Inhibition of Histone Deacetylases in Inflammatory Bowel Diseases

Rainer Glauben and Britta Siegmund

Medizinische Klinik I, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany

This review, comprised of our own data and that of others, provides a summary overview of (HDAC) inhi- bition on intestinal inflammation as well as inflammation-mediated carcinogenesis. Experimental colitis in mice represents an ex- cellent in vivo model to define the specific cell populations and target tissues modulated by inhibitors of HDAC. Oral administra- tion of either suberyolanilide hydroxamic acid (SAHA) or ITF2357 results in an amelioration in these models, as indicated by a significantly reduced colitis disease score and histological score. This effect was paralleled by suppression of proinflammatory cy- tokines at the site of inflammation as well as specific changes in the composition of cells within the lamina propria. In addition, tumor number and size was significantly reduced in two models of inflammation-driven tumorigenesis, namely interleukin (IL)- 10–deficient mice and the azoxymethane–dextran sulfate sodium (DSS) model, respectively. The mechanisms affected by HDAC inhibition, contributing to this antiinflammatory and antiproliferative potency will be discussed in detail. Furthermore, with regard to the relevance in human inflammatory bowel disease, the doses of ITF2357 considered safe in humans and the corresponding serum concentrations are consistent with the efficacious dosing used in our in vivo as well as in vitro experiments. Thus, the data strongly suggest that HDAC inhibitors could serve as a therapeutic option in inflammatory bowel disease. © 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.org Online address: http://www.molmed.org doi: 10.2119/molmed.2011.00069

INTRODUCTION bowel disease (IBD) (1). The results ob- inflammatory condition represents a pre- Inflammatory bowel disease is a tained from the genome-wide association disposing factor for developing cancer is chronic disorder that accompanies pa- studies not only confirmed the relevance well known, and other examples are tients throughout their lives. Two main of earlier characterized pathways, but chronic hepatitis, pancreatitis and forms can be distinguished, namely equally opened novel avenues. If one esophagitis (4). Crohn’s disease and ulcerative colitis. must suggest a hypothesis on the etiology In the example of colorectal cancer, the Whereas the treatment options have dra- of IBD, one could simply say that the mu- mechanism by which inflammation in- matically improved over the last decade, cosal immune system is hyperresponsive duces cancer development has not been in particular because of the introduction to luminal antigens (for example, dietary elucidated; however, it is clear that the of anti–tumor necrosis factor (TNF)-α factors, commensal bacteria) in genetically sequence differs from the pathway acti- strategies (infliximab and adalimumab), predisposed individuals (2). vated in “non-inflammatory” colorectal a relevant subset of patients still exists in A critical long-term complication of cancer (5). Several lines of evidence, ex- whom satisfactory therapy is required. chronic inflammation (in particular, of perimental as well as clinical data, sug- To identify novel therapeutic targets, the colon) is the development of colorec- gest that antiinflammatory strategies we must focus on the pathogenesis of the tal cancer. The risk increases significantly and, as a consequence, mucosal healing disease first. The genome-wide associa- with duration as well as with extension contribute to preventing colorectal can- tion studies of the last years have con- of disease; thus, a chronically active pan- cer. Thus, the identification of novel ther- tributed significantly to the understand- colitis is associated with the highest risk apeutic strategies that exert antiinflam- ing of the pathogenesis of inflammatory (3). Historically, the idea that a chronic matory and possibly antiproliferative properties is desirable. In addition to the identification of ge- Address correspondence and reprint requests to Britta Siegmund, Medizinische Klinik I, netic polymorphisms associated with ei- Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, ther Crohn’s disease or ulcerative colitis, 12200 Berlin, Germany. Fax: +49-30-8445-4017; Phone: +49-8445-4039; E-mail: this review will serve to shed light on a [email protected]. novel way of regulation that might result Submitted February 20, 2011; Accepted for publication February 22, 2011; Epub in a relevant impact in chronic intestinal (www.molmed.org) ahead of print February 22, 2011. inflammation, (namely epigenetic modifi-

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cations and in particular changes driven known for other protein modifications, IgE responses as well as eosinophil and by histone deacetylases [HDACs]). are still missing (9). These studies should mast cell activation. Ulcerative colitis Our own in vivo data from models of be considered when discussing or inves- was thought to represent a Th2-driven acute and chronic intestinal inflamma- tigating the effects of HDAC or HDAC disease, but the absence of IL-4 in colonic tion in mice as well as inflammation- inhibitors, respectively. Because inhibi- tissue from ulcerative colitis patients and driven tumorigenesis strongly indicate tion of deacetylases produces open chro- the observation that both IL-13 and inter- that inhibition of HDAC represents a matin sites all over the genome, changes feron (IFN)-γ are found at elevated levels highly effective therapeutic option. Re- in the expression of distinct proteins are in ulcerative colitis mucosa changed this cent human data (6) provide solid evi- not necessarily attributed to these chro- dogma (20). Recent data suggest that dence that the novel HDAC inhibitor matin changes but are more likely attrib- IL-13 originates from a natural killer ITF2357 can be administered in doses uted to the modulation of the cellular (NK) T cell and targets the epithelial cell sufficient to exert this antiinflammatory acetylome. to become dysfunctional (10,21). capacity; thus, it seems that these com- Lastly, there are regulatory T-cell sub- pounds might in fact provide a future T Helper Cells populations. This is of particular impor- pharmacological strategy for patients T lymphocytes represent the key cell tance, since the immunological defense suffering from IBD. population of the adaptive immune of the intestine, in contrast to the sys- system. Once activated, T cells secrete temic immune system, is one of suppres- REVIEW OF THE LITERATURE and affect all other cell types sion. Healthy individuals generally do Before analyzing the various pathways within a local environment (macro - not develop systemic immune responses of how HDAC inhibition might affect the phages, dendritic cells, neutrophils, ep- against the commensal flora or dietary mucosal immune response, an overview ithelium, endothelial cells, stromal ele- antigens. Regulatory cells are responsible of the cell populations known to con- ments). Both human and murine studies for the immunologically suppressed mi- tribute to intestinal inflammation will be revealed that different T-cell subpopula- lieu in the intestinal mucosa. One group provided. For defined cell populations as tions are aberrantly activated in Crohn’s of regulatory cells contributing to this well as affected mechanisms, the current disease versus ulcerative colitis (10,11). A milieu are regulatory T cells (Tregs) literature will be reviewed of how dominant role for the T helper cell type 1 (22,23). Various subsets have been de- HDAC inhibition might influence their (Th1)-mediated immune response could scribed as being involved in maintaining respective function and thus prevent or be described early on for models of intes- a condition of tolerance within the intes- enhance the effector response. tinal inflammation (12). Recently, an tinal mucosa. With regard to HDACs and additional Th subset was identified (so- the available data, we restrict the view Acetylation of Nonhistone Proteins called Th17 cells) (13). These cells pro- here on the FoxP3+ Treg population that It is well known that histone acetyl duce interleukin (IL)-17 and IL-22, both requires the transcription factor FoxP3 transferases (HATs) do not acetylate his- proinflammatory cytokines capable of (22,23). Remarkably, the absence of tones exclusively, and equally HDACs promoting local tissue destruction. Al- FoxP3 in humans (IPEX syndrome) do not only deacetylate histones. Thus, though Th17 cells are generated from (24,25) and mouse (Scurfy) is followed the term “histone” in this context is Th0 cells by the combined effects of IL-6 by an autoimmune endocrine disorder, merely historical, and the use of lysine and transforming growth factor (TGF)-β, immunodeficiency and an enteropathy deacetylases (KDACs) best represents the there is an absolute requirement for IL-1 mainly affecting the small bowel. In the biological activity of these molecules. (14). In mice deficient in IL-1RI, there is following sections, we will describe in While it has been described that distinct no Th17 response (15). Once generated, detail of how HDAC inhibition can mod- nonhistone proteins (for example, p65, Th17 cells are maintained by IL-23 (16). ulate Treg number and function. STAT3 and HIF1α) are affected by HAT IL-23 belongs to the IL-12 family and T helper cells type 1 and 2. In the nu- and HDAC, Choudhary et al. (7) recently shares the p40 subunit with IL-12. IL-17 cleus of Th cells, class I HDAC proteins demonstrated that various cellular net- as well as IL-22 can be found at increased are present within SIN3 (yeast Switch works, for example, RNA processing, levels in inflamed Crohn’s disease INdependent) or NuRD (Nucleosome- DNA damage repair, cell cycle control mucosa, suggesting that these cytokines Remodeling and histone Deacetylation and nuclear transport, are widely af- contribute to the disease pathogenesis complex) complexes (26) and are ubiqui- fected by protein acetylation. Analysis of (17,18). Functional data from mice and tously expressed (27). Here, the HDACs the so-called “acetylome” (8) revealed a men are in support of this hypothesis, as provide the deacetylase capability, complex regulatory network of cellular described in more detail below. An addi- whereas SIN3 provides the functionality protein acetylation with several thou- tional Th subset is the Th2 cell. These to interact with various repressor mole- sand identified acetylation sites. Yet cells secrete IL-4, IL-5 and IL-13 (19). Th2 cules. NuRD complexes also promote hi- clearly defined signaling pathways, as cells promote atopy with induction of stone deacetylation via HDAC1 and -2,

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but are, in addition, responsible for nu- acid (SAHA), this scenario was attrib- demonstrated for bone marrow–derived cleosome remodeling via Mi-2 proteins, uted in cell lines to chromatin remodel- DCs and the HDAC inhibitors which build the central part of these ing at the IL-2 promoter as well as acety- (41) as well as LAQ284 (42). The produc- complexes (28). Proliferating CD4+ T cells lation of transcription factors such as tion of proinflammatory cytokines IL-12 recruit Sin3-HDAC to the IFNγ locus. nuclear factor (NF)-κB (33,34). and TNFα was suppressed, paralleled by T-bet, the key transcription factor of Th1 Regulatory T cells and Th17 cells. an inhibited mitogen-activated protein cells, displaces Sin3-HDAC and allows Koenen et al. (35) reported that TSA as an (MAP) kinase activation and NF-κB HAT to acetylate the IFNγ locus, result- HDAC inhibitor induces a profound neg- translocation to the nucleus. The treated ing in an IFNγ transcription and conse- ative effect on the emergence of IL-17- DCs were not able to induce a Th1 re- quently a Th1 polarization (29). Thus, producing cells from Tregs. Whereas sponse or Th1 cell migration. Recently, HDAC inhibitors are able to acetylate the sorted regulatory T cells can be polarized pan-HDAC inhibitor (SAHA and IFNγ locus in naïve T cells, leading to a from the Treg phenotype to Th17 cells in ITF2357) treatment was shown to pro- recruitment of transcription factors such a defined milieu, this can be mote STAT3 acetylation in DCs (43,44), as T-bet or STAT4. These mechanistic prevented by the presence of TSA (35). paralleled by an induction of in- data are controversial, since the domi- A more specific concept was described doleamine 2,3-dioxygenase, which is nant effect of HDAC inhibitors is antiin- for regulatory T cells (36). Administra- known to be an inhibitor of T-cell flammatory and thus not driven by Th1 tion of pan-HDAC inhibitors, but not activation, another link to the modulated cells. class I–specific inhibitors in vivo in mice, DC effect on T cells. The particular effect In addition, T-bet has been described increased Foxp3 gene expression, as well of HDAC inhibition on DCs and macro - to interact indirectly with HDAC3 and as the production and suppressive func- phages is further discussed within this HDAC5, a member of class II HDAC tion of regulatory T cells in vitro. HDAC9 issue by Pavan Reddy. (30). Here, T-bet demonstrates a binding proved particularly important in regulat- Alternatively, macrophage activation capacity to GATA-3, the respective key ing Foxp3-dependent suppression, as via lipopolysaccharide (LPS) alters the ex- transcription factor of Th2 cells, demonstrated by in vivo data in HDAC9- pression of HDAC in these cells. Class I CBP/P300 and Sp1 to form a transcrip- knockout mice (37). Optimal function of as well as class II HDAC expression was tion factor complex. From within this regulatory T cells required acetylation of induced after LPS stimulation of bone complex, GATA-3 regulates FUT7 tran- several lysines in the forkhead domain of marrow–derived macro phages (45). While scription by phosphorylation-dependent Foxp3 (38), connecting the results of T-cell activation and polarization class II recruitment of HDAC3/HDAC5 and by Koenen et al. to the HDAC9/FoxP3 data. HDACs seem to play a dominant role, in competing with CBP/P300 in binding to The same group recently suggested a macrophages, it was shown that class I the N terminus of T-bet (30). This tran- link between the presence of HDAC9, HDACs abolish LPS-induced Cox-2 scriptional regulation by GATA-3/T-bet Treg functionality and heat shock protein expression in RAW264.7 macrophages and HDAC3/HDAC5 interactions is a 70 (HSP70) expression. Although there (45,46). In addition, HDAC2 was re- possible way to explain lineage-specific was a clear link between the Treg func- quired for glucocorticoid-induced sup- (Th1/Th2) protein expression, which is tion and HSP70 upregulation in HDAC9- pression of inflammatory mediator of particular interest in IBD, where the knockout experiments, the direct interac- production from human alveolar Th1/Th2 balance was taken for years as tion between HDAC9 and HSP70 has not macrophages (47). Thus, the inhibition one feature of Crohn’s disease (Th1) or been confirmed yet (39). of class I HDACs might provide a thera- ulcerative colitis (Th2). Although this peutic target for the modulation of exact discrimination is not further sup- Dendritic Cells and Macrophages macrophage responses in inflammatory ported according to the recent literature Besides the described direct effects on diseases. (31), the cytokine milieu at the site of in- Th cells, HDAC inhibitors are able to af- As in DCs, LAQ284 was reported to in- flammation represents a crucial charac- fect the influence of antigen-presenting hibit monocyte chemotaxis (via MCP1-3 teristic, defining progress and severity of cells on Th cells. Two different HDAC in- or CCL15/23) as well as Th1 molecules IBD, thus important both for diagnosis as hibitors, TSA and SAHA, have been de- (CD40, IL12, IP10), whereas Th2 mole- well as for therapy. scribed to modulate human dendritic cell cules remained unchanged (42). For LPS- A variety of HDAC inhibitors, such as (DC) function and particularly, the pro- mediated migration of macrophages butyrate, (TSA) or Script - duction of proinflammatory cytokines in themselves, butyrate and TSA reduced aid, induce anergy in Th cells in vitro vivo, namely IL-12/IL-23. Additionally, migration by suppression of Src enhance- (32), as characterized by an inhibition of HDAC inhibitors were found to block ment and focal adhesion kinase activity proliferation accompanied by a reduction the production of the Th1-attracting in both RAW264.7 and rat peritoneal of IL-2 production in Th1 cell cultures. chemokines CXCL9, CXCL10 and macrophages (48). For synovial For TSA and suberyolanilide hydroxamic CXCL11 in vitro (40). A similar effect was macrophages, an inhibition of class I/II

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HDACs or class III adenine vestigate the mode of action of these How do HDACs participate in this dinucleotide (NAD)-dependent sirtuin compounds, since today, miRNAs are critical signaling pathway? Chen et al. HDACs can suppress inflammatory acti- emerging as key regulators of the im- (61) suggested that the NF-κB subunit vation of these cells from patients with mune response (54). Thus, a recent study p65 acts as a substrate for either HAT or rheumatoid arthritis. The HDAC in- focused on the alteration of miR-146 by HDAC within the nucleus of an acti- hibitors were reported to promote apo- the HDAC inhibitors SAHA and ITF2357. vated cell. While the acetylated form in- ptosis as well as modulation of the ex- This miRNA is widely known as a nega- teracts weakly with IκBα, p65 can be pression of various proinflammatory tive regulator of LPS-driven innate im- deacetylated through a specific interac- cytokines (49). Cytokine inhibition mune responses (55). At least in murine tion with HDAC3 (61). This deacetyla- equally shown for the HDAC inhibitor glia cells, this miRNA undergoes no dif- tion promotes effective binding to IκBα KBH-A42 in RAW 264.7 cells is paral- ferential expression in the presence of the and allows the nuclear export. Although leled by a suppressed MAP kinase HDAC inhibitors analyzed (56). this would implicate a translocation of phosphorylation (50). p65 into the nucleus, and a subsequent Regulation of Transcription Factors: hyperactivation of NF-κB by HDAC inhi- Methylation NF-κB bition, there have been various studies Because there are many ways for Acetylation and deacetylation represent describing contradictory results regard- HDAC inhibitors to modulate cellular regulatory mechanisms for the activation ing the application of HDAC inhibitors phenotypes, one should not underesti- or inactivation of selected transcription in vitro (62–65). These conflicting data mate the field of origin for these com- factors, such as NF-κB. In parallel to his- might in part be related to different cell pounds, the epigenetic modification at tones, this process is mediated by the bal- lines, inhibitor concentrations as well as the DNA or the histones. While it is ance of HAT/HDAC and can thus be experimental design. Our own studies, known that various cross-talks between modulated by HDAC inhibitors (57). where we could prove an antiinflamma- (histone) methylation and histone acety- NF-κB represents a key transcription tory potency in addition to a tumor- lation exist, their impact on T-cell polar- factor at the border between inflamma- preventing effect in multiple murine ization was shown recently by Lal et al. tion and cancer (4). Blocking the NF-κB models, equally demonstrated a reduced (51). They identified a highly conserved pathway in myeloid cells in the azoxy - translocation of p65 into the nucleus in CpG island within a FoxP3 enhancer re- methane (AOM)–dextran sulfate sodium vivo at the site of inflammation and tu- gion that is methylated in peripheral (DSS) model resulted in an inhibition of morigenesis in the colon as well as in CD4+ T cells but not in natural regula- intestinal inflammation as well as in a vitro using U937 cells (66). One study tory T cells. Here, this region was histone decrease in tumor number and size. In suggested a concept behind this effect by H3 acetylated and bound by Sp1 and contrast, blocking the NF-κB pathway in focusing on the transcription factor Stat1, TGF-inducible early gene-1 (TIEG1). Re- intestinal epithelial cells did not affect in- which exists in an acetylated and moving this methylation in non-Tregs re- flammation but did equally prevent tu- deacetylated state. Inhibitors of HDAC sulted in an increased and stable FoxP3 morigenesis (58). This ambivalent charac- induce Stat1 acetylation, and only acety- expression. This effect is similar to other ter of NF-κB in intestinal epithelial lated Stat1 interacts with p65, resulting reported modulations of FoxP3 expres- versus myeloid cells within the inflamed in a NF-κB inactivation by translocation sion via methylation (52). lamina propria was supported by the of p65 to the cytoplasm (67). data from other groups. Here, oral treat- MicroRNA ment of mice with the NF-κB essential Impact of HDAC in the Regulation of Posttranscriptional regulation of gene modulator (NEMO)-binding peptide, an Experimental Colitis expression by microRNA (miRNA) is an- inhibitor of NF-κB, resulted in an amelio- As described in detail below, inhibition other phenomenon among the epigenetic ration of experimental colitis (59). In ad- of HDAC results in an amelioration in mechanisms functionally affected by dition, selective inhibition of NF-κB in various models of experimental colitis, as HDAC inhibitors. LAQ824 is followed by intestinal epithelial cells via cell-specific indicated by data from our group (66,68). rapid and significant changes in 40% of NEMO knockout was followed by severe As already introduced above, there are the different miRNA species expressed in chronic intestinal inflammation, thus fur- several classes and thus even more SKBr3, a cancer cell line, with 22 miRNA ther supporting the concept that NF-κB HDACs (69). The majority of currently species downregulated and 5 miRNAs is essential in the control of epithelial in- available HDAC inhibitors are so-called upregulated (53). Although these experi- tegrity. In detail, NF-κB deficiency led to pan-HDAC inhibitors, which inhibit the ments were not performed in immune apoptosis of intestinal epithelial cells, im- activity of a broad spectrum of HDACs cells, it is obvious that miRNA expres- paired expression of antimicrobial pep- (70). The ultimate goal will be to define sion modulated by HDAC inhibitors tides and facilitated translocation of bac- the relevance of a single HDAC within represents a promising new field to in- teria into the mucosa (60). the process of intestinal inflammation. At

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this point, there are only limited data on we will focus on intestinal inflammation ITF2357 (10 mg/kg) when compared this issue. and carcinogenesis. with SAHA (50 mg/kg) did suffice to The group of Wayne Hancock pro- exert the antiinflammatory effect in our vided data characterizing the impact of REVIEW OF OUR DATA in vivo models. In any of our in vivo stud- HDAC9, a member of the class II ies, either substance was administered HDACs (37). Background on their work Antiinflammatory In Vitro orally. This dosing resulted in a pro- showed that pan-HDAC inhibitors, but The described antiinflammatory proper- found suppression of the production of not class I–specific HDAC inhibitors, in- ties could be confirmed by our group for proinflammatory cytokines at the site of creased the function of Foxp3+ Tregs. As various HDAC inhibitors independent of inflammation for ITF2357 as well as for a consequence, these inhibitors pre- their structural classes in different human SAHA. Considering the human data vented and reduced established colitis and murine cell types. Compounds in- available, here a dose of 50 mg orally re- in mice. These pan-HDAC inhibitor– cluded in these studies were SAHA, sulted in a serum concentration of mediated effects were associated with ITF2357, valproic acid, TSA and apicidin 104 nmol/L, sufficient to inhibit cytokine increased numbers of Foxp3+ Tregs (68). They all proved to induce a suppres- synthesis (6). From a study in children within the lamina propria. Because coli- sion of proinflammatory cytokines. How- with juvenile idiopathic arthritis, we tis was in addition associated with an in- ever, they differed significantly in their ef- know that 1.5 mg/kg in two divided creased local expression of HDAC9, fective active concentration (for example, doses of ITF2357 is safe (76). Thus, the HDAC9-knockout mice were subse- SAHA is 1,000 times more effective than concentrations required for mediating quently investigated in models of exper- valproic acid). Although the precise mech- the antiinflammatory potency in humans imental colitis, where they proved to be anism behind the antiinflammatory effi- can be reached, thus indicating that these resistant. Tregs from HDAC9-knockout cacy is still unknown, several studies offer agents might become a therapeutic op- mice expressed increased levels of possible explanations. tion in treating IBD. HSP70 compared with controls. Im- These macroscopic data were paral- munoprecipitation experiments indi- Antiinflammatory In Vivo leled by a reduction of proinflammatory cated an interaction between HSP70 and There are several models of chemically cytokines at the site of inflammation in Foxp3. Inhibition of HSP70 reduced the induced experimental colitis, each re- colon cultures and further underlined by antiinflammatory efficacy of HDAC9- flecting specific characteristics of the a reduced histological inflammation knockout Tregs, while Tregs that overex- human disease. As an example, the mod- score (68). The effect on trinitrobenzene- pressed HSP70 exerted an enhanced els of trinitrobenzene- and oxazolone- induced colitis was comparable. In both suppressive function (39). induced colitis represent Th1- or Th2- models, the tested hydroxamic acids The therapeutic potency of manipulat- mediated models of colitis, respectively were superior to valproic acid with re- ing FoxP3 acetylation via class II HDAC (74,75). In contrast, the model of acute, gard to the antiinflammatory potency. inhibition could be confirmed in other short-duration DSS-induced colitis is not Although nearly all of the effects de- inflammatory models such as transplant a model of immunological origin, but scribed above might contribute to the rejection or arthritis (37,39,71,72). rather a model of mucosal inflammation amelioration colitis, we observed an in- resulting from the disruption of the ep- duction of apoptosis in the isolated lam- Other Models of Inflammation ithelial barrier. Nevertheless, this model ina propria mononuclear cells from HDAC inhibition reduces cytokine pro- is well established and widely applied to treated animals. This observation corre- duction, particularly cytokines relevant to evaluate novel pharmacological com- sponds to that in healthy humans, where autoimmune/inflammatory diseases. Of pounds for their antiinflammatory lamina propria mononuclear cells are particular importance is the effect of capacity. highly susceptible to Fas-mediated apo- nanomolar concentrations of ITF2357 on The antiinflammatory potency of ptosis, whereas lamina propria mononu- TNFα and IFNγ gene expression and syn- HDAC inhibitors was evaluated in these clear cells from patients with Crohn’s thesis as well as the secretion of IL-1β in different models of colitis (68). HDAC in- disease have been shown to be resistant peripheral blood mono nuclear cells in hibitors of different classes (the hydrox- to multiple apoptotic pathways (77). vitro (73). These results were confirmed in amic acids SAHA and ITF2357 as well as Here, an established treatment is repre- vivo investigating inflammatory models the short-chain fatty acid valproic acid) sented by infliximab, an anti-TNFα such as concanavalin A–induced hepatitis suppressed the inflammatory parameters antibody, resulting in monocyte- and or LPS-induced shock in mice. This issue in acute DSS-induced colitis, as evalu- caspase-3–dependent T-cell apoptosis (78). of Molecular Medicine discusses the effects ated by monitoring weight loss, bleeding of HDAC inhibitors on various inflam- and colon shortening as well as repeated Anti–NF-κB matory diseases and models such as examinations via lower endoscopy (66). As stated above, the effect on the acti- transplant rejection or arthritis; however, Remarkably, five times lower doses of vation of NF-κB has been discussed con-

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troversially (62–65). p65 is acetylated within the nucleus by the HAT p300 and deacetylated by HDAC3 (61), allowing for the relocation of p65 into the cytoplasm. Affecting this fundamental process with HDAC inhibitors should result in an ac- cumulation of p65 in the nucleus and sub- sequently a hyperactivation of NF-κB. Al- though some groups could confirm this effect, others, including our own group, reported the opposite: in these studies, NF-κB activation was reduced and the translocation of p65 into the nucleus was inhibited. We could observe this effect on HDAC inhibitor–treated cell lines in vitro as well as in whole colon samples taken from in vivo–treated mice (66). Our data suggest that the compounds affect the pathway upstream of the separation of IκB and the p50/p65 complex. Figure 1. HDAC inhibitors ameliorate intestinal inflammation. Orally administered HDAC in- + Inflammation/Cancerogenesis hibitors affect mucosal immune cells such as CD4 T cells, DCs or macrophages. Whereas in T helper cells, class II HDACs act as the main targets for the antiinflammatory effect, in Given the profound antiinflammatory DCs or macrophages, both classes are relevant. In all three cell types, proinflammatory effect in the models of experimental colitis markers were downregulated (arrows at the center) and more protective markers were as well as the generally known anti - upregulated (arrows on the outside) leading to an overall protective effect within the in- proliferative capacity of HDAC inhibitors, flamed mucosa. one would assume a strong tumor- preventing capacity of HDAC inhibition gressed to infiltrating carcinomas. This comprises the proven efficacy in models in cases where chronic inflammation is progression of tumor development was of experimental colitis when given orally. clearly associated with a subsequent tu- accelerated by a 5-day treatment with the The HDAC inhibitors reach the site of in- morigenesis. To approach this hypothesis, cyclooxygenase 2 inhibitor celecoxib (80). flammation and exert their therapeutic two models of inflammation-associated Although HDAC inhibitors (ITF2357) potency, whereas there are no side effects cancerogenesis, mediated by chronic coli- were not administered until week 12, the related to the gastrointestinal tract. tis, where tumors develop at the site of in- treatment was associated with an inhibi- One characteristic that one has to bear flammation, were investigated. tion of tumorigenesis within the follow- in mind is the global effect of most of The first model is the AOM/DSS ing 4 weeks, demonstrating a high effi- the HDAC inhibitors on HDACs in a liv- model, which is based on a single injec- cacy of ITF2357 in this therapeutic model ing cell. They interfere with a plethora of tion of the carcinogen azoxymethane of inflammation-mediated carcinogenesis cellular proteins that are involved in (AOM) followed by induction of a (66) (Figure 1). central processes, regarding survival, ho- chronic DSS colitis (79). The develop- meostasis or differentiation. Even ment from initial dysplasia to full-grown CONCLUSION though RNA expression assays demon- adenomas was monitored by lower en- There is solid evidence that in multiple strated that only a small percentage of doscopy in the living mice. Here, the models of inflammation and in various genes are actually affected by these com- continuous treatment with either SAHA colitis models in particular, HDAC in- pounds, the outcome of an HDAC in- or ITF2357 attenuated both tumor hibitors exert an antiinflammatory capac- hibitor treatment is still unpredictable on growth and development. Again, ity. This effect was proven from different the cellular level. The dose on which ITF2357, as seen in the colitis models, groups in different species and a multi- apoptosis or anergy or a diminished cy- was more potent than SAHA (66,80). tude of cell types. Additionally, as far as tokine expression occurs, might differ In the second model, the IL-10 knock- these simplified studies could reveal, between cell types, tissues and the given out mice were evaluated. In this model, most of the cell types involved in the in- HDAC inhibitors. Additionally, various no chemical carcinogen was required and flammatory process are affected in an an- HDAC inhibitors, such as valproic acid, the mice spontaneously developed colitis tiinflammatory manner. Beyond that, one show effects beyond the inhibition of hi- and, with age, dysplasia, which pro- promising feature of these compounds stone deacetylases (81).

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