Induction of AP-1 activity by perillyl alcohol in breast cancer cells

Yoshiko Satomi, Shigeki Miyamoto1 and Michael N. Gould2

McArdle Laboratory for Cancer Research and
1 Department of Pharmacology, University of Wisconsin–Madison, Madison, WI 53792, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Monoterpenes display chemopreventive and therapeutic activity in rat mammary tumor models. Monoterpenes can also inhibit cell growth and induce apoptosis of cultured cells. In this study, the monoterpene perillyl alcohol (POH) was found to induce transient expression of the c-jun and c-fos genes transcriptionally. POH also transiently induced phosphorylation of c-Jun protein. These events were associated with transcriptional activation of an AP-1-dependent reporter gene. These results suggest that POH might affect c-Jun activity via the Jun N-terminal kinase/stress-activated protein kinase pathway and modulate expression of AP-1 target genes.

Abbreviations: ACD, actinomycin D; AP-1, activator protein 1; CHX, cycloheximide; JNK, Jun N-terminal kinase; M6P/IGF2R, mannose 6-phosphate/insulin-like growth factor 2 receptor; PBS, phosphate-buffered saline; POH, perillyl alcohol; TGFß1, transforming growth factor ß1; TNF{alpha}, tumor necrosis factor {alpha}; TPA, 12-O-tetradecanoylphorbol-13-acetate.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Limonene and perillyl alcohol (POH) are examples of monoterpenes that are found in many foods, such as citrus fruits, herbs and spices, and display anticancer activity (1,2). Dietary administration of monoterpenes was shown to be effective for prevention and therapy of 7,12-dimethylbenz[a]anthracene- and N-methyl-N-nitrosourea-induced rat mammary tumor models (3). POH is currently in phase 2 cancer trials. The mechanism of anticancer activity of monoterpenes is still unknown.

Several molecular activities of monoterpenes that may be relevant to their anticancer activity have been reported. Monoterpenes inhibit isoprenylation of small G proteins in vitro (4,5). This suggests that monoterpenes may affect growth-related pathways by inhibiting the activity of G proteins, since isoprenylation is critical for the activities of G proteins (6). In addition, monoterpenes induce the expression of mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and transforming growth factor ß1 (TGFß1) genes in rat models (7). M6P/IGF2R activates latent-TGFß1, a mammary tumor growth inhibitor (8,9), and degrades IGF2, a mammary tumor growth stimulator (10,11). Monoterpenes were also shown to induce apoptosis in rat liver tumors induced by diethylnitrosoamine (12). The level of apoptosis correlated with increased expression of M6P/IGF2R and TGFß1, 2 and 3 receptor levels (12). Lipocortin 1, which is a marker of apoptosis in the process of mammary epithelial regression (13), was induced in rat mammary tumor models (14). Thus, monoterpenes may inhibit cell growth and/or induce apoptosis via inhibition of isoprenylation of G proteins, down-regulation of the TGFß1 pathway and/or degradation of the IGF2 mitogen.

Among growth-associated genes, the c-jun proto-oncogene is one of the immediate early genes and encodes a component of the activator protein 1 (AP-1) transcription factor. Recent observations show that c-Jun is involved in not only cell proliferation but also apoptosis (1518). Whether or not the activity of c-Jun is affected by POH treatment is unknown. Here, we report that POH induces expression of the c-jun gene and transcriptional activation of an AP-1-dependent reporter gene. We also show that POH treatment is associated with rapid phosphorylation of c-Jun protein at the N-terminal sites normally phosphorylated by the Jun N-terminal kinase (JNK). These observations suggest that POH may modulate AP-1 activity in part by activation of the JNK/c-Jun pathway.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
POH was purchased from Aldrich (Milwaukee, WI). Cycloheximide (CHX), actinomycin D (ACD) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma (St Louis, MO). Recombinant human tumor necrosis factor {alpha} (TNF{alpha}) was obtained from Calbiochem (San Diego, CA).

Cell culture
Human breast cancer cell line T47D-C4-2W cells were cultured in RPMI-1640 (Gibco BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum, glutamine and gentamicin and maintained in a humidified 37°C incubator containing 5% CO2.

Northern blot analysis
Cells were treated with POH alone or with other reagents as specified in each figure. After various time points, cells were harvested and total RNA was isolated using RNeasy Kits (Qiagen, Chatsworth, CA). Cells that were treated with POH plus CHX were serum starved for 48 h before treatment. Total RNA (15 µg) was denatured, electrophoresed on a 1% formaldehyde–agarose gel and transferred to a Hybond N nylon membrane (Amersham, Arlington Heights, IL). After UV crosslinking, the membranes were hybridized with the corresponding specific probes (for c-jun, the EcoRI fragment from a mouse c-jun clone; for c-fos, the BamHI–XhoI fragment from a mouse c-fos clone) in a hybridyzation buffer containing 0.2 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA and 1% bovine serum albumin at 65°C. After hybridization, the membranes were washed with a wash buffer (0.5x SSC and 0.09% SDS) at 65°C and then analyzed with a Phosphorimager (Molecular Dynamics). Gene expression levels were normalized to ribosomal phosphoprotein P0 mRNA (36B4). All probes were labeled with [{alpha}-32P]dCTP by random priming. Mouse c-jun, jun-B and jun-D clones were obtained from ATCC.

Western blot analysis
Cells were serum starved for 24 h and then treated with POH. The treated cells were washed once with cold phosphate-buffered saline (PBS) without Mg2+ and Ca2+ (PBS–), scraped with cold PBS– and centrifuged at 2000 r.p.m. for 5 min at 4°C. The cell pellets were washed once with cold PBS– and solubilized with cell lysis buffer containing 10 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.5 mM EDTA, 1 mM EGTA, 1% SDS, 0.5 µg/ml leupeptin, aprotinin and pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 20 mM ß-glycerophosphate, 50 mM NaF, 1 mM sodium orthovanadiate and 20 mM p-nitrophenyl phosphate. The cell lysates were sonicated for 10 s on ice and then heated at 95°C for 5 min. The lysates were centrifuged at 14 000 r.p.m. for 20 min at 4°C and the supernatant was stored at –80°C. Protein concentrations were determined using BCA Protein Assay Reagent (Pierce, Rockford, IL). Equal amounts of protein lysate (50 µg/lane) were run on 10% SDS–PAGE and transferred to nitrocellulose Hybond ECL membrane (Amersham) or Trans-Blot Transfer Medium (Bio-Rad, Hercules, CA). The membranes were first blocked with 5% non-fat dry milk in TBST buffer containing 20 mM Tris–HCl, pH 7.6, 137 mM NaCl and 0.1% Tween-20 and then incubated with primary antibody [Ser(P)-63-specific c-Jun or c-Jun, 1:1000] (New England BioLabs, Beverly, MA) in TBST buffer including 5% bovine serum albumin overnight at 4°C. Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2500) (Pierce) and visualized using SuperSignal Substrate (Pierce).

Luciferase assay
Cells were transiently co-transfected with plasmids Col-luc (–73/+63) and pCMVß using Super Transfection Reagent (Qiagen). Forty-eight hours later, cells were treated with POH for 2 h and harvested into lysis buffer. The supernatant was analyzed for luciferase and ß-galactosidase activities using the Luciferase Assay System and ß-Galactosidase Assay System, respectively (Promega, Madison, WI). The relative luciferase activity of each sample was normalized to the ß-galactosidase activity. The Col-luc (–73/+63) plasmid, which contains the minimal promoter region of the human collagenase gene fused to a luciferase gene, was kindly provided by Dr T. Dang (University of California). The pCMVß plasmid, which contains the ß-galactosidase gene, was purchased from Clontech (Palo Alto, CA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of c-jun and c-fos expression by POH
To investigate the effect of POH on the expression levels of c-jun- and c-fos-mRNA, T47D-C4-2W human breast cancer cells were treated with POH at 0.5–2 mM for various durations. Total RNA was prepared from these cells and analyzed by northern blot analysis using c-jun and c-fos cDNA as probes. Rapid induction of c-jun expression was observed at 1.5 h after exposure to POH (Figure 1AGo). This induction was transient, since the level of c-jun mRNA peaked at 1.5 h and returned to the basal unstimulated level by 4 h following POH exposure (data not shown). Similarly, c-fos expression was transiently induced following treatment with POH (Figure 2BGo). The induction of c-jun and c-fos expression was dose dependent and reached maximum levels of 31- and 8-fold with 2 mM POH over the control levels, respectively (Figure 1B and CGo). Under the conditions utilized in Figure 1Go, the effect of POH on c-fos expression in T47D-C4-2W cells was clear only at 2 mM treatment. When these cells were serum starved, which lowers the basal c-fos expression level, the effect of POH on c-fos expression was seen at 0.5 mM (Figure 2BGo). Other members of the jun (junB and junD) and fos (fosB, fra1 and fra2) families were not affected by POH treatment at the time points tested (data not shown).



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Fig. 1. Induction of c-jun and c-fos by POH. (A) Time course of the effect of POH on c-jun expression. Cells were treated with 1 mM POH for 5–120 min. (B and C) Dose–response curves of the effect of POH on c-jun and c-fos expression. Cells were treated with 0–2 mM POH for 1.5 h. Total RNA of treated cells was isolated and subjected to northern blot analysis and hybridized to 32P-labeled c-jun or c-fos probes. Gene expression levels were normalized to ribosomal phosphoprotein P0 mRNA. Data show the averages (A) or averages ± SD (B and C) of two experiments.

 


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Fig. 2. Effect of CHX and ACD on c-jun and c-fos induction by POH. (A and B) Effect of CHX on c-jun and c-fos induction by POH. Cells were treated with 0.5 mM POH plus 10 µg/ml CHX for 1.5 h after serum starvation for 2 days. As a control, cells were treated with POH alone for 0.5–8 h or CHX alone for 1.5 h or 50 ng/ml TPA alone for 0.5 h. The same blot was used to detect ribosomal phosphoprotein P0 mRNA. (C) Effect of ACD on c-jun induction by POH. Cells were treated with 1 mM POH plus 1 µg/ml ACD for 0.5–2 h. Total RNA was isolated and analyzed as described in the legend to Figure 1Go. 0T, time 0; C, control; P, POH; CX, CHX; A, ACD; 36B4, ribosomal phosphoprotein P0 mRNA.

 
To determine whether the effect of POH on the expression of c-jun and c-fos genes is at the transcriptional level, T47D-C4-2W cells were treated with 1 mM POH in the presence and absence of an RNA synthesis inhibitor ACD. ACD (1 µg/ml) completely prevented POH induction of c-jun (Figure 2CGo) and c-fos expression (data not shown). To examine if POH-dependent induction of c-jun and c-fos expression was dependent on de novo protein synthesis, these cells were also treated with POH in the presence and absence of 10 µg/ml CHX, a protein synthesis inhibitor. CHX augmented the induction of c-jun and c-fos by POH (Figure 2A and BGo). The CHX treatment alone also induced expression of c-jun and c-fos mRNAs. However, induction of c-jun and c-fos mRNAs by CHX plus POH was greater than that by CHX alone. The effect of CHX on the induction of these genes by POH was additive. TPA, a potent AP-1 inducer, also induced the c-jun and c-fos genes at 30 min. The inductive effect of TPA on c-fos expression was greater than that on c-jun.

Induction of c-Jun phosphorylation by POH
Induction of c-jun expression by many stimuli is accompanied by prior site-specific phosphorylation at Ser63 and Ser73 of pre-existing c-Jun protein by JNK (15). To determine if POH could also modify c-Jun protein by phosphorylation, c-Jun phosphorylation was investigated utilizing an antibody that specifically recognizes c-Jun proteins phosphorylated at the N-terminal Ser63 residue. POH was found to cause transient induction of site-specific c-Jun phosphorylation with maximal induction seen after 1.5–2 h treatment (Figure 3AGo). Using another antibody, which recognizes c-Jun proteins with or without the N-terminal phosphorylation, induction of c-Jun protein expression can also be demonstrated (Figure 3CGo). Interestingly, however, induction of total c-Jun protein lasted longer than that of N-terminal c-Jun phosphorylation (Figure 3A and CGo). The inductive effect of POH on c-Jun phosphorylation and c-Jun total protein levels was dose dependent (Figure 3B and DGo). TNF{alpha}, a potent JNK activator, caused rapid and marked induction of c-Jun phosphorylation within 10 min in these breast cancer cells (Figure 3CGo).



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Fig. 3. Effect of POH on c-Jun phosphorylation. (A and C) Time course of the effect of POH on c-Jun phosphorylation and total c-Jun level. Cells were treated with 1 mM POH for 10–240 min after serum starvation for 24 h. (B and D) Dose–response curves of the effect of POH on c-Jun phosphorylation and total c-Jun level. Cells were treated with 0–2 mM POH for 1.5 h after serum starvation for 24 h. As a control, cells were treated with 80 ng/ml TNF{alpha} for 10 min. Treated cells were harvested and whole cell extracts were analyzed by SDS–PAGE and immunoblotting using antibodies specific to phosphorylated (Ser63) c-Jun and total c-Jun. C, control; P, POH; c-Jun-P, phosphorylated c-Jun; c-Jun, total c-Jun.

 
POH induction of AP-1-dependent transcription
To determine if POH-induced expression of c-jun and c-fos exert an effect on the expression of genes containing AP-1 sites, the effect of POH on AP-1-dependent transcription was examined. T47D-C4-2W cells were transiently transfected with a reporter plasmid containing a luciferase gene driven by a minimal human collagenase gene promoter (–73/+63) that contains a single AP-1 site. To normalize transfection efficiency, a plasmid containing a ß-galactosidase gene under the control of the constitutive CMV promoter was co-transfected as an internal control. Treatment of transfected cells with 0–2 mM POH for 2 h induced luciferase activity (Figure 4Go). Maximum induction was seen with 0.5 mM, but decreased at higher doses. The inductive effect of POH at 0.5 mM was greater than that of TPA, a positive control, under the conditions utilized.



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Fig. 4. Effect of POH on AP-1-dependent transcription. Cells were transiently co-transfected with 2 µg Col-luc (–73/+63) and 0.2 µg pCMVß by lipofection. Forty-eight hours later, the transfected cells were treated with 0–2 mM POH for 2 h and harvested into lysis buffer. The supernatant was subjected to analysis for luciferase and ß-galactosidase activities. The relative luciferase activity was normalized to the ß-galactosidase activity. Data show the averages ± SD of three experiments. As a control, cells were treated with 50 ng/ml TPA for 2 h.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although several molecular activities of the monoterpenes relevant to their anticancer effects have been described (4,5,7,14), the present study describes one of the earliest molecular events induced by POH in human breast cancer cells. We have demonstrated that POH transiently induced c-jun and c-fos expression in a dose-dependent manner in the human breast tumor cell line T47D-C4-2W. The maximum induction occurred around 1.5 h. CHX and ACD treatments demonstrated that this induction did not require de novo protein synthesis, but did need RNA synthesis. Thus, induction of c-jun and c-fos represents a primary effect of POH treatment, acting through pre-existing signaling components. c-jun and c-fos are typical immediate early genes induced by a wide variety of growth factors and extracellular signals and their transient induction triggers biological downstream events relevant to cell cycle progression and cell proliferation (1922). Recent reports also show that c-jun and c-fos are induced both during and before apoptosis induced by several extracellular stimuli (2326). Thus, transient induction of the c-jun and c-fos genes by POH may be an early key event in the signal cascades leading to apoptosis.

The function of c-Jun is regulated by site-specific phosphorylation (27,28). Phosphorylation of Ser63 and Ser73 in the N-terminal domain of c-Jun by JNK stimulates the transactivating activity of c-Jun (27,28). POH was also found to transiently induce phosphorylation of c-Jun, similar to c-jun expression. In control cells, the effect of serum on c-Jun phosphorylation was seen at 30 min and then decreased. POH also induced total c-Jun protein and the inductive effect on total c-Jun protein lasted longer than c-Jun phosphorylation. At 4 h, phosphorylated c-Jun was diminishing, but total c-Jun protein still remained at a high level. Transient induction of c-Jun phosphorylation by POH was similar to induction by other known JNK activators, including UV and TNF{alpha} (2931). However, it contrasted with IR (32) and other cell growth inhibitors such as green tea polyphenols, isothiocyanates and bufalin (25,33,34), which cause sustained c-Jun phosphorylation. Moreover, western blotting against phosphorylated c-Jun and total c-Jun showed several bands, suggesting that POH treatment caused c-Jun phosphorylation at other sites in addition to Ser63. This suggests that other kinases may also be activated by POH treatment. c-Jun is also phosphorylated in the C-terminal region next to the DNA-binding domain by casein kinase II and extracellular signal regulated kinase (3537). Thus, it is possible that the different forms of c-Jun that are phosphorylated at other sites than Ser63 may be more stable than those phosphorylated at Ser63.

c-jun and c-fos, the components of transcription factor AP-1, are well-known proto-oncogenes and immediate early genes and are implicated in cell cycle control (1922). The luciferase reporter assay demonstrated that POH-induced c-Jun and c-Fos formed a transcription-competent AP-1 complex (Figure 4Go). However, intriguingly, AP-1-dependent transcription of the reporter gene was maximal at 0.5 mM POH and decreased at higher POH doses. This is in contrast to the expression levels of the c-jun gene and protein and c-fos gene, which peaked at 2 mM POH (Figures 1 and 3GoGo). The reason for the reduced AP-1 activity at high concentrations of POH is unknown. Ui et al. reported that highly phosphorylated forms of c-Jun protein are not functional in cellular transformation (38). As seen by western blot analyses, c-Jun seems to be hyperphosphorylated at high doses of POH (Figure 3Go). This hyperphosphorylated c-Jun might not be able to participate in formation of a competent AP-1 transcription factor and might account for the decreased AP-1-dependent transcription at high POH doses. As mentioned above, c-Jun may be phosphorylated in the C-terminal region and phosphorylation of C-terminal sites is considered to inhibit DNA-binding activity and also AP-1 activity (3537). Thus, the decreased AP-1 activity at high POH might also arise from phosphorylation in the C-terminal region of c-Jun.

POH has been found to induce apoptosis in vitro and in vivo (12). Although c-Jun activity is normally associated with cell proliferation, recent studies demonstrated that c-Jun can also cause apoptosis in NIH 3T3 cells and sympathetic neurons isolated from rat superior cervical ganglia (1518). Thus, POH-induced c-Jun activity may also be associated with apoptosis of human as well as rodent cells. However, target genes critical for this alternative c-Jun function are yet to be identified (1518).

In summary, we have shown that POH transiently induces c-jun and c-fos expression and c-Jun phosphorylation. These events were associated with AP-1-dependent transcription of a reporter gene. Although we haven't examined the effect of POH on JNK activity yet, POH seems to modulate the JNK cascade in that c-Jun was phosphorylated at the JNK phosphorylation site. Taken together, transient induction of c-Jun by POH treatment of T47D-C4-2W cells may represent an early response to POH treatment relevant to cell death and/or apoptosis.


    Acknowledgments
 
This work was supported by PHS NIH grant R37CA-38128.


    Notes
 
2 To whom correspondence should be addressedEmail: gould{at}oncology.wisc.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 26, 1999; revised June 29, 1999; accepted June 29, 1999.