Expression and Significance of Microsomal Prostaglandin Synthase-1 (mPGES-1) and Beclin-1 in the Development of Prostate Cancer

For the past few years, the incidence of PCa has a rising trend and it remains the most common cause of death among urologic malignance. Majority of PCa patients manifested androgen dependent prostate cancer (ADPC) in prophase of pathogenesis. However, most patients will develop AIPC after the initiation of androgen deprivation. This development could account for most of the morbidity and mortality associated with this disease. There is no any effective therapy for this disease today, so identification of a new effective biology based therapy is therefore mandatory (Hellerstedt and Pienta, 2002; Wingo et al., 2003). Prostaglandin E2 (PGE2) which converted from arachidonic acid has been shown to play important roles in development and progression of PCa (Jain et al., 2008; Jia et al., 2008; Myung and Kim, 2008). Inhibition of PGE2 production could be achieved by blocking the cyclooxygenases (COX) with COX inhibitors such as celecoxib and etoricoxib, respectively. And the trials have showed promising results to some extent. Unfortunately, recently released data has disclosed a significant increase in the incidence of cardiovascular adverse events among users of COX-2 inhibitors (Smith et al., 2006; Srivastava


Introduction
For the past few years, the incidence of PCa has a rising trend and it remains the most common cause of death among urologic malignance. Majority of PCa patients manifested androgen dependent prostate cancer (ADPC) in prophase of pathogenesis. However, most patients will develop AIPC after the initiation of androgen deprivation. This development could account for most of the morbidity and mortality associated with this disease. There is no any effective therapy for this disease today, so identification of a new effective biology based therapy is therefore mandatory (Hellerstedt and Pienta, 2002;Wingo et al., 2003). Prostaglandin E2 (PGE2) which converted from arachidonic acid has been shown to play important roles in development and progression of PCa (Jain et al., 2008;Jia et al., 2008;Myung and Kim, 2008). Inhibition of PGE2 production could be achieved by blocking the cyclooxygenases (COX) with COX inhibitors such as celecoxib and etoricoxib, respectively. And the trials have showed promising results to some extent. Unfortunately, recently released data has disclosed a significant increase in the incidence of cardiovascular adverse events among users of COX-2 inhibitors (Smith et al., 2006;Srivastava et al., 2009). So it is especially important to find other new drugs that target arachidonic acid metabolism and inhibit carcinogenesis.
Jakobsson has confirmed that PGE synthase (PGES) participant in converting PGH2 into PGE2 (Jakobsson et al., 1999). There are three PGESs presenting in human cells: microsomal prostaglandin-E synthase-1 (mPGES-1), microsomal prostaglandin synthase-2 (mPGES-2) and cytosolic PGES (cPGES). MPGES-1 is a very important inducible enzyme, which is functionally coupled with COX-2 and has a predominantly responsible for PGE2 generation in neoplastic tissues. The other two isoforms are constructive expression,and thought to be less important in cancer (Samuelsson et al., 2007;Banning et al., 2008;Kawata et al., 2010). Takasu et al. (2008) has reported that, in vitro mice trials, over-expression of mPGES-1 have been detected in gastric carcinogenesis and may contribute to progression of this carcinogenesis, Katarina (Rask et al., 2006) also found this phenomenon in ovarian epithelial cancer. Supporting that mPGES-1 is of importance for malignant transformation and progression. Hanaka has demonstrated that there also was a significant high expression of mPGES-1 in PCa cells and high expression of mPGES-1 can promote growth and survival in the cancer cells (Hanaka et al., 2009). These results implied us that inhibition of mPGES-1 is a therapeutic option for cancers that express this enzyme. Wu et al. (2009) had reported deletion of mPGES-1 played a protective role in cardiac ischemia in mice trials. Wang et al. (2006) also found that mPGES-1 deletion in mice rarely occured atherogenesis and inhibition of mPGES-1 could avoid cardiovascular risks of COX-2 inhibitor. Beales et al. (2010) had found that mPGES-1 inhibitor can block proliferation of oesophageal adenocarcinoma cells. The effectiveness and advantages of mPGES-1 inhibitor prompted us that mPGES-1 inhibitor may be an ideal therapeutic measure for cancers.
Beclin-1 is considered a very important factor for modulating autophagy and appear significant downregulation in many tumor cells including PCa (Gao et al., 1995;Chen and Karantza-Wadsworth, 2009). Moreover, there was also a negative correlation with pathological, clinical characteristics, such as stage, grade and so on the more malignant and higher stage, the lower-expression Beclin-1 is (Miracco et al., 2007). MPGES-1 also could up-regulate Bcl-2 expression to promote anti-apoptosis of cancer cells. In the meantime, Bcl-2 could inhibit autophagy mediated low-expression of Beclin-1 in cancer cells (Lalier et al., 2007;Ciechomska et al., 2009;Lian et al., 2010). In AIPC cells, Beclin-1 was also low-expresses and this may be related to anti-apoptosis mechanism of AIPC cells (Bhutia et al., 2010;Lian et al., 2011). But, till now, we do not know whether or not mPGES-1 involve in regulating Beclin-1 in AIPC.
We therefore sought to determine the expression of mPGES-1 and Beclin-1 in a series of surgically resected different prostate tissues and DU-145 cell line to examine the associations between these two factors and their impact on PCa.

Tissues specimen
All prostate tissues included in this study were from 40 adenocarcinoma of prostate cases, 40 benign prostatic hyperplasia (BPH) cases and 10 health men who have accepted medical examination that were diagnosed by two pathologists between 2000 and 2005. The median age of the PCa patients and BPH patients was 67 years (rang from 55 to 81 years) and 65 years (ranged from 53 to 82 years). History, transrectal ultrasound, computed tomography, magnetic resonance imaging and isotope scanning of the skeleton were combined to decide the clinical staging of PCa patients. Every PCa patients has received needlebiopsy. Tissue samples were obtained from 17 PCa patients whose clinical tumor staging were T1 or T2 and from other 23 PCa patients who had lost the opportunity to prostatectomy received neoadjuvant complete androgen ablation therapy based on luteinizing hormone-releasing hormone agonist and an anti-androgen treatment for 15 to 30 months (average 20.8 months). 9 PCa cases Gleason scores were ≤ 7, and 31 patients Gleason scores were > 7. Every BPH patient has accepted transurethral resection of the prostate (TURP). All patients were followed up 5 years after therapy: 14 patients who accepted radical prostatectomy did not have metastases and maintained very low PSA levels (below 0.5 ng/mL), with no relapse; and 3 patients who had had a radical prostatectomy had biochemical recurrences (average PSA level 1.7 ng/mL). Antiandrogen therapy was given intermittently to these 3 patients for a short period. We considered these 17 cases to be androgen dependent prostate cancer (ADPC). The other 23 patients who presented with rises in PSA levels or bone metastases were determined hormone independent PCa. The study was conducted with the approval of the ethical committee of Nanjing Medical University (Nanjing, China).

Specimen interpretation
Semi-determination of mPGES-1 and Beclin-1 expression was judged on the basis of the staining intensity and ratio of positive cells in five randomly selected fields under the high power lens.Stand for staining intensity, Integral Optical Density (IOD) was evaluated on a 3-point scale: 0 (no coloring), 1 (yellow), 2 (brown). The scoring criteria for the proportion of positive substances were as follows: 0 (less than 10% of positive cells), 1 (10%-40% of positive cells), 2 (40%-70% positive cells), 3 (more than 70% of positive cells). Sums of the two kind of the scores were defined as -(0 or 1), + (2), ++ (3 or 4) and +++ (5 or 6). Image Pro plus 6.0 software package was used for image analysis.

Cell culture
DU-145 cell lines (American Type Culture Collection, Rockville, MD, USA) were routinely cultured in RPMI 1640-maintained media containing 10% fetal calf serum, 25 U/mL penicillin and 25 μg/mL streptomycin.In certain experiments,cells were treated with mPGES-1 inhibitors (CAY10526, Cayman Chemical Company, USA) All experiments were repeated at least three times.

Quantitative real-time polymerase chain reaction
Total RNA from PCa and BPH issues was extracted using TRIzol (Gibco, Gaithersburg, MD, USA). QRT-PCR testing was performed using a qRT-PCR system according to the manufacturer's instructions (Takara, Shiga, Japan). The primers of mPGES-1 were as follows:up 5'-GAAGAAGGCCTTTGCCAAC-3' and down 5'-GGAAGACCAGGAAGTGCATC-3', and the length of the production was 200bp. The primers of Beclin-1 were as follows: up 5'-CTGAGGGATGGAAGGGTC-3' and down 5'-TGGGCTGTGGTAAGTAATG-3', and the length of the production was 159bp. Thirty-five cycles of amplification were performed under the following conditions:melting at 95ºC; annealing at 56ºC; and extension at 72ºC. The result was analyzed by 7500 Real Time PCR System (Applied Biosystems company, California, USA).

MTT assay
DU-145 cells were cultured till mid-log phase to obtain a stock cell suspension containing 1×10 5 cells/L. The stock cell suspension (100 µL) was then added to the wells of a 96-well plate. After 24h, the cells were treated with CAY10526 at various concentrations (1, 10, 20, 50 μM), then incubated at 37°C in a 5% CO 2 for 12h. Simultaneously, zero group and control group were established. After incubation, 20 µL MTT stock solution (5 mg/mL in PBS) was added to each well. The cells were further incubated at 37°C for 4h. The supernatant was discarded and 150 µL DMSO was added and the culture medium was shaken for 10 min in the darkness. Six replicates were established per group. Similar results were found in at least three repeat experiments. Finally, the optical density (OD) values were detected on a microplate reader (Biotech Instruments µQuant, USA) at a wavelength of 490 nm. The OD values were positively correlated with the cell viability.

Western blotting
Total cell lysates were obtained from the DU-145 cells.The cell lines were incubated with complete medium (CM), serum-free medium (SF) and intervened with CAY10526 (10 μM, 20 μM) for 12h in serum unsupplemented conditions. Equal amounts (35 μg) of protein were resolved by 5% and 10% SDS-PAGE and transferred onto PVDF membranes which was incubated with the appropriate rabbit polyclonal mPGES-1 antibodies (Cayman) with 1:5000 dilution and Beclin-1 antibodies (Epitomics) with 1:500 followed by incubation with peroxidase-conjugated secondary antibodies. The level of β-actin expression was used as the internal control for equal loading. The bands were compared by densitometry of Western blotting using an Eastman Kodak Co. Image Station 440CF, and the data were analyzed using Kodak ID V.3.5.4 (Scientific Imaging System).

Statistical analysis
SPSS 13.0 software packages were used for statistical analysis. Independent-sample t test was employed to analyze the results of immunohistochemistical staining and western blotting. Analysis of variance (ANOVA) was used to analyze the results of MTT assay. Pearson's χ 2 -test was used to analyze mPGES-1, Beclin-1 and associations with age and clinical-pathological stage and Gleason scores. Kendall test was used to demonstrate the rank correlation of the two variables. P value less than 0.05 was considered to be statically significant.

MpGES-1 immunostaining
The cinnamomeous staining mean positive. In our study,intense staining was seen 72.5% in PCa which were predominantly in endochylema. mPGES-1 was also seen in BPH (21%) and normal tissues (10%). There was significant difference in the mPGES-1 expression between the three groups (P=0.001) ( Table 1). A significant association was observed between mPGES-1 expression and higher Gleason scores (p<0.05) and tumor stage (P<0.05). However, there was no significant association between mPGES-1 expression and age (P>0.05) ( Table  2).
There were 9 and 20 mPGES-1 positive expression specimens in ADPC and AIPC respectively. And the positive rate of mPGES-1 in AIPC was significantly higher than that in ADPC (P<0.05) ( Table 3).
We used western blotting to analyze the expression of mPGES-1 and Beclin-1 after intervening by CAY10526 in DU-145 cells. The result revealed that the expression of mPGES-1 down-regulated in a concentration dependent manner.
There was no significant different of mPGES-1 expression between CM and SF group (P>0.05). The expression of Beclin-1 in the SF group was higher than that in the CM group (P<0.05). And there was a decrement of Beclin-1 expression following the increasing concentration of CAY10526. Furthermore, mPGES-1 was associated with Beclin-1 down-regulation in DU-145 cells. In addition, inhibition of mPGES-1 could efficiently upregulate Beclin-1 expression and decrease the viability of DU-145 cells (Figure 4). Microsomal Prostaglandin Synthase-1 (mPGES-1) and Beclin-1 in Prostate Cancer

Discussion
MPGES-1, as the teminal enzyme of synthesizing PGE2, was found coupled with COX-2 and overexpressed in many cancers including PCa (Nakanishi et al., 2010). So inhibiting the expression of mPGES-1 may be a potential therapeutic target in clinical practice (Murakami et al., 2000). Nakanishi has demonstrated that genetic deletion of mPGES-1 could suppress intestinal and lung tumorigenesis in vivo animal trials (Nakanishi et al., 2008;Pecchi et al., 2008). Cheng et al. (2006) has reported that mPGES-1 inhibitor could block PGE2 and make therapeutic effects in many diseases instead of nonsteroidal anti-inflammatory drugs (NSAIDs), meanwhile, no obvious side effects were observed. Recently, Hanaka et al. (2009) has focused on the role of mPGES-1 in PCa. He found that mPGES-1 over-expressed in both PCa tissues and human PCa cell lines. In addition, xenograft tumors deriving from the mPGES-1 knockdown cells performed delayed tumor development and growth. In our study, we have found that mPGES-1 expression was higher in PCa than that in BPH and normal control group. It also has significant association with higher Gleason scores and tumor stage in PCa tissues. Madaan et al. (2000) has reported that there was significantly greater expression of COX-2 in PCa than that in BPH and normal prostate tissues. Lee et al. (2001) also did some researches in above three prostate tissues and indicated that COX-2 correlated with cancer stage. These results provided us that mPGES-1, likewise COX-2, is a major factor in PCa. In our previous trials, mediated COX-2 had a association with the process of ADPC transforming to AIPC (Jia et al., 2008). In this data, mPGES-1 positive rate in AIPC was also significantly higher than that in ADPC (P<0.05). Implying that mPGES-1 may play an important role in androgen independent transformation and progression of PCa.
Autophagy, just like apoptosis, is an important way of cell death. Both of them interacted in many cancers Liu et al., 2009). As a representative of autophagy, Beclin-1 was identified as a protein that interacts with Bcl-2 (Liang et al., 1998;Pattingre and Levine, 2006). In AIPC cells, there is an increment of Beclin-1 with the decrease of Bcl-2 (Lian et al., 2010). In the mean time, Lalier has reported that mPGES-1 could up-regulate Bcl-2 expression in glioblastoma multiforme (Lalier et al., 2007). In our study, we found that Beclin-1 expression was significantly higher in BPH and normal tissues than that in PCa (P<0.05). Meanwhile, a significant association was observed among Beclin-1 expression, lower Gleason scores and tumor stage. The expression of mPGES-1 in PCa was higher than Beclin-1, conversely in BPH and normal group. MPGES-1 has a negative correlation with Beclin-1 in PCa tissues. Inhibiting mPGES-1 could make Beclin-1 up-regulation to mediate the death of AIPC cells. Suspecting that mPGES-1 and Beclin-1 may have a key function in the AIPC.
Lau has reported that mPGES-1 was relatively high expression in DU145 (Lau et al., 2000). So we selected the DU-145 cells for our study exclusively, meantime, applied CAY10526, a specific mPGES-1 inhibitior, to interference the DU-145 cell lines. The results has showed that the DU-145 cells cytoactive descended with the does of CAY10526 increasing from 10 to 20 μM although there is a plateau phase beyond 20 μM. There was a little different from Beales results that the optimal inhibition concentration of CAY10526 was 10μM in esophageal cancer (Beales et al., 2010). Thus, we considered that CAY10526 at 10 to 20 μM could inhibit mPGES-1 predominantly and this can be the consult for our subsequent research.
In mammalian cells, the autophagic process can be initiated by nutrient starvation, inflammation and neoplasm (Qu et al., 2003). In our research, Beclin-1 expression was higher in SF group than that in CM group. Conforming that, in starve circumstances, Beclin-1 expression could up-regulated to maintain DU-145 cells growth. After being intervened by CAY10526 at 10 μM, Beclin-1 expression down-regulated, however, the expression of Beclin-1 remained higher than that in control group (P<0.05). This demonstrated that inhibiting mPGES-1 could upregulate Beclin-1 expression to mediate DU-145 cells death to a certain degree. Nevertheless, with the cytoactive decreasing, mPGES-1 and Beclin-1 both down-regulated significantly (P<0.05). Owing to the cell viability gradually eclipsing, all kinds of protein would low-express in this process. We inferred that Beclin-1 expression associated with not only interaction by mPGES-1 but also cells survival condition. Taken together, these results indicate that mPGES-1 plays an important role in the mediation of Beclin-1 in DU-145 cells.
In summary, this is the first study to examine the relationship between mPGES-1 and Beclin-1 in different prostate tissues and vitro AIPC cell lines. To a certain degree, inhibiting targeted mPGES-1 could up-regulate Beclin-1 expression to induce autophagic death increases of AIPC cells. MPGES-1 may be a new promising effective treatment target in the future. (10μM,20μM). CM, complete medium group; SF, serum free group. Whole cell lysates were analyzed by Western blot using a specific antibody that recognized Beclin-1 and mPGES-1, respectively. B: mPGES-1 and Beclin-1 and β-actin bands were subject to densitometry on an Eastman Kodak Co. Image Station 440 CF, and the ratio of mPGES-1 and Beclin-1 and β-actin were plotted for quantification of the blots. Representative results of three independent experiments