Effects Of Transforming Growth Factor-β Signaling In Development And Progress Of Prostate Carcinoma

Transforming growth factor-β is a big gene family, composing of plenty of structurally related polypeptide growth factor, including the three major category-prototype TGF-β , activin, and BMP (more than 40 members). The superfamily of TGF- β plays an important role in the proliferation and differentiation of cells, formation of basic matrix, apoptosis, and carcinogenesis. Both the type I receptor with serine/threonine activity and type II receptor mediate the signal transduction pathway of the TGF- β superfamily. The Smads family is a signaling molecule for TGF- β , transducing the signal from the cell membrance into the cell. When activated, Smads is phosphylated by specific receptor on the cell surface and form a heterotrimer with Smad4, translocating into nucleus and stimulating the transcription of target gene. Either abnormality or deletion of TGF- β expression in the signaling pathway or dysfuction of BMPs and Smads will have effects on the ultimate transcription of the cell, impacting the growth, development and carcinogenesis.Androgen receptor (AR) belongs to the nuclear receptor superfamily. AR mediates the signaling pathway of androgen by promoting the growth of prostate gland. But TGF- β inhibits the growth of normal epithelium of prostate and cancer cells. Androgen can impact the expression of TGF- βprotein and TGF-β receptor (TPR). So the two signal transduction pathway are closely related in the proliferation and apoptosis of prostate cells.Prostate cancer, being one of the most common malignancy in man, being second only to lung cancer in European and American countries. Like other cancers, the carcinogenesis of prostate cancer is a multiple step event, from precancer cell, in situ proliferation, to metastasis. Androgen and TGF-β signaling pathway play an important role in the carcinogenesis and development of prostate cancer. In this study, we investigated the expression of TGF- β , BMPs and Smads in the signaling pathway of TGF- β to explore its importance in the carcinogenesis, progression, metastasis and prognosis of prostate cancer.There are 3 parts in this experiment. 1. The expression of Smads in the normal prostate gland and prostate cancer and its relationship with staging, phasing and prognosis of prostate cancer were investigated by immunohistochemistry. 2. The variation in the expression of BMP-2 and BMP-4 in different phase, different Gleason stage was studied to reveal the difference of prostate cancer with bone metastasis and prostate cancer without bone metastasis by Western Blot. 3. The influence of BMP on the cell cycle of androgen dependent cell line of LNCap and androgen independent ell line of PC-3 was explored by Flow Cytometry. 4. The mRNA expression of AR and TGF- β in normal prostate gland and prostate cancer and its relationship with staging and phasing of the cancer were investigated by RT-PCR.MethodsMaterials1 .Main instruments: mierotome,slice-baker,pressure-cooker,couveus, Olympus microscope,microscopic photography instruments,homogenateapparatus;ultrasound comminuter,electrophoresis apparatus,rocking bed,high -speed and low-temperature centrifuger,automatic electrophoresis gel imaging analysis apparatusjow-temperature supercentrifuger,spectrophoto-meter.2.Main reagents:Goat Anti-human Smad3 and Smad4 pdyclonal antibody.ABC test kit;Recombinant human BMP2 and BMP4.Pre-staining marker alkaline phosphatase marked horse Anti-goat second antbody,acrylamide methylenehisaacrylamide,TEMED,trans-blot PVDF membrance;TRIZOL,RT-PCR kit,ethyl pyrophosphate,ayarose,ethidium bromide.Methods:1.Select 49 cases of prastates resected by open prostatectomy and TUR-P during 1997-2003,and follow up the patients for 72 months.All the specimens were fixed by formaldehyde solution,and embedded by parafFin.Take 5 normal prastate from total cystectomy as control specimens that were also fixed by formaldehyde solution.The embedded specimens were made into the section of 4 u m..Apply immunohistochemical ABC method to detect the expressions of Smad3 and Smad4 proteins.Smad3 and Smad4 appear brown in normal prostate epithelial cell.Take the intensity and mode of normal prostate epithelial cell as reference.The positive expression rate is 10% definied as (-);10%-25%a as(+);25%-50% as (++) and up 75% (+++).A poly X2 test of enumeration date to campare the differences of Smad3 and Smad4 in variant grade(Gleason score) and stage.The cure of surrival was drawn by kaplan-meier method2.The fresh prostate cacinoma sample from radical prostatectomy TUR-P and biopsy of prostate and normal prastate frome total cystectomy,were transferred to liquid ritrogenjar to reserve.There are 48 cases.Western blot method was used to detect the expression of BMP2 and BMP4 in prostate cancer and normal prostate.Methods:Homogenate disposal,proteinextraction,trans-blot,hybridezation and coloration,result collection with automatic electrophoresis gel imaginganalysis apparatus,Theresults were expressed by 0D +Standard deriation.The difference in BMP2 and BMP4 protein expression between normal prostate and carcinoma tissue and between different Gleason score and stage was compared by T test.3.Select the androgen-sensitive CaP cell line LNCaP and the androgen-independent CaP cell line PC-3,was used for cell proliferation studies and cell-cycle studies.Cell were treated with 0,20,50,100 and 250ng/ml BMP2 and 0,1,3,10 and 30ng/ml BMP4.Cell proliferation was evaluated by the Quick cell proliferation Assay.Statistical significance (P<0.05)was evaluated using a one-way ANOVA.Cell-cycle studies were through flow cytometric analysis.4.Fresh samples mentioned above would be used in RT-PCR detection.All the 34 fresh samples were selected .According to description extract RNA/TGF- P ,primer the upper steam 5'TACTCGACGC CTTTCCAGGAC-3',downs-Stream 5'TGGCCACCGACCTGCTATCC-3',AR premer.the upper stream 5'TCGGTCTCTA GTTTTCTACT,downstream 5'CATCTTTTGA ATCTCCCTT-3', P -actin primer:the upper stream 5'ATCATGTTTGAGACCTTCAA-3',downstream 5'CATCTCTTGCTCGAAGTCCA-3'perform reverse transcription to synthesis cDNA and PCR amplification.After reverse transcription 30 min in 48 °C and 2min in 94°C PCR amplification was performed directly.The circulation conditions were 30sec in 94°C,30sec in 55°C and lmin in 68 °C for 35 circulations.Take lOObpDNA Marker as reference for molecular weight,and observe the results under ultraviolet lamp after 2% agarose gel electrophoresis for amplification products.The optical density of amplificaton band was determined with automatic electrophoreses gel imaging analysis apparatus,and relative content would be obtained after TGF- P 1 and AR value were standardized by P -actin marker.Compare relative contents TGF- P 1 mRNA and AR mRNA between normal prostate tissue and prostate carcinoma and among the carcinoma in variant clinical stage and gleason grade.The results were showed by Xi S,and were compared by t test.The significant level was regarded as 95%.Resultsl.The positive expression rateof Smad4 in the tumors was 83.3%,and were 56.5%,42.8% and 25%,respectively in I . II and III grade;The positive expression of Smad4 were obvious among clinical stage in expression of Smad4.There are no obvious differences in theexpression of Smad3 protein with normal prostate tissue.2.Western bolt results of BMP2 and BMP4:The optical densities of the tumor in Gleason score lower 5,6-8,and higher were 7547.1 + 1964.12, 9657.4 + 2010.54, 12467.7 + 2496.75 and 5174.4+1400.54, 5940.3 + 1587.42, 6332.1 zb 1647.83 respectively.The optical densities of tumor in T1-T2 and T3-T4 stage were 8003.37 + 1889.23 , 12385.55 + 2506.72and5267.41 +1464.19, 6543.75+1668.46 respectivdy .There was obvious difference between Gleason score lower than 5 and higher than 9 (P<0.01) in expression of BMP2 proteimThere was obvious difference between T1-T2 and T3-T4 stage.3.Growth of the androgen-sensitive CaP cell line LNCaP was significantly inhibited (P<0.05)by BMP2 and BMP4.While PC-3 an androgen-independent CaP cell line was not affected.BMP2 treatment resulted accumulation of LNCaP in Gl phase of the cell cycle but BMP2 treatment didn'taffect the cell-cycle distribution of PC-3 cells4.The RT-PCR results:The relative content of TGF- ￡ 1 mRNA in normal prostate tissue : 0.74368 + 0.11. The relative content of TGF- 13 mRNA in prostate carcinoma:0.43721 + 0.08.There are significant differentce between two tissues.The relative contents of TGF- P 1 mRNA of CaP in T1-T2 and T3-T4 stage: 0.68865 + 0.10 and 0.43721 +0.08 o respectively.There are significant difference in T1-T2 and T3-T4,so as in Gleason score.The relative contents of AR mRNA in prostate tissue : 065497+0.11; The relative content of TGF- P mRNA in prostate carcinoma in T1-T2 and T3-T4 stage: 0.69972+0.12 and 0.81172+0.14; There are significant difference in T1-T2 and T3-T4(p<0.05).Conclusinsl.Smad4 protein is expressed lower in prostate cancer which is associated with clinical stage and Gleason score .The abnormal expression suggests a poor prognosis.There is no difference between expression of Smad3 in nomal prostate and CaP.2.There are growth inhibitory effects of BMP2 and BMP4 with various concentrations on LNCaP a androgen-sensitive prostate carcinoma cell lines.While there are no effects on PC-3 a androgen-indepenent prostate carcinoma cell lines.LNCaP cells were arrested in Gl phase of cell cycle with BMP2 treatment,but don't affect PC-3.3.The expressions of BMP2 and BMP4 protin were inctesed along with the elevation of clinical stage and Gleason score comparing normal prostate tissue specially in BMP2.4.The expression of BMP2 protein is signigicantly increased in bone metastasis of prostate carcinoma.Indicated a poor prognosis in this condition.5.The relative contents of TGF-13 1 mRNA were decresed along with clinical stage and Gleason score ,it is a negative regulation.The relative contents of AR mRNA were increased along with stage and Gleason score ,it belongs to a positive regulation.