Up-regulation of Cyclooxygenase-2 and Apoptosis Resistance by p38 MAPK in Hypericin-mediated Photodynamic Therapy of Human Cancer Cells
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m307591200
ISSN1083-351X
AutoresNico Hendrickx, Cédric Volanti, Ugo Moens, Ole Morten Seternes, Peter de Witte, Jackie R. Vandenheede, Jacques Piette, Patrizia Agostinis,
Tópico(s)Inflammatory mediators and NSAID effects
ResumoPhotodynamic Therapy (PDT) is an approved anticancer therapy that kills cancer cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which selectively accumulates in tumors. We report that hypericin-mediated PDT of human cancer cells leads to up-regulation of the inducible cyclooxygenase-2 (COX-2) enzyme and the subsequent release of PGE2. Dissection of the signaling pathways involved revealed that the selective activation of p38 MAPK α and β mediate COX-2 up-regulation at the protein and messenger levels. The p38 MAPK inhibitor, PD169316, abrogated COX-2 expression in PDT-treated cells, whereas overexpression of the drug-resistant PD169316-insensitive p38 MAPK α and β isoforms restored COX-2 levels in the presence of the kinase inhibitor. Transcriptional regulation by nuclear factor-κB was not involved in COX-2 up-regulation by PDT. The half-life of the COX-2 messenger was drastically shortened by p38 MAPK inhibition in transcriptionally arrested cells, suggesting that p38 MAPK mainly acts by stabilizing the COX-2 transcript. Overexpression of WT-p38 MAPK increased cellular resistance to PDT-induced apoptosis, and inhibiting this pathway exacerbated cell death and prevented PGE2 secretion. Hence, the combination of PDT with pyridinyl imidazole inhibitors of p38 MAPK may improve the therapeutic efficacy of PDT by blocking COX-2 up-regulation, which contributes to tumor growth by the release of growth- and pro-angiogenic factors, as well as by sensitizing cancer cells to apoptosis. Photodynamic Therapy (PDT) is an approved anticancer therapy that kills cancer cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which selectively accumulates in tumors. We report that hypericin-mediated PDT of human cancer cells leads to up-regulation of the inducible cyclooxygenase-2 (COX-2) enzyme and the subsequent release of PGE2. Dissection of the signaling pathways involved revealed that the selective activation of p38 MAPK α and β mediate COX-2 up-regulation at the protein and messenger levels. The p38 MAPK inhibitor, PD169316, abrogated COX-2 expression in PDT-treated cells, whereas overexpression of the drug-resistant PD169316-insensitive p38 MAPK α and β isoforms restored COX-2 levels in the presence of the kinase inhibitor. Transcriptional regulation by nuclear factor-κB was not involved in COX-2 up-regulation by PDT. The half-life of the COX-2 messenger was drastically shortened by p38 MAPK inhibition in transcriptionally arrested cells, suggesting that p38 MAPK mainly acts by stabilizing the COX-2 transcript. Overexpression of WT-p38 MAPK increased cellular resistance to PDT-induced apoptosis, and inhibiting this pathway exacerbated cell death and prevented PGE2 secretion. Hence, the combination of PDT with pyridinyl imidazole inhibitors of p38 MAPK may improve the therapeutic efficacy of PDT by blocking COX-2 up-regulation, which contributes to tumor growth by the release of growth- and pro-angiogenic factors, as well as by sensitizing cancer cells to apoptosis. Photodynamic therapy (PDT) 1The abbreviations used are: PDTphotodynamic therapyAREAU-rich elementATFactivating transcription factorCREcAMP response elementAUF1ARE/poly(U) binding factor 1C/EBPCCAAT/enhancer-binding proteinCDK-1cyclin-dependent kinase 1COX-2cyclooxygenase-2DRdrug resistantEGFepidermal growth factorEMSAelectrophoretic mobility shift assayERKextracellular signal-regulated kinaseFACSfluorescence-activated cell sortingHAhemagglutininJNKc-Jun NH2-terminal protein kinaseMAPKmitogen-activated protein kinaseMEKmitogen-activated/ERK kinaseMKKMAPK kinaseNFnuclear factorPARPpoly(ADP-ribose) polymerasePGprostaglandinPI3Kphosphatidylinositol 3 kinaseTCCtransitional cell carcinomaUTRuntranslated regionWTwild typeELISAenzyme-linked immunosorbent assayGSTglutathione S-transferasezbenzyloxycarbonyl. is an accepted therapeutic procedure suitable for the treatment of a variety of tumors and non-malignant disorders. PDT involves the administration and subsequent light activation (600–850 nm) of a photosensitizing compound (photosensitizer), which specifically accumulates in tumors (1Dougherty T.J. Gomer C.J. Henderson B. Jori G. Kessel D. Korbelik M. Moan J. Peng Q. J. Natl. Cancer Inst. 1998; 90: 889-905Crossref PubMed Scopus (4703) Google Scholar, 2Oleinick N.L. Morris R.L. Belichenko I. Photochem. Photobiol. Sci. 2002; 1: 1-21Crossref PubMed Scopus (1089) Google Scholar). This leads, in the presence of molecular oxygen, to the photochemical generation of reactive oxygen species, which ultimately kills the target cells (1Dougherty T.J. Gomer C.J. Henderson B. Jori G. Kessel D. Korbelik M. Moan J. Peng Q. J. Natl. Cancer Inst. 1998; 90: 889-905Crossref PubMed Scopus (4703) Google Scholar, 2Oleinick N.L. Morris R.L. Belichenko I. Photochem. Photobiol. Sci. 2002; 1: 1-21Crossref PubMed Scopus (1089) Google Scholar). photodynamic therapy AU-rich element activating transcription factor cAMP response element ARE/poly(U) binding factor 1 CCAAT/enhancer-binding protein cyclin-dependent kinase 1 cyclooxygenase-2 drug resistant epidermal growth factor electrophoretic mobility shift assay extracellular signal-regulated kinase fluorescence-activated cell sorting hemagglutinin c-Jun NH2-terminal protein kinase mitogen-activated protein kinase mitogen-activated/ERK kinase MAPK kinase nuclear factor poly(ADP-ribose) polymerase prostaglandin phosphatidylinositol 3 kinase transitional cell carcinoma untranslated region wild type enzyme-linked immunosorbent assay glutathione S-transferase benzyloxycarbonyl. Hypericin is a naturally occurring photosensitizer synthesized by Hypericum plants (St. John's wort). The interesting photosensitizing properties of hypericin together with its selective uptake in tumor tissues and its minimal dark cytotoxicity, have encouraged the evaluation of this hydroxylated phenanthroperylenequinone in the photodynamic treatment of cancers (3Agostinis P. Vantieghem A. Merlevede W. de Witte P.A. Int. J. Biochem. Cell Biol. 2002; 34: 221-241Crossref PubMed Scopus (411) Google Scholar). Although hypericin does not preferentially accumulate in mitochondria but localizes mainly to the membranes of the endoplasmic reticulum and Golgi apparatus, a rapid release of cytochrome c into the cytosol and the subsequent activation of the apoptosome, are the signaling events precipitating apoptotic cell death by the photodynamic stress (4Vantieghem A. Assefa Z. Vandenabeele P. Declercq W. Courtois S. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. FEBS Lett. 1998; 440: 19-24Crossref PubMed Scopus (132) Google Scholar, 5Vantieghem A. Xu Y. Declercq W. Vandenabeele P. Denecker G. Vandenheede J.R. Merlevede W. de Witte P.A. Agostinis P. Photochem. Photobiol. 2001; 74: 133-142Crossref PubMed Scopus (62) Google Scholar, 6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 7Vantieghem A. Xu Y. Assefa Z. Piette J. Vandenheede J.R. Merlevede W. de Witte P.A. Agostinis P. J. Biol. Chem. 2002; 277: 37718-37731Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Since apoptosis is a major in vivo response to hypericin-mediated PDT (8Chen B. Roskams T. Xu Y. Agostinis P. de Witte P.A. Int. J. Cancer. 2002; 98: 284-290Crossref PubMed Scopus (82) Google Scholar), modulation of apoptotic vulnerability is critical for the therapeutic efficacy of this drug. Cellular commitment to apoptosis, or the ability to evade apoptosis in response to cell damage, involves the integration of a complex network of both survival and death pathways. The MAPK family a central mediator of survival and cell death pathways, and MAPKs have been implicated in the response of tumor cells to very diverse antitumor signals, including PDT (2Oleinick N.L. Morris R.L. Belichenko I. Photochem. Photobiol. Sci. 2002; 1: 1-21Crossref PubMed Scopus (1089) Google Scholar). The three major MAPK pathways in mammalian cells include the extracellular signal-regulated kinases (ERKs), the c-Jun NH2-terminal protein kinases (JNKs), and the p38 MAPKs (9Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar). ERKs are acutely stimulated by growth and differentiation factors through activated receptor tyrosine kinases, heterotrimeric G protein-coupled receptors, or cytokine receptors (9Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar). The JNKs and p38 MAPKs are activated in response to a variety of stress signals including UV irradiation, chemotherapeutics, osmotic stress, hypoxia/anoxia, hyperthermia, and they have been involved in the regulation of apoptosis (9Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar). In a previous study we have shown that, parallel to the caspase-activation cascade, hypericin-mediated PDT leads to inhibition of ERK2 (6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), and to a sustained activation of the stress-activated protein kinases, JNK1 and p38 MAPK. Activation of JNK1 and p38 MAPK was suggested to have a protective role against PDT-induced apoptosis (6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). However, downstream targets of these signaling cascades still need to be identified. Some recent reports have implicated p38 MAPK in the up-regulation of the inducible cyclooxygenase-2 (cox-2) gene (10Lasa M. Mahtani K.R. Finch A. Brewer G. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2000; 20: 4265-4274Crossref PubMed Scopus (370) Google Scholar, 11Lasa M. Brook M. Saklatvala J. Clark A.R. Mol. Cell. Biol. 2001; 21: 771-780Crossref PubMed Scopus (219) Google Scholar, 12Rousseau S. Morrice N. Peggie M. Campbell D.G. Gaestel M. Cohen P. EMBO J. 2002; 21: 6505-6514Crossref PubMed Scopus (184) Google Scholar, 13Faour W.H. He Y. He Q.W. de Ladurantaye M. Quintero M. Mancini A. Di Battista J.A. J. Biol. Chem. 2001; 276: 31720-31731Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 14Subbaramaiah K. Marmo T.P. Dixon D.A. Dannenberg A.J. J. Biol. Chem. 2003; 278: 37637-37647Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). COX catalyzes the conversion of arachidonic acid to prostaglandin (PG) H2, the immediate substrate for a number of cell specific prostaglandin and tromboxane synthases, leading to the production and release of PGE2 and other prostanoids, that contribute to the development of important immunomodulatory responses (15Smith W.L. De Witt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2464) Google Scholar, 16Williams C.S. Mann M. DuBois R.N. Oncogene. 1999; 18: 7908-7916Crossref PubMed Scopus (1262) Google Scholar, 17Cao Y. Prescott S.M. J. Cell. Physiol. 2002; 190: 279-286Crossref PubMed Scopus (428) Google Scholar). Two isoforms, COX-1 and COX-2, have been identified and their expression is differently regulated. COX-1 is constitutively expressed in most cell types and may be responsible for housekeeping functions. By contrast, the expression of COX-2, which is regulated both at the transcriptional and post-transcriptional level, is barely detectable in normal tissues but is rapidly induced in response to tumor promoters, oncogenes, cytokines, and mitogens (15Smith W.L. De Witt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2464) Google Scholar, 16Williams C.S. Mann M. DuBois R.N. Oncogene. 1999; 18: 7908-7916Crossref PubMed Scopus (1262) Google Scholar, 17Cao Y. Prescott S.M. J. Cell. Physiol. 2002; 190: 279-286Crossref PubMed Scopus (428) Google Scholar). In a number of cellular and animal models, COX-2 has been shown to promote cell growth, to inhibit apoptosis, and to enhance cell motility and adhesion, and COX-2 overexpression is sufficient to cause tumorigenesis (reviewed in Refs. 16Williams C.S. Mann M. DuBois R.N. Oncogene. 1999; 18: 7908-7916Crossref PubMed Scopus (1262) Google Scholar and 17Cao Y. Prescott S.M. J. Cell. Physiol. 2002; 190: 279-286Crossref PubMed Scopus (428) Google Scholar). In agreement with these observations, COX-2 is up-regulated in many tumors, and this up-regulation has been shown to promote cancer progression, recurrence, and invasiveness, as well as angiogenesis (16Williams C.S. Mann M. DuBois R.N. Oncogene. 1999; 18: 7908-7916Crossref PubMed Scopus (1262) Google Scholar, 17Cao Y. Prescott S.M. J. Cell. Physiol. 2002; 190: 279-286Crossref PubMed Scopus (428) Google Scholar). These studies suggest that the use of specific COX-2 inhibitors may be beneficial in the management of these neoplasms. Interestingly, the combination of porphyrin- and chlorin-based PDT with a specific COX-2 inhibitor, was recently reported to increase the tumoricidal potential of PDT in vivo (18Ferrario A. Von Tiehl K. Wong S. Luna M. Gomer C.J. Cancer Res. 2002; 62: 3956-3961PubMed Google Scholar). However, the mechanisms underlying PDT-mediated COX-2 up-regulation and its protective effect were not further explored. In the present study, we show that hypericin-mediated PDT leads to COX-2 up-regulation and secretion of PGE2, which are completely dependent on the specific activation of p38 MAPK. We also provide evidence in support for a post-transcriptional role of p38 MAPK in stabilizing COX-2 mRNA and for a functional involvement of p38 MAPK in counteracting PDT-induced apoptosis. Our results suggest that inhibition of the p38 MAPK signaling cascade, which results in the repression of COX-2 up-regulation and concomitant increase in the susceptibility of the cells to undergo apoptosis, could be of clinical relevance to improve the efficacy of hypericin-mediated PDT of cancers. Materials—Hypericin was synthesized and purified as described in Ref. 8Chen B. Roskams T. Xu Y. Agostinis P. de Witte P.A. Int. J. Cancer. 2002; 98: 284-290Crossref PubMed Scopus (82) Google Scholar. All cell culture products were obtained from Bio-Whittaker (Verviers, Belgium). The p38 MAPK inhibitor PD169316, the MEK inhibitor PD98059 and the selective COX-2 inhibitor NS-398 were purchased from Calbiochem (San Diego, CA), whereas the inhibitor of IκBα kinase BAY117085 and the specific inhibitor of PI3-kinase LY294002 were from BIOMOL Research Laboratories (Plymouth, PA). Sytox Green was purchased from Molecular Probes (Leiden, The Netherlands). Actinomycin D was from Sigma. The fluorogenic caspase substrates Ac-DEVD-amc and Ac-IETD-amc were obtained from Peptide Institute, Inc. (Osaka, Japan). PARP antibody was from BIOMOL Research Laboratories (Plymouth, PA), anti-cytochrome c was obtained from PharMingen (San Diego, CA), and anti-caspase-3 antibody and anti-COX-2 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-p38 MAPK (Thr180/Tyr182) monoclonal antibody, which specifically recognizes the phosphorylated form of the kinase and anti-p38 MAPK antibody were purchased from New England Biolabs, Inc. (Beverly, MA). Monoclonal anti-α-tubulin antibody was from Sigma. Horseradish peroxidase-conjugated secondary antibodies were from DAKO (Denmark). GST-cJun-(1–223) and polyclonal antibodies to the human JNK1 and ERK2 were prepared as described elsewhere (6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Cell Culture—HeLa cells (human cervix carcinoma cells) and T24 (human transitional cell carcinoma of the urinary bladder) were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium containing l-glutamine (2 mm), penicillin (100 IU/ml), streptomycin (100 μg/ml), and 10% fetal calf serum. Stable HeLa transfectants overexpressing mutated HA-tagged IκBα, were obtained as described in Ref. 19Matroule J.Y. Bonizzi G. Morliere P. Paillous N. Santus R. Bours V. Piette J. J. Biol. Chem. 1999; 274: 2988-3000Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar. Cell Photosensitization—The cells were preincubated with hypericin for 16 h in subdued light conditions (< 1μW/cm2) and subsequently irradiated in Dulbecco's modified Eagle's medium by placing the samples on a plastic diffuser sheet 5 cm above a set of seven L18W30 fluorescent lamps (Osram) as described in Vantieghem et al. (4Vantieghem A. Assefa Z. Vandenabeele P. Declercq W. Courtois S. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. FEBS Lett. 1998; 440: 19-24Crossref PubMed Scopus (132) Google Scholar). The maximal emission of the fluorescent lamps was between 530 and 620 nm, which coincides with the major absorption peaks of hypericin (545 and 595 nm). At the surface of the diffuser, the uniform fluence rate was 4.5 mW/cm2, as measured with an IL 1400 radiometer (International Light, Newburyport, MA). All inhibitors used were added to the cell culture medium 1 h prior to photosensitization at concentrations indicated in figure legends. Preparation of Cell Extracts and Western Blotting—Cell extracts were prepared at indicated time points following photosensitization, and analysis of cytochrome c release was performed as described in Refs. 6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar and 7Vantieghem A. Xu Y. Assefa Z. Piette J. Vandenheede J.R. Merlevede W. de Witte P.A. Agostinis P. J. Biol. Chem. 2002; 277: 37718-37731Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar. Protein concentrations were determined using the bicinchonic acid (BCA) method (Pierce). Samples (30–80 μg of protein) were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose (Protran®), and processed as in Refs. 6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar and 7Vantieghem A. Xu Y. Assefa Z. Piette J. Vandenheede J.R. Merlevede W. de Witte P.A. Agostinis P. J. Biol. Chem. 2002; 277: 37718-37731Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar. Caspase Assay—Caspase activity was measured using the fluorogenic substrate Ac-DEVD-amc as described in Ref. 6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar. Immunoprecipitation and Protein Kinase Assay—JNK1, ERK2, and p38 MAPK activities were measured as previously described in Refs. 6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar and 7Vantieghem A. Xu Y. Assefa Z. Piette J. Vandenheede J.R. Merlevede W. de Witte P.A. Agostinis P. J. Biol. Chem. 2002; 277: 37718-37731Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar. Assessment of Cell Viability—Cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolism as previously described (7Vantieghem A. Xu Y. Assefa Z. Piette J. Vandenheede J.R. Merlevede W. de Witte P.A. Agostinis P. J. Biol. Chem. 2002; 277: 37718-37731Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Transient Transfections—The drug-resistant (DR) p38 α was produced by mutating Thr106, His107, and Leu108 to Met, Pro, and Phe, respectively. In the case of the DR p38β, Thr106, Thr107, and Leu108 were changed to Met, Met, and Phe, respectively. Mutagenesis was performed with the QuikChange mutagenesis kit (Stratagene). DR p38α and p38β mutants were generated using complementary primers (only the forward primer is shown) 5′-gacgtgtacctggtgatgcctttcatgggggcggacctgaacaacatcg-3′ and 5′-gaagtgtacttggtgatgatgtttatgggcgccgacctgaacaacatcg-3′, respectively. FLAG-tagged p38α and FLAG-tagged p38β were kindly provided by Dr. J. Han (Scripps Institute, CA). For transient transfection cells were seeded at 1.0 × 106 (T24) or 7.0 × 105 (HeLa) cells/10 cm Petri dish. After an overnight culture, pcDNA3, WT p38, MKK3, DR-p38, and pEGFP-C3 (Clontech, Palo Alto, CA) (4:1) expression vectors were co-transfected using either the Lipofectin® Plus" (Invitrogen, Merelbeke, Belgium) transfection reagent (T24 cells) or the FuGENE 6 transfection reagent (Roche Applied Science, Vilvoorde, Belgium) (HeLa cells) according to the manufacturer's protocol. After 7 h of transfection the medium was changed with complete Dulbecco's modified Eagle's medium. Twenty-four hours after transient transfection, cells were incubated with or without hypericin for 16 h at 37 °C in dark. Cells were then irradiated as described above. Transfection efficiency was determined by calculating the percentage of fluorescent green cells over the total number of cells under a fluorescence microscope. Total lysates were prepared and analyzed by Western blotting. Cell Cycle Analysis—The cell cycle profile was analyzed by flow cytometry after staining of the DNA with Sytox Green according to Vantieghem et al. (7Vantieghem A. Xu Y. Assefa Z. Piette J. Vandenheede J.R. Merlevede W. de Witte P.A. Agostinis P. J. Biol. Chem. 2002; 277: 37718-37731Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Electrophoretic Mobility Shift Assay—Nuclear extracts were obtained, and EMSA was performed as described by Volanti et al. (20Volanti C. Matroule J.Y. Piette J. Photochem. Photobiol. 2002; 75: 36-45Crossref PubMed Scopus (47) Google Scholar). DNA-protein complexes were resolved by nondenaturing PAGE. Gels were vacuum dried and exposed to Fuji x-ray films. The sequence of the probe (Eurogentec, Liège, Belgium) used was: NF-κB probe, 5′-GGTTACAAGGGACTTTCCGCTG-3′. PGE2Measurement—Cells were photosensitized as described before. After the indicated time points, the medium was collected, dead cells were removed by centrifugation, and PGE2 concentrations were measured by the PGE2 ELISA Kit (R&D Systems, Abingdon, UK) according to manufacturer's instructions. RT-PCR—After indicated time points, total RNA was isolated with the RNeasy Mini Kit (Qiagen, West Sussex, UK) and spectrophotometrically quantified, and the RT-PCR reaction was performed with the ThermoScript™ RT-PCR System (Invitrogen) according to manufacturer's instructions. The reactions were heated at 94 °C for 3 min and then immediately cycled 26 times through a 30 s denaturating step at 94 °C, a 30 s annealing step at 55 °C and a 30 s extension step at 72 °C. After the cycling procedure, a final 10 min elongation step at 72 °C was performed. The following primers were used: COX-2, (F) 5′-TTCAAATGAGATTGTGGGAAAAT-3′ and (R) 5′-AGATCATCTCTGCCTGAGTATCTT-3′; GAPDH, (F) 5′-CCATCAACGACCCCTTCATTGACC-3′ and (R) 5′-GAAGGCCATGCCAGTGAGCTTCC-3′. RNA Stability Assay—The cells were treated as described before. 5-h post-irradiation, the medium was changed to fresh medium containing actinomycin D (3 μg/ml) with or without the p38 MAPK inhibitor PD169316. At different time intervals, cells were collected for RNA preparation. RNA was examined by RT-PCR analysis. PDT with Hypericin Induces Apoptosis in Cancer Cells via the Intrinsic Pathway of Caspase Activation—Dose- and time-dependent studies of the cell death response in the human TCC cell line T24, showed an accumulation of apoptotic cells starting at 3 h post-irradiation, using 150 nm as the optimal lethal hypericin concentration in combination with 4 J/cm2 light dose (Fig. 1, A and C). T24 cells treated with 150 nm hypericin and irradiated, showed a time-dependent increase in the amount of cells with apoptotic morphology, which was paralleled by the increase in cytosolic cytochrome c and cleavage of procaspase-3 and its downstream substrate PARP (Fig. 1B). Apoptotic cell death could be blocked by the cell-permeable caspase inhibitors z-VAD-fmk and z-DEVD-fmk (data not shown), confirming that PDT-induced apoptosis in T24 cells is a caspase-dependent process. As shown in Fig. 1C, activation of procaspase-3 was clearly detectable 7 h after irradiation whereas a negligible increase in caspase-8 activity could be measured 24 h after PDT, likely as a result of a caspase-3 feedback activation loop (21Slee E.A. Adrain C. Martin S.J. Cell Death Differ. 1999; 6: 1067-1074Crossref PubMed Scopus (389) Google Scholar). Similar kinetics of cytochrome c release and dependence on procaspase-3 activation for the progression of apoptosis, have been shown in HeLa cells (4Vantieghem A. Assefa Z. Vandenabeele P. Declercq W. Courtois S. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. FEBS Lett. 1998; 440: 19-24Crossref PubMed Scopus (132) Google Scholar, 6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), indicating that the main mechanism triggering apoptotic cell death in response of hypericin-mediated PDT is conserved in a variety of human cancer cells. Hypericin-mediated PDT of Cancer Cells Leads to Up-regulation of COX-2—Cyclooxygenase-2 is an inducible enzyme whose up-regulation has been linked to tumor recurrence and progression in different cancers, including TCC of the human urinary bladder (22Shirahama T. Arima J. Akiba S. Sakakura C. Cancer. 2001; 92: 188-193Crossref PubMed Scopus (119) Google Scholar). Since in an experimental animal system hypericin-based PDT of bladder TCC has been shown to be only partially effective because of recurrence (23Kamuhabwa A.R. Roskams T. D'Hallewin M.A. Baert L. Van Poppel H. de Witte P.A. Int. J. Cancer. 2003; 107: 460-467Crossref PubMed Scopus (40) Google Scholar), we set out to investigate whether photosensitization of T24 and HeLa cells with hypericin could cause COX-2 up-regulation. Fig. 2A shows that while the basal level of COX-2 in the untreated T24 and HeLa cells was hardly detectable, PDT promoted a time-dependent and sustained increase in COX-2 protein levels. In both cancer cell lines the induction of COX-2 protein was detectable within 3 h after irradiation, attained a steady state at 7–16-h post-irradiation, reaching more than a 12-fold induction, and declined very gradually thereafter (Fig. 2A). Dose response experiments (data not shown) revealed COX-2 induction by hypericin concentrations starting from sublethal doses of 25 nm, corresponding to 80% cell survival (Fig. 1C), and increasing up to 150–200 nm, which corresponds to ∼20% cell survival. In all cases this response was totally light-dependent. Since COX-2 is the rate-limiting enzyme in the biosynthesis of PGE2, we also evaluated the release of this prostaglandin into the medium at the post-irradiation time corresponding to maximal COX-2 induction. As compared with control cells, the levels of secreted PGE2 increased severalfold following exposure of the cells to PDT (Fig. 2B). In the presence of the methyl sulfoxide NS-398, a selective COX-2 inhibitor (24Liu X.H. Yao S. Kirschenbaum A. Levine A.C. Cancer Res. 1998; 58: 4245-4249PubMed Google Scholar), the release of PGE2 was totally blocked (Fig. 2B), demonstrating that the induction of PGE2 synthesis coincides with up-regulation of COX-2 protein levels in response to the photodynamic stress. Cyclooxygenase-2 Is Selectively Up-regulated by the PDT-induced p38 MAPK Signaling Pathway—It has been reported that in T24 cells, which bear an oncogenic H-ras mutation, the MEKK1-p38 MAPK and the Raf-ERK cascades are constitutively activated (25Gupta A.K. Bakanauskas V.J. Cerniglia G.J. Cheng Y. Bernhard E.J. Musche l R.J. McKenna W.G. Cancer Res. 2001; 61: 4278-4282PubMed Google Scholar). In agreement with this, we found that in untreated T24 cells the activity state of p38 MAPK and ERKs (Fig. 3) was elevated. However, whereas the activity level of ERKs was rapidly reduced by PDT, the basal phosphorylation level of p38 MAPK dropped drastically to an undetectable level, to be followed by a de novo p38 MAPK activation, which was specifically mediated by PDT stress (Fig. 3). Interestingly, PDT of HeLa cells, with either membrane- or mitochondria-targeted photosensitizers, has been shown to cause an immediate dose-dependent attenuation of the EGF- or inflammatory cytokine-mediated signal through rapid degradation of the EGF receptor or down-modulation of cytokine-responsive signaling pathways (26Wong T.W. Tracy E. Oseroff A.R. Baumann H. Cancer Res. 2003; 63: 3812-3818PubMed Google Scholar). These PDT-mediated early effects could indeed explain why in HeLa (6Assefa Z. Vantieghem A. Declercq W. Vandenabeele P. Vandenheede J.R. Merlevede W. de Witte P. Agostinis P. J. Biol. Chem. 1999; 274: 8788-8796Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and T24 cells (Fig. 3) we observed a down-regulation of the basal activity levels of ERK and p38 MAPK immediately after irradiation. No difference in the protein levels was observed as shown for p38 MAPK (Fig. 3), indicating that PDT effects are due to changes in the phosphorylation/activity state of these signaling enzymes exclusively (data not shown). To delineate the contribution of these protein kinases to the PDT-mediated up-regulation o
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