The first evidence of a potential role for 5-LO in cancer growth was based on expression studies reported by Hong et al. (1999), who reported that 5-LO and FLAP were universally expressed in numerous epithelial cancer cell lines. These findings were in agreement with observations by Hennig et al. (2002), who demonstrated increased expression of 5-LO in a set of human pancreatic cancer cells and a decreased expression in normal pancreatic ductal cells. Subsequently, Gupta et al. (2001) demonstrated 5-LO overexpression in samples taken from prostate carcinoma patients, where the mean level of 5-LO mRNA was sixfold higher in malignant tissues compared to healthy tissues. Overexpression was further documented in malignant pleural mesothelial cells (Romano et al., 2001), in bladder carcinomas (Yoshimura et al., 2003), esophageal tumors (Zhi et al., 2003), and for breast cancer (Jiang et al., 2003).

In support of this hypothesis, an elevated expression of the LTB₄ receptor was detected in human pancreatic cancer tissue (Hennig et al., 2002). Assessing colorectal cancer samples from 84 patients, Ohd et al. (2003) were able to show a correlation between expression of CysLT receptors, 5-LO, an increased viability of the tumor cells and declined prognosis for patient survival. Notably, overexpression not only applies to 5-LO but also to other 5-LO binding enzymes involved in LT biosynthesis. Hong et al. (1999) found universal overexpression of FLAP in a series of epithelial cancer cell lines. Overexpression of FLAP associated with higher tumor aggressiveness and a poor prognosis for survival was demonstrated by analyzing breast cancer tissue samples of patients (Jiang et al., 2006). LTA₄ hydrolase overexpression and activity was found to be an early event in esophageal and oral adenocarcinogenesis (Chen et al., 2004; Sun et al., 2006), whereas a crucial role of LTC₄ synthase and cPLA_2alpha is evident particularly in pathogenesis of leukemia (Stenke et al., 1998; Sjolinder et al., 2000; Runarsson et al., 2007). Recently, simultaneous overexpression of various enzymes and receptors involved in LT biosynthesis and action, including, 5-LO, FLAP, LTC₄ synthase, LTA₄ hydrolase, BLT, and CysLT receptors, was detected in the majority of human primary neuroblastoma tumors as well as in respective cell lines. Whereas 5-LO is well-recognized as pro-carcinogenic, the related enzyme 15-LO-2 is down-regulated in malignant tissues, considered to function as a tumor suppressor and to inhibit carcinogenesis (Shappell et al., 1999; Hsi et al., 2002). The role of 15-LO-1 is still discussed controversially in the literature (Pidgeon et al., 2007). Depending on the tumor cell type, overexpression of one enzyme of the LT synthesizing machinery in tumor cells does not inevitably account for an overexpression of the other enzymes. Thus, granulocytes from acute myeloid leukemia patients showed suppressed LTB₄ formation accompanied by elevated LTC₄ hydrolase expression and LTC₄ synthesis compared to leukocytes from healthy patients (Stenke et al., 1998).

Numerous studies have demonstrated cytotoxic and anti-proliferative effects of 5-LO inhibitors in cultured tumor cells as an important basis for the involvement of 5-LO in tumorigenesis. Tsukada et al. were amongst the first to describe the potent anti-proliferative effects of the 5-LO inhibitor AA-861 in a human leukemic cell line. This was accompanied by a sharp reduction in cellular DNA, RNA, and protein synthesis. It was concluded from this result that LTs potentially play an essential role in cancer cell viability (Tsukada et al., 1986). Similar growth-inhibitory effects were observed with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) in stomach cancer cells (Shimakura and Boland, 1992).

These data agree well with studies reporting that the 5-LO inhibitor AA-861 was capable of abolishing the AA stimulated increase of prostate cancer cell growth (Ghosh and Myers, 1997) and that inhibition of 5-LO by MK-886 (FLAP inhibitor) triggers severe apoptosis in human prostate cancer cells (Ghosh and Myers, 1998). Subsequent studies described that AA-861 is capable of suppressing the growth of esophageal cancer cells in vitro (Hoque et al., 2005) and the proliferation of MCF-7 breast cancer cells (Hammamieh et al., 2007). AA-861 also has been shown to have similar effects in colorectal cancer cells (Ihara et al., 2007). Strong cytotoxic effects in various cancer cell lines were also observed with the 5-LO inhibitor and LTD₄ receptor antagonist Rev-5901 (Ding et al., 1999; Tong et al., 2002; Titos et al., 2003; Hayashi et al., 2006; Melstrom et al., 2008; Sveinbjornsson et al., 2008). Recent findings with the LTB₄ receptor antagonist, LY293111, which demonstrated potent anti-pancreatic cancer effects by inducing tumor cell apoptosis (Ding et al., 2005) and triggering S-phase cell cycle arrest (Tong et al., 2007), further supported the hypothesis that 5-LO and its downstream products, play crucial roles in tumorigenesis. Finally, a chemopreventive activity of Rev-5901 against colorectal adenocarcinoma xenografts was recently demonstrated in an animal tumor model (Melstrom et al., 2008). Taken together, the diverse set of 5-LO inhibitors was shown to suppress the growth of several types of tumor cells by cytotoxic mechanisms, primarily through activation of the intrinsic pathway of apoptosis. However, in addition to the pro-apoptotic activity of the drugs, it should be noted that additional anti-proliferative effects were observed, such as a decrease in DNA synthesis and induction of cell cycle arrest, which could harbor great significance. A summary of the described drug effects can be found in Table 1.

Several AA metabolites synthesized via the 5-LO pathways have been shown to promote tumor cell viability and to exert protective effects toward the 5-LO inhibitor induced cytotoxicity. The precise molecular mechanisms through which these molecules act on cancer cells remain incompletely understood. Direct proliferative and anti-apoptotic stimuli as well as an enhanced tumor angiogenesis may contribute. 5-HETE and LTB₄ increased cell proliferation and viability of pancreatic cancer cells by activating the mitogenic and anti-apoptotic MAPK and Akt kinase signaling pathways (Ding et al., 2003; Tong et al., 2005). A susceptibility toward 5-HETE also was described for several other tumor cell types, including breast cancer (Avis et al., 2001), and cancer of the lung (Avis et al., 1996). The effects of 5-HETE may, at least partially, derive from 5-oxo-ETE, which is formed by the cellular oxidation of 5-HETE (Powell et al., 1992). 5-oxo-ETE acts by binding to the G-protein-coupled OXE surface receptors (Grant et al., 2009). Some tumor cell types, including colorectal cancer cells, displayed specific mitogenic effects in response to LTB₄, but not to other 5-LO products (Qiao et al., 1995; Bortuzzo et al., 1996). A few additional studies report proliferative and anti-apoptotic effects for CysLTs. Accordingly, LTD₄ increased the proliferation and survival of intestinal epithelial cells (Paruchuri et al., 2005) and potentially enhanced tumor growth by up-regulating the transcriptional activity of the oncogenic protein beta-catenin (Mezhybovska et al., 2006). Romano et al. (2001) were able to show that 5(S)-HETE and LTA₄ but not LTB₄, potently up-regulated vascular endothelial growth factor (VEGF) transcription and expression in a human malignant mesothelioma model, which may contribute to the reported pro-angiogenic and anti-apoptotic effects of 5-LO products. Recent findings uncovered a novel function of LTB₄ in driving oncogenic ras-induced metastasis by acting on BLT-2 receptors (Kim et al., 2009). Furthermore, LTB₄-induced breast cancer cell survival was linked to BLT-2 mediated generation of reactive oxygen species. Collectively, the body of research supports a model in which 5-LO products modulate proliferative and anti-apoptotic events through multiple signaling pathways. Table 2 provides a summary of the described mitogenic effects.

Several studies found reduced cell proliferation rates due to down-regulation of 5-LO expression by antisense approaches. Using 5-LO antisense oligonucleotides, Romano et al. (2001) were amongst the first to provide direct evidence for 5-LO participation in the growth of malignant pleural mesothelial cells. Moreover, reduced expression of the 5-oxo-ETE receptor by siRNA approaches significantly impaired the viability of prostate cancer cells, suggesting a tumorigenic function of the 5-LO product 5-oxo-ETE (Sundaram and Ghosh, 2006). Finally, silencing of LT receptors was capable of suppressing growth of colorectal cancer cells (Ihara et al., 2007) and neuroblastoma cells (Sveinbjornsson et al., 2008). Comparatively few studies have investigated the role of 5-LO in mouse models. Chen et al. (2009) demonstrated that 5-LO-deficient knock-out (KO) mice show significantly impaired induction of chronic myeloid leukemia through specific suppression of leukemic stem cell proliferation. A summary of experimental findings accounting for a role of 5-LO in tumorigenesis can be found in Figure 2.

The increasing number of publications that report a crucial role of 5-LO, and its products in tumorigenesis have been accompanied by additional studies that question the correlation between 5-LO and cancer. There is little disagreement that 5-LO inhibitors exert strong cytotoxic activities against 5-LO overexpressing tumor types and cultured tumor cells, which represents a significant basis for concluding that 5-LO products directly stimulate tumor cell proliferation. However, we recently demonstrated that the common 5-LO inhibitors AA-861, Rev-5901, BWA4C, and CJ-13,610 can reduce the viability of pancreatic cancer cells, cervix carcinoma cells, and leukemic cells independently of suppression of 5-LO product formation (Fischer et al., 2010). The hypothesis of 5-LO-independent cytotoxicity and anti-proliferation was substantiated using several experimental approaches. First, the various 5-LO inhibitors were shown to possess highly different abilities to reduce cell viability, to induce cytotoxic effects and to suppress the proliferation of cultured 5-LO-positive Capan-2 pancreas carcinoma cells. While the commonly used inhibitors AA-861, MK-886, and Rev-5901 produced strong cytotoxicity, other, more selective and more potent 5-LO inhibitors, including CJ-13,610, BWA4C, failed in this respect. Notably, zileuton, the only commercialized 5-LO inhibitor, failed to induce an anti-proliferative or cytotoxic response in all types of tumor cells employed. Additionally, the IC₅₀ values of cytotoxicity for AA-861, MK-886, and Rev-5901 exceeded the respective IC₅₀ values for inhibition of 5-LO enzyme activity by more than 20-fold (Rev-5901) and upto 5,000-fold (AA-861). Lastly, well-established 5-LO-negative tumor cell lines exhibited a higher susceptibility toward the 5-LO inhibitors than their morphologically related 5-LO-p counterparts. These observations are in line with a report by Datta et al. in which MK-886 induced severe apoptosis independently of FLAP (Datta et al., 1999; Fischer et al., 2010). Sabirsh et al. (2005) recently described 5-LO-independent effects of various LT synthesis inhibitors on Ca²⁺ signaling in 5-LO-deficient HeLa carcinoma cells. Also the apoptotic effects of licofelone, a dual COX/5-LO inhibitor, were found to occur independently of the ability of the drug to affect the AA cascade (Tavolari et al., 2008).

Recently, the 5-LO inhibitor zileuton was shown to suppress prostaglandin E₂ biosynthesis in macrophages with an IC₅₀ value of 1.94 μM (Rossi et al., 2010), a value which is close to the IC₅₀ of the drug for suppression of LT production in cell-based assays (Carter et al., 1991). Suppression of the tumor-promoting mediator PGE₂ by zileuton was also observed in human whole blood at clinically achievable concentrations and in rats at standard doses. This raises concerns over the relationship between the drugs chemopreventive effects and suppression of 5-LO.

In contrast to this, some non-tumor cell types showed obvious susceptibility toward 5-LO products, such as freshly isolated murine neuronal stem cells, which produced considerable basal amounts of LTB₄ (~7 ng per 10⁶ cells) and whose growth was suppressed by AA-861 at concentrations less than 1 μM (Wada et al., 2006). Furthermore, zileuton, the only drug devoid of anti-proliferative and cytotoxic off-target effects (Fischer et al., 2010), was capable of inhibiting the proliferation of RAW 264.7 macrophages below 1 μM. The macrophages considerably synthesized LTB₄ and addition of physiologically relevant concentrations of LTB₄ reversed the growth-inhibitory effects of zileuton (Nieves and Moreno, 2006). In sum, in many cases, the cytotoxic and chemopreventive effects 5-LO inhibitors in cell culture assays and in animal tumor models may derive from molecular mechanisms other than suppression of LT biosynthesis and warrant reassessment.

Several studies reported accelerated proliferation of tumor cells in the presence of exogenously added 5-LO products and attenuated anti-proliferative effects by 5-LO inhibitors (see Mitogenic Effects of 5-LO Products, page 3, 2nd paragraph). The extreme concentrations frequently required to observe these effects, however, represent one caveat to these experiments; the final concentrations of 5-LO products used often exceeded those present in the medium of untreated cells by up to 10,000-fold (Ghosh and Myers, 1998; Hoque et al., 2005; Sveinbjornsson et al., 2008). Experiments using physiologically relevant concentrations will be one step forward to test the mitogenicity and the pleiotropic effects of 5-LO products. Experiments are also needed to exclude that high concentrations of these 5-LO products induce non-specific anti-apoptotic responses that may counteract diverse stimuli or drugs inducing the intrinsic pathway of apoptosis.

Few clinical trials have assessed the efficacy of anti-LT drugs in clinical cancer therapy. A randomized double-blind phase II study with the LTB₄ antagonist LY293111 in patients suffering from advanced adenocarcinoma of the pancreas did not reveal any therapeutic benefit (Saif et al., 2009). This is a surprising result as a significant body of literature has demonstrated a remarkable susceptibility of pancreatic cancer cells toward anti-LT drugs in cell culture studies (see Cytotoxic Effects by 5-LO Inhibitors) and therefore suggests a certain lack of correlation between the effects of 5-LO inhibitors in cell culture assays and in patients. Notably, the mean C_max plasma concentrations of the drug achieved in patients after a dosage of 600 mg BID were found to be 4.4 μM (Schwartz et al., 2005) and should lead to almost complete suppression of LTB₄ signal transduction (Marder et al., 1995). Consequently, pleiotropic effects of the drug including modulation of PPAR (peroxisome proliferator-activated receptor) signal transduction are currently discussed (Adrian et al., 2008).