The Effect Of HMGB1 And RAGE On Biological Behavior Of Prostate Cancer PC-3 Cells

BackgroundProstate cancer is the most common maligant tumor among males in Europe and United States, and the second leading cancer-related cause of death in men in the United States. According to the American Cancer Society,220,800 prostate cancer cases are projected to occur in the United States in 2015, accounting for 26% of all cancer cases in males. Despite the slight decrease in the number of prostate cancer death cases than before, it is expected to reach 27,540. Although the incidence of prostate cancer in China is lower than that of western countries, due to the westernized lifestyle, population aging and prostate specific antigen (PSA) screening around the world, it shows a clear upward trend during recent decades, which is more rapidly than that in Europe and America.The symptoms of prostate cancer in early stage is non-specificity and hard to diagnose, leading to patients frequently diagnosed with advanced stage and metastasis, thus missing the best chance for radical treatment.With the popularity of digital rectal examination (DRE) combined with PSA screening, the early diagnosis of prostate cancer has been significantly improved. However, due to the limitations about the sensitivity and specificity of PSA, there are still some controversy on its clinical applications. When consider serum PSA> lOng/ml as reference value, the clinical misdiagnosis rate was 25%; when the PSA value is in clinical diagnostic gray zone 4～10ng/ml, only 30% of the patients were diagnosed with prostate cancer. Radical treatments (including radical prostatectomy and external radiation therapy) are currently the most effective treatments for localized prostate cancer, but some prostate cancer patients after early radical treatment still relapse. The treatment of metastatic prostate cancer currently rely mainly on endocrine therapy, but they often develop to castration-resistant prostate cancer (CRPC) because of the gradual indevelopment on hormones, making treatment more difficult. Thus, We need urgently to find a new prostate cancer biomarkers having higher sensitivity and specificity as well as potential new target for treatment, leading to early detection, early diagnosis, early intervention, early treatment, and further improve the efficacy. This is an important issue for prostate cancer research.However, the molecular mechanism of prostate cancer development and progression is still unclear. Previous studies suggest that prostate cancer might relate to the chromosome abnormalities, inactivation of anti-oncogenes,including wild-type p53, p16, PTEN, mn23, NKX3.1 and Rb, the activation of proto-oncogenes,including c-myc, c-met and ras, the gene polymorphism of AR, PSA, VDR and gene fusion of TMPRSS2-ERG. Recently, a large number of studies show that high mobility group protein B1 (HMGB1) and its main receptor for advanced glycation end product receptor (RAGE) are also associated with the development of prostate cancer.HMGB1, a member of high mobility group protein B (HMGB) family, is named for its electrophoretic mobility on polyacrylamide gels. HMGB1, also named amphoterin before, is a non-histone molecular presented in eukaryotic cell. Intracellular HMGB1 involves in the stable of nucleosome structure, DNA recombination and repair, regulation of gene transcription, regulation of steroid hormones and other life activities. It also can be actively secreted by inflammatory cells or passively secreted into the extracellular by necrotic cell. Extracellular HMGB1 is the prototypic damage-associated molecular pattern(DAMP) molecule and has been implicated in tumor-associated inflammation. HMGB1, secreted from nucleus into extracellular, can act as a signal molecule and play an important role on immune, inflammation, autophagy, cell differentiation,cancer cell migration, proliferation and metastasis. Extracellular HMGB1 receptors include RAGE, Toll-like receptor (TLR) family, CD24, IL-1R, and myeloid cells trigger receptor 1 (TREM-1). Extracellular HMGB1 binds to its receptor, mainly through RAGE, and extensively involves in tumorigenesis, growth, invasion and metastasis process.RAGE was first obtained from bovine lung by Neeper. Encoded in the Class Ⅲ region of the major histocompatability complex (MHC), it is a membrane protein belonging to the immunoglobulin superfamily. Human RAGE consists of 404 amino acids, including an extracellular domain, a transmembrane domain and a short cytoplasmic tail. The expression of RAGE is at a low level (except mature type Ⅰ alveolar cells and embryonic cells) in human physiological conditions, but it is significantly higher in diabetes, autoimmune diseases, neurodegenerative diseases, vascular diseases and cancer. RAGE serves as receptor and mediates its ligands binding in monocyte macrophages, endothelial cells, mesangial cells, nerve cells, vascular smooth muscle cells and a variety of tumor cell surface in signal transduction. It activates various intracellular signal transduction, leading to pathological effects. Although RAGE is named due to the interactions with advanced glycation end products (AGE), it binds to the ligands such as HMGB1, β-amyloid peptide and several members of the calcium-binding S100 family of proteins, which interact with RAGE and plays a different role in various diseases.In recent years, the effect of HMGB1 and RAGE on tumor arouses wide concern. It is reported that HMGB1/RAGE axis is closely related to maligant tumor such as colorectal cancer, stomach cancer, pancreatic cancer, liver cancer, squamous cell carcinoma and malignant mesothelioma. Some studies suggested that HMGB1 and RAGE involved in the regulation of their proliferation, migration and invasion, while blocking the interaction of HMGB1 and RAGE could inhibit tumor growth and metastasis. The relationship between HMGB1/RAGE axis and prostate cancer effect hasn’t been discovered until recently. In 2003, Kuniyasu et al. analyzed pT3 prostate cancer specimens on 40 patients receiving luteinizing hormone-releasing hormone (LHRH) inhibitor of androgen deprivation therapy preoperative and found that RAGE and its ligand HMGB1 were high levels of co-expression in the tumor cells and stromal cells, which was particularly obvious among patients with metastatic. Further experiments showed that HMGB1/RAGE promoted the interaction between tumor cells and stromal cells through paracrine pathway in the course of androgen deprivation, thus accelerating the development and metastasis of prostate cancer. Ishiguro et al. found that the mRNA levels of HMGB1 and RAGE in untreated prostate cancer and hormone-refractory prostate cancer tissues was significantly higher than that in normal prostate tissue. Among the three kinds of prostate cancer cell lines (DU145, PC-3 and LNCaP), they found that DU145, which was hormone-independent prostate cancer cell lines, had the highest RAGE expression in 2005. HMGB1 was expressed in all these three cell lines. In 2012, Elangovan et al. found that blocking HMGB1/RAGE axis through specify antibody could inhibit the growth of prostate cancer in vitro and in vivo.In our previous work, we found that the expression RAGE, HMGB1 and their co-expression were significantly higher in prostate cancer than that in the benign prostatic hyperplasia at histological level.The expression RAGE, HMGB1 and their co-expression were significantly associated with T stage, lymph node metastasis, distant metastasis. We further conducted Kaplan-Meier survival analysis and found that co-expression of RAGE and HMGB1 was correlated with poor patient outcome.However, whether the interaction between HMGB1 and RAGE in prostate cancer exists. And their effect on the biological behavior of prostate cancer (such as proliferation, migration, apoptosis) and the mechanisms remain unclear,which need further investigation and verification at the cellular level. These will provide experimental evidence for the significance of HMGB1 and RAGE on prostate cancer diagnosis, treatment and evaluation, and provide new ideas and methods for clinical diagnosis and treatment of prostate cancer.MethodsBased on the above findings, we firstly detected the expression of HMGB1 protein by Western Blot in prostate cancer cell lines and benign prostatic hyperplasia cell line. Then we used the method of immunofluorescence to observe the intracellular location of HMGB1 in prostate cancer cell lines and prostate hyperplasia cell line. We used agarose affinity column (Ni-IDA) for exogenous HMGB1 protein purification, then we observed the proliferation of prostate cancer PC-3 cells on stimulation of exogenous HMGB1 through CCK-8 assay. The migration of prostate cancer PC-3 cells on HMGB1 stimulation was detected through wound scratch assay, and the apoptosis of PC-3 cells on HMGB1 stimulation was tested by Annexin-V-FITC apoptosis detection kit. We observed the transfection efficiency of HMGB1 plasmid through immunofluorescence, and further validated its overexpression model by Western Blot. The influence of HMGB1 overexpression on proliferation, migration, apoptosis ability in prostate cancer PC-3 cells was observed by CCK-8 assay, wound scratch assay, and apoptosis detection kit. We used RNA interference (RNAi) technology to konck down RAGE in prostate cancer PC-3 cells and then observed the its influence on proliferation, migration, and apoptosis ability in prostate cancer PC-3 cells through CCK-8 assay, wound scratch assay, and apoptosis detection kit, trying to explore the interaction between HMGB1 and RAGE in prostate cancer cells.Results1. HMGB1 is highly expressed in prostate cancer cells. It is expressed mainly in the nucleus in benign prostatic hyperplasia and prostate cancer cell lines cells, and also expressed in the cytoplasm.2. We purified His-HMGB 1 fusion protein.3. Exogenous HMGB1 can promote PC-3 cell proliferation in a concentration-dependent and time-dependent manner, promotes prostate cancer cell migration and suppresses cellular apoptosis.4. Observe the transfection efficiency of HMGB1 plasmid in prostate cancer PC-3 cells, and construct overexpression of HMGB1 prostate cancer cell model successfully.5. The overexpression HMGB1 protein may promote prostate cancer PC-3 cell proliferation, migration and anti-apoptotic ability.6. We use RNAi technology to knock down RAGE protein in prostate cancer PC-3 cells successfully.7.Once knocking down RAGE, HMGB1 cannot promote prostate cancer PC-3 cell proliferation, migration and anti-apoptosis.ConclusionsIn this study, we conducted a preliminary study to explore the effect of HMGB1 and RAGE on biological behavior of prostate cancer by means of exogenous protein stimuli and intracellular overexpression. The results indicated that HMGB1 could promote prostate cancer cell proliferation, migration and anti-apoptotic ability through RAGE. These findings not only deepen our understanding on the biological function of RAGE and HMGB1 protein, but also provide theoretical and experimental basis for the diagnosis, treatment and evaluation of advanced prostate cancer, and provide new ideas and methods for clinical diagnosis and treatment of prostate cancer.