bile acids in the colon, from healthy to cytotoxic molecules

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Review

Bile acids in the colon, from healthy to cytotoxic molecules

Juan I. Barrasa, Nieves Olmo, Ma Antonia Lizarbe, Javier Turnay ⇑Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Complutense University, 28040 Madrid, Spain

a r t i c l e i n f o

Article history:Received 27 July 2012Accepted 20 December 2012Available online 27 December 2012

Keywords:ApoptosisBile acidsColonColorectal cancerOxidative stress

a b s t r a c t

Bile acids are natural detergents mainly involved in facilitating the absorption of dietary fat in the intes-tine. In addition to this absorptive function, bile acids are also essential in the maintenance of the intes-tinal epithelium homeostasis. To accomplish this regulatory function, bile acids may induce programmedcell death fostering the renewal of the epithelium. Here we first discuss on the different molecular path-ways of cell death focusing on apoptosis in colon epithelial cells. Bile acids may induce apoptosis in col-onocytes through different mechanisms. In contrast to hepatocytes, the extrinsic apoptotic pathwayseems to have a low relevance regarding bile acid cytotoxicity in the colon. On the contrary, these mol-ecules mainly trigger apoptosis through direct or indirect mitochondrial perturbations, where oxidativestress plays a key role. In addition, bile acids may also act as regulatory molecules involved in differentcell signaling pathways in colon cells. On the other hand, there is increasing evidence that the continuousexposure to certain hydrophobic bile acids, due to a fat-rich diet or pathological conditions, may induceoxidative DNA damage that, in turn, may lead to colorectal carcinogenesis as a consequence of theappearance of cell populations resistant to bile acid-induced apoptosis. Finally, some bile acids, suchas UDCA, or low concentrations of hydrophobic bile acids, can protect colon cells against apoptosisinduced by high concentrations of cytotoxic bile acids, suggesting a dual behavior of these agents aspro-death or pro-survival molecules.

� 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9652. Biochemistry and physiology of bile acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9653. Programmed cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9654. Molecular pathways of apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9665. Apoptosis in the colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9676. Bile acids and apoptosis in colon cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 967

6.1. The death receptor pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9676.2. The mitochondrial pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9696.3. The endoplasmic reticulum pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9706.4. DNA damage and p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970

7. Effect of bile acids in oxidative stress and cell signaling in colon cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9707.1. Oxidative stress induced by bile acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9707.2. Bile acids: activation of nuclear receptors and cell signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971

8. Colorectal carcinogenesis and bile acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9739. UDCA and the protective effects of bile acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973

0887-2333/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tiv.2012.12.020

Abbreviations: BH, Bcl-2 homology; CA, cholic acid (3a,7a,12a-trihydroxy-5b-cholanoic acid); CDCA, chenodeoxycholic acid (3a,7a-dihydroxy-5b-cholanoic acid); DCA,deoxycholic acid (3a,12a-dihydroxy-5b-cholanoic acid); EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; FXR, farnesoid X receptor; GADD153, growtharrest- and DNA damage-inducible gene 153; LCA, lithocholic acid (3a-hydroxy-5b-cholanoic acid); MAPK, mitogen-activated protein kinase; MPT, mitochondrialpermeability transition; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PXR, the pregnane X receptor; ROS,reactive oxygen species; TNF, tumor necrosis growth factor; TRAIL, TNF-related apoptosis inducing ligand; UDCA, ursodeoxycholic acid (3a,7b-dihydroxy-5b-cholanoic acid);VDR, vitamin D receptor.⇑ Corresponding author. Tel.: +34 91 394 4148; fax: +34 91 394 4159.

E-mail address: [email protected] (J. Turnay).

Toxicology in Vitro 27 (2013) 964–977

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10. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974Conflict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974

1. Introduction

The maintenance of the function and structure of the gastroin-testinal mucosa requires a strict controlled balance between cellproliferation, differentiation and apoptosis. Cell behavior and sur-vival are influenced by luminal components including severalphysiological molecules such as butyrate and bile acids (Hazaet al., 2000). It is well known that the content of the lumen de-pends on the diet. A high intake of dietary fiber and low saturatedfats is associated with a reduced incidence of colorectal cancer;this protective effect has been attributed to butyrate, an end-product of colonic fiber fermentation and the major natural regula-tor of the homeostasis of the normal colonic mucosa (Andoh et al.,2003; Velazquez et al., 1997). Other molecules from the intestinallumen, bile acids and salts among them, may also affect thecolorectal epithelium. A proposed mechanism for the influence ofa diet with a high content in saturated fats in carcinogenesis isthe stimulation of bile discharge; secondary bile acids would alterthe growth of the intestinal epithelium acting as tumor promoters(Bernstein et al., 2005; Ou et al., 2012). On the other hand, thesemolecules induce apoptosis and it has been suggested that adecrease in susceptibility to these agents may correlate with anincreased risk for colorectal cancer (Schlottman et al., 2000). In fact,it has been described that bile acids are involved as etiologic agentsin cancer of the gastrointestinal tract, including cancer of theesophagus, stomach, small intestine, liver, biliary tract, pancreasand colon/rectum (Baptissart et al., 2012; Bernstein et al., 2009).

Taking into account that most of the data regarding the effect ofbile acids in apoptosis, cell signaling and carcinogenesis are mainlyrestricted to hepatocytes, in this paper we review the availableinformation about the influence of these agents in the colonichomeostasis and colorectal carcinogenesis. Therefore, we firstbriefly describe the physiology of bile acids, followed by a depic-tion of the different apoptotic pathways triggered by these agentsin colonocytes. Next we discuss their role in oxidative stress andcell signaling, and the relationship between these processes withcolorectal carcinogenesis. Finally, we summarize the potential pro-tective role of bile acids, mainly UDCA, on the colonic tract. Thus,the aim of this review is to highlight the dual role of bile acids inthe intestinal homeostasis, on the one hand as physiological deter-gents and regulators of cell fate and signal, and on the other handas potential tumor promoting agents.

2. Biochemistry and physiology of bile acids

Bile acids are amphipathic and water-soluble end products ofcholesterol metabolism; they have 24 carbon atoms and constitutea major part of the bile. Some of their properties are related to theiramphipathic nature. They are key molecules involved in digestionwhose main physiological role is to facilitate the emulsion andabsorption of dietary fats and liposoluble vitamins in the gut,and the excretion of cholesterol into the intestinal tract. At theintestinal level, bile acids modulate pancreatic enzyme secretionand cholecystokinin release, and they are potent antimicrobialagents that prevent bacterial over-growth in the small bowel. Bileacids also stimulate biliary lipid secretion and are able to formmixed micelles together with biliary phospholipids, which allowsthe solubilization of cholesterol and other lipophilic compounds

in bile (Hofmann and Hagey, 2008). On the other hand, bile acidsare molecules with potentially membrane-damaging propertiesand they act as calcium ionophores; these properties have been re-lated to their hydrophobicity.

Primary bile acids are synthesized in the liver from cholesterolthrough a cascade of reactions catalyzed by enzymes located at thecytosol, microsomes, mitochondria, and peroxisomes. The modifi-cation of the sterol nucleus of cholesterol precedes the oxidativecleavage of its side chain; it begins with the hydroxylation of cho-lesterol at C-7, catalyzed by cholesterol 7a-hydroxylase, the rate-limiting enzyme of the pathway. In humans, the two primary bileacids, cholic (CA) and chenodeoxycholic (CDCA), are synthesizedthrough this pathway. Extensive descriptions of these reactionsand enzymes can be found elsewhere (Hylemon et al., 2009; Monteet al., 2009). After their synthesis, CA and CDCA are conjugatedwith glycine or taurine in the liver, stored in the gall bladder andthen released into the intestinal tract. During the intestinal transit,these molecules are mainly absorbed in the ileum, but a small frac-tion continues its transit into the large bowel where they undergomodifications by intestinal anaerobic bacteria. This biotransforma-tion in the human colon involves mainly deconjugation and oxida-tion/epimerization of hydroxyl groups at C-3, C-7 and C-12 as wellas dehydroxylation at position C-7. Bacterial dehydratases removethe hydroxyl group at C-7 from CA and CDCA yielding the second-ary deoxycholic (DCA) and lithocholic (LCA) bile acids, respectively.Ursodeoxycholic acid (UDCA) is formed in the human colon by bac-terial epimerization of the hydroxyl group in C-7 of CDCA throughthe stable intermediate 7-oxo-LCA (Fukiya et al., 2009). In addition,7-oxo-LCA, among other secondary bile acids, can be reabsorbed inthe distal intestine and transported back to the liver, where it is re-duced to CDCA and, to a lesser extent, UDCA (Odermatt et al.,2011). The structures of some of the most abundant bile acidsare shown in Fig. 1.

The different bile acids exhibit distinct biological effects,although CA does not exert any significant effect on human coloncarcinoma cells (Barrasa et al., 2011; Hofmann and Hagey, 2008;Martinez et al., 1998; Pérez-Ramos et al., 2005). Interestingly,whereas the unconjugated bile acid UDCA is considered an hepato-cyte protector, CDCA is highly cytotoxic (Amaral et al., 2009;Paumgartner and Beuers, 2002). The only structural difference be-tween them is the configuration of the hydroxyl group at C-7 (b inUDCA and a in CDCA; Fig. 1), revealing the importance of the ste-reospecificity in the cytotoxic mechanisms of these agents. In con-trast to the toxic effects of hydrophobic bile acids, UDCA ishydrophilic and is used as a therapeutic drug for patients withcholestatic liver diseases. Moreover, UDCA has been approved bythe FDA for the treatment of primary biliary cirrhosis. Despite itsclinical efficacy, the precise mechanism by which UDCA improvesliver function is still not entirely understood and controversial re-sults have been reported (Wood, 2011).

3. Programmed cell death

Programmed cell death is an essential physiological process inthe homeostasis of the organisms that is triggered by differentdeath stimuli. Rather than necrosis, which is an accidental andnon-regulated cell death caused by extreme conditions,programmed cell death is an active process that is governed by

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signaling pathways, protein activation cascades and modulation ofgene expression. Although programmed cell death has been usu-ally identified as a synonymous of apoptosis, it also includes differ-ent caspase-independent death programs such as autophagy,paraptosis, necroptosis or mitotic catastrophe (Broker et al.,2005; de Bruin and Medema, 2008; Long and Ryan, 2012; Lu andWalsh, 2012). Each of these death processes involves differentorganelles and molecular pathways. For instance, in autophagy,long-lived organelles and other cytoplasmic components are de-graded by lysosomes. Although its main physiological functionsare cellular survival and remodeling, excessive autophagy underpathological circumstances may lead to cell death in a nonapoptot-ic way (Denton et al., 2012; Glick et al., 2010; Wirawan et al.,2012). Paraptosis shares several features with autophagy, and ischaracterized by a cytoplasmic vacuolization and perturbation inmitochondria and endoplasmic reticulum albeit without involve-ment of caspases during the death program (Sperandio et al.,2000). Necroptosis is an alternative receptor-induced form ofprogrammed cell death that is highly regulated and caspase-independent, with morphological resemblance of necrosis (Dunai

et al., 2011; Wu et al., 2012). Finally, mitotic catastrophe occursduring mitosis and is triggered by failures in the regulation of cellcycle checkpoints usually by DNA damage or microtubuledestabilization (Vakifahmetoglu et al., 2008). Although it involvesmitochondrial permeabilization and potential caspase activation,it has been proposed that this death program does not correspondwith apoptosis as it is not prevented by caspase inhibitors or Bcl-2overexpression.

4. Molecular pathways of apoptosis

Apoptotic cells show several characteristic features derivedfrom the activation of the specific processes, such as loss of mem-brane asymmetry, DNA condensation, internucleosomal degrada-tion, and cell shrinkage with subsequent membrane blebbing(Chowdhury et al., 2006). The resulting cellular debris can bedigested by neighboring cells or macrophages of the immunesystem (Nagata, 2010). Apoptosis can occur by several molecularpathways, but the best characterized ones are the intrinsic path-way, that involves mitochondrial perturbations, and the extrinsicpathway, which requires the activation of membrane receptorsthrough different extracellular stimuli. An alternative mechanismof apoptosis occurs through stress of the endoplasmic reticulum(ER) and is characterized by perturbations in protein processingand transport and loss of calcium homeostasis. Despite the differ-ent mechanisms and characteristics of each of these pathways, allof them eventually converge in the activation of the caspasefamily of proteases. These proteases cleave different nuclear andcytoplasmic targets within the cells and constitute the real effec-tors of the apoptotic program. All these apoptotic pathways havebeen extensively described in detail elsewhere (Chipuk et al.,2010; Estaquier et al., 2012; Kilbride and Prehn, 2012; Krammeret al., 2007; Lavrik and Krammer, 2012; Llambi and Green,2011; Ow et al., 2008; Schultz and Harrington, 2003; Tayloret al., 2008; Wong, 2011).

Briefly, the intrinsic pathway is triggered by different intracellu-lar stresses that eventually lead to perturbations in the mitochon-dria with subsequent membrane permeabilization, loss ofmitochondrial membrane potential and release of pro-apoptoticproteins. Several death stimuli and intracellular stresses arethought to trigger the mitochondrial permeability transition(MPT). The increased permeability after MPT induction allowsthe release of pro-apoptotic factors from the mitochondria, suchas cytochrome c (Desagher and Martinou, 2000). On the otherhand, Bcl-2 family members play also an essential role in theintrinsic apoptotic pathway (Colitti, 2012; Chipuk et al., 2010). Thissystem works as a rheostat, where the sensitivity of the cell to adeath stimulus depends on the interaction between both anti-and pro-apoptotic proteins from the Bcl-2 homology (BH) familyto form homodimers or mixed heterodimers. Independently ofthe mechanism involved in the increased mitochondrial perme-ability, the main consequence of the apoptotic stimuli is the releaseof pro-apoptotic factors from the mitochondria to the cytosol thatpromote the formation of a multiprotein complex known as apop-tosome which eventually leads to the activation of caspase-9 andthe onset of cellular disassembly.

In the extrinsic pathway, cell death is triggered following recog-nition and binding of cognate ligands to their death receptors.These cell-surface receptors belong to the tumor necrosis/nervegrowth factor (TNF/NGF) receptor superfamily. Ligand binding pro-motes the oligomerization of the receptors and the assembly of aprotein complex known as death-inducing signaling complex(DISC) that mediates the recruitment of pro-caspases 8 and 10leading to their autoproteolytic cleavage and activation (Mahmoodand Shukla, 2010). Finally, cells that are subjected to a sustainedand irreversible endoplasmic reticulum stress undergo apoptosis.

Fig. 1. Bile acid biosynthesis. Primary bile acids (CA, cholic acid; CDCA, chenode-oxycholic acid) are synthetized from cholesterol in the liver. Once in the largeintestine, bacterial flora catalyzes their biotransformation into secondary bile acids.Thus, dehydroxylation of CA generates deoxycholic acid (DCA), while this reactionproduces lithocholic acid (LCA) from CDCA. Ursodeoxycholic acid (UDCA) derivesfrom epimerization of CDCA through the stable intermediate 7-oxo-lithocholic acid(7-oxo-LCA). This oxo-bile acid can be reabsorbed and transported to the liver viaenterohepatic circulation (discontinuous line) and transformed into CDCA and, to alower extent, UDCA.

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ER can regulate apoptosis not only by its own, but also bycommunicating with mitochondria through the activation of thetranscription factor GADD153 (growth arrest- and DNA damage-inducible gene 153; also known as CHOP) (Rasheva and Domingos,2009), or by a Ca2+-dependent activation of caspase-12.

It is well established that oxidative stress is one of the mainstimuli triggering directly or indirectly some of these apoptoticpathways through different molecular mechanisms (Matés et al.,2012). ROS can induce DNA damage that may trigger cell death(i.e. via p53 activation), but could also activate proto-oncogenes orinactivate tumor suppressor genes, leading to cell transformation.

5. Apoptosis in the colon

Apoptosis is an essential mechanism for the homeostasis oforganisms, being the colonic epithelium one of the best examplesof the regulatory properties of this programmed cell death. Theintestinal epithelium is a rapidly renewing tissue whose homeosta-sis depends on the correct equilibrium between cell proliferationand cell death. The anatomy of the colonic epithelium consists in‘‘hills and valleys’’ shaped structures called crypts, formed by a sin-gle layer of epithelial cells (Fig. 2). It is well established that thesecolon crypts show a polarized topographical organization wherethe position of a specific cell is closely related to its function,behavior, proliferation ability and fate (Radtke and Clevers,2005). The mature colon epithelium is composed of two maintypes of differentiated cells, enterocytes and goblet cells, with aminor population of enteroendocrine cells. Goblet cells are mainlyfound in the middle area of the crypts, and their function is thesecretion of mucin. Endocrine cells are usually confined to the low-er third of the crypts, and absorptive enterocytes, also known ascolonocytes, are found at the top third and surface of the crypt.On the other hand, colon undifferentiated stem cells occupy thebottom of these structures. Thus, the differentiation state of thecells depends on their position along the crypt, with the lessdifferentiated cells at the bottom, and the more differentiatedones at the top. This differentiation process, and its associated

morphological changes, takes place during migration towards thelumen. When colonocytes reach the surface of the crypt thedifferentiation process is complete, but for an efficient and correctfunction of the colon, removal of senescent colonocytes is essential.While some of these cells are mechanically removed by the exfoli-ating action of the passage of lumen components, the main impor-tant process regulating their renewal is apoptosis (Potten, 1998;Potten et al., 1997). In this regard, it has been described thatBcl-2 expression decreases upward along the crypts, showing thehighest expression levels at the bottom and minimal amounts atthe top (Renehan et al., 2001). Thus, the balance between cellrenewal and apoptosis is responsible for the maintenance of thesize and architecture of the colon. Moreover, apoptosis is also anessential defense mechanism against genotoxic damage inducedby diet carcinogens, or potential tumor promoters such as bileacids. When these mechanisms of control are lost, abnormal cellgrowth can occur and colon cancer may arise.

6. Bile acids and apoptosis in colon cells

The apoptotic effect of bile acids has been extensively ana-lyzed in hepatocytes. However, during the last years, an increas-ing interest is raising in the study of the cytotoxic effect of thesecompounds in colon epithelial cells and other gastrointestinalcells, either normal or tumorigenic. We have focused this reviewin colon cells as they are one of the main targets of bile acidswithin the gastrointestinal tract. Different experimental modelshave been used for the analysis of the regulatory and cytotoxic ef-fects of bile acids in colon (Table 1). Most of the studies havebeen developed in colon cancer cell lines, among them HCT-116, HT29, Caco-2 and BCS-TC2, although these effects have alsobeen analyzed in tissues obtained from colon cancer patients,mice models (i.e. azoxymethane AOM murine model), normal col-onocytes and FHC cells. The latter were established from normalfetal colonic mucosa by Siddiqui and Chopra (Siddiqui and Cho-pra, 1984) but later on a mutation in TP53 together with a tumor-igenic phenotype have been described for these cells (Souceket al., 2010).

There are several reasons for a better understanding of theinfluence of bile acids in the colonic tract. Firstly, besides decon-jugation of taurine and glycine, it is important to take into ac-count that one of the main processes during the transformationof bile acids takes place in the colon: the 7a-dehydroxylation ofthe primary bile acids CA and CDCA by the anaerobic colonic bac-terial flora to yield, respectively, the secondary bile acids DCA andLCA. Under physiological conditions, concentrations of bile acidsin the final tract of the colon may be low compared to thosefound in the gallbladder (�300 mM) or the small intestine(�10 mM). However, concentrations as high as 1 mM in cecumor in fecal waters can be reached in humans with fat-rich dietor under pathological circumstances; under these conditions,DCA concentrations can reach values around 700–800 lM(Hofmann, 1999; Longpre and Loo, 2008; Stadler et al., 1988).Secondly, the most relevant reason is the rising evidence for thetumor-promoting role of bile acids in colon by the selection ofcell populations resistant to the apoptotic effects of these agents.Thus, here we will briefly review the different apoptotic pathwaystriggered by bile acids in colon cells, which are summarized inFig. 3.

6.1. The death receptor pathway

Death receptors are cell surface cytokine receptors belonging tothe tumor necrosis/nerve growth factor (TNF/NGF) receptor super-family. Among them, the best studied ones are Fas, TNF-R1 and

Fig. 2. Structure of the colonic crypts. The basic functional unit of the colon is thecrypt. These structures are composed by several types of cells, with differentfeatures and localizations. Thus, at the bottom of the crypts localize stem cells,undifferentiated and highly proliferative. Differentiation degree increases along thecrypt, inversely correlated with the proliferative ability. Interspersed amongcolonocytes, enteroendocrine and globet cells are found confined to the lowerthird and the middle of the crypt, respectively. Colonocytes are distributed all overthe crypt, showing the most differentiated state at the top. These cells eventuallyundergo apoptosis allowing the renewal of the colonic epithelium.



TRAIL-R1. Although it has been previously demonstrated thathepatocytes are able to respond to bile acids via Fas, this deathreceptor-mediated response does not seem to be active in coloncells. In this regard, there are several experimental evidencespointing towards a death receptor-independent apoptotic effectof bile acids in colon cells (Schlottman et al., 2000; Wachs et al.,2005). In this regard, it has been observed that Fas-positive HT-29 and SW480 cells do not suffer apoptosis after treatment withthe agonist anti-Fas antibody CH-11. Moreover, the Fas-negativeCaco-2 and SW620 cells undergo bile acid-induced apoptosis(Schlottman et al., 2000). Interestingly, activation of caspase-8has been detected in different colon cancer cell lines after

treatment with bile acids. These observations contrast with thepreviously mentioned death receptor-independent bile acid-induced apoptosis, though different possible mechanisms ofcaspase-8 activation have been postulated: a direct activation bybile acids or as well by caspases that are downstream of theperturbation of the mitochondrial transmembrane potential suchas the initiator caspase-9 (Schlottman et al., 2000).

In contrast, other reports support the notion that bile acid-in-duced apoptosis is dependent on Fas in HT-29 and HCT-116 cellsbased on the specific activity of different synthetic enantiomersof LCA, DCA and CDCA, and their ability to activate caspase-8(Katona et al., 2009). These authors suggest that, after treatment

Table 1Experimental models for the study of bile acid effects in colon.

Model Features Effects of bile acids/references

HCT-116 cells – Tumorigenic – Hydrophobicity-related membrane alterationsHCT-15 cells – Positive for transforming growth factor beta 1

(TGF beta 1) and beta 2 (TGF beta 2) expression– Bile acids induce apoptosis– DCA induces: intracellular signaling by membrane perturbations, activation of PKC,

mitochondrial and ER stress, ROS, NF-KB activation, DNA damage, activates ERK and p38,decreases p53 levels and induces GADD153 expression

– Low doses of DCA can be protective(Akare and Martinez, 2005; Bernstein et al., 2004; Brossard et al., 2010; Ha and Park, 2010;Jean-Louis et al., 2006; Katona et al., 2009; LaRue et al., 2000; Longpre and Loo, 2008; Loobyet al., 2005; Payne et al., 2005; Payne et al., 2007; Powell et al., 2006; Powell et al., 2001;Powolny et al., 2001; Qiao et al., 2002; Washo-Stultz et al., 2000; Yui et al., 2008, 2009; Yuiet al., 2005)

– Mutation in codon 13 of the ras protooncogene

HT-29 cells – Tumorigenic – Sodium butyrate and bile acids induce apoptosis– Elevated levels of p53– myc+, ras+, myb+, fos+, sis+, p53+, abl�, ros�, src�,

CD4�

– DCA and CDCA induce DNA damage(Haza et al., 2000; Katona et al., 2009; Marchetti et al., 1997; Milovic et al., 2002; Rosignoliet al., 2008; Rosignoli et al., 2001; Schlottman et al., 2000; Wachs et al., 2005;Washo-Stultz et al., 2002; Washo-Stultz et al., 2000)

Caco-2 cells – Tumorigenic – Hydrophobicity-related membrane alterations– Express retinoic acid binding protein I and retinol

binding protein II– ROS generation– DCA activates PCK and PLC and induces apoptosis

(Akare and Martinez, 2005; Araki et al., 2005; Ha and Park, 2010; Lau et al., 2005; Milovicet al., 2002; Schlottman et al., 2000; Wachs et al., 2005)

SW-480 cells – Tumorigenic – Low doses of DCA induce b-catenin activation, proliferation and invasivenessSW-620 cells – Elevated levels of p53 – DCA induces apoptosis

– Matrilysin is not expressed (Schlottman et al., 2000; Wachs et al., 2005)– Mutation in codon 12 of the ras protooncogene– myc+, myb+, ras+, fos+, sis+, p53+, abl�, ros�, src�

BCS-TC2 cells – Non tumorigenic – Hydrophobic bile acids induce concentration-dependent cytotoxicity– Sensitive to the apoptotic effects of butyrate – DCA and CDCA induce apoptosis via oxidative stress

(Barrasa et al., 2011; Olmo et al., 2007; Pérez-Ramos et al., 2005)

BCS-TC2.BR2cells

– Tumorigenic– Resistant to the apoptotic effects of butyrate

– Cytotoxic effect of DCA and CDCA is lower than in their parental butyrate-sensitive cells– Overexpression of Bcl-2 confers resistance to bile acid-induced apoptosis

(Barrasa et al., 2012; Olmo et al., 2007; Pérez-Ramos et al., 2005)LoVo cells – Tumorigenic

– myc+, myb+, ras+, fos+, p53+, sis�, abl�, ros�, src�– DCA induces proliferation and invasiveness through an increase in tyrosine

phosphorylation of b-catenin(Pai et al., 2004)

H508 cells – Tumorigenic – Bile acids induce colon cancer cell survival by Akt dependent NF-jB activation– Contain Dopa decarboxylase, CA19-9 antigen andCEA but do not express the TAG-72 antigen

– Bile acids induce colon cancer cell proliferation by activation of EGFR(Cheng and Raufman, 2005; Shant et al., 2009)

SNUC4 cells – Tumorigenic– Mutations in hMSH3, BAX and TGF-bRII

– Bile acids induce colon cancer cell survival by Akt dependent NF-jB activation(Cheng and Raufman, 2005)

– Microsatellite instability– Overexpression of MDR1

FHC cells – Human fetal colonic mucosa – Analysis of apoptosis after treatment of monolayers with fecal waters(Haza et al., 2000)

Normalcolonocytesor tissue

– Human normal colorectal mucosa and humanprimary cell culture

– DCA and CDCA induce apoptosis and DNA damage(Garewal et al., 1996; Ha and Park, 2010; Rosignoli et al., 2008)

Tumor biopsies – Human mucosa samples from colon resections – DCA and CDCA induce apoptosis and DNA damage– Reduced apoptosis in CRC samples compared to normal tissue

(Bernstein et al., 2002; Holubec et al., 2005)Mice – Wild-type male B6.129PF2/J

– C57BL/6J FXR�/�– DCA is carcinogenic at high-doses and long-term treatments

(Bernstein et al., 2011; Modica et al., 2008)



with different bile acids, the subsequent generation of reactiveoxygen species (ROS) can lead to Fas translocation and oligomeri-zation, dependent on epidermal growth factor receptor (EGFR)activity, with the consequent activation of caspase-8. Although thiseffector caspase is usually related to the extrinsic apoptotic path-way, it can cleave the pro-apoptotic BH-protein Bid, yielding tBid,which can also trigger the mitochondrial intrinsic pathway withthe subsequent generation of ROS. This caspase-8 and Bid-dependent mitochondrial damage is not clear in colon cells;whereas Bid activation by LCA has been detected in HT-29 andHCT-116 cells (Katona et al., 2009), DCA-induced apoptosis inHCT-116 cells is independent of this pro-apoptotic protein (Yuiet al., 2005). In addition, we have observed that apoptosis inducedby DCA and CDCA in BCS-TC2 cells is dependent on activation ofcaspase-9 with no significant activation of caspase-8 (Barrasaet al., 2011). Finally, it has been described that colon cancer cellsmay down-regulate Fas surface expression, or develop signalingstrategies that inhibit the Fas receptor signaling pathway, to escape

the pro-apoptotic killing by cytotoxic tumor-infiltrating lympho-cytes (Wachs et al., 2005).

6.2. The mitochondrial pathway

The apoptotic program triggered by mitochondrial membraneperturbations is probably the best characterized one in colon can-cer cells. Several works report on the ability of bile acids to inducechanges in the mitochondrial membrane permeability with thesubsequent release of pro-apoptotic molecules such as cytochromec or Smac/Diablo. Cytochrome c release is followed by apoptosomeformation and caspase-9 activation. Bile acid-induced apoptosisthrough this mitochondrial pathway has been described in HCT-116, HT-29, Caco-2, SW480, SW620, BCS-TC2 and BCS-TC2.BR2cells (Barrasa et al., 2011, 2012; Payne et al., 2007; Wachs et al.,2005; Washo-Stultz et al., 2002). Regarding HCT-116 cells, it hasbeen proposed that bile acid treatment, mainly DCA, promotesthe release of cytochrome c from the mitochondria by an undefined

Fig. 3. Bile acid-induced apoptosis. Bile acids are able to activate membrane-associated enzymes, such as NAD(P)H oxidases and PLA2, leading to an increased production ofreactive oxygen species (ROS). On the other hand, activation of PLC by these agents can induce the endoplasmic reticulum apoptotic pathway (characterized by the release ofCa2+ and Bak), while membrane receptors such as EGFR or Fas trigger the extrinsic apoptotic pathway, with the subsequent activation of caspase-8. Oxidative stress promotesthe mitochondrial membrane permeability transition (MPT) and the release of pro-apoptotic factors to the cytosol. After the assembly of the apoptosome, caspases-9 and -3execute the intrinsic apoptotic program. Moreover, Bcl-2 cleavage by caspases allows the formation of additional Bax-dependent pores in the mitochondrial membrane.Finally, oxidative stress also produces genotoxic damage with the subsequent activation of transcription factors such as p53 or GADD153, promoting cell cycle arrest and,eventually, apoptosis.



but specific mechanism (Yui et al., 2005). One hypothesis is thatDCA generates increased ROS production by activation of plasmamembrane associated proteins such as NAD(P)H oxidases andPLA2 with the subsequent mitochondrial oxidative stress, loss ofmitochondrial membrane potential and release of pro-apoptoticfactors (Barrasa et al., 2011; Payne et al., 2007). On the other hand,ROS can also be produced by direct bile acid-induced mitochon-drial damage by different mechanisms, such as direct oxidativedamage, endogenous generation of arachidonic acid, truncationof Bid due to ligand-independent activation of the Fas receptor(FasR), release of Bak from the stressed ER, or the decay of mito-chondrial NAD+ levels as a result of ROS induced DNA damageand subsequent increase in poly(ADPribose) polymerase (PARP)activity (Bernstein et al., 2005). Supporting this notion, it has beenobserved that treatment with different inhibitors of mitochondrialcomplexes I–V protects HCT-116 cells against DCA-induced apop-tosis (Payne et al., 2005). Moreover, rottlerin [a well-known gener-ic protein kinase C (PKC) inhibitor] presents antioxidant activityand is able to largely protect HCT-116 cells against the genotoxiceffects of DCA (Longpre and Loo, 2008). It is also interesting tomention that DCA is able to induce the mitochondrial apoptoticprogram in HCT-116 cells in the absence of Bax (Yui et al., 2008).Although Bax is one of the most important pro-apoptotic membersof the Bcl-2 family whose oligomerization promotes the appear-ance of pores in the mitochondrial membrane, it is thought thatthe increased ROS generation after DCA treatment is sufficient topromote the mitochondrial permeability transition with the subse-quent release of cytochrome c in a Bax-independent manner. Sup-porting this idea, we have observed that DCA and CDCA are able totrigger apoptosis in BCS-TC2 cells through an increase in ROS gen-eration that promotes the mitochondrial permeability transitionand subsequent activation of caspase-9. However, this responseis followed by an amplification of the apoptotic signal via Bcl-2degradation and Bax activation, although MPT seems to be an ini-tial step in this bile acid-induced apoptosis (Barrasa et al., 2011). Inthis regard, we have established the BCS-TC2.BR2 cell line that isresistant to the apoptotic effects of butyrate and also partially toother cellular stresses including bile acids (López de Silanes et al.,2004; Olmo et al., 2007; Pérez-Ramos et al., 2005). Although DCAand CDCA still induce apoptosis in BCS-TC2.BR2 cells via oxidativestress, higher concentrations than in the parental cells are re-quired. This is due to overexpression of Bcl-2 that blocks the ampli-fication of the apoptotic response by inhibition of Bax (Barrasaet al., 2012).

6.3. The endoplasmic reticulum pathway

The ER integrity and calcium homeostasis have been previouslyrelated with the triggering of the apoptotic program mainly byactivation of caspase-12. Little is known about this apoptotic path-way in colon cells, although several reports point out to a second-ary role of the ER during bile acid-induced apoptosis. In Caco-2 andHT-29 cells, an increase in the intracellular levels of Ca2+ has beenobserved after DCA treatment, with the subsequent triggering ofapoptosis (Lau et al., 2005; Marchetti et al., 1997). Although themechanisms responsible for this activation are not yet known, ithas been proposed that DCA may activate PLC. The subsequent pro-duction of IP3 and DAG leads to a Ca2+ release from the ER to thecytoplasm that, in turn, can drive the activation of signaling path-ways, such as PKC. On the other hand, it has also been observedthat, in HCT-116 cells, DCA is able to generate dilatation of theER together with mitochondrial stress. Nevertheless, this ER stressseems to be a secondary effect of the mitochondrial alterations, asthe inhibition of mitochondrial electron transport complexes pro-tects against both mitochondrial and ER stresses generated byDCA (Payne et al., 2005).

6.4. DNA damage and p53

Several studies have demonstrated the ability of bile acids to in-duce DNA damage in different colon cells, such as HCT-116, HT-29,Caco-2 and FHC (Haza et al., 2000; Marchetti et al., 1997; Powolnyet al., 2001; Rosignoli et al., 2008). This genotoxic effect has beenrelated to an increase in ROS generation after bile acid treatments(mainly DCA and LCA), as different dietary antioxidants as b-carotene and a-tocopherol exert a protective effect (Rosignoliet al., 2008). Thus, the role of p53 in the apoptotic response to bileacids in colon cells has been considered. As p53 is a tumor suppres-sor gene strongly related with the apoptotic response to cellularstresses, mainly DNA damage, the hypothesis that bile acid-induced apoptosis could be dependent on p53 function wasstrongly suggested. However, data obtained in different coloncancer cells do not support this notion, as DCA is able to triggerapoptosis in a p53-independent manner after DNA damage. DCAinduces DNA damage and apoptosis in human colon epithelial cellsexpressing either mutant (HCT-15, Caco-2 and HT-29 cells) orwild-type p53 (HCT-116 cells) (Powolny et al., 2001). Interestingly,it has also been observed that DCA is able to stimulate theproteasome-mediated p53 protein degradation in HCT-116 cells(Qiao et al., 2001a). This, in turn, could enhance bile acid-inducedmutagenesis and lead to increased cancer risk. Finally, it has beendemonstrated that DCA strongly increases the expression ofGADD153 in HCT-116 cells, being this gene essential for bile-acidinduced apoptosis (Qiao et al., 2002). This type of GADD153-dependent and p53-independent bile acid-induced apoptosis wasalso previously observed in the non-colonic cervical adenocarci-noma HeLa cells (Zheng et al., 1996).

7. Effect of bile acids in oxidative stress and cell signaling incolon cells

During the last years it has become progressively more evidentthat bile acids are able to trigger different responses within thecells (Baptissart et al., 2012). In this regard, several reports pointout towards distinct mechanisms to develop these regulatory ef-fects. Although these processes have been better characterized inhepatocytes (Hylemon et al., 2009; Perez and Briz, 2009), we willfocus this review in colon cells, where bile acids act in similar waysthan in other enterohepatic cells, but differ in some mechanismsand responses (Fig. 4). It has been reported that bile acids are ableto induce alterations in the plasma membrane of HCT-116 andCaco-2 cells leading to changes in its physicochemical propertiesand lipid composition (Akare and Martinez, 2005; Jean-Louiset al., 2006). These effects depend on their hydrophobicity andcan be mimicked by other hydrophobic molecules. DCA and otherbile acids induce an increase in the quantity of cholesterol in theplasma membrane, which is accompanied by an overall decreasein membrane fluidity. It has been hypothesized that this could bea cell response to prevent bile acid-induced cell membrane lysisgenerated by their detergent-like properties (Jean-Louis et al.,2006). These alterations may lead to the activation of membrane-associated enzymes that trigger an increase in ROS, or as well acti-vate raft-associated receptors in a ligand-independent mannerwith a concomitant activation of signaling events leading to a pro-liferative or pro-survival stress-response pathways. These two dif-ferent bile acid-induced events are described in the followingsections.

7.1. Oxidative stress induced by bile acids

It is well established that one of the most important cytotoxiceffects of bile acids is the increased production of reactive



oxygen/nitrogen species (ROS/RNS) and subsequent apoptosis trig-gered by oxidative stress. Bile acid-induced ROS generation is mul-tifactorial. One of the main sources of ROS production by bile acidsin colon cells is the activation of several plasma membrane en-zymes, such as NAD(P)H oxidases and PLA2. For instance, it hasbeen proposed that, in Caco-2 cells, bile acids can increase thePLA2 activity by alteration of the transepithelial permeability; thesubsequent enhanced production of arachidonic acid by PLA2 leadsto an increased generation of ROS by cyclooxygenase and lipoxyge-nase enzymes as by-products of the synthesis of prostaglandins(Araki et al., 2005). Bile acid-induced activation of NAD(P)H oxi-dases has been observed in hepatocytes, and the following increasein ROS production has been shown to be essential for bile acid-induced apoptosis (Reinehr et al., 2005). Although little is knownregarding this process in colon cells, we have recently reportedthat in BCS-TC2 cells, DCA and CDCA induce ROS productionmainly through this mechanism with a minor contribution ofPLA2 (Barrasa et al., 2011).

Increased ROS generation may also be a consequence of bileacid-induced perturbations in the mitochondria, as we have previ-ously mentioned. In connection with the role of pro-apoptotic pro-teins in bile acid-induced mitochondrial damage, there are severalmembers of the Bcl-2 protein family that are able to alter the mito-chondrial membrane structure leading to a malfunction of the elec-tronic transport chain and a loss of the mitochondrial potential. Inaddition, bile acids may induce ER stress that can be associatedwith mitochondrial stress. It is well known that the pro-apoptoticBcl-2 family member Bak is associated with the ER membrane.Thus, bile acid-induced damage could stimulate the release ofBak from the reticulum with the subsequent binding to the mito-chondrial membrane. In this regard, it has been observed that afterER stress in HEK-293T human embryonic kidney cells, the BH3-only molecules Bim and Puma are able to fully induce cytochromec release from the mitochondria followed by apoptosis in the sole

presence of reticular Bak (Klee et al., 2009). Moreover, several re-ports point out the fact that perturbations in the mitochondrialmembrane fluidity by hydrophobic bile acids can lead to a mal-function in the electron transport chain with the consequent gen-eration of ROS (Palmeira and Rolo, 2004). On this idea, it has beendescribed that arachidonic acid promotes ROS generation by inter-action with the mitochondrial electron transport chain (Coccoet al., 1999) and, as we have previously mentioned, bile acids in-crease the intracellular levels of arachidonic acid through the stim-ulation of PLA2 activity.

Closely related to bile-acid induced ROS generation, the redox-sensitive transcription factor NF-jB should be considered. Thistranscription factor is crucial in different cellular processes, suchas inflammation, immunity, cell proliferation and apoptosis. In fact,a constitutive activation of NF-jB in the colon has been associatedto the production of pro-inflammatory cytokines leading to colitisand eventually to colon cancer. NF-jB can be activated by ROS-dependent and ROS-independent signaling pathways, as has beenshown in different cell types (Gloire et al., 2006; Morgan and Liu,2011). In HCT-116 and HT-29 cells, DCA is able to activate NF-jBexpression through different mechanisms, including NAD(P)H oxi-dases, Na+/K+-ATPase, cytochrome P450, Ca2+ and the terminalmitochondrial respiratory complex IV (Glinghammar et al., 2002;Payne et al., 2007).

7.2. Bile acids: activation of nuclear receptors and cell signalingpathways

Bile acids are considered regulatory molecules: they can act ashormones that interact with nuclear receptors and can also triggermembrane perturbations that activate pathways involving mem-brane-associated proteins (Hylemon et al., 2009).

Bile acids trigger nuclear signal events in colon cells by directactivation of nuclear receptors or by regulation of different

Fig. 4. Bile acids signaling pathways. Bile acids induce perturbations in the plasma membrane that eventually activate several receptors and enzymes. Thus, oxidative stressinduced by NAD(P)H oxidases and PLA2 leads to the activation of NF-jB transcription factor. Production of inositol triphosphate (IP3) by PLC promotes the release of Ca2+ fromthe reticulum, and the subsequent stimulation of PKC. Moreover, bile acids are able to directly induce activation, translocation and differential subcellular localization of PKCisoforms. The bile-acid induced G-protein coupled receptor TGR5 increases AMPc levels by stimulation of adenylate cyclase (AC) followed by activation of PKA (but TGR5 inthe colon is only present in enteroendocrine cells). On the other hand, tyrosine kinase receptors such as EGFR, induce phosphorylation of the small GTPase Ras, triggering theAkt, ERK and JNK signaling pathways. Bile acids indirectly stimulate p38 MAPK through PKC activation. Finally, in colon cells, bile acids bind to their nuclear receptors (PXR,pregnane X receptor; FXR, farnesoid X receptor; VDR, vitamin D receptor), inducing transcriptional changes.



transcription factors. In this regard, there are mainly four bile acidnuclear receptors: the farnesoid X receptor (FXR), the pregnane Xreceptor (PXR), the liver X receptor and the vitamin D receptor(VDR). Although the central role of these bile acid receptors isthe control of the metabolism of lipids and carbohydrates, theyare also important regulators of cell proliferation and differentia-tion, cell death, survival, invasion, and metastasis during normaldevelopment and cancer. The expression and activity of thesereceptors have been analyzed in different colon cell lines. In thisregard, FXR expression is down-regulated in colon-derived adeno-carcinoma cell lines, being inversely correlated with the differenti-ation degree. Thus, the highly tumorigenic and metastatic SW480and SW620 cells do not express this receptor, while significant lev-els are detected in the less tumorigenic Caco-2 and HT-29 cells (DeGottardi et al., 2004). Moreover, in two experimental murine mod-els for intestinal cancer, FXR deficiency led to a significant increasein the size and number of the tumors (Maran et al., 2009); in addi-tion, FXR�/� APC+/� mice showed adenomatous-like lesions andnumerous aberrant crypt foci even before carcinomas had actuallyformed (Modica et al., 2008). On the other hand, PXR has a protec-tive effect in HCT-116 cells against the apoptotic effects of DCAaltering the expression of multiple apoptosis-related genes, suchas MCL1, BAK1 or TP53 (Zhou et al., 2008), and constitutive activa-tion of PXR in transgenic mice confers resistance to LCA toxicity(Zhou et al., 2008). Regarding VDR, mice deficient in this receptorshow cellular hyperproliferation, while treatment of APC Min+/�

mice with the active form of vitamin D induce a decrease in tumordevelopment (D’Errico and Moschetta, 2008). On the other hand,VDR expression has been found to be increased in human colorec-tal tumors associated with mutations in PIK3CA and KRAS althoughwithout a significant correlation with patient survival (Kure et al.,2009). In addition, protective effects of nuclear receptors againsttumor development have been related to their ability to inducethe transcription of different detoxifying enzymes that mediatethe biotransformation and excretion of bile acids, thus reducingthe exposition of colonic cells to these agents (Degirolamo et al.,2011).

It is also well documented that bile acids induce membrane per-turbations that activate different signaling pathways, among themthose involving PKC and EGFR. Regarding PKC a, it is thought thatbile acid-induced activation is a consequence of changes in themembrane curvature and in its structure and phospholipid compo-sition, as it has been previously reported that this PKC is stronglyinfluenced by the physiochemical properties of membrane (Akareand Martinez, 2005). Moreover, DCA is able to induce translocationand differential subcellular localization of PKC isoforms b1, e and din HCT-116 cells, playing an essential role in the development andmaintenance of the abnormal morphogenesis of the colonic epithe-lium (Looby et al., 2005). The activation of PKC and PLC by DCA hasbeen reported through the induction of both Ca2+ release frominternal stores and persistent Ca2+ entry at the plasma membrane(Lau et al., 2005). Although the molecular mechanisms that lead tothis Ca2+ release are not fully understood, they could be related tothe ER stress observed in different colon cancer cells after bile acidtreatment.

The ability of bile acids to activate the EGFR pathway in a li-gand-independent manner has also been well analyzed. DCA isable to modify the quantity and positioning of lipid rafts inHCT-116 cells consequently leading to intracellular signaling(Jean-Louis et al., 2006). It has been suggested that many cells re-quire transactivation of EGFR by G-protein coupled receptors, likeM3R, to trigger a mitogenic response. Supporting this notion,DCA-conjugates stimulate signaling and proliferation in H508 cellsthat co-express the muscarinic receptor M3R and EGFR, but not inChinese hamster ovary CHO cells that express only M3R, or SNU-C4cells that express only EGFR (Cheng and Raufman, 2005). Another

G-protein coupled receptor that responds to bile acids is TGR5(Kawamata et al., 2003). This receptor seems to be essential forthe bile acid-induced activation of EGFR in gastric cells and forthe production of ROS by upregulation of the NADPH oxidaseNOX5-S in esophageal cells (Hong et al., 2010; Yasuda et al.,2007). However, in the colon, this receptor is only expressed inenteroendocrine cells but not in colonocytes or in goblet cells(Thomas et al., 2009).

One of the main signaling pathways clearly affected after bileacid treatment in colon cells is the mitogen-activated protein ki-nases (MAPKs) cascade (Kyriakis and Avruch, 2012). This familyof kinases is able to transduce extracellular signals from the mem-brane to the nucleus, and is composed by three members: the p38MAPK, the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK). Several lines of evidence point out to-wards a bile acid-induced MAPK activation downstream of othersignaling cascades in human colon cells. Thus, in HCT-116 andCaco-2 cells, ERK activation after bile acid treatment has been sug-gested to depend on PKC activation (Akare and Martinez, 2005). Ina similar way, it has also been observed that ERK activation takesplace downstream the bile acid-induced phosphorylation of EGFRin H508 cells leading to increased proliferation (Cheng andRaufman, 2005). On the other hand, it has also been proposed thatincreased ROS production in Caco-2 cells after bile acid-inducedactivation of NADH dehydrogenase and xanthine oxidase, leadsto the activation of phosphatidylinositol 3-kinase (PI3K) that, inturn, promotes phosphorylation of p38 and ERK (Araki et al.,2005). A similar activation of p38 and ERK has also been observedin DCA-treated HCT-116 cells and it has been proposed that ERKactivation reduces sensitivity to DCA-induced apoptosis (Qiaoet al., 2001b). Other mechanism by which bile acid-induced EGFRactivation mediates cell survival is via the PI3K/Akt signaling path-way. In HT-29 and H508 cells, activated Akt can phosphorylate IjB,thereby releasing NF-jB that translocates to the nucleus triggeringthe transcription of target genes (Shant et al., 2009).

DCA at low concentration (5 and 50 lM) is able not only to pro-mote cell growth, but also invasiveness by activation of the b-catenin signaling pathway in SW480 and LoVo cells (Pai et al.,2004). The increased bile acid-induced tyrosine phosphorylationdissociates b-catenin from E-cadherin inducing loss of celladhesion; it also stabilizes b-catenin and promotes its transloca-tion into the nucleus and subsequently increases the expressionof urokinase-type plasminogen activator, its receptor, and cyclinD1, among other targets. The mechanism by which DCA increasestyrosine phosphorylation of b-catenin remains unknown, but theauthors suggest that it may involve activation of growth factorreceptors such as EGFR or c-met (Pai et al., 2004). Regarding othercell cycle proteins, different effects have been observed after DCAtreatment of normal colon cells, and Caco-2 and HCT-116 cells.Thus, DCA treatment increases cyclin E levels in HCT-116 cellsbut not in Caco-2 and normal colonocytes, whereas cyclin A iselevated in Caco-2 and normal colon cells but not in HCT-116 cells(Ha and Park, 2010).

One of the most intriguing observations is that although exper-imental evidence demonstrates that bile acids induce oxidativestress that triggers apoptosis, they are also able to promotecompensatory activation of pro-survival pathways, such as theactivation of EGFR, MAPKs, Akt, b-catenin or NF-jB. These stress-response pathways alter the expression of target genes, reducingthe expression of pro-apoptotic factors and promoting that ofanti-apoptotic ones, and activating growth regulatory genes andtranscription factors (Amaral et al., 2009; Qiao et al., 2001a).Regarding the in vitro experiments using cells in culture, cell viabil-ity is always decreased by treatment with hydrophobic bile acids,which are inherently toxic compounds. But cells remaining in cul-ture present activated pro-survival pathways that function as a



compensatory mechanism to avoid, at least partially, the cytotox-icity of bile acids. For example, suppression of ERK increases thesusceptibility of HCT-116 cells to the pro-apoptotic effects of bileacids whereas an artificial overexpression of this kinase signifi-cantly decreased the pro-apoptotic response (Qiao et al., 2001b).The concurrence of these strikingly opposite activities has beensuggested as an important mechanism for the maintenance ofthe colonic homeostasis in which epithelial cells try to overcomethe extensive elimination of cells when bile acid concentration iselevated (Martinez et al., 1998; Riegler et al., 1992). However,the resultant cell populations that maintain a proliferative statusare prone to DNA damage that may result in mutations leadingto malignant transformation. These cells may even become resis-tant to the apoptotic effects of bile acids while maintaining activetheir effects on growth promoting signals. In fact, the appearanceof cell populations resistant to bile-acid induced apoptosis hasbeen observed in vitro as we will comment on the following section(Payne et al., 2008).

8. Colorectal carcinogenesis and bile acids

Attending to incidence and mortality statistics, colorectal can-cer can be considered as the third most common form of cancerand the second most common cause of cancer-related death world-wide, leading to an incidence of 1.2 million new cases and 608,700deaths estimated in 2008 (Jemal et al., 2011). Together with theinherited mutations, environmental factors are strongly involvedin the development of this disease. Among them, diet and nutri-tional habits constitute determinant factors in the appearance ofsporadic colorectal cancer. There is increasing evidence that afat-rich diet is positively correlated with colon cancer incidence(Bernstein et al., 2011; Butler et al., 2009; Carroll, 1991). In this re-gard, a diet with a high content in saturated fats has been associ-ated with elevated levels of bile acids in the colonic lumen as aconsequence of an increased bile discharge (Reddy, 1981; Reddyet al., 1980). In addition, patients with colonic adenomas and car-cinomas present usually elevated concentrations of fecal bile acidsand epidemiologic studies reveal an increased risk of colorectalcancer linked to high serum or fecal bile acids concentrations(Bayerdorffer et al., 1995; Reddy and Wynder, 1977; Tong et al.,2008). Taking into account these facts, bile acids can be consideredas tumor-promoting factors in colorectal cancer development(Bernstein et al., 2011, 2005; Debruyne et al., 2001). The expositionto high levels of bile acids has two important consequences. First,bile acids promote DNA damage, mainly by oxidative stress,inducing mutations that may lead to an aberrant expression ofoncogenes or tumor suppressor genes. Second, the continuousexposure to high levels of bile acids would allow selective growthof cell populations resistant to their apoptotic effects, which is oneof the major risk factors for the appearance of colonic tumors(Payne et al., 2008).

Bile acid-induced apoptosis resistance has been observedin vitro. Tissue specimens obtained from colon cancer patientsundergoing colonoscopy that were incubated in the presence ofDCA showed resistance to apoptosis, whereas biopsies from nor-mal patients showed a high level of induction of bile acid-inducedapoptosis (Payne et al., 1995). The appearance of resistance to bileacids has also been achieved using established cell lines. HCT-116cells have been used to obtain cell populations resistant to DCA.Mutagenesis induced with ethyl methane sulfonate in these cellsfollowed by long term incubation with UDCA rendered several celllines resistant to UDCA-induced growth arrest (named HOMURcells) that were also resistant to DCA-induced apoptosis. Thesecells not only showed a more neoplastic phenotype compared totheir parental cell line, but also exhibited resistance to anti-

carcinogenic agents, such as etoposide, cisplatin or adriamycin(Powell et al., 2006). In addition, HCT-116 cells grown in thepresence of increasing concentrations of DCA yield a cell popula-tion resistant to the apoptotic effects of 500 lM DCA, and showincreased expression of several genes that may play a role in apop-tosis resistance and early stage carcinogenesis, such as NFKB, BCL2or GRP78 among others (Crowley-Weber et al., 2002). Interestingly,these cells show a constitutive activation of NF-jB that couldaccount for a survival mechanism in response to the DNA damageinduced by oxidative stress. It has also been suggested that NF-jBactivation leads to colon cancer cell survival to chemotherapy andradiation (Sakamoto and Maeda, 2010; Shant et al., 2009). A deeperproteomic study of these DCA-resistant cell lines has revealed analtered protein expression profile, with under-expression ofpro-apoptotic proteins, overexpression of anti-apoptotic proteins,and defective expression of proteins related with DNA repair andcell cycle checkpoint functions that could give rise to chromosomeinstability and to accelerate progression to cancer (Bernstein et al.,2004).

In vitro studies with different colon cancer cell lines indicate anapparent ambivalence regarding the effect of bile acids promotingapoptosis or cell growth, as we have previously discussed. Thisdual behavior depends not only in the nature of the bile acid, butalso on other factors such as their concentration or time of expo-sure. On this idea, it has been reported that low concentrations(5–75 lM) of DCA stimulate growth of Caco-2 and HT-29 cells,while high concentrations (>100 lM) induce apoptosis, pointingout to a mechanism of cancer promotion by increasing epithelialcell turnover in the colon (Milovic et al., 2002). In BCS-TC2 cells,we have observed a transitory increase in DNA synthesis at DCAconcentrations below 20 lM (Pérez-Ramos et al., 2005), in agree-ment with results reported for HT-29 cells (Peiffer et al., 1997)and HCT-116 cells (Glinghammar et al., 2002). This effect is notaccompanied by an increase in the cell number as a consequenceof a cell cycle blockage at the G1 phase and in accordance with thatdescribed for other bile acid-treated tumor cell lines (Martinezet al., 1998). In addition, it has been suggested that bile acidsmay exhibit different effects in dependence of the genetic back-ground of the cells considered. For example, DCA induces apoptosisin HCT-116 cells via a PKC-dependent pathway but not in HT-29cells, although apoptosis is also triggered in these cells by a PKC-independent mechanism (Marchetti et al., 1997; Martinez et al.,1998).

9. UDCA and the protective effects of bile acids

Despite all the previously mentioned cytotoxic and pro-tumorigenic effects of bile acids, there are also evidences aboutthe potential protective effects of some of these molecules, mainlyhydrophilic bile acids as UDCA. This compound was initially usedfor gallstone dissolution and is also employed as the first-line ther-apy for primary biliary cirrhosis, as well as for other chronic chole-static liver diseases (Solimando et al., 2011). The anti-apoptoticeffects of UDCA were initially observed in hepatocytes, mainlydue to inhibition of ROS production (Mitsuyoshi et al., 1999;Rodrigues et al., 1998), and protection from mitochondrial perme-ability avoiding the release of pro-apoptotic factors (Rodrigueset al., 1999, 2003). Research on UDCA has continued to bettercharacterize the biochemical and molecular mechanisms underly-ing its beneficial effects in cell survival (Amaral et al., 2009; Romaet al., 2011). Several reports also point out the protective effect ofUDCA against the development of colonic neoplasia, mainly inhigh-risk populations such as patients with inflammatory boweldisease or prior colorectal adenoma or carcinoma (Serfaty et al.,2010; Tung et al., 2001). However, randomized-controlled trialsstill yield controversial results (Solimando et al., 2011).



In this regard, UDCA has been related with a cytoprotective ef-fect in colon epithelial cells, as it is able to induce differentiationand senescence in HCT-116 cells, being these effects related tochanges in the structure of chromatin by histone hypoacetylation.Cell differentiation induced by UDCA is quite different to that in-duced by butyrate, as UDCA is not a histone deacetylase inhibitorbut instead induces hypoacetylation of histones and increasedexpression of HDAC6 (Akare et al., 2006). UDCA also induces cellsenescence as a late reaction after three passages of growth inthe presence of 500 lM UDCA. Senescence is an irreversiblegrowth arrest of cells characterized by morphologic changes suchas enlargement, flattening, increased granularity, and inhibitionof telomerase activity. Senescence is normally a tumor suppressorresponse activated under stresses that would otherwise promotetumorigenic transformation (Saab, 2011). However, senescence in-duced by UDCA in HCT-116 cells is p53, p21 and Rb independent,and it has been suggested that HDAC6 may play a role in this pro-cess (Akare et al., 2006). Shorter treatment times of HCT-116 orHCT-8 cells with UDCA (3 days) do not induce neither senescencenor apoptosis, but also inhibit cell proliferation with a decreaseof the S-phase and an increase of G1. Growth arrest is reversibleunder these experimental conditions and seems to be caused bya suppression of c-Myc at the protein level, which is followed bya decrease in the expression of the cell cycle regulators CDK4and CDK6 (Peiró-Jordán et al., 2012). It has also been reported thatUDCA inhibits Ras mutations, wild-type Ras activation, and cyclo-oxygenase-2 expression in the AOM murine model of colon carci-nogenesis, supporting its chemopreventive role (Khare et al.,2003).

On the other hand, UDCA has also been described as an inhibitorof signaling pathways triggered by hydrophobic bile acids. On thisidea, it has been observed that UDCA inhibits DCA-induced activa-tion of the EGFR/Raf-1/ERK signaling pathway in HCT-116 cells andin the AOM mouse model (Im and Martinez, 2004; Serfaty et al.,2010). However, this is not the only signaling pathway modulatedby UDCA. In HCT-116 and SW480 cells, UDCA is able to inhibit thetranslocation of PKC isoenzymes induced by DCA, being this effectrelated with chemoprevention of colon carcinogenesis (Shah et al.,2005). In addition, UDCA blocks the activation and DNA-binding ofNF-jB induced by DCA in HCT116 cells, although by itself UDCAdoes not alter NF-jB signaling (Shah et al., 2006).

Finally, it has been described that pretreatment of HCT-116cells with 500 lM UDCA or with low concentrations of DCA andCDCA (<200 lM) suppresses the apoptosis induced by high con-centrations of DCA (>500 lM) by inhibition of caspase-9 activationwithout preventing the release of cytochrome c from the mito-chondria (Yui et al., 2009, 2005). More recently, it has been re-ported that not only the release of cytochrome c is unaffected byUDCA, but it also fails to affect the liberation of Smac/DIABLOand XIAP, or their interaction. In addition, UDCA seems to protectHCT-116 cells from DCA-induced apoptosis by inhibition of theassociation of Apaf-1 (apoptotic protease-activating factor-1) andcaspase-9 independently of the survival signals mediated by thePI3K, MAPK, or cAMP pathways (Saeki et al., 2012).

10. Concluding remarks

Here we have reviewed the essential role of bile acids in theintestinal homeostasis and colorectal carcinogenesis. Besides theirmain physiological/healthy role as natural detergents involved inthe correct digestion and absorption of dietary fat, bile acids canregulate different signaling pathways involved in cell growth andsurvival, such as those related with EGFR, PKC or MAPKs. More-over, they can also modulate the expression of different detoxify-ing enzymes by direct interaction with nuclear receptors.

Nonetheless, despite these beneficial effects, bile acids can be con-sidered cytotoxic molecules mainly due to the induction of oxida-tive stress and membrane damage as a consequence of theirhydrophobicity and detergent-like activity. Thus, the continuousexposure to high concentrations of bile acids promotes cytotoxicand genotoxic effects. In this regard, the selective growth of cellsresistant to bile acid-induced cell death, together with the activa-tion of pro-survival stress-response pathways, may generate cellpopulations that accumulate DNA damage which, in turn, may re-sult in malignant transformation. Therefore, bile acids show a dualbehavior being required for a healthy gastrointestinal function but,under a fat-rich diet or pathological conditions, may present cyto-toxic and potential pro-carcinogenic effects.

Conflict of Interest

We wish to confirm that there are no known conflicts of interestassociated with this publication and there has been no significantfinancial support for this work that could have influenced itsoutcome.

Acknowledgment

This work was supported by Grant BFU2008-04758 from theDGES (Spain).

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bile acids in the colon, from healthy to cytotoxic molecules

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