Introduction

Cyclooxygenase (COX) is the enzyme that responsible for the production of prostaglandins from arachidonic acid. A number of studies suggest an important role of COX-2 in patho-physiology of inflammation and carcinogenesis. It plays role in tumorigenesis of several human malignancies. It’s reported that COX-2 stimulates cell proliferation, inhibits epithelial differentiation and apoptosis, mediates immune suppression and increases the production of mutagens (1,2,3,4,5).

Apoptosis is one the most important event at the pathogenesis of varies diseases including neoplastic process. There are several immunohistochemical methods for demonstrating apoptosis at the cells. Among these terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method is the most common method. On the other hand monoclonal antibody (M30) is the alternative method for detecting early stage of apoptosis that is specific for epithelial cells (Sharma) (6).

The objective of this study is to determine if there is relationship between COX-2, apoptosis and angiogenesis in prostate carcinomas (PC).

Materials and Methods

Samples from 49 cases who are at pathologic stage pT2 and undergoing radical prostatectomy, were selected retrospectively between 2005-2010, from the archives of Pathology Department of Çukurova University, Medical Faculty. Histologic slides of each case were reviewed for the diagnostic reassessment and graded by the Gleason scoring system. The excluding criterias were different stages, metastasis and lost follow-up. Approval of the Ethics Committee obtained for the study (Approval date: 21.09.2016; Decision No: 50243401/2016-11).

Immunohistochemical staining was performed on 5-mm sections of formalin-fixed, paraffin-embedded tissue using antibody COX-2 (1/50, Santa Cruz), vascular endothelial growth factor (VEGF) A (1/80, Santa Cruz), M30 (1/50, Roche), bcl-2 (1/50, DAKO). The visualization system used was BenchMark XT with heat-induced epitope retrieval (CC1 solution) and iView DAB detection kit (Ventana, Tucson, AZ). Cytoplasmic and  membranous staining was considered positive for COX-2, VEGF and bcl-2. Nuclear staining was accepted as positive for M30.

Statistical Analysis

For each continuous variable, normality was checked. Since the data was not distributed normally, appropriate non-parametric test was chosen. Correlations between the variables were tested by Spearman or Pearson’s correlation test. The correlation coefficients were interpreted as either excellent relationship r≥0.91; good 0.90 ≤ r≥0.71; modarate- fair 0.70 ≤ r≥0.51; weak 0.50 ≤ r≥0.31; little or none r≤0.3. The rates of mean or median survival were estimated with the use of the Kaplan-Meier method and the curves were compared with the use of the log-rank test. A p value of 0.05 was taken as the level of significance. SPSS ver 22.0 were used for analyses.

Immunohistochemical Evaluation

The results of staining for COX-2, bcl-2 and VEGF were analyzed semiquantitatively by using an immunohistochemical scoring (IHS) system that combines the percentage of immunoreactive cells (quantity score) and an estimate of staining intensity (staining intensity score). Staining quantity was scored by using a modified version of the method described by Kelly et al. (1): no staining is 0, 1-10% of cells stained are scored as 1, 11-50% as 2, 51-80% as 3 and 81-100% as 4. Staining intensity was scored on a scale of 0-3 where 0 is no staining, 1 is weak, 2 is moderate and 3 is strong. The staining intensity score and staining quantity score were multiplied to give the IHS. An IHS of 9-12 was considered strong immunoreactivity; 5-8 moderate, 1-4 weak and 0 was considered as negative.

Apoptosis was figured out by monoclonal antibody M30, which reacts with the product from the cleavage of cytokeratin 18 (CK18). The number of M-30 positive cells per 1000 cells was expressed as apoptotic index (Figure 1).

Results

The age of the patients ranged from 48 to 73 years old (average 63.8±6.4). Twenty-one (42.8%) of the cases had Gleason score ≤6 and 28 (57.2%) had Gleason score ≥7. COX-2 expression was detected in 81.6% of cases. However there was no correlation between COX-2 and M30 and VEGF expression (p>0.05) there was a positive moderate correlation between COX-2 with bcl-2 expression (r=0.49; p<0.0001) (Table 1). No significant relation was found between Gleason score and COX-2, VEGF and bcl-2. There was a negative weak correlation between Gleason score and M30 (r=-0.28; p=0.04).

Negative correlation between COX-2 expression and mean survival was also observed (p=0.041) (Figure 2). The mean survival time was 31.8 months for the patients with no COX-2 expression, 22.5 months for the patients with low and moderate COX-2 and 13.2 months for the strong COX-2 (p=0.041).

Discussion

COX-2 is a stimulated enzyme that transforms arachidonic acid to prostaglandins, which are strong mediators of inflamation. Several studies showed that COX-2 plays a key role in tumorigenesis at the various malignancies by inducing cell proliferation, metastasis, inhibiting epithelial differentiation and apoptosis and mediating factors of immune suppression (2,3,4). COX-2 expression in prostate tissues or PC has been the issue of many recent studies. COX-2 expression also have examined in inflammatory process such as post-atrophic hyperplasia, prostate intraepithelial neoplasia, and PC (5,6,7). But way of their effect is unclear and controversial. Zha et al. (8) found negative COX-2 at 144 samples of PC in their study. On the other hand, there are some reports that COX-2 expression has been analyzedin non-neoplastic prostate tissue such as post-atrophic hyperplasia or proliferative inflammatory atrophy (8,9).

Additionally, a great majority of the studies have identified overexpression of COX-2 in PC and prostate intraepithelial neoplasia (3,10).

Several types of human malignancies overexpress COX-2 along with VEGF (2,10,11,12). Increased expression of angiogenic factors, and decreased expression of anti-angiogenic factors, or a combination of both events is the result of cancer induced angiogenesis (9,13). COX-2 was expressed in newly formed blood vessels within tumors (14). VEGF is the most important tumor angiogenetic factors that cause the proliferation of vascular endothelial cells. In the literature, there are some controversial studies about the COX-2 and angiogenesis at the PC. Wang et al. (15) reported a direct link between COX-2 and angiogenesis. Also Kim et al. (3) showed a significant correlation between the overexpression of COX-2 and VEGF that was associated with inflammation and carcinoma. However Mukherjee et al. (4) couldn’t find significant correlation between COX-2 and angiogenesis in PC’s. Also we couldn’t find statistically significant correlation between COX-2 and VEGF.

An epitope, situated on CK18, becomes exposed at an early caspase cleavage event during apoptosis, and is not seen in viable epithelial cells. A novel M30 recognizes this neo-epitope on CK18 early in apoptosis. CK18 is divided by caspases at two different sites during apoptosis. This division pattern is special for apoptosis. It can be obtained using recombinant caspase-3 and caspase-6 to in vitro studies (16,17,18). Although various techniques such as TUNEL has been described, we used M30 to evaluate apoptotic index which was more simple and easy to perform (12). M30 is much more sensitive and specific for epithelial cells whereas TUNEL identifies all apoptotic cells in addition to epithelial lineage (19). Also M30 positivity comes out at an early stage of apoptosis and remains positive till to the necrosis of the tumor cells (15,18). For that reason, we thought that apoptosis exposed by M30 would be much more suitable for PC on the base of epithelial origin. Yin et al. (20) reported CK18 was downregulated in poorly differentiated PC tissue samples, indicating a potential association of the downregulation of CK18 with tumor aggressiveness. Our data demonstrated Gleason score was negatively correlated with M30. This result can be explained by localization of M30 on CK18 that cause this down regulation at higher Gleason scores.

Bcl-2 is equilibrate between proapoptotic and antiapoptotic members which is a significant factor of tumorigenesis. Bcl-2 gene mutation in tumor cells, results with the increased expression and suppression of the proapoptotic proteins. Proapoptotic protein gen mutation causes a down-regulation of expression, and the cell loses ability of regulation apoptosis-again tumorigenesis (3,11). Kim et al. (3) reported meaningful correlation between the COX-2 and bcl-2 overexpression that their progressive overexpression was dependent to chronic inflammation and carcinoma. On the other hand Tanji et al. (11) found positive, but not significant correlation between bcl-2 and COX-2 expression. In our study bcl-2 and COX-2 was positively correlated.

We found an overexpression of COX-2 in PC. Unlike some reports, we found no relationship between COX-2 and angiogenesis. However, COX-2 was significantly positive correlated with bcl-2. Our results raise the possibility that COX-2 may influence tumor progression in PC through inhibiting apoptosis rather than the promotion of angiogenesis. The use of selective agents targeting apoptosis and COX-2 may play role in the treatment of PC.

Our results of survival analysis showed that expression of COX-2 is associated with a significantly worse survival. In this regard, overexpression of COX-2 may be useful for assessing the biologic behaviour of PC.

Study Limitations

We acknowledge this study has some limitations such as small number of cases lack of using molecular technics to detect apoptosis and angiogenesis. In summary, our results must be supported by further studies to offer new insights into the treatment strategies and to predict clinical outcome.

Ethics

Ethics Committee Approval: Date: 21.09.2016; Decision No: 50243401/2016-11, Informed Consent: It was taken.

Peer-review: Internal peer-reviewed.

Authorship Contributions

Concept: Emine Kılıç Bağır, Arbil Açıkalın, Volkan İzol, Gülşah Şeydaoğlu, Şeyda Erdoğan, Design: Emine Kılıç Bağır, Arbil Açıkalın, Volkan İzol, Gülşah Şeydaoğlu, Şeyda Erdoğan, Data Collection or Processing: Arbil Açıkalın, Volkan İzol, Analysis or Interpretation: Arbil Açıkalın, Literature Search: Gülşah Şeydaoğlu, Şeyda Erdoğan, Writing: Emine Kılıç Bağır, Şeyda Erdoğan.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.