current pharmaceutical design, , 21, 2147 phenylbutyric acid: … · phenylbutyric acid: simple...

Send Orders for Reprints to [email protected]

Current Pharmaceutical Design, 2015, 21, 2147-2166 2147

Phenylbutyric Acid: Simple Structure - Multiple Effects

Magdalena Kusaczuk1, Marek Bartoszewicz2 and Marzanna Cechowska-Pasko*1

1Department of Pharmaceutical Biochemistry, Medical University of Bia!ystok, Bia!ystok, Poland; 2Department of Microbiology, Institute of Biology, University of Bia!ystok, Bia!ystok, Poland

Abstract: Phenylbutyrate (PBA) is an aromatic short-chain fatty acid which is a chemical derivative of butyric acid naturally produced by colonic bacteria fermentation. At the intestinal level butyrate exerts a multitude of ac-tivities including amelioration of mucosal inflammation, regulation of transepithelial fluid transport, improvement in oxidative status and colon cancer prevention. Moreover, increasing number of studies report the beneficial role of butyric acid in prevention or inhibition of other types of malignancies, leading to cancer cell growth arrest and apoptosis. Similarly, phenylbutyrate displays potentially favorable effects on many pathologies including cancer, genetic metabolic syndromes, neuropathies, diabetes, hemoglobinopathies, and urea cycle disorders. The mecha-nisms by which PBA exerts these effects are different. Some of them are connected with the regulation of gene ex-pression, playing the role of a histone deacetylase inhibitor, while others contribute to the ability of rescuing con-formational abnormalities of proteins, serving as chemical chaperone, and some are dedicated to its metabolic characteristic enabling ex-cretion of toxic ammonia, thus acting as ammonia scavenger. Phenylbutyrate may exert variable effects depending on the cell type, thus the term “butyrate paradox” has been proposed. These data indicate a broad spectrum of beneficial effects evoked by PBA with a high potential in therapy. In this review, we focus on cellular and systemic effects of PBA treatment with special attention to the three main branches of its molecular activity: ammonia scavenging, chaperoning and histone deacetylase inhibiting, and describe its particular role in various human diseases.

Keywords: Ammonia scavenger, butyric acid, chemical chaperone, ER stress, histone deacetylase inhibitor, phenylbutyrate.

1. INTRODUCTION

Butyric acid (BA) has become an object of interest of many researchers since the studies of Roedriger conducted on colonic mucosa over thirty years ago [1]. Multiple activities of this sub-stance have been demonstrated. Butyrate is a short-chain fatty acid naturally occurring in human organism. Humans cannot synthesize BA themselves, but it may be produced in the large intestine through bacterial fermentation of the dietary fiber. The exogenous sources of butyric acid are fruits and vegetables, but it is mostly present in milk fat [2, 3]. There are two ways of butyrate transport through the gastrointestinal tract: some part of butyrate is directly absorbed by colonic epithelium, however BA delivery to the cells mainly comprises of two specific carriers including electroneutral H+-coupled monocarboxylate co-transporter 1 (MCT1/SLC16A1) and Na+-coupled co-transporter (SLC5A8) [4, 5]. Once absorbed, colonocytes quickly oxidize about 95% of butyric acid into ketone bodies used for the synthesis of ATP, and in consequence only a minute amount of butyrate reaches the portal system [6].

In general, the production of short chain fatty acids (primarily acetic acid, propionic acid and butyric acid), allows to save the energy derived mostly from carbohydrates such as dietary fiber, which cannot be digested in the small intestine. It is believed that energy produced in this way may provide from 5% up to 15% of the total human requirements for calories. Butyrate is an essential fatty acid which does not only play an exclusively energetic role in the intestine epithelium, but also influences a range of functions re-sponsible for maintaining the homeostasis of colonic cells. Thus, butyric acid may affect the intestinal barrier, modulate the status of the oxidative stress and potentially act as an anti-carcinogenic and anti-inflammatory mediator [7, 8]. Butyric acid has also been known to evoke a variety of effects in various cell types, as well as in whole organisms. BA decreased the expression of estrogen and progesterone receptors, and caused growth arrest and differentiation

*Address correspondence to this author at the Department of Pharmaceuti-cal Biochemistry, Medical University of Bia!ystok, Mickiewicza 2A, 15-222 Bia!ystok, Poland; Tel: (48.85) 748 56 91; Fax: (48.85) 748 56 91; E-mail: [email protected]

in many cell line models of normal and malignant cells [9]. Bu-tyrate has also been postulated to act through other mechanisms. In this respect, BA has been demonstrated to: inhibit protein prenyla-tion, activate the peroxisome proliferator-activated receptors [10], cause hypometylation of the DNA and also reduce the levels of circulating glutamine [11].

In spite of the presented multiple effects of butyric acid, its usefulness as a therapeutic agent in humans has been limited due to its short half-life, rapid metabolism, and fast excretion in vivo [12]. To overcome these limitations stable butyrate derivatives or pro-drugs have been developed. Compounds such as phenylacetate (PA), and its active precursor – phenylbutyrate/sodium phenylbu-tyrate (PBA) have been widely studied [3]. Although BA has mainly been known for its activity in intestine-related areas, stable butyrate derivatives, mainly sodium butyrate, phenylbutyrate, and sodium phenylbutyrate, have been used in clinics to treat plenty of diseases, including urea cycle disorders [13-17], "-thalassemia [18, 19], sickle cell anemia [20] and various cancers [21-23] (Fig. 1). Indeed, unlike colonic bacterial fermentation, PBA administrated orally exposes the stomach and the small intestine to this fatty acid before it is able to reach the colon. In consequence, higher concen-trations of PBA in the liver [24] and the portal vein [25] can be achieved. It has occurred that this way of PBA application can be effective enough to evoke physiological response, and is safe enough not to cause any serious side effects.

From all the pharmacologically available derivatives of BA, PBA has gained the greatest deal of attention. PBA has been ap-proved by the Food and Drug Administration (FDA) as a safe well-tolerated drug for administering in patients with urea cycle disor-ders and hyperammonemia, where it plays the role of an ammonia scavenger [14, 15, 26].

Pharmacologically-received butyrate derivatives, in general, can act at almost exactly the same way as the unmodified BA. The addition of phenyl group has enriched this compound with chap-eron-like properties. Phenylbutyric acid has been demonstrated to bring back the proper conformation of many proteins, playing a role of a chemical chaperone. Its chaperoning activity has been studied

1873-4286/15 $58.00+.00 © 2015 Bentham Science Publishers

2148 Current Pharmaceutical Design, 2015, Vol. 21, No. 16 Kusaczuk et al.

in multiple researches concerning restoration of endoplasmic reticu-lum (ER) homeostasis [27-29]. The cytoprotective properties of PBA have been proven in cases of various ER stress-related dis-eases, as well as those with mutational origin [30, 31]. Unlike phenylbutyrate, butyric acid deprived of phenyl group, does not exhibit any chaperoning activity [28, 32].

Additionally, in case of many cell types, PBA (similarly to BA) displays the activity of a histone deacetylase inhibitor (HDACI), which has already been tested in clinical trials as a cure for recur-rent malignant gliomas [33, 34]. Furthermore, PBA treatment re-sulted in the induction of apoptosis in prostate cancer cells, medul-loblastoma cells, colon cancer cells [35, 36] and was found to cause the regression of tumors derived from hepatocarcinoma cells in rat model system [37].

These characteristics of phenylbutyrate make it an interesting compound for further studies regarding abroad spectrum of dis-eases. The “butyrate paradox”, which is defined as a contradictory effect: cell-destructive in cancer cells and harmless/cytoprotective in normal cell types [5], seems to introduce phenylbutyrate to be a promising candidate for the treatment of plenty of human diseases, either as a single therapeutic or a co-therapy agent.

As presented above, phenylbutyrate displays multiple modes of actions. The plentitude of research conducted on cell line models, animal models and patients provide a plethora of data about PBA effects on cellular as well as organismal level. In this paper, we decided to describe the therapeutic potential of this substance, con-cerning its molecular and systemic effects, and to pay special atten-tion to the three main branches of PBA molecular activity, being: an ammonia scavenger, a chemical chaperone and a histone deace-tylase inhibitor, and the advantages it brings in the treatment of a multitude of human diseases.

2. MOLECULAR AND CELLULAR EFFECTS OF PHE-NYLBUTYRATE

2.1. Ammonia Scavenger

Phenylbutyric acid is converted in vivo into phenylacetate in a process of "-oxidation in the liver and kidney mitochondria [38]. Basically, it is first activated to its CoA ester (phenylbutyryl-CoA), and subsequently metabolized by "-oxidation to the phenylacetyl-CoA. More precisely, "-oxidation of PBA goes probably following this scheme: phenylbutyryl-CoA is converted into phenylbutenoyl-CoA by the medium chain acyl-CoA dehydrogenase. Next, enoyl-CoA hydratase converts phenylbutenoyl-CoA into "-hydroxy-phenylbutyryl-CoA, which is then transformed into "-ketophenylbutyryl-CoA by "-hydroxyacyl-CoA dehydrogenase. "-ketophenylbutyryl-CoA is subsequently converted into phenylace-

tyl-CoA by the acyl-CoA acetyltransferase (thiolase). Finally, phenylacetyl-CoA is hydrolyzed into phenylacetate [39]. In humans phenylacetate formed this way is next conjugated with glutamine forming phenacetylglutamine. Phenacetylglutamine is a final prod-uct excreted with urine. The above mentioned metabolic pattern represents the mechanism which makes PBA an efficient ammonia scavenger in patients suffering hyperammonemia and urea cycle disorders (UCDs) [26] (Fig. 2).

2.2. Histone Deacetylase Inhibitor

Acetylation of the N-termini of histone proteins is one of the most important posttranslational modifications influencing the mechanism of chromatin remodeling and changing the activity of many genes. Nucleosomes comprised of histones showing low level of acetylation are the hallmark of transcriptionally silent chromatin. Histone acetylation neutralizes the positive charge of lysine resi-dues on the N-termini of the protein chain, and therefore causes the disruption in the structure of the nucleosome. This allows the sur-rounded DNA to get unfolded. As a result, transcription factors have easier access to the relaxed chromatin structure, which in con-sequence alters the expression of many genes. Two crucial groups of enzymes regulate the acetylation and deacetylation of histone proteins. Histone acetyltransferases (HATs) catalyze the transfer of acetyl moieties from acetyl-coenzyme A (acetyl-CoA) onto the ! -amino groups of lysine residues of histone proteins [40, 41]. In contrast, histone deacetylases (HDACs) present the opposite activ-ity to the HATs, as they belong to the class of enzymes catalyzing the removal of acetyl groups from the N-termini of lysine in the core histones. This activity is basically connected with chromatin tightening and transcriptional repression. The 18 different HDACs belonging to the four distinct classes have been described in mam-malian cells [40, 42, 43].

HDAC inhibitors are the group of chemical compounds that inhibit Zn2+-dependent HDAC enzymes. HDACIs were the subject of nearly 500 clinical trials in the last 10 years [42]. The efficacy of HDACIs against malignant cells and their potent anticancer activity in pre-clinical studies, protective effects in animal models of diabe-tes mellitus and various neurological, as well as cardiovascular diseases, has already been demonstrated [44, 45].

There are five classes of HDACIs divided according to the structural characteristics: (I) organic hydroxamic acids (e.g., Trichostatin A (TSA), suberoylanilidehydroxamic acid (SAHA), LBH589 (panobinostat) and PXD101 (belinostat)), (II) short-chain fatty acids (e.g., butyrates and valproic acid (VPA)), (III) ben-zamides (e.g., MS-275, CI-994, MGCD0103), (IV) cyclic tetrapep-tides (e.g., trapoxin, romidepsin), and (V) sulfonamide anilides [40, 46].

Fig. (1). Chemical structure of butyrate and its derivatives. A) butyric acid, B) sodium butyrate, C) 4-phenylbutyrate, D) sodium 4-phenylbutyrate !

!

O

OH

O

ONa

OH

O O

ONa

A. B.

C. D.

Phenylbutyric Acid: Simple Structure - Multiple Effects Current Pharmaceutical Design, 2015, Vol. 21, No. 16 2149

Phenylbutyrate is known as reversible inhibitor of HDACs class I and II. It is considered to be the first generation HDAC inhibitor, due to the non-specific inhibitory effect. Moreover, PBA exerts its effects in relatively high, millimolar working concentrations and the effects are pleiotropic. It has been confirmed that sodium butyrate has been able to inhibit most HDACs with the exception of class III HDACs and HDAC-6 and-10 of the class II [46, 47]. Despite these inconveniences, PBA has been widely studied as a potent HDAC inhibitor in many malignancies as well as other diseases and in certain cases has been proven to represent good efficacy in im-provement of pathological conditions.

2.2.1. PBA in Cancer Cells

Cancer transformation is a complicated process influenced by multitude of factors. It has been known that cancer initiation and progression involve an essential change in the expression of both tumor-favoring oncogenes as well as tumor suppressor genes. The latest studies have shown, that apart from the genetic factors, epi-genetic regulation of gene transcription is a major mechanism in carcinogenesis. In mammals and especially in humans, the most common epigenetic modifications are methylations of the DNA and the posttranslational modifications of histones such as: phosphory-lation, methylation and acetylation [41, 43]. In this respect, it seems reasonable to develop cancer-preventive strategies based on modu-lation of epigenetic modifications.

Phenylbutyrate has been shown to up-regulate the expression of epigenetically silenced genes showing therapeutic potential for treatment of certain types of malignancies [48]. In experimental

tumor models, PBA has been shown to alter the expression of genes connected with tumor growth, angiogenesis, invasion and immuno-genicity, affecting cancer differentiation. The mechanisms by which PBA is able to evoke these alterations are most probably: inhibition of histone deacetylation, modification of lipid metabolism and acti-vation of the peroxisome proliferator-activated receptor [21]. In general, tumor-suppressive properties of phenylbutyrate have mostly been related to: stimulation of differentiation and apoptosis [22, 35, 49, 50], inhibition of cell proliferation [51, 52], induction of expression of silenced genes [21], induction of cell cycle arrest [38, 52], and secretion of TGF! (transforming growth factor-!) [53]. However, the exact molecular mechanisms of PBA action have not been fully elucidated yet.

Wang et al. have demonstrated that PBA treatment resulted in time-dependent growth inhibition in hepatocellular carcinoma Bel-7402 cells [52]. A significant decrease in the fraction of the cells in the S phase and increase in G0/G1 phase cells has been observed. Moreover, PBA has significantly decreased the HDAC4 expression, which improved the level of acetylated histone H4, and increased expression of p21 and E-cadherin in Bel-7402 cells. However, no distinct changes in normal liver cell line L-02 after treatment with PBA have been shown [52]. In accordance with this results, the flow cytometry analysis of gastric carcinoma cell lines SGC-7901 and MGC-803 has shown, that after PBA treatment moderately-differentiated SGC-7901cells have been arrested in the G0/G1 and G2/M phase, while lowly-differentiated MGC-803 cells displayed growth arrest in the G0/G1 and S phases [38].

Fig. (2). Schematic representation of phenylbutyrate metabolism and its involvement in waste nitrogen excretion as an ammonia scavenger. Description in the text.


Goh et al. have shown that, in prostate cancer cell lines (PC3, DU-145, and LNCaP), PBA has attenuated the expression level of many proteins such as: an antagonist of apoptosis – Bcl-XL, DNA-dependent protein kinase – DNA-PK, prostate progression marker – caveolin-1, and the pro-angiogenic vascular endothelial growth factor – VEGF [54]. The down-regulation of Bcl-XL and DNA-PK in prostate cancer cells has been correlated with a sensitizing effect toradiation-induced apoptosis. This data may outline the possibility of PBA to be a potent anti-carcinogenicagent when combined with radiation or chemotherapy [54]. Hattori et al. have also suggested that apoptosis-activating properties of PBA may occur via: the up-regulation of Connexin43 (Cx43) expression, down-regulation of anti-apoptotic Bcl-2 expression, and up-regulation of the activity of apoptosis-associated enzymes, such as caspase-3 and -7 [55]. PBA has also been suggested to induce p21-independent cytostatic effect and enhance the sensitivity to radiation in p53-mutant human glioblastoma cells, suggesting the potential application of combined PBA/radiation therapy in treatment of p53-mutant glioblastomas [56].

It has also been speculated that the pro-apoptotic activity of butyrate may be mediated by restoration of the p53-dependent apoptotic signaling pathway [57-59]. It has been demonstrated that butyric and valproic acid has been able to induce apoptotic cell death through the mitochondrial-dependent signaling pathway, with recruitment of the Bcl-2 family members such as BAX, NOXA, !PUMA and Bcl-2 itself. Stimulation of the neuroblastoma cell lines SK-N-BE and SH-SY5Y, by the low concentrations of HDACIs (0, 9 mmol/l), resulted in G2 cell-cycle arrest and a marked up-regulation of the p21 protein. These HDAC inhibitors have also stimulated the activity of thep53 protein by its hyperace-tylation and nuclear re-localization, however without changing its expression on the protein level [57]. These results are consistent with the in vivo studies conducted on Wistar rats supplemented with an oral dose of 2g/kg of a butyrate prodrug–tributyrin. Tributyrin has led to increased acetylation of histones H3 and H4, hyperacety-lation of the nuclear p53 protein and restoration of p21 expression [58, 59]. These changes have been accompanied by the normaliza-tion of the p53-dependent signaling network followed by the up-regulation of the pro-apoptotic genes and a subsequent increase of apoptosis and autophagy in the liver of tributyrin-treated rats [58]. It might be assumed that butyrate-mediated effects are linked with autophagy by the altered status of the cytoplasmic p53, since it has been known that p53 may either inhibit or activate autophagy dependently of the subcellular location [59, 60]. These results sug-gest that apoptosis may not be the only mechanism of programmed cell death evoked by butyrates, and autophagy may also be consid-ered as one of the possible mechanisms indirectly influenced by this substance. However, these results should be treated with caution and require further investigations, since the effect of phenylbutyrate may be slightly different than this evoked by butyrate itself, and in order to get more general results studies should be performed on many types of malignancies.

Another suggestion is that the pro-apoptotic effect of phenylbu-tyrate may be mediated by the activation of JNK (c-Jun N-terminal kinase) and ERK (extracellular signal-regulated kinase) in the mi-togen-activated protein kinase pathway [22]. Additionally, sodium butyrate seems to act through the inhibition of JAK2/STAT (Janus kinase 2/signal transducers and activators of transcription) pathway [47]. JAK2/STAT signaling plays an essential role in the oncogene-sis of myeloproliferative neoplasms and leukemia. It has been re-ported that JAK2/STAT signaling cascade may be inhibited by the HDAC8-mediated up-regulation of SOCS1 and SOCS3 (suppres-sors of cytokine signaling 1 and 3) expression. Both, SOCS1 and SOCS3 are the potent feedback inhibitors of JAK2/STAT pathway. Additionally, sodium butyrate suppressed the clonogenic potential of hematopoietic progenitors in patients with myeloproliferative

neoplasms thus suggesting to be a reasonable therapeutic agent for testing in clinical trials [47].

In the studies of Ammerpohl et al. PBA was able to induce up to 70% of apoptosis in four pancreatic carcinoma cell lines (Panc1, T4M-4, Colo357, and BxPc3). Interestingly, cell cycle arrest has been evoked only in T3M-4 and Colo357 cells, which have been proven to demonstrate higher expression of p21 than in BxPc3 and Panc1 cells [62]. Moreover, PBA treatment resulted in increased gap junction communication between adjacent T4M-4 cells and efficiently inhibited cellular export mechanisms in Panc1, T4M-4, Colo357, and BxPc3 cell lines. The co-treatment with gemcitabine–a drug used in therapy against pancreatic adenocarcinoma resulted in enhanced apoptosis of BxPC3 and T4M-4 cells. This sensitizing effect has been correlated with PBA-mediated increase in the ex-pression of pro-apoptotic protein Bid and caspase-8, and strong intensification of the gemcitabine-mediated activation of JNK. In-terestingly, H6c7 cells, a pancreatic ductal epithelial cell line of non-malignant origin, but characterized by immortality and a high proliferation rate, has also been responsive to PBA stimulation with the concentrations exceeding 2,0 mmol/l. It suggests that phenylbu-tyrate may not only act specifically on malignant cells, but also on other immortalized cells showing a high proliferation rate. In con-trast, it has not affected lowly proliferating peripheral blood mono-nuclear cells and primary human fibroblasts [62].

Furthermore, it has been demonstrated that a differentiation-based therapy for the malignancies of epithelial origin might poten-tially cause alterations in tumor growth and progression, inhibit angiogenesis, delay or inhibit the metastasis process and eventually affect the response to other forms of treatment [63]. Camacho et al. have suggested that the capability of PBA to be a potent differenti-ating and cytostatic agent may be related to: (I) the methylation of the DNA, (II) strong association with the peroxisome proliferator-activated receptor-# and (III) systemic glutamine depletion [34]. Since the acetylation of core histones and methylation of the DNA are tightly involved in the epigenetic control of gene expression and are known to be crucial regulators of cancinogenesis, a few studies exploring the potential effectiveness of 5-aza-2'- deoxycytidine (5-aza-CdR)/PBA co-treatment have been performed [23, 64-66]. 5-aza-CdR is a potent inhibitor of DNA methyltransferases approved as a drug in therapy of hematologic malignancies. 5-aza-CdR has been found to reactivate the expression of silenced tumor suppres-sor genes in Ewing sarcoma [23]. It has been proven that 5-aza-CdR and PBA have shown highly synergetic effect in the inhibition of cell growth, promotion of apoptosis and re-induction of silenced genes [23, 64-66]. 5-aza-CdR/PBA co-treatment has restored the expression of the three miRNAs: hsa-miR-9, hsa-miR-129 and hsa-miR-137 in three colorectal cell lines [66], and two tumor suppres-sor genes such as E-cadherin and tumor suppressor lung cancer-1 (TSLC1) in Ewing sarcoma cell line [23]. Moreover, 5-aza-CdR/PBA co-treatment has enhanced the expression of 14-3-3" protein in melanoma cell lines. Induction of 14-3-3" expression has led to almost complete suppression of cell proliferation, with cells arrested predominantly in G2/M phase. A combination treatment of 5-aza-CdR and PBA has also been studied in normal human fibro-blast cell line (CCD-1070SK) and in a set of cancinoma cell lines: bladder transitional carcinoma cell line (T24), pancreatic carcinoma cell line (CFPAC-1), lung carcinoma cell line (CALU-1), and em-bryonal carcinoma cell line (NCCIT). It has been pointed out that: (I) application of low dosages of drugs combination resulted in cell cycle arrest, while high drugs dosages evoked apoptotic cell death in T24 cells, (II) in normal and cancer cells the expression level of both p16 and p21 genes was induced in similar extend in a dose-dependent manner, after 5-aza-CdR/PBA treatment [65].

Another set of studies concerning PBA as a co-therapy element have also been conducted. PBA has been tested in context of having potentially beneficial effects in alleviating Adriamycin-induced cardiac injury [67]. The influence of phenylbutyrate on Adriamy-


cin-dependent cardiotoxicity in wild type C57BL/6 mice has been investigated. Adriamycin is a potent anticancer drug used as a treatment for both hematological and solid tumors. The results of cardiac functional study have shown that PBA improved cardiac functions in Adriamycin-treated mice. Consistently, phenylbutyrate has attenuated Adriamycin-induced cardiac ultrastructural defects alleviating 70% damages of the cardiac cells and 75% of the mito-chondria. This data has provided evidence that PBA may protect against Adriamycin-dependent cardiotoxicity, suggesting that a combination of PBA and Adriamycin might be a good approach in cancer treatment [67].

To assess the role of PBA in cisplatin treatment, Burkitt and Ljungman conducted their research on a subset of head and neck cancer cell lines (UM-SCC-1, -6, -25) [68]. Cisplatin is a potent chemotherapeutic widely applied against various cancers. Neverthe-less, one of the main flaws of cisplatin-based therapy is the fact, that tumors initially sensitive to this agent over time become resis-tant. It has been demonstrated that cisplatin responsiveness is corre-lated with defective Fanconi anemia DNA damage-response path-way, present in a subset of head and neck cancer cell lines [69]. It has been proven that PBA causes the sensitization of head and neck cancer cells to cisplatin treatment. It has been shown by WST-1 assay according to which cell viability/proliferation has been re-duced about 50% in cisplatin/PBA co-treatment in comparison to the effects of each agent alone (PBA: 0–10% and cisplatin: 20–30% reduction). The possible mechanism of PBA action may be the down-regulation of the BRCA1expression. Indeed, cells with BRCA1 deficiency are known to show cisplatin hypersensitivity [70], while BRCA1 over-expressing cells display increased resis-tance to cisplatin treatment [71]. This study has shown that in three tested head and neck cell lines PBA supplementation resulted in down-regulation of BRCA-1 expression outlining a potential trend in cancer chemotherapy [69].

The efficacy of the administration of butyrates as a co-therapy with the traditional cancer-treating agents has been confirmed in case of astrocytoma and breast cancer cell lines [72, 73]. It has been demonstrated that butyrate enhanced the antitumor activity of 3-bromopyruvate in three breast cancer cell lines: MCF-7, ZR-75-1 and SK-BR3 [72], and potentiated the efficacy of the photodynamic therapy in glioma cell lines U373-MG and D54-MG [73].

The efficacy of (S)-HDAC-42, aphenylbutyrate-derived histone deacetylase inhibitor has been investigated in prostate and mela-noma cell lines [74, 75]. (S)-HDAC-42 has been considered to be more potent than SAHA (another well-known HDAC inhibitor) in suppressing the viability of all investigated cell lines. The apoptotic effect induced by (S)-HDAC42 has been suggested to occur through the activation of the extrinsic as well as mitochondrial apoptotic pathway, as shown by enhanced cleavage of caspases -3, -8 and -9 and increased release of cytochrome c [74]. In addition to HDAC inhibition, (S)-HDAC-42 has also disrupted signal transduction pathways responsible for cell survival, including down-regulation of the Akt and NF-$B signaling [74, 75] and has markedly de-creased the Bcl-XL and survivin expression in PC-3 cells and mice [75], creating a new value in developing therapeutic strategies for malignant diseases.

Lately, a novel mechanism of PBA antitumorigenic activity has been suggested. Kim et al. have shown that PBA is able to evoke cellular senescence in cancer cells, such as MCF7 and HT1080 [76]. Gu and Kitamura have also proven PBA efficacy in triggering senescent phenotype in NRK-52E rat renal tubular epithelial cells [77]. Recently, cellular senescence is considered to be an interesting phenomenon in context of anti-cancer therapy. Cellular senescence is characterized as a state of irreversible proliferation arrest evoked by various factors, including telomere shortening, oxidative dam-age, DNA damage, and oncogene activation [77]. It has been shown that after 72 h-treatment with PBA, trichostatin A and doxorubicin NRK-52E cells developed senescence-characteristic cell morphol-

ogy [77]. Furthermore, only Akt activation, but neither JNK, nor p38 in PBA-treated MCF-7 cells has been detected. Additionally, induction of p21 and any effect on the expression level of p53 has been observed. Therefore, it has been suggested that the induction of p21 is caused by the Akt pathway in p53-independent manner. In this respect, it has been postulated that the cellular senescence may occur via activation of the Akt/p21pathway [76].

Nevertheless, despite of many positive effects in cancer co-treatment, butyrates have also been demonstrated to negatively affect the co-therapy with different drugs [78, 79]. Hauswald et al. have shown that phenylbutyrate induced expression of the P-glycoprotein and BCRP (breast cancer resistance protein) and caused the efflux of drugs in acute myeloid leukemia [78]. Acute myeloid leukemia cell line – KG-1a treated with PBA has devel-oped resistance to a variety of drugs (etoposide, vinblastine, daun-orubicin, topotecan, mitoxantrone, gemcitabine, paclitaxel, and 5-fluorouracil) which resulted in an impairment of the drug-induced apoptosis [78]. Furthermore, Gurtowska et al. have studied the effect of sodium butyrate on the carboplatin-treated B16 melanoma cell line [79]. Both carboplatin and sodium butyrate alone de-creased the viability of B16 cells with an inhibition of the cell pro-liferation in G1/G0 and S phases. Given this, a synergistic effect of PBA/carboplatin co-treatment has been expected. However, at higher concentrations of carboplatin and increasing concentrations of sodium butyrate, the expected synergism did not appear. Moreo-ver, an increase of cell viability under these conditions has been observed [79]. These results indicate that PBA as a co-treating agent in anticancer therapy, still need further evaluation before application in clinical trials.

Summing up, it has been demonstrated that PBA is a potent agent in controlling cancer development (Fig. 3). This makes it an interesting single-agent drug and a potent co-drug in a combinato-rial therapy with already established chemotherapeutics [22, 62]. Nevertheless, due to recent findings [78, 79], further investigations are needed to comprehensively confirm its efficacy in treatment of malignant diseases.

2.2.2. PBA in Inflammation Suppression

It is already well established that inflammatory responses are activated in many types of diseases, including obesity [80, 81], diabetes [82-84], and neurological disorders [85, 86]. Moreover, a linkage between inflammation and endoplasmic reticulum stress exists. The three branches of the unfolded protein response (UPR) intersect with a multitude of stress and inflammatory signaling net-works including the I kappa B kinase (IKK)- and JNK-AP-dependent pathways. The downstream effect of these signal trans-duction pathways is altered metabolism [87]. Recently, the attention of much research has shifted towards a usefulness of the HDAC inhibitors in modulation of inflammation in different models of diseases. It has been known, that butyric acid is able to alleviate inflammation in the pathogenesis of Crohn's disease by decreasing the expression of pro-inflammatory cytokines, such as tumor necro-sis factors (TNF-!, TNF-"), interleukins (IL-1", IL-6) cyclooxy-genase-2 (COX-2) or ICAM-1. This inflammation-reducing activity seems to occur via the inhibition of NF$B activation (an essential modulator of inflammation) and degradation of I$B! (an inhibitor of NF$B) [88, 89]. Analogically to these results, phenylbutyric acid has also been studied in context of remodeling the inflammatory responses.

Park et al. have examined the anti-inflammatory potential of three short chain fatty acids (sodium phenylacetate, sodium bu-tyrate, and sodium phenylbutyrate) in interferon (IFN)-#-stimulated RAW264.7 murine macrophage cell line [90]. They have managed to demonstrate that each of these acids was able to inhibit the IFN- #-induced expression of iNOS (inducible nitric oxide synthase), TNF-! , and IL-6, simultaneously increasing the expression level of anti-inflammatory IL-10. The anti-inflammatory potential has been


arranged as follows: sodium butyrate, PBA, and sodium phenylace-tate. The mechanistic studies have shown that these three fatty acids were capable of repressing the DNA-binding activity and transcrip-tional activity of NF$B. Additionally, these agents suppressed the IFN-#-induced ERK1/2 phosphorylation without affecting the JAK/STAT activity. These results have suggested that NF$B and ERK signaling networks are at least partially engaged in the anti-inflammatory activity of these fatty acids [90].

The involvement of NF$B-dependent pathways in inflamma-tion-suppressive properties of PBA has also been confirmed in case of other reports. Neuroinflammation and oxidative stress have been known as the main hallmarks of neurodegenerative disorders. Hence, the identification of the drugs able to alleviate the produc-tion of pro-inflammatory molecules and reactive oxygen species (ROS) is a crucial direction of investigations. These kinds of drugs might delay or completely stop the progression of neurodegenera-tion. According to this, PBA has been tested in context of possess-ing anti-inflammatory and anti-oxidative properties in glial cells and mouse model of Parkinson’s disease [85]. Indeed, PBA inhib-ited the expression of various pro-inflammatory molecules (iNOS, TNF-!and IL-1") and suppressed the production of ROS in acti-vated glial cells. Moreover, mice after oral administration of PBA

showed reduced activation of p21ras and p21rac, alleviated activation of NF$B, and inhibited expression of pro-inflammatory mediators in the substantia nigra of the brain. Nevertheless, although phenyl-butyrate suppressed the activation of NF$B at both DNA-binding and transcriptional level, the exact mechanism by which PBA ex-erts this effect has not been fully explained. It has been suggested that it might be the result of the inhibition of p21ras, a membrane-associated small guanine nucleotide-binding protein which acts as a crucial mediator transmitting various extracellular stimuli across the cell. Additionally it has been demonstrated that PBA inhibited phosphorylation of I$B!, thus alleviating the activity of NF$B. It has also been proposed that being a HDAC inhibitor, PBA may suppress the acetylation of p65 subunit of NF$B (known to be ace-tylated by HAT), thus inhibiting its transcriptional activity [85].

However, other mechanisms of inflammation-repressing prop-erties of PBA have also been postulated. The direct linkage between ER stress and inflammation has led Kim et al. to perform research considering these associations in context of PBA functioning [91]. They have shown that in normal human bronchial epithelial cells (NHBE) and in mice lung tissues PBA reduced the lipopolysaccha-ride (LPS)-induced increase in the expression of GRP78 and CHOP – known ER stress markers [91]. Consequently, PBA-mediated

Fig. (3). Tentative model of PBA action in cancer. The scheme shows possible mechanisms of action initiated in cancer cells after PBA exposition. After entering the cell PBA inhibits histone deacetylases activity. HDAC inhibition leads to increased histone acetylation and thus, transcriptional regulation of many genes. Altered expression of particular genes initiates a cascade of events leading to downstream activation of effector molecules. As a conse-quence, changes in cellular status result in cytostatic/cell eliminating effect.


alleviation of the ER stress caused marked decrease in the LPS-induced lung inflammation. PBA supplementation led to the evident attenuation of the LPS-induced up-regulation of IFN-%, TNF-", IL-1# and ICAM-1 proteins in lungs of tested animals. Lung tissues of the LPS-treated mice were characterized by the inhibition of the ER stress, lower expression level of the hypoxia-inducible factor-1" (HIF-1"), reduced nuclear translocation of p65 subunit of NF&B and diminished degradation of I&B" protein. These results sug-gested that suppression of the ER stress attenuates the LPS-induced lung inflammation mostly by disrupting the NF&B/I&B and HIF-1" signaling [91]. This might indicate that the PBA-mediated modula-tion of NF&B could perhaps be the downstream effect of PBA-dependent ER stress alleviation, which thus far has not been taken into consideration in other studies.

Interestingly, in spite of many documented anti-inflammatory implications of PBA, its pro-inflammatory potential has also been suggested [92]. The influence of PBA treatment on the chemokine IL-8 expression, modification of NF&B and AP-1 activities and alteration of MAPK signaling in two $F508-CFTR lung epithelial cell lines has been examined. It has been demonstrated that 6 mmol/l PBA markedly increased the expression of the IL-8 in ERK1/2- and JNK-dependent but NF&B-independent manner. It has been noted that PBA stimulation suppressed the NF&B transcrip-tional activity in resting and TNF-"-treated $F508-CFTR lung cells [92]. These results are in agreement with the studies of Vij et al. who demonstrated the down-regulation of COX-2 and IL-6 tran-scripts in IB3-1 cells after 24h of PBA stimulation but did not ob-serve any decrease in IL-8 expression [93].

These results mean that although much is already known in context of PBA-initiated responses in various cell types represent-ing many diseases, there is still much to uncover to fully understand the nature of this compound.

2.2.3. PBA in Other Research

Considering PBA ability to stimulate transcription of #- and %-globin it has been suggested to be used as a treatment for #-thalassemia and sickle cell anemia [94, 95]. Promising results have also been achieved in case of phenylbutyrate supplementation in Huntington’s disease [96-98], and another fatal neurodegenerative disorder – amyotrophic lateral sclerosis (ALS) [109-101]. Admini-stration of PBA to N171-82Q mice – a transgenic animal model of Huntington’s disease led to the significant up-regulation of many genes including: striatincalmodulin-binding protein 3, glutathione S-transferase, ubiquitin-specific protease 29, proteosome subunit " type 3 and the ATPase 3 subunit of 26S proteasome causing reduc-tion in neuronal cell apoptosis [97]. In G93A mice (animal model of ALS) the expression profile of many genes (glutathione-S-transferase, proteasome subunit " type 3, manganese superoxide dismutase, Bcl-2) has also been changed, resulting in reduced oxi-dative stress and apoptosis in neurons [99, 100].

2.3. Chemical Chaperone

The term chemical chaperone originates from the name of a class of proteins functioning in living cells – molecular chaperones. Under physiological conditions, a vast majority of proteins are only marginally stable, so a part of them often stay unfolded or folded improperly. These proteins exhibit a high degree of aggregation with each other or with correctly functioning protein molecules. This in consequence cause unfavorable effects on the cell function-ing. Molecular chaperones protect against these unwanted associa-tions by sequestration of the unfolded and misfolded proteins and facilitation of their proper folding [102, 103]. In pharmacology chaperones have a similar role, however instead of facilitating pro-tein folding, they usually stabilize an already folded molecule by binding it and protecting its structure against proteolytic degrada-tion and thermal denaturation [104, 105].

The synthesis of the molecular chaperones as a part of the UPR pathway, starts in the endoplasmic reticulum during ER stress. The endoplasmic reticulum is a cellular organelle which guards the in-tracellular Ca2+ levels and is responsible for protein biosynthesis, folding and trafficking. Various stimuli have been known to evoke endoplasmic reticulum stress – a state characterized by an accumu-lation of the unfolded and misfolded proteins in the ER lumen [106, 107]. To deal with this, signal transduction cascade termed the un-folded protein response is initiated. The UPR consists of three branches of responses controlled by three ER transmembrane recep-tors: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6). In ER mem-brane, these proteins are normally held in an inactive state as a re-sult of binding to ER molecular chaperone GRP78 (78-kDa glu-cose-regulated protein). In response to the stress-evoking stimuli GRP78 is directed towards misfolded proteins, thus detaching from IRE1, PERK and ATF6. Unbound receptors become active and initiate signal transduction cascades leading to reestablishment of ER homeostasis. This results in enhanced expression of molecular chaperones which assist in folding of the newly-synthesized pro-teins and facilitate the degradation of the unfolded protein mole-cules. Nonetheless, when the injury is excessive, the same ER stress-dependent signaling cascade may also downstream trigger apoptotic cell death, mainly via up-regulation of CHOP (CCAAT/ enhancer-binding protein homologous protein) transcription factor [107-109].

Disruption of ER homeostasis is related to the development of several chronic diseases, caused mainly by misfolded proteins. In humans, defective protein folding is thought to be a primary cause of many neurodegenerations such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Creutzfeldt-Jakob disease, Gaucher’s disease, Prion’s disease, cystic !brosis (CF), "1-antitrypsin de!ciency and others. This fact has led to the assumption that drugs defeating ER stress may become promising candidates for treatment of multiple disorders.

Chemical chaperones are usually characterized as low-molecular-weight compounds known to restore the proper location of mislocalized and/or aggregated proteins usually by stabilizing mutant proteins and facilitating their folding. This group of com-pounds includes substances such as trimethylamine N-oxide, glyc-erol and amino acid derivatives. Recent studies have suggested that other compounds such as phenylbutyrate and membrane-permeable forms of enzyme antagonists, ligands or substrates, can also act as chemical chaperones. A precise mode of action of chemical chaper-ones has not been fully elucidated yet. Most probable mechanisms by which they may act are: stabilization of misfolded proteins, re-duction of protein aggregation, prevention of the nonspecific inter-actions with other proteins and stimulation of the endogenous chap-erones to more efficient protein trafficking [110]. Other activity of chemical chaperones has also been proposed. It has been suggested that they promote proper protein folding by causing the decrease in the energy barrier between conformational states during protein maturation [111].

Recently, a new mechanism of chemical chaperones function-ing has been proposed. Some of the compounds containing hydro-phobic parts, after being solved in a proper liquid were able to bind to the proteins. Therefore, it has been found that lysophosphatidic acids or butyrate derivatives may mask protein mutations and pro-mote native structure stabilization. These hydrophobic molecules have been suggested to act by binding to surface-exposed hydro-phobic segments of unfolded proteins and prevent the aggregate formation or degradation in ER-associated degradation (ERAD) pathway [112].

Although the exact mechanism of chaperoning-like activity of PBA has not been explicitly explained, it has been demonstrated that PBA is able to suppress ER stress-induced but not the mito-chondria-mediated cell death [86], confirming its involvement in


ER stress-dependent pathways of restoring cellular homeostasis. Given this, the purpose of using chemical chaperones seems to be to mimic the functioning of the molecular ones and thus, to support restoration of protein conformation and transport to the proper cel-lular destinations.

2.3.1. PBA in Neurodegeneration Prevention

Lately, numerous factors have been recognized to play a pivotal role in conformational changes leading to the misfolded protein forms. These factors have been found to be structure-destabilizing mutations, fluctuations of environmental conditions (e.g. pH, oxida-tive stress) and changes in certain protein activity (e.g. amyloid P component, apolipoprotein E, and protein X). Once misfolded pro-teins reach a critical concentration, their aggregates may be formed in the cell. This results in an accumulation of the amyloid-like structures, which finally evokes various types of neurodegenerative disorders, ultimately leading to neuronal cell death [108].

Parkinson’s disease (PD) and Alzheimer’s disease (AD) are the most known examples of neurodegenerative conformational pa-thologies. PD is a neuropathological disorder characterized by the selective degradation of dopaminergic neurons and formation of the !-synuclein-containing Lewy bodies in the substantia nigra of the brain [113].

Dopamine cell death in Parkinson’s disease may result from either genetic as well as environmental factors. It has been linked to the dysfunction of six genes including: !-synuclein, Parkin, UCHL1, DJ-1, PINK1 and LRRK2. The !-synuclein mutations cause autosomal dominant forms of PD. The toxicity of mutant forms of !-synuclein is a result of an increased formation of oli-gomeric and fibrillar aggregates. Some reports demonstrate that the expression of A53T mutant !-synuclein results in protein aggrega-tion and cell death in cultured dopamine neurons [114-116]. Zhou et al. have shown that in case of transgenic mouse model of diffuse Lewy body disease, long-term administration of PBA reduced brain levels of !-synuclein aggregates and prevented an age-related im-pairment in motor and cognitive functions [116]. Authors have speculated that as a chemical chaperone, PBA might bind and mask surface-exposed hydrophobic segments of improperly folded !-synuclein in transgenic mice and thereby stabilize the structure of the protein in the native conformation. This may result in allevia-tion of ER stress and prevent the formation of high molecular weight oligomers and fibrils [116]. Neuroprotective properties of PBA have also been confirmed in the study of Inden et al. who have demonstrated positive effects of PBA on dopamine neurons in rotenone-treated C57BL/6 mice [117]. Each oral administration of rotenone has been preceded by intraperitoneal PBA injections. 28-day-long administration of rotenone resulted in specific neurode-generation of dopamine neurons, motor deficits and the up-regulation of !-synuclein in the surviving neuronal cells. In this respect, PBA has been shown to inhibit rotenone-induced neuronal death and decrease protein level of !-synuclein [117].

Moreover, Ono et al. have examined the beneficial role of PBA treatment in transgenic mice overexpressing double-mutated (A30P + A53T) human !-synuclein [31]. Immunohistochemical analyses revealed that PBA reduced the loss of the tyrosine hydroxylase-positive neurons and inhibited an increase in the phosphorylated form of !–synuclein in the substantia nigra, thus preventing the decrease in the striatal dopamine content [31].

The second hallmark of the Parkinson’s disease is the mutation of the Parkin gene which is connected with the autosomal recessive form of PD. The decline in the ubiquitin-protein ligase activity of Parkin is assumed to be the reason of an accumulation of its sub-strates, such as Parkin-associated endothelin receptor-like receptor (Pael-R) expressed in dopaminergic neurons of the substantia nigra in the brain. Given this, the mutant Parkin-induced accumulation of Pael-R evokes ER stress and is suggested to be the cause of neu-ronal apoptosis observed in Parkinson’s disease [107]. Kubota et al.

have proposed that PBA exhibits chaperoning effect on protein aggregation in vitro [28]. To prove this assumption, authors have investigated the influence of phenylbutyrate on the aggregation of denatured lactalbumine. They have found that PBA acts on the suppression of aggregation in time- and dose-dependent manner. Moreover, PBA prevented the overexpression-induced aggregation of Pael-R in the ER and partially restored the location of Pael-R from the endoplasmic reticulum to the plasma membrane. Subse-quent alleviation of ER stress resulted in marked reduction in the number of SH-SY5Y dead cells in comparison with control and butyrate alone-treated groups. Interestingly, butyrate lacking phenyl group, exhibited no effect on aggregation, even at high – 10 mmol/l concentration [28].

These findings are in agreement with the studies of Mimori et al. who have examined four aromatic fatty acids (3-phenyl propion-ate, 4-phenylbutyrate, 5-phenylvaleric acid, and 6-phenylhexanoic acid), and revealed that each protected against lactalbumin and bovine serum albumin aggregation, thus preventing ER stress-induced neuronal apoptosis [29]. Furthermore, the impacts of these substances on the accumulation of Pael-R in neuroblastoma cells have been investigated. It has been demonstrated that 3-phenylpropionate and phenylbutyrate significantly diminished ER stress-induced neuronal cell death caused by Pael-R overexpres-sion. Excessively expressed Pael-R has accumulated in the ER, and 3-phenylpropionate and phenylbutyrate supplementation has shifted the location of the overexpressed Pael-R away from the ER to the cytoplasmic membrane [29]. ER stress-protecting properties of PBA have also been confirmed in the studies of cerebral ischemic injury, where it prevented ER stress-induced neuronal apoptosis [86, 118]. As shown in animal models of cerebral ischemic injury, PBA inhibited the ER-mediated cell death presumably by: inhibi-tion of eIF2! (eukaryotic initiation factor 2 alpha) phosphorylation, suppression of CHOP induction, inhibition of caspase-12 activation and attenuation of GRP78 up-regulation [86, 118].

Alzheimer’s disease (AD) is the most common neurodegenera-tive disorder and the first cause of senile dementia. At the molecu-lar level AD is characterized by formation of the neurofibrillary tangles (NFTs) and aggregation of the insoluble "-amyloid (A") plaques followed by neuronal loss [119, 120].

The identification of the active UPR signaling within pathologi-cally affected brain regions of patients with Alzheimer’s disease has suggested, that the pathological progression of AD may be con-nected with ER stress state in neuronal cells. The latest reports demonstrate that the trafficking of "-amyloid precursor protein (APP) throughout the secretory pathway may be disrupted in ER stress state, suggesting APP to be retained within the ER or early components of the secretory pathway. The connections between Alzheimer’s disease and ER stress most probably contribute to the trafficking impairment, since disrupted trafficking of APP and the cleaving secretases may have a direct influence on APP cleavage – a key component of AD pathogenesis [27]. Furthermore, ER stress can be evoked by pathogenic amyloid production. Wiley et al. have demonstrated that NAG cells treated with three unrelated ER stress-evoking agents showed repressed proteolysis of APP along with the activation of the UPR signaling [27]. Since it is known that the !-, "-, and #-secretases undergo proteolysis and glycosylation during their maturation process, the functioning of these secretases may be suppressed in ER stress state [121, 122]. PBA treatment has re-stored the functioning of APP proteolytic system and protected cells from ER stress-induced apoptosis.

In general, disrupted proteolysis and defective protein quality control might be the mechanisms promoting protein aggregation and induction of ER stress, which in consequence impair #-secretase-dependent processing of APP in AD-suffering patients. The capacity of phenylbutyrate to overcome ER stress and support protein trafficking aligned with the enhancement of !/#-cleavage, justify further examinations of potential therapeutic application of


PBA in AD treatment [27]. Moreover, studies of Ricobaraza et al. have suggested that PBA may contribute to decreased phosphoryla-tion level of tau protein [120]. Aberrant tau phosphorylation is shared by many neurodegenerative diseases. The agent responsible for this improper tau phosphorylation is the over-activated glycogen synthase kinase 3!/# (GSK-3!/#). Phospho-tau aggregates forma-tion is one of the identifying characteristics of AD and other tauopathies. Indeed, decreased phosphorylation of tau has been connected with a forced expression of the inactive form of the GSK-3# in transgenic Tg2576 mouse model of AD [120]. It has also been demonstrated that GSK-3# is an important mediator of downstream signaling of the ER stress effects. The authors have proposed that PBA prevents ER stress and suppress GSK-3# activ-ity in neuronal cells, which result in the prevention of tau phos-phorylation and restoration of neuronal plasticity. The phosphoryla-tion of tau is an integral part of the NFT deposition, which disrupts functioning of neurons. Given this, it seems likely that the deregula-tion of tau phosphorylation in the hippocampus may be connected with memory impairment occurring during AD course. In general, it has been found that 5-week-long PBA supplementation of Tg2576 mice (expressing human APP), improved AD outcome. Interest-ingly, this advantage of PBA treatment seemed to occur independ-ently of the A! peptide content in the brain, since the levels of the A!42, A!40 as well as senile plagues stayed unaffected in Tg2576 animals after treatment [120]. Later reports of the same research group have also shown that the beneficial effects of PBA treatment in Tg2576 mice might occur through the clearance of the intraneu-ronal A! aggregates and subsequent alleviation of the ER stress and restoration of dendritic spine densities of hippocampal CA1 py-ramidal neurons to control levels [123, 124].

Nevertheless, chaperoning-like activity of PBA does not seem to be the only one important in improving the outcome of neurode-generative pathologies. Lately, it has frequently been suggested that PBA may influence neuronal functioning through its HDAC-inhibitory properties [120, 123-125]. According to this, the admini-stration of PBA to Tg2576 mice restored a degree of histone acety-lation in the brain, and as the most probable consequence, activated transcription of synaptic plasticity markers, such as: GluR1 (Gluta-mate receptor 1) subunit of the AMPA (!-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor, PSD95 (postsynaptic density protein 95), and microtubule-associated protein-2 [120]. The expression of the plasticity-related proteins such as the NMDA (N-methyl-D-aspartate) receptor subunit NR2B (N-methyl D-aspartate receptor subtype 2B) and the synaptic scaffold SAP102 (Synapse Associated Protein 102) has also been markedly up-regulated by PBA [123]. This data suggests that the beneficial role of PBA in memory improvement is mediated via both, its chaper-one-like activity, as well as transcription-activating properties [123]. This observation is consistent with others suggesting that PBA-mediated neuroprotection may occur due to its HDACI activ-ity [28, 116]. In this respect, in N27 dopamine cell line, PBA caused a 3-fold increase in the expression of DJ-1 (a protein associ-ated with early-onset autosomal recessive form of PD), and pro-tected cells against oxidative stress and toxic effects of mutant !-synuclein [116].

Summing up, the duality of PBA nature, namely chemical chaperoning and HDAC inhibitory properties, can correspond with a convergence of biological functions. The main drawback to its therapeutic use is that it requires relatively high dosage (up to 15 g/day) [125], but despite this, PBA seems to be a promising agent to restore proper neuronal functioning in neurodegenerative dis-eases.

2.3.2. PBA in Cystic Fibrosis

Cystic fibrosis (CF) develops as a consequence of the loss of function of single membrane glycoprotein – Cystic Fibrosis Trans-membrane Conductance Regulator (CFTR). About 70% of patients suffering from CF, are the carriers of at least one copy of the mu-

tated CFTR gene encoding cAMP-activated Cl- channel. This causes the deletion of a phenylalanine residue at a position 508 (!F508-CFTR) in the CFTR protein chain [30]. As a result, !F508-CFTR is a temperature-sensitive trafficking mutant that retains in the ER where displays extended binding with calnexin and the 70 kDa heat shock protein family (HSP70). Therefore, !F508-CFTR is believed to interact mostly with Hsc70 (70 kDa heat shock cognate protein), which is a cytosolic chaperone involved in targeting a number of cellular proteins for ubiquitination and proteasomal deg-radation [126]. As a consequence, !F508-CFTR is targeted for rapid intracellular degradation, partially via ubiquitin-proteasome pathway, and is unable to reach its proper localization at the apical cytoplasmic membrane [127]. Given this, treatments based on the promotion of !F508-CFTR trafficking beyond the ER, may par-tially restore the CFTR chloride channel activity at the surface of the cell. Phenylbutyrate has been known from its ability to correct the !F508-CFTR trafficking defect as well as restore CFTR activity in the cytoplasmic membrane of cystic fibrosis epithelial cells in vitro [30].

Nevertheless, the exact mode of action of PBA remains un-solved. It has been suggested that PBA may act through the regula-tion of chaperone-mediated !F508 processing [128-130]. Indeed, a visible increase in the expression of Hsp70/!F508-CFTR complex and a decrease in Hsc70/!F508-CFTR complex after PBA treat-ment have been observed [128]. These observations are in partial accordance with the studies of Suaud et al. who demonstrated de-creased expression of Hsc70/!F508-CFTR complexes in PBA-treated airway bronchial epithelial IB3-1 cells [126]. Nonetheless, they failed to confirm stably increased levels of Hsp70 expression, which is believed to directly promote !F508-CFTR trafficking. Given this, PBA induced only transient increase in Hsp70 mRNA expression that returned to the baseline after 24 hours of incubation. In order to explain this fact, a signaling pathway by which PBA may regulate Hsp70 expression has been studied. The authors, have stated that in CF epithelial cells PBA-mediated regulation of Hsp70 expression may act through Elp2 (Elongator protein 2) and STAT-3 (signal transducer and activator of transcription-3) activation, re-sulting in improved !F508-CFTR intracellular trafficking [126].

In order to identify other PBA-dependent proteins that might control the !F508-CFTR trafficking, the IB3-1 cystic fibrosis bron-chiolar epithelial cells exposed to PBA treatment has been sub-jected to differential display RT-PCR screen analysis [131]. PBA has been found to up-regulate a particular mRNA encoding a lumi-nal endoplasmic reticulum-resident protein – ERp29 suspected to be a novel molecular chaperone. The hypothesis of ERp29 being a PBA-induced ER chaperone affecting the !F508-CFTR trafficking has been tested. Indeed, in IB-1 cells the expression of ERp29 on the mRNA and protein level has shown a significant (about 1,5-fold) increase after PBA-treatment. The IB3-1 cells have shown the co-immunoprecipitation of !F508-CFTR and endogenous ERp29. In consequence, enhanced expression of ERp29 led to overexpres-sion of the !F508-CFTR in the plasma membrane. This data has suggested that ERp29 might be a PBA-dependent ER chaperone responsible for the regulation of biogenesis of wild type-CFTR and promotion of !F508-CFTR trafficking in cystic fibrosis epithelial cells [131].

Pruliere-Escabasse et al. have reported that PBA markedly stimulated the activity of amiloride-sensitive Na+-channel in pri-mary cultures of HNEC (human CF nasal epithelial) cells [129]. According to the authors, this stimulation has been accompanied by an enhanced expression of "-, #-, and $-epithelial sodium channel subunits in the apical membrane. These results have suggested that PBA is able to increase functional expression of epithelial sodium channel through the insertion of new "-, #-, and $-epithelial sodium channel subunits into the apical membrane in HNEC cells. Addi-tionally, PBA has been found to modify epithelial sodium channel trafficking by the reduction of the Hsc70 protein expression [129].


The mass spectrometry and two-dimensional gel electrophoretic proteome profiling of PBA-treated IB3-1 cells have been investi-gated in order to identify butyrate-responsive proteins. The authors managed to identify mostly catalytic enzymes, chaperones, and other proteins involved in cellular defense, trafficking activity, ion transport or protein biosynthesis [132]. These observations have been confirmed later by the same group, who detected a subset of PBA-activated ERAD-associated chaperones (HSP84, GRP78, GRP94, GRP75, and GRP58) to make an association with the im-mature form of CFTR in the ER, while Hsp70 and Hsc70 to interact with the mature form of CFTR at the cell surface. These results have suggested that CFTRs undergoing chemical rescue may asso-ciate with a specific set of proteins belonging to HSP70 family, thus indicating therapeutic potential in correcting ion transport and in-flammatory phenotype in cystic fibrosis suffering individuals [130]. It is now postulated that other derivatives of butyrate: arginine bu-tyrate and 2, 2-dimethyl-butyrate, may also functionally correct the $F508-CFTR defect in IB3-1 cells heterozygous for $F508 and W1282X mutations [133].

The above-mentioned results strongly suggest that also in case of cystic fibrosis the mechanism of PBA action seems to be compli-cated and ambiguous. The chemical-chaperoning activity of phen-ylbutyrate seems to interfere with its capability to stimulate the transcription of many genes, but mostly those encoding ER chaper-ones. In this regard, further analyses are needed to comprehensively explain the exact patterns of PBA functioning in cystic fibrosis.

2.3.3. PBA in Diabetes, Obesity and Other Diseases

Phenylbutyrate has been postulated to have beneficial effect in the outcome of diabetes. Nevertheless, the exact molecular mode of action of PBA in diabetes-affected cells has not been precisely elu-cidated. A growing number of data suggests that ER stress is a key mediator in the pathology of diabetes responsible for insulin resis-tance and loss of the pancreatic beta cells [134, 135]. In this respect, investigation whether endoplasmic reticulum stress may be consid-ered as a potential target of therapy for diabetes and obesity, seems to be a reasonable approach. Indeed, alleviation of the cellular ER stress status resulted in improved outcome of diabetes in ob/ob diabetic mice [136, 137]. Positive effects of PBA treatment in ani-mal models of diabetes as well as diabetes-suffering individuals have mostly been proven on systemic level and will be discussed later.

Elevated risk of developing insulin resistance and type 2 diabe-tes is particularly highly associated with obesity [138]. Increasing incidence of obesity is another huge problem of modern societies. The massive expansion of adipose tissue occurring in obese indi-viduals is frequently connected with various pathological condi-tions, such as insulin resistance, type 2 diabetes, cardiovascular diseases, or even cancer [138]. Lately, white adipose tissue has been stated to play a pivotal role in energy balance and regulation. Moreover, adipose tissue may show the activity of the endocrine organ secreting particular hormones (leptin, adiponectin) and in-flammatory mediators (monocyte chemoattractant protein -1, IL-6, and TNF") into the blood [81]. A significant part of these proin-flammatory agents have been demonstrated to impair normal insu-lin signaling. Moreover, suppression of the obesity-induced in-flammation may lead to improvement in insulin sensitivity in mice and humans [83, 84]. Indeed, adipose tissue exposed to insulin-resistance is prone to inflammation and other cellular pathologies like hypoxia, oxidative stress, and hyperthrophy. The accumulation of these perturbations eventually leads to some cellular organelle failure, especially of the ER and the mitochondria. It is believed that adipocytes are exposed to suffer ER stress, since it has been reported that disrupted ER signaling in obesity results in an accu-mulation and retention of proteins normally subjected to proteaso-mal degradation in ERAD-mediated way [139]. Studies conducted on humans have proven the existence of increased ER stress state in adipose tissue of both obese insulin-resistant subjects, as well as

obese non-diabetic individuals [140, 141]. In this respect, it seems worth to try to investigate possible therapeutic strategies based on the reduction of the ER stress in obesity prevention. Hence, Basseri et al. have examined the influence of PBA, on adipogenesis and weight gain in diet-induced obese C57BL/6 mice [81]. In this study, murine 3T3-L1 cells and C57BL/6 mice have been chosen to inves-tigate the activation of the UPR during adipocyte differentiation. It has been demonstrated that PBA is able to attenuate adipogenesis and alleviate ER stress/UPR activation, as shown by decreased expression of certain proteins, such as GRP78, phosphorylated form of eIF2", and spliced form of XBP1 in 3T3-L1 cells. It has also been observed that particularly at the late stage of differentiation of adipocytes, PBA can modulate the accumulation of lipids, indicat-ing that PBA nither affect the preadipocyte growth arrest nor the clonal expansion of cell during the early stages of adipogenesis. Surprisingly, it has been found that PBA supplementation in 3T3-L1 cells resulted in up-regulated CHOP expression. However, the loss-of-function studies failed to confirm that the up-regulation of CHOP is a mechanism of PBA-mediated inhibition of adipogenesis in vitro. Moreover, CHOP has not been up-regulated in the liver or adipose tissue of PBA-treated C57BL/6 mice. According to this, the alternative mechanism of PBA-mediated inhibition of adipogenesis unrelated to UPR signaling has been explored. Its HDAC inhibitory activity has been speculated. While a majority of studies demon-strate that HDACIs contribute to stimulation of adipocyte differen-tiation in 3T3-L1 cells [142-144], the studies of Basseri et al. have proven differentiation-inhibiting properties of PBA on 3T3-L1 cells, therefore excluding the HDAC-related mechanism of PBA action [81]. Consistent with the in vitro !ndings, mice supple-mented with PBA demonstrated down-regulated expression of GRP78 in the adipose tissue, suggesting the attenuated ER stress/ UPR response.

A different case of obesity is the one having the genetic origin. Early-onset morbid obesity develops as a result of mutated melano-cortin-4 receptor (MC4R), a protein playing a key role in the energy homeostasis. In humans, mutations in the MC4R are the most popu-lar cause of severe disease onset resulting in obesity phenotypically similar to homozygousnull-mouse model of early-onset obesity [145, 146]. It has been reported recently, that obesity-linked vari-ants of MC4R are incorrectly folded in the ER and subsequently targeted for degradation [147]. Additionally, some of these MC4R variants show the impaired binding affinity and deregulated signal-ing response to their agonists [148, 149]. The attempts to resolve these abnormalities have recently been made. Some pharmacologi-cal chaperones have been demonstrated to be effective in restoring functioning of some MC4R variants [145, 146, 150]. Phenylbu-tyrate has been shown to increase plasma membrane expression of wild type-MC4R and partially restored some of the obesity-associated MC4R variants retained in the ER [147]. However, PBA occurred to be slightly effective in rescuing MC4R P272L variant in comparison to the treatment with UBE-41 – a specific inhibitor of ubiquitin activating enzyme E1, suggesting that the retention of MC4R P272L in the ER is a result of its intrinsic affinity to ubiquit-ination, rather than a consequence of the misfolding-dependent ubiquitination [151].

As shown in this section, PBA may exert some positive effects in dealing with symptoms of diabetes and obesity. However, the detailed network of PBA associations in treatment of these patho-logical processes is still unknown. It remains to be uncovered, whether chemical chaperoning activity or histone deacetylase inhib-iting properties predominate in case of these pathologies.

The evidence for chaperone-like activity of phenylbutyric acid have also been confirmed in studies of other disease models, includ-ing "1-antitripsin deficiency [152, 153] glaucoma [154], cataract [155], cholestasis [156] and Wilson disease [157]. In "1-antitripsin deficiency, a mutation in "1-ATZ chain results in misfolded but functional protein, with a tendency to be retained in the ER of hepa-


tocytes, rather than liberated into the blood. PBA has been shown to improve the secretion of functionally active "1-antitrypsin in cell culture as well as in animal models [152].

Yam et al. have studied the influence of PBA treatment on the trafficking of secretion-defective primary open-angle glaucoma–associated mutant myocilin, and a putative possibility of PBA to protect cells co-expressing wild-type and mutant myocilin from ER stress and apoptosis [154]. They have found that PBA increased the solubility of mutated myocilin, prevented the interactions of myo-cilin with ER-resident calreticulin, and finally enabled myocilin secretion. Simultaneously, the number of cells containing myocilin aggregates has dropped, resulting in significantly decreased apopto-sis. Interestingly, any changes in the expression levels of cal-reticulin and GRP78 have been observed, suggesting transcription-ally-independent way of PBA acting [154]. The results of these studies are in agreement with the observations of Zode et al. who pointed out that the topical ocular PBA administration shows bene-ficial effects in improving glaucoma phenotype in murine model of myocilin-related glaucoma (Tg- MYOC Y437H mice) [158].

The beneficial role of PBA treatment in pathogenesis of con-genital cataract has been confirmed in the in vitro studies of the mutant #D-crystallin [155]. It has been shown that PBA up-regulates the expression of Hsp70 and significantly increases the solubility of mutant #D-crystallin relieving its mislocalization from the nuclear envelope [155]. In consequence, PBA has improved the aberrant phenotype of mutant #D-crystallin and rescued the affected cells from apoptosis, suggesting to be a good candidate for treat-ment of lens structural protein aggregation and to prevent lens opacity in cataract formation.

A de!ciency infunctional P-type adenosine triphosphatase member 8B1 (ATP8B1) causes a severe hereditary disease distin-guished by intrahepatic cholestasis. ATP8B1 de!ciency develops as an effect of autosomal recessive mutations in the gene encoding ATP8B1, a putative aminophospholipid-translocating P-type adenosine triphosphatase. Five of seven mutations result in a reten-tion of ATP8B1 in the ER lumen. PBA treatment has partially re-stored an impaired expression and localization of ATP8B1 with missense mutations G308V, D454G, D554N, and I661T, known as the most common mutations in benign recurrent intrahepatic cho-lestasis type 1 [156]. It has been concluded that a substantial pro-portion of ATP8B1 mutations results in defective folding and di-minished expression at the plasma membrane and that these effects can be partially restored by PBA treatment [156]. The protein local-ization-restoring skills of PBA are in agreement with the findings of Sorrenson et al. who have found that PBA treatment restored the proper localization of ATP-binding cassette transporter A1 mutants to the plasma membrane and thus increased cholesterol efflux func-tion [159]. Likewise, PBA and curcumin have also been helpful in restoring expression of most ATP7B protein mutants in Wilson’s disease (WD) cell line models [157]. Wilson’s disease is a genetic disorder of the liver and basal ganglia characterized by copper ac-cumulation in the peripheral tissues. It is provoked by the mutations in the gene encoding ATP7B, a protein of the trans-Golgi network responsible for hepatic copper excretion. It has been demonstrated that the majority of the ATP7B mutations resulted in decreased ATP7B protein expression, without affecting transcript abundance. PBA and curcumin have been found to partially restore protein expression of most ATP7B mutants, suggesting that these novel approaches based on direct enhancement of mutant ATP7B expres-sion with residual copper export activity, may become a promising strategy of WD treatment [157].

Another interesting aspect of PBA mode of action is the ubiq-uitination-modulatory activity suggested by Hayashi et al. in the studies of multidrug resistance-associated protein 2 (MRP2-in hu-mans; Mrp2-in rodents) [160]. According to these studies, MDCKII cells and rat livers expressing MRP2 showed improved transport activity and enhanced expression of MRP2/Mrp2 at the cell surface

after PBA treatment. However this treatment did not cause any significant changes in the MRP2/Mrp2 expression on the mRNA level. The in vitro studies have been performed to investigate the mechanism underlying PBA activity. It has been proposed that cell surface-resident MRP2/Mrp2 undergoes degradation through ubiq-uitin-dependent degradation pathway. Indeed, PBA seems to protect MRP2/Mrp2 against degradation by decreasing its susceptibility to ubiquitination (Fig. 4). However, the exact mechanism underlying this process remains unknown [160].

3. SYSTEMIC EFFECTS OF PHENYLBUTYRATE

The abovementioned results clearly prove a wide range of mo-lecular and cellular effects of PBA in a broad spectrum of cell line and animal models of various disease entities. Understanding the precise molecular mechanisms of PBA functioning is highly impor-tant for assessing its therapeutic potential in patients. However, positive results of cellular-level studies are not always reflected in the studies on a higher organismal level. In this regard, systemic effects of PBA supplementation are well known only in case of a few human diseases. One of the diseases in which systemic effects of PBA has been profoundly known are the urea cycle disorders, in case of which the ammonia scavenging property of PBA has been utilized. Sodium phenylbutyrate is known as a drug for urea cycle disorders for almost twenty years. The FDA has also approved its clinical use in patients with hyperammonemia. It is specifically marketed for the treatment of patients with UCDs caused by a defi-ciency in one of the three following hepatic enzymes: carbamoyl phosphate synthetase I (CPS), ornithine transcarbamylase (OTC), or argininosuccinic acid synthetase (AS) [161]. In case of mammals, the ornithine cycle is a common and important way of excreting the waste nitrogen, since disruptions in this pathway occurring in the early development are basically connected with severe hyperam-monemia with poor future prognosis [26]. Defective expression of the urea cycle enzymes, such as: carbamoyl phosphate synthetase I, ornithine transcarbamylase, argininosuccinic acid synthetase and argininosuccinic acid lyase, leads to the accumulation of the precur-sor metabolites such as ammonia. This kind of defect is very dan-gerous, since ammonia cannot be effectively utilized by any secon-dary clearance system. These abnormalities may finally result in acute cerebral edema with serious neurological disruptions [161]. There are two ways in the treatment of this disease: first is focused on the reduction of the dietary nitrogen intake in order to decrease the need for urea biosynthesis, and second is directed at enhancing the excretion of the waste nitrogen. To deal with this, patients are prescribed to take PBA. Therefore, two moles of nitrogen are neu-tralized by each one mole of the given PBA [162].

PBA therapy for infants, children, and adults with UCDs must be undertaken daily during their whole lifetime. Fortunately, this therapy is characterized by good tolerance and significantly im-proves ammonia status and functioning of the liver [163]. PBA has also been proposed to be the treatment of choice in females suffer-ing the most common form of the UCD connected with the defi-ciency of the ornithine transcarbamylase [163]. OTC deficiency usually affects male neonates and causes the hyperammonemic coma which if not treated, leads to death [164]. OTC is an enzyme functioning in mitochondria, catalyzing the reaction of citruline synthesis from ornithine and carbamoyl phosphate (Fig. 2). Defi-cient enzymatic properties of OTC result in an accumulation of urea cycle intermediates such as: ammonia, glutamine, arginine, and citruline, which may lead to encephalopathy, severe brain injuries, and finally be fatal. OTC deficiency is often lethal in neonate boys, however heterozygous girls, may not show any symptoms of the disease or display the episodic encephalopathy and impairment of cognitive functions [26]. Phenylbutyrate has also been proposed to be the substance of choice for pregnant females with OTC defi-ciency. PBA has been taken without causing any deleterious conse-quences for the fetus [13].


Recently, a novel phenylbutyrate derivative has been approved by FDA for UCDs treatment. Glycerol phenylbutyrate (glyceryl-tri-(4-phenylbutyrate); GPB) has been tested to treat UCDs as the al-ternative drug to PBA. Glycerol phenylbutyrate is a pre-pro-drug containing three molecules of PBA linked to glycerol with the ester bond. In the small intestine pancreatic lipases hydrolyze GPB, so the glycerol and phenylbutyrate are liberated. This way of PBA delivery results in the slower absorption than in case of the admini-stration in a typical form of sodium phenylbutyrate, whereas a di-gestion of the glycerol most likely occurs in the same way as for the dietary glycerol present in food triglycerides [15]. The latest studies have investigated the safeness and efficacy of GPB in ammonia control in comparison to PBA. Indeed, these researches confirmed GPB to be at least as safe and efficient as PBA in controlling am-monia levels and indicated that this form of the drug may also be administrated as a treatment for patients with UCDs [14-17].

Histone deacetylase inhibiting properties of PBA have also been tested for evoking systemic effects in some diseases. Encour-aging results of much in vitro research have been convincing enough to perform a set of clinical trials in order to confirm positive results of PBA in anticancer therapy [33, 36, 165, 166]. Clinical trials involving twenty eight patients with refractory solid tumors, have been trying to evaluate some of the most important parameters

connected with oral administration of PBA such as: toxicity profile, maximum tolerated dose, pharmacokinetic parameters, and prelimi-nary efficacy of PBA in patients. Five dose levels, ranging from 9 g/day to 36 g/day, have been investigated. The noted side effects were nausea, vomiting and hypocalcemia at 36 g/day dose, thus 27 g/day has been a dose recommended for phase II trial. PBA treat-ment resulted in only moderate disease stabilization [33]. Another in vitro study has demonstrated that PBA when combined with 5-fluorouracil (FUra) enhanced the growth inhibitory effect of FUra in human colon carcinoma cells [36]. To support these results clini-cal studies have been undertaken [165]. Patients with advanced colorectal cancer in phase I trial of FUra, were supported by a co-treatment with PBA (120 hour continuous intravenous infusion at fixed dose 410 mg/kg/d x 5). It has been demonstrated that three of four patients who received at least 8 weeks of the treatment have shown marked disease stabilization lasting for 12, 25 and 54 weeks. These results, although limited by the number of evaluated patients, provide the clinical data that the introduction of HDACI between dose-dense cycles of cytotoxic agents, can lead to the stabilization of the disease even in heavily pretreated patients, and still can be quite well tolerated [165].

Taking into consideration the fact that phenylbutyrate is a mul-tifunctional substance, its efficacy as a HDACI in other types of

Fig. (4). Putative model of PBA cytoprotective properties (model represents only chemical chaperoning-like activity of PBA without taking into consideration its gene expression modulatory skills). In general, PBA prevents the aggregation of improperly folded proteins contributing to the alleviation of ER stress. This leads to the attenuation of the unfolded protein response (mediated via PERK, ATF6 and IRE1" receptors), and decreased expression of the pro-apoptotic CHOP molecule, resulting in reduced ER-stress mediated apoptosis (1). PBA prevents the increased proteasomal degradation of proteins occurring via ERAD (ER-associated degradation) pathway, by reducing protein susceptibility to ubiquitination (2). PBA restores the correct trafficking of the misfolded proteins into proper cellular destinations, resulting in the increased expression of the particular protein in the appropriate cell compartment (3).


diseases has also been tested. Administration of PBA attenuated gross brain and neuronal atrophy and caused marked 23% im-provement in survival of N171-82Q mice model of Huntington’s disease [97]. In the transgenic mouse model of ALS, PBA signifi-cantly improved motor function and extended survival by 13% [99]. Moreover, PBA has already been tested in clinical trials in patients suffering $-thalassemia, HD and ALS [95, 98, 101]. In subjects with sickle cell anemia PBA treatment resulted in increased per-centage of F-reticulocytes and HbF levels [94, 95]. In HD patients PBA has been shown to be safe and well-tolerated in daily dosage up to 15 g, but the maximal correction of mRNAs expression levels has been observed in the lowest – 12 g/day group [98]. Similarly, in ALS patients when the medication dosage ranged from 9 to 21 g/day, the 9 g/day dose has been identified to be the best tolerated and safest dosage that evoked the desirable biological effects in blood [101]. These findings strongly suggest further examination of PBA efficacy, both as a single therapeutic or a co-therapeutic agent in treatment of many types of diseases. Its safety and relatively good tolerance make PBA a promising drug for testing in clinical trials.

The other role – of chemical chaperone, makes PBA a prone candidate for defeating ER stress-related diseases. In this regard systemic effects of PBA treatment has been evaluated in some dis-eases such as: neurodegenerative diseases, diabetes and obesity, cystic fibrosis or "1-antitripsin deficiency. Chaperoning-like activ-ity of PBA has been used in functional studies of Parkinson's dis-ease. PD is a chronic and progressive movement disorder character-ized mainly by disrupted motor coordination and bradykinesia. A beneficial role of PBA treatment in transgenic mice overexpressing human "-synucleinhas been examined by Ono et al. [31]. To deter-mine therapeutic benefits of PBA supplementation for bradykinesia and motor coordination the pole test and the rotarod treadmill task have been performed. As a result, both of these motor deteriorations have gradually been improving after PBA treatment [31].

Alzheimer’s disease is physiologically characterized by the progressive memory loss and cognitive impairment [119, 120]. It has been proven that Tg2576 mice chronically supplemented with PBA have shown the reversion of the spatial reference memory deficits [120] and restoration of learning de!cits [123, 124]. These results have convinced authors to persuade that PBA supplementa-tion seems to represent a good ef!ciency for treatment of neurode-generative disorders.

Interesting results of the influence of PBA supplementation have also been achieved in context of diabetes and obesity. It has already been reported that in ob/ob diabetic mice PBA-mediated alleviation of ER stress resulted in normalized hyperglycemia and improved insulin sensitivity [136]. Obese and diabetic mice to which PBA and another chemical chaperone – TUDCA (taurour-sodeoxycholic acid) have been orally administrated, showed re-stored systemic insulin sensitivity, normalized hyperglycemia lev-els, enhanced insulin action in muscle, liver, and adipose tissue and improved outcome of fatty liver disease [136]. Moreover, PBA administration significantly inhibited the progress of diabetic neph-ropathy in rats, by an alleviation of the ER stress-dependent oxida-tive status [137]. To minimize the biases connected with incomplete mimicking of human disease conditions in animal models, Xu et al. have decided to explore four different animal models of diabetes: obese type 2 diabetic mice (ob#ob mice), alloxan-induced type 1 diabetic mice, hydrocortisone-induced insulin resistant mice and non-obese type 2 diabetic rats (Goto-Kakizaki (GK) rats) [167]. Phenylbutyric acid (1g/kg per day) has been administered to all four types of diabetic animals as well as to a normal healthy mouse to determine how it affects the glucose levels in mice sera. PBA has markedly lowered serum glucose levels in obese type 2 diabetic ob#ob mice and normoglycemia has been obtained after 4 days of treatment. Nonetheless, PBA had no glucose-lowering effect in the rest three types of diabetic animals. Moreover, healthy control ani-

mals did not show any changes in normal serum glucose levels. This data indicates that PBA treatment does not cause the general reduction of glucose levels in model diabetic animals and it is capa-ble of normalizing glucose levels specifically in blood of the dia-betic ob/ob mice. According to this, anti-diabetic therapy based on restoration of the ER function is limited to certain types of diabetes in which high blood glucose levels seem to be provoked by ER stress [167].

Type 2 diabetes is characterized mainly by insulin resistance and pancreatic $-cell dysfunction. Many stimuli, including chroni-cally increased levels of free fatty acids (FFAs), are related to the development of insulin resistance and $-cell dysfunction. Prolonged increase in FFAs levels has been proven to trigger insulin resistance in animals and humans [136]. The objective of the studies of Xiao et al. was to assess the possible role of PBA in mitigation of lipid-induced insulin resistance and beta cell failure in humans [136]. Studies have been conducted on eight overweight or obese indi-viduals, because of their higher susceptibility to FFA-induced $-cell dysfunction. Patients were obligated, for two weeks, to take an oral dose of PBA (equal 7, 5 g per day), after which intralipid/heparin or saline have been infused. In line with previous reports, it has been shown that PBA pretreatment partially prevented the lipid-dependent insulin resistance [168]. Interestingly, these studies failed to explicitly confirm PBA-dependent improvement of insulin sensitivity and beta cell function in humans, although PBA-mediated prevention of palmitate-induced decrease in $-cell func-tion has already been demonstrated in vitro [169]. These results may suggest that PBA exerts beneficial effects for the outcome of diabetes evoked by prolonged elevation of FFAs levels, mostly through amelioration of the insulin resistance and $-cell dysfunc-tion. However, further examinations are needed to elucidate the mechanism by which PBA protects against lipid-induced impair-ment of insulin clearance.

Gao et al. have examined the metabolic activities of butyric acid in diet-induced obese mice [170]. They have demonstrated that dietary supplementation with BA is able to protect against diet-induced obesity and insulin resistance in C57BL/6 mouse model. After 5-week diet supplementation with butyrate, obese animals have lost 10, 2% of their initial body weight and 10% of body fat content. Furthermore, BA treatment reduced insulin resistance by 50%, fasting glucose by 30%, and significantly improved intraperi-toneal insulin tolerance. Accordingly, butyrate seems to act through the induction of mitochondrial activity and stimulation of energy expenditure. The abovementioned results suggest that butyrate may be potentially applied in the prevention and treatment of metabolic syndrome in humans [170]. It has also been shown that PBA de-creased plasma levels of glucose, triglycerides, leptin and adi-ponectin, however without changing food intake [81]. In conse-quence, C57BL/6 animals fed by a high-fat diet supplemented with PBA demonstrated a remarkable reduction in weight gain and a decrease in fat pad mass in comparison to control high-fat diet-only mice. Furthermore, beneficial effects of PBA in obesity has also been confirmed in the study of Ozcan et al. who demonstrated that PBA was able to alleviate ER stress in the hypothalamus of obese mice, thus increasing leptin sensitivity [134]. These findings may suggest that an additional axis of PBA activity exists, thanks to which this substance may be considered as drug for obesity.

Promising results of the in vitro studies has shifted the attention of some researchers towards the application of PBA as a drug for cystic fibrosis. Thus, two clinical trials have been performed to monitor the effects of PBA treatment in CF-suffering patients [171, 172]. It has been proven that PBA caused small but significant im-provement of nasal potential difference response to perfusion of anisoproterenol/amiloride/chloride-free solution [172]. Neverthe-less, although slight but significant improved of chloride transport in nasal epithelium of $F508-homozygous CF patients has been


observed, these studies failed to explicitly prove the beneficial role of PBA in cystic fibrosis therapy [171, 172].

Systemic effects of PBA administration has also been moni-tored by the studies of "1-antitripsin deficiency. In "1-antitripsindeficiency mutant "1-antitripsin protein accumulates in hepatocytes, rather than being secreted into the blood. Individuals suffering from "1-antitripsin deficiency experience liver damage and emphysema [153]. After oral administration of PBA, mice transgenic for the human "1-antitripsin gene have shown a consis-tent increase of human "1-antitripsin in blood, reaching a 20–50% of the levels present in control non-transgenic mice and normal humans [152]. However, a single study in humans has demonstrated no increase in serum levels of "1-antitrypsin within 14 days of PBA treatment, and in fact has shown that PBA evoked significant side effects [153].

As presented above, a lot of functional studies on systemic level still need to be performed to demonstrate usefulness and therapeutic potential of phenylbutyrate in living organisms. It seems that prom-ising molecular and cellular effects of PBA treatment are not al-ways reflected by improved organism functioning. Despite this, further investigations should be conducted to explicitly determine the potential of PBA to be a potent drug in the treatment of many diseases. Systemic effects of PBA treatment along with its cellular effects are summarized in Table 1.

4. DISCUSSION

Short-chain fatty acids are important end-products of microbial fermentation [2, 3]. Amongst them butyric acid produced in human intestine, has been widely studied and shown to play an important role in the maintenance of colonic health [7, 8]. Butyrate has been proven to be an anti-inflammatory agent and to act as a histone deacetylase inhibitor not only in colonic cells, but also in many other cell types [38, 52, 90]. However, the main drawback of bu-tyrate utility in clinical studies has been its relative instability and short half-life in vivo after oral administration [12]. To deal with this, pharmacologically synthesized butyrate derivatives have been obtained. Phenylbutyrate is characterized by the relatively simple structure. It is a short-chain (C4) butyric acid with the phenyl group attached to the fourth carbon of the fatty chain (4-phenylbutyric acid). The substitution with the phenyl moiety has not only main-tained the transcription-modulatory activity of butyrate, but in fact enriched PBA with the additional property of a chemical chaperone. Given this, PBA has become a molecule with a broad spectrum of activities including being a histone deacetylase inhibitor, chemical chaperone and ammonia scavenger. In line with this, phenylbu-tyrate has been widely studied since it seems to give an opportunity to treat multiple pathological conditions. However, despite many reports considering the positive effects of PBA coming from studies conducted on molecular and cellular level, its systemic effects does not seem to be a direct reflection of cellular-level studies. In this respect, a clear distinction between these two activities should be mentioned. The therapeutic properties of PBA have been unques-tionably confirmed in case of the urea cycle disorders. Given this, FDA approved PBA for treatment of the UCDs. Phenylbutyrate enables the detoxification of the ammonia excess and counteracts the negative effects of hiperammonemia in UCD-suffering patients [40]. In urea cycle disorders PBA plays a role of an ammonia scav-enger and its therapeutic potential lies in the metabolic characteris-tic of this fatty acid. A key step in the conversion of PBA to its active metabolite-phenylacetate is a round of #-oxidation. In this respect, the correct course of #-oxidation seems to be essential in determining PBA efficiency, because the !rst step in #-oxidation of fatty acids is postulated to be rate-limiting. Thus, the metabolism of PBA-based drugs may also be modulated by similar factors that affect energy metabolism at the same step [39]. This metabolic pattern might be one of the reasons of the distinct effects of PBA on cellular and organismal level.

PBA is also known to exert potent anti-tumorigenic effect in vitro. Namely, in cancer cells PBA has been shown to stimulate growth inhibition and differentiation [38, 51-53], enhance apoptosis [22, 35, 37], and presumably to influence cellular senescence and autophagy by altering the expression of genes involved in these pathways [64, 76, 77, 173]. The molecular mechanisms by which PBA is able to exert these effects seem to be variable and cell type-specific and may include modulation of expression of many genes. Given this, PBA has been demonstrated to change the expression of some tumor suppressor genes such as p21, p16 and p53 [38, 56, 57, 62, 65, 174], genes belonging to the Bcl-2 family involved in apop-totic pathway [54, 55, 62], and other genes critical for cancer cell functioning, such as VEGF [54]. These results seem to be linked with the inhibition of histone deacetylases activity. Inhibited HDACs prevent histone deacetylation, thus resulting in increased chromatin relaxation and enhanced transcriptional activity.

Encouraged by the promising cellular effects of the in vitro and animal-model studies, PBA has already been tested for anti-tumor effectiveness in clinical trials [33, 36, 165]. Unfortunately, patients in clinics seem to respond to PBA with not as good efficiency as in model studies. Systemic effects of PBA treatment are rather mod-est, although some beneficial effects in disease stabilization have been observed when PBA was combined with other anticancer drugs [165]. This failure may be caused by the limited achievable concentration of PBA in human cells in vivo, since many of the model studies obtained potent results with doses exceeding 5 mmol/l, while the orally administrated achievable dose in vivo is approximately 2 mmol/l [175].

Gene transcription-modulatory skills related to the HDAC in-hibitory activity of PBA seem to stay in accordance with the effect of butyrate alone, devoid of the phenyl group [47, 73, 88]. How-ever, as mentioned previously, the pharmacologically derived phenylbutyrate has gained an additional advantage of becoming a chemical chaperone. PBA has been known to display the activity of the chemical chaperone mainly on the basis of its capacity to inhibit or alleviate ER stress [27, 117, 120, 123]. One of the suggested explanations of this phenomenon is the possibility of PBA to bind to the stretched hydrophobic surfaces of the unfolded/misfolded proteins, thus preventing their aggregation and allowing to avoid the check of the cellular quality control system [112]. This in con-sequence, allows cells to restore the correct trafficking of the pro-tein into particular cellular destinations, and to escape from the ER-stress mediated apoptotic pathway [28, 29, 116, 175]. However, although the capability of PBA to interfere with ER stress has been proposed as its cytoprotective mechanism of action, the chaperone-like nature of phenylbutyrate seems to be much more complicated. An engagement of the HDAC inhibitory skills in the cytoprotection can neither be excluded. Recent studies also indicate that targeting HDACs is a promising therapeutic strategy for a number of nonma-lignant diseases including neurodegenerative disorders, cardiovas-cular dysfunction, cystic fibrosis or inflammation-connected dis-eases [67, 85, 90, 91, 116, 123, 131]. There are reports suggesting that PBA chaperoning activity may manifest through the modifica-tions in the expression of some genes. It has been shown that phen-ylbutyrate is capable of up-regulating the expression of the molecu-lar chaperone genes [126, 131]. A great deal of attention has been paid to the analyses based on applications of PBA in Alzheimer’s disease [120, 123-125]. It seems to be the reasonable approach, since PBA is known to penetrate well into cerebrospinal fluid and easily pass the blood-brain barrier [86, 120]. According to these results PBA seems to have beneficial effects on AD pathology, however the precise molecular mode of action appears to be com-plex. Indeed, it seems to play a role of the epigenetic regulator al-tering the histone 3 acetylation and tau phosphorylation on the one hand and a chemical chaperone influencing A!42 production by modulation of APP trafficking on the other [27, 32, 120].


Table 1. Molecular and cellular effects of phenylbutyrate treatment.

Molecular activ-

ity of PBA

Disease entity Molecular and cellular effects Systemic effects

ammonia scaven-ger

urea cycle disorders conjugation of the PBA metabolites with glutamine; restoration of the proper urea cycle functioning [38,

39]

reduction in plasma glutamine and ammonia con-centrations [13-17, 163]

alleviated neurological impairment [17]

cancer stimulation of cell differentiation and apoptosis [22, 35, 49, 50, 55, 58, 59, 63, 63]

inhibition of cell proliferation [51, 52, 57, 69]

induction of cell cycle arrest [38, 52]

induction of cellular senescence [76, 77]

increased autophagy [58, 173]

sensitization to radiation- and chemotherapeutic-induced apoptosis [54, 56, 62, 64-66, 72, 73]

stabilization of the disease progress [33, 165]

inflammatory status down-regulation of many pro-inflammatory genes [88-91]

reduced oxidative status of cells [85]

neuronal protection, normalized striatal neuro-transmitters, and improved motor functions [85]

$-thalassemia and sickle cell anemia

stimulation of $- and %-globin transcription [94, 95] increase in the percentage of F-reticulocytes; increased HbF levels [94, 95]

Huntington’s disease up-regulation of many pro-survival genes; reduced neuronal cells apoptosis [97]

attenuation of gross brain atrophy and neuronal atrophy; improvement in overall survival [97]

histone deacety-lase inhibitor

amyotrophic lateral sclerosis

changes in gene expression profiles, reduced oxidative stress and apoptosis of neurons [99, 100]

improvement of motor function and extended survival [99]

Parkinson’s disease reduction of neuronal "-synuclein levels [116]

alleviation of the ER stress; reduced neuronal apopto-sis [28, 29, 86, 116-118]

restoration of the Pael-R localization from the ER into the plasma membrane [28, 29]

prevention of age-related impairment in motor and cognitive functions [116]

improvement of bradykinesia and motor coordina-tion [31]

Alzheimer’s disease restoration of the proper functioning of APP prote-olytic system; protection against ER stress-induced

apoptosis of neurons [27]

reduction of the phosphorylation level of tau protein; alleviation of ER-stress induced cell death [120]

clearance of the intraneuronal A! accumulation; alleviation of the ER stress [123, 124]

restoration of neuronal plasticity [120]

reversion of the spatial reference memory deficits [120]

restoration of learning de!cits [123, 124]

chemical chaper-one

cystic fibrosis improvement of the $F508-CFTR intracellular traf-ficking; enhanced expression of $F508-CFTR in the

plasma membrane of CF epithelial cells [30, 126, 128-131]

increase of the expression of "-, $-, and %-epithelial sodium channel subunits in the apical membrane ,

enhanced activity of amiloride-sensitive Na+-channel [129]

improvement of the nasal potential difference response to perfusion of an isoprotere-

nol/amiloride/chloride-free solution; improved chloride transport in nasal epithelium [171, 172]


(Table 1) Contd….

Molecular activ-

ity of PBA

Disease entity Molecular and cellular effects Systemic effects

diabetes and obesity alleviation of the cellular ER stress status; reduced apoptosis of pancreatic beta cells [136, 137]

attenuation of adipogenesis and alleviation of ER stress/UPR activation [81]

modulation of lipids accumulation in adipocytes [81]

restoration of the obesity-associated MC4R variants from the ER to the plasma membrane [147, 151]

improvement in insulin sensitivity, normalization of hyperglycemia levels, enhancement of insulin action in muscle, liver, and adipose tissue, im-

proved outcome of fatty liver disease [136, 167-169]

inhibition of the progress of diabetic nephropathy [137]

weight reduction [170]

reduction in plasma levels of glucose, triglyc-erides, leptin and adiponectin [81]

augmentation of leptin sensitivity [134]

"1-antitripsin defi-ciency

restoration of the functionally active "1-antitrypsin from the ER to the plasma membrane of hepatocytes

[152]

increase of "1-antitripsin level in blood [152]

Additionally, PBA seems to cause notable effects on systemic lev-els by improving memory and learning deficits in Tg2576 [120, 123, 124].

One of the most intriguing issues connected with the PBA mode of action is the ability to evoke relatively contradictory effects in healthy and cancer cells. This phenomenon is termed as “butyrate paradox” and is generally defined as the capacity of PBA to be cell-destructive in cancer cells and safe, or even cell-protective in nor-mal cell types [5]. This feature is presumably connected with the metabolic characteristic of these cells. In normal cell, nearly all of the BA may be used in ATP synthesis, which does not disturb cell functioning and results in cell proliferation. In contrast, in tumor cells characterized by predominantly anaerobic metabolism, bu-tyrate seems to be inefficiently oxidized, and able to reach the nu-cleus where regulates gene expression via butyrate-mediated HDAC inhibition and histone acetylation [12]. This theory how-ever, does not seem to be sufficiently clarifying since HDAC in-hibitory properties of butyrate/phenylbutyrate have been demon-strated also in case of many nonmalignant diseases [62, 123, 125]. Accordingly, PBA has been shown to up-regulate the expression of DJ-1 in Parkinson’s disease [116], GluR1 and PSD95 in Alz-heimer’s disease [123], PGC-1" in obesity [170] or ERp29 in cystic fibrosis [131]. Moreover, it has been able to act as an anti-inflammatory agent in many types of inflamed cell, independently of the inflammation-evoking stimuli [85, 90, 91]. This activity has mainly been mediated via modulation of the NF!B expression – a key regulator of the inflammation-response signaling pathway [88, 89]. In a light of this data, it seems impossible to explicitly explain the “buryrate paradox” phenomenon. This may indicate a slightly different, less aerobic/anaerobic metabolism-dependent mode of PBA action. However, the exact mechanism explaining why in malignant cells PBA stimulate mainly transcription of the “cy-tostatic” genes, while in nonmalignant it seems to activate the “beneficial” ones, has not been comprehensively elucidated yet. Given this, it seems impossible to categorically define the exact and universal mechanism of PBA action in various cell types and in different diseases, since it looks like these effects can merge with each other. Thus, while exploring chaperoning activity it should be

advised to simultaneously examine the level of histone acetylation and the expression of critical genes of interest to obtain more com-prehensive results. Nevertheless, PBA is still extensively studied and it seems likely that perhaps in the future we will get to know all the complicated nature of this compound. Hopefully, this knowl-edge will let us overcome the limitations of its efficiency in human treatments.

Although, much information about PBA has already been avail-able, it is still a subject of intensive investigations. Data from many clinical and laboratory studies have shown a wide spectrum of pos-sibilities for potential therapeutic use of phenylbutyrate. Growing numbers of investigations constantly reveal the new effects and molecular mechanisms underlying PBA activity. Nevertheless, since cell line and animal model studies are not always accurately mirrored in patients, more clinical trials are still needed to fully elucidate the role of PBA in human health and disease.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

Declared none.

REFERENCES

[1] Roediger WE. Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa in man. Gut 1980; 21: 793-8.

[2] Parodi PW. Cows’ milk fat components as potential anticarcino-genic agents. J Nutr 1997; 127: 1055-60.

[3] Newmark HL, Young CW. Butyrate and phenylacetate as differen-tiating agents: practical problems and opportunities. J Cell Bio-chem 1995; 22: 247-53.

[4] Goncalves P, Araujo JR, Martel F. Characterization of butyrate uptake by nontransformed intestinal epithelial cell lines. J Membr Biol 2011; 240: 35-46.

[5] Berni Canani R, Di Costanzo M, Leone L, Pedata M, Meli R, Ca-lignano, A. Potential beneficial effects of butyrate in intestinal and extraintestinal diseases. World J Gastroenterol 2011; 17(12): 1519-28.


[6] Macfarlane GT, Macfarlane S. Fermentation in the human large intestine: its physiologic consequences and the potential contribu-tion of prebiotics. J Clin Gastroenterol 2011; 45: S120-7.

[7] Hamer HM, Jonkers D, Venema K, Vanhoutvin S, Troost FJ, Brummer RJ. Review article: the role of butyrate on colonic func-tion. Aliment Pharmacol Ther 2008; 27: 104-19.

[8] Hamer HM, Jonkers DM, Bast A, et al. Butyrate modulates oxida-tive stress in the colonic mucosa of healthy humans. Clin Nutr 2009; 28: 88-93.

[9] Davis T, Kennedy C, Chiew YE, Clarke CL, DeFazio A. Histone deacetylase inhibitors decrease proliferation and modulate cell cy-cle gene expression in normal mammary epithelial cells. Clin Can-cer 2000; 6: 4334-42.

[10] Pineau T, Hudgins WR, Liu L, et al. Activation of a human perox-isome proliferator-activated receptor by the antitumor agent pheny-lacetate and its analogs Biochem Pharmacol 1996; 52: 659-67.

[11] Darmaun D, Welch S, Rini A, Sager BK, Altomare A, Haymond MW. Phenylbutyrate-induced glutamine depletion in humans: ef-fect on leucine metabolism. Am J Physiol 1998; 274: 801-7.

[12] Leonel AJ, Alvarez-Leite JI. Butyrate: implications for intestinal function. Curr Opin Clin Nutr Metab Care 2012; 15: 474-9.

[13] Redonnet-Vernhet I, Rouanet F, Pedespan JM, Hocke C, Parrot F. A successful pregnancy in a heterozygote for OTC deficiency treated with sodium phenylbutyrate. Neurology 2000; 54: 1008.

[14] Lee B, Rhead W, Diaz GA, et al. Phase 2 comparison of a novel ammonia scavenging agent with sodium phenylbutyrate in patients with urea cycle disorders: safety, pharmacokinetics and ammonia control. Mol Genet Metab 2010; 100(3): 221-8.

[15] Lichter-Konecki U, Diaz GA, Merritt JL, et al. Ammonia control in children with urea cycle disorders (UCDs); phase 2 comparison of sodium phenylbutyrate and glycerol phenylbutyrate. Mol Genet Metab 2011; 103(4): 323-9.

[16] Smith W, Diaz GA, Lichter-Konecki U, et al. Ammonia control in children ages 2 months through 5 years with urea cycle disorders: comparison of sodium phenylbutyrate and glycerol phenylbutyrate. J Pediatr 2013; 162(6): 1228-34.

[17] Diaz GA, Krivitzky LS, Mokhtarani M, et al. Ammonia control and neurocognitive outcome among urea cycle disorder patients treated with glycerol phenylbutyrate. Hepatology 2013; 57(6): 2171-9.

[18] Dover GJ, Brusilow SW, Samid D. Increased fetal hemoglobin in patients receiving sodium 4-phenylbutyrate [letter 29]. N Engl J Med 1992; 327: 569.

[19] Perrine SP. Fetal globin stimulant therapies in the beta-hemoglobinopathies: principles and current potential. Pediatr Ann 2008; 37(5): 339-46.

[20] Dover GJ, Brusilow SW, Charache S. Induction of HbF production in subjects with sickle cell anemia by oral sodium phenylbutyrate. Blood 1994; 84: 339.

[21] Gilbert J, Baker SD, Bowling MK, et al. A phase I dose escalation and bioavailability study of oral sodium phenylbutyrate in patients with refractory solid tumor malignancies. Clin Cancer Res 2001; 7: 2292-300.

[22] Zhang X, Wei L, Yang Y, Yu Q. Sodium 4-phenylbutyrate induces apoptosis of human lung carcinoma cells through activating JNK pathway. J Cell Biochem 2004; 93(4): 819-29.

[23] Hurtubise A, Bernstein ML, Momparler RL. Preclinical evaluation of the antineoplastic action of 5-aza-2’-deoxy-cytidine and differ-ent histone deacetylase inhibitors on human Ewing’s sarcoma cells. Cancer Cell Int 2008; 8: 16.

[24] Guilloteau P, Martin L, Eeckhaut V, Ducatelle R, Zabielski R, Van Immerseel F. From the gut to the peripheral tissues: the multiple ef-fects of butyrate. Nutr Res Rev 2010; 23: 366-84.

[25] Miyoshi M, Sakaki H, Usami M, et al. Oral administration of tribu-tyrin increases concentration of butyrate in the portal vein and pre-vents lipopolysaccharide-induced liver injury in rats. Clin Nutr 2011; 30: 252-8.

[26] Iannitti T, Palmieri B. Clinical and experimental applications of sodium phenylbutyrate. Drugs R D 2011; 11(3): 227-49.

[27] Wiley JC, Meabon JS, Frankowski H, et al. Phenylbutyric acid rescues endoplasmic reticulum stress-induced suppression of APP proteolysis and prevents apoptosis in neuronal cells. PLoS ONE 2010; 5(2): e9135.

[28] Kubota K, Niinuma Y, Kaneko M, et al. Suppressive effects of 4-phenylbutyrate on the aggregation of Pael receptors and endoplas-mic reticulum stress. J Neurochem 2006; 97(5): 1259-68.

[29] Mimori S, Okuma Y, Kaneko M, et al. Protective effects of 4-phenylbutyrate derivatives on the neuronal cell death and endo-plasmic reticulum stress. Biol Pharm Bull 2012; 35(1): 84-90.

[30] Rubenstein RC, Egan ME, Zeitlin PL. In vitro pharmacologic resto-ration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic "brosis epithelial cells containing #F508-CFTR. J Clin Invest 1997; 100: 2457-65.

[31] Ono K, Ikemoto M, Kawarabayashi T, et al. A chemical chaperone, sodium 4-phenylbutyric acid, attenuates the pathogenic potency in human alpha-synuclein A30P + A53T transgenic mice. Parkin-sonism Relat Disord 2009; 15(9): 649-54.

[32] Cuadrado-Tejedor M, Ricobaraza AL, Torrijo R, Franco R, Garcia-Osta A. Phenylbutyrate is a multifaceted drug that exerts neuropro-tective effects and reverses the Alzheimer´s disease-like phenotype of a commonly used mouse model. Curr Pharm Des 2013; 19(28): 5076-84.

[33] Phuphanich S, Baker SD, Grossman SA, et al. Oral sodium phen-ylbutyrate in patients with recurrent malignant gliomas: a dose es-calation and pharmacologic study. Neuro Oncol 2005; 7(2): 177-82.

[34] Camacho LH, Olson J, Tong WP, Young CW, Spriggs DR, Malkin MG. Phase I dose escalation clinical trial of phenylbutyrate sodium administered twice daily to patients with advanced solid tumors. Invest New Drugs 2007; 25: 131-8.

[35] Carducci MA, Nelson JB, Chan-Tack KM, et al. Phenylbutyrate induces apoptosis in human prostate cancer and is more potent than phenylacetate. Clin Cancer Res 1996; 2: 379-87.

[36] Huang Y, Horvath CM, Waxman S. Regrowth of 5- fluorouracil-treated human colon cancer cells is prevented by the combination of interferon gamma, indomethacin, and phenylbutyrate. Cancer Res 2000; 60: 3200-6.

[37] Svechnikova I, Gray SG, Kundrotiene J, Ponthan F, Kogner P, Ekström TJ. Apoptosis and tumor remission in liver tumor xenografts by 4-phenylbutyrate. Int J Oncol 2003; 22: 579-88.

[38] Li LZ, Deng HX, Lou WZ, et al. Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest. World J Gastroenterol 2012; 18(1): 79-83.

[39] Kormanik K, Kang H, Cuebas D, Vockley J, Mohsen AW. Evi-dence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate. Mol Genet Metab 2012; 107(4): 684-9.

[40] Takai N, Narahara H. Histone deacetylase inhibitor therapy in epithelial ovarian cancer. J Oncol 2010; 2010: 458431.

[41] Pan LN, Lu J, Huang B. HDAC inhibitors: a potential new category of anti-tumor agents. Cell Mol Immunol 2007; 4(5): 337-43.

[42] Wang X, Wei X, Pang Q, Yi F. Histone deacetylases and their inhibitors: molecular mechanisms and therapeutic implications in diabetes mellitus. Acta Pharm Sin B 2012; 2(4): 387-95.

[43] Ropero S, Esteller M. The role of histone deacetylases (HDACs) in human cancer. Mol Oncol 2007; 1(1): 19-25.

[44] Kim HJ, Rowe M, Ren M, Hong JS, Chen PS, Chuang DM. His-tone deacetylase inhibitors exhibit anti-in!ammatory and neuropro-tective effects in a rat permanent ischemic model of stroke: multi-ple mechanisms of action. J Pharmacol Exp Ther 2007; 321: 892-901.

[45] Wang Z, Leng Y, Tsai LK, Leeds P, Chuang DM. Valproic acid attenuates blood–brain barrier disruption in a rat model of transient focal cerebral ischemia: the roles of HDAC and MMP-9 inhibition. J Cereb Blood Flow Metab 2011; 31: 52-7.

[46] Koutsounas I, Giaginis C, Theocharis S. Histone deacetylase in-hibitors and pancreatic cancer: are there any promising clinical tri-als? World J Gastroenterol 2013; 19(8): 1173-81.

[47] Gao SM, Chen CQ, Wang LY, et al. Histone deacetylases inhibitor sodium butyrate inhibits JAK2/STAT signaling through upregula-tion of SOCS1 and SOCS3 mediated by HDAC8 inhibition in mye-loproliferative neoplasms. Exp Hematol 2013; 41(3): 261-70.

[48] Lin J, Gilbert J, Rudek MA, et al. A phase I dose-finding study of 5-azacytidine in combination with sodium phenylbutyrate in pa-tients with refractory solid tumors. Clin Cancer Res 2009; 15(19): 6241-9.

[49] Ohta M, Abe A, Ohno F, et al. Positive and negative regulation of podoplanin expression by TGF-" and histone deacetylase inhibitors in oral and pharyngeal squamous cell carcinoma cell lines. Oral Oncol 2013; 49(1): 20-6.

[50] Witzig TE, Timm M, Stenson M, Svingen PA, Kaufmann SH. Induction of apoptosis in malignant B cells by phenylbutyrate or


phenylacetate in combination with chemotherapeutic agents. Clin Cancer Res 2000; 6(2): 681-92.

[51] Kennedy C, Byth K, Clarke CL, deFazio A. Cell proliferation in the normal mouse mammary gland and inhibition by phenylbu-tyrate. Mol Cancer Ther 2002; 1(12): 1025-33.

[52] Wang CT, Meng M, Zhang JC, et al. Growth inhibition and gene induction in human hepatocellular carcinoma cell exposed to so-dium 4-phenylbutanoate. Chin Med J (Engl) 2008; 121(17): 1707-11.

[53] Liu L, Hudgins WR, Miller AC, Chen L-H, Samid D. Transcrip-tional up-regulation of TGF-alpha by phenylactate and phenylbu-tyrate is associated with differentiation of human melanoma cells. Cytokine 1995; 7: 449-56.

[54] Goh M, Chen F, Paulsen MT, Yeager AM, Dyer ES, Ljungman M. Phenylbutyrate attenuates the expression of Bcl-X(L), DNA-PK, caveolin-1, and VEGF in prostate cancer cells. Neoplasia 2001; 3(4): 331-8.

[55] Hattori Y, Fukushima M, Maitani Y. Non-viral delivery of the connexin 43 gene with histone deacetylase inhibitor to human na-sopharyngeal tumor cells enhances gene expression and inhibits in vivo tumor growth. Int J Oncol 2007; 30: 1427-39.

[56] Lopez C, Feng FY, Herman JM, Nyati MK, Lawrence TS, Ljung-man M. Phenylbutyrate sensitizes human glioblastoma cells lacking wild-type p53 function to ionizing radiation.Int J Radiat Oncol Biol Phys 2007; 69(1): 214-20.

[57] Condorelli F, Gnemmi I, Vallario A, Genazzani AA, Canonico PL. Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells. Br J Pharmacol 2008; 153(4): 657-68.

[58] de Conti A, Tryndyak V, Koturbash I, et al. The chemopreventive activity of the butyric acid prodrug tributyrin in experimental rat hepatocarcinogenesis is associated with p53 acetylation and activa-tion of the p53 apoptotic signaling pathway. Carcinogenesis 2013; 34(8): 1900-6.

[59] Kuroiwa-Trzmielina J, de Conti A, Scolastici C, et al. Chemopre-vention of rat hepatocarcinogenesis with histone deacetylase inhibi-tors: efficacy of tributyrin, a butyric acid prodrug. Int J Cancer 2009; 124(11): 2520-7.

[60] Maiuri MC, Galluzzi L, Morselli E, Kepp O, Malik SA, Kroemer G. Autophagy regulation by p53. Curr Opin Cell Biol 2010; 22(2): 181-5.

[62] Ammerpohl O, Trauzold A, Schniewind B, et al. Complementary effects of HDAC inhibitor 4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensi-tivity of PDAC cells. Br J Cancer 2007; 96: 73-81.

[63] Carducci MA, Gilbert J, Bowling MK, et al. A phase I clinical and pharmacological evaluation of sodium phenylbutyrate on an 120-h infusion schedule. Clin Cancer Res 2001; 7: 3047-55.

[64] Schultz J, Ibrahim SM, Vera J, Kunz M. 14-3-3# gene silencing during melanoma progression and its role in cell cycle control and cellular senescence. Mol Cancer 2009; 8: 53.

[65] Egger G, Aparicio AM, Escobar SG, Jones PA. Inhibition of his-tone deacetylation does not block resilencing of p16 after 5-aza-2' -deoxycytidine treatment. Cancer Res 2007; 67(1): 346-53.

[66] Bandres E, Agirre X, Bitarte N, et al. Epigenetic regulation of microRNA expression in colorectal cancer. Int J Cancer 2009; 125: 2737-43.

[67] Daosukho C, Chen Y, Noel T, et al. Phenylbutyrate, a histone deacetylase inhibitor, protects against Adriamycin-induced cardiac injury. Free Radic Biol Med 2007; 42(12): 1818-25.

[68] Burkitt K, Ljungman M. Phenylbutyrate interferes with the Fanconi anemia and BRCA pathway and sensitizes head and neck cancer cells to cisplatin. Mol Cancer 2008; 7: 24.

[69] Burkitt K, Ljungman M. Compromised Fanconi anemia response due to BRCA1 deficiency in cisplatin-sensitive head and neck can-cer cell lines. Cancer Lett 2007; 253: 131-7.

[70] Quinn JE, James CR, Stewart GE, et al. BRCA1 mRNA expression levels predict for overall survival in ovarian cancer after chemo-therapy. Clin Cancer Res 2007; 13: 7413-20.

[71] Sylvain V, Lafarge S, Bignon YJ. Dominant-negative activity of a Brca1 truncation mutant: effects on proliferation, tumorigenicity in vivo, and chemosensitivity in a mouse ovarian cancer cell line. Int J Oncol 2002; 20: 845-53.

[72] Queirós O, Preto A, Pacheco A, et al. Butyrate activates the mono-carboxylate transporter MCT4 expression in breast cancer cells and enhances the antitumor activity of 3-bromopyruvate. J Bioenerg Biomembr 2012; 44(1): 141-53.

[73] Bueno-Carrazco J, Castro-Leyva V, García-Gomez F, et al. Sodium butyrate increases the effect of the photodynamic therapy: a mechanism that involves modulation of gene expression and differ-entiation in astrocytoma cells. Childs Nerv Syst 2012; 28(10): 1723-30.

[74] Bai LY, Omar HA, Chiu CF, Chi ZP, Hu JL, Weng JR. Antitumor effects of (S)-HDAC42, a phenylbutyrate-derived histone deacety-lase inhibitor, in multiple myeloma cells. Cancer Chemother Phar-macol 2011; 68: 489-96.

[75] Kulp SK, Chen CS, Wang DS, Chen CY, Chen CS. Antitumor effects of a novel phenylbutyrate-based histone deacetylase inhibi-tor, (S)-HDAC-42, in prostate cancer. Clin Cancer Res 2006; 12(17): 5199-206.

[76] Kim HD, Jang CY, Choe JM, Sohn J, Kim J. Phenylbutyric acid induces the cellular senescence through an Akt/p21WAF1 signal-ing pathway. Biochem Biophys Res Com 2012; 422: 213-8.

[77] Gu L, Kitamura M. Sensitive detection and monitoring of senes-cence-associated secretory phenotype by SASP-RAP assay. PLoS ONE 2012; 7(12): e52305.

[78] Hauswald S, Duque-Afonso J, Wagner MM, et al. Histone deacety-lase inhibitors induce a very broad, pleiotropic anticancer drug re-sistance phenotype in acute myeloid leukemia cells by modulation of multiple ABC transporter genes. Clin Cancer Res 2009; 15(11): 3705-15.

[79] Gurtowska N, Kloskowski T, Olkowska J, et al. Carboplatin and sodium butyrate, separate-yes, but combined-never. Acta Pol Pharm 2013; 70(1): 153-61.

[80] Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu Rev Immunol 2011; 29: 415-45.

[81] Basseri S, Lhoták S, Sharma AM, Austin RC. The chemical chap-erone 4-phenylbutyrate inhibits adipogenesis by modulating the un-folded protein response. J Lipid Res 2009; 50(12): 2486-501.

[82] Akash MS, Rehman K, Chen S. Role of inflammatory mechanisms in pathogenesis of type 2 diabetes mellitus. J Cell Biochem 2013; 114(3): 525-31.

[83] Wellen KE, Hotamisligil GS. Inflammation, stress, and diabetes. J Clin Invest 2005; 115: 1111-9.

[84] Shoelson SE, Lee J, Goldfine AB. Inflammation and insulin resis-tance. J Clin Invest 2006; 116: 1793-801.

[85] Roy A, Ghosh A, Jana A, et al. Sodium phenylbutyrate controls neuroinflammatory and antioxidant activities and protects dopa-minergic neurons in mouse models of Parkinson’s disease. PLoS ONE 2012; 7(6): e38113.

[86] Qi X, Hosoi T, Okuma Y, Kaneko M, Nomura Y. Sodium 4-phenylbutyrate protects against cerebral ischemic injury. Mol Pharmacol 2004; 66(4): 899-908.

[87] Hotamisligil GS. Endoplasmic reticulum stress and the inflamma-tory basis of metabolic disease. Cell 2010; 140(6): 900-17.

[88] Segain JP, Raingeard de la Blétière D, Bourreille A, et al. Butyrate inhibits inflammatory responses through NFkappaB inhibition: im-plications for Crohn's disease. Gut 2000; 47(3): 397-403.

[89] Russo I, Luciani A, De Cicco P, Troncone E, Ciacci C. Butyrate attenuates lipopolysaccharide-induced inflammation in intestinal cells and Crohn's mucosa through modulation of antioxidant de-fense machinery. PLoS ONE 2012; 7(3): e32841.

[90] Park JS, Lee EJ, Lee JC, Kim WK, Kim HS. Anti-inflammatory effects of short chain fatty acids in IFN- ! -stimulated RAW 264.7 murine macrophage cells: Involvement of NF- " B and ERK signal-ing pathways. Int Immunopharmacol 2007; 7: 70-7.

[91] Kim HJ, Jeong JS, Kim SR, Park SY, Chae HJ, Lee YC. Inhibition of endoplasmic reticulum stress alleviates lipopolysaccharide-induced lung inflammation through modulation of NF-kB/HIF-1a signaling pathway. Sci Rep 2013; 3: 1142.

[92] Roque T, Boncoeur E, Saint-Criq V, et al. Proinflammatory effect of sodium 4-phenylbutyrate in deltaF508-cystic fibrosis transmem-brane conductance regulator lung epithelial cells: involvement of extracellular signal-regulated protein kinase 1/2 and c-Jun-NH2-terminal kinase signaling. J Pharmacol Exp Ther 2008; 326(3): 949-56.

[93] Vij N, Amoako MO, Mazur S, Zeitlin PL. CHOP transcription factor mediates IL-8 signaling in cystic fibrosis bronchial epithelial cells. Am J Respir Cell Mol Biol 2008; 38: 176-84.

[94] Dover GJ, Brusilow S, Charache S. Induction of fetal hemoglobin production in subjects with sickle cell anemia by oral sodium phenylbutyrate. Blood 1994; 84: 339-343.


[95] Collins AF, Pearson HA, Giardina P, McDonagh KT, Brusilow SW, Dover GJ. Oral sodium phenylbutyrate therapy in homozy-gous beta thalassemia: a clinical trial. Blood 1995; 85: 43-9.

[96] Ebbel EN, Leymarie N, Schiavo S, et al. Identification of phenyl-butyrate-generated metabolites in Huntington disease patients using parallel liquid chromatography / electrochemical array / mass spec-trometry and off-line tandem mass spectrometry. Anal Biochem 2010; 399(2): 152-61.

[97] Gardian G, Browne SE, Choi DK, et al. Neuroprotective effects of phenylbutyrate in the N171-82Q transgenic mouse model of Huntington’s disease. J Biol Chem 2005; 280(1): 556-63.

[98] Hogarth P, Lovrecic L, Krainc D. Sodium phenylbutyrate in Huntington’s disease: a dose-finding study. Mov Disord 2007; 22(13): 1962-4.

[99] Petri S, Kiaei M, Kipiani K, et al. Additive neuroprotective effects of a histone deacetylase inhibitor and a catalytic antioxidant in a transgenic mouse model of amyotrophic lateral sclerosis. Neurobiol Dis 2006; 22(1): 40-9.

[100] Del Signore SJ, Amante DJ, Kim J, et al. Combined riluzole and sodium phenylbutyrate therapy in transgenic amyotrophic lateral sclerosis mice. Amyotroph Lateral Scler 2009; 10(2): 85-94.

[101] Cudkowicz ME, Andres PL, Macdonald SA, et al. Northeast ALS and National VA ALS Research Consortiums. Phase 2 study of so-dium phenylbutyrate in ALS. Amyotroph Lateral Scler 2009; 10(2): 99-106.

[102] Ringe D, Petsko GA. Q&A: What are pharmacological chaperones and why are they interesting? J Biol 2009; 8: 80. Available at (http: //jbiol.com/content/8/9/80).

[103] Hartl FU, Hayer-Hartl M. Converging concepts of protein folding in vitro and in vivo. Nat Struct Mol Biol 2009; 16: 574-81.

[104] Leandro P, Gomes CM. Protein misfolding in conformational dis-orders: rescue of folding defects and chemical chaperoning. Mini Rev Med Chem 2008; 8: 901-11.

[105] Arakawa T, Ejima D, Kita Y, Tsumoto K. Small molecule pharma-cological chaperones: From thermodynamic stabilization to phar-maceutical drugs. Biochim Biophys Acta 2006; 1764: 1677-87.

[106] Cechowska-Pasko M, Ba#kowski E, Chene P. The effect of hy-poxia on the expression of 150 kDa oxygen-regulated protein (ORP 150) in HeLa cells. Cell Physiol Biochem 2006; 17(1-2): 89-96.

[107] Kusaczuk M, Cechowska-Pasko M. Molecular chaperone ORP150 in ER stress-related diseases. Curr Pharm Des 2013; 19(15): 2807-18.

[108] Zhao J-H, Liu H-L, Lin H-Y, et al. Chemical chaperone and inhibi-tor discovery: potential treatments for protein conformational dis-eases. Perspect Medicin Chem 2007; 1: 39-48.

[109] Ben Mosbah I, Alfany-Fernández I, Martel C, et al. Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia-reperfusion. Cell Death Dis 2010; 8(1): e52.

[110] Perlmutter DH. Chemical chaperones: a pharmacological strategy for disorders of protein folding and traf$cking. Pediatr Res 2002; 52(6): 832-6.

[111] Cohen FE, Kelly JW. Therapeutic approaches to protein-misfolding diseases. Nature 2003; 426: 905-9.

[112] Papp E, Csermely P. Chemical chaperones: mechanisms of action and potential use. Handb Exp Pharmacol 2006; 172: 405-16.

[113] Teraoka M, Nakaso K, Kusumoto C, et al. Cytoprotective effect of chlorogenic acid against $-synuclein-related toxicity in catechola-minergic PC12 cells. J Clin Biochem Nutr 2012; 51(2): 122-7.

[114] Kaul S, Anantharam V, Kanthasamy A, Kanthasamy AG. Wild-type alpha-synuclein interacts with pro-apoptotic proteins PKCdelta and BAD to protect dopaminergic neuronal cells against MPP+-induced apoptotic cell death. Brain Res Mol Brain Res 2005; 139(1): 137-52.

[115] Baulac S, Lu H, Strahle J, et al. Increased DJ-1 expression under oxidative stress and in Alzheimer's disease brains. Mol Neurode-gener 2009; 4: 12.

[116] Zhou W, Bercury K, Cummiskey J, Luong N, Lebin J, Freed CR. Phenylbutyrate up-regulates the DJ-1 protein and protects neurons in cell culture and in animal models of Parkinson disease. J Biol Chem 2011; 286(17): 14941-51.

[117] Inden M, Kitamura Y, Takeuchi H, et al. Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone. J Neurochem 2007; 101(6): 1491-504.

[118] Srinivasan K, Sharma SS. Sodium phenylbutyrate ameliorates focal cerebral ischemic/reperfusion injury associated with comorbid type 2 diabetes by reducing endoplasmic reticulum stress and DNA fragmentation. Behav Brain Res 2011; 225(1): 110-6.

[119] Xu K, Dai XL, Huang HC, Jiang ZF. Targeting HDACs: A promis-ing therapy for Alzheimer’s disease. Oxid Med Cell Longev 2011; 2011: 143269.

[120] Ricobaraza A, Cuadrado-Tejedor M, Perez-Mediavilla A, Frechilla D, Del Rio J, Garcia-Osta A. Phenylbutyrate ameliorates cognitive deficit and reduces tau pathology in an Alzheimer’s disease mouse model. Neuropsychopharmacology 2009; 34(7): 1721-32.

[121] Kim J, Kleizen B, Choy R, Thinakaran G, Sisodia SS, Schekman RW. Biogenesis of gamma-secretase early in the secretory path-way. J Cell Biol 2007; 179: 951-63.

[122] Dries DR, Yu G. Assembly, maturation, and trafficking of the gamma-secretase complex in Alzheimer’s disease. Curr Alzheimer Res 2008; 5: 132-46.

[123] Ricobaraza A, Cuadrado-Tejedor M, Marco S, Pérez-Otaño I, Gar-cía-Osta A. Phenylbutyrate rescues dendritic spine loss associated with memory de$cits in a mouse model of Alzheimer disease. Hippocampus 2012; 22(5): 1040-50.

[124] Ricobaraza A, Cuadrado-Tejedor M, García-Osta A. Long-term phenylbutyrate administration prevents memory deficits in Tg2576 mice by decreasing Abeta. Front Biosci (Elite Ed) 2011; 3: 1375-84.

[125] Cuadrado-Tejedor M, García-Osta A, Ricobaraza A, Oyarzabal J, Franco R. Defining the mechanism of action of 4-phenylbutyrate to develop a small-molecule-based therapy for Alzheimer's disease. Curr Med Chem 2011; 18(36): 5545-53.

[126] Suaud L, Miller K, Panichelli AE, Randell RL, Marando CM, Rubenstein RC. 4-Phenylbutyrate stimulates Hsp70 expression through the Elp2 component of elongator and STAT-3 in cystic fi-brosis epithelial cells. J Biol Chem 2011; 286(52): 45083-92.

[127] Rubenstein RC, Zeitlin PL. Sodium 4-phenylbutyrate downregu-lates Hsc70: implications for intracellular trafficking of %F508-CFTR. Am J Physiol Cell Physiol 2000; 278: C259-67.

[128] Lim M, McKenzie K, Floyd AD, Kwon E, Zeitlin PL. Modulation of deltaF508 cystic fibrosis transmembrane regulator trafficking and function with 4-phenylbutyrate and flavonoids. Am J Respir Cell Mol Biol 2004; 31(3): 351-7.

[129] Prulière-Escabasse V, Planès C, Escudier E, Fanen P, Coste A, Clerici C. Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells. J Biol Chem 2007; 282(47): 34048-57.

[130] Singh OV, Pollard HB, Zeitlin PL. Chemical rescue of %F508-CFTR mimics genetic repair in cystic fibrosis bronchial epithelial cells. Mol Cell Proteomics 2008; 7(6): 1099-110.

[131] Suaud L, Miller K, Alvey L, et al. ERp29 regulates DeltaF508 and wild-type cystic fibrosis transmembrane conductance regulator (CFTR) trafficking to the plasma membrane in cystic fibrosis (CF) and non-CF epithelial cells. J Biol Chem 2011; 286(24): 21239-53.

[132] Singh OV, Vij N, Mogayzel PJ, Jozwik C, Pollard HB, Zeitlin PL. Pharmacoproteomics of 4-phenylbutyrate-treated IB3-1 cystic fi-brosis bronchial epithelial cells. J Proteome Res 2006; 5(3): 562-71.

[133] Nguyen TD, Kim US, Perrine SP. Novel short chain fatty acids restore chloride secretion in cystic fibrosis. Biochem Biophys Res Commun 2006; 342(1): 245-52.

[134] Ozcan L, Ergin AS, Lu A, et al. Endoplasmic reticulum stress plays a central role in development of leptin resistance. Cell Metab 2009; 9: 35-51.

[135] Sundar Rajan S, Srinivasan V, Balasubramanyam M, Tatu U. En-doplasmic reticulum (ER) stress & diabetes. Indian J Med Res 2007; 125: 411-24.

[136] Xiao C, Giacca A, Lewis GF. Sodium phenylbutyrate, a drug with known capacity to reduce endoplasmic reticulum stress, partially alleviates lipid-induced insulin resistance and beta-cell dysfunction in humans. Diabetes 2011; 60(3): 918-24.

[137] Luo ZF, Feng B, Mu J, et al. Effects of 4-phenylbutyric acid on the process and development of diabetic nephropathy induced in rats by streptozotocin: regulation of endoplasmic reticulum stress-oxidative activation. Toxicol Appl Pharmacol 2010; 246(1-2): 49-57.

[138] Gregor MF, Hotamisligil GS. Thematic review series: Adipocyte Biology. Adipocyte stress: the endoplasmic reticulum and meta-bolic disease. J Lipid Res 2007; 48(9): 1905-14.


[139] Schroder M, Kaufman RJ. The mammalian unfolded protein re-sponse. Annu Rev Biochem 2005; 74: 739-89.

[140] Boden G, Duan X, Homko C, et al. Increase in endoplasmic reticu-lum stress-related proteins and genes in adipose tissue of obese, in-sulinresistant individuals. Diabetes 2008; 57: 2468-44.

[141] Sharma NK, Das SK, Mondal AK, et al. Endoplasmic reticulum stress markers are associated with obesity in nondiabetic subjects. J. Clin. Endocrinol. Metab 2008; 93: 4532-41.

[142] Fu M, Rao M, Bouras T, et al. Cyclin D1 inhibits peroxisome pro-liferator-activated receptor gamma-mediated adipogenesis through histone deacetylase recruitment. J Biol Chem 2005; 280(17): 16934-41.

[143] Yoo EJ, Chung JJ, Choe SS, Kim KH, Kim JB. Down-regulation of histone deacetylases stimulates adipocyte differentiation. J Biol Chem 2006; 281(10): 6608-15.

[144] Zuo Y, Qiang L, Farmer SR. Activation of CCAAT/enhancer-binding protein (C/EBP) alpha expression by C/EBP beta during adipogenesis requires a peroxisome proliferator-activated receptor-gamma-associated repression of HDAC1 at the C/ebp alpha gene promoter. J Biol Chem 2006; 281(12): 7960-7.

[145] Fan ZC, Tao YX. Functional characterization and pharmacological rescue of melanocortin-4 receptor mutations identified from obese patients. J Cell Mol Med 2009; 13: 3268-82.

[146] René P, Le Gouill C, Pogozheva ID, et al. Pharmacological chap-erones restore function to MC4R mutants responsible for severe early-onset obesity. J Pharmacol Exp Ther 2010; 335(3): 520-32.

[147] Granell S, Mohammad S, Ramanagoudr-Bhojappa R, Baldini G. Obesity-linked variants of melanocortin-4 receptor are misfolded in the endoplasmic reticulum and can be rescued to the cell surface by a chemical chaperone. Mol Endocrinol 2010; 24: 1805-21.

[148] Xiang Z, Litherland SA, Sorensen NB, et al. Pharmacological characterization of 40 human melanocortin-4 receptor polymor-phisms with the endogenous proopiomelanocortin-derived agonists and the agouti-related protein (AGRP) antagonist. Biochemistry 2006; 45: 7277-88.

[149] Tao YX. The melanocortin-4 receptor: physiology, pharmacology, and pathophysiology. Endocr Rev 2010; 31: 506-43.

[150] Ward NA, Hirst S, Williams J, Findlay JB. Pharmacological chap-erones increase the cell-surface expression of intracellularly re-tained mutants of the melanocortin 4 receptor with unique rescuing efficacy profiles. Biochem Soc Trans 2012; 40(4): 717-20.

[151] Granell S, Serra-Juhé C, Martos-Moreno GÁ, et al. A novel mela-nocortin-4 receptor mutation MC4R-P272L associated with severe obesity has increased propensity to be ubiquitinated in the ER in the face of correct folding. PLoS ONE 2012; 7(12): e50894.

[152] Burrows JA, Willis LK, Perlmutter DH. Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency. Proc Natl Acad Sci 2000; 97: 1796-801.

[153] Teckman JH. Lack of effect of oral 4-phenylbutyrate on serum alpha 1-antitrypsin in patients with alpha 1- antitrypsin deficiency: A preliminary study. J Pediatr Gastroenterol Nutr 2004; 39: 34-7.

[154] Yam GH, Gaplovska-Kysela K, Zuber C, Roth J. Sodium 4-phenylbutyrate acts as a chemical chaperone on misfolded myocilin to rescue cells from endoplasmic reticulum stress and apoptosis. Invest Ophthalmol Vis Sci 2007; 48(4): 1683-90.

[155] Gong B, Zhang LY, Lam DS, Pang CP, Yam GH. Sodium 4-phenylbutyrate ameliorates the effects of cataract-causing mutant gammaD-crystallin in cultured cells. Mol Vis 2010; 16: 997-1003.

[156] van der Velden LM, Stapelbroek JM, Krieger E, et al. Folding defects in P-type ATP 8B1 associated with hereditary cholestasis

are ameliorated by 4-phenylbutyrate. Hepatology 2010; 51(1): 286-96.

[157] van den Berghe PV, Stapelbroek JM, Krieger E, et al. Reduced expression of ATP7B affected by Wilson disease-causing muta-tions is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin. Hepatology 2009; 50(6): 1783-95.

[158] Zode GS, Bugge KE, Mohan K, et al. Topical ocular sodium 4-phenylbutyrate rescues glaucoma in a myocilin mouse model of primary open-angle glaucoma. Invest Ophthalmol Vis Sci 2012; 53(3): 1557-65.

[159] Sorrenson B, Suetani RJ, Williams MJA, et al. Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate. J Lipid Res 2013; 54: 55-62.

[160] Hayashi H, Mizuno T, Horikawa R, et al. 4-Phenylbutyrate modu-lates ubiquitination of hepatocanalicular MRP2 and reduces serum total bilirubin concentration. J Hepatol 2012; 56(5): 1136-44.

[161] Summar M. Current strategies for the management of neonatal urea cycle disorders. J Pediatr 2001; 138: S30-9.

[162] Leonard JV. Disorders of the urea cycle and related enzymes. 4th rev ed. Springer: Berlin 2006.

[163] Burlina AB, Ogier H, Korall H, Trefz FK. Long-term treatment with sodium phenylbutyrate in ornithine transcarbamylase-deficient patients. Mol Genet Metab 2001; 72: 351-5.

[164] Gordon N. Ornithine transcarbamylase deficiency: a urea cycle defect. Eur J Paed Neurol 2003; 7: 115-21.

[165] Sung MW, Waxman S. Combination of cytotoxic-differentiation therapy with 5-fluorouracil and phenylbutyrate in patients with ad-vanced colorectal cancer. Anticancer Res 2007; 27(2): 995-1001.

[166] Maslak P, Chanel S, Camacho LH, et al. Pilot study of combination transcriptional modulation therapy with sodium phenylbutyrate and 5-azacytidine in patients with acute myeloid leukemia or myelo-dysplastic syndrome. Leukemia 2006; 20: 212-7.

[167] Xu TY, Chen RH, Wang P, Zhang RY, Ke SF, Miao CY. 4-Phenyl butyric acid does not generally reduce glucose levels in rodent models of diabetes. Clin Exp Pharmacol Physiol 2010; 37(4): 441-6.

[168] Ozcan U, Yilmaz E, Ozcan L, et al. Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 2006; 313: 1137-40.

[169] Choi SE, Lee YJ, Jang HJ, et al. A chemical chaperone 4-PBA ameliorates palmitate-induced inhibition of glucose-stimulated in-sulin secretion (GSIS). Arch Biochem Biophys 2008; 475: 109-14.

[170] Gao Z, Yin J, Zhang J, et al. Butyrate improves insulin sensitivity and increases energy expenditure in mice. Diabetes 2009; 58: 1509-17.

[171] Zeitlin PL, Diener-West M, Rubenstein RC, Boyle MP, Lee CK, Brass-Ernst L. Evidence of CFTR function in cystic fibrosis after systemic administration of 4-phenylbutyrate. Mol Ther 2002; 6(1): 119-26.

[172] Rubenstein RC, Zeitlin PL. A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in deltaF508-homozygous cystic fibro-sis patients: partial restoration of nasal epithelial CFTR function. Am J Respir Crit Care Med 1998; 157: 484-90.

[173] Shao Y, Gao Z, Marks PA, Jiang X. Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci 2004; 101(52): 18030-5.

[174] Svechnikova I, Ammerpohl O, Ekstr#m TJ. p21waf1/Cip1 partially mediates apoptosis in hepatocellular carcinoma cells. Biochem Biophys Res Commun 2007; 354(2): 466-71.

[175] Hayashi H, Sugiyam Y. 4-Phenylbutyrate enhances the cell surface expression and the transport capacity of wild-type and mutated bile salt export pumps. Hepatology 2007; 45: 1506-16.

Received: November 11, 2014 Accepted: January 1, 2015

current pharmaceutical design, , 21, 2147 phenylbutyric acid: … · phenylbutyric acid: simple...

Documents