| Prostate cancer is the most common cancer and the second leading cause ofcancer-related deaths in the male population of the United States. Meanwhile, with thechanges of life style and the developing of diagnosis technique, detection rate of PCAincreases significantly in recent years in China. If the cancer is detected early and isconfined within the prostatic capsule, it can be cured by surgery, radiotherapy orbrachytherapy. However, the prognosis is often poor if metastasis has already occurred atthe time of diagnosis, and the mainstay of therapy for metastatic prostate cancer iscastration. Castration can be accomplished by either orchectomy orandrogen-antagonistic agents. Although androgen withdrawal prolongs the period free of disease progression, prostate tumor cells eventually become castration-resistant, resultingin relapse and25months patients’ median survival time. Consequently, there is a needfor understanding more about castration-resistant prostate cancer (CRPC) and findingeffective treatments.Androgen receptor (AR), a ligand-dependent transcription factor, regulates thegrowth and development of cancerous prostate grand. CRPC retain AR and selectAR-regulated gene expression in the absence of or with low levels of circulatingandrogens, demonstrating that AR signaling continues to play a significant role inpatients with castration-resistant disease.The human NDRG2gene (AF159092) was first identified in our university’sbiochemistry laboratory. Some previous research has indicated that the expression ofNDRG2decreased in various carcinomas and upregulation of NDRG2could reverse themalignant phenotype of human cancer cells. Therefore, we demonstrated it as a candidatetumor suppressor gene. In prostate cancer, our recent researches indicated that NDRG2expression levels were lower in PCA tissues than the benign ones. Upregulating theexpression level of NDRG2could inhibit proliferation and invasion in PC3cells.Moreover, previous studies in our university verified that NDRG2was repressed byc-Myc. Then after that, other research proofed that c-Myc was required forandrogen-independent PCA cell growth and c-Myc was a downstream target of AR and isregulated at a posttranscriptional level. Therefore, there was a reason to believe thathuman NDRG2protein play crucial role in CRPC tumorigenesis and progession and itmight be a crucial molecule in AR signal pathway.In this research, firstly, we dected the expression levels of NDRG2, c-Myc and ARin prostate cancer tissues and tried to analyze the relationship among the three molecules.Sencondly, we constructed the CRPC cell lines stably expressing NDRG2usinglentivirus carrier. After that, we evaluated the effects of NDRG2overexpression onCRPC cells’ proliferation. Finally, we changed the AR activity using certain reagents,then dectected effects of the changes on NDRG2expression. The object of all of our efforts was to find out the function and molecular mechanisms of NDRG2in CRPCtumorigenesis and progression. Our research was divided into two parts.1. Analyzing the relationship of NDRG2, c-Myc and AR in prostate cancertissuesWe dectected the expression levels of NDRG2, c-Myc and AR in PCA tissues usingimmunohistochemistry. The positive expression percentages of the three molecules were29.91%,33.64%,71.96%, respectively. Moreover, expression levels of NDRG2werenegatively correlated with the expression levels of c-Myc (r=-0.252, p<0.01) and AR(r=-0.277, p<0.01)(Figure1B). However, the relationship between expression levels ofc-Myc and AR was positive correlation (r=0.610, p<0.01).2. Exploring the function and molecular mechanism of NDRG2overexpressionin CRPC cellsWe firstly constructed the LNCaP and22RV1cell lines stably expressing NDRG2protein using lentivirus carrier. Then using cell growth curves, EdU stain and flowcytometry, we evaluated the effect of NDRG2overexpression on the two types of CRPCcells. And western blot was used to detect the influences of NDRG2overexpression on itsup-stream and down-stream molecules. Secondly, we changed the activity of AR usingcertain agents’ stimulti and evaluated the effects of the changes on NDRG2exprssion.Finally, we constructed22RV1xenograft nude mouse model and dectected the function ofNDRG2on CRPC cells in vivo. Cell growth curves showed that NDRG2overexpressioncould inhibit the proliferation of CRPC cells (P＜0.05). Edu stain showed that thenumber of proliferative cells increasing (P＜0.01). Flow cytometry indicated NDRG2overexpression made more CRPC cells arrested in G1phase and made the percentages ofapoptotic cells increase (P＜0.01). Western blot and immunofluorescence showed thatthe overexpresson of NDRG2could not influence the expression level and the expressionpattern of AR. But the overexpression could downregulate the expression level and thesecreting level of prostate specific antigen (PSA) in western blot and ElISA.Dihydrotestosterone (DHT) could promote the proliferation of the CRPC cells and could upregulate the AR expression level and the ratio of AR nunclear localization. However, theeffects of bicalutamide were negative to those of AR. Moreover, the changes of ARactivity could negatively regulate the expresson of NDRG2protein. And the western blotshowed that NDRG2regulated the expression of G1phase-related molecules(downregualed: cyclinD1, cyclinE, CDK2, CDK4; upregulated: p27). The zoopery resultsshowed that overexpression of NDRG2could inhibit the growth of22RV1xenografts atthe castration level of androgen and improve the physical condition of mice withinoculated tumors(P＜0.05).In conclusion, our research firstly explored the function and possible molecularmechanism of NDRG2in CPRC. Overexpression of NDRG2could inhibit the growth ofCRPC cells both in vitro and in vivo. NDRG2overexpression could down regulate PSAexpression, and the changes of AR anctivity could negatively regulate the expression ofNDRG2. All of the results implied that human NDRG2protein acted as a tumorsuppressor in CRPC and it might be a crucial inhibitor in AR signal pathway,and the genemight prove to be a powerful tool in CRPC therapy in the future. |
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