Advances in Molecular Signaling Mechanisms of β-Phenethyl

Mar 23, 2015 - β-Phenethyl isothiocyanate (PEITC) is an important phytochemical from cruciferous vegetables and is being evaluated for chemotherapeut...
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Advances in Molecular Signaling Mechanisms of β‑Phenethyl Isothiocyanate Antitumor Effects Chong-Zhen Qin,†,‡ Xue Zhang,§ Lan-Xiang Wu,§ Chun-Jie Wen,§ Lei Hu,†,‡ Qiao-Li Lv,†,‡ Dong-Ya Shen,†,‡ and Hong-Hao Zhou*,†,‡ †

Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha 410008, P. R. China Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha 410078, P. R. China § Institute of Life Sciences, Chongqing Medical University, Chongqing, Chongqing 400016, China ‡

ABSTRACT: β-Phenethyl isothiocyanate (PEITC) is an important phytochemical from cruciferous vegetables and is being evaluated for chemotherapeutic activity in early phase clinical trials. Moreover, studies in cell culture and in animals found that the anticarcinogenic activities of PEITC involved all the major stages of tumor growth: initiation, promotion, and progression. A number of mechanisms have been proposed for the chemopreventive activities of this compound. Here, we focus on the major molecular signaling pathways for the anticancer activities of PEITC. These include (1) activation of apoptosis pathways; (2) induction of cell cycle arrest; and (3) inhibition of the survival pathways. Furthermore, we also discussed the regulation of drugmetabolizing enzymes, including cytochrome P450s, metabolizing enzymes, and multidrug resistance. KEYWORDS: β-phenethyl isothiocyanate, angiogenesis, apoptosis, cycle arrest, survival pathways

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INTRODUCTION

Quick absorption and high oral bioavailability are particularly interesting features of the PEITC pharmacokinetics in both humans and animals.19−21 One possible explanation for these may be attributed to the PEITC compound having a low molecular weight (MW = 163.2 g/mol) and being fairly lipophilic (logP = 3.47).1 Indeed, a pharmacokinetics study including 50 subjects failed to detect PEITC both in volunteers who had ingested 40 g servings of watercress (releasing 6−12 mg of PEITC) with serial blood samples taken up to 4 h postinjection and in subjects who participated in the phase I PEITC single-dose study at the 40 mg dose level.20 Similarly, results from a rat animal model showed that the maximal plasma concentrations occurred rapidly at 0.4 ± 0.1 and 2.0 ± 1.0 h following doses of 10 μmol/kg and 100 μmol/kg with a ka of 1.8 h−1 after oral administration of PEITC.21 In addition to high oral bioavailability, PEITC showed another feature of low clearance. Interestingly, the metabolic disposition of PEITC is similar in rats and humans.22,23 Following absorption, PEITC is rapidly metabolized mainly through the mercapturic acid pathway.24 PEITC reacts with glutathione (c-glutamatecysteine-glycine; GSH; Table 2), which results in the formation of a PEITC−GSH conjugate catalyzed by glutathione Stransferase in the cytosol of cells. PEITC−GSH is successively converted to S-(N-phenylethyl thiocarbamoyl) cysteinylglycine by γ-glutamyl transferase, and to PETC-Cys by dipeptidase. Finally, the PEITC−cysteine conjugates are converted to Nacetylcysteine (NAC) conjugates by N-acetyltransferase and excreted in the urine (Figure 1).

Cancer, the leading cause of death, remains a serious health problem in all parts of the world. Epidemiologic studies suggest that consumption of fruit and vegetable can reduce the incidence of cancer.1−3 Cruciferous vegetables belong to the Brassica genus and were named for their cross-shaped flower petals, including cabbage, watercress, cauliflower, and broccoli.4,5 Recently, a meta-analysis combined results from a total of 4306 cases in 375,562 controls in 11 independent studies, identifying that cruciferous vegetable intake was associated with a reduced risk of ovarian cancer.3 Moreover, a systematic review showed a statistically significant inverse association between cruciferous vegetable intake and colon cancer.4 In addition, a case-control study including 2141 cases and 2168 controls of lung cancer patients found that weekly consumption of cruciferous vegetables protected against lung cancer.6 In animal models, studies showed that the plants could inhibit carcinogenesis in many cancerous tissues, including lung, liver,7 prostate,8,9 and colorectal cancer.10 Isothiocyanates (ITCs) are present in cruciferous plants and produced by myrosinase when cells are injured, such as from cutting and chewing.11 β-Phenethyl isothiocyanate (PEITC; Table 1) is one of the most extensively studied ITCs focusing on their inhibitory functions in different cancer cells.12 And, the anticancer mechanisms by PEITC have been shown to involve the deletion of carcinogenesis cells through induction of apoptosis pathways,13−15 cell cycle arrest,16−18 and inhibition of the survival pathways.17 In this review, we will discuss the diverse anticancer mechanisms focusing on molecular signaling pathways and the cellular targets regulated by PEITC. Moreover, we integrate these pathways as potential examples to provide how PEITC could prevent carcinogenesis. © 2015 American Chemical Society

Received: Revised: Accepted: Published: 3311

October 18, 2014 March 23, 2015 March 23, 2015 March 23, 2015 DOI: 10.1021/jf504627e J. Agric. Food Chem. 2015, 63, 3311−3322

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Journal of Agricultural and Food Chemistry Table 1. Abbreviations and Structural Formulas of Some Uncommon Chemicals

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ACTIVATION OF APOPTOSIS PATHWAYS

THE INTRINSIC (MITOCHONDRIAL) PATHWAY

Mitochondria have been shown to have a vital role in apoptotic signal transduction.28,29 Emerging evidence suggests that PEITC can cause oxidative mitochondrial damage by enhancing the intracellular ROS to a toxic level.30−32 Possible explanations for these findings are that (1) PEITC conjugates with GSH, and the conjugate compounds export from cancer cells;33 (2) time-dependent ROS accumulation induced by PEITC could induce ROS-mediated lipid peroxidation of mitochondrial membrane, such as oxidation of cardiolipin,30 followed by the loss of membrane integrity (ΔΨm), and release apoptosisinducing factor (AIF) as well as apoptogenic cytochrome c; (3) cytokines influx into the cytosol, and then activate caspase family members that are executionary arm of apoptotic

Apoptosis, a form of programmed cell death, is a pivotal defense against the occurrence of cancer and is essential for metazoans in maintaining tissue homeostasis. In principle, there are two alternative pathways that initiate apoptosis: one is mediated by mitochondria, referred to as the “intrinsic pathway”;25 the other is mediated by death receptors on the cell surface, referred to as the “extrinsic pathway”.26 In addition, a third apoptotic pathway, the “endoplasmic reticulum (ER) stress” pathway, has been described recently.27 All of these mechanisms of apoptosis are associated with caspase activation damage sensing and proapoptotic signaling. 3312

DOI: 10.1021/jf504627e J. Agric. Food Chem. 2015, 63, 3311−3322

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activating the caspase-dependent cell death.41,42 Moreover, in ovarian cancer OVCAR-3 cells, PEITC decreased the level of prosurvival Bcl-2 protein while simultaneously increasing the level of proapoptotic Bax protein, which in turn caused activation of caspase-9 and -3 leading to cell apoptosis.43

Table 2. Other Abbreviations Used abbreviation GSH NAC ΔΨm AIF PTP Bcl-2 TRAIL DR4 DR5 Fas L FADD NF-κB PI3K TLR3 JNK MKK7 MAPK CDK ATM/ATR DMEs GSTs QR γGCS HO-1 NQO1 MDR DNM VBL ABCG2 MRP-1 Pgp-1 NPEITC

name c-glutamate-cysteine-glycine N-acetylcysteine membrane integrity apoptosis-inducing factor permeability transition pore B-cell leukemia/lymphoma-2 tumor necrosis factor-related apoptosis-inducing ligand death receptor 4/TNF-related apoptosis-inducing ligand death receptor 5/TNF-related apoptosis-inducing ligand Fas ligand Fas-associated death domain nuclear factor kappa B phosphatidylinositol 3 kinase toll-like receptor 3 Jun N-terminal kinase mitogen-activated protein kinase 7 p38 mitogen-activated protein kinase cyclin-dependent kinase ataxia telangiectasia mutated/ataxia telangiectasia and Rad3related drug metabolizing enzymes S-transferases quinonereductase γ-glutamyl cysteine-synthetase heme oxygenase1 NAD(P)H quinone oxidoreductase1 multidrug resistance daunomycin vinblastine breast cancer resistance protein multidrug resistance associated protein-1 P-glycoprotein-1 novel alkyne-tagged ITC

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THE EXTRINSIC (RECEPTOR-MEDIATED) PATHWAY The death receptor pathways for caspase activation are referred to as the extrinsic apoptosis pathways.35 Tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) has the unique activity of activating cell death exclusively in tumor cells. TRAIL can bind with two death receptors (DRs), DR4 (TRAIL-R1) and DR5 (TRAIL-R2), both of which are type I transmembrane proteins.44,45 Studies have shown that PEITC can enhance TRAIL-induced apoptosis through downregulation of cell survival proteins and upregulation of the death receptors (DR4 and DR5). For example, in human oral cancer HN22 cells, PEITC activates p38 MAPK through increasing phospho-p38 MAPK. As p38 MAPK are the upstream targets of DR5, the function of DR5 regulating apoptosis induced by PEITC is strongly associated with the p38 MAPK pathway.46 However, another study found that PEITC increased the expression of DR4 and DR5 by downregulating the phosphorylation of ERK 1/2, neither phospho-JNK nor phospho-p38 MAPK. One possible explanation for these contrary findings is that the different kind of cell types might have different pathway responses when cells are exposed to PEITC.47 Previous studies have demonstrated that PEITC can enhance the intracellular ROS and induce apoptosis. And, a recent study demonstrated that PEITC enhanced TRAILinduced apoptosis through the upregulation of DR5 receptors on the ROS-induced-p53.48 Fas is another cell surface death receptor involved in the transduction of apoptotic signals in both normal and cancer cells. Fas ligand (Fas L) binding followed by Fas-receptor (Fas) oligomerization leads to formation of a death-inducing signal complex and recruitment of the adapter molecule Fas-associated death domain (FADD). FADD recruits and aggregates the pro form of caspase-8, leading to the activation of caspase-8, and then cleaved procaspase-3 and procaspase-9.49 PEITC can sensitize the human bladder carcinoma cells leading to Fas-mediated apoptosis. The intracellular GSH out of the cells provides a potential mechanism for PEITC-induced apoptosis.15 But the concise pathogenesis is still not clear.

machinery.34,35 The permeability transition pore (PTP) is an unselective voltage-dependent mitochondrial channel and is central to mitochondrial vital functions. PTP opening can dissipate the transmembrane inner potential, trigger matrix swelling, release cytochrome c, and subsequently elicit cell apoptosis.36 Studies from our own laboratory have shown that cell apoptosis of MCF-7 induced by PEITC is associated with ROS-mediated PTP opening. Ca2+ can also open PTP, leading to the rapid loss of transmembrane inner potential.37 Further studies are needed to define whether calcium overload contributes to PEITC-induced PTP activity. The Bcl-2 (B-cell leukemia/lymphoma-2) family proteins include both antiapoptotic members (such as Bcl-2 and Bcl-xL) and proapoptotic members (such as Bax and Bid). They are distributed not only in the mitochondrial outer membrane but also in the cell membrane, ER, and nuclear membrane. Studies have found that Bcl-2 proteins reside upstream of irreversible cellular damage and focus the greatest efforts at the level of mitochondria.38 PEITC can regulate intracellular location of proapoptotic proteins or antiapoptotic proteins promoting the release of cytochrome c from the mitochondria.39 For example, PEITC induced apoptosis via downregulation of Bcl-2/XIAP and triggering the mitochondrial pathway in MCF-7 cells.40 Similar results have shown that PEITC triggered apoptosis through promotion of Bax and Bid expression, leading to the decreased levels of ΔΨm and release of cytochrome c, and then

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THE “ENDOPLASMIC RETICULUM (ER) STRESS” PATHWAY Recently, the “ER stress” pathway has been recognized as the third apoptotic pathway. 50 ER stress, induced by the accumulation of unfolded or malfolded proteins, activates various apoptotic pathways.27 Very recent studies demonstrated that PEITC induced cell apoptosis in a concentration-dependent, ROS/Ca2+-independent manner.51,52 Similar results showed that PEITC promoted Ca2+ movement by inducing phospholipase C-dependent Ca2+ release from endoplasmic reticulum and Ca2+ entry via storeoperated Ca2+ channels in PEITC-treated human prostate cancer cells (PC3).14,51 Moreover, the rapid increase of cytosolic Ca2+ may cause elevation of mitochondrial Ca2+ and decrease of Ca2+ in endoplasmic reticulum, and such imbalance of Ca2+ may trigger a variety of cascades leading to cell death.53 3313

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Figure 1. Metabolism of phenylethyl isothiocyanate by the mercapturic acid pathway. (I) PEITC (①) initially diffuses into cells and (II) and is rapidly conjugated to glutathione (GSH) via the action of glutathione S-transferases (GST). (III) The GSH conjugates (②) are exported from the cells via efflux pumps, and then (IV) the conjugates are hydrolyzed to the free PEITC, which is able to re-enter the cell. (V) But the intracellular PEITC−GSH is subsequently converted to the cysteinylglycine conjugate (③) by the action of γ-glutamyl transpeptidase and then (VI) converted to the cysteine conjugate (④) by the action of γ-glutamyl transpeptidase. (VII) Finally, the PEITC−cysteine conjugates are converted to NAC conjugates by the action of N-acetyltransferase and (VIII) excreted in the urine.

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In addition to Ca2+ accumulation, inhibition of proteasomal activity and disruption of protein disulfide bond formation may trigger ER stress response. Various studies showed that PEITC significantly inhibited proteasome activities, including chymotrypsin-like, trypsin-like, and caspase-like, by inhibition of rapid accumulation of p53 and IκB nuclear factor-kappa B.54,55 For example, PEITC can selectively induce degradation of α- or βtubulin proteasome in different cancers, including non-small lung cancer A549 cells,56 prostate cancer cells,57 and breast cancer cells.58 Interestingly, PEITC inhibits proteasome activity presumably through direct binding, suggesting that the proteasome, like tubulin, may serve as a molecular target of PEITC. Taken together, the predominant apoptotic pathway targeted by PEITC may differ between cell types. The exact mechanism is difficult to determine because of the cross-talk between the different apoptotic pathways (Figure 2).

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THE NF-κB SURVIVAL PATHWAY The NF-κB survival pathway plays a key role in cancer chemoprevention due to its involvement in tumor cell growth, proliferation, invasion, and survival.59−61 NF-κB is sequestered in the cytoplasm in resting cells by combining with inhibitor proteins (IκB proteins). In response to stimulating factors, IκB was phosphorylated by the inhibitor of κB (IKK) kinase complex and further degraded, thus releasing active NF-κB dimers and allowing NF-κB to translocate to nucleus.62 PEITC has been shown to have promising anti-inflammatory properties through the ubiquitous NF-κB signaling pathway.16,63,64 For example, Xu et al. demonstrated the role of PEITC on NF-κB transcriptional activation and NF-κB-regulated gene expression in vitro.59 A similar result showed that PEITC induced cell death by inhibition of NF-kB and EGFR activities in prostate cancer cells.65 In these studies, PEITC exhibited its antiinflammatory activity via the direct suppression of IκB kinase α/β (IKKα/β) phosphorylation, IκBα phosphorylation, and subsequent p65 NF-κB nuclear translocation.66 Studies have demonstrated that Toll-like receptor 3 (TLR3) is one of the primary sensors of double stranded (ds) RNA and mediates NF-κB activation.18 More recently, another study showed that PEITC could activate prosurvival NF-κB pathway by inhibiting TLR3 mediated IRF3 signaling.67 However, the biochemical details of PEITC on IRF3 signaling are needed in further step. Interestingly, PEITC and curcumin can exert their additive inhibitory effects on cell proliferation and ultimately lead to

INHIBITION OF THE SURVIVAL PATHWAYS

The following section will summarize studies that investigated the effect of PEITC on survival pathways (Figure 3). Nuclear factor kappa B (NF-κB), phosphatidylinositol 3 kinase (PI3K), and p38 mitogen-activated protein kinase (MPKC) pathways are important for cell survival, proliferation, and differentiation. 3314

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Figure 2. Signaling and molecular pathway of apoptosis in carcinoma cells. (1) In the mitochondrial pathway, PEITC-induced cell apoptosis interacts with the Bcl-2 family of proteins and then releases cytochrome c, AIF, and endo G, thereby forming the apoptosome which activates caspase-9 and is capable of proteolytically processing caspase-3 to induce apoptosis. (2) In the death receptor pathway, PEITC targets upregulation of death receptors which can interact with their ligands and recruit adaptor protein and initiator procaspases-8. Then, the active initiator caspase-8 activates the effector caspase-3 to induce apoptosis or to initiate degradation of Bid and to yield cytochrome c releasing from mitochondria. (3) In the ER pathway, PEITC induces cytoplasmic Ca2+ concentration, as a result of ER stress, resulting in the activation of caspase-12. Also, PEITC can induce degradation of proteasome, which may disrupt protein disulfide bond formation and trigger ER stress response.

expression in human glioma cells, at least partly through the PI3K/AKT pathway.73 Similar results showed that PEITC could target the Akt/JNK/Mcl-1 pathway. An in vivo study showed that the activity of the Akt is inhibited by PEITC, leading to Jun N-terminal kinase (JNK) activation, and culminating in Mcl-1 downregulation.17 Moreover, PEITC can suppress the activating of Akt/mTOR, a downstream of Akt pathway.74 Data from treatment of LNCaP and PC-3 cells with PEITC showed that levels of S2448-phosphorylated mTOR and phosphorylation of mTOR downstream target p70s6k (T389) decreased in both cell lines.75

programmed cell death of tumor cells by significant inhibition of NF-kB cell survival signal pathway and EGFR signaling pathway.65 By contrast, others showed that MAPK/ERK signaling pathway, not NF-κB signaling pathway, was associated with apoptosis induced by PEITC plus cisplatin in HeLa cells.68

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THE AKT SURVIVAL PATHWAY Akt (also known as protein kinase B or PKB) comprises three closely related isoforms, Akt1, Akt2, and Akt3 (or PKBα/β/γ respectively).69 It is a serine-threonine kinase intimately involved in the regulation of cell survival and is activated by recruitment to the cell membrane through PI3K. Several research teams have demonstrated that PEITC can inhibit the PI3K/Akt signaling pathway and the downstream in recent years.17,70 For example, PEITC inhibited ovarian tumor growth in vivo by suppressing the EGFR-AKT pathway.71 Also, Xiao et al. demonstrated that PEITC treatment caused a decrease in survival of human umbilical vein endothelial cells by suppression of VEGF, downregulation of VEGF receptor 2 protein levels, and inactivation of prosurvival serine-threonine kinase Akt.72 Very recent studies demonstrated that PEITC inhibited hypoxia-induced accumulation of HIF-1α and VEGF

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THE MAPK SURVIVAL PATHWAY Various studies have shown that PEITC can inhibit invasion and migration in cancer cells by MAPK (JNK, ERK1/2, and p38) pathways.74,76 Indeed, in ovarian cancer cells, PEITC can inhibit Akt, ERK1/2 survival signaling, and c-Myc while simultaneously activating proapoptotic p38 and JNK1/2.43 A similar result showed that PEITC exerted an inhibitory effect on the ERK1/2, mitogen-activated protein kinase 7 (MKK7), and MAP kinase3 (MEKK3) in human gastric cancer cells. Thus, suppressing MAPK pathways might be one of the 3315

DOI: 10.1021/jf504627e J. Agric. Food Chem. 2015, 63, 3311−3322

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Figure 3. Regulation of survival signaling pathways by phenethyl isothiocyanate. Inactivation of survival pathways, including NF-κB, AKT, and MAPK pathways by PEITC, is considered as one important chemopreventive mechanism. (1) NF-κB is normally sequestrated by IκB which is subjected to phosphorylation by IκK. Phosphorylation of IκB by IκK increases degradation of IκB, and then the NF-κB translocates into the nucleus and activates the transcription of target genes involved in cell survival. PEITC has been shown to inhibit activation of NF-κB through inhibiting IκK. (2) PEITC can suppress the activating of AKT/PKB pathway and AKT/mTOR, a downstream of Akt pathway, by suppression of activating phosphorylations of Akt and mTOR. (3) MAPK signaling pathways such as ERK, JNK, and PKC can regulate the transcriptional activities of transcription factors that are induction of transcription of target genes. PEITC can downregulate the protein levels of MAPK pathways, such as ERK1/2, protein kinase C (PKC), and JNK1/2, and inhibit transcription survival genes.

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INDUCE OF CELL CYCLE ARREST Cell cycle progression is a sequential process that directs dividing carcinoma cells through the G1, S, G2, and M phases.82 The ability of PEITC to regulate the cell cycle and inhibit proliferation contributes to its chemopreventive capacity and is mediated through indirect targeting of cyclins, proteins responsible for the progression of the cell cycle, cyclindependent kinase (CDK) molecules, and their inhibitors (Figure 4).83

mechanisms of migration and invasion of cancer cells inhibited by PEITC.16 Previous research has demonstrated that some agents inducing PKC activation can also stimulate MAPK, suggesting that there is a crosstalk between PKC and MAPK signaling pathway.77,78 More recently, it has been shown that the functions of PEITC in migration and invasion of human gastric cancer AGS cells might be via inhibiting PKC/MAPK pathway by molecular targeting of PKC.77 Similarly, in androgenindependent PC-3 cells, PEITC exhibited remarkable efficacy in sensitizing the cells to undergo cell death partly by modulating the activities of PKC.79 Interestingly, some studies had demonstrated that upregulation of Nrf2/ARE-dependent gene expression by PEITC is likely mediated by the MAPK pathway.78 Similar results have shown that PEITC inhibits the HIF-1α expression through inhibiting the MAPK signaling pathway.73 As HIF-1 and Keap1/Nrf2 are involved in the expression of more than a hundred genes,80,81 it is urgent to know whether cellular HIF-1α, Nrf2, and/or Keap1 proteins are direct targets of PEITCs. In conclusion, PEITC have the potential function to prevent invasion and metastasis in a wide range of tumor types by inhibition of the MAPK signaling pathway.

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INDUCE OF G1/S PHASE CELL CYCLE ARREST Recent studies have demonstrated the effect of PEITC on suppressing cell proliferation of cancer cells by causing G1/S phase cell cycle arrest in many cancers, such as human colorectal cancer,84 oral squamous carcinoma,42 and prostate cancer.85 One possible explanation for these findings was that PEITC could lead to G0/G1 phase arrest by substantial reduction levels of cell cycle related proteins, such as CDC25A, CDK6, phospho-Rb, cyclin D, CDK2, and cyclin E proteins, but increase the expression of cyclin-dependent kinase inhibitors p15, p53, p27, and p21.42 For example, in PC-3 cells, PEITC effectively blocked the G1-phase progression by 3316

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Figure 4. The proposed model of PEITC mechanisms of action for cell cycle arrest. The cell cycle is broadly divided into four phases (G1, S, G2, and M) culminating in cell duplication. Each phase of the cell cycle is regulated by a different complement of CDK−cyclin complexes. PEITC can lead to cell cycle arrest by direct reductions of the levels of cell cycle related proteins and indirect activation of the ATM/ATR pathway.

the combined activity of cyclin D1/CDK4, -6, and cyclin E/ CDK2. In addition, decreasing phosphorylated Rb proteins activated the cell cycle progression.85 However, PEITC arrested the cells in the G1 phase by downregulation of cyclins through the activation of the p38 MAPK signaling pathway.84 One possible explanation for these findings was that the p38MAPK stress kinase pathway was activated in response to DNA damage, and that the cell cycle checkpoint function was essential in cells with defective p53.86 Moreover, several studies have demonstrated that the BAG family molecular chaperone regulator 3 (BAG3) belongs to a family of cochaperones and has been shown to play a relevant role in the survival, growth, and invasiveness of different tumor types.87,88 Interestingly, a recent study found that PEITC promoted BAG3 silencing which sensitized EqS04b cells death and cell cycle arrest in G0/G1.89

Another study found that the antiproliferative effects of PEITC in Caco-2 cells were attributed to the activation of the G2/M DNA damage checkpoint and sustained G2/M phase cell cycle arrest through upregulation of p21.93 Several studies have demonstrated that the ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related (ATM/ATR) pathway plays important roles in maintaining genome integrity by triggering cell cycle arrest and DNA damage repair.94,95 It indicated that the ATM/ATR pathway might be a therapeutic target in cancer therapy. Indeed, in oral cancer cells, PEITC induced G2/M phase arrest via a GSH redox stress and oxidative DNA damage-induced ATM-Chk2-p53-related pathway.90 A particularly interesting feature of PEITC was that PEITCNAC, one of the metabolic products, had equal effectiveness with PEITC as an inhibitor of tumorgenesis by NNK while being less irritating and pungent than PEITC.96−98 Therefore, PEITC-NAC, a prodrug of PEITC, has been introduced in a number of preclinical studies.99 A recent study observed that PEITC-NAC had an effect on cell-cycle progression by downregulation of Cdk1 and cyclin B1 protein expression.100 However, more detailed mechanism is needed to identify the chemopreventive activity and metabolic products of PEITC in humans.

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INDUCTION OF G2/M PHASE CELL CYCLE ARREST Furthermore, in a myeloma xenograft mouse model, PEITC caused G2/M cell cycle arrest by decreasing expression of key G2/M-regulating proteins including cyclin B1, p-cdc2, Cdc25C, p53, p27, and 14-3-3ε.90 In vitro cell culture studies showed that PEITC induced time- and dose-dependent G2/M arrest by decline of cell cycle-related proteins, Cdk1 and Cdc25C.91 Similarly, PEITC induced cell cycle arrest at the G2/M phase by inhibiting the levels of cell cycle regulatory proteins such as cyclin A and B1, while promoting the level of Chk1 and p53.92

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INFLUENCE OF DMES Drug metabolizing enzymes (DMEs) play central roles in xenobiotics (such as drugs) metabolism, elimination, and 3317

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(VBL).122,123 Moreover, breast cancer resistance protein (ABCG2) is an ATP-binding cassette transporter that plays an important role in drug absorption and disposition in the development of multidrug resistance in cancer cells. Studies have shown that PEITC is a substrate for BCRP and is a specific inhibitor of BCRP.124 On the other hand, the cellular accumulation or the drugresistance effect of PEITC is regulated by multidrug resistance associated protein-1 (MRP-1) and P-glycoprotein-1 (Pgp-1). When the overexpression of MRP1 and P-gp cells is exposed to PEITC, the agent was rapidly exported, depending on accumulation in cells mainly in forms of GSH and cysteinylglycine conjugates.105 In a word, PEITC can inhibit MDR proteins, but on the other hand, PEITC is regulated by these proteins. Overall, this review summarized the work performed over the past 20+ years on PEITC absorption, metabolism, and cancer chemoprevention. Taken together, we have discussed the molecular signaling mechanisms of PEITC actions on some well-established mechanisms such as the following pathways: induction of apoptosis by intrinsic, extrinsic, and ER stress pathways; inhibition of the survival pathways, and cell cycle arrest; and reduction of the incidence of cancer by modulation of drug-metabolizing enzymes, including phase I and phase II. Recently, a novel alkyne-tagged ITC (NPEITC) was synthesized for the development of a “click” chemistry-based method to identify the protein targets for PEITC, which may pave the road for understanding the biological functions of PEITC as chemopreventive agents.125 In addition to the well characterized effects on apoptosis, cell proliferation, and cell cycle, PEITCs can target angiogenesis, cell attachment,126 and autophagic cell death.9 Further investigation is required to answer whether normal dietary intake or pharmacological administration of PEITC is sufficient to modulate these pathways in vivo.

detoxification in the human body. In general, DMEs protect or defend the body against the potential harmful insults from the environment.101 PEITC has been attributed to its ability to disrupt multiple steps of the carcinogenic process: preventing genetic damage by the inhibition of carcinogen-activating phase I enzymes102,103 and induction of carcinogen-detoxifying phase II enzymes.10,15,104 Moreover, it could accumulate membrane drug transporters, such as MRP-1 and Pgp-1.105

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INHIBITION OF PHASE I CYP DMES PEITC could influence several CYP families by binding with their amino acid residues. Specifically, PEITC can activate or inhibit the expression of CYPs including CYP1A1, CYP1A2,106 CYP2B1, CYP2E1,107 CYP1A2, CYP2C9, CYP2D6, CYP2C19, CYP2B6, CYP2E1, CYP2A6, CYP3A4, and CYP3A5.102,108 There can be many reasons behind the difference in their impacts. The main mechanism might be that PEITC could influence several CYP families by binding with their amino acid residues, and they had difference in binding affinity with the active group of PEITC. PEITC may react with α-amino groups in N-terminal residues and ξ-amino containing lysines through alkylation, thus forming thiourea.109 Also, several recent studies have shown that PEITC reacts with the N-terminal of proline, and binds to hydroxyl group-containing residues of tyrosine.110

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INDUCTION OF PHASE II DMES The phase II metabolizing or conjugating enzymes consist of many superfamily of enzymes such as glutathione S-transferases (GSTs) and quinonereductase (QR).101,111 PEITC was a substrate of GSTs, including Al-1, M1-1, M4-4, and P1-1. GSTs promote the thiol group of GSH to the electrophonic central carbon of the PEITC group to form dithiocarbamates (PEITC−GSH).112 In animal models, treatment with PEITC resulted in an increase of NQO-1, GST, and UGT activities in the rat liver.113 An earlier animal experiment had a similar result that PEITC could largely enhance UGT activities in liver.114 Nrf2, the key mediator of drug-induced changes in phase II metabolizing, can also be activated by ITCs.115 When cells are exposed to oxidative stresses, chemicals, and UVR, Nrf2 dissociates from Keap1 and translocates into the nucleus, thereby activating ARE. PEITC can induce Nrf2-ARE-mediated phase II enzyme and its downstream gene expression, including γ-glutamyl cysteine-synthetase (γGCS), heme oxygenase1 (HO-1), and NAD(P)H quinone oxidoreductase1 (NQO1).80,104 One of the mechanisms might be that PEITC induces the expression of ARE-mediated phase II enzymes via the c-Jun N-terminal kinase-1 (JNK1) and Nrf2-dependent pathways.116

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AUTHOR INFORMATION

Corresponding Author

*Department of Clinical Pharmacology, Xiangya Hospital; Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha, Hunan 410078, P. R. China. Tel: +86 731 84805380. Fax: +86 731 82354476. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank Weijing Gong and Mouze Liu for their helpful advice during the development of this project.

REVERSE OF MULTIDRUG RESISTANCE Multidrug resistance (MDR) is largely responsible for ineffective chemotherapy. P-glycoprotein/MDR1 (ABCB1) and MRP2 (ABCC2) are the uptake and efflux transporters of drugs. P-gp and MRP1 both transport a number of natural product chemotherapeutic agents, existing substrate preferences. Increased expression of P-gp, encoded by MDR1 gene, might account for MDR.117,118 PEITC is effective in reducing and eliminating drug resistance including paclitaxel,119 fludarabine,31 metformin,120 and cisplatin68,121 in many types of tumor. The potential mechanism of PEITC reversing MDR might be inhibiting MRP1 by increasing the cellular accumulation of daunomycin (DNM) and vinblastine

(1) Brown, K. K.; Hampton, M. B. Biological targets of isothiocyanates. Biochim. Biophys. Acta 2011, 1810 (9), 888−94. (2) Suzuki, R.; Iwasaki, M.; Hara, A.; Inoue, M.; Sasazuki, S.; Sawada, N.; Yamaji, T.; Shimazu, T.; Tsugane, S. Japan Public Health Centerbased Prospective Study, G. Fruit and vegetable intake and breast cancer risk defined by estrogen and progesterone receptor status: the Japan Public Health Center-based Prospective Study. Cancer Causes Control 2013, 24 (12), 2117−28. (3) Han, B.; Li, X.; Yu, T. Cruciferous vegetables consumption and the risk of ovarian cancer: a meta-analysis of observational studies. Diagn. Pathol. 2014, 9 (1), 7.

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