Somatostatin Effects On The Proteome Of Prostate Cancer Cells

Background: Prostate cancer is the most common solid tumor in the United States. In 2005, there will be approximately 232,090 men diagnosed with prostate cancer and 30,350 deaths. The lowest rates are usually in Asia, especially in China, but the incidence of prostate cancer has been increasing in recent years. The standard initial systemic therapy for locally advanced or metastatic disease is hormonal or androgen deprivation therapy (ADT) that may be bilateral orchiectomy or luteinizing hormone-releasing hormone (LHRH) administration. The androgen-dependent period in patients with metastatic disease lasts a median of 14-30 months. The disease then progresses to a phase when ADT alone fails to control the malignancy despite castrate testosterone levels, and is termed androgen-independent prostate cancer (AIPC). Some patients with AIPC respond to secondary hormonal manipulations. Subsequently, the disease no longer responds to further hormonal therapy and is referred to as hormone refractory prostate cancer (HRPC). AIPC and HRPC comprise patients with diverse presentations including a rising PSA (prostate specific antigen) alone with or without metastases, radiological progression (bone scan or soft tissue progression) and clinical progression (worsening pain, urinary obstruction). Historically, metastatic AIPC and HRPC are highly resistant states and chemotherapy has yielded poor response rates with median survivals of up to only 12 months.The molecular mechanisms underlying the transition from androgen-sensitive to androgen-resistant prostate cancer are not fully understood. The acquisition of an androgen-independent phenotype is the most serious issue of prostate cancer treatment. Although several experimental cell models have been reported for studying androgen independence, they have limited applications related to hormone-refractory prostate cancer. LNCaP is a human prostate cancer cell-line and often used in various in vitro studies. It has been established from supraclavicular lymph node metastases. These cells express androgen receptors (ARs) and exhibit androgen-sensitive cell growth. Although there is a physiologically equivalent level of androgenic activity in the regular culture condition, LNCaP cells gradually lose their androgen requirement for growth upon passage, mimicking tumor progression in patients.Somatostatin (sms) and its analogs, has been found to inhibit the growth of experimental prostate cancer via several mechanisms, as indirect antihormonal and direct antimitogenic actions, mainly due to inhibition of somatostatin receptor subtypes (sstr1-5). Hormone refractory prostate cancer does express sstrs. Sms analogues (octapeptides such as octreotide) have been tested however only with limited success. HRPC expresses mainly sstr subtype1 and since the octapeptides lack affinity to this subtype (affinity only to sstr2 and sstr5) it could be one explanation to the so far disappointing results. Sms derivatives with high affinity to sstr-1 have therefore been considered to have interest as potentially useful agents for the treatment of HRPC. Recently we reported about a sms derivative based on natural sms (sms 14) with high affinity to all five sstr subtypes, smsdx. Several studies (in vitro, animal studies) have shown that sms analogs can inhibit the growth of experimental prostate cancers. In vitro studies on human HRPC cell lines deriving from the LNCaP cell line, suggest that sms exerts a direct inhibitory effect on the cell proliferation and protein secretion through activation of phosphotyrosyl protein phosphatases.Oncoproteomics is the study of proteins and their interactions in a cancer cell by proteomic technologies. Proteomic research first came to the fore with the introduction of two-dimensional gel electrophoresis. At the turn of the century, proteomics has been increasingly applied to cancer research with the wide-spread introduction of mass spectrometry and proteinchip. There is an intense interest in applying proteomics to foster an improved understanding of cancer pathogenesis, develop new tumor biomarkers for diagnosis, and early detection using proteomic portrait of samples. Oncoproteomics has the potential to revolutionize clinical practice, including cancer diagnosis and screening based on proteomic platforms as a complement to histopathology, individualized selection of therapeutic combinations that target the entire cancer-specific protein network, real-time assessment of therapeutic efficacy and toxicity, and rational modulation of therapy based on changes in the cancer protein network associated with prognosis and drug resistance. Besides, oncoproteomics is also applied to the discovery of new therapeutic targets and to the study of drug effects.Aims: Proteomic analysis using classical high-resolution two-dimensional electrophoresis (2-DE) combined with highly sensitive mass spectrometry is capable of simultaneous analysis and characterization of complex mixture of protein expression patterns.My aims of this current study listed below: 1. we attempted to determine the quantitative similarities and differences of native sms and smsdx effects on LNCaP proteome by proteomic analysis. 2. We will identify proteins affected by sms and smsdx in the LNCaP cells, the possible effects of sms/smsdx in LNCaP cells and potential candidate biomarkers. 3. The study aims to elucidate some of the molecular mechanism of prostate cancer progression from androgen responsive to androgen refractory. Also we hope to be able to identify some significant mitochondrial mediated apoptosis pathways where sms/smsdx might be able to curb the progression of prostate cancer.Materials and Methods: In order to elucidate and learn more about the cellular effects of sms and smsdx, we have used proteomic analysis and the protein expression was subsequently analyzed. The LNCaP cell line was incubated with sms and smsdx. Proteomic analysis using classical high-resolution two-dimensional electrophoresis (2-DE) combined with highly sensitive mass spectrometry is capable of simultaneous analysis and characterization of complex mixture of protein expression patterns. To further investigate the molecular mechanism of androgen-independent growth of prostate cancer, we established a new useful LNCaP cell model that resembles the clinical scenario of hormone-refractory prostate cancer.Results:1. Significant quantitative differences were observed in the protein expression patterns between negative control LNCaP cells and smsdx treated cells after analysis with PDQuest software (version7.1.1). A set of 63 protein spots was≥2-fold up regulated in smsdx treated cells compared to negative control samples (P < 0.05). Additionally, 489 protein spots were≥2-fold down regulated in smsdx treated cells compared to negative control cells. When similar analysis was done between control and sms, the expressions of a set of 44 protein spots were≥2-fold up regulated in sms treated cells compared to negative control samples (P < 0.05 Mann Whitney analysis). On the other hand 386 protein spots were≥2-fold down regulated in sms 14 treated cells compared to negative control cells. The intersections of the datasets i.e. the changes between negative control versus sms treated and the changes between negative control versus smsdx treated were analyzed. The set of 63 protein spots were≥2-fold up regulated in smsdx treated cells. Twenty-four (33.33%) of these were also present among the 46 protein spots that were up regulated by more than 2-fold in sms treated cells. A total of 295 protein spots were common between the 489 and 386 that were up were≥2-fold down regulated in smsdx- and sms14 treated cells respectively compared to negative control cells. The overlapping in the expressed proteins indicates certain similarity between the sms and smsdx treated cells.2. Three replicates were run for each group, control, sms and smsdx. The sms and smsdx were compared separately with the control group using a Mann-Whitney test. 172 spots were identified using MALDI-MS/MS, 140 different proteins were identified and consequently the same protein was sometimes found in multiple spots. The proteins were classified using the Swiss-Prot website, the Genome Browser website, and the National Center for Biotechnology Information (NCBI) Locuslink website. They were categorized on the basis of their known biochemical functions including metabolism, signalling transduction, maintenance of cell structure, cell-cell interactions, transport/trafficking, transcriptional regulation and cell cycle regulation. Catalytic mitochondrial and mitochondrial-associated proteins were significantly affected (fold change ~2 or higher) and they were in general up-regulated. Apoptosis-related proteins were both up-regulated (VDAC1, VDAC2) and down-regulated (PRDX2, TCTP). The fold change was >2 for PRDX2 and <2 for the others. There was a strong agreement between sms and smsdx on the up- and down-regulation of proteins. Sms/smsdx triggered up-regulation of catalytic mitochondrial proteins and seemed to affect apoptotic related proteins. Twenty-two mitochondrial proteins and 22 cancer and apoptosis-related proteins were identified. Referring to literatures, 16 potential candidate marker were identified to related to cancer. 11 proteins were differentially expressed hormone dependent cell line LNCaP and hormone independent cell line.3. Based on the identified proteins in LNCaP cells, we established a "proteome reference map" of LNCaP. Total 222 proteins were identified in the reference map in the LNCaP cells through differential quantitative analysis, several proteins were identified to be regulated by androgens and its receptor. According to strict statistical criteria, 150 spots were found to be significantly differentially expressed in up and down regulation manner in LNCaP-s compared with the parental LNCaP cells, of which we have successfully matched 75 correspondingly. Only 7 protein spots in 150 were up-regulated in LNCaP-s cells. The rest of them were all down regulated. After qualitative change analysis of protein expression, we found 6 new proteins form 75 present in the parental LNCaP cells. We didn't find new proteins in LNCaP-s cell line compared to parental LNCaP cells. Referring to the LNCaP protein map, we found 26 proteins were up and down regulated in LNCaP-s cells, and these proteins were affected by sms/smsdx treatment. Identified proteins were involved in diverse processes including the stress response, intracellular signaling and apostosis. The potential contribution to disease of these processes and identified constituent proteins are discussed.Conclusions:1. The results showed sms-like effects on the protein expression of LNCaP cells are preserved in the smsdx construct. The results can elucidate effects and cellular responses to smsdx treatment. The proteomic approach allows analysis of multiple polypeptides simultaneously with the possibility of identification of protein markers of importance in prostate cancer treatment using sms derivatives.2. Sms and smsdx up/down regulate a number of important proteins in LNCaP. The up/down- protein regulation was in concordance between sms and smsdx. Catalytic mitochondrial and mitochondrial-associated proteins were significantly affected. Sms/smsdx triggered up-regulation of the catalytic mitochondrial proteins and seemed to affect apoptotic related proteins. The results show new qualitative information about smsdx.3. The results might lead to the development of markers and therapeutic targets potentially useful for the treatment of HRPC that currently are resistant to most therapies.Significance: Theproteomic analysis has yeilded quantitative and qualitative results elucidating the effect of androgen deprivation on the proteome of prostatic cancer cells. The results verify sms activity of smsdx and indicate "mechanisms of actions" that might be of importance in the clinical setting.