searched and quality controlled,and the retrieval formula of “Brucea javanica”,“Brucea javanica oil” and “Java brucea”.The retrieved time was from January 1,1993,to December 31,2021.1.2.Bibliometric analysis: Cite Space V software,and VOSviewer software were used to perform visual analysis on the journal citations,publication years,publication journals,authors,the affiliation of correspondence,countries of correspondence,and research types of the included articles.1.3.Network Pharmacology analysis: We learned from the Traditional Chinese Medicine database and analysis platform System Pharmacology(TCMSP,http://tcmspw.com/tcmsp.php)of the Java brucea fruit of natural chemical composition of products and their corresponding targets.A protein interaction network was constructed using STRING online database(https://www.string-db.org/).GO functional enrichment analysis of gene ontology and KEGG pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes were analyzed by R software(3.6.3).Part 2.To investigate the biological behavior and molecular mechanism of the natural product monomers(luteolin,β-sitosterol)of brucea javanica affecting colon cancer2.1 Through MTT and colony formation experiments,the inhibition of colon cancer cells by natural product monomers(luteolin,β-sitosterol)of brucea javanica was detected.2.2The effects of luteolin and β-sitosterol on apoptosis and cell cycle of colon cancer cells were detected by flow cytometry.2.3 Transwell method was used to detect the migration potential of colon cancer cells treated with luteolin and β-sitosterol.2.4 It was predicted that luteolin and β-sitosterol could directly interact with GPSM2 by molecular docking.The signaling pathway regulated by luteolin and β-sitosterol was predicted by bioinformatics.2.5 To elucidate the molecular mechanism of luteolin and β-sitosterol in colon cancer,we used western blot,gene overexpression,si RNA transfection,real-time quantitative PCR,and xenograft model.Part 3.To study the expression and clinical significance of GPSM2 in colon cancer patients.3.1 Data of GPSM2 m RNA in 33 of tumor tissue expression mode were downloaded from UCSC Xena(https://xena.ucsc.edu/).3.2 The expression levels of GPSM2 m RNA in normal tissues,tumor tissues and cancer cell lines were analyzed.3.3Immunohistochemistry assay was used to detect the expression level of GPSM2 protein in tissue of 158 colon cancer patients.We analyzed the relationship between GPSM2 expression and clinical pathological parameters.Results:Part 1.Bibliometric analysis and Network Pharmacology Analysis of Brucea javanica related literature.1.1 Between 2001 and 2021,my country published 1884 Chinese articles related to Brusea Javanica,and reached the peak in 2021.Yang Lihui from the Third Hospital of Xingtai city published 12 papers,11 by He Fengjie from the Affiliated Hospital of the Shaanxi University of Chinese Medicine,10 by Wang He from the Second Affiliated Hospital of Air Force Military Medical University,Tian Huaqin from Foshan Hospital of Chinese Medicine and Zeng Tao from the Institute of Plant Protection,Guangxi Academy of Agricultural Sciences.These articles were most closely related to oncology.Guangzhou University of Chinese Medicine and Beijing University of Chinese Medicine and their corresponding journals,published more relevant literature.Matrix analysis results also suggested that brucea brucea mainly plays an anti-tumor role in apoptosis,cell cycle,and cell proliferation in related reports.And it has been studied mostly in non-small cell lung cancer.We used Cite Space software to analyze brucea javanica,nearly five years of 414 keywords were included which suggested brucea javanica has an anti-tumor effect for stomach cancer,colorectal cancer,liver cancer,lung cancer,esophageal cancer.And the analysis of pharmacodynamics on brucea javanica also suggests its function in cell proliferation,apoptosis,migration,immune function.1.2 From 1993 to 2021,a total of 231 English articles related to brucea javanica were published.The analysis of time distribution started in 2013,and the number of articles published per year increased to 24 articles per year in 2021,reaching a historical high point.A total of 1024 authors participated in the research of brucea javanica.The authors were mainly distributed in 313 research institutions.We ranked them according to the number of publications,and the top five publications were the Chinese University of Hong Kong,Guangzhou University of Chinese Medicine,University of Illinois,Shenyang Pharmaceutical University,And Hokkaido University.China ranked first with136 articles.Though the analysis of keyword,we found that in the early years(1993 to2007),these keywords were mainly the main components of brucea javanica,including luteolin,bruceine,brusatol,etc.Since 2007,researchers began to focus on the anticancer effects of brucea javanica,mainly in practical application,including apoptosis,mitochondrial signaling pathway,cytotoxicity,cancer.These keywords suggest that exploring the antitumor mechanism of Brucea javanica may be one of the future trends of Brucea javanica research.A total of 55 enrichment functions were identified by GO enrichment analysis of brucea javanica targets.We noticed that brucea javanica is mainly enriched in the following functions: Cysteine endopeptidase involved apoptosis,G-protein-coupled amine receptor activity,G-protein-coupled serum receptor activity,and G-protein-coupled neurotransmitter receptor activity.KEGG pathway enrichment analysis showed that a total of 101 compliant results were screened,and the PI3K-Akt signaling pathway ranked first.Part 2.To investigate the biological behavior and molecular mechanism of the natural product monomers(luteolin,β-sitosterol)of brucea javanica affecting colon cancerBased on the previous network pharmacology analysis of brucea javanica,we selected two monomers with good solubility(luteolin and β-sitosterol)among the main components of brucea javanica for subsequent cytological experiments.2.1 Luteolin andβ-sitosterol can inhibit the growth of colon cancer cells in a dose-and time-dependent manner.The IC50 of luteolin on RKO cells was 19.32 u M at 24 h and 6.1u M at 48 h.The IC50 of SW480 cells was 20.67 um at 24 h and 14.45 um at 48 h.The IC50 of β-sitosterol on RKO cells was 93.36 u M at 24 h and 53.08 u M at 48 h.The IC50 of SW480 cells was175.05 u M at 24 h and 84.68 u M at 48 h.2.2 Luteolin and β-sitosterol induced apoptosis and G2/M phase arrest in colon cancer cells,and in a dose-dependent manner.2.3Luteolin and β-sitosterol can inhibit the invasion of colon cancer cells.2.4 Molecular docking showed that luteolin and β-sitosterol directly interact with GPSM2.And bioinformatics analysis suggested that luteolin and β-sitosterol might regulate the Fox O signaling pathway.2.5 With si GPSM2 and the overexpression of GPSM2,we found that luteolin and β-sitosterol were involved in the biological behavior of colon cancer cells by down-regulating GPSM2.2.6 In vivo mouse tumorigenesis experiments confirmed that BJOEI can further regulate the Fox O signaling pathway by down-regulating the expression of GPSM2,significantly reducing tumor growth in mice,and has a synergistic effect with 5-fluorouracil.Part 3.To study the expression pattern and clinical significance of GPSM2 in colon cancer patients.3.1 GPSM2 expression levels are highly variable in different normal tissues.The top three tissue with the highest GPSM2 expression were the vagina,sigmoid colon,and nerve-tibia.While the top three tissue with the lowest GPSM2 expression were the pancreas,liver,and blood.3.2 Among tumor tissues,the top three cancer tissues with the highest GPSM2 expression are rectal adenocarcinoma,colorectal adenocarcinoma,and low-grade glioma.The top three cancers with the lowest GPSM2 expression were uveal melanoma,thyroid cancer,and hepatocellular carcinoma.Amplification is the main altered form of GPSM2,followed by mutation and deep deletion.Colorectal cancer had the highest frequency of mutations.3.3 In the differential expression analysis between cancer tissues and adjacent tissues,GPSM2 expression in colon cancer was significantly higher than that in adjacent tissues.3.4 GPSM2 m RNA levels were higher in colorectal cancer,myeloid cell tumor,and small cell lung cancer cell lines.3.5 Immunohistochemical analysis of the pathological tissues collected from 158 colon cancer patients showed that GPSM2 was an independent risk factor for the prognosis of colon cancer patients.Conclusions:1.In this study,text mining and bioinformatics were combined to comprehensive analysis of brucea Javanica,and comprehensively and specifically analyzed the literature related to brucea Javanica,and found that the researchers’ current research focus is to explore the mechanism of action of brucea Javanica and its natural product monomers.Brucea javanica inhibits a variety of biological behaviors of cancer cells by regulating PI3K/AKT,P53,HIF-1,Fox O,and TNF signaling pathways.Through the quantitative analysis and statistics of the literature on brucea javanica in the past 20 years,there are few reports on the mechanism of brucea javanica in colon cancer,which shed light on our study.2.Brucea Javanica and its natural product monomers(luteolin,β-sitosterol)can effectively inhibit the growth of colon cancer cells in vitro and in vivo.The mechanism is to downregulate GPSM2,mediate the Fox O signaling pathway,inhibit cell proliferation,induce cell phase arrest and apoptosis,and inhibit cell invasion.Our results suggest that the natural product monomers(luteolin,β-sitosterol)of brucea Javanica can be used as a potential therapeutic agent to improve survival in colon cancer patients.3.GPSM2 expressed higher in the tumor tisue than paratumor tissue.And it is an independent prognostic factor for colon cancer patients.