Protective Effects of Long-Term Usage of Cyclo-Oxygenase-2 Inhibitors on Colorectal Cancer in Genetically Predisposed Individuals and Their Overall Effect on Prognosis: A Systematic Review

Colorectal cancer (CRC) is a major global health concern, accounting for significant cancer-related morbidity and mortality worldwide. Despite advancements in early detection and treatment modalities, the prevention of CRC remains a critical goal. Cyclo-oxygenase-2 (COX-2) is an inducible enzyme involved in the production of pro-inflammatory prostaglandins, which play a crucial role in various cellular processes, including inflammation, cell proliferation, apoptosis, and angiogenesis. Elevated COX-2 expression has been consistently observed in colorectal tumors, indicating their role in the pathogenesis of cancer. COX-2 inhibitors, such as celecoxib and rofecoxib, have been studied as potentially effective treatment modalities due to their ability to decrease prostaglandin levels, which are generally higher in cancer patients. Aberrant prostaglandin production is linked to the adenoma-carcinoma sequence, during which adenomas turn dysplastic and accumulate enough damage to become malignant. COX-2 inhibitors have also been shown to modulate various signaling pathways involved in CRC development, such as wingless-related integration site/β-catenin (Wnt/β-catenin), mitogen-activated protein kinase (MAPK), and phosphoinositide-3-kinase-protein kinase B/Akt (PI3K/Akt) pathways. This systematic review aimed to evaluate the protective effects of long-term usage of COX-2 inhibitors on CRC in genetically predisposed individuals and their overall effect on the prognosis of the disease. The researchers conducted a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines and collected data from several databases, including PubMed, PubMed Central, Cochrane Library, and Web of Science. The search strategy combined keywords related to CRC, COX-2 inhibitors, protective effects, and prognosis. They identified 1189 articles and shortlisted 26 full-text articles that met the eligibility criteria. Quality assessment tools, such as the Assessment of Multiple Systematic Review (AMSTAR) for systematic reviews, the Cochrane bias assessment tool for randomized control trials, the scale for the assessment of narrative review articles (SANRA) checklist for narrative reviews, and the Joanna Briggs Institute (JBI) tool for cross-sectional studies and case reports, are used. This review's conclusions will assist in determining the effectiveness of COX-2 inhibitors to prevent CRC. This review may also contribute to developing guidelines for clinicians to manage genetically predisposed individuals with CRC. Furthermore, the results of this review will shed light on the potential of COX-2 inhibitors as a preventive measure against CRC in genetically predisposed individuals.


Introduction And Background
The third most prevalent cancer overall and the most common gastrointestinal (GI) cancer in the United States of America (USA) is colorectal cancer (CRC). Additionally, it is listed as the third-leading cause of cancer-related morbidity and mortality in the USA [1]. A significant proportion of CRC cases are associated with genetic predisposition. About five percent of new-onset CRC cases have a genetic component associated with carcinogenesis [2,3]. It affects men more than women and is more common in developed countries, such as the USA, due to lifestyle changes, including a lack of exercise and dietary habits predominantly involving meat and alcohol [4]. Fruits and dietary fiber are important components of a healthy diet and have been associated with various health benefits, including potential protective effects against CRC [5,6]. Eating fruit and consuming enough fiber may reduce the risk of CRCs, while smoking may raise the risk of colorectal tumors in populations at risk [5]. The high content of antioxidants, vitamins, minerals, and dietary fiber in fruits may contribute to their protective effects by reducing oxidative stress, promoting bowel regularity, and providing essential nutrients that support overall GI health. Studies have consistently demonstrated a positive association between obesity and CRC [2].
Most tissues have a constitutive expression of cyclo-oxygenase-1 (COX-1), which produces prostaglandins in 1 1 1 1 1 physiological quantities. On the other hand, cyclo-oxygenase-2 (COX-2) is routinely expressed in activated macrophages and is raised in malignant cells to support angiogenesis in tumors. It is also highly upregulated after exposure to growth factors or inflammatory stimuli [7]. In CRC, COX-2 overexpression has been linked to more aggressive characteristics, such as increased depth of invasion, a higher tumor stage, and lymph node metastasis. While several treatment options exist for CRC, there is still room for improvement [8].
Adenomas typically precede CRC in most instances. Most colorectal malignancies are believed to arise from adenomas through the adenoma-carcinoma sequence [1,9]. Initially, a small adenoma forms, becoming dysplastic and eventually accumulating enough genomic damage to become an aggressive cancer. This process may take many years to complete and is likely associated with aberrant prostaglandin production [9]. Additionally, overexpression of COX-2 mRNA (messenger ribonucleic acid) contributes to the prevalence of CRC [10].
Researchers currently focus on the prevention and regression of adenomas, often utilizing NSAIDs such as aspirin to reduce the occurrence of CRC precursors. However, NSAIDs are associated with gastritis and GI bleeding [11][12][13][14]. COX-2 inhibitors have been shown to have chemopreventive properties in both preclinical and clinical studies [15][16][17][18]. Consequently, studies have been conducted to evaluate how specific COX-2 inhibitors, such as celecoxib and rofecoxib, affect the growth of adenomas and assist in disease management [19][20][21][22][23][24]. Elevated prostaglandin levels, particularly prostaglandin E2 (PGE2), are commonly observed in individuals with cancer. Prostaglandin synthesis involves the catalytic action of cyclo-oxygenase-1 (COX-1) and COX-2 enzymes, and the upregulation of COX-2 results in the release of prostaglandins, which significantly contribute to tumor cell proliferation. Lynch syndrome and familial adenomatous polyposis (FAP) are two genetic conditions associated with an increased risk of developing CRC [2,5]. The adenomatous polyposis coli (APC)-catenin pathway is activated, B-cell leukemia/lymphoma 2 protein (bcl-2 protein) activity is increased, increased levels of vascular endothelial growth factor (VEGF) are produced, and Fas-induced apoptosis is decreased through the action of COX-2 [15].
In this systematic review, we aimed to evaluate the protective effects of long-term usage of COX-2 inhibitors in genetically predisposed individuals for CRC and their overall impact on the prognosis of the condition.

Search Sources and Search Strategy
We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines to conduct the research. PubMed MeSH articles available in English. We included only human participants of all age groups. We did not include pregnant women; those articles whose full text was not retrieved were excluded; grey literature was also excluded.

Process of Selection
We collected the articles using the keyword search strategy, transferred them to the endnote, and recovered the duplicates. We screened each article with titles, abstracts, and eligibility criteria such as inclusion and exclusion criteria and assessed for full-text articles, and then the articles were selected accordingly.

Quality Check of Articles
All the shortlisted articles are screened for quality checks using the relevant quality assessment tools. Systematic reviews were checked by the assessment of multiple systematic review (AMSTAR) tools, randomized controlled trials were checked by the Cochrane bias assessment tool, narrative reviews by scale for the quality assessment of narrative review articles (SANRA) checklist, and cross-sectional studies and case reports are assessed by the Joanna Briggs Institute (JBI) tool.

Results
We identified a total of 1189 articles using the databases. From them, we removed 132 duplicates through endnote, and later the articles were screened by looking into the titles, abstracts, and full-text articles, and of them, 45 articles were shortlisted. These shortlisted full-text articles were assessed for eligibility criteria and were undergone quality checks using the quality assessment tools, and finally 26 articles were finalized for the review. Figure 1 shows the selection process of studies in detail.

Outcome Measures
This study systematically reviewed data from multiple studies to evaluate the effects of long-term usage of COX-2 inhibitors on CRC in genetically predisposed individuals. It compared the incidence rates of CRC and the presence and size of adenomas in individuals treated with COX-2 inhibitors versus control or placebo groups. Additionally, the study analyzed the characteristics of colorectal tumors, including stage, invasion depth, metastasis, and differentiation, concerning COX-2 inhibitors. It also investigated the expression levels of COX-2 in CRC tissue and adjacent normal mucosa. This study aimed to provide insights into the potential preventive role of COX-2 inhibitors.

Study Characteristics
We conducted a comprehensive review that included 26 research papers. Four randomized controlled trials, three cross-sectional studies, two case-control studies, 10 narrative reviews, three systematic reviews and meta-analyses, one systemic review and one meta-analysis, one case report, and one clinical trial collectively comprise the research studies that were selected.
The sample sizes of the included studies were reported, detailing the number of participants in each study group, such as the COX-2 inhibitor group, control group, or placebo group. The duration of the studies, including the length of follow-up or treatment with COX-2 inhibitors, was also provided. Participant characteristics, such as age, gender distribution, genetic predisposition to CRC, and inclusion/exclusion criteria, were described to assess the generalizability of the findings. The specific COX-2 inhibitors used, including dosage, frequency, and duration of treatment, were outlined. The control groups employed, such as placebo, no-treatment, or alternative treatment groups, were mentioned. The statistical methods used for data analysis, encompassing descriptive and inferential statistics and measures of effect size, were reported to facilitate result interpretation and determine the significance of the findings.
The articles were assessed for eligibility using different quality appraisal tools. Table 2 below shows the quality appraisal for the case reports, Table 3 for the narrative reviews, Table 4 for cross-sectional studies, Table 5 for systematic reviews and meta-analyses, and Table 6 for studies relevant to this review.

Genetic Predisposition for CRC
Genetic factors are crucial in CRC and FAP. In more than 80% of spontaneous CRCs, mutations occur in the APC tumor suppressor gene [24]. Changes in tumor suppressor genes (e.g., APC, TP53: tumor protein 53) and oncogenes (e.g., KRAS: Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) can cause CRC, while FAP is caused by inherited germline mutations in the APC gene, which is located on chromosome six. Multiple colorectal adenomas, ranging in number from a few polyps to several thousand, grow over time, often starting in childhood. At a mean age of 44 years, at least one of the polyps will develop into cancer if left untreated, about 20 years earlier than the average age at which malignancies in those who do not have the condition first emerge [6]. The interaction with these genetic factors becomes significant when considering COX-2 inhibitors, which selectively block the COX-2 enzyme. COX-2 inhibitors may affect inflammation, cell proliferation, and dysregulated signaling pathways in individuals with specific genetic alterations associated with CRC. Synchronous primary CRC may develop from FAP. This can occasionally result in the inability to achieve remission despite therapy and present as metachronous cancer, requiring close monitoring. Unfortunately, there is no research on the best practices for managing synchronous and metachronous CRCs based on aberrant mutational analyses [1]. In the case of FAP, COX-2 inhibitors show the potential to reduce polyp formation and growth. The response to COX-2 inhibitors may vary based on specific genetic mutations and individual factors. Genetic profiles can optimize treatment strategies for better outcomes in genetically predisposed individuals [2,3,5].

COX: The Housekeeping Enzyme and Its Various Functions
COX-1 and COX-2 are involved in prostaglandin production. Several organs, most notably the glomeruli of the kidney and the cortex of the brain, physiologically and functionally express COX-2 [7]. These enzymes convert arachidonic acid to prostaglandin H2, which is further converted to various prostaglandins, thromboxanes, and prostacyclins [24]. COX-2 is an enzyme that plays an important role in the body's inflammatory response. COX-2 increases the production of prostaglandins, especially PGE2, which plays a major role in inflammation and causes fever during injuries. COX-2 is important in decreasing Fas-induced apoptosis, increasing B-cell leukemia/lymphoma 2 protein (bcl-2) activity, activating the APC-β catenin pathway, and producing a high amount of VEGF. PGE2, produced due to the increased activity of COX-2 in colon cancer, promotes the growth of cancer cells by activating a signaling pathway involving G proteins [15]. This pathway links the prostaglandin EP2 receptor to beta-catenin regulation.

COX-2 Enzyme in Colorectal Carcinogenesis
Two hereditary diseases linked to a higher chance of developing CRC include Lynch syndrome and FAP. Studies have shown that using COX-2 inhibitors, such as celecoxib, can reduce the incidence of colorectal polyps in individuals with Lynch syndrome and FAP [16,17].  [25].
The primary target of cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNFα) are fibroblasts from the mesenchymal (stromal) layer. Fibroblasts make up the majority of mesenchymal cells, and fibroblasts from non-neoplastic colorectal tissue are an important source of COX-2 expression, which is well-established as a crucial process in the development of CRC [8]. Aspirin and other NSAIDs have been investigated in numerous recent trials for chemoprevention of CRC [15][16][17][18][19][20][21][22][23][24]. It is possible that COX-1 activity in activated platelets serves as a signal to induce COX-2 expression by blocking the release of paracrine lipid and protein mediators that induce COX-2 expression. This may help explain why aspirin and NSAIDs have chemopreventive effects even at doses that make it impossible to reduce COX-2 expression in nucleated cells [9]. However, recent investigations have found that, by precisely targeting the COX-2 enzyme, inhibiting it, and limiting the generation of prostaglandins, COX-2 inhibitors significantly impact inflammation and cancer. This also explains their anti-inflammatory and antipyretic effects. NSAIDs, which also block the COX-1 enzyme, can result in GI side effects such as bleeding and ulcerations due to the loss of mucosal protection effect of prostaglandins. COX-2-specific inhibitors, on the other hand, spare COX-1 and are less likely to cause GI side effects. A study by Harewood et al. showed a protective effect of aspirin usage (OR = 0.80, 95% CI = 0.73-0.89), but no correlations between hormone replacement therapy (RR = 0.92, 95% CI = 0.83-1.02), oral contraceptives (RR = 1.06, 95% CI = 0.98-1.14), or statin use (RR = 0.94, 95% CI = 0.67-1.31) and the incidence of proximal colon cancer compared to never/non-use. There was no correlation between the anti-hypertensives and metformin in either study. However, COX-2 inhibitors have been associated with increased cardiovascular events, such as stroke and heart attack, because the prostaglandins produced by COX-2 have protective effects on the cardiovascular system. A cross-sectional study by Roelofs et al. measured COX-2 mRNA levels in 60 paired samples of non-cancerous tumor tissue and comparable normal colon mucosa taken at least 5 cm away from the tumor [8]. COX-2 mRNA levels are reported to be overexpressed in over 80% of colorectal malignancies when compared with paired adjacent normal colorectal mucosa, implying a function for COX-2 as a possible marker for the likelihood of cancer, whereas COX-2 inhibitors could be useful in colon cancer prevention [10].
Several studies have investigated the potential use of COX-2 inhibitors as chemopreventive agents for Lynch syndrome and FAP [16,17]. Steinbach et al. conducted a randomized trial that demonstrated a significant reduction in colorectal polyp burden in FAP patients treated with celecoxib, a COX-2 inhibitor, compared to placebo [16]. It was discovered that celecoxib might increase mitochondrial oxidation, activate the mitochondrial apoptosis process, promote endoplasmic reticulum stress, and promote autophagy to facilitate the apoptosis of tumor cells [22]. A key mechanism for tumor metastasis is angiogenesis. VEGF is a powerful angiogenic factor that affects the growth and survival of endothelial cells, the growth of new blood vessels, and the suppression of the immune response of the host, among other things that help tumors grow. Compared to the folinic acid, fluorouracil, and irinotecan (FOLFIRI) regimen alone, celecoxib and FOLFIRI together caused a statistically significant drop in serum VEGF levels. This finding suggests that celecoxib may have anti-angiogenic properties. The ability of celecoxib to inhibit the COX-2 enzyme with subsequent decreased PGE2, which plays a crucial role in the generation of VEGF, may be the cause of the suppression of angiogenesis translated by decreased VEGF levels by the concurrent use of celecoxib with FOLFIRI [23]. When compared to sporadic forms, COX inhibition has shown statistically significant results in lowering the risk of familial CRC [4].
This case-control study has studied the effects of selective and non-selective COX-2 inhibitors on colon cancer risk [14]. Selective COX-2 inhibitors, regular aspirin, ibuprofen, or naproxen all significantly reduced risk, according to the findings. Acetaminophen and low-dose aspirin did not significantly alter colon cancer risk. These findings highlight the potential of selective and non-selective COX-2 inhibitors for the chemoprevention of colon cancer [21]. Venkatachala et al. conducted a cross-sectional study using 65 instances of CRC. The malignancies were classified as low or high positives according to overall COX-2 scores and were linked with clinicopathological characteristics. Overexpression of COX-2 was linked to a deeper invasion (p = 0.021), a higher stage of the tumor (p = 0.05), a higher rate of spread to lymph nodes, and less differentiation. This shows that the link between COX-2 overexpression and rising degree and depth of invasion may support using COX-2 inhibitors as a chemo and radiation adjuvant [14].

Cardiovascular Events Linked to COX-2 Inhibitors: A Therapeutic Roadblock
In a randomized, double-blind, placebo-controlled trial involving 1,561 subjects who had previously undergone adenoma removal, daily treatment with the COX-2 inhibitor celecoxib at a dose of 400 mg decreased detection through year three by 35.7% [10]. In the celecoxib group, the cumulative rate of advanced adenomas was also markedly decreased. However, the celecoxib group experienced major cardiovascular events slightly more frequently than the placebo group (2.5% vs. 1.9%, RR = 1.30, 95% CI = 0.65-2.62) [17]. In a study of 2,586 individuals with colorectal adenomas, rofecoxib use (25 mg daily) was associated with an increased risk of thrombotic cardiovascular events. Over 3,059 patient-years, 46 rofecoxib-treated patients experienced events compared to 26 patients in the placebo group. Mortality rates were similar in both groups [18]. Moreover, 2,587 individuals with a history of adenomas participated in a trial by Baron et al., and they were randomly assigned to receive either a placebo or 25 mg of rofecoxib [19]. When compared to placebo, rofecoxib significantly reduced the recurrence of adenomas (41% vs. 55%). In the first year, the protective impact was more noticeable. However, rofecoxib was linked to higher risks of GI and cardiovascular problems [19]. In conclusion, even though rofecoxib reduced the risk of adenoma, its possible toxicity needs to be taken into account. However, Ye et al.'s investigation indicated that the number of cardiovascular events had not significantly increased and that only one out of five studies had to be stopped due to a marginal increase in the number of cardiovascular events [26].
Ma et al. conducted a systematic review and meta-analysis of 32 randomized controlled trials involving 278,694 participants. The results showed that cyclo-oxygenase inhibitors significantly reduced the risk of colorectal, advanced adenoma, and metachronous adenoma compared to placebo, but they also increased the risk of serious side effects. Other interventions, including low-dose aspirin, folic acid, ursodeoxycholic acid (UDCA), vitamin D, and calcium, did not significantly reduce adenoma risk. They concluded that the evidence does not strongly support the regular use of COX inhibitors or low-dose aspirin for colorectal adenoma prevention [20]. Although using low doses of aspirin is linked to a lower incidence of cardiovascular events, the effectiveness of this medication in preventing CRC is constrained by extremely variable outcomes caused by the length of follow-up, the duration of usage, and the timing of outcome evaluation [11].

Limitations
It is worth noting that our review has certain limitations. Firstly, the available evidence is primarily based on observational studies and a limited number of randomized controlled trials. This may introduce bias and affect the strength of the conclusions drawn. Additionally, variations in study design, dosages, and duration of COX-2 inhibitor use make it challenging to establish consistent recommendations. Further well-designed randomized controlled trials are needed to validate the findings and determine the optimal dosage and duration of COX-2 inhibitor therapy for CRC prevention in genetically predisposed individuals.

Conclusions
This systematic review explored the long-term usage of COX-2 inhibitors in treating CRC in most genetically predisposed patients, particularly in patients with Lynch syndrome and familial adenomatosis polyposis. Based on the articles reviewed, we found that COX-2 inhibitors are protective against colorectal malignancy. Early recognition of COX-2 m-RNA helps improve the prognosis of the disease. However, the evidence is limited and conflicting. Long-term usage of COX-2 inhibitors has been a topic of debate and research, especially regarding their effects on cardiovascular health. While these drugs have shown promise in reducing inflammation and preventing the growth of cancer cells, the risk of cardiovascular events associated with their use cannot be ignored. Therefore, using COX-2 inhibitors should be carefully considered on a case-by-case basis, considering the patient's medical history and risk factors. Healthcare professionals should closely monitor and encourage patients to report any new symptoms or changes in their health status while taking these medications. We believe further studies are needed to confirm the protective effects of COX-2 inhibitors on colorectal malignancy incidence in genetically predisposed individuals, to identify the optimal duration and dose of COX-2 inhibitor usage for chemoprevention, and to minimize the risk of adverse events.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.