Study On XAF1 Expression And Regulatory Effects Of Somatostatin On XAF1 In Human Prostate Cancer

BackgroundProstate cancer is the most common cancer and the leading cause of cancer death among men. The incidence is the fifth in the world and the second in the male. It was estimated that approximately 217,730 new cases and 32,050 prostate cancer-related deaths occurred in the US in 2010. Although epidemiological studies showed that the incidence of prostate cancer in Asians is much lower than that in African-Americans, the occurrence of the disease has rapidly increasing in China. So it is urgently necessary to explore new methods to prevent and to treat the human prostate cancer. Initially, almost all metastatic prostate cancers require testosterone for growth, and the role of androgen deprivation as a first-line therapy for metastatic prostate cancer has been recognized for more than sixty years. Androgen-deprivation therapy (ADT) is the mainstay of therapy for metastatic disease and is principally palliative in nature. Most prostate cancers are initially androgen-dependent but become androgen-independent and refractory to hormone withdrawal therapy. It is important to find out the mechanisms of the development of prostate cancer.Like all other human malignancies, prostate cancer cells escape apoptotic death through highly efficient pathways involving multiple mechanisms. X-linked inhibitor of apoptosis protein-associated factor-1 (XAF1) was first identified as an interacting protein of X-linked inhibitor of apoptosis (XIAP). XIAP suppresses apoptotic cell death by binding to caspases and inhibiting their functions. XAF1 antagonizes XIAP activities, thereby promoting apoptosis. XAF1, a potent apoptosis-inducer, plays a significant role in the process. A number of studies have shown that XAF1 can sensitize cancer cells to TRAIL, TNF-α, IFN-βand MEK inhibitor-induced apoptosis in vitro. Moreover, some researchers have recently indicated the effect of XAF1 combination with these factors on inhibition of tumour growth in vivo. XAF1 is therefore believed to play an important role in the major apoptosis-related pathways. XAF1 also serves as a candidate tumour suppressor gene. XAF1 mRN A is expressed at low or undetectable levels in most cancer cell lines, and transcriptional down-regulation in tumour cells as opposed to corresponding normal tissues and has been shown to occur at different frequencies in gastric adenocarcinomas, colorectal cancer, urothelial carcinomas, malignant melanomas, clear-cell renal cell carcinomas, non-small cell lung cancer, bladder cancer and B chronic lymphocytic leukemia. However, little is yet known about its potential implication in prostate cancer.In this current study, prostate cancer specimens and normal prostate samples adjacent to the tumour site were obtained from 20 prostate cancer patients treated with radical prostatectomy, we examined XAF 1 mRN A and protein expression using RT-PCR and Western blot in those human prostate tumours and their adjacent normal prostate tissues. To perform Immunohistochemistry, prostate tumour and benign prostatic hyperplasia (BPH) paraffin blocks were obtained from the archives of the Division of Surgical Pathology with approval from the Ethics Committee of Qilu Hospital, Shandong University. Forty-nine patients diagnosed with BPH and twenty-six patients diagnosed with PCa were included in the present study and we examined XAF1 stainings in them. Through the study, we want to reveal the relationship of XAF1 and human prostate cancer furthermore to determine the potential implications of XAF1 on human prostate cancer.Objective This study was performed to evaluate the potential implications of tumour suppressor gene XAF1 on human prostate cancer.Methods1. Prostate cancer specimens and normal prostate samples adjacent to the tumour site were obtained from 20 prostate cancer patients treated with radical prostatectomy. The fresh samples were embedded in TissueTec OCT medium (Sakura, Tokyo) immediately after surgical section and stored at -80℃until use.2. To perform Immunohistochemistry, prostate tumour and BPH paraffin blocks were obtained from the archives of the Division of Surgical Pathology with approval from the Ethics Committee of Qilu Hospital, Shandong University. Forty-nine patients diagnosed with BPH and twenty-six patients diagnosed with PCa were included in the present study.3. To compare prostate cancer specimens and normal prostate samples adjacent to the tumour site. We analyzed 20 primary prostate cancer samples and their adjacent normal prostate tissues for XAF1 mRNA and protein expression using RT-PCR and western blot.4. To compare benign prostatic hyperplasia (BPH) tissues and prostate cancer specimens. We analyzed 49 benign prostatic hyperplasia tissues and 26 primary prostate cancers (PCa) for XAF1 stainings with Immunohistochemistry.Results1. RT-PCR:We analyzed 20 primary prostate cancer samples and their adjacent normal prostate tissues for XAF1 mRNA expression. The tumour samples derived from 18/20 patients expressed substantially lower amounts of XAF1 mRNA compared to their paired normal prostate tissues (P<0.05), the tumour samples derived from 1/20 patients expressed the similar amounts of XAF1 mRNA compared to their paired normal prostate tissues, and the tumour samples derived from 1/20 patients expressed the higher amounts of XAF1 mRNA compared to their paired normal prostate tissues.2. Western blot:We analyzed 20 primary prostate cancer samples and their adjacent normal prostate tissues for XAF1 protein expression. The tumour samples derived from 16/20 patients expressed substantially lower amounts of XAF1 protein compared to their paired normal prostate tissues (P<0.05), the tumour samples derived from 2/20 patients expressed the similar amounts of XAF1 protein compared to their paired normal prostate tissues, and the tumour samples derived from 2/20 patients expressed the higher amounts of XAF1 protein compared to their paired normal prostate tissues.3. Immunohistochemical staining:Immunohistochemical analyses were carried out in 49 benign prostatic hyperplasia (BPH) tissues and 26 prostate cancers (PCa). XAF1 stainings were observed in epithelial cells of BPH and prostate cancer tissues. XAF1 expression was detected in 45 of 49 BPH samples, with a strong cytoplasm and a weak nucleus expression pattern. In contrast,23 of 26 prostate cancers are shown with negative XAF1 stainings in cytoplasm and in nucleus. (P<0.05)ConclusionsThe low expression of XAF1 mRNA and protein may contribute to the development of human prostate cancer. BackgroundProstate cancer is the most common cancer among men. The incidence is the fifth in the world and the second in the male. Recently, epidemiological studies showed the incidence of prostate cancer was a rising trend in the developing country, especially in China. Many research focused on the mechanism of prostate cancer development and the strategy of therapy. Androgen-deprivation therapy (ADT) was the standing method of therapy in the early stage of prostate cancer because the almost all metastatic prostate cancers dependent on the testosterone. ADT played an important role in the therapy by means of luteinizing hormone releasing hormone or bilateral orchiectomy. ADT removed androgen stimulation, and initially induced apoptotic involution of prostate cells. The disease eventually progresses to an androgen-independent (AI) state with poor prognosis for a survival of approximately 15-20 months. Hormone-refractory prostate cancer (HRPC) is a clinically heterogeneous disease that is uniformly defined by persistent cancer-cell growth and progression in spite of low testosterone concentrations. HRPC was no significant relation with the concentration of testosterone. The method of HRPC therapy was single in the current. And the prognosis of prostate cancer was poor as HRPC was treated completely. Chemotherapy had the significant side effects although it was the major therapy. So it was urgent to search a new strategy.Somatostatin was a neuropeptide, and included 14 amino acids. Somatostatin is widely present in central and peripheral human cells/tissues including prostate. Octreotide is an analogue of somatostatin and has been used in clinical practice since data emerged in the 1980s confirming its ability to palliate carcinoid syndrome. Somatostatin exerted a potent anti-tumour action by affecting tumour cell proliferation, apoptosis, angiogenesis and the host's immune response. Our previous results have shown that somatostatin may affect the mitochondria of LNCaP and DU145 cells in a way that eventually triggers mitochondrial-mediated apoptosis and exert its effects on prostate cancer cells via MAPK pathway and by regulating the activities of phosphotyrosine phosphatases. Some oncogenes or anti-oncogenes were abnormal in the development of prostate cancer. More and more people focused on the gene therapy of prostate cancer. The gene therapy was that anti-oncogene was transferred to prostate cancer cells and expressed, so as to achieve the purpose of inhibiting tumor cell growth. First of all, the strategy gene should be available for the prostate cancer gene therapy, and X-linked inhibitor of apoptosis protein-associated factor-1 (XAF1) was important.The expression of XAF1 was lower in the prostate cancer. XAF1 was considered as a candidate strategy gene. The growth of gastric adenocarcinomas was inhibited when XAF1-adenovirus was used to treat the animal model of gastric adenocarcinomas in the nude mice. The transplanted tumor was not detected with the combination of XAF1-adenovirus and TRAIL, which showed that XAF1 was related closely with the development of cancer. The high expression of XAF1 might induce the apoptosis of prostate cancer cells. However, the mechanism was still unclear. The report about the relation between XAF1 and somatostatin was not found. Whether somatostatin regulated the expression of XAF1 and played its therapeutic role was still unclear. In the current study, we detected tumour suppressor gene XAF1 mRNA and protein expression and XAF1 in human prostate epithelial cells RWPE-1, androgen dependent prostate cancer LNCaP, and androgen independent DU145 and PC3 cells. We found low expression of tumour suppressor gene XAF1 in the three prostate cancer cell lines. Thereafter, to explore the affection of somatostatin on the expression of XAF1, we detected regulatory effects of somatostatin and Octreotide on XAF1 mRNA and protein expression in the three prostate cancer cell lines before stimulation or after stimulation. This study offered a new method for the therapy of prostate cancer, and established a basement for the potential strategy XAF1.ObjectiveThis study was performed to evaluate the potential regulatory effects of somatostatin and Octreotide on tumour suppressor gene XAF1 during the development of prostate cancer cells.MethodsA human prostate epithelial cell line (RWPE-1) and prostate cancer cell lines (LNCaP, DU145 and PC3) were obtained from the American Type Culture Collection (ATCC). To compare human prostate epithelial cell line (RWPE-1) and prostate cancer cells (LNCaP, DU145 and PC3), we detected XAF1 mRNA and ptotein expression among them with RT-PCR and Western blot, besides, we detected XAF1 stainings in human prostate epithelial cell line (RWPE-1) and prostate cancer cell lines (LNCaP, DU145 and PC3) with immunocytochemistry. Then we used somatostatin and its analogue Octreotide to stimulate prostate cancer cells different time (0,1h,12h,24h,72h), thereafter, the regulation of XAF1 mRNA and protein expression by somatostatin and its analogue Octreotide in prostate cancer cells were evaluated with RT-PCR and Western blot.Results1. RT-PCRSubstantial levels of XAF1 mRNA were detected in RWPE-1 cells, whereas prostate cancer cells LNCaP, DU145 and PC3 exhibited lower XAF1 expression. (P<0.05). Somatostatin and Octreotide up-regulated XAF1 mRNA expression in all prostate cancer cell lines.2. Western blotSubstantial levels of XAF1 protein were detected in RWPE-1 cells, whereas prostate cancer cells LNCaP, DU145 and PC3 exhibited lower XAF1 expression. (P<0.05). Somatostatin and Octreotide up-regulated XAF1 protein expression in all prostate cancer cell lines. 3. Immunocytochemical stainingXAF1 staining was observed strongly in cytoplasm and nucleus in human prostate epithelial cells RWPE-1, while neither in cytoplasm nor in nucleus of XAF1 staining was detected in prostate cancer cells LNCaP, DU145 and PC3.ConclusionsThe enhanced tumour suppressor gene XAF1 mRNA and protein expression by somatostatin and Octreotide indicates a promising strategy for prostate cancer therapy, which offered a new method for prostate therapy.