Phorbol Ester-induced Apoptosis in Prostate Cancer Cells via Autocrine Activation of the Extrinsic Apoptotic Cascade

It is well established that activation of protein kinase C (PKC) by phorbol esters promotes apoptosis in androgen-dependent prostate cancer cells. However, there is limited information regarding the cellular mechanisms involved in this effect. In this report we identified a novel autocrine pro-apoptotic loop triggered by PKCδ activation in prostate cancer cells that is mediated by death receptor ligands. The apoptotic effect of phorbol 12-myristate 13-acetate in LNCaP cells was impaired by inhibition or depletion of tumor necrosis factor alpha-converting enzyme, the enzyme responsible for tumor necrosis factor α (TNFα) shedding. Moreover, the apoptogenic effect of conditioned medium collected after phorbol 12-myristate 13-acetate treatment could be inhibited by blocking antibodies against TNFα and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not FasL, as well as by RNA interference depletion of TNFα and TRAIL receptors. Moreover, depletion or inhibition of death receptor downstream effectors, including caspase-8, FADD, p38 MAPK, and JNK, significantly reduced the apoptogenic effect of the conditioned medium. PKCδ played a major role in this autocrine loop, both in the secretion of autocrine factors as well as a downstream effector. Taken together, our results demonstrate that activation of PKCδ in prostate cancer cells causes apoptosis via the release of death receptor ligands and the activation of the extrinsic apoptotic cascade.

It is well established that activation of protein kinase C (PKC) by phorbol esters promotes apoptosis in androgen-dependent prostate cancer cells. However, there is limited information regarding the cellular mechanisms involved in this effect. In this report we identified a novel autocrine pro-apoptotic loop triggered by PKC␦ activation in prostate cancer cells that is mediated by death receptor ligands. The apoptotic effect of phorbol 12-myristate 13-acetate in LNCaP cells was impaired by inhibition or depletion of tumor necrosis factor alpha-converting enzyme, the enzyme responsible for tumor necrosis factor ␣ (TNF␣) shedding. Moreover, the apoptogenic effect of conditioned medium collected after phorbol 12myristate 13-acetate treatment could be inhibited by blocking antibodies against TNF␣ and tumor necrosis factor-related apoptosisinducing ligand (TRAIL), but not FasL, as well as by RNA interference depletion of TNF␣ and TRAIL receptors. Moreover, depletion or inhibition of death receptor downstream effectors, including caspase-8, FADD, p38 MAPK, and JNK, significantly reduced the apoptogenic effect of the conditioned medium. PKC␦ played a major role in this autocrine loop, both in the secretion of autocrine factors as well as a downstream effector. Taken together, our results demonstrate that activation of PKC␦ in prostate cancer cells causes apoptosis via the release of death receptor ligands and the activation of the extrinsic apoptotic cascade.
Protein kinase C (PKC) 2 isozymes, a family of at least 10 related serine-threonine kinases, play important roles in the regulation of various cellular processes, including differentiation, proliferation, and malignant transformation, and have been widely implicated in the progression of cancer. This family of signaling kinases comprises the classical (␣, ␤I, ␤II, and ␥), novel (␦, ⑀, , and ), and atypical ( and /i) PKCs, which have differential patterns of cell and tissue distribution and unique modes of regulation (1). Phorbol esters, natural compounds that potently activate the classical and novel PKCs, trigger a plethora of cellular responses that vary depending on the cell type and the relative expression of individual PKC isozymes. Whereas phorbol esters are capable of promoting mitogenic or survival responses, many cell types undergo growth arrest or apoptosis in response to PKC activation. A major reason for such heterogeneity is the diversity of pathways activated by each PKC isozyme, their distinct relocalization, and their differential access to substrates upon activation (2). Such functional diversity is exemplified by the novel PKCs: whereas in most cases PKC⑀ acts as a mitogenic or anti-apoptotic kinase, PKC␦ generally inhibits proliferation, or in some cell types it triggers an apoptotic response and is required for drug-induced apoptosis (3). Dissecting the signaling events regulated by individual PKCs still represents a major challenge and will certainly help to understand the functional roles of PKC isozymes in normal and cancer cells.
Androgen-dependent prostate cancer cells, such as LNCaP cells, represents one of the most studied models for phorbol ester-induced apoptosis via PKC activation (4,5). Phorbol 12-myristate 13-acetate (PMA) stimulates apoptosis in LNCaP cells and xenografts, and it sensitizes LNCaP tumors in mice to the apoptotic effects of ionizing radiation (6,7). We and others have assigned a key role to PKC␦ as a mediator of phorbol ester-induced apoptosis in LNCaP cells (8 -10). Unlike observed in other cell types, activation of PKC␦ in prostate cancer cells is independent of its cleavage to a catalytically active form, but it rather depends on allosteric mechanisms upon translocation to membranes (8). PKC⑀, on the other hand, was shown to stimulate proliferation in LNCaP cells and cause progression to an androgen-independent state (11). Signaling studies have determined an essential role for p38 MAPK and ceramide as mediators of phorbol ester-induced apoptosis in LNCaP cells (6,12), and in addition, a modulatory role for the JNK interacting protein 1 (JIP-1, an inhibitor of JNK) has been recently postulated (13), suggesting complex modes of regulation downstream of PKC in prostate cancer cells. The relative contribution of the intrinsic and extrinsic apoptotic cascades in this context remains to be determined. However, as many of the aforementioned pathways are known effectors of death receptor ligands, such as TNF␣, TRAIL, or FasL, it is reasonable to speculate that phorbol ester-induced apoptosis in LNCaP cells might involve the activation of the extrinsic apoptotic pathway.
It has been known for years that phorbol esters are capable of stimulating the release of autocrine or paracrine factors that modulate PKC cellular responses. For example, conditioned medium (CM) collected from PKC⑀-overexpressing R6 fibroblasts stimulates DNA synthesis and causes morphologic transformation. Transforming growth factor-␤ release has been associated, at least in part, to the growth abnormalities caused by PKC⑀ overexpression (14). Limited information, however, is available on the potential contribution of autocrine factors to apoptotic responses caused by PKC activation. One attractive, yet unexplored hypothesis, is that prostate cancer cell death upon phorbol ester stimulation involves the activation of an apoptotic autocrine loop that stimulates death receptors and the extrinsic apoptotic pathway.
In the present study we demonstrate that the induction of apoptosis in LNCaP prostate cancer cells by PKC involves the secretion of death ligands. Moreover, by means of a series of pharmacological and molecular approaches, we have established that the novel PKC␦ is crucial in this autocrine regulation, playing roles both in autocrine factor release as well as downstream of death receptor activation. The identification of this novel autocrine mechanism highlights the complexities of PKC signaling and may have great implications in the identification of novel targets for prostate cancer therapy.
Collection of CM-Cells (ϳ70% confluence) were treated with PMA (100 nM) or vehicle (ethanol) for 1 h, and then washed twice with medium to remove the phorbol ester or vehicle. After incubation for different times, CM was collected, filtered, and added to fresh LNCaP cells (ϳ70% confluence). When indicated, CM was dialyzed using 12-14-kDa cut-off membranes for 36 h at 4°C against RPMI medium, which was changed each 12 h.
Western Blot Analysis-Cells were harvested into lysis buffer containing 50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 5% ␤-mercaptoethanol, and then lysed by sonication. Equal amounts of protein (20 g/lane) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk or 5% bovine serum albumin in 0.05% Tween 20/phosphate-buffered saline and then incubated with the primary antibody for 1 h. After washing three times with 0.05% Tween 20 in phosphate-buffered saline, membranes were incubated for 1 h with either anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase (1:3000, Bio-Rad). Bands were visualized with the enhanced chemiluminescence (ECL) Western blotting detection system. Densitometric analysis was performed using Image software (National Institutes of Health) under conditions that yielded a linear response.
Apoptosis Assays-Cells were trypsinized, mounted on glass slides, and then fixed in 70% ethanol. Morphological changes in chromatin structure were assessed after staining with DAPI, as described previ-ously (8,9). Apoptosis was characterized by chromatin condensation and fragmentation when examined by fluorescence microscopy. The incidence of apoptosis was analyzed by counting 500 cells followed by the determination of apoptotic cells in each preparation. We have previously determined that results observed by these methods essentially matched those observed by flow cytometry and correlate with DNA laddering analysis (8).
Retroviral Infection of LNCaP Cells-Retroviral vectors encoding for short hairpin RNAi sequences to knock-down FADD or caspase-8, or encoding a mutated sequence for caspase-8 were kind gifts from Dr. Wafik el-Deiry (University of Pennsylvania). Empty vector p-SUPER Retro was used as a control. Retroviruses were obtained after transfection of Phoenix-Ampho packaging cells with the corresponding retroviral constructs by using Lipofectamine (Invitrogen) and collection of the supernatant. Subconfluent LNCaP cells in 6-or 12-well plates were infected with the different retroviruses for 14 h in RPMI 1640 medium supplemented with 10% fetal bovine serum and Polybrene. After removal of the retrovirus by extensive washing, cells were incubated for 24 h in RPMI 1640 medium supplemented with 10% fetal bovine serum. Selection was carried out with 1 mg/ml puromycin.
RNA Isolation and cDNA Synthesis-LNCaP cells were treated for 1 h with either PMA or vehicle. At different time points, cells were lysed and RNA was extracted using TRIzol (Invitrogen). Five micrograms of RNA per sample were reverse transcribed using the First-Strand cDNA Synthesis Kit (Amersham Biosciences). Each reverse transcription reaction was performed in a total volume of 50 l.
Real-time PCR-PCR primers and fluorogenic probes for human TNF␣ were purchased from Applied Biosystems. The probes were 5Ј end-labeled with 6-carboxyfluorescein (FAM). Each PCR amplification was performed in a total volume of 12.5 l, containing 6.25 l of 2ϫ TaqMan Universal PCR Master Mix (Applied Biosystems), commercial target primers (300 nM), the fluorescent probe (200 nM), and 1 l of cDNA. PCR were performed with an ABI PRISM 7700 Detection System (TaqMan; Applied Biosystems) using the following conditions: 2 min at 50°C and 10 min at 94°C, followed by a total of 40 cycles at 95°C for 15 s and 60°C for 1 min. The PCR product formation was continuously monitored during the PCR using Sequence Detection System software version 1.7 (Applied Biosystems). The FAM signal was normalized to the endogenous glyceraldehyde-3-phosphate dehydrogenase. Results were expressed as -fold increase relative to those in untreated cells.
Enzyme-linked Immunosorbent Assay (ELISA)-TNF␣ levels were determined by ELISA using 2 g/well of coating anti-TNF␣ antibody. Nonspecific binding sites were blocked with 10% fetal bovine serum in phosphate-buffered saline. CM (50 l) was added in each well and incubated overnight at 4°C. Subsequently, 100 l of a biotin-labeled anti-TNF␣ antibody (0.3 g/ml) was added for 2 h at room temperature.
PKC Translocation-Translocation was determined by Western blot using a subcellular fractionation technique, as previously reported (8).
Protein Determination-Protein determinations were performed with the Micro BCA Protein Assay from Pierce Biotechnology, Inc., using bovine serum albumin as a standard.

PMA Stimulates the Release of Apoptogenic Factors from Prostate
Cancer Cells-LNCaP prostate cancer cells undergo apoptosis upon PMA treatment (4, 5, 8 -10). To determine whether autocrine mechanisms could be involved in this effect, we first compared the activity of conditioned CM collected from PMA-treated cells (CM-PMA) and vehicle-treated cells (CM-vehicle). LNCaP cells were treated with PMA (100 nM, 1 h), and then washed extensively to remove the phorbol ester. When added to fresh LNCaP cells, CM-PMA caused a significant apo-ptotic response, as determined by apoptotic cell counting after DAPI staining. On the other hand, CM-vehicle did not cause cell death (Fig. 1,  A and B). Flow cytometry analysis revealed a significant increase in the population of cells in sub-G 0 /G 1 24 h after treatment either with PMA or CM-PMA (Fig. 1C). To determine that the effect was not because of PMA remaining in the CM despite the extensive washings, CM-PMA was dialyzed using 12-14-kDa cut-off dialysis membranes (molecular mass of PMA is 617). The apoptogenic activity of the CM-PMA was indeed retained after dialysis (Fig. 1D). CM-PMA collected from androgen-independent DU-145 and PC3 cells, as well as from Tsu-Pr1 cells (originally classified as a prostate cancer cell line but later re-classified as a bladder cancer cell line) also triggered an apoptotic response when added to fresh LNCaP cells. On the other hand, CM collected from NIH-3T3 cells treated with PMA in a manner similar to the prostate cancer cells was unable to cause apoptosis when added to fresh LNCaP cells (Fig. 1E). This experiment suggests that the effect is cell typespecific, and it also serves as a control for the absence of PMA in the CM after the washings. To determine whether the apoptotic effect of PMA was entirely dependent on the release of apoptotic factors, we per- formed consecutive washings every 15 min during the first 6 h after PMA treatment. We reasoned that this would prevent the accumulation of released autocrine factors in the CM. Remarkably, cells did not undergo apoptosis under this experimental condition (Fig. 1F), suggesting that the apoptotic effect of the phorbol ester was entirely dependent on the autocrine loop. Apoptotic activity was detected with CM from LNCaP cells collected shortly (1 h) after PMA treatment (data not shown), suggesting that the effect was probably independent of the synthesis of apoptotic factors. To further explore this issue, we collected CM from LNCaP cells treated with the protein synthesis inhibitor cycloheximide (50 mM). A significant fraction of the apoptogenic effect of CM-PMA (ϳ70%) was retained after "de novo" protein synthesis inhibition (Fig. 1G), suggesting that the effect was largely caused by the release of pre-formed factors.
PKC␦ Has a Dual Role Both in Apoptotic Factor Release and as an Effector of CM-induced Apoptosis-PKC␦ has been established as a key mediator of PMA-induced apoptosis in LNCaP cells (8,10). To determine whether PKC␦ is involved in the release of apoptotic factors, we collected CM from LNCaP cells treated with either the pan-PKC inhibitor GF109203X or the PKC␦ inhibitor rottlerin ( Fig. 2A). We found that CM collected after pharmacological inhibition of PKC␦ had a significantly lower apoptotic effect when added to fresh LNCaP cells. GF109203X totally blocked the PMA effect. Because rottlerin is known to cause nonspecific effects unrelated to PKC␦ inhibition (15), we used a RNAi approach, as we have previously described (9). PKC␦ levels were reduced by ϳ90% in LNCaP cells upon delivery of a specific dsRNA. This dsRNA specifically reduces the levels of PKC␦ without affecting the expression of other PMA-responsive PKC isozymes present in LNCaP cells (Fig. 2C). Remarkably, CM-PMA collected from PKC␦depleted cells has remarkably less apoptotic activity ( Fig. 2A), arguing that PKC␦ is required for the release of apoptogenic factors in response to PMA. PKC␦ also plays a role as an effector of the apoptotic effect of CM-PMA. Indeed, when CM-PMA was added to PKC␦-depleted LNCaP cells, apoptosis was basically undetected. GF109203X and rottlerin also impaired the apoptotic effect of the CM-PMA. The inhibitory effect of rottlerin was not complete, probably because of its ability to cause apoptosis through nonspecific mechanisms (9,15). Interestingly, the CM-PMA caused translocation of PKC␦ in LNCaP cells (Fig. 2D).
Thus, PKC␦ appears to have a dual role, both in the release of apoptogenic factors as well as an effector.
A Role for TNF␣ in the PKC-induced Autocrine Loop-As TNF␣ is known to cause cell death in LNCaP cells (12,16), we sought to investigate whether this cytokine was involved in the PKC-mediated autocrine effect. As a first approach we used TAPI-2, an inhibitor of TACE/ADAM17 (TNF␣-converting enzyme), a metalloprotease responsible for TNF␣ shedding (17). CM was collected from LNCaP cells treated with PMA in the presence of increasing concentrations of TAPI-2, and then added to fresh LNCaP cells. As shown in Fig. 3A, pretreatment with the TACE inhibitor significantly impaired the apoptotic activity of the CM-PMA. The involvement of TACE was further confirmed using RNAi. We could achieve a 63 Ϯ 8% reduction (n ϭ 3) in TACE expression in LNCaP cells upon delivery of a specific TACE dsRNA (Fig. 3B). CM-PMA collected from TACEdepleted cells has indeed significantly lower apoptogenic activity (ϳ60% inhibition) when added to fresh LNCaP cells (Fig. 3C). To assess the involvement of TNF␣ we used a specific anti-TNF␣ neu- tralizing antibody, which dose-dependently impaired the ability of the CM-PMA to cause apoptosis. At a concentration of 10 mg/ml, a 48 Ϯ 3% inhibition (n ϭ 3) was observed (Fig. 4A). Real-time PCR assays showed that PMA treatment caused a marked increase in TNF␣ mRNA levels (357 Ϯ 92-fold after 3 h, and 1992 Ϯ 146-fold after 6 h). Because our results using cycloheximide (Fig. 1G) suggest that the effect is largely independent of protein synthesis, we believe that the contribution of newly generated TNF␣ is probably less important than the released of a pre-formed pool of the cytokine.
PKC␦ Is Required for PMA-induced Secretion of TNF␣-In the next series of experiments we explored whether PKC activation could promote the secretion of TNF␣ from LNCaP cells. TNF␣ levels in CM-PMA (as determined by ELISA) were remarkably higher compared with those in CM-vehicle (Fig. 4B). As expected, TNF␣ levels were significantly reduced by preincubation with the TACE inhibitor TAPI-2 as well as by TACE RNAi. The PKC inhibitor GF109703X completely blocked the secretion of TNF␣ caused by PMA. In LNCaP cells subject to PKC␦ RNAi, there was also a marked reduction in TNF␣ levels in the CM upon PMA treatment. Taken together, these results support a critical role for PKC␦ in phorbol ester-induced secretion of TNF␣.
TRAIL Is Also Involved in the PKC-mediated Autocrine Effect-Other cytokines in addition to TNF␣ are pro-apoptogenic in prostate cancer cells, including TRAIL and FasL (18 -21). When a neutralizing TRAIL antibody was added to the CM-PMA, its apoptogenic activity was significantly reduced. Maximum inhibition (ϳ30%) was achieved at an antibody concentration of 1 g/ml (Fig. 5A). On the other hand, a blocking anti-FasL antibody was not effective, even at concentrations of 10 g/ml (Fig. 5B). When neutralizing antibodies for TNF␣ (1 g/ml) and TRAIL (0.1 g/ml) were added together to the CM-PMA, the inhibitory effect was higher than with each antibody alone, as both antibodies  together suppressed apoptosis by ϳ65% (Fig. 5A). Determination of TRAIL levels by ELISA revealed a 2-3-fold increase in CM-PMA relative to CM-vehicle (Fig. 5C). Thus, these results suggest that TNF␣ and TRAIL, but not FasL, are involved in the PKC-mediated autocrine loop in LNCaP cells. When LNCaP cells were treated with TNF␣ and TRAIL at concentrations similar to those found in the CM-PMA (1 ng/ml TNF␣ and 0.1 ng/ml TRAIL), the percentage of apoptotic cells was only 7 Ϯ 1%. Moreover, LNCaP cells treated with medium containing concentrations of TNF␣ and TRAIL 100-fold higher than those in CM-PMA led to 13 Ϯ 2% of apoptosis. Thus, TNF␣ and TRAIL are required but not sufficient to cause the full apoptotic response.
Involvement of the Extrinsic Apoptotic Pathway in the PKC-mediated Autocrine Loop-Death receptors mediate the actions of TNF␣ and TRAIL, leading to the activation of the extrinsic apoptotic cascade. TNFR1 and TNFR2 receptors mediate the effects of TNF␣, and TRAIL acts via the activation of D4 and D5 receptors (22). The relative contribution of each of these receptors was assessed using RNAi. In the case of the D4, D5, and Fas receptors, reductions in expression of 75 Ϯ 5 (n ϭ 3), 71 Ϯ 2 (n ϭ 3), and 80 Ϯ 10% (n ϭ 3), respectively, were achieved (Fig. 6A). TNFR1 and TNFR2 mRNA levels were also significantly reduced (in this later case reverse transcriptase-PCR was used because of the low sensitivity of available TNFR antibodies in our experimental model). We found that the apoptogenic effect of CM-PMA was significantly reduced when added to LNCaP cells in which TNFR1, TNFR2, D4, or D5 were individually depleted. On the other hand, Fas receptor RNAi did not affect the apoptotic effect of CM-PMA (Fig. 6B).
The involvement of TNF␣ receptors was further confirmed using blocking antibodies for TNF␣ receptors. As shown in Fig. 6C, incubation of LNCaP cells with either anti-TNFR1 or anti-TNFR2 specific blocking antibodies (10 g/ml) significantly inhibited the apoptotic effect of CM-PMA. The response was somehow higher when both antibodies were used together.
Involvement of the Extrinsic Apoptotic Pathway in the PKC-mediated Autocrine Loop-Stimulation of death receptors activates the extrinsic apoptotic pathway. TNF␣ and TRAIL receptors activate various signaling cascades, including the JNK, p38 MAPK, and NF-B pathways. Whereas NF-B mediates mainly anti-apoptotic signals in prostate cancer cells, including LNCaP cells (23,24), emerging evidence suggests a prominent pro-apoptotic role for p38 MAPK and JNK in prostate cancer cells (9,13,25,26). We found that CM-PMA significantly activates JNK and p38 MAPK in LNCaP cells, as revealed by Western blot using phospho-specific JNK and p38 MAPK antibodies. Other well known downstream effectors of death receptors, such as caspase-8 and NF-B, also become activated, as determined by antibodies against cleaved caspase-8 and phospho-IB, respectively. In agreement with previous studies (19,27,28), TNF␣ activates these pathways in LNCaP cells (Fig.  7, A-C). Neutralizing antibodies for TRAIL and TNF␣ blocked the activation of caspase-8 by CM-PMA (Fig. 7D). To determine the relative contribution of the p38 MAPK and JNK pathways in the context of the apoptotic autocrine loop, we used pharmacological inhibitors of p38 MAPK (SB203580) and JNK (SP600125). Pretreatment of LNCaP cells with either inhibitor significantly blocked the apoptotic effect of CM-PMA; inhibition was almost complete by pretreating cells with both inhibitors together (Fig. 8A). On the other hand, neither of these inhibitors affected the release of the apoptotic factors by PMA (Fig. 8B), arguing for a role for p38 MAPK and JNK as effectors of the apoptotic response. Caspase-8 and FADD RNAi Impair the Apoptotic Effect of the CM-PMA-Ligand binding to death receptors recruits FADD and procaspase-8 to the death-inducing signaling complex (DISC). FADD is a key adaptor for transmitting death receptor signals upon receptor activation, and activated caspase-8 is critical for initiating the apoptosisexecuting caspase cascade (22). To further assess the contribution of the extrinsic cascade in PMA-induced apoptosis, LNCaP cells were infected with retroviruses encoding short hairpin RNAs specifically designed to knock-down caspase-8 or FADD. Using this approach we achieved a reduction of 54 Ϯ 10 (n ϭ 3) and 73 Ϯ 6% (n ϭ 3) in caspase-8 and FADD expression, respectively (Fig. 9, A and C). We observed that FADD-depleted cells were markedly resistant to apoptosis caused by the CM-PMA (Fig. 9B). Similar results were observed in caspase-8-depleted LNCaP cells. On the other hand, LNCaP cells transduced with a mismatched short hairpin RNA sequence for caspase-8 retained their sensitivity to the apoptogenic effect of CM-PMA (Fig. 9D). Taken together, these results suggest a critical role for the extrinsic apoptotic pathway in the autocrine effect triggered by PKC activation in LNCaP cells.

DISCUSSION
Several laboratories, including ours, have shown that phorbol esters trigger an apoptotic response in androgen-dependent prostate cancer cells via PKC activation (4 -10), but the mechanisms involved have been partially understood. A distinctive aspect of our studies is that they underscore the existence of a PKC-activated autocrine loop that mediates phorbol ester-induced apoptosis that involves the autocrine pro-duction of death factors, including TNF␣ and TRAIL. Neutralizing antibodies for these cytokines, as well as inhibition or depletion of their receptors in LNCaP cells, impairs the apoptotic effect of CM collected upon PMA stimulation. The involvement of the extrinsic apoptotic cascade further supports the contribution of death receptors to the apoptotic effect. An important observation was that PKC␦ plays a dual role both as a key mediator of apoptogenic factor release and as an effector.

Essential Role for PKC␦ in Phorbol Ester-induced Apoptosis in LNCaP
Prostate Cancer Cells-The identification of the roles of individual PKC isozymes and their downstream effectors in apoptotic responses has been a subject of intense investigation. Considerable evidence supports the involvement of PKC␦ as mediator of the apoptotic effect of phorbol esters and other agents, including in prostate cancer models (8 -10). The role of PKC⑀, the other novel PKC present in LNCaP cells, still remains controversial, as studies have shown that PKC⑀ renders cells resistant to apoptosis by PMA, whereas others have suggested a proapoptotic role for this PKC isozyme (10,11). In some cell types PKC␦mediated apoptosis is independent of its phosphorylating activity and probably involves a scaffolding function (29), however, its kinase activity is required for apoptosis in LNCaP cells (8). Whereas PKC␦-mediated apoptosis involves its proteolytic cleavage and the generation of a constitutively active catalytic fragment in several cell types, such as hemopoietic cells, keratinocytes, and salivary epithelial cells (30 -32), it strictly depends on allosteric activation and is cleavage-independent in prostate cancer cells (8). Such heterogeneity in the molecular mechanisms of activation of PKC␦ emphasizes its complex regulation and cell-context dependence (33). Our results showing that PKC␦ was required for the secretion of TNF␣ in LNCaP cells underscores a previously unknown role for this novel PKC in the context of the apoptotic response. As cells depleted from PKC␦ have also an impaired response to death factors secreted to the CM, it appears that the involvement of this PKC in response to PMA occurs at multiple levels (see below).
TNF␣ Is Required for PMA-induced Apoptosis in LNCaP Prostate Cancer Cells-Studies from the laboratory of Weinstein (14) have initially established the potential contribution of PKC isozymes to autocrine factor release, although in those studies PKCs were shown to stimulate pro-mitogenic and transforming events. CM collected from PKC⑀-overexpressing R6 fibroblasts causes abnormal proliferation and malignant transformation, effects that are mediated, at least in part, by TGF-␤ (14). Also in the context of transformation, recent studies have shown that TNF␣ levels are elevated in the skin of PKC⑀ transgenic mice and contribute to the development of metastatic squamous cell carcinomas (34). We have determined that factors known to cause apoptosis in LNCaP cells, which include TNF␣ and TRAIL, are released as a consequence of PKC activation. TACE/ADAM-17, a metalloprotease that promotes the shedding of the pro-form of TNF␣ into its mature soluble form, can be activated by PKC (35). Our results showing that TACE depletion (using RNAi) or inhibition (using TAPI-2) impaired PMAinduced apoptosis in LNCaP cells further support this hypothesis. Emerging evidence in hemopoietic cells suggests that PKCs could regulate pro-apoptotic autocrine responses (36), and a role for PKC⑀ in LPS-induced apoptosis and TNF␣ secretion has been reported (37). Enhanced TNF␣ release in response to the PKC activator bryostatin I (in conjunction with the cdk inhibitor flavopiridol) has also been observed in human myeloid leukemia cells, an effect that is attenuated by the PKC inhibitor GF109203X. In LNCaP prostate cancer cells PKC␦ seems to be critical for TNF␣ release in response to PMA, as determined by pharmacological inhibition of PKC␦ and PKC␦ RNAi. Whereas the detailed mechanisms by which PKC␦ regulates TACE activity/function in prostate cancer cells are yet unknown, studies in keratinocytes have suggested that TACE activation by PKC could be mediated by reactive oxygen species (34).
Other Factors Contributing to PMA-induced Apoptosis in LNCaP Prostate Cancer Cells-Whereas our results clearly show that TNF␣ is a mediator of the PKC-activated autocrine loop in prostate cancer cells, it is likely that this cytokine alone is not sufficient to cause the apoptotic response. This could be inferred from the partial inhibitory effects of TAPI-2, TNF␣ neutralizing antibody, TNFRs blocking antibodies, and TNFRs RNAi (although in this later case complete depletion of the receptors could not be achieved). Moreover, the concentrations of TNF␣ released to the CM upon PMA stimulation would be insufficient to cause apoptosis in LNCaP cells. Whereas our results do not support a role for FasL, experiments using a TRAIL blocking antibody and RNAi for TRAIL receptors would argue for a role for TRAIL in the autocrine effect. TRAIL is elevated in CM from PMA-treated LNCaP cells. Nevertheless, complete inhibition could not be achieved even when both TNF␣ and TRAIL blocking antibodies were used together, which might suggest the involvement of additional factors. Indeed, when LNCaP cells were treated with TNF␣ and TRAIL at concentrations similar to those found in the CM-PMA, the apoptotic index was very small. Preliminary studies using a cytokine array suggest that several other cytokines/chemokines are elevated in CM-PMA from LNCaP cells, 3 although it is premature at this stage to conjecture on their relative contribution to phorbol ester-induced apoptosis.
LNCaP cells are partially sensitive to TNF␣ and insensitive to TRAILinduced cell death when they act as single agents. However, chemotherapeutic drugs and irradiation sensitize LNCaP cells to TRAIL or TNF␣induced cell death (12,19,38,39). This led us to speculate that the autocrine regulation should involve additional mechanisms. Yet unidentified factors released to the CM and/or alternative PKC-regulated pathways, such as phosphatidylinositol 3-kinase/Akt, may sensitize cells to the effects of TNF␣/TRAIL. We support this second hypothesis. Akt plays a major role in prostate cancer cell survival. LNCaP cells have high constitutive Akt activity because of loss of phosphatase and tensin homologue deleted by chromosome ten (PTEN) function, and inhibition of this pathway has been shown to sensitize prostate cancer cells to a number of apoptotic agents (40 -42). We have previously shown that in LNCaP cells PMA treatment leads to the dephosphorylation and inactivation of Akt via a PP2A phosphatase. Moreover, an activated Akt mutant (Myr-Akt) protects LNCaP cells from PMA-induced apoptosis (9). It is likely that this dephosphorylation event might sensitize LNCaP cells to the effects of autocrine factors released upon PKC activation, a hypothesis supported by numerous studies showing that suppression of Akt activity makes LNCaP cells sensitive to death factors (19,41,42). Interestingly, treatment of LNCaP cells with CM-PMA causes a significant Akt dephosphorylation (data not shown). Although further studies would be required to understand the mechanistic basis of this effect, it is likely to believe that this suppression of Akt activity contributes to apoptosis sensitization.
A Role for the Extrinsic Apoptotic Pathway in PMA-induced Apoptosis in LNCaP Cells-Cell surface death receptors, which belong to the TNF receptor superfamily, contain a cytoplasmic death domain that enables the receptors to engage the cell apoptotic machinery. Stimulation of TNFRI and TNFRII receptors by TNF␣, or D4 and D5 receptors by TRAIL, results in the activation of the initiator caspase-8, which can propagate the apoptotic signal by direct cleavage of downstream effector caspases (22). We determined that the apoptotic effect of the CM-PMA was impaired in caspase-8-depleted LNCaP cells. In addition, depletion of the adaptor protein FADD also inhibited the apoptotic response. The involvement of the extrinsic cascade could also be inferred from the observed induction of caspase-8 cleavage, as well as by the observed phosphorylation (and therefore activation) of p38 MAPK, JNK, and IB, well established death receptor effectors. The differential kinetics of JNK activation by TNF␣ and CM-PMA might relate to the effect of still unknown factors in the CM.
The involvement of JNK and p38 MAPK in various forms of cell death has been documented extensively. Whereas JNK is required for apoptotic cell death elicited by stimuli such as oxidative stress and UV, recent evidence supports a role for JNK in TNF␣-induced apoptosis (43,44). Using pharmacological inhibitors of JNK and p38 MAPK we determined that these two pathways are not involved in the release of apoptotic factors but instead they are effectors of the autocrine factors in prostate cancer cells. Previous studies have determined that p38 MAPK and JNK become activated by phorbol ester treatment in LNCaP prostate cancer cells (9,13,26), and that p38 MAPK inhibitors interfere with PMA-induced apoptosis in these cells (9), which support our experimental conclusions.
As discussed briefly above, our experiments show that the apoptogenic effect of CM-PMA was substantially reduced when added to PKC␦-depleted cells, suggesting a role for PKC␦ as a death receptor effector. This observation was at first puzzling, because death receptors do not couple directly to effectors that generate diacylglycerol and activate PKC. However, some recent reports have suggested a potential link between death receptors and PKC. A paper by Kilpatrick et al. (45) showed that in neutrophils PKC␦ forms a signal complex with TNFR1 and phosphatidylinositol 3-kinase in response to TNF␣ that greatly influences the recruitment of receptor adaptors. TNF␣ activation of PKC␦ has been recently reported in pancreatic acinar cells (46). In intestinal cells, TNF␣ could cause membrane translocation of PKC isozymes, including PKC␦, an effect that correlates with increased apoptosis (47,48). Ongoing studies in our laboratory are aimed at understanding the mechanisms of death receptor regulation of PKC␦ function in prostate cancer cells.
Final Remarks-In summary, our studies have introduced a novel paradigm in phorbol ester-induced apoptosis in LNCaP prostate cancer cells that involves the autocrine production of death factors and the activation of the extrinsic apoptotic pathway. PKC␦ is required both for the release of autocrine factors and for transducing apoptotic signals downstream of death receptors. In addition to the mechanistic implications, our studies may have significant therapeutic relevance. Indeed, PKC activators (including phorbol esters) are in clinical trials for various types of cancers (49 -51) and greatly enhance the effectiveness of other antitumor agents and radiation in prostate cell tumor-bearing mice (6,7).