| OBJECTIVE:Castration-resistant prostate cancer(CRPC)is an inevitable stage of prostate cancer(PCa)after Androgen deprivation therapy(ADT).Enzalutamide is an effective drug for the treatment of CRPC,but acquired Enzalutamide resistance is usually unavoidable within the short term in many patients.Lycopene,a safe and effective phytochemical,has been documented to have anticancer activity in a variety of tumors,especially for prostate cancer.The aim of this study was to provide data support for the combination of lycopene and enzalutamide in the treatment of CRPC and to identify novel treatment strategies for clinicians to delay the progression of CRPC patients.METHODS:(1)To investigate the influence of lycopene on CRPC cells,22RV1 and C4-2B cells would expose to different concentrations(5μM,10μM,15μM,and 20μM)and different time periods(12h,24 h,36h,48 h,and72h).Then cell viability was measured at different treatment time or different dosages of lycopene using CCK-8 assay.Then,we chose 15μM as the experimental concentration for present study.After the CRPC cells treated with lycopene were subjected to colony formation assay and Transwell assay.(2)To further explore the mechanism of lycopene affecting CRPC cells,lycopene related proteins were analyzed by protein-chemical database STITCH(Version 5.0,https://stitch.embl.de/),Gene Ontology(GO)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were performed for the selected genes by R software(3.6.3),western blotting(WB)was used to detect the levels of AKT related proteins and the proteins associated with proliferation(PCNA)and invasion(MMP-2 、 MMP-9)after treated with lycopene.In addition,CRPC cells were treated with lycopene and AKT agonist,and then NC group,lycopene group,SC79 group,and lycopene +SC79 group were assigned.The cells proliferation were detected by colony formation assay and EDU,cells invasion ability was analyzed by transwell assay,and the protein expression levels of AKT,p-AKT(Ser473),EZH2,p-EZH2(Ser21),AR and other proteins(PCNA,MMP-2 and MMP-9)were examined by western blotting.(3)A total of 50 PPC tissue samples and 50 CRPC tissue samples were collected from the patients were underwent needle biopsy of the prostate or performed radical excision of prostate cancer at Department of Urology of the First Affiliated Hospital of Chongqing Medical University from January2017 to April 2021.The proteins expression of p-AKT(Ser473)and its downstream factors p-EZH2(Ser21)and AR in the tumor tissues was analyzed using immunohistochemistry(IHC),and its correlation between demographic and clinical characteristics was also be assessed.After the Chi-square test was used to compare the p-AKT-positive and p-EZH2-positive ratio between the PPC group and CRPC group.Meanwhile,the progression-free survival(PFS)was compared between ‘p-AKT-positive’versus ‘p-AKT-negative’ and ‘p-EZH2-positive’ versus ‘p-EZH2-negative in CRPC patients was estimated with the Kaplan Meier method.(4)In order to explore the effects and mechanisms of lycopene combined with enzalutamide on CRPC,we performed experiments in vitro and in vivo.The CCK-8 assay was applied to measure cell proliferative ability after combination treatment of lycopene and enzalutamide in 22RV1 and C4-2B cells.For in vivo studies,22RV1 cells were selected to establish subcutaneous xenograft tumor model and bone metastasis model in nude mice.Nude mice about 6 weeks old were divided into four groups(NC group,lycopene group,enzalutamide group and lycopene + enzalutamide group).Then bone metastasis was examined by X-ray imaging weekly and xenograft tumors were checked every week.Lastly,the expression of related proteins in four group of tumor tissues were detected by IHC,and the bone metastasis were determined by H&E staining.RESULTS:(1)To investigate the effect of lycopene on biological function of22RV1 and C4-2B cell lines,CCK-8,colony formation assay and transwell assay were applied.The results showed that the viability of lycopene group was significantly decreased,but interestingly,there was no further increase in inhibition of cell viability when lycopene concentration increased to20μM compared with 15μM in both 22RV1 and C4-2B cells.Therefore,we chose 15μM as the experimental concentration for present study.Moreover,according to the colony formation assay,lycopene showed markedly reduced colony number and size of 22RV1 and C4-2B cells.Moreover,transwell assay indicate that lycopene could inhibit the invasive ability of22RV1 and C4-2B cells.Our results showed that lycopene could suppress the proliferation and invasive ability of CRPC cells.(2)There were 20 target proteins of lycopene identified by STITCH database(minimum interaction score 0.7),they are: XRN1,RHOA,RB1,PTEN,PPARG,NOX4,NME1,MMP9,MMP7,MAPK8,KLF2,IGF1,HMGB1,GJA1,EIF2AK3,EFNA5,CASP3,BCO2,AKT1,ABCA1.In addition,the GO and KEGG pathway analysis of the selected genes found that lycopene was closely related to Ras signaling pathway,prostate cancer and PI3K-AKT signaling pathway.WB results confirmed that the protein levels of p-AKT(Ser473),p-EZH2(Ser21)were significantly reduced compared with the control group,but did not significantly change the total levels of AKT and EZH2.Moreover,lycopene downregulated protein level of AR,MMP-2,MMP-9,and PCNA in two CRPC cells.This result indicated that AKT/EZH2/AR maybe a critical signaling pathway for the inhibitory effect of lycopene on the proliferation and invasion of CRPC cells.Then,we treated 22RV1 and C4-2B cells with AKT phosphorylation agonist SC79 and lycopene in combination or alone.WB results showed that SC79 increased the level of p-AKT and related signal molecules downstream of AKT,such as p-EZH2,AR,MMP-2,MMP-9 and PCNA,after lycopene treatment of 22RV1 and C4-2B cells.Colony formation experiment and EDU assay suggested that lycopene and SC79 co-treatment could partially abolish the affection of lycopene on 22RV1 and C4-2B cells proliferation.Moreover,transwell assay results showed that invasive ability of 22RV1 and C4-2B cells was increase in the lycopene and SC79 co-treated group than lycopene group.(3)After specimen types were determined by H&E staining,PPC and CRPC tissue samples were analyzed by immunohistochemistry.The results showed that the expression of p-AKT,p-EZH2 and AR was increased in both PPC and CRPC tissues,but the positive rate of p-AKT,p-EZH2 was higher in CRPC than PPC tissues.In addition,the expression trend of AR was consistent with p-AKT and p-EZH2.The expression of p-AKT and pEZH2 among various clinical parameters in PPC and CRPC was positively correlated with bone metastasis,but was positively correlated with Gleason score of tumors only in CRPC.Kaplan-Meier survival analysis showed that the progression-free survival(PFS)was 16 months for patients with pAKT-positive CRPC [95% confidence interval(CI)=13-19 months]compared with p-AKT-negative CRPC 28 months(95%CI=21-35 months).In addition,PFS of p-EZH2-positive CRPC patients was 18 months(95%CI=15-21 months),while PSF of p-EZH2-negative CRPC patients was 27 months(95%CI=17-33 months).This suggests that p-AKT-positive and p-EZH2-positive tumor are associated with shorter PFS.Taken together,these data suggests that the high expression of p-AKT and p-EZH2 may connected with poor prognosis of CRPC patients.(4)The CCK-8 was applied and the results showed that the combination of lycopene and enzalutamide significantly inhibited the growth of 22RV1 and C4-2B cells more than those treated with lycopene or enzalutamide alone.In addition,WB results showed that lycopene plus enzalutamide could inhibit the expression of p-AKT,p-EZH2,AR and PCNA more than those treated with lycopene or enzalutamide alone.Minor treatment effects were observed on total protein expression levels of AKT and EZH2.The results demonstrated that combination of lycopene and enzalutamide has an enhanced effect on the proliferation of CRPC cells which may be related to the AKT/EZH2/AR signaling pathway.In vivo,enzalutamide plus lycopene reduced tumor growth and weight more strongly than enzalutamide alone.IHC was applied and the results revealed that the expression of p-AKT,p-EZH2 and Ki-67 show more less in cotreated with lycopene and enzalutamide group than others.Base on X-ray images and H&E staining,we noticed that lycopene or enzalutamide treated alone slightly prevented 22RV1 cells bone metastasis,while a strong inhibition of bone metastasis was achieved by co-treated with lycopene and enzalutamide.CONCLUSION:In summary,our study demonstrated the anticancer effect of lycopene on CRPC,and it was confirmed for the first time in vivo and in vitro that lycopene could increase the anti-CRPC effect of enzalutamide.In-depth mechanistic studies suggest that lycopene may exert its effect by inhibiting AKT/EZH2/AR signaling pathway.As such,our findings may provide new therapeutic strategies for delaying the progression of CRPC patients.However,the reliability and usefulness of these strategies need to be further evaluated in clinical trials. |
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