Sulforaphane inhibition of TPA-mediated PDCD4 downregulation contributes to suppression of c-Jun and induction of p21-dependent Nrf2 expression

European Journal of Pharmacology 741 (2014) 247–253

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Sulforaphane inhibition of TPA-mediated PDCD4 downregulation contributes to suppression of c-Jun and induction of p21-dependent Nrf2 expression Jong-Ho Cho a, Young-Woo Kim a, Bu Young Choi b, Young-Sam Keum a,n a b

College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do, 410-773, Republic of Korea Department of Pharmaceutical Science and Engineering, Seowon University, Cheongju, Chungbuk, 361-742, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 10 March 2014 Received in revised form 18 August 2014 Accepted 18 August 2014 Available online 26 August 2014

Programmed cell death 4 (PDCD4) is a bona fide tumor suppressor protein and plays a critical role in controlling the rate of protein synthesis. Here, we show that TPA selectively activated the S6K1 and ERK1/2 kinases, contributing to PDCD4 proteolysis and Pdcd4 mRNA degradation in HepG2 cells, respectively. In addition, we observed that sulforaphane suppression of TPA-induced S6K1 and ERK1/2 activation played a critical role in attenuating PDCD4 poly-ubiquitination and Pdcd4 mRNA downregulation. Moreover, we observed that silencing Pdcd4 led to not only an increased expression of c-Jun, but also a decreased expression of p21, the latter of which contributed to suppression of Keap1dependent Nrf2 poly-ubiquitination. Finally, we demonstrate that the expression of PDCD4, p21 and Nrf2 is higher, but that of c-Jun is lower in normal human liver tissues, compared with hepatoma tissues. Collectively, our study illustrates that attenuating the rate of PDCD4 proteolysis and Pdcd4 mRNA degradation serves as a novel anti-inflammatory and cytoprotective mechanism of sulforaphane. & 2014 Elsevier B.V. All rights reserved.

Keywords: Sulforaphane PDCD4 S6K1 ERK1/2 Nrf2

1. Introduction Eukaryotic translational initiation factors (eIFs), including the eIF4F complex, participate in the translation initiation process. The eIF4F complex is a multiple-subunit complex that consists of the scaffold protein (eIF4G), the cap-binding protein (eIF4E), and the RNA helicase enzyme (eIF4A) (Sonenberg and Hinnebusch, 2009). The eIF4A catalyzes the unwinding of secondary structure in the 50 -untranslated region (50 –UTR) of mRNA, contributing to the cap-dependent protein translation (Linder et al., 1989). Using yeast two-hybrid assay, Yang et al. (2003a) have previously identified Programmed Cell Death 4 (PDCD4) as a novel protein that directly binds to and inhibits the helicase activity of eIF4A . Follow-up studies have demonstrated that PDCD4 suppresses neoplastic transformation, anchorage-independent growth, and invasion of mouse epidermal JB6 cells (Yang et al., 2003b). Transgenic mice that overexpress Pdcd4 gene in the epidermis exhibited a significant resistance against 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin papilloma formation (Jansen et al., 2005), whereas Pdcd4 knock-out mice showed a greater susceptibility of skin papilloma formation in the same experimental

n

Corresponding author. Tel.: þ 82 31 961 5215; fax: þ 82 31 961 5206. E-mail address: [email protected] (Y.-S. Keum).

http://dx.doi.org/10.1016/j.ejphar.2014.08.007 0014-2999/& 2014 Elsevier B.V. All rights reserved.

model (Schmid et al., 2008). Together, these facts suggest that PDCD4 acts as a tumor suppressor in vivo, at least in part, by controlling the rate of protein translation and cell growth. Epidemiologic studies suggest that dietary consumption of cruciferous vegetables, such as broccoli, Brussels sprouts, cauliflower, and cabbage lowers overall cancer risks, including that of colon and prostate cancer (Seow et al., 1998). Sulforaphane, an isothiocyanate isolated from broccoli, has first been identified as a chemopreventive agent by monitoring its ability to induce quinone reductase in murine hepatoma cells (Zhang et al., 1992). Sulforaphane exists as a precursor form of thioglucoside, e.g. glucoraphanin, in cruciferous vegetables. Following consumption, glucoraphanin is hydrolyzed into sulforaphane by the enzymatic action of plant-specific myrosinase or unidentified gut microflora (Keum et al., 2004). Chemopreventive mechanisms of sulforaphane include the induction of phase II cytoprotective enzymes, cell cycle arrest and apoptosis, inhibition of histone deacetylase activity, and the blockade of inappropriate amplification of intracellular signaling kinases (Zhang, 2004). Nonetheless, the mechanisms by which sulforaphane controls mitogenstimulated cell growth have not been fully understood. In the present study, we have identified that sulforaphane inhibits TPA-induced transcriptional and post-translational downregulation of PDCD4 in HepG2 cells by blocking extracellular signal-regulated kinase-1/2 (ERK1/2) and ribosomal S6 kinase-1 (S6K1) phosphorylation. In addition, we observed that the silencing of Pdcd4 increased the

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expression of c-Jun, but diminished that of NF-E2-related factor-2 (Nrf2), p21 and heme oxygenase (HO-1). Finally, we observed that the expression of PDCD4, p21 and Nrf2 is higher, but that of c-Jun is lower in normal human liver tissues, compared with hepatoma tissues. Taken together, we have identified that sulforaphane protection against PDCD4 downregulation constitutes a new mechanistic basis for its anti-inflammatory and cytoprotective effects.

2. Materials and methods 2.1. Cell culture, chemicals, plasmids, and antibodies Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Welgene Corporation (Daegu, Republic of Korea). All cancer cell lines, used in the study were purchased from Korean Cell Line Bank (Seoul, Republic of Korea). Primary mouse embryonic fibroblasts (MEFs) were provided by Dr. Hyuk-Wan Ko (College of Pharmacy, Dongguk University, Republic of Korea). HepG2 cells and MEFs were grown in DMEM, supplemented with 10% heatinactivated FBS, penicillin (100 U/ml) and streptomycin (100 U/ml). Cells were incubated at 37 1C in a humidified atmosphere of 95% air and 5% CO2. Sulforaphane, 12-O-tetradecanoylphorbol-13-acetate (TPA), puromycin, HA-agarose bead and the lentiviral PDCD4 short hairpin RNA (shRNA) constructs were purchased from Sigma (St. Louis, MO). Polybrene was purchased from Millipore (Billerica, MA). Lentiviral helper plasmids (pMD.2G and psPAX.2) and pRK5HA-Ubiquitin plasmid were acquired from Addgene (Cambridge, MA). Ubiquitin cDNA was amplified by PCR and subcloned to pcDNA3-FLAG vector. pGL6-TA-ARE-firefly luciferase vector was provided by Dr. Siwang Yu (Peking University, China). Renilla luciferase reporter plasmid (pGL4.74) was acquired from Promega (Madison, WI). Human Pdcd4 and Nrf2 cDNAs were purchased from Korea Human Gene Bank (KRIBB, Daejeon, Republic of Korea) and subcloned into pcDNA3 plasmid by PCR amplification. Polyclonal PDCD4 and HO-1 antibodies were purchased from Abcam (Cambridge, MA). Nrf2 rabbit polyclonal antibody was purchased from Santa Cruz Technology (Santa Cruz, CA). U0126, ubiquitin (P4D1) mouse monoclonal antibody and rabbit polyclonal antibodies against c-Jun, c-Fos, c-Abl, p21, Akt1, phospho-Akt1, S6K, DYKDDDDK (FLAG) tag were purchased from Cell Signaling Technology (Beverly, MA). Total actin antibody was acquired from Developmental Studies Hydridoma Bank (Iowa City, IA). 2.2. Western blotting and immunoprecipitation After treatments, cultured cells were collected and washed twice with ice-cold 1  phosphate-buffered saline (PBS). After centrifugation at 12,000 rpm (9500 g) for 5 min, cells were resuspended with 200 μl RIPA buffer [50 mM Tris–HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na3VO4, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF)] and kept on ice for 30 min. After centrifugation at 12,000 rpm for 10 min, protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Cell lysates (30 μg) were then resolved by SDS-PAGE and transferred to PVDF membranes (BioRad, Hercules, CA). The membranes were incubated in blocking buffer (5% skim milk in 1  PBS-0.1% Tween20, PBST) for 1 h and hybridized with the appropriate primary antibodies in 1  PBS containing 3% bovine serum albumin (BSA) or 3% skim milk overnight at 4 1C. After washing thrice with 1  PBST for 30 min, the membrane was hybridized with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature and washed thrice with 1  PBST solution for 30 min. The membrane was

visualized by using an enhanced chemiluminescence (ECL) detection system. Actin was used as the control for an equal loading of samples in Western blots. The poly-ubiquitination of endogenous PDCD4 was examined by immunoprecipitation, followed by Western blot analysis. In brief, HepG2 cells were lysed with 200 μl NP-40 buffer [50 mM Tris–HCl at pH 7.5, 150 mM NaCl, 0.5% NP-40, 50 mM NaF, protease inhibitors cocktail (Roche, Indianapolis, IN)] for 30 min on ice and cell lysates were immunoprecipitated with PDCD4 polyclonal antibody and protein A/G-plus agarose bead (Santa Cruz Technology) overnight at 4 1C. The protein A/G beads were washed with 1  PBS thrice and denatured in 1  sample buffer. In order to examine the poly-ubiquitination of Nrf2, HepG2 cells, transduced with mock or Pdcd4 shRNA constructs were transfected with pcDNA3-HA-Nrf2 and pc-DNA3-FLAG-ubiquitin plasmids. After 48 h, cells were lysed by 200 μl NP-40 buffer, followed by immunoprecipitation with HA-agarose bead and Western blot with FLAG antibody. 2.3. PathScan RTK signaling antibody array and immunofluorescence assay The PathScan RTK Array (Cell Signaling Technology, Beverly, MA) was conducted as recommended by the manufacturer. The resulting chemiluminescent array images were captured by the Chemidoc XRS system (BioRad, Hercules, CA). Human tissue microarrays were purchased from SuperBioChips (Seoul, Republic of Korea). Human tissue slides were deparaffinized, incubated with blocking serum (1% BSA) for 30 min and immunostained with primary PDCD, p21, Nrf2 or c-Jun antibodies overnight at 4 1C. The slides were washed with 1  PBS (1% BSA) thrice and probed with fluorescein isothiocyanate (FITC)-conjugated rabbit secondary antibody (Jackson Immunoresearch, West Grove, PA). The fluorescent images were obtained with a C2 confocal microscope (Nikon Korea, Seoul, Republic of Korea). 2.4. Generation of stable cells by lentiviral infection Generation of stable knock-down cells was conducted with lentiviral transduction. Briefly, 293T packaging cells were transfected with 3 μg putative Pdcd4 shRNA construct together with lentiviral helper vectors (3 μg pMD2.G and 3 μg psPAX.2), using JetPEI reagent (Polyplus-Transfection, New York, NY). After 72 h, viral supernatant was collected and filtered, using a 0.45 μm syringe filter. HepG2 cells were transduced with viral supernatant containing 10 μg/ml polybrene for 12 h at 37 1C. After viral infection, lentivirally-transduced cells were selected with 3 μg/ml puromycin for 48 h. 2.5. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis HepG2 cells were collected and total RNA was extracted by Hybrid-R RNA extraction kit (GeneAll, Seoul, Republic of Korea). 1 μg of total RNA was subject to cDNA synthesis, using PrimeScript RT-PCR kit (TaKaRa Korea, Seoul, Republic of Korea). The real-time RT-PCR analysis was conducted by using SYBR premix EX taq (Takara Korea, Seoul, Republic of Korea) as recommended by the provider in quadruplicate on CFX384 Real-time system (BioRad, Hercules, CA). The PCR protocol comprises incubations of cDNA for 5 min at 95 1C, followed by 40 cycles of each cycle consisting of 10 s at 95 1C, 10 s at 55 1C, and 20 s 72 1C with a final cycle of 10 s at 95 1C. The expression level of human PDCD4 gene was normalized with that of human GAPDH. The specific real-time PCR primer pairs are as follows: human PDCD4 primers [50 –AGAAAATGCTGGGACTGAGGA-30 (Forward) and 50 –AGTCCCGGGATGAGTTTTTCC-30 (Reverse)] and human GAPDH primers [50 –ATTCCATGGCACCGTCAAGG-30 (Forward) and 50 –GGACTCCACGACGTACTCAG-30 (Reverse)].

J.-H. Cho et al. / European Journal of Pharmacology 741 (2014) 247–253

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Fig. 1. TPA-induced S6K1 Activation Contributes to PDCD4 proteolysis and sulforaphane inhibits TPA-induced PDCD4 Poly-ubiquitination by blocking S6K1 activity in HepG2 cells. (A) Primary mouse embryonic fibroblasts (Left Panel) and a variety of cancer cell lines (Right Panel) were exposed to TPA (100 ng/ml) for 8 h and Western blot was conducted against PDCD4 and actin antibodies. (B) HepG2 cells were exposed to TPA (100 ng/ml) alone or in combination with sulforaphane (10 μM) at different times, and Western blot (Left Panel) and real-time RT-PCR (Right Panel) analyses were performed. (C) HepG2 cells were exposed to TPA (100 ng/ml) at different times and the PathScan receptor tyrosine kinase (RTK) analysis was conducted as recommended by the provider. (D) HepG2 cells were exposed to TPA (100 ng/ml) alone or in combination with sulforaphane (10 μM) at different times and Western blot was conducted against phospho-S6K1, S6K1, and actin antibodies. (E) The endogenous PDCD4 poly-ubiquitination level was assessed by the immunoprecipitation of PDCD4, followed by Western blot against ubiquitin antibody.

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2.6. Dual luciferase assays Mock- or shPDCD4-transduced HepG2 cells were seeded on 70% confluence in six-well plate and transfected with 3 μg pGL6-TA-AREfirefly luciferase plasmid and 3 μg Renilla luciferase reporter plasmid. After 48 h, cells were lysed with luciferase lysis buffer [0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT, 2 mM EDTA] and the dual luciferase activity was measured by GLOMAX

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Multi-system (Promega, Madison, WI). The data is depicted as a fold ratio of the firefly luciferase activity, compared with Renilla luciferase activity. Statistical analysis was conducted by Student t-test with n¼6.

3. Results 3.1. Sulforaphane inhibits TPA-induced PDCD4 proteolysis by blocking phosphorylation of S6K1 in HepG2 cells We first attempted to examine the changes in the expression of PDCD4 in response to treatment of TPA, a prototypic tumor promoter. As a result, we observed that an exposure of TPA significantly decreased the PDCD4 expression in mouse embryonic fibroblasts (MEFs) (Fig. 1A, Left Panel). We then examined the changes in PDCD4 level in different cancer cell lines (HepG2, MCF7, HeLa, DU145, PC-3, HT-29 and U2OS cells), stimulated with TPA. Incubation of TPA for 8 h and a subsequent Western blot analysis revealed that TPA selectively reduced PDCD4 expression in HepG2 and MCF7 cells, but not in other cancer cell lines (Fig. 1A, Right Panel). Next, we examined the effect of sulforaphane, a wellknown cancer chemopreventive agent, on PDCD4 expression in HepG2 cells stimulated with TPA. As a result, treatment with sulforaphane (10 μM) inhibited TPA-mediated downregulation of PDCD4 at both protein (Fig. 1B, Left Panel) and mRNA level (Fig. 1B, Right Panel). In order to explore the potential intracellular signaling mechanisms responsible for TPA-mediated PDCD4 downregulation, HepG2 cells were exposed to TPA for various times (0.5, 1 or 2 h) and the analysis of PathScan receptor tyrosine kinase (RTK) array was conducted. As a result, we observed that TPA selectively induced phosphorylation of S6 and ERK1/2 proteins in a timedependent manner (Fig. 1C). Western blot analysis also showed that treatment with TPA for 1–2 h caused an increased phosphorylation of S6K1, which was attenuated by incubation with sulforaphane (Fig. 1D). It is known that the activation of S6K1 induces PDCD4 phosphorylation at Ser67, thereby facilitating the proteasomal degradation of PDCD4 by the E3 ubiquitin ligase, ß-TrCP (Dorrello et al., 2006). We, therefore, speculated that the inhibitory effect of sulforaphane on TPA-induced depletion of PDCD4 might be mediated through the blockade of proteasomal degradation of PDCD4. In line with this idea, we observed that sulforaphane significantly attenuated TPA-induced poly-ubiquitination of endogenous PDCD4 protein in HepG2 cells (Fig. 1E). Together, these results imply that sulforaphane suppresses the proteasomal degradation of PDCD4 by inhibiting the activation of intracellular S6K1.

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Fig. 2. TPA-induced ERK1/2 Activation Contributes to Pdcd4 mRNA downregulation and sulforaphane inhibits TPA-induced Pdcd4 mRNA downregulation by blocking ERK1/2 activity in HepG2 cells. (A) HepG2 cells were exposed to TPA (100 ng/ml) alone or in combination with sulforaphane (10 μM) at different times and Western blot was conducted against PDCD4, phospho-ERK1/2, ERK1/2, and actin antibodies. (B) HepG2 cells were exposed to TPA (100 ng/ml) alone or in combination with U0126 (10 μM) at different times and Western blot analysis was conducted against PDCD4 and actin antibodies. (C) HepG2 cells were exposed to TPA (100 ng/ml) alone or in combination with U0126 (10 μM) at different times and a semiquantitative RT-PCR analysis with human PDCD4 and GAPDH primers were conducted.

3.2. Sulforaphane protects against TPA-induced downregulation of Pdcd4 mRNA by blocking phosphorylation of ERK1/2 in HepG2 cells Since TPA increased the phosphorylation of ERK1/2 in HepG2 cells (Fig. 1C), we attempted to test the hypothesis that TPA-induced ERK1/2 activation might be responsible for the Pdcd4 mRNA stability and that sulforaphane would be able to modulate TPA-mediated Pdcd4 mRNA downregulation by blocking the ERK1/2 activity. Concordant with the results of PathScan RTK array, treatment of HepG2 cells with TPA showed an increased phosphorylation of ERK1/ 2 starting from 45 min post-treatment, which was completely diminished by incubation with sulforaphane (Fig. 2A). This inhibitory effect of sulforaphane on TPA-induced ERK1/2 phosphorylation was

Fig. 3. PDCD4 is a negative regulator of c-Jun and a positive regulator of p21, contributing to suppression of Keap1-dependent Poly-ubiquitination of Nrf2 in HepG2 cells. (A) Endogenous Pdcd4 gene expression was silenced by lentiviral transduction and Western blot analysis was conducted against PDCD4, c-Jun, c-Fos, c-Abl, p21, Nrf2, HO-1 and actin antibodies. (B) HepG2 cells, stably infected by mock or shPdcd4 lentiviral vectors were transiently transfected with pGL6-TA-ARE-firefly luciferase and Renilla luciferase reporter plasmids. The resulting ARE-firefly luciferase activity was measured and normalized by Renilla luciferase activity. (C) HepG2 cells (Left Panel) or HepG2 cells, stably infected by mock or shPdcd4 lentiviral vectors (Right Panel) were exposed to sulforaphane (10 μM) at different times and Western blot analysis was conducted against Nrf2, HO-1, p21 and actin antibodies. (D) HepG2 cells were transfected with pc-DNA3-HA-Nrf2 and pcDNA3-FLAG-Ubiquitin (FLAG-Ub) plasmids. After 48 h, cell lysates were then immunoprecipitated by HA-agarose bead and Western blot analysis was conducted against FLAG antibody.

sh PD C D 4

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J.-H. Cho et al. / European Journal of Pharmacology 741 (2014) 247–253

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Fig. 4. The expression of PDCD4, p21, Nrf2 is higher, but that of c-Jun is lower in human normal hepatocytes, compared with human hepatoma. The immunofluorescence analysis was conducted in commercially available human normal liver and hepatoma tissues slides, using (A) PDCD4, (B) p21, (C) Nrf2 and (D) c-Jun antibodies. The immunofluorescence signal spots were manually counted under the confocal microscope. M and F denote male and female, respectively. Statistical analysis was conducted by Student t-test and symbols indicate a statistically significance with nPo 0.05 and nnP o 0.01.

also correlated with the time-dependent protective effect of the compound against TPA-mediated downregulation of PDCD4 (Fig. 2A). Moreover, treatment with U0126, a pharmacological inhibitor of ERK1/2, restored TPA-depleted PDCD4 at both protein (Fig. 2B) and mRNA (2C) level, suggesting the involvement of ERK1/2 in decreasing the Pdcd4 mRNA level upon TPA stimulation. These findings suggest that sulforaphane attenuation of TPA-induced Pdcd4 mRNA downregulation is achieved by modulating the ERK1/2 activity. 3.3. PDCD4 is a negative regulator of c-Jun and mediates p21-dependent Nrf2 induction by sulforaphane Next, we have generated HepG2 cells, in which Pdcd4 was selectively silenced by the lentiviral transduction and conducted Western blot against a number of marker proteins that are closely associated with cell cycle, proliferation and inflammation. We noticed that knocking-down PDCD4 increased the expression of c-Jun, but not that of other proto-oncogenes, such as c-Fos, c-Abl and p65 (Fig. 3A). On the other hand, we observed that knockingdown Pdcd4 decreased the p21, Nrf2 and HO-1 expression (Fig. 3A). Nrf2 is responsible for transcriptional activation of the antioxidant response element (ARE)-dependent phase II cytoprotective enzymes, including heme oxygenase-1 (HO-1) (Keum, 2011). Complying with this observation, we observed that the ARE-dependent luciferase activity was attenuated in HepG2 cells, when Pdcd4 gene was silenced (Fig. 3B). Zhang and colleagues have previously provided

evidence that p21 can increase the cellular Nrf2 level by directly interfering with Keap1 recognition of Nrf2 (Chen et al., 2009). These facts raise a possibility that PDCD4 might be necessary for p21dependent Nrf2 induction by sulforaphane. Supporting this notion, we observed that sulforaphane elicited a time-dependent induction of p21 and that it was well correlated with the induction of Nrf2 and HO-1 (Fig. 3C, Left Panel). In addition, sulforaphane induction of p21, Nrf2 and HO-1 was lost or significantly attenuated when Pdcd4 was silenced (Fig. 3C, Right Panel). Moreover, we observed that Nrf2 polyubiquitination was significantly increased when Pdcd4 gene was silenced (Fig. 3D). Collectively, these facts illustrate that PDCD4 might be necessary for sulforaphane induction of p21, which in turn contributes to interfering with Keap1-mediated Nrf2 proteolysis and a subsequent induction of HO-1. 3.4. The level of PDCD4, p21 and Nrf2 is higher, but that of c-Jun is lower in human normal liver, as compared with human hepatoma After the identification of PDCD4 as a negative regulator of c-Jun and a positive regulator of p21, Nrf2 and HO-1, we examined the levels of PDCD4, c-Jun, Nrf2 and p21 in human normal and hepatoma samples. To this end, commercial human tissue microarray slides containing four matched liver samples (normal vs. cancer) and six unmatched liver samples (normal vs. cancer) were purchased and used for conducting immunofluorescence assay with PDCD4, p21, Nrf2 and c-Jun polyclonal antibodies. It should be noted that we have

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conducted the statistical analysis with a combination of matched and unmatched samples in order to provide a sufficient statistical power. Our data indicate that the level of PDCD4 (Fig. 4A), p21 (Fig. 4B), and Nrf2 (Fig. 4C) was significantly higher in human normal hepatocytes, compared with human hepatoma. On the contrary, the level of c-Jun was significantly higher in human hepatoma, compared with normal human liver (Fig. 4D). Collectively, our data indicate that PDCD4 is a bona fide tumor suppressor protein in human liver, whose expression is positively correlated with p21 and Nrf2, but negatively correlated with c-Jun in human clinical hepatic samples. 4. Discussion In the present study, we sought to examine the molecular mechanisms underlying anti-tumorigenic effects of sulforaphane. Previous studies have demonstrated that sulforaphane exerts chemopreventive effects, at least in part, by blocking the activation of activator protein-1 (AP-1) (Keum et al., 2005), an eukaryotic transcription factor composed of proteins from the Jun and Fos family, and inducing phase II cytoprotective genes via activation of Nrf2 (Das et al., 2013). Here, we report that the tumor suppressor protein PDCD4 plays a critical role in sulforaphane modulation of c-Jun expression and Nrf2 activation in HepG2 cells (Fig. 3). Our study also revealed that sulforaphane protects TPA-induced downregulation of PDCD4 at both protein and mRNA level and that this restoration of PDCD4 is mechanistically linked to the Nrf2 induction by this compound (Figs. 1 and 2). It appears that sulforaphane protection of TPA-induced PDCD4 downregulation occurs in two-ways: (i) sulforaphane attenuates TPA-induced S6K1 activation, thereby suppressing PDCD4 proteolysis, and (ii) suppresses TPA-induced Pdcd4 mRNA downregulation by blocking ERK1/2 phosphorylation. While sulforaphane suppression of S6K1 activity contributes to attenuating phosphorylation-dependent proteolysis of PDCD4 by the E3 ubiquitin ligase, ß-TrCP, the detailed biochemical mechanisms how sulforaphane suppression of ERK1/2 activity is linked to transcriptional regulation of Pdcd4 is unclear at present. There is some evidence that transcriptional regulation of Pdcd4 might be associated with microRNA-21 (miR-21) and/or mitogen-activated protein kinase (MAPK) pathway, including ERK1/2 (Young et al., 2010). Thus, it would be worthwhile to investigate whether sulforaphane protection of TPA-induced Pdcd4 transcriptional downregulation occurs via the ERK1/2-mediated regulation of miR-21. We have observed that knocking-down of PDCD4 increased c-Jun expression in HepG2 cells. While it is unclear whether PDCD4 serves as a direct translation repressor of c-Jun mRNA, it is interesting to note that the direct target genes of PDCD4 are related to cell survival or apoptosis, including XIAP and Bcl-XL (Liwak et al., 2012), procaspase-3 (Eto et al., 2012) and p53 (Wedeken et al., 2011). However, we noticed that sulforaphane barely exerted cytotoxic effects on the viability of HepG2 cells (data not shown). Since sulforaphane prevented TPA-induced downregulation of PDCD4, it may be conferred that the compound inhibits c-Jun expression via restoration of PDCD4. An inverse correlation between PDCD4 and c-Jun expression in human normal liver and hepatoma tissues also support this notion (Fig. 4). Thus, it is possible to presume that the protective effect of sulforaphane against PDCD4 degradation may be implicated in the anti-proliferative and anti-inflammatory effects of the compound. We also found that knock-down of PDCD4 decreased the expression of p21, Nrf2, and HO-1 in HepG2 cells (Fig. 3A). Moreover, the induction of p21, Nrf2 and HO-1 by sulforaphane was abrogated in cells transduced with shPdcd4 plasmid (Fig. 3C, Right Panel). These findings suggest that PDCD4 is critical for the induction of p21, Nrf2 and HO-1 by sulforaphane. Although the detailed molecular mechanisms need to be fully elucidated, it seems that sulforaphane induction of Nrf2 and phase II cytoprotective enzymes,

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including HO-1 requires PDCD4-dependent p21 induction. In addition, we have demonstrated that PDCD4 is positively correlated with Nrf2 and HO-1 in human hepatocytes (Fig. 4). Based on these observations, we propose that PDCD4 is a bona fide tumor suppressor that might suppress hepatic carcinogenesis via the inhibition of selected pro-inflammatory genes, including c-Jun and the induction of Nrf2-dependent phase II cytoprotective enzymes through regulation of p21, and that sulforaphane exerts chemopreventive effects partly through the restoration of PDCD4.

Acknowledgment The authors thank Dr. Joydeb Kumar Kundu (School of Pharmacy, Keimyung University, Republic of Korea) for his kind help of editing the manuscript. This research was supported by Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Korean Ministry of Education, Science and Technology (2011–0013733).

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