Send Orders of Reprints at [email protected] 1720 Current Drug Targets, 2012, 13, 1720-1729 Anticarcinogenic Actions of Tributyrin, A Prodrug

Renato Heidor, Juliana Festa Ortega, Aline de Conti, Thomas Prates Ong and Fernando Salvador Moreno*

Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, Brazil

Abstract: Bioactive food compounds (BFCs) exhibit potential anticarcinogenic effects that deserve to be explored. Bu- tyric acid (BA) is considered a promising BFC and has been used in clinical trials; however, its short half-life considera- bly restricts its therapeutic application. Tributyrin (TB), a BA prodrug present in milk and honey, has more favorable pharmacokinetic properties than BA, and its oral administration is also better tolerated. In vitro and in vivo studies have shown that TB acts on multiple anticancer cellular and molecular targets without affecting non-cancerous cells. Among the TB mechanisms of action, the induction of apoptosis and cell differentiation and the modulation of epigenetic mecha- nisms are notable. Due to its anticarcinogenic potential, strategies as emulsions, nanoparticles, or structured containing TB are currently being developed to improve its organoleptic characteristics and bioavailability. In addition, TB has minimal toxicity, making it an excellent candidate for combination therapy with other agents for the control of cancer. Despite the lack of data available in the literature, TB is a promising molecule for anticancer strategies. Therefore, additional preclinical and clinical studies should be performed using TB to elucidate its molecular targets and anticarcino- genic potential. Keywords: Bioactive food compounds, butyrate, butyric acid, cancer, cellular targets, chemoprevention, molecular targets, tributyrin.

INTRODUCTION BUTYRIC ACID (BA) AND CANCER Cancer is a major health problem worldwide, with ap- The hypothesis that the intake of the dietary fiber present proximately 12 million new cancer cases in 2008 [1, 2] and in fruits and vegetables may modulate the development of approximately 8 million deaths. Approximately 13.1 million several pathological conditions is not new. In the 1960s, deaths are predicted to occur in 2030 [3]. According to re- Burkitt found a lower incidence of diseases such as diver- cent estimates, the risk of developing cancer may be reduced ticulitis, appendicitis, and colon cancer among populations 30-40% when preventive measures such as the promotion of that consumed large amounts of fruits and vegetables com- appropriate nutrition including the use of specific foodstuffs pared with populations that consumed refined foodstuffs [16, and regular physical activity are adopted [4]. In this regard, 17]. This dietary pattern of consuming large amounts of several epidemiologic and preclinical studies have convinc- fruits and vegetables is associated with a greater production ingly argued for a definitive role of bioactive food com- of short-chain fatty acids (SCFA) such as acetic, propionic, pounds (BFCs) for the prevention and treatment of some and BA in the colon. Each of these acids has been tested in cancers [5-10]. BFCs generally exhibit low toxicity, are well transformed human cell lines, and BA exhibited the strongest tolerated by humans, and are pleiotropic i.e., they act on inhibitory effects on cell growth compared to other SCFAs multiple targets involved in carcinogenesis [11]. Some BFCs [18]. In the colon, BA is the main source of energy and pro- may be toxic in high doses [12]. However, its use can be vides 70 to 90% of the energy consumed by colonocytes considered as a therapy option for individuals that are prone [19], thus playing a major role in the normal development of to developing cancers, whereas the toxic effects may be tol- this organ [20]. The remarkable anticarcinogenic potential of erated [13, 14]. Therefore, future translational studies focus- BA was further observed in several in vitro and in vivo can- ing on the clinical relevance and mechanism of BFCs are cer models including prostate [21, 22], breast [23, 24], stom- needed to further assess the applicability of dietary factors as ach [25], and liver [26] cancer. Leder and Leder observed chemopreventive agents and/or adjuvant chemotherapeutic that BA and/or its salt sodium butyrate (NaBu) induced cul- agents [15]. tured erythroleukemic cell differentiation due to their struc- tural characteristics rather than through an exclusive function

of their role as energy sources or the action of their metabo- *Address correspondence to this author at the Department of Food and lites [27]. Because it exhibits potential for chemotherapy use, Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of NaBu has been considered a potential molecule for cancer São Paulo, 580 Prof. Lineu Prestes Avenue, Building 14, 05508-900, São therapy molecule [28]. Such observations led to the devel- Paulo, SP, Brazil; Tel: 00 55 (11) 3091-3630; Fax: 00 55 (11) 3815-4410; E-mail: [email protected] opment of pharmacologic-clinical studies using parenteral NaBu in patients with acute leukemia [29, 30] that resulted

1873-5592/12 $58.00+.00 © 2012 Bentham Science Publishers Anticarcinogenic Actions of Tributyrin Current Drug Targets, 2012, Vol. 13, No. 14 1721 in partial remission [29]. It is worth emphasizing that the nisms of action in cells Fig. (1), the induction of apoptosis intravenous administration of BA up to 1.5 g/kg/day during [21] and cell differentiation [52] are notable. In addition, TB 17 days [29] is not associated with noticeable toxicity [29, also modulates epigenetic mechanisms such histone acetyla- 30]. Nevertheless, the therapeutic efficacy of NaBu may be tion, whose dysregulation is currently acknowledged as an affected when ideal serum concentrations similar to those important molecular feature of carcinogenesis [15, 43, 44]. observed in in vitro studies (i.e., 0.5 mM) are not achieved [30]. NaBu is quickly absorbed [31] through the plasma TB and Apoptosis Induction membrane by simple diffusion [32], SCFA/HCO - co- 3 The induction of apoptosis is an important mechanism transport [33], and the active transport of dissociated forms. for the inhibition of carcinogenesis and cancer growth by Effective butyrate blood concentrations are difficult to main- TB. Numerous studies have documented the ability of this tain because it is quickly metabolized into ketone bodies, which mainly include 3-hydroxybutyrate [34] and butyryl- compound to induce several programmed cell death mecha- nisms in different cancer cell lines [48-53] even more in- CoA, enoyl-CoA, L-hydroxyacyl-CoA, acetoacetyl-CoA, tensely than BA [21]. The intrinsic apoptosis pathway is in- and acetyl-CoA [35] as well. Bioavailability studies have duced by TB in a caspase-3-dependent or caspase-3- demonstrated that the butyrate half-life exhibits a biphasic independent manner. Thus, in HT-29 colon cancer [46] and behavior as a consequence of its pattern of elimination from B16-F10 melanoma cells [53], TB increased apoptosis by the body. During the first minutes, butyrate clearance is fast and results in a serum half-life of just 30 seconds. Butyrate activating caspase-3 activity. In MCF-7 human mammary carcinoma cells, TB induced a transient increase in mito- then binds to serum proteins, its clearance decreases, and its chondria-associated Bax, dissipation of the mitochondrial half-life increases up to 14 minutes [36]. In rodents, the bu- membrane potential, and caspase-3-independent cleavage of tyrate serum half-life was less than five minutes after intrap- PARP [54]. These events were followed by a transient in- eritoneal or intravenous administration [36]. crease in cytosolic cytochrome c, and finally, through the The butyrate half-life may be considerably increased generation and accumulation of cells with subdiploid DNA when it is administered as its natural prodrug, namely tribu- content, a terminal apoptosis event [54]. TB inhibited the tyrin (TB) [37-39]. TB, which is found in a variety of food- growth of SGC-7901 gastric cancer cells by decreasing DNA stuffs such as milk fat and honey [40, 41], is a triacylglycerol synthesis and inducing cell death, which was associated with composed of three BA molecules esterified with . the down-regulation of Bcl-2 and up-regulation of Bax ex- Therefore, the full hydrolysis of 1 mole of TB may generate pression [55]. NaBu increased the susceptibility of hepatoma 3 moles of BA. After oral administration to rodents, the TB cells to apoptosis by inducing the signaling of tumor necrosis half-life was approximately 40 minutes [42]. In addition, the factor [56]. In an experimental hepatocarcinogenesis study in oral administration of TB to rodents produced detectable rats, TB inhibited the development of persistent preneoplas- serum butyrate levels five minutes later, and the levels tic lesions (pPNLs), which are considered sites of hepatocel- reached their peak 15 to 60 minutes after administration [39] lular carcinoma (HCC) progression. In addition, TB in- and could be maintained above 0.1 mM for up to 120 min- creased PNL remodeling, which tends to make the PNLs utes [39, 41]. Mice exhibited serum butyrate levels between disappear. This protective effect was partially associated 0.8 and 1 mM for up to one hour after administration of a with the induction of apoptosis in PNLs undergoing remod- 7.75 g/kg body weight (b.w.) dose of TB. A 8.2 g/kg b.w. eling [43]. dose of TB did not exhibit toxic effects in rodents treated via Alterations in the p53 tumor suppressor gene have been the oral or intraperitoneal routes [39]. The oral administra- linked to reduced apoptosis in cancer cells and chemotherapy tion of 2 g/kg b.w. of TB to animals subjected to an experi- mental hepatocarcinogenesis model increased the BA level resistance [57]. In three phenotypically and genotypically divergent human prostate cancer cell lines, PC-3 (p53- in the liver up to five times, compared to animals that did not deficient), TSU-PR1 (p53-negative), and LNCaP (p53- receive TB, but were submitted to the same experimental normal), TB strongly induced growth inhibition, cell cycle procedure [43, 44]. The fact that TB may be administered arrest, and apoptosis. These data indicated that the com- per the oral route represents an advantage because, in addi- pound acts independently of the p53 status [22]. Similarly, tion to more favorable pharmacokinetic properties, it exhibits minimal toxicity [45], is better tolerated than butyrate [39, TB administration for four consecutive weeks to mice after the implantation of prostate cancer cell lines resulted in 42], and is better accepted by patients [38, 39]. Thus, TB is strong cell growth inhibition and increased apoptotic activ- quite promising, and its use as a food additive has been ap- ity. These effects also appeared to be independent of the p53 proved in several countries including the United States, Can- status [21]. The pathologic accumulation of cytoplasmic ada, and Brazil [46-50]. In addition, the administration of TB wild-type p53 has been related to alterations in differentia- to mice reduced the circulating levels of triacylglycerols and cholesterol and attenuated the increase in serum leptin and tion, an increase in malignancy, tumor metastasis, and poor cancer prognosis [58]. Hepatic pPNLs, considered to be resistin. These effects indicate that TB can protect against more aggressive and the sites of origin for HCC, exhibit a diet-induced obesity and the associated insulin resistance cytoplasmic accumulation of p53 [59], which may be associ- [51]. ated not only with increased genomic instability during the early stages of hepatocarcinogenesis but also with the eva- TB CELLULAR AND MOLECULAR TARGETS FOR CANCER CONTROL sion of apoptosis [60]. Interestingly, TB reduced the fre- quency of hepatic pPNLs with aberrant cytoplasmic p53 ac- The anticarcinogenic potential of TB was shown in sev- cumulation, indicating that TB may act on lesions exhibiting eral in vitro and in vivo studies (Table 1). Among its mecha- more aggressive phenotypes and greater potential to evolve 1722 Current Drug Targets, 2012, Vol. 13, No. 14 Heidor et al.

Table 1. The anticarcinogenic potential of TB.

Study Type Feature of the Study Most relevant Findings Literature

In vitro Use of human myeloid leukemia cells (HL-60) and murine erytro- TB induced differentiation Chen and Breitman. leukemia cells (MEL-cells) 1994 [37]

In vitro Use of transformed human liver cells (Hep G2) TB was a potent apoptotic agent Watkisn, et al. 1999 [20]

In vitro Use of human prostate cancer cells lines (LNCaP, PC-3, TSU-PrI) TB inhibited cell growth and induced Maier, et al. 2000 [22] apoptosis

In vitro Use of human colon cancer cells (HT-29) TB induced apoptosis mediated through Clarke, et al. 2001 [46] the activation of caspase-3

In vitro Use of human colonic adenocarcinoma cells (LS 174T) TB reduced the cell proliferation and Schröder, et al. 2002 [68] induced apoptosis by up-regulation of caspases 3 and 8

In vivo Use of prostate cancer cell lines (PC3 and TSU-PrI) seeded on the TB reduced the cell tumor cell prolifera- Kuefer, et al. 2004 [21] chorioallantois membrane and implanted in a xenograft model tion and possibly involved a p21/Rb/c- using nude mice myc pathway

In vivo Use of “resistant hepatocyte” model of hepatocarcinogenesis in TB inhibited development of hepatic Kuroiwa-Trzmielina, Wistar rats preneoplasic lesions, increased apoptosis, et al. 2009 [43] hepatic nuclear histone H3K9 hyperacety- de Conti, et al. 2012 [44] lation and p21 protein expression and reduced aberrant cytoplasmatic p53 ac- cumulation

O

O O O O

O O O- + O- + O-

O O Tributyrin butyrate butyrate butyrate

cyt c Apoptosis butyrate Diferentiation

Bax caspase-3 dependent or independent Bcl-2 ERK

PARP p53 p53 PARC p38 MAP kinase p53 p53 P38 MAP Kinase

importin α HDAC Epigenetics

p53 p53 p53 CRM1 NF-κB dependent genes ARC p53 p53 NDRG1 histone hyperacetilation Ac Ac β-casein Ac Ac miRNA c-myc

AcAcAc Ac Ac Ac p21

Fig. (1). TB Anticarcinogenic Actions. The full hydrolysis of 1 mole of TB generates 3 moles of butyrate. Once absorbed butyrate can exert different inhibitory effects on cancer cells. The mechanisms of action may involve: 1) Apoptosis induction through a caspase-3 dependent or independent pathway involving Bcl-2 down-regulation and Bax up-regulation; PARP cleavage and p53 cytoplasmic-nuclear translocation; 2) Cellular differentiation acting in ERK inhibition, p38 MAP kinase activation, up and down regulation of NF-B dependent genes, and in NDRG1, -casein and c-myc regulation; and 3) Epigenetic mechanisms acting as a HDACi, in induction of histone hiperacetylation on the promoting region of some tumor suppressor genes, such as p21; as well as, in the alteration of miRNAs profile. Anticarcinogenic Actions of Tributyrin Current Drug Targets, 2012, Vol. 13, No. 14 1723

into cancer [43]. Unpublished results by our group suggest up- and down-regulated genes were associated with the TB- that TB modulates proteins such as chromosomal-region- induced cell differentiation process in a prostate cancer line, maintenance 1 (Crm1), apoptosis repressor with caspase re- some of which are nuclear factor kappa-B (NF-B)- cruitment domain protein (Arc), parkin-like ubiquitin ligase dependent [52]. Treatment with NaBu induced the in vitro (Parc), and importin-, which are involved in the regulation differentiation of U138B human glioblastoma cells as de- of p53 nuclear-cytoplasmic translocation. Such nuclear- noted by alterations in their morphology, inhibition of cell cytoplasmic machinery may become a viable and promising proliferation, decreased c-myc expression, and the increased chemopreventive (therapeutic) TB target [61, 62]. expression of glial fibrillary acidic protein [71]. These posi- tive results reappeared in preclinical studies of brain tumor Interestingly, multiple cell signaling pathways leading to models with rodents [72]. apoptosis are induced by TB at concentrations to which nor- mal cells are not affected [43, 44]. The mechanisms that de- The assessment of PNL persistence or remodeling is used termine the resistance against apoptosis, induced by TB, in in chemoprevention studies in experimental hepatocarcino- normal cells, is still unknown. However, it is suggested that genesis [73-75]. Although the cellular and molecular mecha- the rapid reversion of the AB linkage to the histone deacety- nisms involved in both phenotypes i.e., PNL persistence and lase (HDAC) of classes I and II may provide normal cells remodeling, have not been sufficiently elucidated, it is be- with the ability to compensate for the inhibitory effects of lieved that remodeling may in fact involve the redifferentia- these agents [63]. The transformed cells do not present the tion of the hepatocytes in the nodules into a phenotype simi- same type of compensatory response, because they often lar to that of the adult liver [76, 77]. TB inhibited the devel- present a defect on the Checkpoint kinase (Chk1). The Chk1 opment of persistent PNLs that evolve into cancer and in- plays an essential role in the correction of damages in normal duced the remodeling of PNL towards a “normal” liver. cells, which in part gives them resistance to the HDACi (his- Thus, the induction of cell differentiation also appears to be tone deacetylase inhibitor)-induced apoptosis [64]. As a re- involved in the mechanism of the chemopreventive activity sult, cells harboring neoplastic and preneoplastic lesions of TB in experimental hepatocarcinogenesis [43]. would be more susceptible to the cytotoxic effects of TB. These actions represent important features for establishing TB and Epigenetic Mechanisms Modulation safe and clinical relevant strategies for the prevention and Cancer is considered a genetic and epigenetic disease treatment of cancer. [78], and increased attention has been given to the role of epigenetic alterations during cancer development [79]. Epi- TB and Cellular Differentiation Induction genetics, which was first described in 1942 [80], generally The induction of cell differentiation is considered an at- refers to heritable changes in gene expression and chromatin tractive mechanism for cancer control. NaBu was classified organization that are not due to alterations in the DNA se- as a powerful inducer of cell differentiation in several trans- quence [78, 79]. Epigenetic modifications, including changes formed lines including K562 and HL60 leukemia cells, in DNA methylation, histone covalent modifications, and PANC1 pancreatic carcinoma cells [65], HT-29 colorectal small RNAs, are of particular interest in the field of cancer cancer cells [65, 66], SKG-IIIa uterine cervical cancer cells research because their impact on the epigenome is involved [67], androgen-sensitive and androgen-resistant human pros- in cell proliferation, differentiation, and survival [78, 79, 81]. tate cancer cells [22], and PLC/PRF/5 hepatoma cells [56]. The reversibility of epigenetic chromatin modifications by TB treatment induces cell differentiation in HL60 and MEL synthetic and natural compounds has opened new avenues leukemia cells at a level that is three times more powerful for the control of cancer [82-85] with great potential for than that induced by BA [37]. In addition, TB induced dif- clinical translation [86]. ferentiated phenotypes for LS 174T colon cancer cells [68] Vorinostat (suberoylanilide hydroxamic acid, SAHA, and prostate cancer cell lines [22] regardless of androgen ® Zolinza , Merck & Co Inc., Whitehouse Station, New Jer- sensitivity [52], and it is considered a promising compound sey) was the first commercially available HDACi approved for clinical trials in patients with prostate cancer [22]. Thus, for oncology use [87]. It has been approved for use in pa- phase I clinical trials were performed mostly based on the tients with cutaneous T-cell lymphoma who have progres- notion that butyrate and its derivatives may play a role in the sive, persistent, or recurrent disease, or those following two treatment of cancer by inducing cell differentiation [65]. systemic therapies [87]. NaBu was the first compound found Nevertheless, the exact mechanism by which such com- to cause an increase in histone acetylation [88, 89]. Butyrate pounds induce the differentiated phenotype of neoplastic acts as a weak ligand for HDAC and inhibits most HDACs cells is poorly understood. as class I HDAC and class II HDAC 4, 5, 7 and 9 [90, 91], Thus, for example, the induction of the differentiation of most likely because it contains a short three-carbon spacer K562 leukemia cells was associated with extracellular sig- attached to a carboxylic acid group, which likely enters into nal-related kinase (ERK) inhibition and activation of the p38 the active site of the enzyme and forms a bidentate ligand MAP kinase pathway [69]. In addition, p38 activation com- with a zinc atom [85]. Two butyrate molecules could possi- bined with the induction of stress signaling pathways plays bly occupy the hydrophobic pocket and inhibit the enzyme an important role in the butyrate-induced differentiation of [90]. PANC1, HT29, and HL-60 cells [69]. It was reported that Histone hyperacetylation was shown in a variety of ver- NaBu induces breast cancer cell differentiation by regulating tebrate cell lines after butyrate treatment [37]. NaBu induces beta-casein and N-myc downstream-regulated gene 1 the sensitization of HeLa cells towards the action of cis- (NDRG1) in breast cancer cells [70]. A substantial number of platin, and it was associated with hyperacetylation of the 1724 Current Drug Targets, 2012, Vol. 13, No. 14 Heidor et al. histone core and abrogation of the cisplatin-imposed cell this transcription factor and induces the expression of genes cycle arrest [92]. Genome-wide analyses demonstrated that involved in the apoptotic pathway induced by p53. NaBu-induced enterocytic differentiation is associated with miRNA expression can contribute to cancer development hyperacetylation on lysine 9 of histone 3 (H3K9) at some and progression, and miRNAs are differentially expressed in gene promoters, indicating that this epigenetic pattern seems normal tissues and cancers [108]. Growing evidence docu- to be an important marker of distinct differentiation path- ments the emerging role of miRNAs in the identification of ways [93]. BA administration reduces the incidence of colo- therapeutic targets of specific cancers, and this may be a rectal cancer induced by 1,2-dimethylhydrazine (DMH) in promising approach for molecular cancer therapy [109]. ICR mice, and this action is involved with the epigenetic NaBu treatment induced the differentiation of human embry- mechanism of histone acetylation [94]. TB suppressed hepa- onic stem cells, which was related with alterations in the tocarcinogenesis in rats by increasing histone acetylation miRNA profile [110]. In addition, NaBu inhibited several [43, 44] specifically in the nuclei of PNL hepatocytes, which miRNAs induced in HCT-116 human colon cancer cells. reflects a specific and direct epigenetic action on such PNL cip/waf One such miRNA is mir106b, whose target is p21 [111]. that may be relevant for its chemopreventive activity [43]. In The clinical anticancer potential of molecules that act by addition, this epigenetic modulation occurred specifically modulating the miRNA expression must be explored [112]. H3K9, which is considered an important target for the Thus, the identification of microRNAs modulated by TB reactivation of gene expression [43, 44]. Similar actions represents another interesting molecular mechanism to inves- were observed in transformed MCF-7 breast cells treated tigate. with NaBu [95]. Several studies have shown that TB, as an HDACi, BIOMARKERS strongly activated p21cip/waf expression [96, 97], a tumor sup- TB acts in several cellular mechanisms, and hence it is pressor gene that influences multiple functional processes such as cell division, cell death, repair, and differentiation. necessary to find a biomarker that is capable of monitoring cip/waf its clinical effectiveness. Moreover, once the HDAC activity p21 -deleted cells failed to undergo growth-inhibition by is linked to carcinogenesis, it is possible that the measure- NaBu in the HT29 human colon carcinoma cell line [98]. ment of its expression or activity may be considered as a Histone acetylation in the promoter of this gene after bu- response biomarker. Therefore, patients that present distinct tyrate treatment has been reported [99]. Butyrate administra- cip/waf patterns of HDAC expression and activity may be divided tion increases the expression of p21 in the colon mucous membranes of rats, and this effect was associated with in- into subgroups so that the therapy with HDACi, such TB, may be more potent [113, 114]. creased histone acetylation [100]. Increased p53-independent p21cip/waf induction and G2-M arrest was observed in the TB- The biomarker which is the most used in clinical studies mediated growth arrest and apoptosis of wild-type p53 MCF- is the acetylation of histones H3 and H4, once this epigenetic 7 cells [54]. The chemopreventive actions of TB in hepato- modification is directly regulated by HDACs. However, the carcinogenesis were also accompanied by the restoration of acetylation of H3 and H4 does not seem to be related to the p21cip/waf expression, which indicates the potential of this treatment response [113]. The utilization of the p21 expres- substance for gene reactivation [43, 44]. In this regard, stud- sion as a biomarker of HDAC inhibition has been investi- ies indicate that as much as 10% of genes may be affected by gated [115]. NaBu either directly or indirectly. Interestingly, a similar HDACis are involved with the transcriptional regulation number or even more genes are down-regulated than up- of a small number of genes; it is possible that a group of regulated by this drug. This can be explained by the specific genes is identified as biomarkers of therapy response. One of genomic regions that can be deacetylated after butyrate ex- these genes, HR23B, has been validated as a promising bio- posure [101]. The promoter regions of butyrate-responsive marker [116]. Studies are being conducted in this way and genes contain butyrate response elements that control genes will allow a better comprehension of the HDACi clinical that are induced or repressed by this compound [24, 102- effects. 105]. In this context, a bipartite butyrate-responsive element was identified in the human promoter region of calretinin PHARMACEUTICAL PERSPECTIVES (CALB2), a gene negatively regulated by butyrate and ex- pressed in the majority of poorly differentiated colon carci- Translational cancer research aims to translate scientific noma cells [106]. discoveries into new methods of cancer control. Thus, TB may be considered a promising molecule because it often However, histones are not the only substrates that can acts on known critical mechanisms of carcinogenesis [109]. undergo post-translational modification. High-mobility group proteins, including multiple transcription factors, can In addition, TB exhibits better bioavailability compared to BA and can be administered via the oral route; thus, its clini- be acetylated, which has a wide range of effects on their cal potential should be better explored. function. HDACis including NaBu reverted the resistance to chemotherapy by inducing p53 acetylation and its conse- Conley et al. (1998) conducted the first TB clinical trial quent migration towards the nucleus, thus restoring its func- with the following aims: to establish its tolerable single daily tion in neuroblastoma cells [107]. Unpublished data obtained dose; to establish its toxic and therapeutic effects; to docu- by our group suggest that the chemopreventive activity of ment its pharmacokinetics; to establish the dose that results TB is not only associated with histone acetylation but also in effective serum butyrate concentrations (i.e., 0.5-3 mM); with p53 acetylation, which increases the nuclear stability of and to define the dose that may maintain such serum concen- trations [38]. Thus, 13 patients with different types of cancer Anticarcinogenic Actions of Tributyrin Current Drug Targets, 2012, Vol. 13, No. 14 1725 were given TB gelatin capsules in a single daily dose varying tendency for OR and inhibited the viability of HT-29 colon between 50 and 400 mg/kg b.w. The patients did not exhibit cancer cells more efficiently than an emulsion containing TB any significant toxicity signs and symptoms. With a 200 alone [120]. That same study further observed that TB, but mg/kg TB dose, the serum concentration reached a level of not the other components of the system, is responsible for the 0.45 mM, which is close to the concentration used in in vitro effects of such emulsions on cell viability. These results are studies (i.e., 0.5 mM). Based on the serum butyrate concen- important for the development of systems that increase and trations measured every 30 minutes during 4 hours after ad- improve the organoleptic characteristics and bioavailability ministration of the compound, it was concluded that a single of TB to facilitate its incorporation in foods and beverages daily dose of TB is unable to maintain serum concentrations [119] and its use in food/drug delivery systems for anticancer at a constant and sufficient level needed for its therapeutic agents [47]. activity, and the dose suggested was 200 mg/kg three times Recently, nanotechnology has become an interesting per day [38]. technology applied to cancer treatment [121-124], particu- A subsequent clinical trial [45] performed with 20 pa- larly for the development of drug delivery systems that may tients with different types of cancer administered 200 mg/kg improve the bioavailability of lipophilic molecules such as TB gelatin capsules to patients three times per day, and no TB [120]. Nanoparticles obtained from cholesterol and bu- relevant toxic effects were observed. In addition, the large tyrate ester i.e., cholesteryl butyrate, inhibited the prolifera- number of capsules each patient needed to take did not re- tion of lung carcinoma cell lines [125] and induced apoptosis duce the compliance with treatment, which lasted over sev- in melanoma cells in a dose-dependent manner [126]. It was eral months and succeeded in stabilizing the disease in sev- further observed that the treatment of myeloid or lympho- eral cases [45]. The average serum butyrate concentration in cytic leukemia cells with these nanoparticles more power- such individuals was higher compared with leukemia pa- fully inhibited the cell proliferation and expression of the c- tients treated with intravenous NaBu [30, 45]. myc oncogene than NaBu [127]. Thus, the incorporation of TB into nanoparticles may enhance the anticarcinogenic ef- These TB studies found significant variations in the se- fects of BA and consequently be used in strategies for cancer rum butyrate concentration between the individuals who chemoprevention [124]. were given TB capsules, which may be related to individual metabolic variations or an inherent instability of the prodrug The combination of TB with other agents also exhibiting [38, 45]. Therefore, several different strategies aimed at in- anticarcinogenic potential may increase biological responses creasing the TB bioavailability are currently being investi- and thus be used as a promising anticancer strategy. A com- gated. The administration of a TB lipid emulsion to rats in- bination of TB and all-trans-retinoic acid (ATRA) enhanced creased the bioavailability of butyrate and/or TB up to four the effects of the retinoid on NB4 acute promyelocytic leu- times compared with the administration of pure TB [42]. kemia cells [128]. In addition, ATRA-loaded TB submicron This TB emulsion was synthesized with synthetic low- emulsions with affinity for apolipoprotein(s) could improve density lipoprotein (LDL) fractions that exhibited serum the uptake and exert a larger antiproliferative effect than that kinetic behavior similar to native LDLs, i.e. the ability to observed with ATRA in Caco-2 and HepG2 cells that ex- bind to LDL receptors and intracellular incorporation [117]. press LDL receptors [42]. A combination of retinoids and Thus, a TB emulsion exhibiting such properties has the po- HDACis was assessed in preclinical and clinical trials [129- tential to act on neoplastic cells hyperexpressing LDL recep- 131]. TB and its analogs restored the response to retinoids in tors and to induce apoptosis in transformed colon and liver cases of leukemia that were resistant to retinoid acid [128] cells [42]. An oil-in-water emulsion with TB as internal oil and in prostate [132] and breast [133] cancer by inducing the phase inhibited the growth and colony formation of mela- hyperexpression of the retinoic acid receptor  (RAR-), a noma cells in vitro and in vivo [53]. Because the internal tumor suppressor gene. The combined administration of TB phase of such an emulsion provides an environment appro- and vitamin A (VA) to rats subjected to a model of experi- priate for the solubilization and transport of lipophilic mole- mental hepatocarcinogenesis exhibited inhibitory effects on cules, these molecules can be combined with the emulsion, PNL, induced H3K9 acetylation, and restored the expression thus producing a drug delivery system with anticarcinogenic of p21cip1/waf1. In addition, this combination induced apopto- and even additive properties [53]. Celecoxib, which is a se- sis in PNLs more intensely than TB administered alone [44]. lective cyclooxygenase-2 (COX-2) inhibitor, exhibits several The epigenetic reactivation of the genes involved in the me- anticancer effects. However, because celecoxib is a highly tabolism of retinoids, which are silent in several types of hydrophobic molecule, its clinical use is problematic. Incor- preneoplastic and neoplastic lesions, may possibly explain poration of celecoxib into TB-based emulsions allowed the synergism between those compounds and their use in overcoming limitations inherent to its solubility. In addition, strategies for the control of cancer. It has been observed that the anticarcinogenic properties of celecoxib in transformed treatment with VA and NaBu inhibited the growth of MCF-7 cells improved when it was incorporated into the TB-based breast cancer cells in addition to restoring RAR- expression emulsion, which was due to its improved solubility and the [95]. The effect of the combination of ATRA and TB was addition of its anticarcinogenic effects with those of TB investigated in the FTC-133 poorly differentiated follicular [118]. Emulsions containing TB are unstable due to its low thyroid carcinoma cells, which do not respond to radioiodine molecular weight, and they induce the Ostwald ripening ablation. This study found that the combination of ATRA (OR) phenomenon [119], which makes its liposolubility and and TB synergistically induced the expression of sodium- incorporation in foodstuffs difficult. In this regard, TB in- iodide symporter (NIS), which is one of the most important corporated into different emulsions containing long-chain thyroid differentiation markers regulating iodide trapping. In triacylglycerols mostly derived from corn oil reduced the addition to increasing the NIS expression in a dose- 1726 Current Drug Targets, 2012, Vol. 13, No. 14 Heidor et al. dependent manner, this combination also significantly in- CONCLUSIONS creased the radioiodine uptake in FTC-133 cells compared TB, the natural BA prodrug, is quickly absorbed into the with ATRA treatment alone, which points to a novel treat- serum and is chemically stable. The hydrolysis of TB gives ment to restore radioiodine uptake and inhibit cell prolifera- rise to butyrate molecules that exert inhibitory effects on tion in poorly differentiated thyroid carcinomas [134]. The data reported by those studies support the effectiveness of several types of cancer. Pathways involving the induction of apoptosis and cell differentiation and the modulation of epi- TB as a HDACi, which should be considered for cancer con- genetic mechanisms represent the main cellular and molecu- trol combination strategies including those with retinoids lar TB targets. In addition, TB can specifically target multi- [44]. A combination of TB and dihydroxycholecalciferol ple pathways in preneoplastic and neoplastic cells, and it has (OH) D inhibited the proliferation and induced the differen- 2 3 no deleterious effects on normal cells. This feature may play tiation of Caco-2 colon cancer cells. That effect was attrib- uted to an increased expression of vitamin D receptors, an important role in its anticarcinogenic properties. Options for improving the organoleptic characteristics and bioavail- which exhibited higher affinity for their ligand [135]. These ability of TB aiming at its clinical application are being in- data likely suggest a novel therapeutic option for patients vestigated, e.g., the development of structured lipids. In addi- with colon cancer. A combination of NaBu and folic acid tion, TB may be used alone or in combination with other (FA) was tested on DMH-induced colon carcinogenesis in compounds as adjuvant therapy alongside conventional can- mice. This study found that the investigated combination inhibited the development of colon cancer compared with its cer treatments. Therefore, the use of TB may become a promising strategy in the fight against cancer. More preclini- separate components. In addition, epigenetic mechanisms cal and clinical studies should be conducted using TB to bet- including DNA methylation and the post-translational modi- ter elucidate its anticarcinogenic potential. fication of histones explained the protective action of the NaBu and FA combination [94]. Unpublished results ob- CONFLICT OF INTEREST tained by our group indicate that the combination of TB and FA exhibits chemopreventive effects in hepatocarcinogenesis The authors confirm that this article content has no con- during the promotion stage in rats. In this regard, the in- flicts of interest. volvement of epigenetic mechanisms is currently being in- vestigated. ACKNOWLEDGEMENTS The physicochemical properties of triacylglycerols may Research in the Diet, Nutrition and Cancer Laboratory is be altered by restructuring or modifying the composition supported by Fundação de Amparo à Pesquisa do Estado de and/or positional distribution of fatty acids on the glycerol São Paulo [FAPESP], Coordenação de Aperfeiçoamento de molecule. Specific lipases allow for the synthesis of struc- Pessoal de Nível Superior [CAPES], and Conselho Nacional tured lipids containing fatty acids derived from different tria- de Desenvolvimento Científico e Tecnológico [CNPq]. R.H. cylglycerols within a single glycerol molecule [136-138]. is a researcher/specialist of Laboratory of Diet, Nutrition and These may be considered as a new generation of substances Cancer of Faculty of Pharmaceutical Sciences of University with chemopreventive or therapeutic potential for several of São Paulo, J.F.O. and A.C. are recipient of FAPESP pathological conditions including cancer [139]. In this re- scholarship, T.P.O. and F.M. are professors of Faculty of gard, rats were given Yoshida sarcoma cell xenografts and Pharmaceutical Sciences of University of São Paulo. While treated with structured lipids obtained through the interesteri- we have attempted to highlight key findings in the field of fication of fish oil and medium-chain fatty acids. Treatment anticarcinogenic actions of tributyrin, we apologize for the with structured lipids resulted in smaller tumors compared inevitable and inadvertent omissions of important work. with rats treated with short-chain fatty acids or fish oil alone [140]. Recently, triradylglycerols were suggested as potential REFERENCES vehicles for BA in the form of alkylglycerols such as 2,3- dibutyroil-1-O-octadecyl glycerol (D-SCAKG) [141]. It is [1] Boyle P, Boffetta P, Autier P. Diet, nutrition and cancer: public, media and scientific confusion. 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Received: July 27, 2012 Revised: October 05, 2012 Accepted: November 03, 2012

PMID: 23140283