Prostate cancer is the most common adenocarcinoma and second most common cause of cancer death in men in the Western world. In fact prostate cancer has its own ranking in the 6th most common cancer incidence occurred in Malaysian population. The management of patients with prostate cancer depends on an accurate assessment of the biological potential of the tumor. Unfortunately, the current examination techniques are mostly inadequate to have a precise and correct clinical staging, and assessment of the tumor grade itself. There is an urgent requirement for better understanding of molecular abnormalities that define this tumor is needed, to help identify more precise biomarkers in order to give proper clinical outcome prediction of the disease. Recently, epidemiological, experimental and clinical studies have demonstrated that non-steroidal anti-inflammatory drugs (NSAIDs) can decrease the incidence of cancer. The effects of NSAIDs are mediated mainly by their inhibition of prostaglandin synthesis. Cyclooxygenase also known as prostaglandin (PG) endoperoxide synthase is essential in the conversion of arachidonic acid to proinflammatory prostaglandins. COX-2 is typically undetectable in most tissues but can be induced rapidly by proinflammatory signal, endotoxin, and some growth factor. COX-2 expression plays an important role in inhibiting apoptosis, stimulating angiogenesis and promoting tumor cell metastasis and invasion. Loss of expression of cyclin-dependent kinase inhibitor p27Kip1 has been implicated in malignant development. p27Kip1 gene encodes a small protein that have capacity for negatively regulating cell cycle progression. p27Kip1 is present in abundance in quiescent cells, and its expression diminishes, primarily by proteosome degradation, immediately before S-phase entry. Loss of p27Kip1 has been shown to be negatively prognostic marker in mammary, gastric, pulmonary, esophageal, and colorectal carcinoma.
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Purpose of this study is to evaluate the expression of COX-2 and p27Kip1 in malignant and benign prostate tissue and investigate the relationship between the expression of marker with clinicopathological parameters by immunohistochemistry and gene expression method.
BPH, prostate cancer, cyclooxygenase, expression, gene expression, p27, NSAIDs
Prostate Cancer is one of the commonest cancers in men. In Malaysia it is the sixth most frequent cancer and it accounts for 5.7 per cent of cancer cases in males. Very few prostate cancers occur in men under 50 years of age. The rate increases sharply with age and is highest in the oldest age group. In Malaysia, the Chinese record the highest incidence of prostate cancer compared to Malays and Indians. Research is being conducted to identify genes that may predispose people to develop the disease (National Cancer Registry, 2003). Benign prostatic hyperplasia is disease which results from the uncontrolled growth of the prostate gland. This could be due to age-related hormonal changes and is common among senior citizens. Increased prostaglandins (PGs) are associated with many inflammatory pathophysiological conditions; and are synthesized from arachidonic acid by either of 2 enzymes, cyclooxygenase-1 (COX-1) or -2 (COX-2). Recent epidemiologic, expression, and pharmacologic studies suggest COX-2 derived metabolites also play a functional role in the maintenance of tumor viability, growth and metastasis (Koki et al., 2002). Cyclooxygenase-2 (COX-2) plays an important role during oncogenesis. This enzyme is expressed in cells shown to promote tumorigenesis, such as the neoplastic and surrounding stromal cells; including blood vessels, smooth muscle, and inflammatory cells. Numerous studies in relevant syngeneic, chemically-induced, and xenograft animal models of various epithelial cancers report compelling data that COX-2 inhibitors may have antitumor and antimetastatic activities, and imply COX-2 activity may play a pivotal role throughout the pathophysiological progression to cancer (Koki et al., 2002). Several epidemiological studies have demonstrated that chronic NSAID use leads to reduced risk for developing many types of human tumors, including colon (Sandler et al., 1996; Reeves et al., 1996; and Marnett et al., 1995) lung (Schreinemachers et al., 1994), stomach (Farrow et al., 1998), and esophageal (Funkhouser et al., 1995) neoplasias. Retrospective analyses suggested a 40ââ‚¬"50% reduction in the relative risk of death by colon cancer in persons regularly taking aspirin or other NSAIDs (Giovannucci et al., 1995 and Rigas et al., 1994) indicating that inhibition of COX in humans has a chemo preventive effect. Taken together, the epidemiological data, COX-2 expression and the chemoprevention studies in animal models imply that COX-2 plays a critical role during initiation, proliferation, and or transformation from normal to malignant disease (Koki et al., 2002). Deregulation of the tumor suppressor p27kip1 (p27) has been implicated in a variety of human cancers, suggesting it might be a viable therapeutic target. Developing p27-specific intervention strategies requires understanding its role and regulation in normal and pathologic states (Seonghun et al., 2003). Although p27 has been extensively characterized as an inhibitor of cyclin-dependent kinases, disruption of this function is inadequate to explain its role in tumorigenesis. A more comprehensive understanding of p27 biology would facilitate development of therapeutic responses to p27 disruption in human cancers. The protein p27kip1 (p27) has emerged as a prognostic indicator in many different types of cancer, with abnormally low protein levels indicating more aggressive tumors with poorer prognosis and decreased long-term survival for the patient (Tsihlias et al., 1999 and Lloyd et al., 1999). Several studies show that low p27 levels are associated with poor prognosis in many different tumors including nonsmall cell lung carcinoma (Espisito et al., 1997), breast carcinoma (Porter et al., 1997), prostate carcinoma (Cote et al., 1998), gastric carcinoma (Mori et al., 1997), head and neck (Erminia et al., 2005) carcinoma and laryngeal carcinoma (Pruneri et al., 1999). In vivo show that, mice lacking p27 protein developed pituitary hyperplasias, confirming p27 is a tumor suppressor protein in that tissue type (Fero et al., 1996; Nakayama et al., 1996; and Kiyokawa et al., 1996). p27 can thus be considered a viable therapeutic target, since restoring its protein levels or replacing its biological function might halt expansion of cancerous cells.
Materials and Methodology
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233 patients are retrospectively selected for analysis, ethical approval will obtained from the local research and ethical committee for use of tissue in this study. Patients selected for analysis included: those with BPH, prostate cancer and normal sample. Prostate-specific antigen (PSA)
values and Gleason scores were available for each patient with prostate cancer.
Tissues sections (4 um) are dewaxed in xylene and rehydrated through graded alcohols. Antigen retrieval is carried out by heating in TBS buffer (pH9) for 20 min under high power in a microwave. The specimen then is block with Hydrogen peroxidase (H2O2). The tissue is stained with a COX2 and p27 (human) monoclonal antibody (DAKO, Denmark) at 1:100 and 1:50 dilution respectively and a biotinylated secondary antibody, streptavidin peroxidase complex and enhanced 3,3-diaminobenzidine (DAB) as a substrate. Finally, the tissues were counter-stained with haematoxylin and dehydrated through graded alcohols and xylene. Negative control sections were incubated with distilled water as replacement of primary antibody.
Total RNA was extracted from specimes by using the WaxFree RNA Kit (TrimGen, USA) method. For polymerase chain reaction (PCR) analysis of RNA, complementary DNA (cDNA) was made by reverse-transcription (RT) using cDNA synthesis kit (Bioline, UK). PCR reactions were performed with 3 uL of each cDNA, 3 uL of each sense and antisense primer (20 mM), and 1 unit of Taq polymerase (Bioline, Uk). Thirty-five cycles of denaturation, annealing, and extension (94 ÂÂ°C for 45 seconds, 54 ÂÂ°C for 45 seconds, and 72 ÂÂ°C for 2 minutes) were performed on an automatic thermocycler (Eppendorf, Australia). Human glyceraldehyde 3-phosphate dehydrogenase is used as housekeeping gene to internalize the variation of expression between the COX-2 and p27 gene.
For each tissue specimen, the extent and intensity of staining with COX-2 and p27 antibodies will graded on a scale from 0 to 3+ by two blinded observers and an average score was calculated (Sano et al., 1995). Staining was classified into five grades from 0 to 3+ according to the intensity of staining (Sano et al., 1995). A 3+ grade implies that all staining was maximally intense throughout the specimen, whereas a 0 grade implies that staining was absent throughout the specimen. The COX-2 and p27 expression pattern then will correlated with clinicopathological parameters.
Gene expression level of the genes tested will interpreted as any significant (defined as >2 fold ) up-or down-regulation (Cheng et al., 2007). The COX-2 and p27 expression pattern then will correlated with clinicopathological parameters. All data including immunohistochemistry and gene expression results will be analyze using SPSS software for Windows, version 16.0. (Chicago, USA). The Wilcoxon rank sum test for nonparametric method will be use to analyzed the differences in COX-2 and p27Kip1 immnunoreactivity between normal prostate, BPH, and prostate cancer. Mean value will considered significant at P < 0.05. A possible association with clinicopathological parameter (age, PSA, Gleason score and stage) will assessed by ANOVA test
There is significant difference of COX-2 and p27 expression level between prostate carcinoma and benign prostatic hyperplasia. There are also association between expression of both markers with clinicopathological parameters.
Study of project
Patient data collection
Result analysis and processing