| Prostate cancer is the most common malignant tumor in men,with the second highest incidence of cancer of male cases and the fifth highest mortality worldwide.The treatment methods of prostate cancer are mainly including endocrine therapy and prostatectomy,among which endocrine therapy is easy to cause castration resistance,the treatment method is relatively limited,and the overall prognosis is poor.Therefore,to searching for new drugs for the treatment of prostate cancer has become an important topic of prostate cancer-related research.Ethnic drugs are important sources for developing anti-cancer drugs or their leading compounds.Carpesium cernuum L.is a perennial herb of the Compositae family.It is used as a folk medicine in Guizhou Miao area.It tastes bitter,pungent and cool.The whole herb is used as medicine to treat urinary tract infections.Modern pharmacological studies have found that the Carpesium cernuum L.contains a variety of chemical components,which can inhibit the growth of tumors such as liver cancer,colon cancer,breast cancer and gastric cancer et al.It has been reported that extracts of Carpesium cernuum L.exhibiting anti-prostatic hyperplasia effects,but the research aganist prostate cancer has not been reported yet.Based on this,the prsent study was aimed to explore the active constituents against prostate cancer from Carpesium cernuum L.,as well as their underlying mechanisms,which can provide scientific basis for the development of new anti-prostate cancer drugs.This research includes the following 6 parts.(1)Study on the active constituents of the Carpesium cernuum L against prostate cancerThe 95% ethanol extract which was refluxed heating extraction was extracted with petroleum ether,ethyl acetate and n-butanol to obtain different polar fractions,respectively.MTT method was used to screen the activity.The results showed that both the ethanol extract and ethyl acetate fractions had significant anti-prostate cancer activity.The ethyl acetate was isolated by silica gel column,Sephadex LH-20 and HPLC.The results showed that 1 new compound and 10 known compounds were isolated from the ethyl acetate part and their structures were respectively identified by mass spectrometry(MS)and nuclear magnetic resonance(NMR)as ineupatolide(1)carpescernolides D(2)ineupatolide E(3)carpescernolide C(4)2,9-epoxy-5,9-dihydroxy-8-angeloyloxy-11-methoxymethyl-germacran-4(15)-en-6,12-olide(5)carpescernolide B(6)2 α,5-Epoxy-5,10-dihydroxy-6 α,9 β-diangeloyloxygermacran-8 α,12-olide(7)4E-9β-hydroxy-7α,10α H-germacra-4,11(13)-dien-12,6α-olide(8),incaspitolide A(9)carpescernolides E(10)carpescernolide A(11).Compound 5 is a new compound.The results showed that compounds 1,7,9 and 10 had significant anti-prostate cancer activity.Based on the cellular activity and number of compounds,ineupatolide(1)and incaspitolide A(9)will be selected in the later stage of this paper to conduct experimental studies on the anti-prostate cancer effect and mechanism in vivo and in vitro.(2)Network pharmacology analysis of ineupatolide and incaspitolide A against prostate cancerIneupatolide and incaspitolide A were analyzed the mechanism of action against prostate cancer by coexisting gene targets with prostate cancer via a network pharmacology approach.The corresponding gene targets of ineupatolide,incaspitolide A and prostate cancer were obtained through Pharm Mapper and Uni Prot databases.There were 119 overlapping targets between prostate cancer and ineupatolide while 124 overlapping targets between ineupatolide and incaspitolide A,and the corresponding target proteins formed an interacting protein network.From the network,it was found that the molecular mechanism of ineupatolide and incaspitolide A against prostate cancer included proteins related to PI3K/Akt signaling pathway.Through GO enrichment analysis,incaspitolide A is involved in the metabolic process and catalytic activity of prostate cancer cytoplasm,and these related functions are closely related to the occurrence and development of prostate cancer.In order to further study the molecular mechanism of ineupatolide and incaspitolide A against prostate cancer,the overlapping targets were found to be involved in PI3K/Akt,FOXO,HIF-1,IL-17,estrogen,and relaxin through KEGG pathway enrichment analysis.Through the analysis of signaling pathways and proteins interaction network,PI3K/Akt is closely related to other signaling and plays an intermediate role,indicating that the mechanism of action of ineupatolide and incaspitolide A against prostate cancer may be accomplished through the PI3K/Akt signaling pathway.(3)Transcriptomics study of incaspitolide A and ineupatolide against prostate cancerThe mechanism of ineupatolide and incaspitolide A against prostate cancer was fuether studied by using transcriptome method.PC-3 cells were treated with 0 and 20μM ineupatolide and incaspitolide A for 24 h,and total RNA was extracted.Differentially expressed genes were detected by RNA-seq technology.The original base was 76.97 Gb,and the clean base was 74.14 Gb after quality control.Q20 > 97.71%,Q30 > 93.44%,GC content in 48.52%-52.64%,indicating that the quality of sequencing data is qualified,in line with the requirements of transcriptome sequencing.There were15,811 differentially expressed genes(7,058 up-regulated genes and 8,753 downregulated genes)before and after incaspitolide A.A total of 10,407 differentially expressed genes(5,110 up-regulation and 5,297 down-regulation)were detected before and after administration of ineupatolide.The transcriptome changes of prostate cancer cells treated with incaspitolide A were significantly different,which may be related to the types of cells and compounds.Further GO function analysis and KEGG signal pathway analysis showed that PC-3 cells treated with ineupatolide and incaspitolide A respectively differentially expressed genes were involved in cell metabolism,protein binding,and cell nucleus and organelles formation.It is involved in RNA synthesis,protein synthesis,proteasome,cell cycle and apoptosis,and regulates tumor necrosis factor signaling pathway,m TOR pathway and PI3K/Akt signaling pathway.Among which 84.38% of differentially expressed genes related to PI3K/Akt signaling pathway.The results indicated that PI3K/Akt signaling pathway is an important signaling pathway of ineupatolide and incaspitolide A against prostate cancer.(4)Study on the anti-prostate cancer effect and mechanism of ineupatolide in vivoThe 5-week-old female BALB/c-nu nude mice were inoculated with prostate cancer PC-3 cells in vivo and subcutaneously to establish human prostate cancer xenograft tumor model.0,20,40,80 mg/kg ineupatolide were respectively injected intraperitoneally.After administration,the indexes of diet,water intake and activity level of tumor-bearing nude mice were normal,and there was no significant difference in body weight,indicating that ineupatolide treatment exhibit low toxicity and good safety.The growth and weight of the transplanted tumor were observed and measured.The volume and average weight of the transplanted tumor decreased significantly with the increase of the concentration(P<0.05),further HE staining results of transplanted tumor showed that chromatin in the nucleus and nucleic acid in the cytoplasm decreased significantly with the increase of concentration,presenting a dose-dependent relationship,suggesting that ineupatolide can inhibit the growth of prostate cancer cells and has anti-prostate cancer activity.Using cell biology,molecular biology,and biochemistry method further research on the biological mechanism of ineupatolide inhibiting prostate cancer cells.The transcription level of related genes was measured by q RT-PCR,and it was found that the gene expression level of PI3 K did not change significantly,while the gene expression level of Akt increased significantly(P<0.05).The gene expression level of cell cycle-related genes CDK1、CDK2 and CCNA2 were down-regulated(P<0.05),while the gene expression level of P53 was significantly increased(P<0.05).Meanwhile,the apoptosis-related genes Bcl-2,x IAP were significantly down-regulated,while Bax,PARP,Caspase-3 was significantly upregulated.Autophagy-related gene m TOR did not change significantly,and the expression level of P70-S6 K increased significantly(P<0.05).Further Western blot protein expression levels showed that PI3 K,Akt,m TOR,and S6 K protein phosphorylation levels were significantly down-regulated(P<0.05),indicating that cell growth-related pathways were inhibited;CDK1、CDK2,and CCNA2 protein expression levels were down-regulated(P<0.05),the protein expression level of P53 increased significantly(P<0.05);Bcl-2 and x IAP were significantly down-regulated,while cleaved-Caspase-3,cleaved-PARP and Bax were significantly up-regulated(P<0.05).These results suggested that ineupatolide may inhibit the growth of prostate cancer,promote apoptosis and autophagy through the PI3K/Akt signaling pathway.(5)Study on the mechanism of action of ineupatolide against prostate cancer in vitroTo further explore the anti-prostate cancer mechanism of ineupatolide,prostate cancer PC-3 and DU-145 cells were used to study.CCK-8 experiment showed that ineupatolide inhibiting the activity of prostate cancer cells.Ed U proliferation experiment results showed that ineupatolide can effectively inhibit the proliferation of PC-3 and DU-145 cells(P<0.05);flow cytometry analysis showed ineupatolide may block the PC-3 and DU-145 cell cycle in S or G2/M phase.Further detection of protein expression levels by Western blot showed that PI3 K,Akt protein phosphorylation levels and CDK1,CDK2,CNNA2 expression levels were significantly down-regulated(P<0.05),and P53 protein expression was significantly up-regulated.These results indicate that ineupatolide may block the cell cycle by inhibiting the PI3K/Akt/P53 signaling pathway;Hoechst staining and flow cytometry showed that PC-3 and DU-145 cells appeared apoptotic bodies and the apoptotic rate increased significantly.The protein expression level of cleaved-PARP and Bax increased significantly by Western blot(P<0.05),while PI3 K,Akt protein Phosphorylation and pro-caspase-3,x IAP and Bcl-2 protein expression levels were significantly down-regulated(P<0.05).These results indicate that ineupatolide may induce apoptosis by inhibiting PI3K/Akt/x IAP signaling pathway.Lyso Tracker results showed intracellular autophagolysosomes and autophagosomes increased significantly with the increase of ineupatolide concentration(P<0.05).Further Western blot results showed that the protein expression level of LC3-II/LC3-I increased significantly(P<0.05),while PI3 K,Akt,m TOR,PRAS40,P70-S6 K protein phosphorylation levels and SQSTM1/p62 protein expression levels were significantly down-regulated(P<0.05)with dose-dependent manners.In order to further study whether the inhibitory effect of ineupatolide was worked through PI3K/Akt sigling pathways,PC-3 cells with overexpressing of PI3 K was treated by ineupatolide,The Western blot results showed that PI3 K,Akt,m TOR protein phosphorylation levels and x IAP protein expression levels were significantly down-regulated(P<0.05),The expression level of p53 and cleaved-caspase-3 protein were significantly up-regulated(P<0.05),indicating that ineupatolide may regulate the growth and apoptosis of prostate cancer by inhibiting the PI3K/Akt signaling pathway.Through scratch and transwell cell invasion experiments,it was found that ineupatolide can significantly inhibit the migration and invasion of PC-3 and DU-145 cells(P<0.05).In summary,ineupatolide may block the prostate cancer cell cycle by inhibiting the PI3K/Akt signaling pathway,inducing prostate cancer cell apoptosis and autophagy,and inhibiting the migration and invasion of prostate cancer cells.(6)Study on the mechanism of action of incaspitolide A against prostate cancer in vitroIn order to further study the anti-prostate cancer activity and the mechanism of Carpesium cernuum L,incaspitolide A was studied in prostate cancer PC-3 and DU-145 cells by CCK-8 assay,Ed U proliferation test,flow cytometry cycle analysis,apoptosis experiments,Hoechst staining experiments,apoptotic protein chip experiments,lysotracker staining experiments,scratches and transwell experiments.It was showed that inaspitolide A may significantly inhibit both PC-3 and DU-145 cells growth and proliferation,cell cycle arrest,suppress the migration and invasion,as well as induction of cell apoptosis and autophagy.Using modern biological technology to do further research on the mechanism of inaspitolide A inhibiting prostate cancer cells.Western blot detection of protein expression levels showed that PI3 K,Akt,m TOR,PRAS40,P70-S6 K protein phosphorylation levels and protein CDK1,CDK2,CNNA2,SQSTM1/p62,pro-caspase-3,x IAP and Bcl-2 expression The level was significantly down-regulated(P<0.05),the ratio of protein LC3-II/LC3-I was significantly increased,and the expression levels of protein cleaved-PARP,p53,and Bax were significantly increased(P<0.05),all in a dose-dependent manner.These results indicated that ineupatolide may block the cell cycle in S or G2/M phase through PI3K/Akt/P53 signaling pathway,induce cell apoptosis through PI3K/Akt/x IAP signaling pathway,and induce autophagy through PI3K/Akt/m TOR signaling pathway.Further through PI3 K overexpression experiments,Western blot detection of protein expression levels showed that PI3 K,Akt,m TOR protein phosphorylation levels and x IAP protein expression levels were significantly down-regulated(P<0.05),protein cleaved-Caspase-3,p53 expression levels were significant increase(P<0.05).The above research indicated that incaspitolide A may inhibit the cell cycle,induce apoptosis and autophagy,and inhibit the migration and invasion of tumor cells through regultation of the PI3K/Akt signaling pathway in vitro.Taken together,the present study found that Carpesium cernuum L exhibited antiprostate cancer activity in vivo.11 compounds were isolated and identified from the active ethyl acetate extracts.Among them,ineupatoide(1)and incaspitolide A(9)exhibitd significant anti-prostate cancer activity.Through network pharmacology and transcriptome studies,it was found that the mechanism of ineupatoide and incaspitolide A against prostate cancer may be related to PI3K/Akt signaling pathway.In vivo experiments showed that ineupatoide can inhibit the growth of prostate cancer cells,block cell cycle,induce apoptosis and autophagy by inhibiting the PI3K/Akt signaling pathway.Further research showed that cell cycle could be blocked by inhibiting the PI3K/Akt/P53 signaling pathway,cell apoptosis could be induced by inhibiting the PI3K/Akt/x IAP signaling pathway,and autophagy could be induced by inhibiting PI3K/Akt/m TOR.Incaspitolide A,the analogues of ineupatoide was applied to the similar in vitro study and showed similar anti-cancer effect and mechamism with ineupatoide.The study provided potential candidate compounds for the treatment of prostate cancer,and surpported a theoretical basis for the resource development of Carpesium cernuum L... |
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