Study On The Molecular Mechanism Of Matijin-su Derivatives For Regulating Inflammatory Cytokines In Prostate Cancer Cells

Objective:To study the anti-prostate cancer activity of the Matijin-Su（MTS）derivatives and the molecular mechanism of their regulation of inflammatory cytokines in prostate cancer cells.Methods:MTT assay was used to screen MTS derivatives against PC3 cells of prostate cancer.In vivo experiment:Nude mice exograft experiment was carried out to investigate the anti-proliferative activity of active MTS derivatives.In vitro experiments:Hoechst 33258 staining was used to detect the effects of compounds on DNA replication of cancer cells.The effects of the compounds on PC3 apoptosis and PC3 cycle of prostate cancer were detected by flow cytometry.Molecular mechanism:Combining the results of inflammation-related factors and tumor-related factors in differential proteins by proteomics sequencing,the proteomics results were verified by PCR and q PCR experiments from m RNA level.Western Blot assay was used to verify the proteomic results and detect the expression of five NF-κB family proteins.Cellular thermal transfer assay（CETSA）was used to explore the targeting protein of MTS derivatives binding to prostate cancer.Molecular docking technology simulates the binding sites between MTS derivatives and targeted proteins and visualizes the results.si RNA gene silencing assay binding protein immunoblotting assay was used to detect the silencing protein of MTS derivatives,and gene silencing cell lines were obtained.EDU staining was used to detect the sensitivity of silenced prostate cancer cells to compound 7c.Results:The results of MTT assay showed that compound 7c had the best inhibitory effect on PC3 of prostate cancer cells at the concentration of 5mmol/L,and the inhibitory rate reached about 85%.The results of in vivo experiments showed that compound 7c could inhibit the proliferation of tumor in nude mice,with statistically significant difference;The results of in vitro experiments showed that the IC₅₀value of compound 7c was 3.63±0.43mmol/L after 12 h treatment,1.57±0.28mmol/L after 24 h treatment,and 1.38±0.18mmol/L after 48 h treatment.Hoechst 33258 staining showed that compound 7c could induce apoptosis of PC3 cells by affecting chromatin and DNA replication;The results of flow cytometry showed that compound 7c inhibited apoptosis of prostate cancer cells in a concentration-dependent manner.However,it does not affect the occurrence and development of PC3 cell cycle in prostate cancer;Molecular mechanism:Proteomic sequencing results showed that compound 7c acted on prostate cancer cells.Under the condition of differential ratio of 1.3,446 differential proteins were regulated,among which 150 differential proteins were up-regulated and 296 were down-regulated.Under the condition of differential multiple of 2.0,20 differential proteins were regulated,all 20 differential proteins were up-regulated,and no proteins were downregulated.Molecular Function,biological process,cellular component and bioinformatics analysis results;Twelve differential proteins were selected:LAMB3,OMR,CYR61,STC1,OLR1,SDC4,IL6,PLAU,JUN,TNFRSF12A,CXCL8,TNFSF9.They belong to inflammation-related factors,transcription-related factors and apoptosis-related factors respectively,and these differential proteins mainly regulate inflammation-related and apoptosis-related pathways.The results of phosphorylated proteomics sequencing indicated that compound 7c May affect the adhesion function of PC3 cells and inhibit the proliferation of prostate cancer.PCR and RT-q PCR results showed that LAMB3,OMR,CYR61,STC1,OLR1,SDC4,IL6,PLAU,JUN,TNFRSF12A,CXCL8 and TNFSF9 were upregulated on m RNA level after compound 7c treatment of prostate cancer cells,which was consistent with the protein sequencing results;Western Blot results showed that LAMB3,OMR,CYR61,STC1,OLR1,SDC4,IL6,PLAU,JUN,TNFRSF12A,CXCL8 and TNFSF9 were upregulated in prostate cancer cells after compound 7c treatment,which was consistent with the protein sequencing results.At the protein level,compound 7c did not affect the expression of Rel B,C-Rel,P50,P52 and their phosphorylation in the NF-κB family of prostate cancer cells,but only affected the expression of P65phosphorylation;Cell thermal shift assay showed that compound 7c played a role by binding to Jun,CYR61 and TNFSF9 proteins.Molecular docking technology simulates the active sites of compound 7c binding Jun,Cy R61 and TNFSF9.The results of si RNA silencing assay showed that the protein expression of Jun,CYR61and TNFSF9 gene decreased compared with the blank group.EDU staining results showed that compound 7c could bind JUN,CYR61 and TNFSF9,and inhibit the proliferation of prostate cancer.Conclusions:1.Among all the Matijin-Su（MTS）derivatives,7a,7b and 7c showed strong inhibitory activity against PC3 cells,especially compound 7c.2.Compound 7c can inhibit the proliferation of prostate cancer cells in vivo and in vitro,inhibit the proliferation of prostate cancer cells by regulating the inflammatory and apoptotic pathways,and affect the expression of inflammatory factors（IL6,CXCL8,CYR61,PLAU,TNFRSF12A and OSMR）and tumor factors（JUN,TNFSF9,TNFRSF12A,CYR61,CXCL8,LAMB3,OSMR,STC1,OLR1 and SDC4）.3.Compound 7c induces apoptosis of prostate cancer cells by targeting Jun,CYR61 and TNFSF9.