Neuroendocrine Differentiation In Prostate Cancer And The Relationship With Chemotherapy Resistance

Present study is planning to evaluate expression of NSE,P-gp,MRP1 and Bcl-2 in the surgical samples of prostate cancer and BPH.EGF and TGF-αwould be added to the medium of androgen-independent prostate cancer cell line DU145.NED would be confirmed by light microscope, electron microscope and examination of NSE protein and mRNA. Whether EGF and TGF-αcould induce NED and whether NED could mediate chemoresistance of prostate cancer would be investigated.Part one Expression of NSE,P-gp,MRP1 and Bcl-2 in prostate cancer and BPHAim:To investigate expression of NSE,P-gp,MRP1 and Bcl-2 in prostate cancer and BPH,and relationship between expressions and prostate cancer differentiation.Materials and Methods:ten cases of BPH(55-75 years old,averagely 61.2 years old)and 44 cases of prostatic cancer paraffin-embeded tissues were provided by urological department of XiangYa Hospital during 2000-2007.Prostatic cancer tissues were categorized according to Gleason scoring.No prostate cancer patients received endocrine therapy or other treatment.Consecutive resection specimens of 4μm were performed. Immunohistochemistry was carried out to detect NSE,P-gp,MRP1 and Bcl-2 expression.Count of positive cells in ten representive high power fields was performed.Less than 5%of positive cells were defined as negative expression,while more than 5%as positive. Results:No P-gp expression was detected in both cancer and BPH resections,while NSE,MRP1 and Bcl-2 showed expression.There was significant difference in MRP1 between two tissues(p＜0.01),and expression in cancer was higher than in BPH.There was no difference in NSE and Bcl-2 expression between two tissues(p＞0.05).Expression of NSE, MRP1 and Bcl-2 increased with decreasing of cancer differentiation levels. Coefficient of determination of NSE was 0.438.MRP1 was 0.472.Bcl-2 was 0.375.Prostate cancer tissues were categorized into two groups according to whether NSE expression was positive.Expression of MRP1 and Bcl-2 showed significant difference between two groups(p＜0.05). Expression in positive group was higher than negative group.Conclusions:(1)Expressions of NSE,MRP1 and Bcl-2 were detected in prostate cancer.And expression was correlated with cancer differentiation, Gleason scores.The worse differentiated cancer showed higher expression. This cancer showed no P-gp expression.(2)Prostate cancer with NSE positive expression showed higher expression of MRP1 and Bcl-2.It was suggested that NED was related to MDR and increasing ability of anti-apoptosis.(3)Expressions ofNSE,MRP1 and Bcl-2 were detected in PBH.It was indicated that the three proteins participated in development of BPH.Part two NED induction of androgen-independent prostate cancer cell line DU145 and detection of P-gp,MRP1 and Bcl-2Aim:To investigate NED after DU145 was exposed to EGF and TGF-α.Expressions of P-gp,MRP1 and Bcl-2 were examed.Materials and Methods:DU145 cells were cultured under various conditions for three days.Morphology was observed under light microscope. Cells were collected after three days.Western blotting was used to exam expression of P-gp,MRP1 and Bcl-2.Real-time semiquantitiy PCR was used to detect NSE mRNA.Electron microscope was utilized to observe neuroendocrine differentiation particles.Results:(1)light microscope observation:no apparent morphological changes were observed in cells with only 2%BCS.More apparent changes in cells with 10ng/ml EGF were observed than those with 5 ng/ml EGF.The largest number of NED cells was counted at first day.The number increased everyday.At the third day,morphology changes were much more apparent. Cells shape changed from oval to triangle or polygon-shaped.NED cells had long branching dendrite-like processes.The effect in cells treated by TGF-αwas similar to EGF.But morphological changes were less apparent.No morphological changes were observed in cells treated with AG825.(2)Western blotting results:NSE expression:mild expression was detected in cells with only 2%BCS.Expression increased after exposed to EGF.Cells with 10ng/ml showed stronger expression than 5ng/ml.Those added with AG825 showed no apparent expression.The effect in cells treated by TGF-αwas similar to EGF.Bcl-2 expression:Expression was detected in cells with only 2%BCS.Expression increased strongly after exposed to growth factors.Those with AG825 showed mild expression. P-gp expression:No expression was detected in any cells.MRP1 expression: Expression was detected in cells with only 2%BCS.Expression increased strongly after exposed to growth factors.Those with AG825 showed no apparent expression.(3)Real-time semiquantitiy PCR:compared with cells with only 2%BCS,expression of NSE mRNA in cells with EGF 10 ng/ml and TGF-α 10 ng/ml increased 10.7 and 10.1 fold respectively.It was suggested that level of NED increased.Those with AG825 decreased 81%and 77%.(4)Electron microscope:No neuroendocrine particles were detected in cells treated only with 2%BCS.After exposed to EGF and TGF-α,numbers of particles increased dramatically.No particles were detected in cells with AG825Conclusions:(1)NED could be induced by EGF and TGF-αin DU145 cells.The effect could be counteracted by AG825.(2)After induction,expression of NSE,MRP1 and Bcl-2 increased.It was suggested that ability of anti-apoptosis and MDR increased.Part three Chemoresistance of DU145 to cisplatin after NED inductionAim:To investigate chemoresistance of DU145 to cisplatin after NED induction.Materials and Methods:Cells of exponential phase were implanted in 96-cell plate after induced by EGF and TGF-α.Each group cells were added into various concentrations of cisplatin(0.1,0.5,1,5,10,50,100μg/ml). After 48 hours culture,MTT assay was used to test effect of cisplatin on viability.With similar treatment cells were exposed to 5μg/ml cisplatin for 48 hours.Flow cytometry was used to test percentage of apoptotic cells and cell cycle.Results:(1)Cisplatin could suppress growth of DU145.At every concentration point,especially higher concentration,viability treated by EGF 10 ng/ml was higher than treated by EGF 5 ng/ml.Viability treated by TGF-α10 ng/ml was higher than treated by TGF-α5 ng/ml.The effect of EGF was similar to TGF-α.Viability apparently decreased for those treated with AG825.(2)After exposed to 5μg/ml cisplatin for 48 hours,those treated with growth factors showed less percentage of apoptosis,and those treated with EGF 10 ng/ml showed less percentage than EGF 5 ng/ml(p＜0.05).It was similar for TGF-α.Changers in cell cycles were observed.Cells number of G₀/G₁ decreased.Cells of S and G2/M phase increased.Conclusions:(1)Chemoresistance of DU145 to cisplatin increased after NED induction by EGF and TGF-α.(2)Cells treated with higher concentration growth factor showed stronger chemoresistance.The effect was similar comparing one growth factor with each other.