Oncogenes (Ras & src) Interleukin 1 Hypoxia Benzo[a]pyrene uv light

Epidermal growth factor Transforming growth factor beta TNF alpha

Dexamethasone Antioxidants p53

PGE2 production

FIGURE 1 Induction and suppression of cyclo-oxygenase-2. Abbreviations: ras, reticular activating system; TNF, tumor necrosis factor.

metastases, IL-6 with cancer cell invasion and haptoglobin with implantation and angiogenesis. These processes, some of which are discussed in more detail later in this chapter, provide a basis for our understanding of both selective and nonselective COX-2 inhibitors in carcinogenesis.

Cyclo-Oxygenase-2 Inhibition in the Chemoprevention of Esophageal Cancer: Scientific Data

COX-2 overexpression has been reported in patients with both BE and esophageal adenocarcinoma. Several studies have demonstrated that COX-2 expression is increased serially along the BE-dysplasia-adenocarcinoma sequence. They consistently show absence of or negligible COX-2 in normal esophageal epithelium (26-30). While the Zimmerman study also demonstrated absence of COX-2 in Barrett's metaplasia cells, the other four studies reported expression of COX-2 in between 50% and 81% of Barrett's metaplasias. All five studies reported COX-2 expression in between 78% and 100% of esophageal adenocarcinomas. Interestingly, high COX-2 expression has also been shown as an independent prognostic variable in patient survival, suggesting that tumors with a high COX-2 content have a more aggressive course (30).

Most research into the role of COX-2 inhibition in carcinogenesis has concentrated on animal studies and human colonic cancers. The hypothesis for COX-2 inhibition as a preventa-tive strategy for esophageal adenocarcinoma originated from supportive evidence for its role in colonic cancer (31,32). The implication is that the studies and mechanisms discussed in this chapter may be transferable in our understanding of esophageal carcinoma. Indeed, COX-2 inhibition has been shown to reduce the development of esophageal adenocarcinoma in animal models (33).

The role of COX-2 in carcinogenesis appears to be in resisting apoptosis, increasing cell proliferation, stimulating angiogenesis, modulating a cancer cell's invasive properties and increasing inflammation (Fig. 2). The molecular role of COX-2 inhibitors in these individual processes is discussed as follows:

Reduced Resistance to Apoptosis

Inhibition of programmed cell death can lead to the proliferation of abnormal cells. This results in clonal expansion of tumor cells. Most research to date has focused on human colonic cancer cells, and there are various mechanisms by which COX-2 has been implicated in this process. First, malignant cells expressing COX-2 have been shown to contain higher levels of the anti-apoptotic protein bcl-2 (34). NSAIDs have been seen to reverse the resistance to apoptosis by down regulation of bcl-2 (35). Second, COX inhibition has been shown to both actively induce apoptosis (36,37) and to reduce the expression of the transcriptional factor, nuclear factor kappa B (NF-kappa B), which prevents apoptosis (38).

Inhibition of Cell Proliferation

The Ras/Raf/MAPK pathway is a key growth-stimulating cascade which results in cellular proliferation. COX-2 is induced by activation of this pathway (39) and NSAIDs can inhibit this process. Selective COX-2 inhibitors can prevent epithelial cells progressing from the G0/G1 quiescent stage in the cycle to the S-phase of DNA replication, thereby reducing cell proliferation.

Inhibition of Angiogenesis

The growth of a tumor is dependent on its blood supply. A tumor's secretion of vascular growth factors is increased by its overexpression of COX-2 (40). Selective COX-2 inhibitors therefore inhibit angiogenesis (41). Selective COX-2 inhibitors also reduce angiogenesis via MAPK pathway inhibition (42).

Reduction in Invasion

COX-2 overexpression is shown to increase the invasive properties of cancer cells by increasing PG production (43), activating metalloproteinases 1 and 2 and by increasing the cell-surface receptor CD44 (44). It has also been seen to increase tumor dissemination in animals (43).

Core Values
FIGURE 2 Role of cyclo-oxygenase-2 in carcinogenesis. Abbreviations: MAPK, mitogen-activated protein kinase; PG, prostaglandin; NF-kappa B, nuclear factor-kappa B; PGE2, prostaglandin E2; MHC Ag, major histocompatibility antigens.

Inhibition of Immunosuppression

Tumor cells release colony-stimulating factors that result in PGE2 production. PGE2 works at a cellular level allowing the tumor cells to escape normal immune surveillance. PG synthesis can by inhibited by NSAIDs, indirectly enhancing immune responses (45). They may also upregu-late expression of major histocompatibility complex antigens (MHC Ag) (46).

Inhibition of Inflammation

The chronic inflammation of BE is a recognized risk factor for malignant change (47). It would therefore follow that reduction in cytokine-mediated induction of COX-2 and subsequent reduced synthesis of PGs would result in reduced carcinogenesis.

As an exeption, despite the evidence to support NSAID-associated chemoprevention as a COX-2-mediated phenomenon, several factors are likely to play a part in the process. Genetic predisposition is among these. Recently published data indicated that the role of NSAIDs is influenced by protein expression of the cyclin D1 gene in tumors (48). It was concluded that the risk for developing esophageal adenocarcinoma was reduced by aspirin and other NSAIDs only in patients with cyclin D+ tumors and not in cyclin D- tumors. Of note, this data was not specific to patients with previous BE.

Cyclo-Oxygenase-2 Inhibition in the Chemoprevention of Esophageal Cancer: Clinical Data

Many epidemiologic studies have shown a reduction in the development of malignant disease with the long-term use of NSAIDs, especially of gastrointestinal cancers (49). Statistically significant studies looking at the use of aspirin favor the drug as a chemopreventative agent in esophageal cancer. For example, Thun et al. demonstrated an approximately 40% lower risk of esophageal carcinoma in patients who used aspirin at least 16 times per month for at least a year (50). Occasional aspirin use has also been shown to reduce the risk of esophageal adeno-carcinoma by 90%, according to data from the National Health and Nutrition Examination Survey and the National Epidemiological Follow-up Studies, which looked at over 14,000 patients (evidence level 2b) (51). This data was supported by a case-controlled study by Farrow et al., who reviewed 650 cases and 695 controls (52). They also reported a reduced incidence of esophageal squamous-cell carcinoma in aspirin users.

Two further case-controlled studies have looked at the role of NSAIDs in esophageal ade-nocarcinoma, but results differed. The first, a population-based study, found a protective effect of NSAIDs based on their 12,174 cases and 34,934 controls (53). These findings however were not supported by a single hospital-based study which found no significant reduction in cancer risk with NSAIDs (54).

The most conclusive data so far for the use of COX-2 inhibitors in the prevention of esopha-geal adenocarcinoma comes from a systematic review with meta-analysis of observational studies evaluating the association of aspirin/NSAID use and esophageal cancer (55). Nine studies (two cohort, seven case control) containing 1813 cancer cases were identified and independently reviewed. Pooled results support a protective association between aspirin and NSAIDs and esophageal cancer (of both histologic types). Results also provided evidence for a dose effect. They concluded that their findings would support the evaluation of these agents in clinical trials of high-risk patients (evidence level 2b).

One example of a high-risk group might include those patients with BE. The specific role of COX-2 inhibition in BE is discussed as follows.

Cyclo-Oxygenase-2 Inhibition and Chemoprevention of Malignant Change in Barrett's Esophagus

As the aforementioned in vivo studies have looked at the overall incidence of esophageal cancer, irrespective of the presence of Barrett's metaplasia, the role of COX-2 inhibitors in Barrett's metaplasia has also been studied in both in vitro and animal studies. In primary cultured endoscopic biopsy specimens of Barrett's tissue, selective COX-2 inhibitors were found to significantly decrease both COX-2 activity and proliferation of epithelial cells by 55% (56). Buttar's team also demonstrated a 55% reduced relative risk of developing esophageal adenocarcinoma and 79% reduction postesophagojejunostomy in rat models compared with controls. The prevalence of BE in the two groups was not significantly different (57).

A single completed in vivo study has demonstrated possible chemopreventative qualities of selective COX-2 inhibitors. Kaur et al. compared biopsy specimens of Barrett's epithelium with samples taken after 10 days of rofecoxib 25-mg daily. Rofecoxib resulted in a 77% reduction in COX-2 expression, 59% decrease in PGE2 content, and 62.5% reduction in proliferating-cell nuclear antigen expression (58).

These findings have initiated the start of two ongoing randomized clinical trials. One multicenter trial by Forastiere et al. (Baltimore, U.S.A.) is looking at the effect of selective COX-2 inhibitors in low- and high-grade dysplasia. The second is a European study being conducted by Attwood et al. comparing the effect of PPIs alone with the effect of a PPI plus a selective COX-2 inhibitor in the prevention of esophageal adenocarcinoma in Barrett's patients. Their outcomes are awaited.

Whether a selective or nonselective COX-2 inhibitor is preferable for chemoprevention in Barrett's patients is uncertain. Selective COX-2 inhibitors appear to have a lower side-effect profile than NSAIDs when used long term. In one study, the effects of a selective COX-2 inhibitor (rofecoxib) were compared with aspirin in BE patients on varying doses of the PPI esomeprazole (59). Biopsies of the BE segment and esophageal mucosa found that only the combination of PPI and aspirin resulted in a significant reduction in COX-2 and PGE2.

The Specific Role of Aspirin in Chemoprevention of Esophageal Cancer

It has been recognized for over a decade that aspirin, a nonselective COX inhibitor, reduces the risk of developing oesophageal cancer. Corley et al.'s meta-analysis referred to earlier concluded that there exists a protective association between aspirin and NSAIDs and oesophageal cancer of both histologic types, the effect of aspirin being greater (55). It also provided evidence for a dose effect, with greater protection being afforded by frequent compared with intermittent drug use. A similar conclusion for the chemopreventative role of aspirin was reached in recently published data of three case-controlled studies (60).

Whether this data can be accurately extrapolated to cover the role of aspirin in Barrett associated adenocarcinoma is of major importance in the management of BE. While aspirin is known to be associated with several complications, including gastrointestinal haemorrhage and strokes, a study looking at the cost-effectiveness and safety of aspirin in the prevention of Barrett's adenocarcinoma supported its use (61). It concluded that, regardless of whether the patient undergoes endoscopic surveillance, enteric-coated aspirin used in the management of BE is a cost-effective strategy for preventing malignant change.

The exact mechanism by which aspirin exerts its chemoprotective effect is uncertain. Various hypotheses exist and these are discussed in the following (Table 3). Some have already been outlined in relation to specific COX-2 inhibition.

Induction of Apoptosis

As previously stated, programmed cell death is essential for the prevention of clonal expansion of abnormal or malignant cells. The role of aspirin in this process was demonstrated by examining its effect on the growth and apoptosis of ten oesophageal cancer cell lines (62). Results showed that: (i) growth inhibition by aspirin was dose and time dependent and associated with the induction of apoptosis; (ii) bile acids could induce COX-2 expression in six out of eight cell lines tested, correlating to PGE2 production (a product of COX-2); (iii) aspirin could inhibit the enzymatic activity of COX-2 induced by bile acids; (iv) bcl-2 was downregulated by aspirin in the two cell lines tested. They thereby surmised that induction of apoptosis by aspirin may be one mechanism by which the drug interferes in esophageal carcinogenesis.

Inhibition of Prostaglandin E2

Both human and experimental esophageal tumors contain increased PGE2, probably resulting from activation of COX-2 in response to mitogens and growth factors. The increased level of PGE2 is thought to accelerate cell proliferation within the malignant tissue (63). The optimal dose of aspirin to suppress such an effect remains unclear.

The E-cadherin-Catenin Complex

Beta-catenin has an essential role in intercellular adhesion and signal transduction, functioning as a transcriptional activator downstream in the wnt signaling pathway. It has been implicated in the development of several cancers, including gastric (64), oesophageal squamous cell (65), and colorectal (66).

The E-cadherin-catenin complex is involved in adhesion of epithelial cells. Disruption of a part of this complex results in poor differentiation and increased invasiveness of cancers, as

TABLE 3 Hypothesized Chemotherapeutic Actions of Aspirin

Induction of apoptosis bcl-2 down regulation Inhibition of PGE2 Beta-catenin expression E-cadherin expression Transcription factor inhibition NF-kappa B AP-1

MAPK pathways

Abbreviations: MAPK, mitogen-activated protein kinase; NF, nuclear factor; PGE2, prostaglandin E2; AP-1, activating protein-1.

has been seen in gastric carcinoma (64). Tumors retaining normal membranous beta-catenin have a survival advantage, suggesting abnormal beta-catenin to be a poor prognostic marker. One study indicated that altered subcellular distribution of beta-catenin occurs frequently in dysplastic BE and possibly reflects the signaling function of this molecule (67).

Specific evidence for a relationship between aspirin and beta-catenin in esophageal adenocarcinoma has not been established. However, aspirin and indomethacin have been shown to downregulate beta-catenin/T-cell factor (Tcf) signaling in colorectal cancer cells via enhanced phosphorylation (66). The role of aspirin therefore remains a promising key target in esophageal anticancer therapy.

Nuclear Factor-Kappa B and AP-1 Inhibition

NF-kappa B and AP-1 are two important transcription factors governing the expression of many early response genes involved in inflammation and carcinogenesis. Environmental or occupational exposure to certain chromium particles can cause inflammation and malignancy. It has been suggested that these particles activate NF-kappa B and AP-1 expression and that aspirin substantially inhibits this process (68). The data suggests that the activation of AP-1 or NF-kappa B is through involvement of the MAPK or I kappa B kinase (IKK) pathways, respectively. This provides a further possible explanation for the molecular role of aspirin in the chemoprevention of esophageal cancer. Similar data in skin cancer supports the promising role of aspirin in chemoprevention by inhibition of ultraviolet light-induced AP-1 activity (69).

Mitogen-Activated Protein Kinase Pathways

The role of MAPK pathways have been discussed earlier in reference to acid exposure and are clearly not specific to aspirin. Both selective and nonselective NSAIDs inhibit angiogenesis through direct effects on epithelial cells. Angiogenesis is necessary for the growth and metastasis of solid tumors. The role of NSAIDs in this process involves inhibition of MAPK (ERK2) activity and interference with extracellular signal regulated kinase ERK nuclear translocation. It is independent of protein kinase C and has PG-dependent and PG-independent components (42).

Selective Cyclo-Oxygenase-2 Inhibitors as Neoadjuvant Therapy for Esophageal Cancer

In contrast to COX-1 inhibition, administration of a selective COX-2 inhibitor significantly suppresses cell growth and increases apoptosis in human esophageal cancer cell lines expressing COX-2 (70). In addition, survival benefit has been shown in patients with low COX-2 expression in esophageal adenocarcinoma after intentionally curative resection (30). These studies provide an experimental basis for clinical studies into the role of COX-2 inhibitors in the treatment of adenocarcinoma related to BE. They also provide supportive data for the initiation of therapeutic trials with selective COX-2 inhibitors as neoadjuvant therapy for esophageal adenocarcinoma. A small number of such trials are in process, although no large randomized control trials have yet published data with hard endpoints.

Helicobacter pylori and Esophageal Adenocarcinoma

It has been hypothesized that the increasing incidence of esophageal adenocarcinoma may in part reflect the decreasing prevalence of H. pylori in the developed world. A meta-analysis of 17 studies (published only in abstract) examined the prevalence of H. pylori and cagA+ strains of H. pylori in patients with BE or esophageal adenocarcinoma, along with controls (71). A study was made on 2162 patients and 3132 controls with a pooled H. pylori prevalence of 37.7% and 51.2%, respectively. The conclusion was that the prevalence of H. pylori and cagA+ H. pylori is negatively associated with BE and esophageal adenocarcinoma. The proposed mechanism is that H. pylori infection may lead to gastric body atrophy. This in turn would lead to reduced gastric acid secretion and its complications. Conversely, H. pylori infection would result in reduced acid secretion. It should be noted however that, despite this association, no protective effect has been proven between H. pylori infection and esophageal adenocarcinoma and the question of "friend or foe?" still remains.

Combination Drug Therapy

A PPI with a COX-2 inhibitor has been suggested as a promising combination in the prevention of adenocarcinoma secondary to BE. Preliminary results of a multicenter, randomized controlled trial however demonstrated no reduction in the cell proliferation index or development of dysplasia, despite a reduction in COX-2 expression (level 1b evidence) (72). Sixty-two patients with BE were followed up over a six-month period having been randomized to take either a PPI alone or in combination with rofecoxib. It was found that although initial introduction of rofe-coxib reduced COX-2 expression, it did not significantly reduce cell proliferation. These results challenge the theory that COX-2 inhibition reduces malignant change, although the short follow-up period is acknowledged. Further work looking at combination drug therapy in esophageal chemoprevention is required.

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