Potential of Phenylbutyrate as Adjuvant ... - ACS Publications

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Potential of Phenylbutyrate as Adjuvant Chemotherapy: An Overview of Cellular and Molecular Anticancer Mechanisms Maha S. Al-Keilani*,† and Nour A. Al-Sawalha† †

Jordan University of Science and Technology, College of Pharmacy, Department of Clinical Pharmacy, P.O. Box 3030, Irbid 22110, Jordan ABSTRACT: Despite the advancement in cancer therapy, a high number of patients fail treatment because of drug resistance. Several preclinical in vitro data suggest that phenylbutyrate has antiproliferative, antiangiogenic, antimetastatic, immunomodulatory, and differentiating properties. Moreover, phenylbutyrate administration in vivo provided an oncoprotective effect. However, the results of clinical trials indicate that the antineoplastic potential of phenylbutyrate is hindered by its pharmacokinetic and pharmacodynamic properties. Thus, understanding the exact mechanisms of the anticancer effect of phenylbutyrate could assist in the selection of patients who will best benefit from this drug. The present review discusses the proposed mechanisms of antineoplastic effect of phenylbutyrate and the preclinical and clinical evidence suggesting its potential role as anticancer in different types of cancer.

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CONTENTS

1. Introduction 2. Antitumor Effects of Phenylbutyrate and Derivatives 2.1. Inhibition of HDAC 2.2. Inhibition of Tumor Cell Growth 2.3. Induction of Apoptosis 2.4. Induction of Differentiation 2.5. Inhibition of Angiogenesis 2.6. Inhibition of DNA Repair 2.7. Inhibition of Metastasis 2.8. Immunomodulatory Effects 2.9. Miscellaneous Activities 3. Phenylbutyrate and Cancer Treatment and Prevention 4. Conclusions and Future Perspectives Author Information Corresponding Author Funding Notes Biographies Abbreviations References

eliminated in urine as phenylacetylglutamine, thereby mediating elimination of waste nitrogen. However, the beneficial effects of phenylbutyrate are not limited to patients with urea cycle disorders. Phenylbutyrate is one of the first drugs encountered in cancer therapy as a histone deacetylase inhibitor (HDACI).2 In malignant cells, HDACs are dysregulated in terms of expression or activity,3,4 resulting in an imbalance between the acetylation and deacetylation and hence aberrant gene expression,5 such as abnormal downregulation of tumor suppressor genes or activation of oncogenes resulting in cancer development and progression.6−8 HDACIs are emerging as a new group of drugs with antineoplastic potential. The proof-of-concept for HDACIs therapies is the approval of vorinostat, romidepsin, and belinostat by the U.S. Food and Drug Administration (FDA) for use in hematological cancers.9 The results of numerous preclinical and clinical studies suggested the beneficial effect of phenylbutyrate and its derivatives [sodium butyric acid, sodium butyrate, and sodium phenylbutyrate (NaPB)] for cancer treatment or prevention through their interaction with essential cellular functions.2 For example, both in vitro and in vivo studies have demonstrated that phenylbutyrate and derivatives possess dose-dependent antitumor effects, including inhibition of growth, invasion, and migration, and induction of differentiation and apoptosis in different types of solid tumors, such as malignant glioma, prostate cancer, breast cancer, and lung cancer, and hematological tumors such as acute myelogenous leukemia.1,2,10−13 Moreover,

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1. INTRODUCTION Phenylbutyrate, an ammonia scavenger, is a commonly used drug for treatment of urea cycle disorders and hyperammonemia.1 In human body, phenylbutyrate is oxidized to phenylacetate, which is in turn conjugated with glutamine and © 2017 American Chemical Society

Received: June 3, 2017 Published: September 20, 2017 1767

DOI: 10.1021/acs.chemrestox.7b00149 Chem. Res. Toxicol. 2017, 30, 1767−1777

Chemical Research in Toxicology

Review

family consists of four classes; class I, IIa, IIb, and IV, while the sirtuin family consists of class III enzymes.6 The HDACIs that are under current investigation are either selective to class I or II enzymes or act as pan-inhibitors of HDACs class I, IIa, IIb, and IV.6 Phenylbutyrate is a competitive inhibitor of class I and IIa HDAC enzymes.22 The cellular uptake of phenylbutyrate is mediated by mainly two transporters: monocarboxylase transporter 1 (MCT1) and sodium-coupled monocarboxylase transporter 1 (SMCT1).23 Phenylbutyrate binds to the substrate binding site of the HDAC enzymes with four hydrogen bonds in total and hence blocks the substrate access to the enzyme’s active site.24 The active site of class I and IIa enzymes consists of a tubular pocket with a chelating zinc ion moiety at the bottom, which is essential for the enzyme catalysis. Phenylbutyrate binds to the zinc moiety and thus inhibits the enzyme activity.24 Consequently, promoting a hyperacetylation status of histones leads to chromatin decondensation and genetranscription activation subsequently due to enhanced accessibility of the transcription factors. On the other hand, hyperacetylation of nonhistone proteins, such as transcription factors, and post-translational modifications were also evident with phenylbutyrate, resulting in regulation of several cellular functions including protein−protein and protein−nucleotide interactions, protein translocation, and modulating various signal transduction pathways via affecting kinases activities.2 2.2. Inhibition of Tumor Cell Growth. Modulation of the cell-cycle related proteins is the most commonly studied mechanism of cell-type-specific antitumor effects of phenylbutyrate treatment that results in reduced proliferation and cellcycle arrest in G1 or G2 phases. A gene expression microarray analysis revealed that sodium butyrate downregulated 100 cell cycle-associated genes in nonsmall cell lung cancer (NSCLC) cells.25 A common sequela of phenylbutyrate treatment is the upregulation of p21, also known as p21WAF1/Cip1, a cyclin-dependent kinase inhibitor and key regulator of cell cycle progression. DNA-damage induced checkpoints, such as p21, activate several tumor-suppressor pathways and consequently induce cell cycle

these anticancer effects were shown to be relatively selective to tumor cells,2,14,15 which encouraged their recruitment in clinical trials. Additionally, the recruitment of phenylbutyrate and derivatives in the anticancer regimen enhanced the cytotoxicity of several anticancer agents such as bortezomib in glioblastoma,16 5-aza-2′-deoxycytidine in lung cancer,17 and topotecan in neuroblastoma.18 Several clinical trials have assessed the antineoplastic activity of phenylbutyrate and derivatives especially in refractory, relapsed, and resistant cancer cases.19,20 However, the major focus was on phenylbutyrate and other newly developed derivatives because of their higher potency and specificity and better pharmacokinetic and safety profiles compared to the parent compound, N-butyrate. However, the molecular mechanisms underlying the response to phenylbutyrate and derivatives and selectivity toward cancer cells and tissues are not yet fully understood. In this review, the possible molecular mechanisms of the anticancer action of phenylbutyrate and derivatives as well as the preclinical and clinical data are discussed in depth.

2. ANTITUMOR EFFECTS OF PHENYLBUTYRATE AND DERIVATIVES 2.1. Inhibition of HDAC. The current proposed anticancer molecular mechanism of phenylbutyrate is mainly associated with the inhibition of HDAC enzymes. Histone modification via acetylation and deacetylation of lysine residues in histone tails is one type of epigenetic alterations that is regulated by histone acetyltransferases (HAT) and HDAC enzymes as shown in Figure 1. The acetylation status controls the expression of genes. Moreover, HATs and HDACs are responsible for regulating the acetylation status of nonhistone protein substrates; thus, their role is far beyond regulating gene expression and includes controlling cellular processes such as apoptosis, cell-cycle, differentiation, angiogenesis, necrosis, and immune responses.3,21 There are two families of HDACs, the classical and the sirtuin that differ in structure and catalytic activity.6 The classical

Figure 1. Epigenetic modification via histone acetylation and deacetylation of histone tails. 1768



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arrest at certain stages to fix the errors encountered by various stimuli and eventually inhibiting the growth. One of the mechanisms by which p21 inhibits growth is via reducing the kinase activity of various cyclin-dependent kinase (CDK)−cyclin complexes, primarily CDK1 and CDK2. This results in reduced retinoblastoma protein (Rb) phosphorylation and E2F inactivation.26 The inhibition of cell cycle progression promoted by p21 is mediated by p53-dependent gene repression of important cell cycle related proteins, such as cdc25c, cdc2, cyclin B1, telomerase, and survivin, and p53-independent pathways, such as via downregulating Sp1 and upregulating Sp3 transcription factors.26,27 In prostate cancer, phenylbutyrate at clinically achievable concentrations (0.1 mM-8 mM), repressed cell growth via p53-dependent and -independent pathways, and it resulted in upregulation of p21 and downregulation of survivin.13,28,29 In neuroblastoma cells, the upregulation of p21 by butyrate was due to the activation of the p53-pathway via nuclear translocation and hyperacetylation of p53.30 Cell-cycle arrest in the G1 or G2 phase upon treatment with butyrate or its derivatives was also evident in several other types of cancers and was associated with loss of telomerase activity and modulation of protein kinase c activity, upregulation of p19, p21, p27, and other tumor suppressor genes, and downregulation or modulation of enzymatic activity of cyclin B1, cyclin D1, cdc2, cdc25C, CDK2, CDK6, hTERT, Sp1, c-myc, and p45SKP2 (F-box protein responsible for p27KIP1 degradation and subsequent cell cycle progression to S-phase).12,31−37 Upregulation of insulin-like growth factor binding protein 3 (IGFBP-3) is another unique antiproliferative mechanism of sodium butyrate in breast cancer cells that was independent of p53 and was dependent on Sp1 and Sp3.38 2.3. Induction of Apoptosis. As part of the plethora of antitumor mechanisms of phenylbutyrate, induction of apoptosis is the major process of cytotoxicity. Apoptosis is programmed cell death that is mediated by two distinct pathways, intrinsic (mitochondrial) pathway and extrinsic (death-receptor pathway).2 Several experimental cancer models have shown that phenylbutyrate and its derivatives upregulate pro-apoptotic proteins and downregulate antiapoptotic proteins. Administration of (S)-HDAC-42, a phenylbutyrate derivative, enhanced apoptosis in prostate cancer cells via reducing phosphorylated AKT and phosphorylated Bad, and downregulating Bcl-XL, survivin, cIAP-1, and cIAP-2.38 Phenylbutyrate and its derivatives upregulated p21, gelsolin, phosphorylated p38, JNK, and ERK (MAPK pathway members), Bax, caspases-3, -8, -9, and -10, NOXA and αPUMA, Bid, and DAPK1/2, and downregulated Bcl-X L, Bcl-2, cytochrome c, FAK, and survivin.12,18,29,30,33,37,39−46 The apoptotic activity induced by phenylbutyrate in NSCLC was dependent on the JNK status, indicating that JNK is a key mediator of phenylbutyrate-induced apoptosis.40 Combining NaPB with retinoid, all-trans retinoic acid (ATRA; prototype of differentiation therapy) and Ro 41−5253, enhanced the antiproliferative action against breast cancer cells compared to monotherapy.47 This was partially explained by the induction of pro-apoptotic mechanisms, as revealed by the accumulation of cells in the G0/G1 phase.47 Furthermore, in HER2-positive breast cancer cells, sodium butyrate synergized with trastuzumab, a monoclonal antibody against HER2 receptors, in a dose- and time-dependent manner in terms of enhanced inhibition of cellular proliferation, cell

cycle arrest, and apoptosis. This synergism was associated with an increased expression of p27Kip1.48 On the other hand, the use of caspase-8 and caspase-10 inhibitors and PDTC, an inhibitor of transcription factor NF-kB, attenuated the sodium butyrateinduced cell death in breast cancer cells due to counter effects on sodium butyrate-mediated changes in the expression and activity of caspase-3 and caspase-10.43 The effect of PDTC was not due to inhibition of NF-kB since the application of other NF-kB inhibitors did not affect the response to sodium butyrate.43 Autophagic cell death was also induced upon treatment with butyrate in Hela cells with Apaf-1 knockout or Bcl-XL overexpression.49 Therefore, the cytotoxic effect of butyrate is mediated by several mechanisms of action. 2.4. Induction of Differentiation. Poor differentiation is a hallmark of aggressive tumors, and the development of differentiation-based agents is a promising strategy for cancer therapy. Several cellular morphological changes were induced by NaPB and these are indicative of its differentiation activity.50 In NSCLC, phenylbutyrate treatment modulated the differentiation status that favors a less aggressive tumor behavior, and this was evident by the increased expression levels of the differentiation markers; gelsolin, Mad, and p21.51 Moreover, sodium butyrate induced differentiation in a series of breast cancer cells through upregulating the level of N-Myc Downstream Regulated 1 (NDRG1) and β-casein; differentiation factors.52 Additionally, butyrate treatment increased the activity of alkaline phosphatase, a differentiation marker in colon cancer cells.53 Inhibition of phosphoinositide 3-kinase (PI3K) pathway was associated with increased antitumor effects of sodium butyrate in terms of enhanced differentiation and apoptosis via upregulating caspase-3 and caspase-9 and the subsequent cleavage of poly(ADP-ribose) polymerase (PARP) and DNA fragmentation.54,55 Administration of NaPB to primary myeloid leukemia cell increased expression of differentiation stage-specific cell surface antigens, CD14, CD34, and HLA-DR, suggesting that NaPB promotes monocytic differentiation in acute myelogenous leukemia.56 The differentiating activity of phenylbutyrate was further potentiated when combined with ATRA.57 Furthermore, in promyelocytic leukemia cells treated with retinoic acid, pretreatment with NaPB enhanced the antileukemic activity through increasing the accumulation of cells in the G0/G1 phase and increasing H4 histone acetylation and subsequent H3 phosphoacetylation, both of which were associated with enhanced granulocytic differentiation of cells that was evident by the increased expression of the early myeloid differentiation factor CD11b.58 Moreover, treatment of promyelocytic leukemia cells with sodium butyrate induced hyperacetylation of H4 and increased levels of trimethyl H3 at K4 (H3K4me3) that eventually increased the gene expression and protein levels of E-cadherin and subsequent induction of granulocytic differentiation.59 2.5. Inhibition of Angiogenesis. Angiogenesis, formation of new blood vessels, is an important process for tumor growth and metastasis. Phenylbutyrate and its derivatives have been shown to inhibit angiogenesis in various types of cancers, and therefore, they could be used as effective adjuvant therapy to inhibit tumor metastasis. Butyrate treatment reduced the level of vascular endothelial growth factor (VEGF)28 and VEGF165 as well as the nuclear translocation of hypoxia inducible factor 1 alpha (HIF-1α), a key regulator of VEGF expression, in prostate cancer cells.60,61 In oral cancer cells, sodium butyrate treatment decreased the 1769



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tongue cancer cells.35 Additionally, phenylbutyrate enhanced the cytotoxicity of temozolamide in malignant glioma cells via suppression of the endoplasmic reticulum stress revealed by the decreased expression of GRP78 and GADD153.72 Sodium butyrate treatment of prostate cancer cells was associated with downregulation of androgen receptor and decreased transcription activity, which was explained by an upregulation of the SMRT, an androgen receptor transcription corepressor.73 In breast cancer cells, MDA-MB-231, a group of cells revealed resistance to sodium butyrate. The resistant cells possessed stem cell characteristics with 40% of them expressed CD133 marker, and they expressed high levels of c-MET, an oncogene.74 Additionally, knockdown of c-MET enhanced the apoptotic effect of sodium butyrate.74 Consequently, mouse models transplanted with MDA-MB-231 cells with c-MET knockdown and treated with sodium butyrate showed lower incidence rates as measured by tumor volume due to decreased tumorigenicity of the transplanted cells compared to animal models transplanted with control cells or sodium butyrate-resistant cells.74 This points toward the suggestion that sodium butyrate may not be effective as monotherapy for treatment of cancer since it is not effective for eradication of cancer stem cells. The various antitumor effects of phenylbutyrate therapy are summarized in Figure 2.

level of lymphangiogenic and angiogenic markers, platelet derived growth factor subunit B (PDGF-B), angiopoietin-2, VEGF-C, and VEGF-D.62 2.6. Inhibition of DNA Repair. Phenylbutyrate and derivatives are not known to possess any mutagenic/genotoxic effects on cells.63 On the other hand, they have been revealed to affect DNA repair, for example, treatment of prostate cancer cells with phenylbutyrate downregulated DNA-dependent protein kinase (DNA-PK), an enzyme involved in nonhomologous end joining that is required for double strand break repair.28 Moreover, sodium butyrate and phenylbutyrate enhanced the radiosensitivity of human melanoma cell lines through affecting DNA repair mechanisms.64 This was evident by the decreased gene and protein expression of DNA repair proteins, Ku70 and Ku86, and the prolonged accumulation of phosphorylated H2AX foci, a marker of reduced repair of DNA double strand breaks induced by irradiation.64 In head and neck cancer cells, phenylbutyrate enhanced the sensitivity of cells to cisplatin via inhibiting the repair of cisplatin-induced DNA interstrand breaks through the inhibition of FA/BRCA1 pathway that was evident by the decreased formation of Fanconi anemia complementation group D2 (FANCD2) nuclear foci and downregulation of BRCA1.65 2.7. Inhibition of Metastasis. Several lines of evidence suggest that phenylbutyrate and derivatives inhibit tumor metastasis via downregulating several proteins that are involved in cell invasion, migration, and metastasis such as caveolin-1, urokinase, c-myc, decay-accelerating factor, and pro-matrix metalloproteinases.10,28,66,67 A unique proposed antineoplastic mechanism of NaPB in malignant glioma was the delivery of functional acid-sensing ion channels (ASIC) type 2 to the plasma membrane thus conversion of cells from high-grade glioma phenotype to nontransformed normal astrocyte phenotype that resulted in the loss of the amiloride-sensitive inward Na+ current which was subsequently associated with reduced cell migration and proliferation.68 2.8. Immunomodulatory Effects. A study by Melichar et al. suggested that the antitumor activity of phenylbutyrate against ovarian carcinoma cells was due to enhanced antitumor immunological response assayed by increased human leukocyte antigen (HLA) class I expression and CD58 and reduced TGF-β2, beside its effect on mevalonate incorporation.69 NaPB had synergistic effect to ganciclovir in EBV+ Burkitt’s lymphoma cells by upregulating thymidine kinase activity.31 Other proposed immunological response induced by sodium butyrate therapy in cancer is the induction of lymphokineactivated killer cell sensitivity of rat tumor cells, with complete cure of tumor-bearing rats when combined with IL-2.70 2.9. Miscellaneous Activities. In colorectal cancer cells, butyrate treatment induced a metabolic shift toward increased glutamine utilization with reduced lactate production, a wellrecognized phenotype of cancer cells.53 This effect was associated with reduced activity of pyruvate dehydrogenase complex (PDC) and upregulation of pyruvate dehydrogenase kinases (PDK), PDK2, PDK3, and more prominently PDK4.53 Consequently, this resulted in inhibition of growth of colorectal cancer cells in dose- and time-dependent manners.53 Moreover, butyrate treatment in colorectal cancer cells resulted in an acute stress response that was associated with HSP27 activation, activation of ASK1 (MAP3K) and p38 MAPK pathway consequently.71 Also it resulted in elevated cellular levels of reactive oxygen species (ROS) in oral and

3. PHENYLBUTYRATE AND CANCER TREATMENT AND PREVENTION Several in vivo studies illustrated the promising effect of phenylbutyrate and derivatives in different animal models. Administration of NaPB inhibited tumor growth of human prostate cancer xenografts29,75 as well as hepatocarcinoma and hepatoblastoma in laboratory animals.76 Additionally, sodium butyrate and tributyrin, a triglyceride derivative of butyric acid, at doses 0.1−5 mM, induced cell apoptosis of human prostate cancer cells in prostate microtumors.77 Moreover, sodium butyrate and tributyrin inhibited tumorigenesis of prostate cancer xenografts in nude mice.77 Svechnikova and colleagues found that 4-phenylbutyrate induced cell apoptosis, increased the acetylation of histones H3 and H4, and reduced the expression level of α-fetoprotein.76 A previous study by Fadeev and colleagues showed that NaPB attenuated the tumor growth, prolonged mice survival in a low-toxicity profile in Ehrlich ascites carcinoma mice model.78 Administration of butyrate, either as tributyrin or from diet, inhibited mammary tumorigenesis in nitrosomethylureainduced animal model of breast cancer.79 The benefits of NaPB have been extended beyond the cancer treatment to include cancer prevention. Kennedy and colleagues found that the proliferation of epithelial cells, expression of estrogen receptor as well as cyclin D1 were inhibited and acetylated H3 was increased in normal mammary glands of mice by NaPB.80 In addition, enriched butyrate diet resulted in the development of fewer tumors as compared to conventional diet in dimethylhydrazine induced animal model of large bowel cancer.81 Downregulating the level of survivin is one of the postulated anticancer mechanisms of action. The colon cancer cells with APC mutations in APCmin/+ mice, that have the tendency to develop multiple intestinal neoplasia, were resistant to the effect of butyrate due to the inability of butyrate to downregulate survivin in these cells.82 This downregulation of survivin was proposed to be p53-independent because HT-29 colon cancer cells express mutant p53.82 However, administration of 3,3′-diindolylmethane was able to decreased survivin levels through 1770



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Figure 2. Antitumor effects of phenylbutyrate and derivatives.

the level of stearic and oleic acids as well as docosahexaenoic acid in abdominal fat and in liver respectively in dimethylhydrazine-induced colon cancer.84 These changes in lipid metabolism are suspected to be involved in the process of carcinogenesis.85,86 However, butyrate administration did not reduce the incidence or the number of intestinal tumors as well as the level of apoptosis and p21 expression in azoxymethane -induced intestinal tumor in rats.87 This inconsistent result could be due to variations in the animal models of cancer as well as the mode of drug administration. Other studies focused on examining the effect of combinational regimens that include histone deacetylase inhibitors (Table 1). Sodium butyrate in combination with wild-type p53 gene therapy induced tumor regression in xenografted human hepatocellular carcinoma cells and gastric cancer cells in nude mice and this response was not observed with any agent alone.87 NaPB in combination with selenium, rosiglitazone, and hydralazine inhibited the progression as well as volume of adenoma and hyperplasia of lung cancer in mice.63 Although

increased degradation and thus enhanced the sensitivity of HT-29 cells to the apoptotic effect of butyrate.82 The combined treatment of butyrate and 3,3′-di-indolylmethane also resulted in increased the expression level of Bax and Bak, and the subsequent release of the proapoptotic proteins, cytochrome c and Smac, and eventually activating apoptosis cascade.82 In vivo, this was associated with reduced tumorigenesis APCmin/+ mice when treated with combined therapy of butyrate and 3,3′-di-indolylmethane compared to each therapy alone, thus indicating a beneficial role of this therapy in cancer prevention.82 In dimethylhydrazine-induced colon cancer, high level of intracolonic butyrate concentration, either from diet or butyrate administration, increased the gene and protein levels of caspase-1, enhanced the expression of cleaved PARP product, and reduced the expression level of Bcl-2.83 These changes resulted in reduced tumor volume and aberrant crypt number and hence inhibited the tumorigenesis in dimethylhydrazine induced animal model of colon cancer.83 In addition, butyrate decreased

Table 1. Partial List of in Vivo Studies That Involved Phenylbutyrate and Derivatives as Single and Combination Therapiesa animal study

animal model

administered drug

Monotherapy 81 Dimethylhydrazine induced Butyrate enriched diet large bowel cancer in male Sprague−Dawley rats 75 Human prostate cancer was PB induced using LNCaP and LuCaP cell lines in 6−8 weeks old Male BALB/c nu/nu mice 79 Nitrosomethylurea-induced tributyrin or dietary butyrate mammary tumors in female from anhydrous milk fat, Sprague−Dawley rats added to the diet 83 Dimethylhydrazine induced Butyrate enriched diet or colon cancer in rats intracolonic butyrate administration

dose

duration

biomarkers

Diet containing 10% fiber

33 weeks

Total tumor mass

Fiber-enriched diet reduced total tumor mass

600 mg/kg

21 days

Proliferative and apoptotic activity in vivo

Tumor growth and apoptotic activity were inhibited by PB

1% tributyrin, or 3% dietary butyrate

118 days

Tumor incidence and mammary tumorigenesis Aberrant crypt number, apoptotic index, the expression of proapoptotic proteins; caspase-1 and cleaved poly(ADP-ribose)

Butyrate inhibited mammary tumorigenesis and tumor incidence Pectin-fed and butyrate-instilled groups showed lower crypt number, tumor volume, and higher expression of caspase-1 and cleaved poly(ADP-ribose) poly-

Diet enriched with 15% citrus pectin or 50 mM sodium butyrate solution instilled intrarectally

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Table 1. continued animal study

animal model

administered drug

dose

duration

biomarkers

results

polymerase and the antiapoptotic protein Bcl-2 Incidence and number of intestinal tumors, the level of apoptosis and expression of p21CIP. Epithelial cell proliferation, estrogen receptor α (ERα) protein level, and cyclin D1 expression Tumor size, apoptosis, differentiation and histones acetylation

merase product, and lower Bcl-2 expression

Monotherapy

87

Azoxymethane- induced in- Sodium butyrate testinal carcinogenesis in F344 rats

150 mg of sodium butyrate/day mixed with diet

33 weeks

80

Normal mammary glands in NaPB virgin BALB/c mice

250 mg/kg/day and 500 mg/kg/day

7 days

76

Hepatocarcinoma (Hep3B) PB and hepatoblastoma (HepT1) cell lines were induced in nude rats

77

Human prostate cancer was Sodium butyrate and tribuinduced using PC3 and yrin TSU-PR1 cell lines in 7-week-old NMR/−nu/nu male mice

84

1.2-dimethylhydrazine in- Sodium butyrate duced colon cancer in rats Sodium butyrate

Mini-pump with an intratumor catheter, pumping 20 μmol of PB per cm3 of tumor volume per day i.p. administration of 24 mg of sodium butyrate or 7.9 mg of tributyrin was used to achieve final plasma concentration of 10 mM A solution containing 3.4% sodium butyrate with a final concentration of 372 mmol/L

78

Female mice with transplanted Ehrlich ascites carcinoma.

29

Human prostate cancer was NaPB induced using DU145 and PC3 in 8-week-old male Sprague−Dawley rats

None of the measured biomarkers were affected by sodium butyrate

NaPB inhibited epithelial cell proliferation, and reduced the protein levels of ERα and cyclin D1 PB caused xenografts regression, increased apoptosis and histone acetylation, and induced differentiation

4 weeks

Tumor growth and Ki67 protein level as a proliferation marker

Sodium butyrate and tributyrin inhibited tumor growth, and reduced Ki-67 expression

4 weeks

Aberrant crypt foci, colonic expression of cyclins D1 and E, and fatty acid profile in liver, colon, intra-abdominal fat and feces

400, 800, and 1200 mg/kg with drinking water daily

21 days

Tumor growth inhibition

2.5 mmol/kg

28 day

Tumor formation in vivo

Sodium butyrate reduced aberrant crypt foci, with no effect on expression of cyclins D1 and E. Also it decreased the level of stearic and oleic acids in the intra-abdominal fat and docosahexaenoic acid in the liver, increased level of linoleic acid in the intraabdominal fat. NaPB inhibited the growth of tumor in a dose-dependent manner with the effective therapeutic dose was 800 mg/kg Decreased tumorigenicity as the tumor sizes in NaPBtreated group stayed the same as those of first day of the experiment

Prostate carcinoma was in- PB and 13-cis retinoic acid duced in 4−6 week-old (CRA) male athymic nude mice NaPB ± 5-aza-2′-deoxycytiDNMT+/+ and DNMT± mice dine (DAC)

600 mg/kg/day of PB, i.p. ± 30 mg/kg/day of CRA 0.25 mg/kg DAC for DNMT± mice or 0.5 mg/kg for DNMT+/+ mice, i.p. ± 300 mg/kg NaPB, i.p.

4 weeks

Tumor volume and microvessel density

4 weeks

number of pulmonary lesions

100

Xenografted human gastric Combination of sodium bucancer cells (KATO-III) tyrate and adenoviral vecand hepatocellular carcitor carrying wild type p53 noma cells (HuH7) in gene therapy nude mice

500 mM of sodium butyrate administered i.p.

6 days

Tumor regression, necrotic changes, vascularity, and level of brain specific angiogenesis inhibitor-1

82

Intestinal tumors from APCmin/+ mice

Butyrate +3,3′-di-indolylmethane

10 mg/kg/day 3,3′-di-indolylmethane in days 1, 3, and 5 followed Butyrate at a dose of 24 mg/ day for 1 week started on day 7

1 week

Number of tumors and apoptosis

63

4-methylnitrosamino-1-(3- NaPB in combination with pyridyl)-1-butanone-inselenium, rosiglitazone duced preinvasive lung and hydralazine cancer in female A/J mice

300 mg/kg NaPB, i.p.

6 weeks

Size of hyperplasias and adenomas

NaPB

Combinational Therapy 89

88

a

The combined treatment decreased tumor volume and the microvessels density In DNMT+/+ mice DAC + NaPB decreased the number of lesions higher than each drug alone, and no additional effect of NaPB was observed in DNMT± mice Combined therapy induced complete regression of the tumor, induced necrotic changes and reduced tumor vascularity, and induced level of brain specific angiogenesis inhibitor-1 Butyrate and 3,3′-di-indolylmethane combination therapy reduced the number of tumors and enhanced apoptosis higher than mice treated with each drug alone Combined treatment reduced the progression as well as volume of adenoma and hyperplasia of lung cancer in mice

Abbreviations: PB, phenylbutyrate; NaPB, sodium phenylbutyrate; i.p., intraperitoneal. 1772



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excessive somnolence, and confusion were the most common dose limiting toxicities of continuous infusion of NaPB in patients with hormone refractory prostate cancer.95 Administration of oral phenylbutyrate to patients with refractory solid tumor malignancies was tolerable with dyspepsia, grade 1−2, and fatigue were the most common seen adverse effect.96 Although no remission was seen, around one-quarter of enrolled patients had stable disease for more than 6 months.96 In addition, oral NaPB was well tolerated in patients with supratentorial recurrent malignant gliomas and the patients developed fatigue and somnolence when they received high doses.20 However, these patients showed limited clinical response.20 The promising results of the in vitro and in vivo studies for combinational anticancer regimens containing phenylbutyrate, and the results of the previously discussed clinical trials that indicated a modest anticancer activity of phenylbutyrate or derivatives when given as monotherapy, suggest a greater benefit when phenylbutyrate is administered with other anticancer agents. Synergism was proposed that could reduce the serum level requirements of phenylbutyrate and enhance the antineoplastic efficacy. Combination of subcutaneous injections of 5-azacytidine followed by intravenous NaPB in patients with acute myeloid leukemia or myelodysplastic syndrome revealed a clinical beneficial response in half of the enrolled patients.97 However, fatigue was the most common adverse effect of NaPB.97 Furthermore, combination of continuous infusion of NaPB and 5-azacytidine in patients with refractory solid tumors was well tolerated and safe and yet minor neutropenia and anemia were observed.98 However, the combination failed to show any clinical benefit.98 Additional clinical studies were conducted to test the efficacy of NaPB in several types of cancers such as metastasis solid tumors, brain tumors, and hematological tumors among others in adult and pediatric population (Table 2).99

NaPB alone did not inhibit the development of lung tumor in murine lung cancer, the combined treatment of NaPB with 5-aza-2′-deoxycytidine, a demethylating agent, was efficient in reducing the development of lung tumor.88 Further, the combined regimen of phenylbutyrate and 13-cis retinoic acid decreased the density of microvessels and tumor cells’ proliferation and increased the rate of apoptosis in prostatic tumor xenografts in mice.89 The clinical role of phenylbutyrate in cancer has been the focus of several clinical studies for years. Phase I studies revealed the well tolerability of phenylbutyrate and derivatives when they were administered by different routes in patients with a variety of cancer types. Oral and parenteral administration of phenylbutyrate have been tried as well as the administration of high doses over a short time versus low doses over a long time, as proposed mechanisms to overcome the unfavorable pharmacokinetics of the drug and to reduce the adverse effects and enhance the efficacy. Continuous prolonged intravenous infusion of phenylbutyrate was well tolerated in patients with myelodysplastic syndrome and acute myeloid leukemia.90 However, one patient (out of 23 treated patients) developed central nervous system toxicity.90 Although the continuous infusion of NaPB did not show any complete or partial remission in patients with myelodysplasia and acute myeloid leukemia, some patients had improved hematological response.91 Patients with acute leukemia who received continuous infusion of sodium butyrate did not show any toxicity or clinical response due to short half-life in vivo and hence low plasma level.92 Administration of intravenous infusion of pivaloyloxymethyl butyrate, a prodrug of butyric acid, in patients with solid tumors resulted in partial response in chemotherapy naive and chemotherapy pretreated patients with metastatic and advanced nonsmall cell lung cancer and mild-moderate adverse events.93,94 Gore and colleagues showed that large doses of NaPB induced reversible neurocortical type of toxicity.91 Neuro-cortical toxicity,

Table 2. List of Clinical Trials That Studied the Pharmacokinetics of Phenylbutyrate and Derivativesa clinical trial

type of cancer

intervention

dose

101

Solid tumor

Tributyrin

50−400 mg/kg/day of tributyrin orally once daily for 3 weeks, followed by a 1-week rest.

91

MDS and AML

NaPB

125, 250, 375, 500, 750, and 1000 mg/kg/day of NaPB as 7-day continuous infusion repeated every 28 days with the MTD of 375 mg/kg/day.

95

Refractory solid tumors

NaPB

Continuous i.v. infusion of NaPB at 150, 225, 285, 345, 410, and 515 mg/kg/day for 120 h every 21 days (5 days infusion and 16 days rest).

96

Refractory solid tumor

NaPB

Oral NaPB at doses of 9, 18, 27, 36 g/ day, and 45 g/day.

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PK parameters

efficacy and toxicity

Median time to reach peak plasma concentration: 0.5 h. For two patients at doses of 200 mg/kg, peak plasma butyrate concentrations were 0.1−0.45 mM and AUC values on day 1 were 0.91 and 1.52, mM h, respectively. At the MTD: Mean Css: 0.287 ± 160 nM Mean Vd: 11.03 ± 3.05 L Mean AUC 40817 ± 18848 μmol h/L Mean t 1/2: 0.52 ± 0.25 h PB exhibited saturable metabolism to the active metabolite PA. PB: mean. Vd: 0.169 ± 0.043 L/kg, Vmax: 148 ± 23 μmol/h/kg, PA: Vd: 0.720 ± 0.562 L/kg, Vmax: 77 ± 10 μmol/h/kg.

Efficacy: no response. DLT: grade 3 nausea and vomiting at 50 mg/kg and myalgia at 150 mg/kg.

PB: mean Tmax: 1.8 ± 0.72 h, bioavailability: 78% ± 24, Cmax, AUC, and time PB concentrations sustained about 500 μM were increased in a linear fashion with increased dose, t1/2: 1 h (day 1 or 2), apparent clearance: 15 L/h. For PA: Tmax: 4.4 ± 1.6 h, t1/2: 1.8 (1−5.3).

DLT: 2/16 AML and 1/11 MDS neurocortical toxicity at 500 mg/ kg/day. This was correlated with elevated PA concentrations (3.3 mM). Efficacy: no patients achieved partial or complete remission. Efficacy: No clinical response, and two patients had stable disease for 168 days. DLT: neurocortical toxicity, hypokalemia, hyponatremia, hypocalcemia, hyperuricemia, nausea, fatigue. DLTs: nausea and vomiting, and hypocalcemia at 36 g/day, fatigue and edema and serious neurotoxicity at 45 g/day; all resolved after reduction of dose to 27 g/day. Efficacy: no patients achieved partial or complete remission. 12/23 patients had stable disease; 7 of them had stable disease for >6 months. DOI: 10.1021/acs.chemrestox.7b00149 Chem. Res. Toxicol. 2017, 30, 1767−1777


Review

Table 2. continued clinical trial

type of cancer

intervention

90

MDS and AML

NaPB

20

Recurrent malignant gliomas

NaPB

98

Refractory solid tumors

5-Azacytidine (5-AC) + NaPB

dose

PK parameters

efficacy and toxicity

Schedule 1 (7/14): 375 mg/kg/day of NaPB by ambulatory infusion pump for 1 week followed by 1 week of drug holiday (for total of 12 weeks; 6 cycles) Schedule 2 (21/28): 375 mg/kg/day of NaPB by ambulatory infusion pump for 3 weeks followed by 1 week of drug holiday (for total of 12 weeks; 3 cycles). Oral NaPB at 9, 18, 27, 36, and 45 g/day.

Median end of infusion concentration of PB 0.4 mM and 0.43 mM in the 7/14 and 21/28 dose schedules, respectively.

Efficacy: no measurable clinical benefit was observed in both dosing schedules. DLT: Neurocortical toxicity in one patient who received schedule 1 and it resolved 2 days after discontinuation of NaPB.

At the recommended dose of 27 mg/m2: PB: Cmax: 1225 ± 415 μM/h, Tmax: 1.5 ± 0.49, AUC: 2487 ± 972 μM/h, CL: 23 ± 13 L/h. For PA: Cmax: 617 ± 246 μM/h, Tmax: 3.3 ± 0.65 μM, AUC: 2437 ± 1180 μM/h. PA:PB AUC ratio: 1.0 ± 0.35. 24-h infusion of NaPB at a dose level of 400 mg/kg/d: Cmax: 775 ± 467 μM, AUC: 17722 ± 8345 h μM, Vd: 14.0 ± 8.0 L. For PA: Cmax: 1395 ± 594 μM, AUC: 26680 ± 11236 h μM PB concentrations were sustained above 500 μM for 16.46 h PA:PB AUC ratio: 1.71 ± 0.92. 7-day infusion of NaPB: PB Css: 210 ± 73.8 μM at 200 mg/kg/d and 446 ± 211 μM at 400 mg/kg/d. Only 1 patient had PB concentrations sustained about 500 μM. PA Css: 184 ± 85.2 μM at 200 mg/kg/d and 1464 ± 1285 μM at 400 mg/kg/d.

Efficacy: one complete response and five patients had stable disease for 1.9−5.7 months. DLTs: headache, light-headedness, CNS toxicity consisting of fatigue and somnolence, and anemia and neutropenia.

Regimen A: 14 days of 10−25 mg/m2/d of 5-AC and continuous infusion of NaPB at a dose of 400 mg/m2/d on days 6 and 13 (24-h infusions). Regimen B: 7 days of 75 mg/m2/d of 5-AC and continuous infusion of NaPB at a dose of 200−400 mg/m2/d on days 7−14 (7 days infusion). Regimen C: 21 days of 10−12.5 mg/m2/d of 5-AC and continuous infusion of NaPB at a dose of 400 mg/m2/d on days 6, 13, and 20 (24-h infusions).

Efficacy: No clinical response at any dose regimen, and only one patient on dose regimen B had stable disease for 4.5 months. DLT: grade 3 and 4 neutropenia resolved after dose reduction by 25%, grade 3 hyponatremia, grade 3 somnolence and confusion (neurocortical toxicity) resolved after discontinuation of NaPB.

a

Abbreviations: PK, pharmacokinetic; DLT, dose-limiting toxicity; MDS, myelodysplastic syndrome; AML, acute myelogenous leukemia; MTD, maximum tolerated dose; PB, phenylbutyrate; NaPB, sodium phenylbutyrate.

4. CONCLUSIONS AND FUTURE PERSPECTIVES Phenylbutyrate is an FDA approved drug for treatment of urea cycle disorders. However, increasing evidence suggests that phenylbutyrate exerts pleiotropic anticancer effects independent of the ammonia reduction. Preclinical data, in vitro and in vivo, suggest that phenylbutyrate has antiproliferative, antiangiogenic, antimetastatic, immunomodulatory, and differentiating properties. Nevertheless, the results of numerous clinical trials with phenylbutyrate and derivatives as single agents in solid tumors are disappointing and indicate that the antineoplastic benefit from phenylbutyrate is hindered by the unfavorable pharmacokinetic and pharmacodynamic properties of the drug. This could be explained by the inability to identify the optimum drug dose and administration schedule. On the other hand, the promising results from in vitro and in vivo studies of combinational regimens containing phenylbutyrate are spurring more interest.

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clinical pharmacology, pharmacogenomics, and other related courses. Her main research interest is the role of pharmacogenomics for medicine personalization especially in cancer. She is now an associate member in the AACR and a faculty member in the scientific research review committee, conferences and scientific days, and clinical pharmacy committees. Dr. Nour A. Al-Sawalha received her PhD degree in Pharmacology from University of Houston, 2013, and currently is an assistant professor at the Clinical Pharmacy Department at Jordan University of Science and Technology. She lectures pharmacology, advanced pharmacology, drug information resources, pharmacotherapy of respiratory system, and comprehensive therapeutics. The research focus of Dr. Al-Sawalha is determining the pharmacological mechanism of action of several drugs and the signaling cascade that is involved in the development of several diseases. In addition, she focuses on elucidating the effect of waterpipe tobacco smoke exposure on the function of different organs utilizing appropriate animal models.

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AUTHOR INFORMATION

Corresponding Author

ABBREVIATIONS AKT, protein kinase B; Apaf-1, apoptotic peptidase activating factor 1; APCmin/+, adenomatous polyposis coli; ASIC, acidsensing ion channels; ASK1, apoptosis signal-regulating kinase 1; ATRA, all-trans retinoic acid; Bad, BCL2 associated agonist of cell death; Bak, BCL2 antagonist/killer 1; Bax, BCL2 associated X, apoptosis regulator; Bcl-2, BCL2, apoptosis regulator; Bcl-XL, B-cell lymphoma-extra large; Bid, BH3 interacting domain death agonist; CD11b, cell surface glycoprotein MAC-1 subunit alpha; CD133, hematopoietic stem cell antigen; CD14, myeloid cell-specific leucine-rich glycoprotein; CD34, hematopoietic progenitor cell antigen; CD58, lymphocyte function-associated antigen 3; cdc2, cyclin dependent kinase 1; cdc25c, cell division cycle 25C; CDK, cyclin-dependent kinase; cIAP-1, inhibitors of

*E-mail: [email protected]. Phone: +962 (0) 798005279. Funding

This research was funded by the Deanship of Scientific Research at Jordan University of Science and Technology. Research Grant No. 20150158. Notes

The authors declare no competing financial interest. Biographies Dr. Maha Al-Keilani finished her PhD in Clinical Pharmaceutical Sciences from University of Iowa, 2013. After finishing her PhD, she became an assistant professor at Jordan University of Science and Technology. She lectures pharmacology, pharmacotherapy of endocrine and renal diseases, and pharmacotherapy of infectious diseases, 1774



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(9) Mottamal, M., Zheng, S., Huang, T. L., and Wang, G. (2015) Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents. Molecules 20 (3), 3898−3941. (10) Engelhard, H. H., Homer, R. J., Duncan, H. A., and Rozental, J. (1998) Inhibitory effects of phenylbutyrate on the proliferation, morphology, migration and invasiveness of malignant glioma cells. J. Neuro-Oncol. 37 (2), 97−108. (11) Dyer, E. S., Paulsen, M. T., Markwart, S. M., Goh, M., Livant, D. L., and Ljungman, M. (2002) Phenylbutyrate inhibits the invasive properties of prostate and breast cancer cell lines in the sea urchin embryo basement membrane invasion assay. Int. J. Cancer 101 (5), 496−499. (12) Choi, Y. H. (2006) Apoptosis of U937 human leukemic cells by sodium butyrate is associated with inhibition of telomerase activity. Int. J. Oncol. 29 (5), 1207−1213. (13) Carducci, M. A., Nelson, J. B., Chan-Tack, K. M., et al. (1996) Phenylbutyrate induces apoptosis in human prostate cancer and is more potent than phenylacetate. Clin. Cancer Res. 2 (2), 379−387. (14) Gore, S. D., and Carducci, M. A. (2000) Modifying histones to tame cancer: Clinical development of sodium phenylbutyrate and other histone deacetylase inhibitors. Expert Opin. Invest. Drugs 9 (12), 2923−2934. (15) Papeleu, P., Vanhaecke, T., Elaut, G., et al. (2005) Differential effects of histone deacetylase inhibitors in tumor and normal cells-what is the toxicological relevance? Crit. Rev. Toxicol. 35 (4), 363−378. (16) Asklund, T., Kvarnbrink, S., Holmlund, C., et al. (2012) Synergistic killing of glioblastoma stem-like cells by bortezomib and HDAC inhibitors. Anticancer Res. 32 (7), 2407−2413. (17) Boivin, A. J., Momparler, L. F., Hurtubise, A., and Momparler, R. L. (2002) Antineoplastic action of 5-aza-2′-deoxycytidine and phenylbutyrate on human lung carcinoma cells. Anti-Cancer Drugs 13 (8), 869−874. (18) Calvaruso, G., Carabillo, M., Giuliano, M., et al. (2001) Sodium phenylbutyrate induces apoptosis in human retinoblastoma Y79 cells: The effect of combined treatment with the topoisomerase I-inhibitor topotecan. Int. J. Oncol. 18 (6), 1233−1237. (19) Bhalla, K., and List, A. (2004) Histone deacetylase inhibitors in myelodysplastic syndrome. Best Pract. Res. Clin. Haematol. 17 (4), 595−611. (20) Phuphanich, S., Baker, S. D., Grossman, S. A., et al. (2005) Oral sodium phenylbutyrate in patients with recurrent malignant gliomas: A dose escalation and pharmacologic study. Neuro. Oncol. 7 (2), 177− 182. (21) Kroesen, M., Gielen, P., Brok, I. C., Armandari, I., Hoogerbrugge, P. M., and Adema, G. J. (2014) HDAC inhibitors and immunotherapy; a double edged sword? Oncotarget. 5 (16), 6558−6572. (22) Sekhavat, A., Sun, J. M., and Davie, J. R. (2007) Competitive inhibition of histone deacetylase activity by trichostatin A and butyrate. Biochem. Cell Biol. 85 (6), 751−758. (23) Lee, N. Y., and Kang, Y. S. (2016) In vivo and in vitro evidence for brain uptake of 4-phenylbutyrate by the monocarboxylate transporter 1 (MCT1). Pharm. Res. 33 (7), 1711−1722. (24) Abou-Zeid, L. A., El-Mowafy, A. M., Eikel, D., Nau, H., and ElMazar, M. (2007) Mechanism of butyrate binding to histone deacetylase (HDAC) correlation with chemopreventive effects and ligand selectivity. J. Basic and Applied Sci. 3, 73−85. (25) Jin, X., Wu, N., Dai, J., Li, Q., and Xiao, X. (2017) TXNIP mediates the differential responses of A549 cells to sodium butyrate and sodium 4-phenylbutyrate treatment. Cancer Med. 6 (2), 424−438. (26) Abbas, T., and Dutta, A. (2009) P21 in cancer: Intricate networks and multiple activities. Nat. Rev. Cancer 9 (6), 400−414. (27) Gartel, A. L., and Tyner, A. L. (2002) The role of the cyclindependent kinase inhibitor p21 in apoptosis. Mol. Cancer Ther. 1 (8), 639−649. (28) Goh, M., Chen, F., Paulsen, M. T., Yeager, A. M., Dyer, E. S., and Ljungman, M. (2001) Phenylbutyrate attenuates the expression of bcl-X(L), DNA-PK, caveolin-1, and VEGF in prostate cancer cells. Neoplasia. 3 (4), 331−338.

apoptosis protein-1; cIAP-2, inhibitors of apoptosis protein-2; c-MET, MET proto-oncogene, receptor tyrosine kinase; c-myc, MYC proto-oncogene, BHLH transcription factor; DAPK1/2, death associated protein kinase 1/2; DNA, deoxyribonucleic acid; DNA-PK, DNA-dependent protein kinase; E2F, E2 transcription factors; EBV, Epstein−Barr virus; ERK, extracellular signal-regulated kinase; FA/BRCA1, Fanconi anemia/ breast cancer 1, early onset; FAK, focal adhesion kinase; FANCD2, Fanconi anemia complementation group D2; FDA, Food and Drug Administration; GADD153, growth arrest and DNA damage-inducible protein; GRP78, heat shock protein family A (Hsp70) member 5; H2AX, H2A histone family member X; HAT, histone acetyltransferase; HDAC, histone deacetylase; HDACIs, histone deacetylase inhibitors; HER2, human epidermal growth factor receptor 2; HIF-1α, hypoxia inducible factor 1 alpha; HLA, human leukocyte antigen; HLADR, human leukocyte antigen−antigen D related; HSP27, heat shock protein 27; hTERT, telomerase reverse transcriptase; IGFBP-3, insulin-like growth factor binding protein 3; IL-2, interlukin-2; JNK, c-Jun N-terminal kinase; Ku70, X-ray repair cross complementing 6; Ku86, X-ray repair cross complementing 5; Mad, MAX dimerization protein 1; MAPK, mitogenactivated protein kinase; NaPB, sodium phenylbutyrate; NDRG1, N-Myc downstream regulated 1; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; NOXA, immediate-early-response protein APR; NSCLC, non-small cell lung cancer; p21WAF1/Cip1, cyclin-dependent kinase inhibitor 1; p27Kip1, cyclin-dependent kinase inhibitor 1B; p45SKP2, F-box protein responsible for p27KIP1; PARP, poly(ADP-ribose) polymerase; PDC, pyruvate dehydrogenase complex; PDGF-B, platelet derived growth factor subunit B; PDK, pyruvate dehydrogenase kinase; PDTC, ammonium pyrrolidinedithiocarbamate; PI3K, phosphoinositide 3-kinase; Rb, retinoblastoma protein; ROS, reactive oxygen species; Smac, second mitochondria-derived activator of caspases; SMRT, nuclear receptor corepressor 2; Sp1, specificity protein 1; Sp3, specificity protein 3; TGF-β2, transforming growth factor beta 2; VEGF, vascular endothelial growth factor; WHO, World Health Organization; αPUMA, P53 up-regulated modulator of apoptosis

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