| BackgroundOn a global scale, the incidence of prostate cancer ranks five of all malignant tumors and two among men. In the United States, prostate cancer is the most common cancer and the second leading cancer-related cause of death in men. Currently, the incidence of prostate cancer is ten times lower in our country than in the United States. But due to the changing lifestyle, aging population and new screening technology for early prostate cancer, such as PSA, PSMA, PSCA, DD3, hK-2, AMACR and uPA, the incidence and diagnosis of prostate cancer in our country is increasing.As many previous studies been taken out, a variety of gene and protein as well as their mechanism involved in prostate cancer has been reported. There are p53, p16, PTEN, NKX3.1, Rb and LKB1inactivation or missing, C-MYC, met, ras upregulation, TMPRSS2:ERG fusion, abnormal expression of AR and PSCA, promoter methylation and defective DNA damage response pathway. Yet, we still have a very rudimentary understanding of the molecular mechanisms leading to prostate cancer. Recent studies have found that the receptor for advanced glycation end products (RAGE) is closely related to prostate cancer. Though the receptor for advanced glycation end products was initially identified as a receptor for AGEs, it is well known as a multi-ligand receptor which interacts with various ligands including amphoterin, amyloid-p peptide, members of the S100/calgranulin protein family, immunoglobulin light chain, prions, and so on. The ligand/RAGE interaction plays a significant role in carcinogenesis, tumor progression and metastasis while blockade of RAGE reverse those effects. And the relationship between RAGE and prostate cancer has reported recently. In prostate tissues, prostate cancer tissue, especially in high-grade adenocarcinomas and those with metastasis, showed higher RAGE expression than normal prostate tissue. Among RAGE ligands, amphoterin, AGEs and S100A8/A9play an important role in cancer. Many reports point out the significance of ligand/RAGE interactions in carcinogenesis, tumor progression and metastasis. What’s more, the RAGE-amphoterin system induction in prostatic stromal cells by androgen deprivation is associated with metastatic prostate cancer. Allmen et al have already reported V domain of RAGE interacts with AGEs on prostate carcinoma cells, which is necessary for tumor progression. Targeting RAGE by RNAi induces apoptosis, so as to inhibit prostate tumor growth.However, the molecular basis leading to the activation of RAGE signaling pathways after RAGE/ligand ligation remains to be elucidated. In our previous studies, we have constructed the expression vector for intracellular domain of RAGE and obtained that fusion protein. Since then twenty-five clones of encoding proteins were found to interact with the fusion protein with T7select phage display system. After using ELISA and BLAST, nine kinds of protein were obtained, one of which is the retinoblastoma protein (Rb protein).Rb protein has known to be responsible for the inhibitation of cell proliferation and tumor suppression. Tyagi A et al have reported that silibinin causes growth inhibition of the androgen-dependent human prostate carcinoma LNCaP cells by the inhibition of Rb protein phosphorylation at serine sites and an increase in Rb-E2F complex formation. Rb protein can specially bind to the SV40T antigen, E1A and E7resulting in tumor suppression. Yet, RB function is disrupted in a multitude of tumor types, including prostate cancer. In prostate cancer, allelic loss has been mostly attributed to loss of Rb function. Bookstein et al has reported that one of prostate cancer cell lines, DU145, contained an abnormally small protein translated from an RB messenger RNA transcript that lacked105nucleotides encoded by exon21. They also reported that cells that maintained stable exogenous RB expression lost their ability to form tumors in nude mice, although their growth rate in culture was apparently unaltered. In addition, Taneja SS et al reported that the reduction in Rb protein level stimulated by androgen occured through Rb degradation via the ubiquitin/proteasome pathway, and was preceded by selective Rb phosphorylation by cyclin A/Cdk2and cyclin B/Cdkl, resulting in cellular proliferation in the human prostate cancer cell line LNCaP. But it was also reported that Rb could function as a co-activator to induce AR transcriptional activity in prostate cells, which may represent the first data to link a negative growth regulatory protein function in a positive manner.Since we have proved the combination between RAGE and Rb protein, the interaction between them need more verification, like co-immunoprecipitation. There is also a question that the way how the two protein combine. Rb protein is known to be nucleoprotein. How does that interaction play in prostate cancer? The answers might not only clarify the biological function of RAGE and Rb protein, but also raise new idea for the prevention of disease including prostate cancer.MethodsTo work these out, we investigated RAGE and Rb protein expression in prostate cancer cell lines (DU145, LNCaP, PC-3). Then prostate cancer cell lines stimulated with AGE-BSA were examined by CCK-8assay. Moreover, co-immunoprecipitation and immunofluorescence were selected to confirm the interaction between RAGE and Rb protein. After that, prostate cancer cell lines were stimulated with AGE-BSA in different concentration and time to observe the variation of Rb protein expressed in prostate cancer cell lines. Finally, we constructed eukaryotic expression vectors for different domains of the extracellular region of RAGE, in order to investigate the combining domains between RAGE and Rb protein. ResultsFirst, all prostate cancer cell lines expressed RAGE, but Rb protein was only detected in LNCaP and PC-3, not in DU145. Second, LNCaP and PC-3cell growth were significantly enhanced by AGE-BSA stimulation compared to BSA stimulation and control (P<0.05). On the other hand, the cell growth stimulated by BSA did not show any significant difference compared to control. Third, RAGE was expressed in both cytoplasm and nuclei of the prostate cancer cells, LNCaP and PC-3. RAGE was showing a co-localization with Rb protein in these two cell lines suggesting the probability of the interaction between them. Fourth, direct association was tested by immunoprecipitation of RAGE and Rb protein, and detection by WB for the putative associated protein. Both Rb protein and RAGE could be detected in LNCaP and PC-3suggesting the interaction between them. Then, AGE-BSA induced the decreased expression of Rb protein in a concentration-and time-dependent manner, with significant effect observed at800ng/ml and maximal effect achieved at400ng/ml with an incubation of72hours. Finally, the expression vectors containing V and VC1domains of RAGE were successfully constructed. The V and VC1domains of RAGE were highly expressed and showed a cytoplasmic distribution in transfected PC-3cells.ConclusionsIn this study, we have obtained the following conclusions:First, the AGE-RAGE interaction induced the growth in prostate cancer cell lines. Second, RAGE was detected in the cytoplasm and nucleus of prostate cancer cell lines LNCaP and PC-3, in which RAGE and Rb protein showed co-localization. Third, the interaction between RAGE and Rb protein indeed existed in prostate cancer cells. Furthermore, AGEs decreased the Rb expression through a concentration-and time-dependent manner. AGE-RAGE interaction might induce that outcome. At last, the eukaryotic expression vectors for different domains of the extracellular region of RAGE were successfully constructed and efficiently expressed in PC-3cells. |
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