Tumoral Markers in Prostate Cancer

In Mexico, in 70% of cases, the prostate cancer (PCa) is found in advanced stage. PCa currently occupies second place in frequency of cancer in men, surpassed only by skin cancer, and is the second principal cause of death in men after of lung cancer (Hall et al., 2005). Reactive oxygen species (ROS) such as superoxide (O2 −) and hydrogen peroxide (H2O2) are found in a large number of tumors and in high levels they induce cell death, apoptosis, senescence and angiogenesis (Ushio-Fukai & Nakamura, 2008). One of the major sources of ROS is NADPH oxidase (NOX). The NOX are a family of enzymes that are found in various tissues. The NOX receives an electron from NADPH generating O2 (Bánfi et al., 2001). Xia et al, Lim et al. and Brar et al. found that some NOX isozymes increase in association with ROS-production and tumor progression in ovarian and human colon cancer and in DU-145 cells of PCa, respectively (Brar et al., 2003; Lim et al., 2005; Xia et al., 2007). Cells have different antioxidant systems including low molecular weight antioxidant molecules and various antioxidant enzymes. Superoxide dismutase (SOD) catalyses the dismutation of O2  into H2O2 that can be transformed into H2O and O2 by catalase (CAT) (Genkinger et al., 2006). Mn-SOD is the major antioxidant in the mitochondria and is essential to the vitality of mammalian cells. In many types of tumor cells has been found to contain high levels of Mn-SOD, Cu/Zn-SOD or CAT expression compared to their nonmalignant counterpart such as in human tumor cancer cells of esophageal, gastric, ovary, breast, neuroblastoma, osteosarcoma, melanoma, pleura and leukemia (Grigolo et al., 1998; Janssen et al., 2000; Starcevic et al., 2003; Qian et al., 2005, López Laur et al., 2008). However, the role of these enzymes in carcinogenesis remains unclear. On the other hand, iNOS or NOS-2 is an inducible isoform of nitric oxide synthases (NOS). All isoforms of NOS catalyze the reaction of L-arginine, NADPH and oxygen to nitric oxide (NO), L-citrulline and NADP. NO is a lipophilic physiological messenger wich regulate a variety of cellular responses and may exert its cellular action by cGMP-dependent as well as by cGMP-independent pathways (Stamler, 1994). The expression of iNOS has been found to be increased in a variety of human cancers such as colon, stomach, brain and breasts cancers (Alderton et al., 2001; Church & Fulton, 2006) by multiple mechanisms that control their activity (Stamler, 1994; Friebe & Koesling, 2003).


Introduction
In Mexico, in 70% of cases, the prostate cancer (PCa) is found in advanced stage. PCa currently occupies second place in frequency of cancer in men, surpassed only by skin cancer, and is the second principal cause of death in men after of lung cancer (Hall et al., 2005).  (Genkinger et al., 2006). Mn-SOD is the major antioxidant in the mitochondria and is essential to the vitality of mammalian cells. In many types of tumor cells has been found to contain high levels of Mn-SOD, Cu/Zn-SOD or CAT expression compared to their nonmalignant counterpart such as in human tumor cancer cells of esophageal, gastric, ovary, breast, neuroblastoma, osteosarcoma, melanoma, pleura and leukemia (Grigolo et al., 1998 On the other hand, iNOS or NOS-2 is an inducible isoform of nitric oxide synthases (NOS). All isoforms of NOS catalyze the reaction of L-arginine, NADPH and oxygen to nitric oxide (NO), L-citrulline and NADP. NO is a lipophilic physiological messenger wich regulate a variety of cellular responses and may exert its cellular action by cGMP-dependent as well as by cGMP-independent pathways (Stamler, 1994). The expression of iNOS has been found to be increased in a variety of human cancers such as colon, stomach, brain and breasts cancers (

Patients and methods
We obtained 62 samples of prostate tissue through of various surgical procedures (transurethral resection and biopsy transrectal and diaminobenzidine (Vector Laboratories, Burlingame, CA). After of intensive washing in PBS, slides were counterstained with hematoxylin. Sections were dehydrated in graded alcohols, treated with xylene and subsequently mounted. All specimens were examined by light microscopy (Axiovert 200 M, Carl Zeiss, Germany), photographs were taken with a digital camera (Axiocam HRC, Carl Zeiss, Germany). The number of positive cells (brown) was determined with a computerized image analyzer KS-300 3.0 (Carl Zeiss, Germany). The percentage of damaged area with histopathological alterations was obtained (400x magnification). Five random fields were studied (total area 1,584,000  2 ). The results were expressed as a percentage.

Statistics
Findings were expressed as the mean ± SD. The statistical significance of the protein expression levels of p22 phox subunit of NOX, Mn-SOD, Cu/Zn-SOD, CAT, iNOS and COX-2 between PCa and BPH groups glands or stroma, was determined using the software Prism version 3.32 (GraphPad Prism 4.0 Software, San Diego, CA, USA) with "student t-test". It was considered a p <0.05 as statistical difference between groups.

Results
The results obtained in PCa and BPH groups are summarized in Table 1. NOX, Mn-SOD, Cu/Zn-SOD and CAT protein immunohistochemistry were significantly higher (1.76, 1.7, 1.78 and 5.88 fold, respectively) in stroma and were significantly higher (3.74, 1.69, 4.76 and 1.59 fold ,respectively) in gland of patients with PCa than that in patients with BPH. Moreover, NOX, Mn-SOD and CAT protein expressions were significantly higher in gland than in stroma, while as Cu/Zn-SOD protein expression was significantly higher in stroma than in gland in patients with BPH. NOX and Mn-SOD protein expression were significantly higher in gland than in stroma in patients with PCa. However, iNOS and COX-2 protein expressions were significantly higher in stroma and gland of BPH (1. 47    In both groups was determined % area marked by field (400x) and was analized the values with significative increase in gland PCa immunoreactivity.

Discussion
Recently, a new hypothesis has been proposed for prostate carcinogenesis. It suggested that exposure to environmental factors such as infectious agents and dietary carcinogens, and hormonal imbalances lead to injury of the prostate and to the development of chronic inflammation and regenerative 'risk factor' lesions, referred to as proliferative inflammatory atrophy (PIA). PCa is associated with oxidative stress, which stimulates the production of reactive oxidative species (ROS) and reactive nitrogen species. Oxidative stress derived from endogenous and exogenous sources are associated with DNA damage that occurs with aging and plays a role in carcinogenesis (Klein et al., 2006). The results obtained, for the first time, in this study showed an increased in the expression of p22 phox subunit of NOX, Mn and Cu/Zn-SOD and CAT in stroma and gland of PCa.
In previous studies concluded that NOX has a role as a signaling mechanism that regulates the cell growth and apoptosis in PCa (Vignais, 2002). The exact signaling pathways of NOX are uncertain and may be tissue specific. COX-1 and COX-2 regulate a key step in prostanoid (i.e., tromboxanes and prostaglandins) synthesis. Prostaglandins regulate various pathophysiological processes such as inflammatory reaction, gastrointestinal cytoprotection and ulceration (Smith & Langenbach, 2001). COX-1 is the constitutive isoform and COX-2 is the inducible isoform. COX-1 is expressed in most tissues and plays a role in the production of prostaglandins that control normal physiological processes. COX-2 is undetectable in most normal tissues (except for the central nervous system, kidneys and seminal vesicles), but is induced by various inflammatory and mitogenic stimuli (growth factors, pro-inflammatory cytokines and tumor necrosis factor) and other regulatory factors (Peppelenbosch et al., 1993;Zhang et al., 1998, Chen et al., 2001Dempke et al., 2001). Although the mechanism of COX-2 upregulation is not fully understood, it could result from activation of Ras and mitogen-activated protein kinase (MAPK) pathway. It has been recognized that Akt/PKB activity is implicated in Rasinduced expression of COX-2. COX-2 is regulated at transcriptional and post-transcriptional levels by proinflamatory agents. These pathways lead to the activation of regulatory factors that eventually bind the promoter region of the COX-2 gene. (Sheng et al., 1998(Sheng et al., , 2000.
In this study, we found that exist strong expression of COX-2 in stroma and gland of HPB, in comparison with PCa. There are conflicting data regarding whether COX-2 is increased in the epithelial , gland or the stromal component of tumors (Horsman et al., 2010). Liu et al were the first to describe tumorigenesis induced by COX-2 over-expression. In their study, the murine COX-2 gene was inserted downstream of a murine mammary tumor virus promoter. As a consequence, hyperplasia and carcinoma of the mammary gland were observed and associated with strong COX-2 expression in mammary gland epithelial cells with increase prostaglandin E2 levels. (Liu et al., 2001). The role of COX-2 in tumor promotion is more strongly supported by previous studies in colorectal tumor models describen by Oshima et al (Oshima et al., 1996). These findings have been confirmed analyzing many tumors including pancreas, skin, gastric, bladder, lung, head, and neck cancers, suggesting that COX-2, but not COX-1, may play a pivotal role in tumor formation and growth (Thun et al., 2002). COX-2-derived prostaglandins contribute to tumor growth by inducing angiogenesis that sustain tumor cell viability and growth. COX-2 is expressed within human tumor neovasculature as well as in neoplastic cells present in human colon, breast, prostate and lung cancer biopsy tissue. (Kerbel & Folkman, 2002). The proangiogenic effects of COX-2 are mediated primarily by three products of arachidonic metabolism: Tromboxane A2, Prostaglandins I2 and E2 and selective inhibition of COX-2 activity has been shown to suppress angiogenesis in vitro and in vivo (Tsujii et al., 1998;Masferrer et al., 2000;. We suggested that COX-2 overexpression in stroma inhibit apoptosis and promote angiogenesis in prostate gland. Our results suggest that iNOS and COX-2 play a key role in tumorigenesis and indicate that iNOS and COX-2-selective inhibitors could be a novel class of therapeutic agents for PCa.

Conclusions
We suggested that the O 2  /H 2 O 2 balance regulated by the over-expression of NOX, Cu/Zn-SOD, Mn-SOD and CAT is actively involved in tumor environment, cell proliferation, differentiation, tumor progression and angiogenesis of PCa. On the other hand, iNOS and COX-2 may promote blood vessel formation in gland from its overexpression in stroma by multiple mechanisms that involve reactive oxygen species, transcription factors, cytokines, growth factors and tumor necrosis factor. Moreover, we suggested that the NOX, Cu/Zn-SOD, Mn-SOD, CAT, iNOS and/or COX-2 in combination with PSA, could be a molecular markers or prognostic indicators for the early diagnosis and post-treatment monitoring of PCa.

Future research
Our research group is determining the gene expression and activity of nitric oxide synthases isoforms (eNOS, nNOS and iNOS), Mn-SOD, Cu/Zn-SOD, Glutathione peroxidase, Glutathione reductase, Glutathione-S-transferase, Catalase and Ciclooxygenase-2 to integrate the effect of the regulation of the antioxidant system in the development of prostate cancer and recently in breast cancer. Actually, we begin a new line of research where we studied the gene expression and polymorphisms of some components of the cytochrome P450 system as well as its association with the risk of developing prostate cancer and breast cancer. We found a protein over-expression of CYP2W1, 4F11 and 8A1, orphans cytochromes, in prostate cancer. We hope to find a molecular marker or prognostic indicator for prostate cancer and breast cancer.