Correlations Between Skp2, P27～(kip1), PTEN Protein Expressions And The Clinicopathological Features Of Prostate Cancer

INTRODUCTIONIn the Western world, adenocarcinoma of prostate is the most common malignant neoplasia of human males. In recent year, it increased dramatically in China . However, little is known about its molecular process of development and progression in prostate cancer. Decreased expression of the cyclin-dependent kinase inhibitor p27 is frequently observed in many human cancers, including breast carcinoma, gastric carcinoma, colorectal carcinoma, and prostate cancer, where p27 loss provides independent prognostic information. The level of p27 is regulated primarily by the ubiquitin E3 ligase SCF(Skp2) through ubiquitin-dependent proteolysis. Expression of the F-box protein Skp2 is inversely correlated with p27 in many cancers. Overexpression of Skp2 also serves as an excellent malignant marker for prostate and other human cancers. Furthermore, some studies indicated that loss of PTEN leads to increased levels of skp2 through an unknown mechanism. The purpose of this study was to investigate the correlation between Skp2. p27kip1 ,PTEN protein expression and the clinicopathological features of prostatic carcinoma, and the relationships between expressions of Skp2 , p27 kip1 and PTEN in prostate cancer.MATERIALS AND METHODS Patients and tissue samplesThe tissue specimens examined were taken from 41 prostate cancer patients who underwent open surgery at our hospital from August 1999 to March 2003. No patientreceived any treatment for cancer before surgery. Resected specimens were fixed in 10% formalin and embedded in paraffin. Histological diagnoses of prostate cancer were done on hematoxylin and eosin (H&E)-stained sections. The mean age was 73 years (range. 50-82). Five patients had preoperative serum prostatic-specific antigen (PSA) levels less than 4ng/ml, eight had PSA between 4 ng/ml and 10ng/ml. twenty-eight had PSA higher than 10ng/ml. Three patients had stage A, 20 stage B, 11 stage C, and 7 stage D cancers according to Jewett-Whitmore clinical staging system. Eight patients had Gleason score 2-4, 22 Gleason score 5-7, and 11 Gleason score 8-10 according to Gleason grade system. The cancers were reviewed according to the rules of clinical and pathological studies on prostate cancer for preoperative PSA, invasion, clinical stage, histological grade. The tissue specimens examined as control were taken from 20 benign prostatic hyperplasia (BPH) patients who underwent open surgery at our hospital in 2002. The mean age was 68 years (range, 56-75).ImmunohistochemistryThe EnVision?method was used for immunohistochemical staining. Five-micrometer-thick sections were deparaffinized, and endogenous peroxidase activity was blocked with 0.3% H2O2 in Tris-buffered saline (TBS) for 30minutes. Sections were then boiled (pressure-cooking) in citrate buffer, pH6.0, for 3 minutes for antigen retrieval. Nonspecific binding was blocked with 5% goat serum in TBS for 15 minutes, and the tissues were incubated with monoclonal antibody to Skp2 (1:50; Zymed Laboratories, Inc. South San Francisco, CA. Clone No. 2C8D9) and p27kip1(1:50. DakoCytomation. Denmark, Clone No. SX53G8) monoclonal antibody to PTEN(1:50, Neomarkers USA.Clone No. 28H6) in TBS containing 2% rabbit serum and 1% bovine serum albumin for 1 hours. The sections were washed with TBS. This was followed by incubation with EnVision?(Goat Anti-Mouse/ HRP. DakoCytomation) for 60minutes .The color was developed indiaminobenzidine solution (Sigma Chemical Co., St. Louis, MO). The sections were then counterstained with Mayer's hematoxylin. Tissues were incubated with TBS containing 2% rabbit serum and 1% bovine serum albumin without the primary antibody as control. Furthermore, each run included a positive control side, which had been shown previously to be strongly positive for COX-2. Those who performed the immunohistochemical analysis were blinded to patient. Nuclear Skp2, p27 and PTEN irnmunostaining in tissue was evaluated microcopically and recorded as the percentage of positive cells (labeling frequency %)Statistical AnalysisAnaly...