Luteolin , a Bioflavonoid Inhibits Colorectal Cancer through Modulation of Multiple Signaling Pathways : A Review

Flavonoids are biologically active polyphenolic compounds widely distributed in plants. More than 5000 individual flavonoids have been identified, which are classified into at least 10 subgroups according to their chemical structure. Flavonoids of 6 principal subgroupsflavonols, flavones, anthocyanidins, catechins, flavanones, and isoflavonesare relatively common in human diets. Flavonoids are a large and diverse group of phytochemicals and research into their anti-carcinogenic potential with animal and cellular model systems supports a protective role (Kocic et al., 2013). Luteolin, 3’, 4’, 5, 7-tetrahydroxyflavone, belongs to a group of naturally occurring compounds called flavonoids that are found widely in the plant kingdom. Belonging to the flavone group of flavonoids, luteolin has a C6-C3-C6 structure and possesses two benzene rings (A, B), a third, oxygencontaining (C) ring, and a 2-3 carbon double bond. Luteolin also possesses hydroxyl groups at carbons 5, 7, 3’, and 4’ positions (Figure 1) (Ross and Kasum, 2002). The hydroxyl moieties and 2-3 double bonds are important structure features in luteolin that are associated with its biochemical and biological activities (Chan et al., 2003). Vegetables and fruits such as celery, parsley, broccoli, onion leaves, carrots, peppers, cabbages, apple skins, and chrysanthemum flowers are rich in luteolin (Neuhouser, 2004; Miean and Mohamed, 2001; Gates et al., 2007; Sun et al., 2007; Mencherini et al., 2007). As in other flavonoids, luteolin is often glycosylated in plants, and the glycoside is hydrolyzed to free luteolin during


Introduction
Flavonoids are biologically active polyphenolic compounds widely distributed in plants.More than 5000 individual flavonoids have been identified, which are classified into at least 10 subgroups according to their chemical structure.Flavonoids of 6 principal subgroups-flavonols, flavones, anthocyanidins, catechins, flavanones, and isoflavones-are relatively common in human diets.Flavonoids are a large and diverse group of phytochemicals and research into their anti-carcinogenic potential with animal and cellular model systems supports a protective role (Kocic et al., 2013).Luteolin,3',4',5,belongs to a group of naturally occurring compounds called flavonoids that are found widely in the plant kingdom.Belonging to the flavone group of flavonoids, luteolin has a C6-C3-C6 structure and possesses two benzene rings (A, B), a third, oxygencontaining (C) ring, and a 2-3 carbon double bond.Luteolin also possesses hydroxyl groups at carbons 5, 7, 3', and 4' positions (Figure 1) (Ross and Kasum, 2002).The hydroxyl moieties and 2-3 double bonds are important structure features in luteolin that are associated with its biochemical and biological activities (Chan et al., 2003).
Vegetables and fruits such as celery, parsley, broccoli, onion leaves, carrots, peppers, cabbages, apple skins, and chrysanthemum flowers are rich in luteolin (Neuhouser,
Chemoprevention refers to the use of natural or synthetic compounds to prevent, reverse, or delay the development of cancer (Swan and Ford, 1997).Because food derived products exist universally and are expected to be safe, they are highly interesting for development as chemopreventive agents to treat cancer (Sengupta et al., 2002;Chihara et al., 2010).Luteolin act as a strong anticancer agent against many types of malignancies including liver, lung, breast, esophageal squamous carcinoma, colon, prostate and melanoma (Zhou et al., 2009;Hwang et al., 2011;Tang et al., 2011;Wang et al., 2012a;Wang et al., 2012b;Ruan et al., 2012;Pandurangan et al., 2014a).This review is aimed to emphasize the molecular action of luteolin on molecular targets of colorectal cancer.

Luteolin Effects on Colon Cancer
Cancers of the large and small intestine are major contributors to worldwide cancer morbidity and mortality (Greenlee et al., 2000).Although CRC was well studied, the progress in the field of preventing or curing this disease has not been significant.CRC is a multi-step process involving three distinct stages, initiation that alters the molecular message of a normal cell, followed by promotion and progression that ultimately ends up with a phenotypically altered cancer cell (Pitot, 1986).Many signaling pathways are deregulated during the progression of colon cancer (Pandurangan, 2013;Pandurangan and Esa, 2013;Pandurangan and Esa, 2014;).Epidemiological and experimental studies suggest that colon cancer is strongly influenced by nutritional factors, including the amount and composition of dietary fat (Willet et al., 1990;Pandurangan et al., 2013c;He et al., 2014).
Oxidative stress is defined as a disturbance between pro-oxidant and antioxidant balances in favor of the former, leading topotential damage.Oxidative stress can result in cell injury due to lipid peroxidation, DNA damage, mutagenesis and has been associated with various stage of tumor formation process (Halliwell and Gutteridge, 1989).Lipid peroxidation (LPO) is a free radical mediated process.It is involved in the formation of lipid radicals, a rearrangement of the unsaturated lipids that consequences in a variety of degraded products like alkanes, malondialdehyde (MDA), conjugated dienes and lipid hydroperoxides and eventually damage to cells (Upsani et al., 2001).Published reports showed that luteolin reduces tumor number, inhibits the lipid peroxidation and restores the antioxidant enzymes during 1, 2-Dimethyl hydroxide-induced colon cancer in rats (Manju et al., 2005;Manju and Nalini, 2005;Manju and Nalini, 2007).Administration of luteolin significantly reduced the levels of LPO and OH-in plasma and colonic mucosa as well as increases the antioxidant enzymes which might be due to the strong antioxidant property of luteolin (Ashokkumar and Sudhandiran, 2008).
Glycoproteins play crucial role in mediating cell surface function, such as cell-cell recognition, cellular adhesion, binding and clearance of serum glycoproteins and metabolic transport among others.Elevated levels of glycoprotein contents are valuable markers of carcinogenic process and these changes alter the rigidity of cell membrane (Selvam and Nagini, 1995).
Glycoconjugates are necessary for the assembly of the oligosaccharide moieties of the glycoprotein chains and their levels have been found to be elevated in neoplastic conditions and can therefore be designated as non-specific markers of malignancy (Sen et al., 1983).Its levels are high in tumor tissue due to increased lipid peroxidation resulting in lowered antioxidant status (Ashokkumar and Sudhandiran, 2008) and aberrant glycosylation (Hakomori, 1996).Pandurangan et al. (2012) reported that luteolin have the ability to control glycoproteins such as hexose, hexosamine, fucose and sialic acid in AOMinduced CRC.The multiple action of Luteolin on different models of colon cancer was represented in Table 1.

Luteolin Effects on Preneoplastic Lesions
Aberrant crypt foci (ACF) is considered as putative pre-neoplastic lesion as an end point marker have been used to assess the influence of various modulatory factors (Bird, 1995;Bird and Good, 2000).ACF are characterized by one or more crypts that appear as a single focus but are larger than normal crypts.It is characterized by thickened epithelia, altered luminal openings, have an increased pericrytal area between them (McLellan et al., 1991).Finally, these can develop into polyps and eventually into CRC.Plant constituents and their derivatives may exert significant effects on decreasing the incidence of ACF in the colon (Waly et al., 2012;Tammasakchai et al., 2012;Ansil et al., 2013;Guizani et al., 2013;Madrigal-Bujaidar et al., 2013).Treating the animals with luteolin during the AOM-induced CRC animals showed reduced incidence of ACF (Ashokkumar and Sudhandiran, 2008).

Luteolin Effects on iNOS
Nitric oxide (NO) is considered as one of the smallest signaling molecules that can diffuse into the cell.NO is present in all cells in the body, synthesized through several enzymatic and non-enzymatic pathways.As a free  radical with complex redox chemistry, NO can modify all biological molecules and is, therefore, implicated in all biological functions in living systems.NO is an important bioregulatory mediator involved in a variety of biological processes in both normal and pathophysiological conditions.
Nitric oxide synthase (NOS), particularly inducible NOS (iNOS or NOSII) and endothelial forms are overexpressed in various cancers in both humans and rodents (Rao et al., 1999;Rao et al., 2002).NO can exert its effects directly by forming reactive nitrogen-oxygen species and indirectly by post-translational modifications of proteins via S-nitrosylation or tyrosine nitration (Gow et al., 2002;Hess et al., 2005).Previous studies in both carcinogen-induced and genetic models support a role for iNOS in the promotion of colon carcinogenesis (Rao et al., 1999;Rao et al., 2002).Increased iNOS expression and activity were observed in carcinogen-induced rat dysplastic aberrant crypt foci (ACF), adenomas and adenocarcinomas, but not in hyperplastic ACF (Takahashi et al., 2000).The tumor-enhancing effects of iNOS in the colon may be associated with the ability of NO to increase the expression/activity of the enzyme COX-2 (Thun et al., 2002;Somchit et al., 2014).To support further the role iNOS in colon tumor promotion, mice from an Apc Min/+ -iNOS-knockout genetic background showed decreased intestinal tumor formation (Ahn and Ohshima, 2001).In humans, iNOS expression is up-regulated in carcinomas compared with patient's normal-appearing colonic mucosa (Yagihashi et al., 2000).Luteolin has an anti-inflammatory role was already reported by modulating the inflammatory mediators (Choi, 2007).But in chemically induced model luteolin decreased the expression of iNOS in mice (Pandurangan et al., 2014b), but the mechanism unknown.

Luteolin Effects on COX-2
COX-2 is considered as an enhancer of carcinogenesis in many organs including the colon.Henceforth, assays of COX-2 expression may be used to monitor the process of carcinogenesis, and the suppression of COX-2 expression has become an important target for treatment and prevention of CRC (Tanaka et al., 2001;Turini and DuBois, 2002;Hamiza et al., 2012).COX-2 is induced by inflammatory cells, by various stimuli including cytokines, growth factors and tumor inducing factors.Overexpression of COX-2 results in dedifferentiation, adhesion to extracellular matrices and inhibition of programmed cell death in untransformed rat intestinal epithelial cells (Tsujii and DuBois, 2002).Inducibility of COX-2 in response to mitogenic stimuli, oncogenes and tumor promoters link it to cell proliferation.Increased COX-2 expression leads to the elevated levels of PGE2 that is correlated with increased MDA, which forms adducts with DNA in human colon leads to carcinogenesis (Hanif et al., 1996) or inhibit apoptosis in epithelial tumor cells (Sheng et al., 1998).
Treating with Non-steroidal anti-inflammatory drugs was the potential way to control the production of COX-2 during tumorigenesis.In this context, natural sources and flavonoids also a right approach to inhibit COX-2 (Shafie et al., 2013a).Especially plant polyphenols are the strong inhibitors of COX-2 (Banerjee et al., 2013;Wang et al., 2013) and Pandurangan et al., (2014b) reported that luteolin inhibits COX-2 during AOM-induced colorectal cancer in BALB/c mice.

Luteolin Effects on the Nrf2/keap1 Pathway
NF-E2-related factor 2 (NRF2) transcription factor belongs to the Cap 'n' Collar subfamily of basic leucine zipper family of transcription factor.Under normal condition Kelch-like ECh-associated protein 1 (keap 1) play a central role and regulate NRF2 activity.NRF2 bound with KEAP1 due to an interaction between single NRF2 protein and a KEAP1 dimer through cysteines residue (Itoh et al., 1997).KEAP1 serves as a substrate linker protein for interaction of Cul3-based E2ubiquitin ligase complex with NRF2 and its proteosomal degradation.NRF2 is activated by number of stressors such as ROS, reactive nitrogen species lipid aldehydes and certain variety of natural agents' results in the dissociation of one or both NRF2-interacting motifs from NRF2.The activated NRF2 translocate into the nucleus and transcribes GST, NQO1 which enables the cytoprotection (Holland et al., 2008;Yamamoto et al., 2008).
It is difficult to discuss the cancer chemoprevention without mentioning Nrf2 transcription factor, since the discovery of Nrf2 is attributed greatly to studies with anti-carcinogenic compounds (Zhang, 2006).There are many reports stating that Nrf2-deficient mice are more susceptible to toxicity, DNA adduct formation and cancer development in several models of chemical-induced carcinogenesis (Xu et al., 2006;Khor et al., 2008).Nrf2null mice have decreased basal and inducible expression of antioxidant genes, increased oxidative stress, and decreased reducing activity and antioxidant capacity (Chan et al., 2000;Hirayama et al., 2003), suggesting that the Nrf2/ARE pathway is crucial in the regulation of intracellular redox status.During CRC the expression of Nrf2 was limited (Patel et al., 2008).However, treatment with flavonoid compounds such as EGCG and PBT has the potential to activate the Nrf2 by dissociating the Nrf2-keap1 complex (Chiou et al., 2012).Luteolin is reported to activate the Nrf2 in AOM-induced colon cancer (Pandurangan et al., 2014a).On the other hand luteolin sensitizes two oxaliplatin-resistant colorectal cancer cell lines to chemotherapeutic drugs via inhibition of the nrf2 pathway (Chian et al., 2014).This phenomenon was further confirmed by the elevated expression of GST-α and GST-µ (Summart et al., 2014).On other hand, supplementation of Luteolin elevates the intracellular reduced glutathione (GSH) (Pandurangan and Ganapasam, 2013a).Since GSH plays an intracellular radical scavenger and is the substrate of many xenobiotic elimination reactions (Gregus et al., 1996), an increased level of GSH also postulates that it activates GSH dependent enzyme GST (Pandurangan et al., 2014a).

Luteolin Effects on IGF-1
Insulin-like growth factors (IGFs) are polypeptides that stimulate the growth of a variety of mammalian cells (Baserga et al., 1997).The IGF system (IGF-I, IGF-II, IGF-binding protein, and IGF-IR) performs an important role in the growth of various cancer cells, including colon cancer cells (Frasca et al., 2008;Jung et al., 2008).Luteolin, dose-dependently reduced the IGF-2 secretion of HT-29 cells.IGF-1 stimulated HT-29 cell growth but did not abrogate luteolin-induced growth inhibition.Luteolin reduced the levels of the IGF-IR precursor protein and IGF-1R transcripts.Luteolin reduced the IGF-1-induced tyrosine phosphorylation of IGF-1R and the association of p85 with IGF-IR.Additionally, luteolin inhibited the activity of PI3K activity as well as the phosphorylation of Akt, ERK1/2, and CDC25c in the presence and absence of IGF-1 stimulation (Kim et al., 2012).

Luteolin Effects on the Wnt-β-catenin Pathway
The wnt/β-catenin pathway plays an essential role in embryonic development and contributes to tissue homeostasis and tumorigenesis.Frequent mutations in the Wnt pathway are considered an early, important step in human CRC and are responsible for colon tumor formation in patients with familial polyposis (FAP) (Moser et al., 1990;deLau et al., 2007).The oncogenic potential of the Wnt pathway derives from β-catenin protein stabilization and relocation from the cell membrane to the nucleus, where it is recruited into T-cell factor/lymphoid enhancer factor 1 (TCF/LEF) transcriptional regulatory complexes (Fuchs et al., 2005).TCF/LEF complexes bind to enhancer regionsof target genes involved in proliferation, invasion, and inhibition of apoptosis, including c-Myc and cyclin D1.These effects contribute directly to the development CRC (Kumar et al., 2012).In our laboratory we shown that luteolin inhibits the translocation of β-catenin from the cytosol to nucleus in CRC in vitro and in vivo (Pandurangan et al., 2013a;Ashokkumar and Sudhandiran, 2011).Inhibition of β-catenin is mediated by modulating the expression of p-GSK3β.Luteolin also shown that; it inhibits the expression of cyclin D1, a downstream target of wnt/β-catenin pathway (Pandurangan et al., 2013a).Baskar et al., (2011) reported that luteolin-7-O-glucoside isolated from ophiorrhizamungos Linn showed to decrease the expression of β-catenin in COLO 320 DM cells.

Luteolin Effects on Apoptosis
Apoptosis is termed as a programmed cell death, which is characterized by cell shrinkage, chromatin condensation, DNA fragmentation, and the activation DOI:http://dx.doi.org/10.7314/APJCP.2014.15.14.5501 Luteolin, a Bioflavonoid, Inhibits Colorectal Cancer through Modulation of Multiple Signaling Pathways of specific cysteine proteases known as caspases (Zou et al., 1997).In particular, caspase-3 is the most widely studied of effector caspases.It plays an important role in both death pathways and cleaves a wide range of cellular substrates, including structural proteins and DNA repair enzymes (Fernandes-Alnemri et al., 1995).Caspase-3 is a critical component of the cell death machinery, being regarded as the most downstream enzyme in the apoptotic process due to its location in the protease cascade pathway (Fernandes-Alnemri et al., 1995a).The ratio of Bax/Bcl-2 is a critical determinant of the overall predisposition of a cell to undergo apoptosis.An increase in Bax relative to Bcl-2 promotes release of cytochrome C from the mitochondria with subsequent activation of caspase-3, thereby inducing mitochondrial mediated apoptosis (Gupta and DuBois, 2001).Natural compounds has the ability to induce cytotoxicity thereby protects against cancer and many researcher's developing chemotherapeutic by based on its ability to induce apoptosis (Sriram et al., 2008;Acebedo et al., 2014;Shafie et al., 2013b;Zou et al., 2013).Lim et al. (2012) reported that luteolin induce cell cycle arrest and apoptosis in HT-29 human colon cancer cells.Many reports stating that Luteolin arrest cell cycle and induce apoptosis in colon cancer cells (Attoub et al., 2011;Pandurangan and Ganapsam, 2013).Another study from Attoub et al. (2011) showed that Luteolin induces apoptosis by activating caspase 3 in HT-29 colon cancer cells.A study from our laboratory showed that luteolin induces apoptosis in colon cancer by modulating the expressions of bax, Bcl-2 and caspase 3 in vitro and in vivo (Lim et al., 2007;Pandurangan et al., 2013;Pandurangan and Ganapasam, 2013b).On the other hand Luteolin acts against DNA damage and activates DNA repair mechanism in caco-2 colon cancer cells (Ramos et al., 2010).

Conclusion
Epidemiological studies have indicated that colorectal cancer is strongly related with diet, and thus it may be possible to prevent the incidence of this cancer through dietary modification.Chemoprevention refers to the use of the naturally derived or synthetic compounds to prevent, reverse or delay the development of cancer.Because food-derived products exist universally and are expected to be safe, they are highly interesting for development as chemopreventive agents.To summarize the present scenario on luteolin against colon cancer, so far an ample of evidences available, that luteolin potentially controls colon cancer in multiple aspects.

Table 1 . Chemopreventive and Chemotherapeutic Effects of Dietary Flavonoid Luteolin on Colorectal Cancer
Luteolin reduces the incidence of aberrant crypt foci (ACF).Inhibits lipid peroxidation and Hydroxyl radical formation.Increased the activities of enzymic and non-enzymic antioxidants.