Cancer, also called malignancy, is characterized by an abnormal growth of cells. There are more than 100 types of cancer, including breast cancer, skin cancer, lung cancer, colon cancer, prostate cancer, and lymphoma. Cancer is caused by uncontrolled proliferation and the inappropriate survival of damaged cells, which results in tumour formation. Cells have developed several safeguards to ensure that cell division, differentiation and death occur correctly and in a coordinated fashion, both during development and in the adult body.
Cancer is derived from single benign cell which undergone mutation. The initial mutation is carried on to genetically homogenous clones when transformed cell divides. In normal cell proliferation occurs only when required. With additional genomic alteration a cell population that can escape normal control of proliferation may ultimately evolve into cancer. This multistep process can occur in any of mutated subclones that initially were derived (Helena, 2006).
Many regulatory factors switch on or off genes that direct cellular proliferation and differentiation. Damage to these genes, which are referred to as tumour-suppressor genes and oncogenes, is selected for in cancer.
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Most tumour-suppressor genes and oncogenes are first transcribed from DNA into RNA, and are then translated into protein to exert their effects. Recent evidence indicates that small non-protein-coding RNA molecules, called microRNAs (miRNAs), might also function as tumour suppressors and oncogenes. There are several mechanisms that leads to cancer development such as activation of oncogenes, over expression of growth factors, inactivation of tumour suppressor genes, tumoral pathway, tumoral epigenetics, and inflammatory - mediated tumorigenesis. National Cancer Institute, 2008).
Biological function of the prostate
The prostate is a walnut-sized gland surrounding the urethra at the base of the bladder. It is surrounded by a fibroelastic capsule that penetrates the gland to divide it into lobes. As part of the male reproductive system, it produces a milky white fluid that helps to protect and transport sperm. The gland also helps to control the flow of urine (urination). The prostate contributes to the seminal fluid an alkaline liquid which is rich in spermine, phosphlipids, cholesterol, fibrinogenase, citric acid, fibrinolysin, zinc and acid phosphatase and other proteins. The seminal fluid consists further of the fluid produced in the seminal vesicles and the sperm.The sperm, produced in the testis, enters the upper portion of the prostate through the vas deferens. Sperm and fluid from the seminal vesicles then mix with secretions emitted from the prostate to form the seminal fluid that is expelled at the time of ejaculation.
Interestingly, the prostate is neither required for viability nor for basal levels of fertility. It is widely discussed that this might be the reason for its high incidence of cancer as other vitally important organs of the urogenital system, such as the seminal vesicles and bulbourethral glands, are nearly immune to neoplasias (Abate, 2000).
History of prostate cancer
Prostate cancer is defined as a cancer that forms in tissues of the prostate gland in the male reproductive system found below the bladder and in front of the rectum. Prostate cancer usually occurs in older men. Prostate cancers are generally of multifocal nature and belong the most heterogeneous tumors in humans (Macintosh et. al., 98). 70% of the tumors arise in the peripheral zone, whereas 15-20% arise in the central zone, and 10 -15% arise in the transitional zone.
Most of the prostate tumors are adenocarcinomas (95%), only about 4% of cases have transitional cell morphology and are thought to arise from the uroehelial lining of the prostatic urethra. Few cases have neuroendcrine morpholgy. These cells are believed to arise from the neuroendocrine stem cells normally present in the prostate as shown in figure 2.1.
Prostate cancer was first described in medical literature in 1817 by London physician George langstaff (Helena, 2006). The first time a prostate was surgically removed by radical perineal prostatectomy was in 1904 at the Johns Hopkins hospital by Young. This technique was used as a standard method for prostatectomy for 40 years and only minor modifications were made in order to reduce the morbidity of the operation.
Figure 2.1 . Tumor development in prostate cancer (Hanahan et. al., 2000)
According to American National Cancer Institute in 2008, it is estimated that 186 320 new cases and 28 660 deaths from prostate cancer in the United States. Prostate cancer is one of the most common male cancers and is the second most common cause of death by cancer in Western countries (Landis (a), 1998). Since early diagnosis of prostate cancer is difficult, it is usually found in the progressive form, about 33-40% of total prostate cancers (Landis (b), 1998).
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Based on National Cancer Registry Malaysia 2003, prostate cancer is the sixth common male cancer in Malaysia. According to the World Health Organisation (WHO) there was 679 023 new cases and 221 002 deaths from prostate cancer world wide in year 2002. Its pathologic characteristics and prognostic factors, such as serum prostate-specific antigen level (sPSA), Gleason score, tumor volume or size, DNA ploidy, chromosome abnormality and p53 mutation, have been studied extensively in Western countries (Bostwick et. al., 1997), (Rosai et. al., 1996). During the past 5 years, its significance in the incidence and cancer-related death of prostate cancer patient has now been increasing almost twice in Korea like other Asian country such as Singapore (Cancer Statistics in Korea, 2002). Thus, it is important to understand the pathologic characteristics and prognostic factors of prostate cancer in Malaysia because Malaysia is one of the Asian country. In addition to incidence, are there any more differences between the Western prostate cancer and the Asian one? A couple of studies have answered 'yes' to this question. Data on Japanese and Singaporean patients have shown that p53 over expression and mutation occur in prostate cancer with a considerably lower frequency than those found in Western populations (Chia, 2000, Watanabe, 1994, Uchida, 1993).
The number of males diagnosed each year with PCa is not similar among different racial populations. Bernstein and Ross (Bernstein, et. al., 1991) observed that PCa is most commonly diagnosed in African-Americans (116/100,000 persons per year). Intermediate incidence rates are found in Caucasians (71/100,000) and lowest rates among Asians (Japanese, 39/100,000; Chinese, 28/100,000). Thus, this particular registration found a more than 4 fold difference in PCa incidence worldwide. This difference is a conservative estimate when compared to cancer registration studies that found a 30-fold difference or more in PCa incidence between African-Americans and Asians (Nomura et. al., 1991).
The underlying causes of the development of prostate cancer remain largely unknown. The disease is heterogenous, probably reflecting a complex interaction between environmental and genetic factors. The only known risk factor for prostate cancer are age, family history and ethnicity.
Gene alterations on chromosome 1, 17, and the X chromosome have been found in some patients with a family history of prostate cancer. The hereditary prostate cancer 1 (HPC1) gene and the predisposing for cancer of the prostate (PCAP) gene are on chromosome 1, while the human prostate cancer gene is on the X chromosome (Verhage et. al., 2003). In addition, genetic studies suggest that a strong familial predisposition may be responsible for as many as 5-10% of prostate cancer cases. Recently, several reports have suggested a shared familial risk (inherited or environmental) for prostate and breast cancer (Stanford and Ostrander 2001). Men with a family history of prostate cancer have a higher risk of developing prostate cancer and are also likely to present 6-7 years earlier.
African American men have a higher prevalence and more aggressive prostate cancer than white men, who, in turn, have a higher prevalence than men of Asian origin (American Cancer Society, 2005). Studies have found that young African American men have testosterone levels that are 15% higher than in young white men. Furthermore, evidence indicates that 5-alpha reductase may be more active in African Americans than in whites, implying that hormonal differences may play a role. The independent contribution of race alone is difficult to qualify when the effects of health care access, income, education, and insurance status are also considered.
A high-fat diet may lead to increased risks, while a diet rich in soy may be protective. These observations have been proposed as reasons for the low prevalence of this cancer in Asia. Rates of prostate cancer are much greater in Japanese American men than in native Japanese men, supporting the association of a high-fat diet with cancer. Cell culture studies have shown that omega-6 fatty acids are positive stimulants of prostate cancer cell growth, while omega-3 fatty acids are negative stimuli (Chan et. al., 2001). These fats may exert their effects by alterations of sex hormones or growth factors or through effects on 5-alpha reductase.
Soy seems to decrease the growth of prostate cancer cells in mouse models; however, apart from epidemiologic factors, no direct evidence supports a beneficial effect in humans (Adlercreutz et. al., 1995). Vitamin E may have some protective effects because it is an antioxidant. Decreased levels of vitamin A may be a risk factor because this can promote cell differentiation and stimulate the immune system. Vitamin D deficiency was suggested as a risk factor, and studies show an inverse relationship between ultraviolet exposure and mortality rates for prostate cancer. However, a specific correlation between 1,25-dihydroxyvitamin D levels and palpable disease, well-differentiated tumors, or mortality is inconclusive.
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Selenium may have a protective effect based on epidemiologic studies and is also believed to extend its effect via its antioxidant properties. The Selenium and Vitamin E Cancer Prevention Trial (SELECT) is an ongoing intergroup, phase 3, randomized, controlled trial designed to test the efficacy of selenium and vitamin E alone and in combination in the prevention of prostate cancer (Shirai et. al., 2002).
Hormonal causes have also been postulated. Androgen ablation causes a regression of prostate cancer. In addition, as indirect evidence of hormonal causes, eunuchs do not develop adenocarcinoma of the prostate.
Hsing and Comstock (1993) performed a large study comparing patients with prostate cancer with controls and found no difference in levels of testosterone, dehydrotestosterone, prolactin, follicle-stimulating hormone, or estrone.
The Prostate Cancer Prevention Trial studied the prevalence of prostate cancer between a control group and a group given a 5-alpha reductase inhibitor (finasteride) (Thompson et. al., 2003). While the 5-alpha reductase inhibitor appeared to decrease the prevalence of tumors, those that did arise appeared histologically more aggressive. Only long-term follow-up of these patients will determine whether this more aggressive histology accurately reflects the underlying biology of these tumors or whether it is an artifact of the treatment.
Diagnosis and prediction of prognosis
Standard methods for diagnosis and assessment of prognosis in PCa include digital rectal examination (DRE), serum prostate specific antigen (PSA) and ultrasound-directed biopsies. Digital rectal examination (DRE), measurement of the prostate specific antigen (PSA) in the blood and the transrectal ultrasonography (TRUS) are the main parameter used in prostate cancer diagnosis. Nowadays prostate cancer is not diagnosed by symptoms, but because of increased levels of PSA in the blood and abnormal findings in the DRE. Thus it was possible to diagnose more and more patients at earlier stages of the disease, hoping to increase the probability of a cure. A biopsy is currently the only method to make a definitive diagnosis of PCa. Most commonly, transrectal needle biopsies are taken according to a standardized schedule. In order to improve the PCa detection rate multiple biopsy cores are taken (Davis et. al., 2002) and recent studies suggests that 8 to 12-cores are optimal (Presti et. al., 2003).
Prostate cancer antigen (PSA) is a tissue specific tumor marker routinely used to diagnose prostate cancer and to monitor treatment response, prognosis and progression of prostate cancer (Sadar et. al., 1999) It is a single-chain glycoprotein with a molecular mass of about 33 kD which functions in the liquefaction of seminal coagulum. Serum levels of PSA of healthy patients are between 0 - 4 ng/ml. In prostate tumor patients the PSA levels can raise up to 100 ng/ml. Generally, PSA levels rise with tumor volume, but it is expressed in all stages of cancer (Caplan et. al., 2002). Although PSA is the best marker for prostate cancer existing today, it is still far from being perfect. For example, PSA tends to increase with age and rises in men with evidence of benign prostatic hyperthrophy. Thus many men are diagnosed falsely positive for prostate cancer. On the other hand PSA levels do not increase in some patients with prostate cancer which leads to a false negative diagnosis. Additionally, preoperative PSA cannot be used to predict capsular penetration or seminal vesicle invasion. Further, PSA is not able to predict progression in adenocarcinomas of the prostate following radical prostatectomy (Sauvageot et. al., 1998)
Findings from the DRE are crucial. An irregular firm prostate or nodule is typical, but many cancers are found in prostates that feel normal. Pay careful attention to the prostate consistency, along with the seminal vesicles and adjacent organs, to detect spread of the disease to these structures.
Overdistended bladder due to outlet obstruction
Neurologic findings secondary to cord compression: Other subtle findings, such as paresthesias or wasting, are uncommon.
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TRUS is used to examine the prostate for hypoechoic areas, which are commonly associated with cancers but are not specific enough for diagnostic purposes. At least 6 or, more recently, 10 or more systematic biopsy specimens of peripheral and, occasionally, transitional zones are taken under ultrasonographic guidance. Samples should include most areas of the gland, irrespective of ultrasonographic abnormalities
For histopathological grading of PCa, the Gleason grading system is now universally acknowledged (Gleason and Mellinger 1974). The Gleason grading separates the architectural features of the cancerous glands into 5 histologic patterns of decreasing differentiation, pattern 1 being most differentiated and pattern 5 being least differentiated. The Gleason sum (score) is obtained by adding the dominating (primary) grade and the next most common (secondary) grade. The Gleason grade is one of the strongest predictors of outcome (Egevad et. al., 2001). However, a limitation of this grading system is that a majority of newly diagnosed cancers are Gleason score 6 tumors which can be either aggressive or indolent.
For accurate treatment of PCa, it is necessary to determine the stage of the disease. If the tumor is no longer confined to the prostate, the cancer is defined as non-curable and the treatment will be palliative. The most widely used system for staging PCa is called the TNM system (Grenee et. al., 2002). It describes the extent of the primary tumor (T stage), the absence or presence of spread to nearby lymph nodes (N stage) and the absence or presence of distant metastasis (M stage).
Not palpable or visible
Diagnosed on needle biopsy only
Confined to prostate
One-half of one lobe or less
>one-half of one lobe but not both lobes
Through prostatic capsule
Fixed or invading adjacent structures: bladder neck, external sphincter, rectum, levator muscles, pelvic wall
Regional lymph node(s)
Non-regional lymph nodes(s)
Table 2.1 : TNM staging criteria
Although especially in older patients with early stage cancers it is enough to carefully watch the tumor growth as these cancers usually grow at a very slow rate and the possible risks and side effects of therapy may outweigh the possible benefits, many prostate tumors need treated through surgery, radiation or hormones.
The radical prostatectomy and the radiation therapy are the most commonly used treatment forms for clinically localized prostate cancer (T1 and T2). The surgery involves removal of the entire prostate and in some cases of the surrounding tissues as part of the urethra and the seminal vesicles. Radiation may be used to destroy cancer cells that may have remained in the area after surgery, but it is also used as a stand alone therapy.
Tumors which have spread out of the prostate gland (T3) and are thus beyond the reach of a local treatment by surgery or radiation, are treated by hormonal therapy. Although hormonal therapy cannot cure, it usually shrinks or stops the advance of the disease. Drugs which are used nowadays for treatment are either antiandrogens, which block the action of the androgens (for example flutamides and bicalutamide) or drugs which block the testicals from producing testosterone (e.g. luteinizing hormone-releasing hormone (LH-RH) agonists as leuprolide and goserelin). Finally aminoglutethimide and ketoconazole are used to prevent the adrenal glands from producing androgens.
Chemotherapy is seldom used for prostate cancer treatment as the response rate is very low. Usually these unspecific systemic drugs are given when hormone therapy has failed. Today drugs such as Docetaxel, Doxorubicin or Estramustine phosphate are used for treatment.
In the last years the development of target drugs for the treatment of cancers has dramatically increased, a progress that is likely to continue in the future. This approach is based on the targeting of genes found to be overexpressed in tumors or other disease by monoclonal antibodies, small-molecules, immunotoxins and antisense oligonucleotides. This form of therapy has considerable advantage over unspecific systemic drugs such as the chemotherapy. They are more specific, thus less toxic, and more effective in the treatment of cancer (Stockwin and Holmes (a) 2003). Antibodies (150 kD) are used to target the extracellular portion of membrane proteins, whereas small-molecules can also inhibit the function of intracellular localized proteins as they can penetrate through the membrane (smaller than 1 kD) (Seemann et. al., 1990).
For example antibodies are used to treat indications as diverse as cancer, inflammation and infectious disease. They can be used as cell targeting reagents and thus tag specific cells for complement- or effector-mediated lysis. Antibodies can further be modified to deliver toxic or modulatory payloads (radionuclides or enzymes) (Stockwin and Holmes (b) 2003).
Benign Prostatic Hyperplasia
As men age, it is common for the prostate to get larger. This enlargement is called benign prostatic hyperplasia (BPH). BPH does not lead to prostate cancer. BPH affects more than half of men in their sixties and most men in their seventies and eighties (Adrian et. al., 2005).
Several large population studies have shown that progressive enlargement of the prostate gland is extremely common, and is seen in most men aged >70 years. The most studied populations are those of Baltimore (Maryland, USA), the Forth valley (Stirling, Scotland) and Olmsted County (Minnesota, USA). (Kirby et. al., 2005) They have provided much information about the complex interactions between BPH, lower urinary tract symptoms, concentration of prostate-specific antigen in serum, risk of urinary retention, urinary flow rate and risk of surgery over time.
In general, with ageing, there is a gradual increase in lower urinary tract symptoms, an increase in prostate size, and a decrease in peak urinary flow rate. In the Forth valley, there was a 25% overall incidence of a combination of reduced urinary flow, urinary symptoms and 'measurable BPH' (i.e. volume >20 cm3), rising to 43% in men aged >60 years. In Olmsted County, median urinary flow rate dropped by about 2% per year, and more rapidly in those presenting with restricted flow and in those aged >70 years (Maderschabcher et. al., 2005) The Baltimore Longitudinal Study of Aging showed that men with prostatic enlargement and obstructive symptoms were eight times more likely to undergo prostatectomy within ten years than those of the same age without prostatic enlargement.
Cellular and molecular pathology of BPH
The pathogenesis of BPH is incompletely understood. A multifactorial pathogenesis is likely because of the great variation in macroscopic, microscopic and molecular changes seen in BPH. Hyperplasia of the epithelial component of BPH is at least partly linked to age-related changes within the cells. Cells that are regularly lost are replaced by replication of stem cells hence, with differentiation, identical cell layers are created. Evidence suggests that this process gradually changes with ageing. This 'senescent phenotype' has been shown in BPH epithelial cells, resulting in abnormal cellular response to peptide growth factors and other cellular signalling, allowing the development of BPH (Clifford et. al., 2000) The prostatic stroma produces fibroblast growth factor-2, -7 and -10, which exert a stimulatory effect on epithelial growth.
The senescent epithelial cell phenotype overexpresses interleukin-1α and -8, which stimulate the production of fibroblast growth factor-2, -7 and possibly -10. Thus, the senescent prostatic epithelium is indirectly responsible for the production of its own stimulatory peptide growth factors. Fibroblast growth factor-2 is also stimulatory to stromal cells, thereby stimulating the growth of the cells that produce the factor. The senescent epithelial cell may lose the ability to respond to negative growth signalling such as transforming growth factor-β which, in health, inhibits epithelial growth (Kirby et. al., 2005). Increased concentrations of transforming growth factor-β as seen in BPH tissue promotes differentiation of stromal cells, which partly accounts for the increased amount of smooth muscle seen in BPH.
Age is associated with increasing prostate size related to BPH. Hyperplasia is initially seen in about the fifth decade, and increases with age. About 40% of men aged ≥50 years and >90% of men aged ≥80 years have microscopic histopathological evidence of BPH (McNicholas et. al., 2000).
Genetic - in studies of inherited factors predisposing to the development of BPH, candidate genetic polymorphisms are the androgen receptor and the SRD5A2 gene coding for the 5-alpha reductase enzymes responsible for the conversion of testosterone to dihydrotestosterone (which is about five times more potent at the androgen receptor) (McConnell et. al., 2003). Dihydrotestosterone appears to be necessary for the development of BPH.
Racial - the prevalence of and surgery rates for BPH are lower in Asian men than Caucasian controls. Analysis of the Olmsted County cohort with respect to racial origin showed a higher prevalence of moderate-to-severe lower urinary tract symptoms in Afro-Caribbean men than Caucasians. However, other studies have shown a similar rate of diagnosis of BPH and hospitalization rate for BPH-related surgery in Afro-Caribbean and Caucasian men (Kirby et. al., 2005).
Diet - modest associations with total energy and total intake of animal protein and risk of BPH have been reported. A 'western' diet seems to be a risk factor for BPH and prostate cancer (McNicholas et. al., 2000).
Diagnosis and assessment
Many men are anxious that they may have prostate cancer or may be going into renal failure; they are often aware of the risks of painful and unexpected acute urinary retention. Rapid estimation of these risks can be achieved by assessment incorporating, a history of symptoms, comorbidity and age careful examination of the prostate and lower abdomen measurement of creatinine and prostate-specific antigen in serum, and urinary flow rate (McNicholas et. al., 2000).
Severe, life-threatening urinary infection and renal failure are extremely unusual in men with lower urinary tract symptoms and BPH in western countries.
Accuracy is improved with transrectal ultrasound and measurement of prostate-specific antigen, but digital rectal examination may reveal palpable abnormalities. Studies have shown a prevalence of prostate cancer in up to 20% of men with a prostate-specific antigen (PSA) of 0-4 ng/ml. Assess whether the prostate feels healthy and regular in shape and consistency, or if hard nodules or irregularities are present, suggesting prostate cancer.
Men with relatively poorly functioning bladder detrusor muscle (detrusor underactivity) do not tolerate surgery well, and some authors suggest all surgical candidates should undergo pressure-flow studies to prove obstruction and a functioning detrusor before surgery.
There is little evidence for examination of the upper urinary tract by radiology or ultrasound unless there is a large residual urine, palpable bladder or raised creatinine. Transrectal ultrasound of the prostate is valuable if the digital rectal examination and or prostate-specific antigen are abnormal, and gives useful information on prostatic volume, but is most important for guiding the acurate placement of biopsy needles into the prostate gland.
Pharmacotherapy is used to relieve excessive smooth muscle tone within the lower urinary tract or to reduce prostatic size. Over-the-counter remedies for urinary symptoms are extremely popular with patients. The most common active ingredient is saw palmetto (extracted from the American dwarf palm tree), which is taken by more than two million American men.
α-blockers block, α-adrenoceptor sites found particularly at the bladder neck and trigone and within the prostate. They work quickly and on all sizes of prostate glands. They are usually well tolerated, but can affect blood pressure.
5-α reductase inhibitors work more slowly on established BPH to achieve moderate improvement in symptoms (over 4-6 months, though earlier changes have been recorded) by blocking the enzymes metabolizing testosterone to the much more potent intracellular dihydrotestosterone (McConnell et. al., 2003). An approximate shrinkage of 30% and a halving of concentrations of prostate-specific antigen can be expected. They are safe agents with an expected hormonal side-effect profile (altered libido and sexual function) in all age groups, but which is usually more problematic in younger, sexually active men.
Minimally invasive therapies usually involve heating the prostate gland by various means (electrical, microwave, laser). Insertion can be directly into the prostate via a needle or into the urethra via a catheter or probe.
Surgical procedures characteristically incise or remove obstructing tissue, usually requiring anaesthesia, a catheter and at least a short stay in hospital. Surgery relieves obstruction; other therapies do not appear to do so, but sufficiently relieve symptoms for many men (McNicholas et. al., 2000).
Although benign prostatic hyperplasia (BPH) is one of the most common disease processes affecting the aging male, surprisingly little is known about its pathophysiology. Cause-and-effect relationships have not been established, despite intense research efforts in the last four or five decades aimed at elucidating the underlying etiology of prostatic growth in older men. Previously held notions that the clinical symptoms of BPH (prostatism) are due simply to a mass-related increase in urethral resistance are too simplistic. It is now clear that a significant portion of the symptoms are due to obstruction-induced detrusor dysfunction.
COX-2 and cancer
Cyclooxygenase-2 (COX-2) is the inducible isoform of rate limiting enzyme that convert arachidonic acid to proinflammatory prostaglandins as well as a primary target for nonsteroidal anti-inflammatory drugs (NSAID). The PGs are a diverse group of autocrine and paracrine hormones that mediate many cellular and physiological processes (Turini et. al., 2002).
COX-2 expression may be a universal during carcinogenesis. In fact, the COX-2 expression is over expressed in several cancer tissues, such as breast carcinoma (Hwang et. al., 1998), gastric carcinoma (Ristimaki et. al., 1997), esophageal carcinoma (Zimmerman et. al. 1999), pulmonary carcinoma (Hida et. al., 1998), hepatocellular carcinoma (Koga et. al., 1999), squamous cell carcinoma of head and neck (Chan et. al., 1999) and pancreatic carcinoma (Tucker et. al., 1995).
The COX-2 signaling pathway is important in cancer because when it's activated, it can stimulate many key steps in cancer development, including cell division, inhibition of cell death, angiogenesis (the creation of new blood vessels to nourish growing tumors) and metastasis (Harris et. al., 2004).
Cyclooxygenase-2 (COX-2) is also formally called prostaglandin H2 synthase-2 (PGHS-2), mostly in the American literature. COX-2 and COX-1 are isoforms of an enzyme which catalyses the first stage in the oxidation of arachidonic acid to the prostanoids. COX exists in two isoforms commonly known as COX-1 and COX-2, the order in which they were identified.
Although both isoforms catalyze the same enzymatic reactions and have similar Km and Vmax values for arachidonic acid, significant differences exist between them. COX-1 is constitutively expressed in majority of the cells performing the housekeeping functions that require immediate generation of prostanoids related to vascular homeostasis, water reabsorption, gastric acid secretion, platelet aggregation and renal blood flow.
COX-1 is located on human chromosome 9q32-q33.3 spanning 25 kb in size containing 11 exons and produces 2.8kb mRNA which synthesizes about 68 kDa protein (Bakhle et. al., 2001). In contrast, COX-2 is a pro-inflammatory and inducible enzyme which can be induced by mitogens, tumor promoters, cytokines and growth factors in different cell types and controlled at both the transcriptional and post-translational levels (Herchman, 1996).
COX-2 in involved in differentiative processes, such as inflammation, ovulation, and labor, in situation where only transient PG production is required. COX-2 is an 8 kb gene with 10 exons located on human chromosome 1q25.2-q25.3 and transcribes a 4.1-4.5 kb mRNA which encodes a protein of about 68 kDa. Although, genes for COX-1and COX-2 are located on two separate chromosomes but they are highly related at theDNA,RNA, and protein level.COX-1 and COX-2 consist of 576 and 587 amino acids, respectively, and they share approximately 60% primary sequence homology (Kosaka et. al., 1994). Both these enzymes exist as integral membrane glycoprotein homodimers and are found on the luminal surfaces of the endoplasmic reticulum and nuclear envelop (Otto et. al., 1995).
In recent years, overexpression of COX-2 has been implicated in the progression of cancer (DuBouis et. al., 1998). Aberrant or increased expression of COX-2 has been found in most of the cancers of the body sites (Lipsky et. al., 1999). Compelling evidence from genetic and clinical studies indicates that COX-2 upregulation is one of the key steps in carcinogenesis (Prescott et. al., 2000).
Several studies have shown that overexpression of COX-2 is sufficient to cause tumorigenesis in animal models and subsequently inhibition of the COX-2 pathway results in reduction in tumor incidence and progression (Liu et. al., 2001).
Madaan et. al., (2000) have determined COX-1 and COX-2 expression in 30 BPH and 82 prostate cancer specimens. In this study a significant COX-2 overexpression in tumor cells was found compared to benign glands; however, COX-1 expression in tumor cells was similar to benign glands. A significant positive correlation between COX-2 expression was found with increasing tumor grade suggesting that COX-2 may play an important role in prostate carcinogenesis. These studies are in agreement with the study by Lee et. al., (2001) where COX-2 was found to be over-expressed in 15 out of 18 (83%) prostate cancer samples and was detected in only 22% (4/18) of paired benign tissues.
The important biological difference between the isoforms is that COX-1 is normally present in most types of cells and is a constitutive, housekeeping enzyme. The latter characteristic is derived from its DNA and RNA structures and in practical terms implies that amounts of COX-1 protein remain virtually constant (about 2 ± 5 fold variation) under either physiological or pathological conditions. By contrast, COX-2 protein is normally absent from most cells ± with some notable exceptions ± but appears rapidly (2 ± 4 h) in large amounts in a range of pathological, often inflammatory, situations and in many cell types.
One further aspect of COX biochemistry must be emphasised. Both COX-1 and COX-2 form the same product, PGH2. This PG is the common precursor for the biosynthesis of thromboxane A2 (TxA2), prostacylin (PGI2) and the other prostaglandins PGD2, PGE2, PGF2a. These `post COX' transformations are catalysed by quite separate enzymes. It is these prostanoids that determine the final biological response to the action of COX.
Aberrant or increased expression of COX-2 has been implicated in the pathogenesis of many diseases, including carcinogenesis. Evidence for this role has come from epidemiologic, in vitro pharmacologic, in vivo animal, and human clinical studies. The molecular and clinical consequences of COX-2 overexpression in neoplasia are currently being elucidated. Increased expression has been shown to correlate with increased angiogenesis, decreased apoptosis, increased tumor invasiveness, and immunosuppression in a variety of tumors.
Preclinical findings have also implicated COX-2 in PCa. Immunohistochemical, protein, and RNA studies have shown that human PCa consistently overexpresses COX-2. Increased COX-2 expression is also seen in lymph node metastases, suggesting that in the prostate COX-2 may act early in disease progression. Several studies have observed that COX-2 expression correlates with PCa stage, grade, and progression (Raj et. al., 2006).
Growing interest in the use of nonsteroidal anti-inflammatory drugs (NSAIDs) as chemopreventive agents has been spawned by epidemiological and animal-model studies showing the effectiveness of NSAIDs in reducing cancer risk (Taketo, Dubois, Ziegler, 1998). However, despite recent advances in the clinical application of NSAIDs in cancer prevention, the biochemical basis by which these anti-inflammatory agents exert their anti-tumor activity remains elusive.
COX-2 has been found to be dramatically upregulated in various types of cancer cells. The premise that COX-2 is involved in the pathological process of cancer growth and progression is supported by animal studies indicating that tumorigenesis is inhibited in COX-2 knockout mice (Taketo, 1998, Oshima, 1996). Furthermore, overexpression of COX-2 led to phenotypic changes in intestinal epithelial cells that could enhance their tumorigenic potential (Tsujii et. al., 1995).
Selective inhibitors of COX-2 have been demonstrated to induce apoptosis in a variety of cancer cells, including those of colon (Erickson et. al., 1999), stomach (Glaser et. al., 1997), and prostate (Hsu et. al., 2000).
Moreover, evidence suggests that there exists a correlation between COX-2 expression and susceptibility to the apoptotic effect of COX-2 inhibitors. Currently, a number of selective COX-2 inhibitors are clinically available, and some of them show promising chemopreventive functions (Kellof, 1999).
Several epidemiological studies suggest that extensive NSAID use can decrease prostate cancer risk (Nelson, 2000). To accurately assess the molecular pathway by which COX-2 inhibitor may prevent prostate cancer, a better understanding of the level and location of COX-2 expression in normal prostate and various prostatic lesions is critical.
Results from a new, five-year study show that regular use of popular prescription pain relievers may reduce the risk of breast cancer by up to 71 percent and may offer similar benefit in the prevention of prostate, colon and lung cancers (Michelle et. al., 2006).
Harris et. al., (2006) discovered that women who used NSAIDs on a regular basis had less breast cancer. Specifically, they found that those who used celecoxib or rofecoxib for at least two years appeared to benefit the most, experiencing a 71 percent reduction in risk of breast cancer. Ibuprofen use over the same period was associated with a 64 percent reduction, while regular aspirin offered a 51 percent reduction in risk of the disease.
NSAIDs have long been known for their analgesic, antipyretic and anti-inflammatory function (Taketo, 1998). Since inflammation is closely related to tumor promotion, agents with potent anti-inflammatory activities are anticipated to exert chemopreventive effects. These observations led to a wide variety of investigations to determine whether or not these drugs have an ability to reduce the riskof progression of severalhumancancers. NSAIDs and their mode of action in cancer chemoprevention has been the focus ofmanyepidemiologicaland experimental studies, which support the importance of these drugs in cancer chemoprevention (Moran, 2002).
Epidemiological studies have shown a decreased risk of some cancers in people who regularly take aspirin or other NSAIDs (Moran, 2002) and (Xu, 2002). Many subsequent studies in several human cancers have established the anti-tumorigenic effect of thesedrugs.These studies haveshown that antitumorigenic action of NSAIDs is mediated by selective inhibition of COX, particularly COX-2.
Non-specific NSAIDs such as aspirin, sulindac and indomethacin inhibit not only the enzymatic action of inducible and pro-inflammatory COX-2, but also the constitutively expressed, cytoprotective COX-1 as well. Consequently, non-selective NSAIDs can cause platelet dysfunction, gastrointestinal ulceration, and kidney damage (Murray et. al., 1997). For this reason, selective inhibition of COX-2to treat neoplastic proliferation is preferred over non-selective inhibition. Selective COX-2 inhibitors such as melaxicam, celecoxib and rofecoxib are NSAIDs that have been modified chemically to preferentially inhibit COX-2 without affecting COX-1.
The risk of prostate cancer was also significantly reduced in men who were reported to be taking prescription NSAIDs (odds ratio = 0.35, 95% confidence interval = 0.15-0.84, P < 0.05). Another recent study (Roberts et. al., 2002) employing 1362 white men ranging from 50-79 year-old from the Olmsted County, Minnesota using prescription and nonprescription NSAIDs has shown that daily use of NSAIDs may be associated with a lower incidence of prostate cancer in men aged 60 years or older. The stronger effect among older men suggests that NSAIDs may prevent the progression rather than early stages of prostate cancer from latent to clinical disease.
Immunohistochemistry is the localization of antigens in tissue sections by the use of labeled antibody as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold.
Albert H. Coons and his colleagues were the first to label antibodies with a fluorescent dye, and use it to identify antigens in tissue sections. With the expansion and development of immunohistochemistry technique, enzyme labels have been introduced such as peroxidase and alkaline phosphatase. Colloidal gold label has also been discovered and used to identify immunohistochemical reactions at both light and electron microscopy level. Other labels include radioactive elements, and the immunoreaction can be visualized by autoradiography.
Since immunohistochemistry involves specific antigen-antibody reaction, it has apparent advantage over traditionally used special enzyme staining techniques that identify only a limited number of proteins, enzymes and tissue structures. Therefore, immunohistochemistry has become a crucial technique and widely used in many medical research laboratories as well as clinical diagnostics.
There are numerous immunohistochemistry methods that may be used to localize antigens. The selection of a suitable method should be based on parameters such as the type of specimen under investigation and the degree of sensitivity required.
Special controls must be run in order to test the protocol and for the specificity of the antibody being used.
Positive control is to test a protocol or procedure and make sure it works. It will be ideal to use the tissue of known positive as a control. If the positive control tissue showed negative staining, the protocol or procedure needs to be checked until a good positive staining is obtained.
Negative control is to test for the specificity of an antibody involved. First, no staining must be shown when omitting primary antibody or replacing an specific primary antibody with normal serum (must be the same species as primary antibody). This control is easy to achieve and can be used routinely in immunohistochemical staining.
Direct method is one step staining method, and involves a labeled antibody (i.e. FITC conjugated antiserum) reacting directly with the antigen in tissue sections. This technique utilizes only one antibody and the procedure is short and quick. However, it is insensitive due to little signal amplification and rarely used since the introduction of indirect method.
Indirect method involves an unlabeled primary antibody (first layer) which react with tissue antigen, and a labeled secondary antibody (second layer) react with primary antibody (Note: The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised). This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. In addition, it is also economy since one labeled second layer antibody can be used with many first layer antibodies (raised from the same animal species) to different antigens.
The second layer antibody can be labeled with a fluorescent dye such as FITC, rhodamine or Texas red, and this is called indirect immunofluorescence method. The second layer antibody may be labeled with an enzyme such as peroxidase, alkaline phosphatase or glucose oxidase, and this is called indirect immunoenzyme method.