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Originally published In Press as doi:10.1074/jbc.M702938200 on June 15, 2007

J. Biol. Chem., Vol. 282, Issue 33, 24364-24372, August 17, 2007
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Modulation of Intracellular Signaling Pathways to Induce Apoptosis in Prostate Cancer Cells*

Jinjin Guo{ddagger}, Tongbo Zhu{ddagger}, Zhi-Xiong J. Xiao§, and Chang-Yan Chen{ddagger}1

From the {ddagger}Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215 and the §Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, April 6, 2007 , and in revised form, June 13, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
An understanding of the molecular pathways defining the susceptibility of prostate cancer, especially refractory prostate cancer, to apoptosis is the key for developing a cure for this disease. We previously demonstrated that up-regulating Ras signaling, together with suppression of protein kinase C (PKC), induces apoptosis. Dysregulation of various intracellular signaling pathways, including those governed by Ras, is the important element in the development of prostate cancer. In this study, we tested whether it is possible to modulate the activities of these pathways and induce an apoptotic crash among them in prostate cancer cells. Our data showed that DU145 cells express a high amount of JNK1 that is phosphorylated after endogenous PKC is suppressed, which initiates caspase 8 cleavage and cytochrome c release, leading to apoptosis. PC3 and LNCaP cells contain an activated Akt. The inhibition of PKC further augments Akt activity, which in turn induces ROS production and the accumulation of unfolded proteins in the endoplasmic reticulum, resulting in cell death. However, the concurrent activation of JNK1 and Akt, under the condition of PKC abrogation, dramatically augment the magnitude of apoptosis in the cells. Thus, our study suggests that Akt, JNK1, and PKC act in concert to signal the intracellular apoptotic machinery for a full execution of apoptosis in prostate cancer cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been well documented that oncogenes (such as myc or ras) and their downstream effectors cannot only promote cell growth, but also, under certain circumstances, elicit programmed cell death or apoptosis (1-4). Mutational activation of the Ras gene is a key event in human cancer development (5-10). In the process of transformation, a persistent increase in Ras activity up-regulates its downstream effector pathways, leading to the phosphorylation and activation of pro-growth transcriptional factors (5-10). Despite its central involvement in cell growth and differentiation, we and others have demonstrated that treatment with PKC2 inhibitors can induce apoptosis in various types of cells overexpressing oncogenic v-Ha or Ki-ras (11-16). In addition, stress-related kinases, such as c-Jun kinase (JNK)/p38 functions as Ras downstream effectors to initiate apoptosis (4).

Ras governs multiple downstream effector pathways, such as Raf/MAP/ERK, PI3K/Akt, JNK/p38, and RalGSD (5-10). In response to mitogenic stimulation, Ras is activated, which in turn causes the plasma membrane translocation of the Raf/MAPK/ERK cascade, resulting in phosphorylation of transcription factors and other proteins (17, 18). The activation of PI3K or RalGDS has been suggested to be involved in the regulation of the rearrangement of cytoskeleton to promote growth-related signal transduction (19-23). Furthermore, it has been demonstrated that in response to PKC inhibition, JNK1 acts as an intermediator to redirect Ras-mediated signaling for the initiation of caspase cascade (16). However, the suppression of JNK1 only partially blocked the apoptotic process, indicating that other apoptotic factors are involved in this process. The molecular mechanisms of apoptosis regulated by Ras remain unclear.

PI3K cannot only be activated by Ras, but also by receptors or nonreceptor protein-tyrosine kinases (24-27). Through activating Akt, PI3K has been shown to be involved in pro-survival activities (28-30). PTEN is an antagonist of PI3K, which is often mutated or deleted in various types of tumors, including prostate carcinoma (31-35). Increases of Akt activity have been observed to be associated with the development of prostate cancer, through affecting cell growth and angiogenesis (36). It has also been reported that the PI3K/Akt pathway regulates apoptosis, in which the kinases are either pro-apoptotic or anti-apoptotic, depending upon the types of stimuli and circumstances (37, 38). For example, Akt controls the status of several enzymes (such as NADPH oxidase) to promote the generation of ROS, the levels of which determine the outcomes (cell proliferation or death) (39, 40).

The ER serves as the site for synthesis, folding, modification, and trafficking of proteins, and plays a critical role in the maintenance of homeostasis (41-44). Pharmacological interference with ER function triggers the accumulation of misfolded proteins, resulting in the adaptive ER stress response program named the unfolded protein response (UPR) (45, 46). Activation of oncogenes has been demonstrated to be able to trigger the UPR (47-49). Studies also showed that, under persistent ER stress, the UPR plays a significant role in the initiation of apoptosis (50-52). The emerging evidence indicates that the ER, like other subcellular compartments, is a focal site for the initiation of programmed cell death pathways (50-52). Multiple ER stress-induced factors sensitize different intracellular targets to execute apoptotic programs. For example, GADD153 has been shown to heterodimerize with other CAAT/enhancer-binding protein family members and be able to induce apoptosis or regulate growth arrest (53-55). In mouse embryotic fibroblasts, the up-regulation of GADD153 correlates with the onset of PUMA- or NOXA-induced cell death (51).

We previously demonstrated that Ras mutation, together with abrogation of PKC, are synthetically lethal (11-16). Therefore, in this study, we tested whether abnormalities in Ras downstream effectors are able to induce apoptosis under PKC-deficient conditions in prostate cancer cells. The results demonstrated that the cooperation of JNK1, Akt, and PKC sensitizes prostate cancer cells to apoptosis through the activations of caspase 8 and the up-regulation of ROS.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents—LNCaP, PC3, and DU145 cells are human prostate cancer cells (obtained from Dr. Z. Lou, Boston University School of Medicine) and cultured in RPMI1640 medium containing 10% fetal calf serum (Invitrogen). The v-Ha-ras, JNK, or active Akt are inserted in a retroviral vector.

For the suppression of GADD153, 20 µM sense (as control) or antisense oligos were added into cell cultures for 48 h, and subsequently the cells were re-fed with media containing a half-dose of the oligos every 2 days. Antibodies used in the study were from Santa Cruz Biotechnology. The anti-Ras and caspase 12 antibodies were from Cell Signaling Technology.

GO6976 is a PKC inhibitor that specifically suppresses the activity of the phorbol ester-dependent PKC isoforms (Calbiochem). JNK inhibitor I is a cell-permeable, biologically active peptide, which inhibits the activation domains of JNK (Calbiochem).

DNA Fragmentation Analysis—A flow cytometric analysis was performed using a FACScan (BD Biosciences). The data analysis was performed using the Cell-Fit software program (BD Biosciences). Cell-Fit receives data from the flow cytometer and provides real-time statistical analysis, computed at 1-s intervals, and also discriminates doublets or adjacent particles. Cells with sub-G0-G1 DNA contents after staining with propidium iodide were counted as apoptotic cells. In brief, following treatments, cells were harvested by trypsinization, washed with 1x cold phosphate-buffered saline, and then fixed in 70% cold ethanol. Afterward, cells were stained with 0.1 mg/ml propidium iodide containing 1.5 µg/ml RNase. Following incubation at room temperature for 2-4 h, DNA contents of cells were measured by a BD FACScan machine (BD Biosciences) and evaluated with BD FACStation software CellQuest.

Immunoblotting Analysis—After treatment, cells were washed in 1x phosphate-buffered saline and then lysed in the lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-114, 0.5% sodium deoxycholate, 0.1% SDS, containing 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin A) on ice for 30 min. The total protein concentrations in the cell lysates were normalized and adjusted to 0.4 M NaCl, 0.5% deoxycholate, and 0.05% SDS for immunoblotting (56). The samples were separated on a 10% SDS-PAGE gel and subsequently transferred to a nitrocellulose membrane.

Measurement of Ral Activation—After treatments, cells were lysed in a buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Iegpal CA-630, 0.25% sodium deoxycholate, 10% glycerol, and 25 mM NaF (56). Protein was normalized to 1 mg/ml and precipitated by mixing 1 mg of cell lysate with 20 µg of Ral-RBD overnight at 4 °C. The complexes were washed 5 times in lysate buffer, and then separated on a 12.5% SDS-PAGE gel. Proteins were transferred onto a polyvinylidene difluoride membrane and immunoblotted with a pan-Ras antibody (Oncogene Research Products).

Measurement of ROS—Cells, after the treatments, were washed with ice-cold phosphate-buffered saline and resuspended in 5 µg/ml of 2', 7'-dichlorodihydrofluorescein diacetate (Molecular Probes). Samples were incubated for 10 min at room temperature and analyzed immediately by a flow cytometer (57).

Preparation of Subcellular Fractions—After treatments, cells (1 x 108) were washed twice with 1x phosphate-buffered saline and resuspended in 1 ml of 1% Triton X-114 lysis buffer (11). The cell suspensions were transferred to a 1-ml syringe and sheared by being passed 40 times through a 25-gauge needle. The lysates were centrifuged at 280 x g for 10 min, and the supernatant was centrifuged at 16,000 x g for 30 min. Afterward, the supernatant was collected as the cytosolic fraction.

Statistics—Mean ± S.D. of the results of the experiments were computed. Standard deviations are displayed as error bars in the figures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of Apoptosis in Prostate Cancer Cells after Down-regulation of PKC—We and others have demonstrated that oncogenic Ras is able to induce human or mouse fibroblasts or lymphocytes to undergo apoptosis once endogenous PKC is suppressed (11-15). In this process, multiple Ras downstream effectors are involved for transmitting apoptotic signaling. Here, we tested whether it is possible to induce apoptosis in various prostate cancer cells with or without expressing v-Ha-ras following treatment with GO6976. A v-Ha-ras was transiently introduced into prostate epithelial PrEC cells or prostate cancer LNCaP, PC3, DU145, and HPV7 cells. After transfection, a high level of Ras expression was detected in all transfectants by immunoblotting (Fig. 1a). Subsequently, the cells with or without expressing v-ras were treated with GO6976 (0.1 µM) for 24 h and a DNA fragmentation assay was conducted to determine the occurrence of apoptosis (Fig. 1b). More than 40% of PrEc/ras and prostate cancer cell lines expressing v-ras underwent apoptosis in response to PKC down-regulation. Interestingly, 25% of PC3 or DU145 cells and 15-18% of LNCaP cells had fragmented DNA following treatment. In comparison, apoptosis did not occur in PrEC or HPV7 cells after the addition of GO6976. The results indicate that persistent activation of Ras, together with suppression of PKC, can induce apoptosis in normal prostate epithelial or cancer cells.

Because some prostate cancer cell lines were moderately susceptible to apoptosis in response to GO6976 treatment, it indicates that the Ras-related, apoptotic pathway(s) is/are activated in these cells. Therefore, we examined the expression or activation status of Ras downstream effectors in PrEC and prostate cancer cell lines by immunoblotting (Fig. 2). ERK1/2 was detected in all cell lines and the phosphorylated forms of the kinases were only present in DU145 cells, but not in PrEC or other prostate cancer cell lines (Fig. 2a). Anti-JNK1 antibody revealed a high level of this stress-related kinase in DU145 cells only (Fig. 2b). It is known that PTEN mutation or deletion is often detected in prostate cancer cells, which leads to the up-regulation of PI3K/Akt signaling (31-33). We then tested the activation status of Akt in these cells using the anti-phosphorylated Akt antibody (Fig. 2c). Although a similar amount of Akt was present in all cell lines. However, the antibody detected a high level of the phosphorylated Akt in PC3 cells and a moderate amount of the active kinase in LNCaP cells, which may reflect that PC3 cells are in more advanced stages of the malignancy than LNCaP cells. The active form of Akt was undetectable in PrEC, DU145, or HPV7 cells. The expression of p38 or activation of Ral was also tested using either anti-p38 antibody or Ral/RBD-glutathione S-transferase fusion protein. Only a basal-line expression of p38 was present in all cell lines tested, and there was no association of Ral with the fusion protein (Fig. 2, d and e).


Figure 1
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  		FIGURE 1.
Induction of apoptosis in response to PKC inhibition. a, after introducing v-Ha-ras into PrEC and different prostate cancer cell lines, the level of Ras expression was examined by immunoblotting. b, after treatment with GO6976 (0.1 µM) for 24 h, the percentage of DNA fragmentation in the cells with or without expressing v-ras was measured by a flow cytometer. Error bars represent the S.D. of over 15 samples (five independent experiments and triplicates for each treatment).

 


Figure 2
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  		FIGURE 2.
Expression of Ras downstream effectors in PrEC and various prostate cancer cell lines. Cell lysate from each cell line was prepared and subsequently immunoblotted with anti-p-ERK1/2 (a), JNK1 (b), p-Akt (c), and p-p38 (d) antibodies, or immunoprecipitated with the Ral/RBD-glutathione S-transferase fusion protein (e). The even loading of total proteins was normalized by re-probing the gels with the corresponding antibodies.

 
Down-regulation of PKC Elicits JNK Activation and Caspase Cascade in DU145 Cells—Activation of JNK has been shown to be involved in apoptosis induced by various apoptotic stimuli (16). We previously reported that JNK1 functions as an intermediator during Ras-mediated apoptosis to initiate caspase 8-regulated caspase cascade (16). Because the addition of GO6976 elicited a moderate magnitude of apoptosis in DU145 cells that expressed an elevated level of JNK1, we tested the activation status of JNK1 in the cells in the presence or absence of GO6976 treatment using the anti-phosphorylated JNK antibody (Fig. 3a). JNK1 was not activated in all prostate cancer cell lines, including DU145 cells under normal growth conditions. However, the antibody detected a high level of the phosphorylated form of JNK1 in DU145 cells following treatment with GO6976. It appears that down-regulation of PKC activates a kinase responsible for JNK1 activation in DU145 cells. It is also possible that other prostate cancer cell lines express a very low amount of JNK1 that could not be detected by the antibody.


Figure 3
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  		FIGURE 3.
JNK1 and caspase 8/cytochrome c are activated after PKC suppression in DU145 cells. a, the cells, with or without GO6976 treatment, were immunoblotted with the anti-phosphorylated JNK antibody. Equal loading of total proteins was verified by beta-actin expression. b and c, after being treated with GO696 (0.1 µM) for 24 h, the cleaved, active fragment of caspase 8 (b) and cytochrome c in the cytosol fraction (c) were immunoblotted with the corresponding antibodies. Equal loading of total proteins was verified by beta-actin or heat shock protein 70.

 
JNK1 activity has been linked to caspase 8 activation in Ras-mediated apoptosis (16). We then examined if the cleavage of caspase 8 occurred under the current experimental settings, using immunoblotting (Fig. 3b). Under normal growth conditions, caspase 8 was not cleaved in LNCaP, PC3, or DU145 cells. After PKC inhibition, the cleaved, active caspase 8 was revealed by the antibody only in DU145 cells. It is known that following caspase 8-initiated caspase cascade, cytochrome c is released from the mitochondria to the cytosol for the execution of apoptosis (16). Therefore, the cytosol fractions from the prostate cancer cell lines with or without GO6976 treatment were isolated and immunoblotted (Fig. 3c). There was no cytochrome c released in all untreated cells. Consistently, the protein was detected by the antibody in GO6976-treated DU145 cells only. The data indicate that caspase cascade occurs in DU145 cells upon PKC down-regulation, which accompanies JNK1 activation.

JNK1 Activation Is Required for the Induction of Apoptosis in DU145 Cells after PKC Suppression—Because ERK1/2 were activated in DU145 in Fig. 2, despite the overexpression of JNK1, it raised the possibility that apoptosis occurred in the cells in response to PKC down-regulation might be through the MAPK/ERK1/2 pathway or cooperation between ERK and JNK. To clarify the role of ERK1/2 in the regulation of apoptosis and to define the requirement of JNK1 in this process, PD98059 (an inhibitor for MAPK/ERK1/2 pathway) or JNK inhibitor was used prior to PKC suppression in DU145 cells (Fig. 4). The cleaved, active caspase 8 was present in GO6976-treated DU145 cells after suppression of the MAPK/ERK1/2 pathway, but was absent following the addition of JNK inhibitor (Fig. 4a). Furthermore, cytochrome c was released into the cytosol of DU145 cells after addition of GO6976 in the absence of MAPK/ERK1/2 signaling, which did not happened in the same cells after abrogating JNK1 activity (Fig. 4c). The results of the DNA fragmentation assay were consistent, in which apoptosis occurred in GO6976-treated DU145 cells in the presence of PD98059, but was completely blocked by the addition of JNK inhibitor (Fig. 4c).

To further determine the role of JNK in the induction of Ras-related apoptosis, a JNK1 expression vector was introduced into LNCaP or PC3 cells. Subsequently, the release of cytochrome c in GO6976-treated or untreated LNCaP or PC3 cells with or without overexpressing JNK1 was examined (Fig. 4d). After introducing JNK1, cytochrome c was present in the cytosol fraction in the GO6976-treated cells, but was absent in untreated cells. However, these cells with overexpressed JNK1 became more susceptible to GO6975-induced apoptosis than their parental cells (Fig. 4e). The data suggest that JNK1 is a crucial factor for the induction of apoptosis triggered by PKC down-regulation.

Down-regulation of PKC Causes the Accumulation of ROS and UPR in LNCaP and PC3 Cells—Increase of Ras activity in cells has been linked to the augmentation of ROS production, which is required for the transformation process (58). Studies have also shown that oncogenic Ras, under the condition of PKC abrogation, dramatically up-regulates ROS production that in turn participates in the apoptotic process (59, 60). Because JNK1 expression and activity were not increased in LNCaP and PC3 cells and these cells were still susceptible to PKC suppression-induced apoptosis, it led us to test the redox state in these cells (Fig. 5a). A moderately elevated ROS was present in untreated LNCaP and PC3 cells. In contrast, DU145 and HPV7 cells had a very low amount of ROS. The introduction of v-ras into HPV7 cells moderately increased ROS production, which is consistent with others' findings that Ras is able to up-regulate ROS production (59, 60). After treatment with GO6976, the amount of ROS in LNCaP or PC3 cells as well as in HPV7 cells expressing v-ras, was further increased. We then tested whether the expression of heme oxygenase (HO-1, a ROS modulator) is altered after treatment using immunoblotting (Fig. 5b). HO-1 could not be detected in LNCaP, PC3, or HPV7/ras cells under normal growth conditions and induced after treated with GO6976. The data indicate that a moderate increase of ROS under normal growth conditions is unable to induce HO-1 in these cells, but the ROS modulator is up-regulated by GO6976 treatment. These findings also pointed to the possible role of Akt signaling in the perturbation of the equilibrium of the intracellular redox state.


Figure 4
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  		FIGURE 4.
Apoptosis induced by PKC inhibition requires JNK1. a-c, DU145 cells were treated with either PD98059 or JNK inhibitor I prior to the addition of GO6976. Subsequently, the cleaved caspase 8, cytosolic cytochrome c, and the percentage of DNA fragmentation were examined, respectively. d and e, a JNK1 expression vector was transiently transfected into LNCaP or PC3 cells. After treatment with GO6976, the expression of the cytosolic cytochrome c and percentage of DNA fragmentation were examined, respectively. Error bars represents the S.D. over 15 samples (5 independent experiments and triplicates for each treatment). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Activation of PI3K/Akt or MAPK, under certain circumstances or in different types of cells, can cause ER stress and subsequent unfolded protein accumulation, which triggers the UPR (49). Under such conditions, cells induce ER chaperone proteins to relieve protein aggregation and activate the proteasome machinery to degrade misfolded proteins. However, the UPR induced by persistent ER stress often switches on the apoptotic machinery (49). GADD153 is often up-regulated during ER stress, the induction of which is closely correlated with the onset of apoptosis (53-55). To determine whether PKC suppression could induce GADD153 expression in prostate cancer cells, Western blot analysis was performed (Fig. 5c, upper panels). GADD153 was detected by the antibody in LNCaP or PC3 cells, but not in DU145 cells following GO6976 treatment. Subsequently, the expression of the ER protein BiP was examined after treatment with GO6976. Consistently, the level of this ER protein was increased in LNCaP or PC3 cells, but not in DU145 cells (Fig. 5c, lower panels).

Akt Is Required for the Induction of Apoptosis in GO6976-treated LNCaP or PC3 Cells—LNCaP or PC3 cells, in the absence of JNK1 activation, were still sensitive to GO6976 treatment for the induction of apoptosis. Also, PC3 cells with a high amount of phosphorylated Akt are more susceptible to apoptosis than LNCaP cells that express a moderately elevated level of active Akt (see Figs. 1 and 2). These led us to further investigate the role of Akt activation in the initiation of apoptosis. We first examined whether GO6976 treatment further affects the level of Akt phosphorylation in PC3 or LNCaP cells by immunoblotting. The phosphorylated Akt was present in untreated cells, in which the level of the activated kinase in PC3 cells was higher than that in LNCaP cells (Fig. 6a). The addition of the inhibitor further proportionally increased the amounts of phosphorylated Akt in both cell lines. Next, we tested the susceptibility of GO6976-treated LNCaP, PC3, and DU145 cells to apoptosis following suppressing the Akt signaling pathway by the Akt inhibitor, ROS production by NCA (a ROS inhibitor), or GADD153 by GADD153 antisense oligos (Fig. 6b). These chemical or genetic inhibitors blocked the apoptotic process in LNCaP and PC3 cells, and played no role in DU145 cells. To further test the effect of Akt on the onset of apoptosis induced by PKC suppression and define the possible role of MAPK signaling in this process, a constitutively active Akt or V12S35ras that preferentially binds to and activates Raf/MAP kinase was transiently introduced into prostate cancer cells. Following PKC down-regulation, the constitutive activation of the Akt pathway had no further influence on the induction of apoptosis in LNCaP or PC3 cells. In contrast, the up-regulation of Akt signaling dramatically augmented the magnitude of apoptosis in DU145 cells. Again, activation of the MAPK pathway by V12S35ras did not affect apoptosis triggered by GO6976 treatment. These data suggest that Akt, but not MAPK, takes part in the regulation of apoptosis triggered by PKC suppression in LNCaP and PC3 cells.


Figure 5
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  		FIGURE 5.
Perturbation of the redox state in LNCaP and PC3 cells upon PKC suppression. a, following treatment with GO6976 (0.1 µM) for 24 h, the cells were stained with 2', 7'-dichlorodihydrofluorescein diacetate, and analyzed for reactive oxygen species levels using a flow cytometer. Error bars represents the S.D. over 15 samples (5 independent experiments and triplicates for each treatment). b and c, expression of HO-1 (b) or GADD153 and BiP (c) in the cells, after PKC suppression, was tested by immunoblotting. Equal loading proteins was verified by beta-actin expression.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is known that cells expressing oncogenic Ha- or Ki-ras are susceptible to apoptosis following the suppression of endogenous PKC (11-15). In this Ras-mediated apoptotic process, JNK1 activation is required (16). It has also been reported that multiple Ras downstream effector pathways are involved in the regulation of this cell death program (13). In this study, using various prostate cancer cell lines with different expression levels or activation status of Ras downstream effectors, we tested whether it is possible to initiate the Ras-mediated apoptotic process by modulating the activities of these signaling pathways, and define which Ras-governed effectors are required for the induction of apoptosis. We identified JNK1 and Akt as critical mediators in the induction of apoptosis initiated by PKC inhibition in prostate cancer cells. By knocking-out or overexpressing JNK1, we demonstrated that JNK1, together with the suppression of PKC, induce apoptosis through eliciting caspase 8 cleavage and cytochrome c releasing. Using prostate cancer cells with different a activation status of Akt, we also defined that the Akt pathway is one of the essential elements to sensitize prostate cancer cells to apoptosis following PKC suppression through a mechanism that perturbs the intracellular redox equilibration that in turn triggers the UPR, resulting in apoptosis. Thus, the data presented here suggest that Akt, JNK1, and PKC not only participate in the regulation of cell growth-related activities, but also function as important apoptotic mediators in the initiation of apoptosis in prostate cancer. Moreover, the concurrent activation of JNK1 and Akt, under the condition of PKC abrogation, achieves a full execution of programmed cell death.

Persistent increases of Ras activity trigger a wide spectrum of cellular responses, leading to transformation. These responses are often related to the oncogenic activities of Ras in the promotion of human malignancy. Recently, we and others have discovered that, under certain circumstances, oncogenic Ras is able to induce apoptosis, which has drawn attention for its therapeutic potential. Ras has been known to function through binding to multiple effector proteins that subsequently activate distinct downstream signaling pathways (5-10). It has also been shown that, upon PKC suppression, JNK1 activity is required for the formation of the death-inducing complex (16). However, the suppression of JNK only accounted for 40% reduction of Ras-mediated apoptosis (16). In this study, using various prostate cancer cell lines with different expression levels or activation status of several important signal transducers, we assessed the contribution of each Ras-related pathway to the induction of apoptosis. The study discloses a regulatory network for the induction of apoptosis in prostate cancer, in which the concurrent activation Akt and JNK1 pathways synergize with PKC suppression to induce a full execution of programmed cell death. In this process, Akt signals to a machinery controlling the intracellular redox state, which in turn elicit ER stress and the UPR. JNK1 functions in concert with PKC down-regulation on a separate pathway to induce apoptosis through recruiting caspase activity.

JNK, as one of Ras downstream effectors, phosphorylates the transcriptional factor c-Jun, which further participates in regulation of cell growth, differentiation, or apoptosis. The increase of JNK1 activity by tumor necrosis factor or Fas receptor has been shown to be responsible for the induction of apoptosis (61, 62). In response to the ligation between the ligand and receptor, a death-induced signaling complex is formed and subsequently activates JNK1, leading to caspase 8-mediated caspase cascade. A similar pattern of caspase cascade triggered by JNK1 occurs during Ras-mediated apoptosis (16). Studies also demonstrated that JNK1 is able to inactivate the ASK1 inhibitor, thioredoxin, which in turn mobilizes ASK1 signaling to up-regulate ROS for the induction of apoptosis (63). Using DU145 containing a high amount of JNK1 as well as LNCaP and PC3 cells that express a very low or undetectable level of the protein, we demonstrated that JNK1 activation plays no role in ROS accumulation during this PKC suppression-triggered apoptosis. Instead, like in Fas-induced apoptosis, this kinase is crucial for the initiation of caspase 8 cleavage and cytochrome c releasing.


Figure 6
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  		FIGURE 6.
Akt activation is required for GO6976-induced apoptosis in LNCaP and PC3 cells. a, after treatment with GO6976 (0.1 µM) for 24 h, the level of phosphorylated Akt in LNCaP or PC3 cells were examined by immunoblotting. b, LNCaP, PC3, or DU145 cells were cultured in the growth medium containing GADD153 antisense or sense oligos and GO6976, or treated with NCA or wortmannin (an Akt inhibitor) prior to the addition of GO6976. Subsequently, the percentage of the cells with fragmented DNA was measured. Error bars represents the S.D. over 15 samples (5 independent experiments and triplicates for each treatment). c, a constitutively active Akt or ras mutant V12S35ras that preferentially binds to and activates the Raf/ERK pathway was introduced into prostate cancer cells. Subsequently, the cells were treated with GO6976 and the percentage of DNA fragmentation was measured. Error bars represent the S.D. over 15 samples (5 independent experiments and triplicates for each treatment).

 
ROS is an important intracellular signal transducer of growth factors (58-60). In response to abnormally persistent ligations of growth factor receptors, a moderate increase of intracellular ROS was induced, which has been shown to be required for cellular transformation by altering the structure of the cytoskeleton and further inducing a transformed phenotype in a Rac-depependent fashion (58). In PC12 cells, Ras has also been suggested to up-regulate ROS production upon epidermal growth factor stimulation (64). Furthermore, studies demonstrated that oncogenes can perturb the equilibrium of the intracellular redox state and cause DNA single strand breaks, which in turn disrupts genetic integrity (57). Despite regulating cell proliferation and transformation, the increase of ROS production has also been suggested to play an obligatory role in the induction of apoptosis by various apoptotic stimuli (59, 60). In NF{kappa}B-regulated programmed cell death, ROS accumulation precedes mitochondrial depolarization and caspase activation (65). Our study demonstrated that the activation of Akt in LNCaP or PC3 cells moderately induces ROS production, which may be utilized for tumor maintenance or growth (57). Notably, the inhibition of PKC further augments the level of Akt phosphorylation in these tumor cells, which coincided with a further up-regulation of ROS production. It is possible that PKC and Akt function in an opposite way to maintain the intracellular redox state. After lifting the negative control rendered by PKC, activated Akt disrupts the balance of the intracellular redox state, leading to high amounts of ROS accumulation.

ER stress has been shown to be activated by alternations in proteins and lipid metabolism that can cause the accumulation of unfolded proteins (50-52). Dysregulation of ER stress programs, such as the UPR, can elicit cytoprotective or cytotoxic reactions, depending upon the severity of the stress or other cellular regulators. GADD153 is a transcriptional factor and often induced upon ER stress (36-38). Under the GADD153-deficient condition, cells presented a resistance to ER stress-induced apoptosis, indicating the importance of GADD153 in this process (50-52). Our study showed that ER stress chaperons, including GADD153, are induced in response to PKC suppression in LNCaP and PC3 prostate cancer cells. Using the antisense oligos, we identified that GADD153 functions downstream of Akt and acts as a crucial apoptotic modulator. In this process, a significant accumulation of unfolded proteins occurred, which subsequently elicited apoptosis. It appears that genetic or epigenetic mechanisms in these cells, in response to PKC inhibition, surpass the buffering capacity of the ER and in turn trigger the cell death program. Our study also indicates that the ER functions as a sensor to detect changes in the cellular microenvironment.

Taken together, the results of our study demonstrate that the up-regulation of the JNK1 or Akt pathways can moderately induce apoptosis upon PKC suppression. However, the collaboration among these three signaling pathways (JNK1, Akt, and PKC) sufficiently initiate the cell death program in prostate cancer cells. Because the abnormality of various growth-related signaling pathways often accompanies prostate cancer, our study supports the notion that these signaling molecules can serve as molecular targets for the development of new prostate cancer therapies. However, the further elucidation of the regulatory network of these signaling pathways in prostate cancer cells for the induction of apoptosis is undoubtedly required.


   FOOTNOTES
 
* The work was supported by National Institutes of Health Grant RO1CA100498 and Department of Defense Grant W81XWH-04-1-0246. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: 21-27 Burlington Ave., Rm. 553C, Boston, MA. Tel.: 617-632-8513; Fax: 617-632-0635; E-mail: cchen6{at}bidmc.harvard.edu.

2 The abbreviations used are: PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; ER, endoplasmic reticulum; UPR, unfolded protein response; HO, heme oxygenase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Z. Luo (Boston University School of Medicine, Boston, MA) for reagents.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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