| Objective Prostate cancer is one of the most common malignant tumors in clinical men,and it ranks fifth in cancer deaths.In recent years,with the improvement of quality of life and medical level in my country,the incidence of prostate cancer has also increased year by year.Many clinically advanced prostate cancer patients develop androgen resistance after androgen deprivation therapy,that is,castration-resistant state,and eventually progress to castration-resistant prostate cancer.The metastatic probability of prostate cancer patients is about 28.7%,and the prognosis of patients with metastatic prostate cancer is poor,with a median survival rate of only 13 years.At present,invasion and metastatic growth are still the main causes of death in patients with prostate cancer.Therefore,it is of great significance to find new intervention targets and treatment methods,and to provide new ways for the treatment of prostate cancer.Zeylenone is a polyoxy-substituted cyclohexenone compound.As a pure natural plant extract,Zeylenone is isolated from the ethanolic extract of the leaves of the sansho cotyledon of the genus Ansopaceae.It exhibits good activity in anti-tumor,immunosuppression,insecticidal,anti-protozoal,anti-microbial and anthelmintic aspects,especially in anti-tumor,with a unique mechanism of action.Our research idea is to use Zeylenone to inhibit androgen-independent prostate cancer cells PC3 and DU 145,in addition to detecting the expression and transcription of key target protein genes in each group of Wnt/β-catenin signaling pathway and PI3K/Akt signaling pathway In addition,focus on detecting the expression and transcription of the GSK-3β protein gene,a possible cross-talk common target in the two signaling pathways,and whether there is a synergistic effect on the expression and transcription of the related protein genes of the EMT mechanism in the nucleus..In this way,we can evaluate the value of the combined effect of existing inhibitors,with a view to discovering and researching new inhibitors for these two pathways,which will provide valuable potential therapeutic drugs or lead compounds for clinical practice,and also provide a basis for revealing these two signals.The regulatory mechanism of the pathway provides a good research tool.Chapter 1 Inhibitory effect of Zeylenone on normal prostate epithelial cells RWPE1,androgen-independent prostate cancer cells PC3 and DU145Objective To investigate the inhibitory effect of Zeylenone on normal prostate epithelial cells RWPE1,androgen-independent prostate cancer cells PC3 and DU 145,and preliminarily explore the in vitro effects of Zeylenone on prostate cancer cells.Methods Prostate cancer cells PC3 and DU 145 and normal prostate epithelial cell RWPE1 were selected and cultured in RPMI1640 and DMEM medium,respectively.The effects of Zeylenone on the survival rate of prostate cancer cells,the effect of DAPI staining on the nuclei of prostate cancer cells,and the clonogenicity assay were detected.Effects on colony formation ability of prostate cancer cells,cell cycle detection by flow cytometry,cell scratch test,Transwell test.Results ①The growth of PC3 and DU 145 cells treated with different concentrations of Zeylenone for 12,24,48 and 72 h was detected by MTT assay.The inhibitory effect of Zeylenone on PC3 and DU 145 cell growth showed a good timeand dose-dependent relationship.At 12,24,48 and 72h,the IC50 values of Zeylenone on PC3 cells were(5.89±0.24)M,(4.32±0.30)M,(2.02±0.15)M,(1.38±0.19)M;inhibition on DU145 cells The IC50 values of(5.02±0.20)M,(4.19±0.27)M,(1.58±0.17)M,(1.05±0.14)M.When Zeylenone treated PC3 and DU145 cells for 24h,the apoptosis caused by it was more significant,so 24h was used as the drug treatment time in the follow-up study,and the concentration of zeylenone on PC3 cells was 2.5g/ml,5g/ml and 10g/ml.The concentration of action on DU 145 cells is 1.25g/ml,2.5g/ml and 5g/ml.②PC3 cells were treated with Zeylenone and then stained with DAPI.It can be seen that the cells undergo nuclear disintegration,nuclear fragmentation,and chromatin pyknosis after drug treatment,and the above conditions are more significant with the increase of Zeylenone content.It is most obvious when the content is 10g/ml,which shows a good dose-dependent relationship.After treatment with Zeylenone,DU145 cells were stained with DAPI,and it could be seen that the cells had nuclear disintegration,nuclear fragmentation and chromatin pyknosis after drug treatment,and the above conditions were more significant with the increase of Zeylenone content.It is most obvious when the content is 5g/ml,which shows a good dose-dependent relationship.③ Zeylenone showed a strong inhibitory effect on PC3 cells colony formation,and the inhibitory effect was gradually strengthened with the increase of Zeylenone content.is 57.5%.Zeylenone showed a strong inhibitory effect on colony formation on DU 145 cells,and the inhibitory effect was gradually strengthened with the increase of Zeylenone content.is 60.5%.④ After PC3 cells were treated with 2.5g/ml,5g/ml and 10g/ml of Zeylenone for 12h,24h and 48h,respectively,compared with the control group,the proportion of cells in G0/G1 phase increased significantly;DU 145 cells were treated with 1.25g/ml Zeylenone.,2.5g/ml and 5g/ml Zeylenone were treated for 12h,24h and 48h,respectively,compared with the control group,the proportion of S phase cells was significantly increased,and the effect was strengthened with the increase of drug concentration and time..⑤ After PC3 cells were treated with 2.5g/ml,5g/ml and 10g/ml of Zeylenone for 24h,the cell invasion rates were(62.34±5.11)%,(30.65±3.82)%,(26.94±2.43)%,all lower than(100.00±0.00)%of the control group(P<0.05);after DU 145 cells were treated with Zeylenone of 1.25g/ml,2.5g/ml and 5g/ml for 24h,the cell migration rates were(82.64)±5.30)%,(42.88±4.19)%,(30.10±4.25)%,which were all lower than(100.00±0.00)%of the control group(P<0.05).Conclusion Zeylenone can significantly inhibit the migration and invasion of prostate cancer cells,change the cell cycle changes,and promote cell apoptosis.Chapter 2 Effects of Zeylenone on the expression of related signaling pathways in prostate cancer PC3 and DU145 cellsObjective To investigate the effect of Zeylenone on Wnt/β-catenin signaling pathway,PI3K/AKT/mTOR signaling pathway and GSK-3β in prostate cancer PC3 and DU 145 cells,and further explore the mechanism of Zeylenone on prostate cancer.Methods Prostate cancer cells PC3 and DU 145 were cultured in RPMI1640 and DMEM medium,the protein expression level was detected by Western blot,the mRNA level was detected by real-time quantitative PCR,and Wnt5a overexpression plasmid was constructed.Results ① After PC3 cells were treated with 2.5g/ml,5g/ml and 10g/ml of Zeylenone for 24h,the Wnt/β-catenin signaling pathway wnt5a protein,β-catenin protein and cyclin protein were significantly decreased compared with the control group,and with the effect The expression of related proteins gradually decreased with the increase of drug dose(P<0.05).Compared with the control group,the wnt5a protein,β-catenin protein and cyclin protein of the signaling pathway were significantly decreased,and the expression of related proteins gradually decreased with the increase of the dose of the acting drug(P<0.05).② After PC3 cells were treated with 2.5g/ml,5g/ml and 10g/ml of Zeylenone for 24h,PI3K/AKT signaling pathway PI3K protein,AKT protein,mTOR protein,EMT-related proteins E-cadherin protein,N-cadherin protein,Vimentin protein Compared with the control group,the α-SMA protein and GSK-3β protein were significantly decreased,and the expression of related proteins gradually decreased with the increase of the dose of the acting drug(P<0.05).PI3K/AKT signaling pathway PI3K protein,AKT protein,mTOR protein,EMT-related proteins E-cadherin protein,N-cadherin protein,Vimentin protein,α-SMA protein after 24h treatment with ml,2.5g/ml and 5g/ml Zeylenone Compared with the control group,the GSK-3βprotein was significantly lower,and the related protein expression gradually decreased with the increase of the acting drug dose(P<0.05).③In PC3 cells,the expression level of Wnt5a in the blank group was(1.01 ±0.04),the expression level of Wnt5a in the blank plasmid control group was(1.03±0.05),and the expression level of Wnt5a in the Wnt5a overexpression group was(1.68±0.23)(P<0.05);DU145 cells The expression level of Wnt5a in the blank group was(1.03±0.07),the expression level of Wnt5a in the blank plasmid control group was(1.04±0.08),and the expression level of Wnt5a in the Wnt5a overexpression group was(1.72±0.25)(P<0.05).④In PC3 and DU 145 cells,the expressions of Wnt,N-cadherin,Vimentin,α-SMA,β-catenin,p-GSK3β,p-mTOR,p-PI3K,p-AKT protein and mRNA in Zeylenone treatment group were lower than those in blank control group.The expressions of GSK3β,E-cadherin protein and mRNA were higher than those in the blank control group(P<0.05);the expressions of Wnt,N-cadherin,Vimentin,α-SMA,β-catenin,p-GSK3β protein and mRNA in the Wnt5a overexpression group were higher than those of the blank control group In the control group,the expressions of E-cadherin,p-AKT protein and mRNA were lower than those in the blank control group(P<0.05);Wnt5a overexpression+Zeylenone treatment group Wnt,N-cadherin,Vimentin,α-SMA,β-catenin,p-The expressions of mTOR,p-PI3K,p-AKT protein and mRAN were lower than those in the blank control group(P<0.05).Conclusion PI3K/Akt pathway,Wnt pathway and common factor GSK-3β may have a synergistic effect in the mechanism of intranuclear EMT of prostate cancer cells,and Zeylenone inhibits the proliferation and migration of prostate cancer cells by inhibiting the expression of related factors of the above pathways.Chapter 3 In vivo mechanism of action of Zeylenone on prostate cancer mouse modelObjective In this chapter,the mouse tumorigenesis model was further adopted on the basis of the above research,and the in vivo mechanism of Zeylenone on prostate cancer mouse model was verified in vivo.Methods Thirty female BALB/c nude mice aged 4-6 weeks and weighing 16-20 g were selected,12 hours white and 12 hours black,temperature 25±1℃,humidity 45-55%,free food and water.Adaptive feeding for one week.A nude mouse tumorigenic model was constructed,the tumor volume and tumor mass were detected,HE staining,and Western blot were used to detect the expression of AKT/GSK-3β/β-catenin signaling pathway and EMT key target proteins in mouse tumorigenic tissues.Results ①From the 8th day,the tumor volume in the experimental group was lower than that in the control group in nude mice inoculated with PC3 and DU145(P<0.05).②In the tumor tissue of the nude mice inoculated with PC,the tumor mass of the experimental group was(0.35±0.06)g,which was lower than that of the control group(1.42±0.16)g;in the tumor tissue of the nude mice inoculated with DU 145,the tumor mass of the experimental group was(0.39±0.08)g,lower than the control group(0.92±0.14)g(P<0.05).③The tumor tissue was pathologically analyzed by HE staining.The experimental results are shown in Figure 14.In the PC3 and DU145 cells of the blank group and the control group,the tissue cells were normal and neatly arranged,the staining distribution was uniform,and the size of the nuclei was uniform.In the group,the tissue cells showed extensive necrosis,the nuclei were broken,and some nuclei appeared lysed.④Compared with the control group,the expressions of PI3K protein,AKT protein,GSK-3β protein,β-catenin protein,E-cadherin protein,N-cadherin protein,Vimentin protein and α-SMA protein in the tumor tissue of the experimental group were significantly decreased(P<0.05).Conclusion Zeylenone can inhibit tumor mass in mouse tumorigenic models of prostate cancer,and reduce the expression of AKT/GSK-3β/β-catenin signaling pathway and EMT key target proteins in tumorigenic tissues.Conclusion ① The Wnt/β-catenin signaling pathway and PI3K/Akt signaling pathway may affect the development of prostate cancer through the tandem common target GSK-3β protein,and the three synergistically affect the EMT mechanism in the nucleus.②Zeylenone can inhibit the proliferation,migration and invasion of prostate cancer cells and other biological behaviors,and its mechanism of action may be related to the inhibition of Wnt/β-catenin signaling pathway,PI3K/Akt signaling pathway and GSK-3β to inhibit prostate cancer cell EMT. |
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