| ObjectiveProstate cancer(PCa)represents the most commonly diagnosed malignancy in men and the fifth cause of male cancer death worldwide.Currently,androgen-depletion therapy(ADT)is one of the major clinical treatments for prostate cancer.Although the achievement of this treatment is significantly effective,almost all PCa patients will inevitably progress to recurrent castration-resistant prostate cancer(CRPC).China is one of the main countries that applied marine natural products,and the application of marine drugs to control diseases has been explored for a long time.Shennong’s Classic of Materia Medica,Compendium of Materia Medica(BencaoGangmu),Compendium of Materia Medica(Bencao Gangmu Shiyi)have collected more than 100 kinds of traditional Chinese medicine derived from marine natural products.Consequently,this study focused on the discovery of bioactive natural products from marine natural products,and further investigate the potential mechanism.In order to further provide candidate new lead compounds for clinical drug development of CRPC.Improve therapeutic effect in the treatment of PCa and for the treatment of resistant cancers offer new therapeutic strategies.MethodsPart 11.Ilicicolin A was screened from the Marine natural product library by cell viability assay(CCK-8 method).We performed CCK-8 and cell counting to validate the anti-tumor effects of human prostate cancer cell lines C4-2B and 22Rv1.2.Western blot were used to determine the effects of Ilicicolin A on expression of apoptotic proteins of human prostate cancer cell lines C4-2B and 22Rv1.3.Caspase3/7 activity assay kit was used to detect the activity of Ilicicolin A against human prostate cancer cell lines C4-2B and 22Rv1 Caspase3/7 expression.4.Colony formation were performed to verify the anti-tumor effect of Ilicicolin A in prostate cancer cells.Part 21.RNA-sequencing(RNA-Seq)and GSEA gene enrichment were performed followed by transcript annotation.2.The gene expression and relationship between the expression level and prognosis of EZH2 were further analyzed using GEPIA and TCGA database.For downstream analyses,the enrichment in cell cycle downstream region toward the enrichment in the EZH2.3.RT-PCR analysis were used to delineate the enrichment gene AURKA,PLK1 and cell cycle related genes including CCNB2,FoxM1.At the same time,western blot were used to delineate the expression of AURKA,PLK1 and cell cycle related proteins expression including CCNB2,FoxM1.4.RT-PCR analysis were used to delineate AR targets gene KLK2、KLK3 and western blot were used to verify cell proliferation and survival related proteins expression including AR,c-Myc,cyclinD1.Part 31.Molecular docking analysis were performed to to investigate the interaction of Ilicicolin A with EZH2 protein.2.Explore Ilicicolin A whether inhibit of EZH2 by enzyme assays;The intervention pathway of Ilicicolin A on EZH2 was verified by proteasome inhibitor response assay.3.ChIP analysis were used to delineate the mechanism of EZH2 regulating cell cycle genes including AURKA and PLK1 by Ilicicolin A;ChIP analysis were used to verify the mechanism of H3K27me3 regulating cell cycle genes including AURKA and PLK1 by Ilicicolin A;And ChIP assay detect EZH2 regulate AR gene by Ilicicolin A.4.Using siRNA to knock down EZH2 in prostate cancer cells,the effects of knocking down EZH2 in prostate cancer cells with Ilicicolin A were examined by cell proliferation.Part 41.The effects of Ilicicolin A on C4-2B/ENZR and 22Rv1 cells were examined by cell viability assay(Cell-Titer Glo methods).2.Cell counting,colony formation were performed to verify the anti-tumor effect of Ilicicolin A on C4-2B/ENZR and 22Rv1 cells.And further investigate the anti-tumor effects of Ilicicolin A combined with ENZ in C4-2B/ENZR cells and 22Rv1 cells.3.Western blot were used to detect apoptotic related proteins expression including Cleave-PARP-1 and Cleave-Caspase-7 to verify apoptosis effects of Ilicicolin A combined with ENZ in C4-2B/ENZR cells and 22Rv1 cells.And further investigate AR-variant expression.Part 51.We established 22Rv1 cell xenograft model to investigate the anti-tumor effects of Ilicicolin A combined with enzalutamide in vivo.2.Western blot were used to verify the mechanism in vivo.Results1.Ilicicolin A possessed a potent anti-tumor effects and induced apoptosis effects in prostate cancer cells.2.Analysis of RNA-seq data showed that differential genes were enriched in the downstream of cell cycle pathway and EZH2 targets.3.Western Blot and qPCR analysis showed that Ilicicolin A decreased the common genes AURKA and PLK1 expression of cell cycle pathway and downstream of EZH2,and CCNB2 and FoxM1 were also significantly inhibited in a dose-dependent manner at mRNA level and protein level.4.Western Blot and RT-QPCR analysis showed that the expression of AR and its downstream KLK2 and KLK3 were significantly inhibited by Ilicicolin A in a dosedependent manner.AR protein expression and the expression of c-Myc and cyclinDl related to proliferation and survival were inhibited at the same time.5.Compared with adjacent normal samples,EZH2 were significantly upregulated in prostate cancer,and their expression level significantly increased in the late clinical stage of prostate cancer.The high expression of EZH2 was closely associated with the poor prognosis in prostate cancer patients.6.Compared with positive compound EPZ6438,Ilicicolin A showed weak inhibition of histone methylation transferase EZH2 activity by enzyme activity assay.Ilicicolin A degrades EZH2 protein through the protein degradation pathway.7.Molecular docking results showed that Ilicicolin A show affinity to EZH2 binding site.8.In the mechanism study,we found that on the one hand EZH2 directly regulate the gene expression of AURKA and PLK1,which ChIP-qPCR analysis show the binding effects could break by Ilicicolin A,the same to H3K27me3.And EZH2 directly regulate the gene expression of AR,Moreover,ChIP-qPCR analysis showed that the binding ability of EZH2 protein to the promoter region of AR gene was significantly reduced treated with Ilicicolin A.9.knockdown of EZH2,significantly inhibited the proliferation of prostate cancer cells,and combining with Ilicicolin A the effect on the growth of prostate cancer cells was not significantly changed.10.Ilicicolin A combined with enzalutamide significantly inhibited the growth of 22Rv1 and C4-2B/ENZR cells.What’s more,the inhibition effect of AR-variant and the upregulate expression of Cleave-PARP-1 and Cleave-Caspase-7 in the combination group were significantly better than those it the two drugs treated alone group.11.Ilicicolin A combined with enzalutamide significantly inhibited the growth of tumors in tumor-bearing mice;Compared with the control group,the expression of EZH2,AURKA and PLK1 protein in the tumor tissue of each drug treatment group was significantly less,and the combination group was the most obvious.Conclusion1.Marine natural product Ilicicolin A showed the inhibition of CRPC cell viability.2.Ilicicolin A,which possessed potent anti-tumor effects through EZH2 targets and cell cycle,thus providing a new candidate compound for further clinical drug development.3.Ilicicolin A exerts anti-tumor effect through AR pathway.4.Ilicicolin A plays an anti-tumor effect by participating in the cycle inhibition of prostate cancer cells mediated by EZH2.5.In vivo experiments,Ilicicolin A combined with enzalutamide significantly inhibited the growth of tumor-bearing tumors.And the expression of genes in vitro was also obtained in mouse tumor tissues. |
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