NSAID’s and selectively COX-2 inhibitors as potential chemoprotective agents against cancer

1 Introduction

Internationally there are >10 million new cancer cases and >7 million cancer related death reported each year making cancer research a top priority. As per WHO report 1997 cancer continues to be a major health problem in urban areas and during the next 25 years or so cancer alone will contribute 6.3 million annual deaths (Doreswamy and Darshan, 1999). Cancer is a disease of cells characterized by reduction or loss of effectiveness in normal cellular control mechanism which regulates multiplication. Carcinogenesis is a multi-step process as was first described in 1965 by Leslie Foulds, who deduced that there were multiple pathological processes of cancer induction and tumor progression for many human epithelial cancers.

Cancerogenesis is of a three stage process. The first stage is initiation which involves in the mutation by physical, chemical or viral exposure and occurs rapidly with high frequency. The second stage is tumor promotion, a low frequency event that requires sustained chronic exposure to tumor promoters, such as growth factors, hormones or ultraviolet radiation. The third stage is tumor progression in which tumor becomes malignant. Many cancer cells have defects in their progression through the cell cycle or their regulation of cell death. In particular, the distinct feature of tumors is lack of regulation of the cell cycle, resulting in uncontrolled proliferation. Although the goal of cell division is for each daughter cell to inherit a copy of each intact chromosome, defects in this process can lead to aneuploidy, genetic instability and ultimately, metastatic tumorigenesis. A list of various chemotherapeutic drugs is summarized in Table 1 (Goodman and Gilman’s, 2001).

Table 1 List of chemoprotective agents.

Class	Type of agent	Drugs	Disease	Structure
Alkylating agents	Nitrogen mustard	Mechlorethamine	Hodgkin’s disease, non-Hodgkin’s lymphomas
		Cyclophosphamide, Ifosfamide	Acute and chronic lymphocytic leukemia; Hodgkin’s disease, non-Hodgkin’s lymphomas; multiple myeloma
		Melphalan	Multiple myeloma; breast, ovarian cancer
		Chlorambucil	Chronic lymphocytic leukemia, primary macroglobulinemia, Hodgkin’s disease, non-Hodgkin’s lymphomas
	Ethylenimines and methylmelamines	Hexamethylmelamine	Ovarian cancer
		Thiotepa	Bladder, breast, ovarian cancer
	Alkyl sulfonates	Busulfan	Chronic granulocytic leukemia
	Nitrosoureas	Carmustine	Hodgkin’s disease, non-Hodgkin’s lymphomas; primary brain tumors, multiple myeloma, malignant melanoma
		Lomustine	Hodgkin’s disease
	Triazenes	Dacarbazine	Malignant melanoma, Hodgkin’s disease
Antimetabolites	Folate antagonist	Methotrexate	Acute lymphocytic leukemia; choriocarcinoma; breast, head and lung cancer
	Pyrimidine analogs	Fluorouracil	Breast, colon, stomach, pancreas, ovarian, head and neck, urinary bladder cancer
		Cytarabine	Acute granulocytic and lymphocytic leukemia
	Purine analogs	Mercaptopurine	Acute granulocytic and lymphocytic leukemia
Natural products	Vinca alkaloids	Vinblastine	Hodgkin’s disease, non-Hodgkin’s lymphomas, breast and testis cancer
		Vincristine	Acute lymphocytic leukemia, neuroblastoma, Wilm’s tumor
	Taxanes	Paclitaxel, Docetaxel	Ovarian, breast, lung, head and neck, urinary bladder cancer
	Epipodophylotoxins	Etoposide, Teniposide	Testis, small-cell lung, breast cancer; Hodgkin’s disease, non-Hodgkin’s lymphomas, acute granulocytic leukemia
	Camptothecins	Topotecan, Irinotecan	Ovarian cancer, small-cell lung cancer, colon cancer
	Antibiotics	Dactinomycin	Choriocarcinoma, Wilm’ tumor, Kaposi’s sarcoma
		Daunorubicin	Acute granulocytic and lymphocytic leukemia
		Doxorubicin	Soft-tissue, osteogenic, Hodgkin’s disease, non-Hodgkin’s lymphomas; breast, thyroid, lung cancer
		Bleomycin	Testis, skin, lung, head and neck, urinary bladder cancer
		Mitomycin	Testis, skin, lung, head and neck and genitourinary tract cancer
Miscellaneous	Platinum coordination complexes	Cisplatin, Carboplatin	Testis, ovary, bladder, head and neck, lung, thyroid, osteogenic sarcoma
	Substituted urea	Hydroxyurea	Chronic granulocytic leukemia
	Methylhydrazine derivatives	Procarbazine	Hodgkin’s disease
	Tyrosine kinase inhibitors	Imatinib	Chronic myelocytic leukemia
Hormones and antagonist	Adrenocorticosteroids	Prednisone	Acute and chronic lymphocytic leukemia, Hodgkin’s disease, non-Hodgkin’s lymphomas
	Progestins	Hydroxyprogesterone caproate, Medroxyprogesterone acetate, Megestrol acetate	Endometrium, breast cancer
	Estrogens	Diethylstilbestrol	Breast, prostate cancer
	Antiestrogen	Tamoxifen	Breast cancer
	Androgens	Testosterone propionate	Breast cancer
	Gonadotropin-releasing hormone analog	Leuprolide	Prostate cancer

In the past, due to poor therapeutic response and high incidence of adverse reactions, chemotherapy was considered as a last resort, after more successful treatments like surgery and radiotherapy had failed. However, even with recent advances, the treatment of cancer continues to be one of the greatest challenges in medicine, as many forms of human cancers still resist effective chemotherapy. A major limitation in cancer therapy is inadequate selectivity of most anti-cancer drugs (Prasanna et al., 2003). COX-2 (also known as prostaglandin H synthase-1) is upregulated in response to inflammatory cytokines, growth factors and tumor promoters (Matthew et al., 2003).

2 Various chronic inflammatory conditions that predispose to cancer

Various chronic inflammatory conditions predispose to the neoplastic transformation. Tumors are mostly of epithelial cell origin (carcinomas). Various cancerous disease associated with inflammatory conditions such as colon carcinoma associated with inflammatory bowel disease (chronic ulcerative colitis and Crohn’s disease), esophageal adenocarcinoma associated with reflux esophagitis (Barrett’s esophagus), hepatitis predisposing to liver cancer, schistosomiasis causing an increased risk of bladder and colon carcinomas, and chronic Helicobacter infection leading to cancer of the stomach. Some increase in the incidence of lymphoma is also seen, particularly mucosa-associated lymphoid tissue (MALT) lymphoma.

3 Non-infectious causes of chronic inflammation lead to cancer

Various non-infectious agents also cause chronic inflammation associated with an increased risk of cancer. For example, esophageal reflux causes chronic exposure of the esophageal epidermis to irritation by gastric acids. This leads to reflux esophagitis, or Barrett’s esophagus, and subsequent development of esophageal carcinoma (Hanahan and Weinberg, 2000). Excess fecal bile acids in patients with primary sclerosing cholangitis and ulcerative colitis are associated with an increased risk of colorectal carcinoma. A recent publication demonstrated that ursodiol, a drug that reduces the colonic levels of deoxycholate and other bile acids (used to treat cholangitis), significantly reduces the incidence of neoplasia (Hennings et al., 1993). Chronic irritation of the liver by alcohol causes cirrhosis and hepatocellular carcinoma (Jackson et al., 1997).

Non-digestible agents such as asbestos, coal and silica dust lead to chronic inflammation in the lung because of the inability of the immune system to remove the substances. Such sterile inflammations increase the incidence of epithelial cancers including mesothelioma and lung carcinoma (Baron and Sandler, 2000). Environmental carcinogen exposure is a requisite for the development of mostly all lung cancers such as asbestos exposure interacts synergistically with tobacco smoke to induce lung cancer. Previous studies have suggested that asbestos was associated with the presence of a k-ras mutation in adenocarcinoma of the lung. k-ras mutation is strongly associated with adenocarcinoma. There were 84 male patients with available questionnaire-derived work history data and paraffin-embedded tumor tissue for determination of k-ras mutation status. Chest radiographic evaluation was done for all of the patients who reported occupational exposure to asbestos. The prevalence of k-ras mutation was higher among those with a history of occupational asbestos exposure (crude odds ratio, 4.8; 95% confidence interval, 1.5–15.4) compared to those without asbestos exposure, and this association remained after adjustment for age and pack-years smoked (adjusted odds ratio, 6.9; 95% confidence interval, 1.7–28.6). These data suggest that asbestos exposure increases the likelihood of mutation at k-ras codon 12 and that this process occurs independently of the induction of interstitial fibrosis (Nelson et al., 1999). Cigarette smoke is a complex pro-neoplastic agent that may act, in part, by inducing a chronic inflammatory condition. Smoking not only causes chronic bronchitis, but also delivers an array of genotoxic carcinogens (e.g. nitrosamines, peroxides) into the lungs. Definitive evidence that chronic inflammation predisposes to cancer requires identification of the causative inflammatory mediators as well as the agents that prevent neoplastic transformation through inhibition of the inflammatory process. The remainder of this review will focus on the mechanisms whereby inflammatory mediators promote neoplastic transformation.

4 Prostaglandins a mediator responsible for development of cancer

Different evidence from human and animal studies suggests that prostaglandins contribute to the development of cancer (Herschman 1996; Giovannucci et al., 1995). Prostaglandins such as prostaglandin E2 (PGE2) are lipid mediators of the inflammatory immune response and are derived from oxidative metabolism of arachidonic acid. These lipids are synthesized in large quantities by inflammatory cells in response to both acute and chronic inflammatory stimuli. Two different cyclooxygenase (COX) enzymes catalyze the rate-limiting first step in prostaglandin synthesis (Labayle et al., 1991). COX-2 is expressed during inflammation. Its primary site of synthesis is inflammatory monocytes and macrophages, but it is also expressed in noninflammatory cells, such as fibroblasts, epithelial cells, and endothelial cells. Bacterial cell products and inflammatory cytokines induce in vitro expression of COX-2. Experimental induction of COX-2 in animal models is accomplished with agents that induce chronic inflammation such as administration of azoxymethane to rats. COX-1 is a constitutive enzyme expressed in most cell types and is associated with regulation of housekeeping functions such as gastric acid secretion.

5 NSAIDs and novel agents

Chronic intake of NSAIDs may reduce carcinogenesis by inhibiting production of prostaglandins, cytokines, and angiogenic factors. Note that NSAIDs do not eliminate inflammation but rather act by reducing the production of selected inflammatory factors. Hence, unlike steroids, they do not suppress elements of the immune response that are necessary for tumor depletion such as T-cells, NK cells, and macrophages. COX-2 selective inhibitors may provide a safer method for chemoprevention than older NSAIDs such as aspirin and indomethacin, which also inhibit COX-1 activity and cause gastric lesions.

NSAIDs exert analgesic, antipyretic, and anti-inflammatory effects through the inhibition of COX-catalyzed biosynthesis of prostaglandin’s (Nakadate et al., 1982). Moreover, the ability of these NSAIDs drugs to prevent cancer is thought to be due, in part, to COX inhibition. Currently, there are two known isoforms of COX, both of which catalyze the metabolism of arachidonic acid to prostaglandin H₂, a precursor to prostaglandins (Earashi et al., 1995). The COX-1 isoform is constitutively expressed and produces the prostaglandins important for normal physiological function. COX-2 can be induced by cytokines, growth factors, and tumor promoters and produces prostaglandins at sites of inflammation (Parrett et al., 1997). In carcinogenesis, over expression of COX is thought to deregulate arachidonic acid metabolism and lead to elevated prostaglandin production (Buckmann et al., 1998). The incidence of COX-2 protein expression gradually increases with the development of esophageal lesions, from 75% in metaplasia, to 83% in low grade dysplasia and up to 100% in high-grade dysplasia (Morris et al., 2001). Increased prostaglandin (PG) levels have been observed in human and animal tumors compared with surrounding normal tissue and are thought to contribute to colon carcinogenesis by influencing cell proliferation, tumor promotion, immune response, and metastasis (Marnett, 1992; Kraemer et al., 1996). Increased PG synthesis caused stimulation of cell proliferation and contributed to the development of dysplasia in Barrett’s epithelium (Zhang et al., 2001).

The role of arachidonic acid metabolites as modulators in the multi-step process of carcinogenesis, particularly in tumor promotion, has often been postulated with supportive evidence from epidemiological and experimental studies. This notion is strengthened by reports on a reduced mortality and a lower incidence of human colon cancer following chronic consumption of non-steroidal anti-inflammatory drugs (NSAIDs), e.g. acetylsalicylic acid (Puga et al., 1997). Sulindac and indomethacin suppress the number and size of colonic polyps in patients with familial adenomatous polyposis (Vogel et al., 1998; Reddy et al., 1987). Moreover, inhibitors of cyclooxygenase and lipoxygenase activities decrease the tumor promoting effects of various structurally unrelated agents and inhibit the growth of tumor cells in vivo and in vitro. The impact of arachidonic metabolism on tumorigenesis is based on the following observations: (i) the levels of specific metabolites of arachidonic acid and the expressions of cyclooxygenases and lipoxygenases are enhanced in various human and rodent tumors and (ii) the cyclooxygenase isoenzyme COX-2 is induced in normal tissues by the treatment with tumor promoters, e.g. TCDD and TPA (Levy, 1997). Interestingly, the induction of COX-2 has been observed in pathological processes including tumor promotion. Thus, in a quest for a more mechanistic risk assessment pertaining to tumor promoters such as dioxin-like compounds, the expression of cyclooxygenase activities may provide an important link necessary for this goal (Rubio, 1984).

6 COX-2 contributes to cancer: as preclinical evidence

Evidence suggests that COX-2 may contribute to esophageal carcinogenesis. Increased amount of COX-2 is commonly found in adenocarcinoma as well as squamous cell carcinoma of the esophagus (Nasser, 2004 and Eling et al., 1990). In addition, over expression of COX-2 has been observed in premalignant condition of the esophagus, such as squamous dysplasia and Barrett’s esophagus (Souza et al., 2000). Shirvani et al. reported a progressive increase in COX-2 expression with increasing histological severity from metaplasia to low-grade and high-grade dysplasia (Li et al., 2001). Increased COX-2 expression has also been associated with decreased survival in patient with esophageal adenocarcinoma. The Approve trial included a randomized multicenter, placebo controlled, double blind trial to investigate whether the chronic use of the coxib (rofecoxib 25 mg daily) would reduce the adenoma recurrence in 2586 patients with a history of colorectal adenomas. Therapy with rofecoxib was associated with a significant reduction in adenoma number and size. An increase in rofecoxib associated cardiovascular adverse events beginning at 18 months was also noted, which led to early study termination (Baron et al., 2006). Similarly, Bertagnolli et al. in a five-year efficacy and safety analysis of the adenoma prevention with celecoxib trial found an inhibitory effect of celecoxib in colorectal adenoma formation but they reported an elevated risk for cardiovascular and thrombotic adverse events[6% (RR, 1.6; 95% CI, 1.0–2.5) and 7.5% (RR, 1.9; 95% CI, 1.2–3.1) in celecoxib 200 and 400 mg twice daily users, respectively, compared to 3.8% in placebo group (Bertagnolli et al., 2009). However, the most direct evidence supporting a cause-and-effect connection between COX-2 over expression and carcinogenesis comes from genetic studies. In a seminal study Oshima et al. reported that knocking out the COX-2 gene significantly reduced the numbers of intestinal polyps in a mouse model of familial adenomatous polyposis. In another study, forced expression of the COX-2 gene in the mammary gland of transgenic mice led to the development of mammary cancer (Langergren et al., 1999). These studies provide the most direct evidence of a cause-and-effect relationship between COX-2 and cancer development.

6.3

6.3 Modification of known indomethacin to improve its specificity for COX-2

The Merck Frosst first reported improving the selectivity of indomethacin for COX-2 by making the larger trichlorobenzoyl analog (Fig. 2). Exchanging the carboxylic acid moiety of indomethacin for a 4-bromophenyle thiazole group (Fig. 3) afforded the highly selective COX-2 inhibitor. It is not known how the 1-benzoylindole (Fig. 4), lacking a side chain at C-3 of the indole nucleus, bind to the active site in COX-2, but in a cell based assay inhibited prostaglandin formation by COX-2 (Kunkel et al., 1986).

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Figure 1

Biosynthetic pathway for eicosanoid derived from arachidonic acid.

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Figure 2

Modification of known indomethacin molecules: (A) indomethacin, (B) trichlorobenzoyl analog and (C) enlarged beta branched and 4-bromobenzyl derivative.

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Figure 3

Exchanging with the carboxylic acid moiety of indomethacin with a 4-bromophenyle thiazole group.

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Figure 4

1-Benzoylindole, lacking a side chain at C-3 of the indole nucleus, bind to the active site in COX-2, but in a cell based assay inhibited prostaglandin formation by COX-2.

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7 Conclusion

This review will summarize the clinical association between chronic inflammation and cancer and will describe the inflammatory factors and pathways that are thought to be pro-neoplastic agents. Emphasis will be placed on examining the role of the reactive oxygen species, nitrogen intermediates, cytokines and prostaglandins. It also indicates that addition of NSAIDs to conventional anti-cancer therapies may enhance their anti-tumor effect.