Role Of ERβ On Proliferation And Apoptosis In Hormone Independent Prostate Cancer Cell Line PC-3 And PC-3M

Morbidity of prostate cancer(PCa) has been the first place of the malemalignant tumour in the USA, Canada and so on the the western countries.The fatality of it has been the second only of lung cancer. With the aging ofpopulation and the change of life style and food ingredient, the morbidity ofPCa has been increasing year by year in our country. Moreover, 72 percent ofthese patients are the middle or advanced cancer. From the August of 1999,our center had been assumed the responsibility of JICA project. And the firstcarried out the basic epidemiological study of PCa's early discovery , earlydiagnosis and prevention and cure. The discovery rate of prostate cancer was1.28% through team examine for 12,027 men over 50 years old until April2002. Among of them, 42% were middle stage or advanced stage cancer and18.8% were osseous metastasis and only 13.1% of them were accepted radicaloperation. It is thus clear that Prostate cancer are becoming one of theserious diseases gradually threatened our countries gerontal men. Therefore, ithas been introduced for us an imminent and urgently solved topic to study themechanism and therapeutic tool of PCa.The operation or anti-androgen therapy were traditional way to cure thePCa and made a good effected. As an chemistry medicine for anti-androgentherapy , Estrogen can indirect down regulate the level of androgen incirculation blood through the hypothalamus and induce the tumor cellsdegenerated. Nevertheless, part of the PCa patients aggravated afteranti-androgen therapy two to there years and even developed metastasis,transformed from hormone dependent to hormone independent prostatecancer. It shown that there were others hormone regulation pathwaypresented in prostate cancer cells. Simultaneously, Treating for the hormoneindependent cancer was yet becoming a new topic.Since 1996,the founding for the second estrogen receptor –ERβ,revoked many researchers attention again. ER-α and ER-? has expressed inmany tissues but they are very different in the tissular and organiz distributionand expressed level. ER-α mainly expressed in prostatic basal cells andstroma cells and no expressed in epithelial cells, but ER-β expressed both inthe stroma cells and in the epithelial cells. It's suggested that estrogen not onlyindirect regulate the androgen hormone in peripheral blood through thehypothalamus but also direct regulate the hormone in prostatic cells throughthe cyto-endocrine pathway.It was reported that no matter dependent or independent prostate cancer,the transcription of ER-β decreased compared with the normal prostatictissues. Lisa etc found that ER-β high expressed in normal prostatic tissuebut no expressed in over 75% of the prostate cancers, yet absenced obviouslyin the invasion prostate cancer tissues. It's found in clinical that the expressionof ER-? had been decreased with the increased of the Gleason grade. So, theexpressed absence of ER-β was intimate correlated with the genesis anddevelopment of prostate cancer, and might be a potential cancer inhibitor.The research of ER?-/-mouse found that the prostate glandularepithelium appeared visible hyperplasy. In the near future, one after anotherscholars reported that ER-? could inhibit the hyperplasy of ovary cancer cell,breast cancer cell, endometrial cancer cell and prostate cancer cell line DU145.It's suggested that ERβ could regulate the cancer cell's proliferation andapoptosis through the intercellular secretion pathway.1.Objective: To rearch the role of ERβon the nosogenesis in prostateceaner and provide new idea and experiment evidence for the PCa therapy.2.Method and results:2.1 The construction and identification of hERβ eukaryotic expression:2.1.1 To construct pcDNA3.1-hERβ and pEGFP-C1-hERβ eukaryoticexpressionplasmid by using gene recombination technique: To take human ERβ(hERβ)complete gene by RT-PCR and then clone it into the eukaryoticexpression plasmid pcDNA3.1 and pEGFP-C1 , identified the result byendonuclease digested and DNA auto sequencing.2.1.2 To construct pGCsilencerTM U6/Neo/GFP-siRNA-ERβ plasmid bySiRNA technique. we use DNA vector-based RNAi approach to producesiRNA in vivo .first, we design three pairs double stranded siRNAoligonucleotide against ERβ gene that contained a sense strand of 19nucleotide equences followed by a short spacer (TTCAAGAGA), the reversecomplement of the sense strand, and five thymidines as an RNA polymeraseIII transcriptional stop signal. oligos were annealed in the anneal buffer(100mM K-actate,30mM HEPES-KOH pH 7.4,and 2 mM Mg-acetate,Incubate the mixture at 900C for 3min and then at 370Cfor 1hr) and cloned intothe BamHI–BamHI site of pGCsilencerTM U6/Neo/GFPPsliencer 1.0-u6vector which can express hairpin siRNAs under the control of the u6promoter .2.2 The identification of hERβ transfected into the prostate cancer cell2.2.1 Observation of GFP: To transfected the pEGFP-C1-hERβ andpGCsilencerTM U6/Neo/GFP-siRNA-ERβ plasmid into the prostate cancer celland then observe the green fluorescent in these cells. It was provided that thesuccessful tronsfected that the strong expression of GFP in these cells .2.2.2 Identify in the protein level: Test the ERβ protein expression bywestern blotting and found that: ① Cell which transfected bypcDNA3.1-hERβ and pEGFP-C1-hERβ has appeared ERβ protein specialstrap in 56KD and the level of protein increased 260% and 30% comparedwith control;② Cell which transfected by pGCsilencerTM U6/Neo/GFP-siRNA-ERβ has't appeared any strap.2.2.3 Identify in the mRNA level: ① Cell which transfected bypcDNA3.1-hERβ and pEGFP-C1-hERβ has appeared special ERβ cDNA strapin 1600bp and the level of mRNA increased 260% and 30% compared withcontrol;② Cell which transfected by pGCsilencerTM U6/Neo/GFP-siRNA-ERβ has't appeared any strap.2.2.4 Immunocytochemistry test hERβ:It was shown that the ERβexpression increased obviously in pcDNA3.1-hERβ and pEGFP-C1-hERβtransfected cells compared with the control. But the cells transfected bypGCsilencerTM U6/Neo/GFP-siRNA-ERβ hasn't any ERβ expression.2.3 The effect of ERβ on cell proliferation in PC3 and PC-3M cell line.The cell number of transfected by pcDNA3.1-hERβ andpEGFP-C1-hERβ cellshas decreased obviously and transfected bypGCsilencerTM U6/Neo/ GFP-siRNA-ERβcells has no different comparedwith control.2.4 The effect of ERβ on cell apoptosis in PC3 and PC-3M cell line2.4.1 The index of apoptosis: ① The results of acridine orange shownthat cells which transfected by pcDNA3.1-hERβ and pEGFP-C1-hERβ hasappeared crocus apoptosis cell ,but cells transfected by pGCsilencerTMU6/Neo/GFP-siRNA-ERβ hasn't .② Test by FCAS found that cells whichtransfected by pcDNA3.1-hERβ and pEGFP-C1-hERβ has appeareddeuto-double peak ahead the G1 stage which named apoptosis peak, but cellstransfected by pGCsilencerTM U6/Neo/GFP-siRNA-ERβ hasn't.③The resultsof TUNEL shown that pGCsilencerTM U6/Neo/GFP-siRNA-ERβ group andcontol group has not the positive staining , but the deep buffy positive stainingwas found in cells which transfected by pcDNA3.1-hERβ andpEGFP-C1-hERβ.2.4.2 The effect of ERβ on genes related with proliferation and apoptosis:RT-PCR and Western blot analysis were used to detect the mRNA and proteinlevel, the results showed the pcDNA3.1-hERβ,pEGFP-C1-hERβ couldsignificantly increased the mRNA and protein level of ERβ expression. Andthe ERβ expression was inhibited by pGCsilencerTMU6/Neo/GFP-siRNA-ERβ. We detected the protein of Caspase-3,Caspase-9and cyclinD1 by western blot and the mRNA of cyclinD1,P21, Bcl-XL, AKt,Survivin,Caspase-3 and Caspase-9 by RT-PCR. The results shown that withthe increasing expression of ERβ the Caspase-3,Caspase-9 were increased andthe cyclinD1 was decreased in protein level, and in mRNA level, only the P21and Caspase-9 were increased , others were decreased.2.5 The effect of ERβ on cells invasion2.5.1 Matrigel invasion assay: The numbers of cell permeated matrigeland enter the under camerula were decreased in transfeced PC-3M cells withpcDNA3.1-hERβ and pEGFP-C1-hERβ plasmid and were increased intransfeced PC-3M cells with pGCsilencerTM U6/Neo/GFP-siRNA-ERβ.2.5.2 Zymogram analysis: Compare with the control, the enzyme activityof MMP2 and MMP9 were decreased in cells transfected withpcDNA3.1-hERβ and pEGFP-C1-hERβ plasmid and increased in cellstransfectd with pGCsilencerTM U6/Neo/GFP-siRNA-ERβ.2.5.3 RT-PCR analysis the mRNA of MMP9 expression: Compare withthe control, the mRNA expression of MMP9 was decreased in cells transfectedwith pcDNA3.1-hERβ and pEGFP-C1-hERβ plasmid and increased in cellstransfectd with pGCsilencerTM U6/Neo/GFP-siRNA-ERβ.3.Conclusion:(1) The construction of pCDNA3.1-hERβ , pEGFP-C1-hERβ andpGCsilencerTM U6/Neo/GFP-siRNA-ERβ were successful detected byendonuclease digested and DNA sequencing.(2)Transfected the pCDNA3.1-hERβ and pEGFP-C1-hERβ to prostatecancer cell line PC-3 and PC-3M to creat the ERβ high expression cyto-model,and transfected pGCsilencerTM U6/Neo/GFP-siRNA-ERβ to creat the ERβsilence model.(3)Growth inhibiting essay proved that pCDNA3.1-hERβ andpEGFP-C1-hERβ could inhibit the proliferation of PC-3 and PC-3M cell, butpGCsilencerTM U6/Neo/GFP-siRNA-ERβ couldn't. It suggested that ERβ caninhibit the grown of prostate cancer cell obviously .(4)We proved that ERβ could target the cell apoptosis of PC-3 andPC-3M cell.(5)The mechanism of apoptosis induced by ERβ was related with thesuppress of cyclinD1 and down regulate of the Bcl-XL , AKt and Survivin andthe expression regulate of P21 and caspase3 , caspase9.(6)Matrigel invasion assay and Zymogram analysis suggested that ERβcould decrease the invasion of prostate cancer cell and ERβ silence couldstrengthen the invasion of cancer cell.