Phorbol Ester-induced Apoptosis in Prostate Cancer Cells via Autocrine Activation of the Extrinsic Apoptotic Cascade
2005; Elsevier BV; Volume: 280; Issue: 47 Linguagem: Inglês
10.1074/jbc.m506767200
ISSN1083-351X
AutoresAnatilde M. González-Guerrico, Marcelo G. Kazanietz,
Tópico(s)Natural product bioactivities and synthesis
ResumoIt 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 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. Protein kinase C (PKC) 2The abbreviations used are:PKCprotein kinase CFADDFas-associated protein with a death domainTACETNFα-converting enzymeMAPKmitogen-activated protein kinaseJNKc-Jun NH2-terminal kinasePMAphorbol 12-myristate 13-acetateTNFαtumor necrosis factor αCMconditioned mediumDAPI4′,6-diamidino-2-phenylindoleRNAiRNA interferencedsdouble-strandedELISAenzyme-linked immunosorbent assayTNFRtumor necrosis factor receptorTRAILtumor necrosis factor-related apoptosis-inducing ligand 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 (α, βI, βII, and γ), novel (δ, ϵ, η, and θ), and atypical (ζ and λ/i) PKCs, which have differential patterns of cell and tissue distribution and unique modes of regulation (1Dempsey E.C. Newton A.C. Mochly-Rosen D. Fields A.P. Reyland M.E. Insel P.A. Messing R.O. Am. J. Physiol. 2000; 279: L429-L438Crossref PubMed Google Scholar). 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 (2Yang C. Kazanietz M.G. Trends Pharmacol. Sci. 2003; 24: 602-608Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Such functional diversity is exemplified by the novel PKCs: whereas in most cases PKCϵ acts as a mitogenic or anti-apoptotic kinase, PKCδ generally inhibits proliferation, or in some cell types it triggers an apoptotic response and is required for drug-induced apoptosis (3Brodie C. Blumberg P.M. Apoptosis. 2003; 8: 19-27Crossref PubMed Scopus (374) Google Scholar). 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. protein kinase C Fas-associated protein with a death domain TNFα-converting enzyme mitogen-activated protein kinase c-Jun NH2-terminal kinase phorbol 12-myristate 13-acetate tumor necrosis factor α conditioned medium 4′,6-diamidino-2-phenylindole RNA interference double-stranded enzyme-linked immunosorbent assay tumor necrosis factor receptor tumor necrosis factor-related apoptosis-inducing ligand Androgen-dependent prostate cancer cells, such as LNCaP cells, represents one of the most studied models for phorbol ester-induced apoptosis via PKC activation (4Powell C.T. Brittis N.J. Stec D. Hug H. Heston W.D. Fair W.R. Cell Growth & Differ. 1996; 7: 419-428PubMed Google Scholar, 5Zhao X. Gschwend J.E. Powell C.T. Foster R.G. Day K.C. Day M.L. J. Biol. Chem. 1997; 272: 22751-22757Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). 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 (6Garzotto M. Haimovitz-Friedman A. Liao W.C. White-Jones M. Huryk R. Heston W.D. Cardon-Cardo C. Kolesnick R. Fuks Z. Cancer Res. 1999; 59: 5194-5201PubMed Google Scholar, 7Zheng X. Chang R.L. Cui X.X. Avila G.E. Lee S. Lu Y.P. Lou Y.R. Shih W.J. Lin Y. Reuhl K. Newmark H. Rabson A. Conney A.H. Cancer Res. 2004; 64: 1811-1820Crossref PubMed Scopus (40) Google Scholar). We and others have assigned a key role to PKCδ as a mediator of phorbol ester-induced apoptosis in LNCaP cells (8Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li L. Yuspa S.H. Kazanietz M.G. J. Biol. Chem. 2000; 275: 7574-7582Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Tanaka Y. Gavrielides M.V. Mitsuuchi Y. Fujii T. Kazanietz M.G. J. Biol. Chem. 2003; 278: 33753-33762Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 10Yin L. Bennani-Baiti N. Powell C.T. J. Biol. Chem. 2005; 280: 5533-5541Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Unlike observed in other cell types, activation of PKCδ 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 (8Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li L. Yuspa S.H. Kazanietz M.G. J. Biol. Chem. 2000; 275: 7574-7582Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). PKCϵ, on the other hand, was shown to stimulate proliferation in LNCaP cells and cause progression to an androgen-independent state (11Wu D. Foreman T.L. Gregory C.W. McJilton M.A. Wescott G.G. Ford O.H. Alvey R.F. Mohler J.L. Terrian D.M. Cancer Res. 2002; 62: 2423-2429PubMed Google Scholar). Signaling studies have determined an essential role for p38 MAPK and ceramide as mediators of phorbol ester-induced apoptosis in LNCaP cells (6Garzotto M. Haimovitz-Friedman A. Liao W.C. White-Jones M. Huryk R. Heston W.D. Cardon-Cardo C. Kolesnick R. Fuks Z. Cancer Res. 1999; 59: 5194-5201PubMed Google Scholar, 12Kimura K. Markowski M. Edsall L.C. Spiegel S. Gelmann E.P. Cell Death Differ. 2003; 10: 240-248Crossref PubMed Scopus (29) Google Scholar), and in addition, a modulatory role for the JNK interacting protein 1 (JIP-1, an inhibitor of JNK) has been recently postulated (13Ikezoe T. Yang Y. Taguchi H. Koeffler H.P. Br. J. Cancer. 2004; 90: 2017-2024Crossref PubMed Scopus (24) Google Scholar), 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 TNFα, 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 PKCϵ-overexpressing R6 fibroblasts stimulates DNA synthesis and causes morphologic transformation. Transforming growth factor-β release has been associated, at least in part, to the growth abnormalities caused by PKCϵ overexpression (14Cacace A.M. Ueffing M. Han E.K. Marme D. Weinstein I.B. J. Cell. Physiol. 1998; 175: 314-322Crossref PubMed Scopus (27) Google Scholar). 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 PKCδ 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. Materials—PMA was purchased from LC Laboratories (Woburn, MA). DAPI (4′,6-diamidino-2-phenylindole) was obtained from Sigma. The p38 MAPK inhibitor SB203580 was purchased from LC Laboratories. The JNK inhibitor SP600125 was obtained from Alexis Biochemicals (San Diego, CA). Cell culture reagents and media were purchased from ATCC (Rockville, MD). Recombinant human TRAIL/Apo2L and sFas ligand were from PetroTech, Inc. (Rocky Hill, NJ). Recombinant human TNFα was from R&D Systems, Inc. 2,2′-Azino-bis(3-ethylbenzyazoline)-6-sulfonic acid was from Roche Diagnostics. The following antibodies were used: mouse monoclonal anti-human TNFα (clone RDI-2C8, Research Diagnostics, Inc., Flanders, NJ); biotinylated anti-human TNFα (Research Diagnostics Inc.); monoclonal anti-human FasL (clone 2C101, Alexis, San Diego, CA); mouse monoclonal anti-human TRAIL, TNFR1, and TNFR2 (R&D Systems), anti-PKCα (Upstate Biotechnology, Inc., Lake Placid, NY); anti-PKCδ and anti-JNK (Transduction Laboratories, Lexington, KY); anti-phospho-JNK (Cell Signaling Technology, Beverly, MA); anti-caspase-8, anti-IκB, and anti-phospho-IκB (Santa Cruz Biotechnologies, Santa Cruz, CA); anti-Fas-associated protein with a death domain (FADD), anti-D4, and anti-D5 (kind gifts from Dr. Wafik el-Deiry, University of Pennsylvania), and anti-TACE (a kind gift from Dr. Marcos Milla, University of Pennsylvania). Cell Culture—LNCaP human prostate cancer cells were purchased from ATCC. Cells (passages 2-10) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin (100 units/ml)-streptomycin (100 μg/ml) at 37 °C in a humidified 5% CO2 atmosphere. Collection of CM—Cells (∼70% 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 (∼70% 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% β-mercaptoethanol, and then lysed by sonication. Equal amounts of protein (20 μg/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 previously (8Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li L. Yuspa S.H. Kazanietz M.G. J. Biol. Chem. 2000; 275: 7574-7582Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Tanaka Y. Gavrielides M.V. Mitsuuchi Y. Fujii T. Kazanietz M.G. J. Biol. Chem. 2003; 278: 33753-33762Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). 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 (8Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li L. Yuspa S.H. Kazanietz M.G. J. Biol. Chem. 2000; 275: 7574-7582Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). RNA Interference (RNAi)—Twenty-one bp dsRNAs were purchased from Dharmacon, Inc. (Lafayette, CO). The following target sequences were used: AAGUGCCACAAAGGAACCUAC (TNFR1), AACAGAGUGAGACCCUGUCUC (TNFR2), AAGCAGAAGAUUGAGGACCAC (DR5), AAGUGCAUGGACAGGGUGUGU (DR4); AAGUGCCACAAAGGAACCUAC (Fas), CCAUGAGUUUAUCGCCACC (PK-Cδ), and AACAUCGCUGUAGCAUCGUCU (control RNAi). TACE RNAi (AAACGAAAGCGAGTACACUGC) was purchased from Qiagen-Xeragon (Germantown, MD). DsRNAs (100 nm) were transfected into LNCaP cells using Oligofectamine (Invitrogen) following the instructions provided by the manufacturer. In each case, the optimal time for protein/RNA depletion after transfection was determined. Unless when indicated, experiments were carried out 48 h after transfection. 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 μl. Real-time PCR—PCR primers and fluorogenic probes for human TNFα were purchased from Applied Biosystems. The probes were 5′ end-labeled with 6-carboxyfluorescein (FAM). Each PCR amplification was performed in a total volume of 12.5 μl, containing 6.25 μl of 2× TaqMan Universal PCR Master Mix (Applied Biosystems), commercial target primers (300 nm), the fluorescent probe (200 nm), and 1 μl 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)—TNFα levels were determined by ELISA using 2 μg/well of coating anti-TNFα antibody. Nonspecific binding sites were blocked with 10% fetal bovine serum in phosphate-buffered saline. CM (50 μl) was added in each well and incubated overnight at 4 °C. Subsequently, 100 μl of a biotin-labeled anti-TNFα antibody (0.3 μg/ml) was added for 2 h at room temperature. Bound antibody was detected by incubation with peroxidase-labeled streptavidine and 2,2′-azino-bis(3-ethylbenzyazoline)-6-sulfonic acid, and absorbance was measured at 405 nm. TRAIL levels were determined using a Quantikine M kit from R&D Systems. PKC Translocation—Translocation was determined by Western blot using a subcellular fractionation technique, as previously reported (8Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li L. Yuspa S.H. Kazanietz M.G. J. Biol. Chem. 2000; 275: 7574-7582Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). 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 (4Powell C.T. Brittis N.J. Stec D. Hug H. Heston W.D. Fair W.R. Cell Growth & Differ. 1996; 7: 419-428PubMed Google Scholar, 5Zhao X. Gschwend J.E. Powell C.T. Foster R.G. Day K.C. Day M.L. J. Biol. Chem. 1997; 272: 22751-22757Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li L. Yuspa S.H. Kazanietz M.G. J. Biol. Chem. 2000; 275: 7574-7582Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Tanaka Y. Gavrielides M.V. Mitsuuchi Y. Fujii T. Kazanietz M.G. J. Biol. Chem. 2003; 278: 33753-33762Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 10Yin L. Bennani-Baiti N. Powell C.T. J. Biol. Chem. 2005; 280: 5533-5541Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). 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 apoptotic 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-G0/G1 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 type-specific, 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 performed 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 (∼70%) was retained after “de novo” protein synthesis inhibition (Fig. 1G), suggesting that the effect was largely caused by the release of pre-formed factors. PKCδ Has a Dual Role Both in Apoptotic Factor Release and as an Effector of CM-induced Apoptosis—PKCδ has been established as a key mediator of PMA-induced apoptosis in LNCaP cells (8Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li L. Yuspa S.H. Kazanietz M.G. J. Biol. Chem. 2000; 275: 7574-7582Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 10Yin L. Bennani-Baiti N. Powell C.T. J. Biol. Chem. 2005; 280: 5533-5541Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). To determine whether PKCδ is involved in the release of apoptotic factors, we collected CM from LNCaP cells treated with either the pan-PKC inhibitor GF109203X or the PKCδ inhibitor rottlerin (Fig. 2A). We found that CM collected after pharmacological inhibition of PKCδ 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 PKCδ inhibition (15Tillman D.M. Izeradjene K. Szucs K.S. Douglas L. Houghton J.A. Cancer Res. 2003; 63: 5118-5125PubMed Google Scholar), we used a RNAi approach, as we have previously described (9Tanaka Y. Gavrielides M.V. Mitsuuchi Y. Fujii T. Kazanietz M.G. J. Biol. Chem. 2003; 278: 33753-33762Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). PKCδ levels were reduced by ∼90% in LNCaP cells upon delivery of a specific dsRNA. This dsRNA specifically reduces the levels of PKCδ without affecting the expression of other PMA-responsive PKC isozymes present in LNCaP cells (Fig. 2C). Remarkably, CM-PMA collected from PKCδ-depleted cells has remarkably less apoptotic activity (Fig. 2A), arguing that PKCδ is required for the release of apoptogenic factors in response to PMA. PKCδ also plays a role as an effector of the apoptotic effect of CM-PMA. Indeed, when CM-PMA was added to PKCδ-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 (9Tanaka Y. Gavrielides M.V. Mitsuuchi Y. Fujii T. Kazanietz M.G. J. Biol. Chem. 2003; 278: 33753-33762Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 15Tillman D.M. Izeradjene K. Szucs K.S. Douglas L. Houghton J.A. Cancer Res. 2003; 63: 5118-5125PubMed Google Scholar). Interestingly, the CM-PMA caused translocation of PKCδ in LNCaP cells (Fig. 2D). Thus, PKCδ appears to have a dual role, both in the release of apoptogenic factors as well as an effector. A Role for TNFα in the PKC-induced Autocrine Loop—As TNFα is known to cause cell death in LNCaP cells (12Kimura K. Markowski M. Edsall L.C. Spiegel S. Gelmann E.P. Cell Death Differ. 2003; 10: 240-248Crossref PubMed Scopus (29) Google Scholar, 16Chopra D.P. Menard R.E. Januszewski J. Mattingly R.R. Cancer Lett. 2004; 203: 145-154Crossref PubMed Scopus (59) Google Scholar), 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 (TNFα-converting enzyme), a metalloprotease responsible for TNFα shedding (17Crowe P.D. Walter B.N. Mohler K.M. Otten-Evans C. Black R.A. Ware C.F. J. Exp. Med. 1995; 181: 1205-1210Crossref PubMed Scopus (245) Google Scholar). 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 ± 8% reduction (n = 3) in TACE expression in LNCaP cells upon delivery of a specific TACE dsRNA (Fig. 3B). CM-PMA collected from TACE-depleted cells has indeed significantly lower apoptogenic activity (∼60% inhibition) when added to fresh LNCaP cells (Fig. 3C). To assess the involvement of TNFα we used a specific anti-TNFα neutralizing antibody, which dose-dependently impaired the ability of the CM-PMA to cause apoptosis. At a concentration of 10 mg/ml, a 48 ± 3% inhibition (n = 3) was observed (Fig. 4A). Real-time PCR assays showed that PMA treatment caused a marked increase in TNFα mRNA levels (357 ± 92-fold after 3 h, and 1992 ± 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 TNFα is probably less important than the released of a pre-formed pool of the cytokine.FIGURE 4Involvement of TNFα in PMA-induced apoptosis.Panel A, LNCaP cells were treated with CM-PMA, either in the presence or absence of increasing concentrations of a TNFα blocking antibody or a normal mouse IgG. The CM was incubated with the antibodies for 30 min and then added to the cells. The incidence of apoptosis was determined 24 h later. Data are expressed as percentage of apoptosis induced by CM-PMA in the absence of antibodies. Results are presented as mean ± S.E. (n = 3). Panel B, LNCaP cells were transfected with specific RNAi duplexes for TACE, PKCδ, or a control RNAi (48 h). Alternatively, cells were pretreated with tumor necrosis factor alpha inhibitor-2 (10 μm) or GF109203X (10 μm), 40 min before and during 100 nm PMA-treatment. CM-PMA or CM-vehicle were collected 24 h later, and TNFα levels were determined by ELISA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PKCδ Is Required for PMA-induced Secretion of TNFα—In the next series of experiments we explored whether PKC activation could promote the secretion of TNFα from LNCaP cells. TNFα levels in CM-PMA (as determined by ELISA) were remarkably higher compared with those in CM-vehicle (Fig. 4B). As expected, TNFα 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 TNFα caused by PMA. In LNCaP cells subject to PKCδ RNAi, there was also a marked reduction in TNFα levels in the CM upon PMA treatment. Taken together, these results support a critical role for PKCδ in phorbol ester-induced secretion of TNFα. TRAIL Is Also Involved in the PKC-mediated Autocrine Effect—Other cytokines in addition to TNFα are pro-apoptogenic in prostate cancer cells, including TRAIL and FasL (18Yu R. Mandlekar S. Ruben S. Ni J. Kong A.N. Cancer Res. 2000; 60: 2384-2389PubMed Google Scholar, 19Kimura K. Gelmann E.P. J. Biol. Chem. 2000; 275: 8610-8617Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 20Nakanishi H. Mazda O. Satoh E. Asada H. Morioka H. Kishida T. Nakao M. Mizutani Y. Kawauchi A. Kita M. Imanishi J. Miki T. Gene Ther. 2003; 10: 434-442Crossref PubMed Scopus (63) Google Scholar, 21Zhang X. Jin T.G. Yang H. DeWolf W.C. Khosravi-Far R. Olumi A.F. Cancer Res. 2004; 64: 7086-7091Crossref PubMed Scopus (134) Google Scholar). When
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