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Carcinogenesis Advance Access originally published online on November 4, 2005
Carcinogenesis 2006 27(3):437-445; doi:10.1093/carcin/bgi251
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Carcinogenesis vol.27 no.3 © Oxford University Press 2005; all rights reserved.

ERK and JNK signaling pathways are involved in the regulation of activator protein 1 and cell death elicited by three isothiocyanates in human prostate cancer PC-3 cells

Changjiang Xu, Guoxiang Shen, Xiaoling Yuan, Jung-hwan Kim, Avantika Gopalkrishnan, Young-Sam Keum, Sujit Nair and Ah-Ng Tony Kong *

Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA

* To whom correspondence should be addressed. Email: kongt{at}rci.rutgers.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many isothiocyanates (ITCs) such as sulforaphane (SFN), phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC) are highly effectively in chemoprevention or reduction of the risk of cancer and possess antitumor activities in vitro and in vivo. The activator protein 1 (AP-1) and MAPK signaling pathways are believed to play an important role in cancer chemoprevention and chemotherapy due to their involvement in tumor cell growth, proliferation, apoptosis and survival. In the present study, we determined the effects of SFN, PEITC and AITC on AP-1 activation, and investigated the roles of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways in the regulation of AP-1 activation and cell death elicited by these ITCs in human prostate cancer PC-3 cells. SFN, PEITC and AITC each induced AP-1 activity potently and caused a significant elevation in the phosphorylation of ERK1/2, JNK1/2, Elk-1 and c-Jun. Transfection with ERK2 and upstream kinase DNEE-MEK1 activated AP-1 activity, and transfection with dominant-negative mutant ERK2 (dnERK2) potently decreased AP-1 activation induced by SFN, PEITC and AITC. Transfection with JNK1 and upstream kinase MKK7 activated AP-1 activity, and transfection with dominant-negative mutant JNK1-APF significantly attenuated AP-1 activation induced by SFN, PEITC and AITC. Pretreatment with MEK1-ERK inhibitor U0126 and JNK inhibitor SP600125 substantially attenuated the decrease in cell viability induced by SFN, PEITC and AITC. Transfection with dnERK2 and JNK1-APF significantly reversed the decrease of Bcl-2 expression elicited by these ITCs. Furthermore, transfection with dnERK2 and JNK1-APF blocked the apoptosis induced by these ITCs in PC-3 cells. Taken together, our results indicate that the activation of the ERK and JNK signaling pathways is important for transcriptional activity of AP-1 and is involved in the regulation of cell death elicited by ITCs in PC-3 cells.

Abbreviations: ITC, isothiocyanate; SFN, sulforaphane; PEITC, phenethyl isothiocyanate; AITC, allyl isothiocyanate; AP-1, activator protein 1; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Prostate cancer is one of the most prevalent malignancies and the second leading cause of cancer deaths in men in the United States. Clinically, it is usually diagnosed in patients over 50 years old. Thus, there could be opportunity for intervention using cancer chemopreventive compounds that prevent or slow the progression of this disease. Recent studies have indicated that using dietary chemopreventive compounds, such as isothiocyanates (ITCs), might be a promising strategy to decrease the incidence of prostate cancer (1,2). Many epidemiological studies have shown that consumption of vegetables, particularly cruciferous vegetables, can reduce the risk of different kinds of cancer in humans (3–5). Some ITCs derived from cruciferous vegetables, such as sulforaphane (SFN), are highly effective in preventing or reducing the risk of cancer induced by carcinogens in animal models (3,5–7). Studies have indicated that SFN and other two other ITCs, phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC), can reduce the risk of prostate cancer in animal models, inhibit prostate cancer cell growth, induce apoptosis and retard growth of prostate cancer cell xenografts in vivo (1,7–12). Our recent studies indicated that SFN and PEITC can also potently inhibit NF-{kappa}B activity and NF-{kappa}B regulated gene expression in PC-3 cells via inhibition of the IKK{alpha}/ß-I{kappa}B{alpha}-p65 signaling pathways (13).

The transcriptional factor activator protein 1 (AP-1) has been proposed to play important roles in carcinogenesis and cancer development (14–19). AP-1 regulates a wide range of cellular processes, including cell proliferation, death, survival and differentiation. AP-1 composes of heterodimeric protein complexes of members of the basic leucine zipper (bZIP) protein families, including the Jun (c-Jun, JunB and JunD) and Fos (c-Fos, FosB, Fra-1 and Fra-2) family, Jun dimerization partners (JDP1 and JDP2) and the closely related activation transcription factor (ATF; ATF2, LRF1/ATF3 and B-ATF) subfamilies (14,15). Jun proteins can form stable homodimers that bind to a type of AP-1 DNA recognition element termed the 12-O-tetradecanoylphorbol-13-acetate (TPA) responsive element (TRE, 5'-TGAG/CTCA-3') present within the regulatory region of a variety of genes (15,20). Fos family proteins can form heterodimers with Jun proteins that are more stable than Jun homodimers and also bind to DNA. ATF family proteins, which can form homodimers as well as heterodimers with Jun proteins, bind preferentially to the cAMP responsive element (CRE, 5'-TGACGTCA-3') (20,21).

AP-1 activity can be regulated by several mechanisms, including through the activation of the mitogen-activated protein kinase (MAPK) pathways (22). The MAPKs are serine/threonine superfamily kinases consisting of four MAPKs that have been reported to involve in the regulation of AP-1: the extracellular signal-regulated kinases (ERK), the c-Jun NH2-terminal kinases (JNK), p38 and c-Fos-regulating kinase (FRK) (20,22–24). Serum response element (SRE) is the major MAPK-response element located within the c-fos promoter and can be bound by a transcriptional factor complex, such as dimeric serum response factor and the ternary complex factors Elk-1, Sap1 and Sap2. Elk-1 can be activated by phosphorylation via ERK, JNK and p38 MAPKs, resulting in enhanced SRE-dependent c-Fos expression (22,25). Activation of JNK leads to the phosphorylation and activation of c-Jun and ATF-2, which form heterodimers and preferentially bind to TRE to activate c-jun transcription; activation of p38 can also phosphorylate and activate ATF-2 (22). c-Fos was also found to be phosphorylated and activated by FRK, and forms heterodimer with c-Jun and binds to AP-1 response element located within the promoters of target genes (23).

AP-1 regulates the transcription of many genes involved in carcinogenesis and is upregulated during tumor promotion and progression. Interestingly, it is also upregulated during apoptosis. Some chemopreventive agents such as theaflavins, (–)-epigallocatechin-(3)-gallate, resveratratol, t-butylhydroquinone and nordihydroguaiaretic acid that can inhibit AP-1 activation induced by UV-radiation or TPA pretreatment have been shown to be effective against carcinogenesis, implying a potential role for AP-1 in carcinogenesis in these system (24,26–29). In contrast, other chemopreventive agents such as PEITC and SFN can activate the basal level of AP-1 activity in the absence of pretreatment of TPA implicating AP-1 in preventing carcinogenesis under these conditions. However, at higher concentrations, PEITC and SFN can also suppress the basal level of AP-1 activity and block TPA-induced AP-1 activity in HT-29 cells (30). Thus, it appears that the activation or inhibition of AP-1 activity by chemopreventive compounds is complex. The effects on AP-1 activity may be dependent on the types and concentrations of the chemopreventive agents, as well as the cell types, and is probably to be affected by the treatments of UV-radiation or TPA in the induction of AP-1.

SFN-induced apoptosis in PC-3 cells is associated with up-regulation of Bax, down-regulation of Bcl-2, and activation of caspases-3, -9 and -8 (7). PEITC can also induce apoptosis in PC-3 cells, and this apoptotic process is thought to be mediated by ERK (8). AITC has also been reported to induce apoptosis, G2/M arrest and down-regulation of Bcl-2 in PC-3 cells (12). We have previously demonstrated that 5 or 10 µM of PEITC can induce the basal constitutive AP-1 in HT-29 cells dramatically, whereas 35 or 50 µM of PEITC can block the basal and TPA-induced AP-1 activity substantially (30). Treatment with 50 µM PEITC induces caspase-3 activity potently and results in apoptosis in HT-29 cells. The JNK inhibitor SP600125 attenuates both cytochrome c release and caspase-3 activation induced by PEITC and suppresses apoptosis induced by PEITC, and that the activation of JNK is believed to be critical for the initiation of the apoptotic processes in HT-29 cells (31).

In view of the important roles of AP-1 and MAPKs such as ERK and JNK in carcinogenesis, transformation, proliferation and apoptosis, it is important to know how chemopreventive agents such as ITCs induce AP-1 and cell death in PC-3 cells. In this study, we investigated the roles of MAPK signaling pathways involving ERK and JNK in the regulation of AP-1 transcriptional activity as well as cell death induced by SFN, PEITC and AITC in PC-3 cells. The results show that ERK and JNK MAPKs signaling pathways are important for the transcriptional activation of AP-1 activity and cell death induced by ITCs in PC-3 cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
SFN was obtained from LKT laboratories. PEITC and AITC were purchased from Sigma. PSN Antibiotic Mixture was bought from Gibco BRL. LipofectamineTM 2000 was bought from Invitrogen Life Technology. MTS Cell Proliferation assay kit, luciferase assay reagent and MEK1-ERK inhibitor U0126 were bought from Promega. Sirus Luminometer was bought from Berthold Detection Systems (GmbH D-75173 Pforzheim, Germany). JNK inhibitor SP600125 (SP) was bought from Alexis Biochemicals. Antibodies against phospho-JNK1/2 (Thr183/Tyr185), -ERK1/2 (Thr202/Tyr204), -Elk-1 (Ser383) and -c-Jun (Ser63) were purchased from Cell Signaling Technology. Antibody recognizing Bcl-2 was bought from Santa Cruz Biotechnology. Enhanced chemiluminescence (ECLTM) western blotting reagents were purchased from Amersham Pharmacia Biotech. The AP-1-luciferase reporter plasmid construct containing AP-1 consensus binding sites and pcDNA3-HA-JNK1 plasmid were kindly provided by Drs Michael Karin and Anning Lin (University of California, San Diego, CA). Constitutively active MEK1 (DNEE-MEK1-pcDNA3) was a gift from Dr Rony Seger (The Weizmann Institute of Science). Plasmids carrying MKK4, pcDNA-Flag-JNK1-APF, and wild-type and mutant ERK2 (dominant negative) were described previously (32,33).

Cell culture
Human prostate cancer PC-3 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2.2 g/l sodium bicarbonate and 10 ml/l PSN Antibiotic Mixture in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The medium was removed prior to treatment when cells were ~80% confluent, and the cells were maintained overnight in low serum MEM containing 0.5% FBS.

Stable transfection and transient transfection
The AP-1 construct was stably transfected into PC-3 cells by LipofectamineTM 2000 following the manufacturer's instruction. Five hours after transfection, the media were replaced with MEM containing 10% FBS, and the cells were selected with G418 to obtain single stable cell clones. The cell clone PC-3 C9 was a single stable clone transfected with the AP-1-dependent luciferase construct and was used for the experiments described here. The transient transfection was performed using the same procedure as stable transfection except without G418 selection.

MTS assay
The MTS [3-(4,5-dimethyltiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay was performed with the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay Kit according to the manufacturer's instruction. Briefly, PC-3 cells were plated in 24-well plates, and after 24 h, the growth media were replaced with media containing 0.5% serum overnight. Cells were then treated with different concentrations of SFN, PEITC and AITC for 24 and 48 h. The media were removed, and the culture media containing MTS and phenazine methosulfate solution were added. After 30 min, the absorbance was measured at 490 nm with a µQuant ELISA reader (BIO-TEK Instruments, Madison, WI). The data were expressed as percent of cell viability compared with that of the control, which was treated with 0.1 % dimethyl sulfoxide (DMSO). The values presented are means (n = 4) ± SD.

AP-1-dependent reporter gene expression assay
AP-1 activity was measured by AP-1-luciferase reporter gene expression. PC-3 C9 cells were treated with different concentrations of compounds for different lengths of time, and AP-1-luciferase activities were measured according to the manufacturer's instruction. Briefly, after treatment, the cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested in 1x reporter lysis buffer. After centrifugation, 10 µl aliquots of the supernatants were measured for luciferase activity by using a Sirus Luminometer The luciferase activity was normalized by protein concentrations or ß-galactosidase activity and expressed as fold of induction of luciferase activity over the control cells, which were treated with 0.1% DMSO. The values presented are means (n = 3) ± SD.

Western blot analysis
PC-3 cells were plated in 6-well plates and after 24 h, the growth media were replaced with media containing 0.5% serum and grown overnight. Cells were then treated with SFN, PEITC and AITC for different time periods and harvested with MAPKs lysis buffer (containing 10 mM Tris–HCl, 50 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 µM sodium orthovandate, 2 mM iodoacetic acid, 5 mM ZnCl2, 1 mM phenylmethylsulfonyl fluoride and 0.5% Triton-X 100) or whole cell lysis buffer (containing 10 mM Tris–HCl, 250 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 0.5% Triton-X 100, 10% glycerol, 1x proteinase inhibitor mixture, 1 mM phenylmethylsulfonyl fluoride, 100 µM sodium orthovandate, 2 mM iodoacetic acid and 5 mM ZnCl2). Protein extracts were prepared and the levels were determined by Bio-Rad protein assay according to the manufacture's instruction. An aliquot of 20 µg of total protein from each sample was mixed with 4x loading buffer and heated at 90°C for 4 min. Proteins were then separated by 10% mini-SDS–PAGE and transferred onto polyvinylidene fluoride membrane using a semi-dry transfer system (Fisher). The membrane was then blocked with 5% BSA or 5% non-fat milk in TBST buffer (2.42 g/l Tris–HCl, 8 g/l NaCl, 1 ml/l Tween-20, pH 7.6), washed with TBST buffer and incubated overnight at 4°C with primary antibodies prepared in TBST buffer. The membrane was then washed with TBST and incubated with secondary antibody conjugated with horseradish peroxidase. After washing with TBST, the detection was performed with ECLTM western blotting reagents.

Transient transfection cell death assay
PC-3 cells were plated in 6-well plates for 24 h before transfection. Cells were transfected with a ß-galactosidase-expression reporter construct together with an empty vector (pCDNA3.1) or the plasmids containing dominant-negative mutants dnERK2 and JNK1-APF using LipofectamineTM 2000. After transfection, the cells were cultured in fresh MEM for 12 h before treatment with ITCs. Cells were stained with 5-bromo-4-chloro-3-indoxyl-ß-D-galactopyranoside (X-gal). Apoptosis was quantitated by measuring the percentage of round cells among the ß-galactosidase-positive blue cells.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
SFN, PEITC and AITC induced AP-1 activity in PC-3 cells
To investigate and compare the effects of the ITCs SFN, PEITC and AITC on AP-1 activity, we assayed dose-responsive and time-dependent induction of AP-1-luciferase activity in PC-3 C9 cells (Materials and methods). Each of the ITCs tested potently induced AP-1 activation after treatments for 6, 12 and 24 h, with the highest induction of AP-1 seen after 24 h treatment (Figure 1). However, activation of AP-1 activity was dependent on the concentrations of ITCs. For instance, at higher concentrations (40 µM SFN, 10 µM PEITC and 100 µM AITC), AP-1 activity was not induced as strongly as at the lower concentrations. In fact, treatment with 40 µM SFN or 10 µM PEITC for 24 h actually inhibited AP-1 activity as compared with the control (Figure 1). After treatment for 6 h, 20 µM SFN, 5 µM PEITC and 50 µM AITC all showed statistically significant induction of AP-1 activity relative to controls. Aliquots containing 5 µM SFN, 2 µM PEITC or 20 µM AITC also induced AP-1 activation significantly but only after 24 h of treatment. The results suggest each of the ITCs may induce AP-1 in a dose- and time-dependent manner and that PEITC is the most potent inducer of AP-1 activity among the three ITCs tested.


Figure 1
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  		Fig. 1. Effect of SFN, PEITC and AITC on AP-1-luciferase reporter gene activity in PC-3 C9 cells. Briefly, PC-3 C9 cells were seeded in 6-well plates for 24 h and starved overnight with MEM containing 0.5% FBS, then treated with different concentrations of ITCs for 6, 12 and 24 h; the AP-1 activity was measured by luciferase activity assay. Data are mean (n = 3); bars, ±SD. Each experiment was repeated twice for reproducibility.

 
SFN, PEITC and AITC decreased the viability of PC-3 cells
The effects of SFN, PEITC and AITC on the viability of PC-3 cells were determined by a colorimetric MTS assay after 24 and 48 h treatment. Both SFN and PEITC exhibited much stronger growth inhibitory effects as compared with AITC. After treatment for 24 h, the cell viability with 20, 30 and 40 µM SFN was 86, 71 and 36%, respectively. For PEITC, the cell viability at 24 h was 85, 69 and 52% at 5, 7.5 and 10 µM, respectively. Treatment with 40 µM SFN and 10 µM PEITC for 24 h resulted in obvious cell death and decrease of cell viability, which contributed to the decrease of the basal AP-1 activities (Figure 1). At 100 µM of AITC, it caused significant growth inhibition with the cell viability of 64% at 24 h. After treatment for 48 h, cell survival was dramatically decreased by SFN and PEITC, and even 50 µM AITC caused significantly inhibition of cell viability as compared with 24 h treatment. Similar to the AP-1 activity, PEITC had the strongest effects on cell viability among the three ITCs. To determine how these ITCs induce AP-1 activity and inhibit cell viability, we performed the following experiments.

SFN, PEITC and AITC increased the phosphorylation of ERK1/2 and JNK1/2 in PC-3 cells
There are some reports that regulation of AP-1 is associated with MAPK-dependent signaling pathways (6,24). To investigate whether ITCs mediate AP-1 via MAPK signaling pathways, we assessed the phosphorylation of MAPKs ERK1/2 and JNK1/2. PC-3 cells were treated with two different concentrations of SFN, PEITC and AITC after overnight serum starvation and the cell extracts were harvested using MAPKs lysis buffer to detect phosphorylated-JNK1/2 and -ERK1/2 by western blotting. After treatment for 2 h, 20 and 30 µM SFN, 5 and 7.5 µM PEITC, and 50 µM AITC each strongly induced the levels of phosphorylated-ERK1/2. An aliquot of 20 µM AITC had no obvious effect on phosphorylated-ERK1/2. PEITC was the most potent activator for the phosphorylation of ERK1/2 in PC-3 cells. The phosphorylation levels of ERK1/2 after treatment for 4 h were still elevated significantly, as compared with the control, but the levels showed slight decrease as compared with the 2 h treatment.

A similar effect of ITC treatment was seen on JNK1/2 phosphorylation. After 2 h of treatment, 20 or 30 µM SFN, 5 or 7.5 µM PEITC and 50 µM AITC each induced the phosphorylation of JNK1/2 strongly, while 20 µM AITC did not show significant elevation of JNK1/2 phosphorylation as compared with the control. As with phosphorylation of ERK1/2, PEITC was the most potent activator of the phosphorylation of JNK1/2 in PC-3 cells. Using another human prostate cancer cell line, the androgen-sensitive LNCaP cells, we found the phosphorylation levels of ERK and JNK were also increased after treatment with these ITCs (data not shown).

Effects of SFN, PEITC and AITC on the phosphorylation of Elk-1 and c-Jun in PC-3 cells
Elk-1 can be phosphorylated and activated by ERK, JNK and p38 MAPKs, which results in enhanced SRE-dependent c-Fos expression (22,25). c-Jun, which can form heterodimers with ATF-2 and preferentially binds to TRE located in AP-1, is mainly phosphorylated by JNK (22,25). We treated PC-3 cells with 20 µM SFN, 5 µM PEITC and 50 µM AITC for 2 h, and harvested the cell extracts using MAPKs lysis buffer to detect phosphorylated-Elk-1 and -c-Jun by western blotting. We found that each ITC strongly elevated the phosphorylation of Elk-1. The phosphorylation of c-Jun was also activated by these three ITCs. An aliquot of 5 µM PEITC had the strongest activation, and 20 µM SFN activated the phosphorylation of c-Jun much less than PEITC, although it was still significantly increased relative to the controls.

Transfection with DNEE-MEK1 and ERK2 elevated AP-1 activity
The above data show that AP-1 activation can be induced by treatment with SFN, PEITC and AITC for 6 h, and that the levels of phosphorylated-ERK1/2 and -Elk-1 were elevated by SFN, PEITC and AITC after treatment for 2 h. To investigate the role of MEK1-ERK in the regulation of AP-1 activation in PC-3 cells, we transfected DNEE-MEK1 and/or ERK2 plasmids into PC-3 C9 cells as described in Materials and methods. After 5 h, the medium was changed to the normal MEM and cultured for additional 6 and 24 h. The results showed that transfection with ERK2 for 24 h increased the AP-1 activity to 1.58 ± 0.15 fold as compared with the cell transfected with control plasmid (Figure 2A). Transfection with DNEE-MEK1 for 24 h increased the activity of AP-1 to 1.47 ± 0.10 fold (Figure 2A). There was an obvious additive effect when DNEE-MEK1 and ERK2 were co-transfected into PC-3 C9 cells, the AP-1 activity was elevated to 2.24 ± 0.14 fold after 6 h, and 3.87 ± 0.44 fold after 24 h (Figure 2A).


Figure 2
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  		Fig. 2. Regulation of AP-1 activity by MEK1-ERK signaling pathway in PC-3 cells. Briefly, PC-3 C9 cells were seeded in 6-well plates for 24 h, (A) transfected with ERK2 and/or DNEE-MEK1 plasmids for 5 h followed by replacement of media with normal MEM for additional 6 and 24 h incubation or (B) transfected with dnERK2 or empty vehicle plasmid for 5 h followed by replacement of media with normal MEM for additional 12 h, then treated with SFN, PEITC and AITC for 6 and 24 h. The AP-1 activity was measured by luciferase activity assay. Data are mean (n = 3); bars, ±SD. Each experiment was repeated twice for reproducibility.

 
Transfection with a dominant-negative mutant of ERK2 inhibited the AP-1 activity induced by SFN, PEITC and AITC
Transfection of DNEE-MEK1 and ERK2 into PC-3 C9 cells increased the activity of AP-1 (Figure 2A). To confirm these findings, we transfected PC-3 C9 cells with a plasmid expressing a dominant-negative mutant of ERK2 (dnERK2) and treated the cells with SFN, PEITC or AITC for 6 and 24 h. We found that dnERK2 blocked the AP-1 activity induced by SFN from 3.89 ± 0.32 fold to 2.39 ± 0.41 fold at 6 h, and from 10.65 ± 0.88 fold to 2.69 ± 0.48 folds at 24 h (Figure 2B). The AP-1 activity induced by PEITC was blocked from 5.42 ± 0.68 fold to 3.91 ± 0.47 fold at 6 h, and from 12.64 ± 1.24 fold to 6.87 ± 0.78 fold at 24 h (Figure 2B). The AP-1 activity induced by AITC was decreased from 3.23 ± 0.39 fold to 2.22 ± 0.35 fold at 6 h, and from 7.38 ± 0.92 fold to 4.06 ± 0.66 fold at 24 h (Figure 2B). We also found that pretreatment with MEK1-ERK inhibitor U0126 could inhibit the AP-1 activity induced by these three ITCs (data not shown).

Transfection with MKK7 and JNK1-HA induced AP-1 activity
We have shown that the levels of phosphorylated JNK1/2 and c-Jun were elevated by treatment with SFN, PEITC and AITC. To further study the potential role of JNK in AP-1 regulation in PC-3 cells, we transfected the PC-3 C9 cells with MKK7 and/or JNK1-HA, and cultured for additional 6 and 24 h. We found that transfection with JNK1-HA for 24 h significantly elevated AP-1 activity to 2.54 ± 0.28 fold, but there is no significant elevation after transfection with MKK7 for 6 and 24 h. After co-transfection with MKK7 and JNK1-HA for 6 and 24 h, AP-1 activity was significantly elevated to 2.24 ± 0.33 and 3.90 ± 0.45 fold, respectively (Figure 3A).


Figure 3
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  		Fig. 3. Regulation of AP-1 activity by MKK7-JNK1 signaling pathway in PC-3 cells. Briefly, PC-3 C9 cells were seeded in 6-well plates for 24 h, (A) transfected with MKK7 and/or JNK1-HA plasmid for 5 h followed by replacement of media with normal MEM for additional 6 and 24 h incubation, (B) transfected with JNK1-APF or empty vehicle control plasmids for 5 h followed by replacement of media with normal MEM for additional 12 h, then treated with SFN, PEITC and AITC for 6 and 24 h. The AP-1 activity was measured by luciferase activity assay. Data are mean (n = 3); bars, ±SD. Each experiment was repeated twice for reproducibility.

 
Transfection with dominant-negative mutant JNK1-APF inhibited the AP-1 activity induced by SFN, PEITC and AITC
To confirm that JNK1 plays a role in AP-1 activation, we transfected PC-3 C9 cells with dominant-negative mutant JNK1-APF, and treated with SFN, PEITC and AITC for 6 h and 24 h. We found that JNK1-APF significantly blocked the AP-1 activity induced by 24 h treatment with SFN (9.47 ± 0.68 fold decreased to 6.46 ± 0.89 fold), but had no effect on the activation after only 6 h of treatment (Figure 3B). The AP-1 activity induced by 24 h treatment with PEITC was inhibited from 11.38 ± 0.90 fold to 6.51 ± 1.01 fold, whereas AP-1 activity elicited by 24 h treatment with AITC was decreased from 7.43 ± 0.54 fold to 3.26 ± 0.87 fold, respectively (Figure 3B).

MEK1-ERK inhibitor U0126 and JNK inhibitor SP600125 blocked the cell viability decrease elicited by SFN, PEITC and AITC
We have demonstrated that these three ITCs can induce AP-1 activity dramatically (Figure 1) and that the ERK and JNK signaling pathways are involved in their regulation of AP-1 activity (Figures 2 and 3). We have also found that SFN, PEITC and AITC strongly inhibited the cell viability of PC-3 cells. Therefore, we performed the next experiment about the roles of ERK, JNK and AP-1 in the regulation of cell growth and death in PC-3 cells. We have already found that the down-regulation of ERK and JNK caused the decrease of ITC-induced AP-1 activity (Figures 2 and 3). We predict that based on these results the down-regulation of ERK and JNK could block the negative effects of the ITCs on cell viability. We therefore pretreated PC-3 cells with 10 µM MEK1-ERK inhibitor U0126 and 10 µM JNK inhibitor SP600125 for 30 min, then treated with SFN, PEITC and AITC for 48 h and then performed cell viability assayed. We found that both significantly blocked the decrease in cell viability elicited by 40 µM SFN, as well as 7.5 and 10 µM PEITC. Cell viability was also slightly elevated by U0126 + SP600125 in AITC-treated cells, although not significantly (Figure 4A).


Figure 4
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  		Fig. 4. Inhibition of JNK1 and ERK2 signaling attenuates cell death elicited by SFN, PEITC and AITC in PC-3 cells. Briefly, PC-3 cells were seeded in 6-well plates for 24 h, (A) starvation overnight with MEM containing 0.5% FBS, pretreated with 10 µM U0126 or 10 µM SP600125 for 30 min, then incubated with SFN, PEITC and AITC for 48 h. Cell viability was then determined by MTS assay. Data are mean (n = 4); bars, ±SD. (B) Cells were treated with SFN, PEITC and AITC for 24 h, or transfected with DNEE-MEK1 + ERK2, MKK7 + JNK1, and cultured for 24 h, or transfected with dnERK2 or JNK1-APF, and cultured with normal MEM for 12 h, then treated with SFN, PEITC and AITC for additional 24 h. Cells were then harvested using whole cell lysis buffer, and western blotting against Bcl-2 were performed. (C) Suppression of ITC-induced apoptosis by dnERK2 and JNK1-APF. Cells were transfected with an expression vector encoding ß-galactosidase (2 µg) with (a and b) empty vector control, or (c) dnERK2 or (d) JNK1-APF (2 µg) 5 h later cells were cultured with normal MEM for 12 h, then treated with 7.5 µM PEITC for additional 12 h (b, c and d). Cells were then fixed and stained with X-gal.

 
Dominant-negative mutants dnERK2 and JNK1-APF reversed ITC-induced decrease of Bcl-2 expression
SFN, PEITC and AITC were reported to induce apoptosis in PC-3 cells (7,8,12). Bcl-2 expression was decreased dramatically after PC-3 cells were treated with 30 µM SFN, 7.5 µM PEITC or 100 µM AITC for 24 h (Figure 4B). Similar results were found in the decrease of Bcl-2 expression when LNCaP cells were treated with these ITCs (data not shown). To determine whether the effects of ITCs on Bcl-2 expression could be mediated via MAPK signaling, PC-3 cells were first transfected with DNEE-MEK1 + ERK2 or MKK7 + JNK1; the expression level of Bcl-2 was decreased as compared with the control (Figure 4B). Then, PC-3 cells were transfected with dominant-negative mutants of ERK2 and JNK1, dnERK2 and JNK1-APF, and treated with SFN, PEITC or AITC for 24 h. We found that both dnERK2 and JNK1-APF strongly reversed the decrease of Bcl-2 expression elicited by SFN, PEITC and AITC (Figure 4B).

Dominant-negative mutants dnERK2 and JNK1-APF suppressed ITCs-induced apoptosis
To obtain further evidence for the role of ERK and JNK signaling pathways in ITC-induced apoptosis, we performed transient transfection cell death assays using dominant-negative mutants of ERK2 (dnERK2) and JNK1 (JNK1-APF). PC-3 cells were transfected with ß-galactosidase and empty vector pCDNA3.1 or with ß-galactosidase and dnERK2 or JNK1-APF as we have described previously. After transfection, cells were treated with ITCs or 0.1% DMSO as control. To ensure that the majority of cells were attached to the plates after ITC treatment, a suitable treatment time period was determined. Following treatment with ITCs for 12 h, the cells were stained with X-gal to identify ß-galactosidase-expressing cells (blue in color) (Figure 4C). Cells transfected with empty vector and treated with 0.1% DMSO solvent had an almost normal appearance (‘a’ in Figure 4C). However when the cells were treated with 7.5 µM PEITC for 12 h, 73 ± 12% of the transfected cells (blue in color) assumed a characteristic apoptotic appearance (round shape, as shown in ‘b’ of Figure 4C). However, when PC-3 cells transfected with dnERK2 or JNK1-APF were treated with 7.5 µM PEITC for 12 h, most cells retained a normal shape with only 25 ± 6%, and 34 ± 8% having the round apoptotic appearance, respectively (‘c’ and ‘d’ in Figure 4C). The effects of dnERK2 and JNK1-APF on SFN- and AITC-induced apoptosis were similar to that of PEITC (data not shown). These data suggest that the down-regulation of ERK and JNK could suppress the apoptosis induced by SFN, PEITC and AITC in PC-3 cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Prostate cancer is the second leading cause of cancer death in men in the United States. The etiology of prostate cancer is not clear but may involve genetic changes, activated oncogenes, growth factors, androgen hormones and/or dietary factors (2,34). Recent studies suggest that high consumption of vegetables, particularly cruciferous vegetables, could be associated with a reduced risk of prostate cancer (2,34,35). Many ITCs, such as SFN, which is found abundantly as glucoraphanin in cruciferous vegetables, have been shown to effectively block chemically induced carcinogenesis in various animal models (3,5,36). SFN is a potent inducer of phase II detoxifying enzymes in human prostate cells, the induction of which could explain the association between high consumption of cruciferae and decreased prostate cancer risk (10). However, in pre-initiated or tumor cells, the ITCs such as SFN, PEITC and AITC could act through inhibition of cell growth and induction of apoptosis in human PC-3 prostate cancer cells (7,8,12), leading to the prevention of cancer progression (37), although the precise molecular and cellular molecular mechanism remain unclear. Previous studies on the mechanism of growth inhibition activities in PC-3 cells have implicated the roles of Bax, Bcl-2, caspases, ERKs and regulation of cell cycle via Bcl-2 (7,8,12). However, the effects of ITCs on AP-1 activation, the roles of ERK and JNK signaling pathways in the regulation of AP-1 activation and cell death elicited by these ITCs in human prostate cancer PC-3 cells remain known.

Under physiologically relevant concentrations of ITCs (38,39), we found that treatment with 20 and 30 µM SFN, 5 and 7.5 µM PEITC, and 20 and 50 µM AITC all significantly induced the basal level of AP-1 activity in PC-3 C9 cells (Figure 1), without greatly impacting on the cell viability. Thus, we chose these concentrations of SFN, PEITC and AITC to investigate the possible signaling pathways involved in the regulation of AP-1 activation and cell death induced by ITCs in PC-3 cells. SFN, PEITC or AITC strongly induced the basal levels of phosphorylated ERK1/2, Elk-1, JNK1/2 and c-Jun. AITC required a higher concentration (50 µM) in general, with 20 µM not sufficient to induce phosphorylation of all targets tested. Elk-1 activation by phosphorylation usually leads to increased SRE-dependent c-Fos expression and MAPKs, such as ERK and JNK are able to phosphorylate and activate Elk-1 (22). c-Jun can be phosphorylated and activated by JNK and forms heterodimers with c-Fos, which preferentially bind to TRE or AP-1 (22,25). In the present study, we found that both Elk-1 and c-Jun were activated by ITCs, suggesting that their activation could contribute to the AP-1 activation (Figure 1) in PC-3 cells.

Previous studies suggested that ERK1/2 and JNK1/2 signaling pathways could be important mediators in the regulation of AP-1 activation elicited by ITCs in PC-3 cells (8,9,22,25,40); however, direct challenges to the system using activated or dominant-negative mutants are lacking. In our current studies, we found that transfection with ERK2 and DNEE-MEK1 enhanced AP-1 activation by ITCs, whereas transfection with dominant-negative mutant dnERK2 blocked the AP-1 activation induced by SFN, PEITC and AITC, demonstrating for the first time, the direct involvement of ERK signaling in AP-1 activation in PC-3 cells. Similarly, transfection of PC-3 cells with JNK1-HA and MKK7 revealed significant activation of AP-1, and that transfection with dominant-negative mutant JNK1-APF strongly blocked the AP-1 activation induced by SFN, PEITC and AITC, also implicating the importance of the JNK signaling pathway in AP-1 regulation in the PC-3 cells. Comparing the three well-known ITCs, there are some interesting differences between the effects of SFN, PEITC and AITC on AP-1 regulation via the ERK and JNK signaling pathways. SFN appears to be more dependent on the MEK1-ERK signaling pathway to regulate AP-1 activity in PC-3 cells. This point is illustrated by the fact that SFN activated ERK more potently than that of JNK, and furthermore, the dominant-negative mutant ERK2 had much stronger suppressive effect on SFN-induced AP-1 activation than the dominant-negative mutant JNK1-APF (Figures 2B and 3B). The definitive role of AP-1-mediated apoptosis would most likely work in concert and/or in collaboration with other signaling pathways such as NF-{kappa}B (13) as well as others (41).

SFN, PEITC and AITC each caused a time- and dose-dependent inhibition of cell survival in PC-3 cells, and each activated AP-1 (Figure 1) as well as ERK and JNK. Previous studies have shown that transfection with dominant-negative mutant JNK1-APF suppresses PEITC-induced apoptosis in 293 cells (40) and that JNK inhibitor SP600125 blocks cytochrome c release and cell death elicited by PEITC in HT-29 cells (31). Pretreatment with ERK2 inhibitor PD98059 suppressed PEITC-induced apoptosis in PC-3 cells (8). Our present data also strongly support a role of ERK and JNK in ITCs-mediated cell death. We found that pretreatment with MEK1-ERK inhibitor U0126 and JNK inhibitor SP600125 greatly attenuated the decrease in cell viability induced by ITCs (Figure 4A). Furthermore, transfection of dnERK2 and JNK1-APF reversed the inhibition of Bcl-2 expression elicited by ITCs (Figure 4B) and concomitantly suppressed ITCs-induced apoptosis (Figure 4C). Taken together these data suggest that ITCs-induced apoptosis is tightly coupled to ERK and JNK signaling, as well as AP-1 activation in human prostate PC-3 cells, and that these signaling mechanisms could contribute to the overall potential cancer chemopreventive effects of these class of dietary compounds in human prostate cancers.


   Acknowledgments
 
The authors thank all the members in Dr Tony Kong's laboratory for their help in the preparation of this manuscript. This work was supported by grant R01-CA-094828 from the National Institutes of Health (NIH).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 14, 2005; revised September 17, 2005; accepted October 26, 2005.


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