Roles of p38- and c-jun NH2-terminal kinase-mediated pathways in 2-methoxyestradiol-induced p53 induction and apoptosis

Keiji Shimada, Mitsutoshi Nakamura, Eiwa Ishida, Munehiro Kishi and Noboru Konishi1

Department of Pathology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, 634-8521, Japan

1 To whom correspondence should be addressed Email: nkonishi{at}naramed-u.ac.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, has been established to cause apoptosis of prostate cancer cells, the downstream effectors of the signaling remain unclear. In the current study, we investigated molecular mechanisms by which 2-ME induces apoptosis in human prostate cancer cell line, LNCaP. It was found that 2-ME mediates apoptosis through p53 induction. Nuclear factor kappaB (NF{kappa}B) was activated by 2-ME and closely regulated by the mitogen-activated protein kinase, p38. Inhibition of p38 or NF{kappa}B resulted in suppression of p53 induction and apoptosis. Moreover, we demonstrated that 2-ME activates the c-jun NH2-terminal kinase (JNK)/activation protein (AP)-1 pathway. Interestingly, inhibition of JNK strongly reduced Bcl-2 phosphorylation by 2-ME as well as p53 induction, and almost completely suppressed 2-ME-induced apoptosis. Androgen stimulation with dihydrotestosterone, a major endogenous metabolite of testosterone, also significantly inhibited p38/NF{kappa}B and JNK/AP-1 activation and apoptosis. The results suggest that not only p53 induction through p38/JNK-dependent NF{kappa}B/AP-1 activation but also JNK-dependent Bcl-2 phosphorylation are required for 2-ME-induced apoptosis; moreover, inhibition of these pathways may be involved in androgen-mediated resistance to apoptosis.

Abbreviations: AP, activation protein; DHT, dihydrotestosterone; Hyg B, Hygromycin B; JNK, c-jun NH2-terminal kinase; 2-ME, 2-methoxyestradiol; MAP, mitogen-activated protein; PARP, poly-ADP ribose polymerase.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prostate cancer, a common disease in western countries, is becoming increasingly common in Japan. Improved procedures for surgical intervention and radiation treatment have significantly reduced the number of fatalities; however, this type of cancer is resistant to chemotherapy, and there is still no effective cure for patients with advanced disease (13). Identification of effective chemical agents would have an important impact on prostate cancer morbidity and mortality (4), and a number of investigators have focused on 2-methoxyestradiol (2-ME) as a novel therapy for advanced prostate cancer. This is a natural metabolic by-product of estrogen that inhibits tubulin polymerization and possesses growth inhibitory and cytotoxic activity (58). Notably, there are no adverse reactions such as hair loss, intestinal damage or infections (9). Despite being a natural derivative of estrogen, 2-ME binds only weakly to the estrogen receptors and its antiproliferative and cytotoxic effects on tumors are not considered to be receptor-mediated (10,11). This has also been demonstrated with prostate cancer cells (5,12,13), the vast majority of which express estrogen receptor {alpha} at the protein and mRNA levels (14). Recent reports showed that 2-ME can potently inhibit superoxide dismutase, resulting in enhanced formation of reactive oxygen species and thus tumor cell toxicity (15,16). Other authors (17) have demonstrated that 2-ME induces cell cycle arrest at the G2/M phase independently of p53. In contrast, induction of wild-type p53 appears to play a critical role in 2-ME-induced apoptosis of human lung cancer cells (18) and colorectal cancer (19). Thus, studies of mechanisms of 2-ME in cancer-derived cell lines have provided conflicting evidence. In the current study, we focused on whether p53 contributes to the execution of 2-ME-induced apoptosis. In an attempt to address this question, we used a human prostate cancer cell line, LNCaP, which carries wild-type p53 and is androgen sensitive. LNCaP cells are reminiscent of early-stage prostate cancer, but they share characteristics of advanced prostate cancers in that they do not undergo apoptosis in response to androgen deprivation (20,21). Because p53 mutations are not very common in primary prostate cancers including advanced stage lesion, the data using LNCaP might be relevant to the clinical use of 2-ME.

The tumor suppressor gene, p53 plays an important role in the induction of apoptosis by various anticancer drugs (22,23). The sequence of p53 response element is almost identical to a NF{kappa}B binding site and upon stimulation of cells, the inhibitory protein I{kappa}B leads to ubiquitin-dependent degradation, then NF{kappa}B translocates into the nucleus and binds to the promoter or enhancer region of the target genes (24), including both the anti-apoptotic gene, Bcl-2 (25) and the pro-apoptotic gene, p53 (26). The results suggest that whether NF{kappa}B functions as a pro-apoptotic or anti-apoptotic factor is dependent on the specific regulation of apoptosis-related genes (27). It has been demonstrated that NF{kappa}B-induced activation of p53 is essential for the induction of apoptosis by genotoxic agents (27,28), whereas others have reported that induction of p53 can lead to activation of NF{kappa}B that correlates with apoptotic activity (29). Thus, NF{kappa}B-dependent signals act either upstream or downstream in the pathway of p53.

Mitogen-activated protein (MAP) kinases transduce signals from the cell membrane to the nucleus and thus contribute to a wide spectrum of cellular processes including cell growth, differentiation and apoptosis. Activation of extracellular stress-regulated kinase contributes to cell differentiation, proliferation and survival (30,31), whereas c-jun NH2-terminal kinase (JNK) and p38 in contrast, are activated by pro-inflammatory cytokines and environmental stresses and promote apoptosis (32,33). There are a number of reports indicating an association of MAP kinases with p53 induction and stabilization: upon activation, JNK phosphorylates several transcriptional factors like c-jun, thereby regulating the expression of the gene including p53. It is well known that post-translational stabilization of p53 is achieved through phosphorylation at multiple sites by protein kinases including JNK or p38, p53 then escaping from ubiquitin-dependent degradation (34,35).

Bcl-2 has been found to be a member of inhibitors of apoptosis, and when inactivated through their phosphorylation, it drives the cells towards apoptosis. The Bcl-2 phosphorylation is a feature of treatment with microtubule-interfering agents including taxol or 2-ME (36). The available findings indicate that p53 activation and Bcl-2 phosphorylation are key signals for the execution of 2-ME-induced apoptosis, and that MAP kinases are also important. However, it remains unclear to what degree each molecule contributes apoptosis and how signals interact. The present study provided an indication that the key up-stream signal is activation of JNK and p38: JNK is essentially associated with Bcl-2 phosphorylation and contributes to p53 induction through the c-jun/activation protein (AP)-1 pathway, and p38-dependent NF{kappa}B activation is required for p53 induction in LNCaP. In addition, we found that androgen stimulation with dihydrotestosterone (DHT) strongly suppresses apoptosis through inhibition of 2-ME-induced JNK/p38 activation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture, plasmids and chemicals
The human prostate cancer cell line, LNCaP, was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI supplemented with 10% fetal bovine serum. Plasmid Myc-tagged dominant-negative MKK7 [MKK7 KL, in which Lys (K) 165 is replaced by Leu (L)] was cloned by PCR and ligated into a Myc-tagged expression vector (Invitrogen, Tokyo, Japan) (3). A dominant-negative I{kappa}B{alpha} vector (the pCMV-I{kappa}B{alpha} mutant) was purchased from Clontech Laboratories (Tokyo, Japan), in which the N-terminal 36 amino acids, including serines 32 and 36 were deleted, and ligated into a FLAG-tagged expression vector (37). The expression vector for the Hygromycin-resistant gene (pTK-Hyg) was also from Clontech. Anti-poly-ADP ribose polymerase (PARP) polyclonal antibodies were from Transduction Laboratories; the anti-c-jun and anti-I{kappa}B{alpha} polyclonal antibodies were from Cell Signaling Technology (Beverly, MA); the anti-actin monoclonal antibody was from Oncogene Research Products (Darmstadt, Germany); control and antisense oligonucleotides of c-jun and p53 were from Biomol Research Laboratories (PA) and DHT was from Nacalai Tesque (Kyoto, Japan). 2-ME and anti-FLAG monoclonal antibodies were from Sigma-Aldrich (Tokyo, Japan); anti-c-Myc antibodies were from Clontech; anti-JNK1, anti-p38 and anti-Bcl-2 antibodies as well as a recombinant glutathione S-transferase fusion protein of the N-terminal peptide of c-jun (amino acids 1–79), ATF-2, were from Santa Cruz Biotechnologies (Santa Cruz, CA); Bay 117082 was from Alexis Biochemicals Japan (Tokyo, Japan). The specific inhibitor of p38, SB203580 was purchased from Calbiochem (San Diego, CA).

Transfection with nonsense or antisense p53 or the c-jun oligonucleotide
The LNCaP line was seeded at 5 x 105 cells/well in 6 well plates and transfected with NSO (5'-GGAGCCAGGGGGGAGCAGGG-3' for p53 and 5'-ACTGCAAAGATGGAAACG-3' for c-jun) or ASO (5'-CCCTGCTCCCCCCTGGCTCC-3' for p53 and 5'-CGTTTCCATCTTTGCAGT-3' for c-jun) (38,39). Each transfection was performed using LipofectAMINE (Invitrogen) according to the manufacturer's protocol. At 48 h thereafter, cells were stimulated with the indicated reagents and the expression of c-jun or p53 was analyzed by western blotting using anti-c-jun and -p53 antibodies.

Stable transfection of expression vectors
To establish cell lines stably expressing vectors, LNCaP cells were seeded at 5 x 105 cells/well in 6 well plates, cultured in fresh medium for 24 h and then co-transfected with the pTK-Hyg vector harboring the Hygromycin B (Hyg B)-resistant gene and the expression vector encoding FLAG-tagged d/n I{kappa}B{alpha} or Myc-tagged d/n MKK7 using LipofectAMINE (Invitrogen). Selection was carried out in Hygromycin B, and resistant colonies were isolated after ~6 weeks as described previously (3,37), and identified by western blotting using anti-FLAG, anti-I{kappa}B{alpha} and anti-Myc antibodies.

Preparation of cell lysates; immunoprecipitation and western blotting analysis
LNCaP cells were washed once with PBS and suspended in lysis buffer [40 mM HEPES, pH 7.4, with 10% glycerol, 1% Triton X-100, 0.5% Nonidet P-40 (NP-40), 150 mM NaCl, 50 mM NaF, 20 mM b-glycerol phosphate, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride and 0.1 mM vanadate] containing a protease inhibitor mixture (1 µg/ml aprotinin, leupeptin and pepstatin). Cells lysates were cleared by centrifugation at 15 000 r.p.m. for 30 min. In preparation for immunoprecipitation assays, cell lysates were incubated with 2 µg of anti-JNK1 and anti-p38 polyclonal antibodies for the JNK and p38 kinase assays, respectively, and precipitated with protein A– or G–Sepharose (Amersham Pharmacia Biotech., Tokyo, Japan). Cell lysates or immunoprecipitates were resolved on SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were then blocked in TBST buffer (20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl and 0.1% Tween 20) with 5% skimmed milk at room temperature for 1 h, and then incubated with anti-JNK1 or anti-p38 antibody for 1 h. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech.), further washed with TBST, and assayed for peroxidase activity using an enhanced chemiluminescence detection system.

Flow cytometry and apoptosis analysis
LNCaP cells were stably transfected with or without FLAG-tagged dominant-negative I{kappa}B{alpha} or Myc-tagged dominant-negative MKK7 (MKK7-KL), or transiently transfected with antisense and nonsense oligonucleotides of c-jun or p53. After incubation, cells were stimulated with or without 2-ME, harvested, centrifuged and fixed in 80% ethanol. The cells were re-suspended in phosphate-buffered saline containing 50 µg/ml propidium iodide, 0.1% NP-40, and 100 µg/ml RNase A (Sigma), and incubated for 1 h. Apoptotic cells were determined by their hypochromic, subG1, staining profiles and cells and their numbers were analyzed by flow cytometry (37).

Reverse transcription–PCR
Total RNA was extracted using Trizol reagent and subjected to reverse transcription (RT) and PCR with Ready-to-Go beads (Pharmacia) according to the manufacturer's protocol. PCR conditions were 95°C for 30 s, 68°C for 30 s and 72°C for 1 min for a total of 36 cycles. The PCR primers were for p53 sense 5'-GCGGACAGGAATTGAAGCGGA-3', and antisense, 5'-TCTGAAGGCTGCAGGCTCTCT-3', and for glyceraldehyde-3-phosphate dehydrogenase sense 5'-ACCACAGTCCATGCCATCAC-3', and antisense, 5'-TCCACCACCCTGTTGCTGTA-3'. PCR products were analyzed on a 1.5% agarose gel and visualized by ethidium bromide staining.

Electrophoretic mobility shift assays
DNA binding activity of NF{kappa}B or AP-1 was analyzed using electrophoretic mobility shift assays as described previously (3,40). In brief, 10 µg of nuclear extracts prepared from cells stimulated earlier with various reagents were incubated with a 32P-end labeled 22mer double-stranded NF{kappa}B oligonucleotide, 5'-AGTTGAGGGGACTTTCCCAGGC-3' or the AP-1 oligonucleotide, 5'-CGCTTGATGAGTCAGCCGGAA-3', for 15 min at 37°C. The DNA–protein complexes formed were separated from free oligonucleotide using 6.6% native polyacrylamide gels. A 1 µg aliquot of antibodies to p65, p50 and c-jun was used to examine the binding of NF{kappa}B or AP-1 to DNA (41). The specificity was also analyzed by competition with unlabeled oligonucleotides.

Transient transfection and NF{kappa}B or AP-1 luciferase activity assays
LNCaP cells were transiently transfected with 1 µg of plasmid containing luciferase and NF{kappa}B or AP-1 response elements (pNF{kappa}B-TA-luc or pAP-1-TA-luc) or a control vector with no response elements (pTA-luc). The plasmids were purchased from Clontech. After 24-h incubation, cells were stimulated with 2-ME for 12 h, harvested and analyzed for luciferase activity, normalized to ß-galactosidase activity, according to the manufacturer's protocol.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Induction of p53 is required for the execution of 2-ME-induced apoptosis of LNCaP cells
As shown in Figure 1A, 2-ME induced apoptosis in a dose-dependent manner, confirmed by the data for the cleavage of PARP. In addition, RT–PCR and western blot analyses showed that 10 µg/ml of 2-ME significantly up-regulated mRNA and protein expression of p53 at 24 h (Figure 1B). To estimate the requirement of p53 for 2-ME-induced apoptosis, we examined the effects of transfection with the p53 antisense oligonucleotide. As shown in Figure 1C, this block inhibited cleavage of PARP and apoptosis of LNCaP cells. The results reveal that 2-ME transactivates and induces p53 expression that plays a critical role in 2-ME-induced apoptosis, consistent with a previous report (18).



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Fig. 1. p53 induction contributes to 2-ME-induced apoptosis in LNCaP cells. (A) LNCaP cells were treated with the diluent, dimethyl sulfoxide, or the indicated concentrations of 2-ME for 48 h, then harvested and the percentage of apoptotic cells was measured by flow cytometric analysis (left panel), and cleavage of PARP determined by western blotting with an anti-PARP antibody (right panel) as described in the Materials and methods. The data are average values with standard deviations (flow cytometric analysis; n = 4). (B) Cells were treated with 10 µg/ml of 2-ME for 24 h, then harvested. After isolation of total RNA, RT–PCR was performed to detect p53 and glycerolaldehyde-3-phosphate dehydrogenase transcripts (upper panel). After lysis of cells, expression of p53 and actin were determined by western blotting with anti-p53 and anti-actin antibodies, as described in the Materials and methods (lower panel). (C) Cells were transfected with nonsense and antisense p53 oligonucleotides. After 24 h cultivation, they were treated with 10 µg/ml of 2-ME for 24 h and p53 expression was determined by western blotting (left panel). Cells were treated with the indicated concentrations of 2-ME for 48 h, then percent apoptosis and cleavage of PARP were determined as described in (A) (middle and right panels).

 
p38-dependent NF{kappa}B activation is necessary for 2-ME-mediated p53 induction and apoptosis
To estimate the key signals involved in p53 induction by 2-ME, we first focused on the role of NF{kappa}B activation. A 10 µg/ml sample of 2-ME increased the DNA binding activity of NF{kappa}B, the transcriptional activation of the NF{kappa}B-responsive promote and I{kappa}B{alpha} degradation in LNCaP as assessed by electrophoretic mobility shift assay, transient transfection luciferase assay and western blotting (Figure 2A–C). We investigated whether inhibition of NF{kappa}B activation by the I{kappa}B{alpha} kinase-specific inhibitor, Bay117082, or stable overexpression of a dominant-negative mutant form of I{kappa}B{alpha} affects 2-ME-induced apoptosis. We selected two clones of LNCaP cells stably overexpressing FLAG-tagged plasmid encoding dominant-negative mutant I{kappa}B{alpha} (clones 2 and 12) and a Hygromycin-resistant clone without the mutant gene as the control (Hyg B). Expression was determined by western blotting using anti-I{kappa}B{alpha} and anti-FLAG antibodies. Bay117082 significantly inhibited the 2-ME-induced increase in DNA binding activity, transcriptional activation of NF{kappa}B and I{kappa}B{alpha} degradation (Figure 2A and B). The same results were demonstrated with both clones 2 and 12, the mutant form of I{kappa}B{alpha} not being degraded in response to 2-ME. As shown in Figure 2D and E, NF{kappa}B inhibition by Bay117082 or stable overexpression of the dominant-negative mutant form of I{kappa}B{alpha} strongly reduced 2-ME-mediated p53 induction and apoptosis in LNCaP cells. The inhibitory effects closely paralleled the degree of NF{kappa}B inhibition.



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Fig. 2. The role of NF{kappa}B activation in 2-ME-mediated p53 induction and apoptosis in LNCaP cells. (A) and (B) LNCaP cells were transfected with the Hyg B resistance gene (control) with an empty vector or a FLAG-tagged vector encoding the dominant-negative mutant form of I{kappa}B{alpha} (d/n I{kappa}B{alpha}). After incubation in the presence of Hygromycin B, one clone for control (Hyg B) and two clones for d/n I{kappa}B{alpha} transfectants (clones 2 and 12) were selected. The expression of endogenous I{kappa}B{alpha} and exogenous d/n I{kappa}B{alpha} were analyzed by western blotting using anti-I{kappa}B{alpha} and anti-FLAG antibodies (A, right panel). LNCaP cells pre-treated with or without 10 µM of Bay 117082 (Bay), or Hygromycin B, clones 2 and 12 were stimulated with 10 µg/ml of 2-ME for 12 h, then harvested and NF{kappa}B activity was determined by electrophoretic mobility shift assay (A) or transient transfection luciferase assay (B) as described in the Materials and methods. For antibody perturbation experiments 1 µg of p50 or p65 supershift antibodies were used. (C) and (D) LNCaP cells pre-treated with or without Bay 117082 (Bay), or Hygromycin B, clones 2 and 12 were stimulated with 10 µg/ml of 2-ME. After 12 h cultivation, degradation of I{kappa}B{alpha}, the expression of d/n I{kappa}B{alpha} and actin were analyzed by western blotting with anti-I{kappa}B{alpha}, anti-FLAG and anti-actin antibodies, respectively (C). After 24 h cultivation, p53 transcripts and protein expression were determined by RT–PCR and western blotting, respectively. (E) LNCaP cells pre-treated with or without 10 µM of Bay 117082 (Bay), or Hygromycin B, clones 2 and 12 were stimulated with 10 µg/ml of 2-ME for 48 h. Percent apoptosis was determined by flow cytometric analysis as described in the Materials and methods. The data are average values with standard deviations (B and E) (n = 4).

 
Next, we examined the effect of p38 activation on 2-ME-induced apoptosis. As shown in Figure 3A, 10 µg /ml of 2-ME activated p38 at 3 h after stimulation, then the activity was decreased. The specific inhibitor of p38, SB203580, strongly inhibited 2-ME-induced p38 activation (Figure 3A, right panel), whereas activation of other MAP kinases such as extracellular signal-regulated kinase and JNK by 2-ME were not affected (data not shown). Treatment with SB203580 also significantly inhibited the 2-ME-induced increase in DNA binding activity of NF{kappa}B and transcriptional activity of the NF{kappa}B-responsive promoter as determined by electrophoretic mobility shift assay and transient transfection luciferase assay, as well as the I{kappa}B{alpha} degradation (Figure 3B). In addition, inhibition of p38 resulted in suppression of 2-ME-induced p53 induction at both mRNA and protein levels, and apoptosis (Figure 3C and D) of LNCaP cells. Taken together, the results indicate that p38 activation is closely associated with 2-ME-mediated p53 induction and apoptosis through regulation of NF{kappa}B activity.



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Fig. 3. 2-ME-induced activation of NF{kappa}B is regulated by p38. (A) LNCaP cells were pre-treated with or without 20 µM of SB 203580 (SB) and stimulated by 10 µg/ml of 2-ME for the indicated times, harvested, lysed and immunoprecipitated with anti-p38 antibody. An in vitro kinase assay of the immunoprecipitated p38 was performed and radioactive glutathione S-transferase (GST)–ATF2 (p-ATF2) was quantified by autoradiography. The immunoprecipitated p38 was analyzed by immunoblotting using the anti-p38 antibody. (B) Cells were stimulated with 10 µg/ml of 2-ME for 12 h, then NF{kappa}B activity was determined by electrophoretic mobility shift assay (left panel) or transient transfection luciferase assay (right panel). Degradation of I{kappa}B{alpha} was analyzed by western blotting using the anti-I{kappa}B{alpha} antibody (left lower panel). (C) Cells were stimulated with 10 µg/ml of 2-ME for 24 h. p53 transcripts and protein expression were determined by RT–PCR and western blotting, respectively. (D) Cells were stimulated with 10 µg/ml of 2-ME for 48 h. Then, percent apoptosis and cleavage of PARP were determined by flow cytometric analysis (right panel) and western blotting (left panel), respectively. The data are the average values with standard deviations (B and D) (n = 4).

 
JNK-dependent activation of c-jun/AP-1 is required for 2-ME-mediated p53 induction and apoptosis
As shown in Figure 4A, JNK was rapidly activated by 10 µg/ml of 2-ME at 1 h, as assessed by in vitro kinase assay. DNA binding activity and transcriptional activity of the promoter of activation protein (AP)-1 were also regulated by 2-ME (Figure 4B). Increase of DNA binding of AP-1 started at 3 h and peaked at 6 h, slightly later than the time of JNK activation. To investigate the roles of JNK and AP-1 activation in 2-ME-induced apoptosis, we performed transfection with antisense oligonucleotide of c-jun or with dominant-negative mutant form of the specific kinase of JNK, MAP kinase kinase 7 (MKK7-KL) (37,42,43). 2-ME-induced JNK activation and increase in DNA binding activity of AP-1 was significantly inhibited in both these clones and in cells transfected with antisense oligonucleotide of c-jun (Figure 4D). The same results were also obtained with transient transfection luciferase assays (data not shown). In addition, strong reduction of c-jun expression or inhibition of JNK resulted in the suppression of p53 induction and apoptosis by 2-ME (Figure 4E and F). These results indicate that JNK activation and phosphorylation of c-jun are centrally involved in AP-1 activation, this playing a critical role in 2-ME-mediated up-regulation of p53 and apoptosis.



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Fig. 4. JNK-dependent c-jun/AP-1 activation contributes to 2-ME-mediated p53 induction and apoptosis in LNCaP. (A) LNCaP cells were stimulated with 10 µg/ml of 2-ME for the indicated times, then harvested, lysed and immunoprecipitated with anti-JNK1 antibody. An in vitro kinase assay of the immunoprecipitated JNK1 was performed and radioactive GST–c-jun (p-c-jun) was quantified by autoradiography. The immunoprecipitated JNK1 was analyzed by immunoblotting using the anti-JNK1 antibody. (B) Cells were stimulated with 10 µg/ml of 2-ME for the indicated times, then AP-1 activation was determined by electrophoretic mobility shift assay (left panel) or transient transfection luciferase assay (right panel; incubation time was 6 h). For antibody perturbation experiments 1 µg of c-jun supershift antibody was employed. (C) Cells were transfected with the Hyg B resistance gene (control) with an empty vector or with a Myc-tagged vector encoding the dominant-negative mutant form of MKK7 (d/n MKK7). After incubation in the presence of Hygromycin B, one clone for control (Hyg B) and two clones for d/n MKK7 transfectants (clones 3 and 5) were selected. The expression of d/n MKK7 was determined by western blotting using the anti-MKK7 antibody (left middle panel). Normal cells in the presence of Hyg B, clones 3 and 5 were stimulated with 10 µg/ml of 2-ME for 1 h, then cells were harvested, lysed and immunoprecipitated by anti-JNK1 antibody. An in vitro kinase assay of the immunoprecipitated JNK1 was performed and radioactive GST–c-jun (p-c-jun) was quantified by autoradiography. The immunoprecipitated JNK1 was analyzed by immunoblotting using the anti-JNK1 antibody (left panel). Cell were transfected with nonsense (NSO) or antisense oligonucleotide (ASO) of c-jun and incubated for 24 h. The expression of c-jun was determined by western blotting using the anti-c-jun antibody (right panel). (D) LNCaP cells treated with c-jun nonsense (NSO) and antisense oligonucleotides (ASO), or Hyg B and clones 3 and 5 were stimulated with 10 µg/ml of 2-ME for 6 h. AP-1 activity was determined by electrophoretic mobility shift assay. (E) and (F) LNCaP cells treated with nonsense (NSO) or antisense c-jun oligonucleotides (ASO) or Hygromycin B and clones 3 and 5 were stimulated with 10 µg/ml of 2-ME. (E) After 24 h cultivation, p53 transcripts and protein expression were determined by RT–PCR and western blotting, respectively. (F) After 48 h cultivation, percent apoptosis was determined by flow cytometric analysis as described in the Materials and methods. The data are the average values with standard deviations (B, right panel and F) (n = 4). (G) and (H) LNCaP cells treated with nonsense (NSO) and antisense c-jun oligonucleotides (ASO) or Hygromycin B and clones 3 and 5 were stimulated with 10 µg/ml of 2-ME for the indicated times. Phosphorylation of Bcl-2 was analyzed by western blotting with anti-Bcl-2 antibody.

 
Inhibition of 2-ME-induced apoptosis was more prominent in the LNCaP cells stably expressing MKK7-KL than in the presence of c-jun antisense oligonucleotide, even though AP-1 activation and p53 induction by 2-ME were similarly suppressed under both conditions. It has been shown that phosphorylation and inactivation of Bcl-2 are essential for apoptosis by microtubule-interfering agents such as taxol and 2-ME (42). Thus, we investigated whether JNK is associated with Bcl-2 phosphorylation by 2-ME in LNCaP cells. As shown in Figure 4G, Bcl-2 was phosphorylated by 10 µg/ml of 2-ME in a time-dependent manner, and this was strongly inhibited in cells expressing MKK7-KL but not in the presence of the c-jun antisense oligonucleotide. The results clearly indicate that JNK activation is closely associated not only with induction of c-jun/AP-1-dependent p53 but also Bcl-2 phosphorylation, and this can explain the data for apoptosis illustrated in Figure 4F.

Androgen inhibits 2-ME-induced JNK/p38 activation and apoptosis
Finally, we examined the effect of androgen stimulation with a major endogenous metabolite of testosterone, DHT, on 2-ME-induced apoptosis (Figure 5A). Co-treatment with 10 nM significantly inhibited 2-ME-mediated up-regulation of p53 transcript and protein expression, and apoptosis by 10 µg/ml of 2-ME in LNCaP cells (Figure 5B). As shown in Figure 5C and D, activation of both JNK/p38 and its dependent p53 induction and Bcl-2 phosphorylation were also significantly inhibited by the treatment with DHT.



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Fig. 5. The effects of DHT on 2-ME-induced apoptosis. (A) LNCaP cells were pre-treated with or without 10 nM of DHT for 12 h then stimulated by 10 µg/ml of 2-ME for 48 h. Percent apoptosis and cleavage of PARP were determined by flow cytometric analysis (left panel) and western blotting (right panel), respectively. (B) Cells were pre-treated with or without 10 nM of DHT for 12 h, then stimulated with 10 µg/ml of 2-ME for 24 h. p53 transcripts and protein expression were determined by RT–PCR and western blotting, respectively. (C) Cells were pre-treated with or without 10 nM of DHT for 12 h, then stimulated with 10 µg/ml of 2-ME. After 1 h cultivation, they were harvested, lysed and immunoprecipitated with anti-JNK antibody. An in vitro kinase assay of the immunoprecipitated JNK1 was performed and radioactive GST–c-jun (p-c-jun) was quantified by autoradiography. The immunoprecipitated JNK1 was analyzed by immunoblotting using the anti-JNK1 antibody (left upper panel). After 6 h cultivation, AP-1 activity was determined by electrophoretic mobility shift assay (left lower panel). After 48 h cultivation, phosphorylation of Bcl-2 was analyzed by western blot with the anti-Bcl-2 antibody (right lower panel). (D) Cells were pre-treated with or without 10 nM of DHT for 12 h, then stimulated with 10 µg/ml of 2-ME. After 3-h cultivation, they were harvested, lysed and immunoprecipitated with anti-p38 antibody. An in vitro kinase assay of the immunoprecipitated p38 was performed and radioactive GST–ATF2 (p-ATF2) was quantified by autoradiography. The immunoprecipitated p38 was analyzed by immunoblotting using the anti-p38 antibody (left panel). After 12 h cultivation, NF{kappa}B activity was determined by electrophoretic mobility shift assay (right upper panel) and degradation of I{kappa}B{alpha} assessed by western blotting (right lower panel).

 

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 Abstract
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 References
 
We demonstrated here for the first time that p38/JNK-dependent NF{kappa}B/AP-1 activation directly contributes to 2-ME-induced apoptosis through regulation of p53. Several studies have indicated that 2-ME-induced activation of JNK is involved in phosphorylation/inactivation of the anti-apoptotic protein, Bcl-2 (13,43). The same mechanism appears to operate in apoptosis by paclitaxel (46), mediated by inhibition of microtubule dynamics. In contrast, other investigators have demonstrated that protection against paclitaxel-induced apoptosis by epidermal growth factor is caused by activation of JNK in cervical cancer cells (47). MAP kinases such as p38 and the extracellular signal-regulated kinase have been shown to be associated with sensitivity to apoptosis by microtubule-interfering drugs. For example, subsequent exposure of a paclitaxel treated human monocytic leukemia cell line, U937, to the MAP kinase kinase kinase inhibitor, PD98059, inhibited paclitaxel-induced activation of extracellular signal-regulated kinase and induced a dramatic increase in p38 activation and apoptosis (46,48). However, Yu et al. (46) found extracellular signal-regulated kinase-dependent I{kappa}B kinase activation to be essential for paclitaxel-induced apoptosis. Recently, it has been suggested that microtubule-interfering agent-induced activation of p38 contributes to transcription of cyclooxygenase-2 (49), which is known to make prostate cancer cells resistant to chemotherapy and inhibition of which has emerged as a novel therapeutic intervention for prostate cancer (5054). Thus, the signaling pathways of MAP kinases mainly involved in 2-ME-induced apoptosis appear to differ with the cancer cell type. The current results clearly indicate that 2-ME-induced apoptosis is mediated through p53 induction, which requires both p38-dependent NF{kappa}B and JNK-dependent c-jun/AP-1 signals in the prostate cancer cell line, LNCaP. Kirch et al. (55) demonstrated that the AP-1 motif in the human p53 promoter binds to c-jun and the NF{kappa}B motif binds to p50/p65, whereas the levels of endogenous p53 mRNA were reduced in the presence of c-jun, p50 and p65 antisense oligonucleotides in various types of tumors. We investigated the effect of extracellular stress-regulated kinase, but inhibition of ERK by overexpression of the dominant-negative mutant form or MAP kinase kinase kinase inhibitors such as PD98059 or U0126 did not significantly affect p53 regulation or the sensitivity to 2-ME-induced apoptosis (data not shown).

In the present study, we established that induction of p53 plays an important role in 2-ME-induced apoptosis, although transfection of an antisense oligonucleotide did not completely abolish cell death. The inhibitory effects on apoptosis were similar to those in the presence of p38 and IKK inhibitors. In contrast, inhibition of JNK activation by transfection with MKK7-KL suppressed not only 2-ME-mediated p53 induction but also Bcl-2 phosphorylation, and almost completely blocked 2-ME-induced apoptosis. Taking into account all the available information, we conclude that Bcl-2 phosphorylation as well as p53 induction is necessary for complete induction of apoptosis by 2-ME, both being closely regulated by 2-ME-induced activation of JNK in LNCaP cells (Figure 6).



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Fig. 6. Schematic presentation of the p38/JNK-mediated regulation of 2-ME-induced apoptosis and the effects of androgen. Activation of p38 and JNK mediates p53 induction through transactivation of NF{kappa}B and c-jun/AP-1, and contributes to 2-ME-induced apoptosis in LNCaP cells. In addition to p53 induction, phosphorylation and inactivation of Bcl-2 by JNK is closely associated with apoptosis by 2-ME. Androgen blocks 2-ME-induced activation of JNK/p38-mediated apoptotic signals and strongly inhibits apoptosis.

 
Determination of whether NF{kappa}B activation functions as an anti-apoptotic or pro-apoptotic factor in anticancer drug-induced apoptosis is a complex problem. NF{kappa}B activation is believed to play an important role as a blocker of different types of apoptosis (29,5658). In the present study, overexpression of a dominant-negative I{kappa}B{alpha} or treatment with the I{kappa} kinase inhibitor, Bay117082 strongly reduced p53 induction and apoptosis by 2-ME in LNCaP cells and we can hypothesize that NF{kappa}B regulates p53 gene expression, although the possibility that 2-ME treatment increases stability of p53 mRNA transcripts must be borne in mind. The same role of NF{kappa}B was recently proposed with regard to paclitaxel-induced apoptosis (46,59). Moreover, a pro-apoptotic function of NF{kappa}B appears essential for nitric oxide-induced apoptosis in chondrocytes, and p38 activation by nitric oxide phosphorylates p53 at serine 15, resulting in stabilization and accumulation (27). In contrast, the 15 serine residue of p53 was not significantly modified by the stimulation with 2-ME at the concentrations used in the current study (data not shown).

We here could show that androgen stimulation with a major endogenous metabolite of testosterone, DHT, strongly suppresses 2-ME-induced apoptosis in LNCaP cells associated with significant inhibition of both JNK and p38 activation and suppression of p53 induction as well as Bcl-2 phosphorylation. Interactions between androgen stimulation and p53 activation have been described earlier, for example, Wang et al. have demonstrated that androgen receptor-mediated signals are linked to silencing of p21, which is one of the major targets (60). The present study indicated for the first time that inhibition of JNK/p38 is involved in blockage of p53-dependent signaling by an androgen. The inhibitory effect of DHT was more prominent for p38 activation than for JNK activation by 2-ME. Kimura et al. (61) have reported inhibition of NF{kappa}B activation and apoptosis induced by tumor necrosis factor {alpha}, with or without irradiation, but the effect of the androgen on cell death was not mediated by interference with the NF{kappa}B signaling pathway in LNCaP cells. Even in the same cancer cell line, it seems that the activation of NF{kappa}B has different effects on apoptosis according to the type of stimulation.

In summary, p53 induction through both p38-dependent NF{kappa}B activation and JNK-dependent c-jun/AP-1 activation plus Bcl-2 phosphorylation are required for the execution of 2-ME-induced apoptosis in the human prostate cancer cell line, LNCaP. In addition, DHT can inhibit 2-ME-induced activation of MAP kinases such as JNK/p38 playing central roles in the execution of apoptosis (Figure 6). We should identify whether the mechanisms are relevant to the majority of human prostate cancer by investigating other lines or primary cultures from prostate cancer patients. As 2-ME is generally regarded to have great potential as a chemotherapeutic agent, our current findings may be significant for development of a novel cancer therapy.


    Acknowledgments
 
This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (14770103), Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 25, 2003; revised March 25, 2003; accepted March 27, 2003.