The Thioxotriazole Copper(II) Complex A0 Induces Endoplasmic Reticulum Stress and Paraptotic Death in Human Cancer Cells
2009; Elsevier BV; Volume: 284; Issue: 36 Linguagem: Inglês
10.1074/jbc.m109.026583
ISSN1083-351X
AutoresSaverio Tardito, Claudio Isella, Enzo Médico, Luciano Marchiò, Elena Bevilacqua, Maria Hatzoglou, Ovidio Bussolati, R. Franchi‐Gazzola,
Tópico(s)RNA regulation and disease
ResumoThe copper(II) complex A0 induces a type of non-apoptotic cell death also known as paraptosis. Paraptosis involves extensive endoplasmic reticulum vacuolization in the absence of caspase activation. A wide panel of human cancer cell lines was used to demonstrate differences in cytotoxicity by the paraptosis-inducing drug A0 and the metal-based pro-apoptotic drug cisplatin. Gene expression profiling of the human fibrosarcoma HT1080 cells showed that, while cisplatin induced p53 targets, A0 up-regulated genes involved in the unfolded protein response (UPR) and response to heavy metals. The cytotoxic effects of A0 were associated with inhibition of the ubiquitin-proteasome system and accumulation of ubiquitinylated proteins, in a manner dependent on protein synthesis. Cycloheximide inhibited the accumulation of ubiquitinylated proteins and hampered A0-induced cell death process. The occurrence of the UPR during A0-induced death process was shown by the increased abundance of spliced XBP1 mRNA, transient eIF2α phosphorylation, and a series of downstream events, including attenuation of global protein synthesis and increased expression of ATF4, CHOP, BIP, and GADD34. Mouse embryonic fibroblasts expressing a mutant eIF2α, which could not be phosphorylated, were more resistant to A0 than wild type cells, pointing to a pro-death role of eIF2α phosphorylation. A0 may thus represent the prototypical member of a new class of compounds that cause paraptotic cell death via mechanisms involving eIF2α phosphorylation and the UPR. The copper(II) complex A0 induces a type of non-apoptotic cell death also known as paraptosis. Paraptosis involves extensive endoplasmic reticulum vacuolization in the absence of caspase activation. A wide panel of human cancer cell lines was used to demonstrate differences in cytotoxicity by the paraptosis-inducing drug A0 and the metal-based pro-apoptotic drug cisplatin. Gene expression profiling of the human fibrosarcoma HT1080 cells showed that, while cisplatin induced p53 targets, A0 up-regulated genes involved in the unfolded protein response (UPR) and response to heavy metals. The cytotoxic effects of A0 were associated with inhibition of the ubiquitin-proteasome system and accumulation of ubiquitinylated proteins, in a manner dependent on protein synthesis. Cycloheximide inhibited the accumulation of ubiquitinylated proteins and hampered A0-induced cell death process. The occurrence of the UPR during A0-induced death process was shown by the increased abundance of spliced XBP1 mRNA, transient eIF2α phosphorylation, and a series of downstream events, including attenuation of global protein synthesis and increased expression of ATF4, CHOP, BIP, and GADD34. Mouse embryonic fibroblasts expressing a mutant eIF2α, which could not be phosphorylated, were more resistant to A0 than wild type cells, pointing to a pro-death role of eIF2α phosphorylation. A0 may thus represent the prototypical member of a new class of compounds that cause paraptotic cell death via mechanisms involving eIF2α phosphorylation and the UPR. Advancement in cancer therapy requires a better understanding of why and how cancer cells are induced to die. Although in the past apoptosis was considered the only way to kill cancer cells, the role of other types of cell death has been increasingly recognized in the tumor response to therapy (see for reviews, Refs. 1.Brown J.M. Attardi L.D. Nat. Rev. Cancer. 2005; 5: 231-237Crossref PubMed Google Scholar, 2.Ricci M.S. Zong W.X. Oncologist. 2006; 11: 342-357Crossref PubMed Scopus (387) Google Scholar). Cisplatin is the most widely used anticancer drug and causes cell death by inducing apoptosis. Nevertheless, the high rate of resistance observed during therapy with cisplatin, as well as the occurrence of non-sensitive cancer cells, prompt the quest for agents endowed with apoptosis-independent mechanisms of action. Moreover, therapies based on the induction of non-apoptotic cell deaths may represent a promising approach to suppress the multidrug-resistant phenotype often associated with resistance to apoptosis. A non-apoptotic cell death, characterized by a specific cellular morphology, was observed during embryo development or neuronal degeneration (3.Pilar G. Landmesser L. J. Cell Biol. 1976; 68: 339-356Crossref PubMed Scopus (225) Google Scholar, 4.Clarke P.G. Anat. Embryol. 1990; 181: 195-213Crossref PubMed Scopus (1530) Google Scholar). This death process is hallmarked by massive cytoplasmic vacuolization and is known as type III cell death (4.Clarke P.G. Anat. Embryol. 1990; 181: 195-213Crossref PubMed Scopus (1530) Google Scholar) or paraptosis (5.Sperandio S. de Belle I. Bredesen D.E. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 14376-14381Crossref PubMed Scopus (780) Google Scholar). Several studies have described paraptotic-like cell death processes in various models (6.Schneider D. Gerhardt E. Bock J. Müller M.M. Wolburg H. Lang F. Schulz J.B. Cell Death Differ. 2004; 11: 760-770Crossref PubMed Scopus (46) Google Scholar, 7.Jadus M.R. Chen Y. Boldaji M.T. Delgado C. Sanchez R. Douglass T. Al-Atar U. Schulz W. Lloyd C. Wepsic H.T. Cancer Gene Ther. 2003; 10: 411-420Crossref PubMed Scopus (37) Google Scholar, 8.Galvan V. Logvinova A. Sperandio S. Ichijo H. Bredesen D.E. J. Biol. Chem. 2003; 278: 13325-13332Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 9.Chen Y. Douglass T. Jeffes E.W. Xu Q. Williams C.C. Arpajirakul N. Delgado C. Kleinman M. Sanchez R. Dan Q. Kim R.C. Wepsic H.T. Jadus M.R. 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Studies from our laboratory have identified A0 as the most active thioxotriazole copper(II) complex among a number of triazole-metal based compounds screened for their cytotoxicity in human cancer cells (15.Dallavalle F. Gaccioli F. Franchi-Gazzola R. Lanfranchi M. Marchiò L. Pellinghelli M.A. Tegoni M. J. Inorg. Biochem. 2002; 92: 95-104Crossref PubMed Scopus (42) Google Scholar, 16.Tardito S. Bussolati O. Maffini M. Tegoni M. Giannetto M. Dall'asta V. Franchi-Gazzola R. Lanfranchi M. Pellinghelli M.A. Mucchino C. Mori G. Marchio L. J. Med. Chem. 2007; 50: 1916-1924Crossref PubMed Scopus (71) Google Scholar, 17.Gaccioli F. Franchi-Gazzola R. Lanfranchi M. Marchiò L. Metta G. Pellinghelli M.A. Tardito S. Tegoni M. J. Inorg. Biochem. 2005; 99: 1573-1584Crossref PubMed Scopus (27) Google Scholar). Although A0 and cisplatin have comparable potencies in HT1080 human fibrosarcoma cells, the latter induces typical caspase-dependent apoptosis, while A0 inhibits caspase-3 activity and elicits a death process lacking the typical features of apoptosis (18.Tardito S. Bussolati O. Gaccioli F. Gatti R. Guizzardi S. Uggeri J. Marchiò L. Lanfranchi M. Franchi-Gazzola R. Histochem. Cell Biol. 2006; 126: 473-482Crossref PubMed Scopus (46) Google Scholar). In contrast, huge vacuoles, derived from the dilatation of the endoplasmic reticulum (ER), 3The abbreviations used are: ERendoplasmic reticulumUPRunfolded protein responseERADendoplasmic reticulum-associated degradationWTwild typeGFPgreen fluorescent proteinZbenzyloxycarbonylFMKfluoromethylketone. 3The abbreviations used are: ERendoplasmic reticulumUPRunfolded protein responseERADendoplasmic reticulum-associated degradationWTwild typeGFPgreen fluorescent proteinZbenzyloxycarbonylFMKfluoromethylketone. are the most evident features of A0-induced cell death, consistently with the induction of a paraptotic process. A possible explanation is that A0, by inhibiting caspase-3 (18.Tardito S. Bussolati O. Gaccioli F. Gatti R. Guizzardi S. Uggeri J. Marchiò L. Lanfranchi M. Franchi-Gazzola R. Histochem. Cell Biol. 2006; 126: 473-482Crossref PubMed Scopus (46) Google Scholar), impairs the execution of the apoptotic program, thus addressing the cells to alternative death pathways. Interestingly, recent evidence suggests that paraptosis becomes preponderant when apoptosis executors are somehow inhibited (19.Ding W.X. Ni H.M. Yin X.M. Apoptosis. 2007; 12: 2233-2244Crossref PubMed Scopus (24) Google Scholar). endoplasmic reticulum unfolded protein response endoplasmic reticulum-associated degradation wild type green fluorescent protein benzyloxycarbonyl fluoromethylketone. endoplasmic reticulum unfolded protein response endoplasmic reticulum-associated degradation wild type green fluorescent protein benzyloxycarbonyl fluoromethylketone. A0 induces copper overload leading to an increase of the cellular oxidized glutathione (16.Tardito S. Bussolati O. Maffini M. Tegoni M. Giannetto M. Dall'asta V. Franchi-Gazzola R. Lanfranchi M. Pellinghelli M.A. Mucchino C. Mori G. Marchio L. J. Med. Chem. 2007; 50: 1916-1924Crossref PubMed Scopus (71) Google Scholar). Moreover, it has been recently demonstrated that other copper complexes inhibit proteasome activity in cancer cells both in vivo and in vitro, indicating that the metal may work as a novel anticancer agent through the accumulation of misfolded proteins (20.Chen D. Cui Q.C. Yang H. Dou Q.P. Cancer Res. 2006; 66: 10425-10433Crossref PubMed Scopus (457) Google Scholar, 21.Ahtoniemi T. Goldsteins G. Keksa-Goldsteine V. Malm T. Kanninen K. Salminen A. Koistinaho J. Mol. Pharmacol. 2007; 71: 30-37Crossref PubMed Scopus (37) Google Scholar, 22.Adsule S. Barve V. Chen D. Ahmed F. Dou Q.P. Padhye S. Sarkar F.H. J. Med. Chem. 2006; 49: 7242-7246Crossref PubMed Scopus (310) Google Scholar). Despite these phenotypic data, very few molecular studies have been aimed to a better comprehension of copper-induced cell death in cancer cells (23.Tardito S. Marchiò L. Curr. Med. Chem. 2009; 16: 1325-1348Crossref PubMed Scopus (206) Google Scholar). However, it has been recently shown that copper induces genes involved in the ubiquitin-proteasome system and in the oxidative stress response (24.Muller P. van Bakel H. van de Sluis B. Holstege F. Wijmenga C. Klomp L.W. J. Biol. Inorg. Chem. 2007; 12: 495-507Crossref PubMed Scopus (64) Google Scholar). In another study, copper complexes, other than A0, have been demonstrated to cause a cell death process with paraptotic features (25.Marzano C. Pellei M. Colavito D. Alidori S. Lobbia G.G. Gandin V. Tisato F. Santini C. J. Med. Chem. 2006; 49: 7317-7324Crossref PubMed Scopus (116) Google Scholar). ER-derived cytoplasmic vacuolization, the hallmark of paraptotic-like cell death, is increasingly recognized as a phenotype indicating a perturbed functional link between ER and the proteasome. Indeed, excluding those of clearly autophagic origin, giant vacuoles derived from the ER are described under conditions such as (i) treatment with proteasome inhibitors (26.Ding W.X. Ni H.M. Gao W. 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Bezrookove V. Verhaegen M. Johnson T.M. Fullen D.R. Pointer J.N. Gruber S.B. Su L.D. Nikiforov M.A. Kaufman R.J. Bastian B.C. Soengas M.S. Nat. Cell Biol. 2006; 8: 1053-1063Crossref PubMed Scopus (265) Google Scholar). In this work, we have compared the differences in sensitivity of cancer cells to A0 and cisplatin, and investigated the mechanism of action of A0, showing a specific transcriptional and translational cellular response that underlie paraptotic cell death. The cell human cancer cell lines Caco-2, Calu-3, HOS, HT1080, HT29, PANC-1, RD, A375, A431, A549, CAPAN-1, CFPAC-1, HeLa, HepG2, Hep2, U2-OS, SAOS-2, SW872, MCF-7, MCF-7/DX, SH-SY5Y, PNT1A, PC3, 2008, C13*, and the mouse embryonal fibroblasts S/S and A/A were obtained and cultured as described under supplemental methods. HT1080PTR were established in our laboratory treating continuously HT1080 parental cells with increasing doses of cisplatin until a cell population able to grow in the presence of 10 μm of the drug was obtained. The procedure for HT1080PTR selection is detailed under supplemental methods. The A0 (cis- [CuCl2(H2L)]Cl) copper(II) complex of HL (4-amino-1,4-dihidro-3-(2-pyridyl)-5-thioxo-1,2,4-triazole) was synthesized as described previously (15.Dallavalle F. Gaccioli F. Franchi-Gazzola R. Lanfranchi M. Marchiò L. Pellinghelli M.A. Tegoni M. J. Inorg. Biochem. 2002; 92: 95-104Crossref PubMed Scopus (42) Google Scholar). A0 was freshly dissolved in dimethyl sulfoxide (DMSO) at a concentration of 25 mm before each experiment. Cisplatin was dissolved at a concentration of 1 mm in 0.154 m NaCl solution and frozen at −20 °C. To complex metals with HL ligand, a 50 mm solution in DMSO was prepared for the ligand and for each metal. An equal volume of the ligand and metal solution were mixed to obtain a 1000× solution (25 mm). The mix was left to react for 2 h at room temperature. A change of the solution color or of the UV-visible spectra indicated the ligand-metal complexation. Caspase inhibitors (Sample Pack from R&D Systems) were dissolved 10 mm in DMSO and used at a final concentration of 50 μm. The inhibitors are abbreviated as followed: Z-VAD-FMK, general caspase inhibitor: pan; Z-WEHD-FMK, caspase-1 inhibitor: 1; Z-VDVAD-FMK, caspase-2 inhibitor: 2; Z-DEVD-FMK, caspase-3 inhibitor: 3; Z-YVAD-FMK, caspase-4 inhibitor: 4; Z-VEID-FMK, caspase-6 inhibitor: 6; Z-IETD-FMK, caspase-8 inhibitor: 8; Z-LEHD-FMK, caspase-9 inhibitor: 9; Z-AEVD-FMK, caspase-10 inhibitor: 10; Z-LEED-FMK, caspase-13 inhibitor: 13. The fluorogenic proteasome substrate Suc-Leu-Leu-Val-Tyr-AMC was purchased by Alexis. Cell viability was assessed with the resazurin assay as previously reported (18.Tardito S. Bussolati O. Gaccioli F. Gatti R. Guizzardi S. Uggeri J. Marchiò L. Lanfranchi M. Franchi-Gazzola R. Histochem. Cell Biol. 2006; 126: 473-482Crossref PubMed Scopus (46) Google Scholar). See supplemental methods for the detailed procedure. Briefly, cells were seeded in multiwell plates 24 h before treatment. Cells were treated with drugs at the selected concentration for 48 h. At the end of the treatment cells were incubated in a resazurin solution and fluorescence recorded. Cytotoxicity was expressed as percent of control, and the value calculated according to the equation reported in supplemental methods. Dose response curves were fitted with nonlinear regression. IC50 values were calculated with GraphPad Prism 4.0TM. HT1080 and HT1080PTR cells were seeded in 6-well plates at a density of 150 cells/cm2 and immediately treated with the indicated concentration of cisplatin. Cells were incubated for 8 days renewing medium every 3 days. Colonies were then washed with phosphate-buffered saline (PBS), fixed with 3.7% paraformaldehyde, and stained with a 0.5% solution (w/v) of methylene blue in PBS supplemented with 1% ethanol. Digital images of colonies were acquired with a Nikon DS5MC digital camera and colonies greater than 100 μm were visually counted. 1 × 106 cells were seeded in 100-mm culture dishes. After 24 h, the cultures were incubated in normal growth medium (12 h) or in the presence of 25 μm A0 (1, 3, 6, 12 h) or 25 μm cisplatin (3, 6, 12 h). At the end of the treatments, cells and culture medium were collected, washed with PBS, and total RNA was extracted and processed as reported in supplemental methods. Raw microarray data were processed for summarization with the BeadStudio software (v. 1.5.1.3), with Rank-Invariant Normalization and filtering by Detection. If the detection value for both samples in at least one replicate was higher than 0.95, the probe was kept for further analysis. Statistical analysis of the processed data were conducted by performing a Dunnett's t test (32.Dunnet C.W. Biometrics. 1964; 20: 482-491Crossref Google Scholar) for multiple comparisons between each treatment condition and the untreated control. 749 probes, corresponding to 734 genes, emerged for being regulated by A0 and/or cisplatin in at least one time point with a false discovery rate below 5%, a minimum log2 ratio of 0.7 and an α of 1.5 (supplemental Table S2). The statistical search for genes differentially regulated genes by A0 and cisplatin (supplemental Table S3) is described under supplemental methods. Gene displaying similar expression patterns were clustered using the FLAME algorithm implemented in the GEDAS software (33.Fu L. Medico E. BMC Bioinformatics. 2007; 8: 3Crossref PubMed Scopus (400) Google Scholar). To interrogate the connectivity map data base, gene symbols from gene expression were mapped to HGU-133A probes IDs according to Biomart Ensembl release 47 and redundant probes were filtered out. Profiles for up-regulated and down-regulated genes were analyzed with the connectivity map resource. Total RNA, obtained from cells treated as described above for microarray analysis, were purified with the RNeasy Mini Kit (Qiagen S.p.a., Milano, Italy). Reverse transcription and real time quantitative PCR were performed as described under supplemental methods. The primers used are reported in supplemental Table S4. Relative quantitative evaluation of transcript levels was calculated by comparing ΔCt normalized on GAPDH transcript levels. Cells grown to subconfluence were incubated in the absence or in the presence of A0 or cisplatin at the indicated concentrations. Culture medium was collected together with the cells and samples processed as described under supplemental methods. Briefly, 30 μg of protein were loaded on 10% or 15% gels for SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane in a 10% blocking solution (Roche Applied Science). Blots were exposed overnight at 4 °C to the antibodies diluted in the blocking solution. See supplemental methods for antibodies suppliers and concentration used. Membranes were exposed to anti-mouse or anti-rabbit horseradish peroxidase-conjugated Western blotting detection reagent (ExactaCruz, Santa Cruz Biotechnology). The blots were visualized using enhanced chemiluminescence (Amersham Biosciences). HT1080 cells were collected in lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.5% Nonidet P-40, 0.5 mm dithiothreitol). The lysates were centrifuged at 14,000 × g for 10 min, and protein content was determined in the supernatant. For the evaluation of the chymotrypsin-like activity, aliquots of 30 μg were incubated for 15 min at 37 °C in 100 μl of 50 mm Tris-HCl, pH 7.5, with 20 μm fluorogenic substrate (Suc-Leu-Leu-Val-Tyr-AMC) in the absence or in the presence of A0, CuCl2, or MG132. After incubation, the AMC release was measured (excitation 355 nm, emission 460 nm) using a Wallac 1420 Victor2 Multilabel Counter. On the day before the experiment, 1·105 cells/well were seeded in a 6-well plate. After treatments, standard culture medium was replaced with MEM without serum, leucine, methionine, and lysine, supplemented with l-[4,5-3H]leucine (5 μCi/ml, Amersham Biosciences). After 30 min, cells were harvested by trypsinization, added to the respective collected medium, centrifuged, and incubated in ice cold 5% trichloroacetic acid. Pellets were washed twice with trichloroacetic acid and proteins suspended in 5% sodium-deoxycholate in 1 n NaOH. Scintillation fluid was added to the protein solution and the incorporated radioactivity counted with a scintillation spectrometer (Wallac Microbeta Trilux counter). In parallel, aliquots were used for Lowry protein determination. Two-tailed Student's t test for unpaired samples was used for statistical analysis. 8 × 104 cells were seeded on chamber slides and grown to subconfluence. Cells were left untreated or treated with 25 μm A0 for 12 h. Cells were then fixed in 3.7% paraformaldehyde, permeabilized with methanol, and blocked with 5% anti-goat serum. The incubation with rabbit anti-calnexin antibody (1:50, Cell Signaling) was performed at 4 °C overnight. After washing, cells were incubated with 488Alexafluor goat anti-rabbit antibody (1:800) and then visualized by means of Nikon Eclipse 300 inverted fluorescence microscope. HT1080 cells were seeded in a 12-well plate at a density of 2 × 10 5 cells/well. The day after cells were treated with A0 in the absence or in the presence of cycloheximide, as detailed in the legend of Fig. 4. At the end of the treatment the culture medium was collected from each well and briefly centrifuged to pellet floating cells and debris. The assay was performed as described in supplemental methods. See supplemental methods for detailed transfection procedure. Cells were transfected with a mixture containing FuGENE6 (Roche Applied Science) and DNA vectors encoding for wild type, S51A eIF2α, or green fluorescent protein. eIF2α S51A and WT vector were kindly provided by Dr. David Ron (NYU School of Medicine, Skirball Institute, NY). After transfection, cells were treated with A0 (20 μm) for 16 h, and viability recorded determined using the resazurin method or propidium iodide to stain dead cells. Images in phase contrast and fluorescence of 10 randomly chosen microscopic fields were taken for each condition. Fluorescent GFP- or PI-positive cells were counted on the digital images. Previous results from our laboratory have demonstrated that the copper(II) complex A0 causes non-apoptotic death of cancer cells (18.Tardito S. Bussolati O. Gaccioli F. Gatti R. Guizzardi S. Uggeri J. Marchiò L. Lanfranchi M. Franchi-Gazzola R. Histochem. Cell Biol. 2006; 126: 473-482Crossref PubMed Scopus (46) Google Scholar). As shown in Fig. 1a, the cytotoxic effect of A0 in HT1080 cells is selectively mediated by copper, since other bivalent metals of the first transition series failed to induce comparable toxic effects when complexed with the A0 ligand. To assess the involvement of caspases in the A0-induced cell death, we used specific inhibitors of caspase-3 as well as of other caspases. Consistently with our previous results (18.Tardito S. Bussolati O. Gaccioli F. Gatti R. Guizzardi S. Uggeri J. Marchiò L. Lanfranchi M. Franchi-Gazzola R. Histochem. Cell Biol. 2006; 126: 473-482Crossref PubMed Scopus (46) Google Scholar), the cytotoxic effect of A0 was not affected by any of the caspase inhibitors tested (Fig. 1b), whereas the cisplatin-induced decrease of viability was significantly decreased. Moreover, a Western blot analysis indicated that A0 activated neither caspase-9 (Fig. 1c, upper panel) nor caspase-3 (Fig. 1c, lower panel). In contrast, both enzymes underwent activation cleavage during cisplatin treatment. We used a panel of 23 human cancer cell lines to determine the half-maximal inhibitory concentrations (IC50) of A0 and cisplatin (Fig. 1d and supplemental Table S1). In all the cell lines tested, the cytotoxic effect of A0 was associated with the appearance of massive vacuoles, typical of type III cell death or paraptosis (4.Clarke P.G. Anat. Embryol. 1990; 181: 195-213Crossref PubMed Scopus (1530) Google Scholar, 18.Tardito S. Bussolati O. Gaccioli F. Gatti R. Guizzardi S. Uggeri J. Marchiò L. Lanfranchi M. Franchi-Gazzola R. Histochem. Cell Biol. 2006; 126: 473-482Crossref PubMed Scopus (46) Google Scholar) (data not shown). Neither correlation nor linear regression between A0 and cisplatin IC50 values were significant (Spearman test r = 0.29, n = 14, and r2 = 0.08), indicating that the sensitivities to A0 and cisplatin are unrelated. Moreover, A0 caused a comparable, dose-dependent response in the ovarian cell line 2008 and in its subline C13*, a well-known cisplatin-resistant cell model (34.Andrews P.A. Albright K.D. Cancer Res. 1992; 52: 1895-1901PubMed Google Scholar) (IC50 values of 9 and 12 μm, respectively, Fig. 1e). To confirm the absence of cross-resistance to the two metal-based drugs, we used the human fibrosarcoma HT1080 cells that are very sensitive to both A0 and cisplatin (18.Tardito S. Bussolati O. Gaccioli F. Gatti R. Guizzardi S. Uggeri J. Marchiò L. Lanfranchi M. Franchi-Gazzola R. Histochem. Cell Biol. 2006; 126: 473-482Crossref PubMed Scopus (46) Google Scholar). Given that no cisplatin-resistant HT1080 cells are commercially available, we generated a cisplatin-resistant population from this cell line. After eight months of selection, we obtained a population (HT1080PTR), which formed colonies in the presence of high cisplatin concentrations (Fig. 1, f and g). HT1080PTR were less clonogenic than the parental cells in control medium (Fig. 1f), but were able to form colonies even in the presence of 10 μm cisplatin. The quantitative results of the assay (Fig. 1g) indicated that the colony-forming competence of HT1080PTR cells was not significantly affected by cisplatin concentrations as high as 5 μm. Consistently, HT1080PTR cells grew more slowly than parental cells under control conditions but actively proliferated even in the presence of 10 μm cisplatin, when parental cells did not survive (Fig. 1h). The dose response curves, obtained for A0 and cisplatin in the two cell lines (Fig. 1i), yielded comparable IC50 values for A0 (14 and 12 μm, for HT1080PTR and HT1080, respectively), while confirmed the difference in sensitivity to cisplatin (IC50 values of 39 μm and 9 μm for HT1080PTR and HT1080 cells, respectively). HT1080PTR cells when incubated with cytotoxic concentrations of A0, exhibited the typical massive cytoplasmic vacuolization with a substantial maintenance of nuclear integrity (Fig. 1j). These data show that acquisition of resistance to cisplatin does not affect the ability of HT1080PTR cells to undergo paraptotic death when treated with A0. To characterize better the differences between the cytotoxic effects of A0 and cisplatin, we performed gene expression profiling on HT1080 cells. Cells were treated with the two drugs for the times indicated in Fig. 2, and two biological replicates were obtained for each condition. Biotinylated cRNA probes, generated from total RNA, extracted from control and treated cells, were hybridized to microarrays covering 24,000 transcripts. Statistical analysis of the microarray data highlighted 749 probes, corresponding to 734 genes, the expression of which was either increased or decreased by A0 and/or cisplatin in at least one time point (detailed under "Experimental Procedures" and supplemental methods). Notably, expression of only 74 genes was significantly changed by both drugs, suggesting distinct transcriptional effects of A0 and cisplatin. A0 produced more changes in gene expression than cisplatin (487 versus 321 significantly regulated genes, respectively). Clustering of the regulated genes using the FLAME algorithm (33.Fu L. Medico E. BMC Bioinformatics. 2007; 8: 3Crossref PubMed Scopus (400) Google Scholar) led to the definition of sixteen clusters (Fig. 2a), plus one cluster of outliers, i.e. genes that cannot be reliably assigned to any cluster. Seven clusters showed responses with opposite signs (Fig. 2a, clusters 1, 6, 7, 11, 14, 15, 17), whereas only clusters 10 and 13 displayed genes exhibiting similar responses to A0 and cisplatin. The statistical search for genes with a differential response to A0 and cisplatin yielded 386 transcripts, corresponding to 359 genes, grouped by FLAME in 5 clusters (Fig. 2b). Cluster 1 was the largest and contained 150 genes induced by A0 and not by cisplatin, while cluster 2 contained genes the expression of which was selectively decreased by A0. Clusters 3 and 4 contained genes up- or down-regulated respectively by cisplatin only. Taken together, these data indicate that the A0-driven transcriptional response was clearly different from that driven by cisplatin. To evaluate the biological significance of the clusters of genes regulated by A0 or cisplatin, we performed a functional keyword enrichment analysis using the DAVID web-tool (see Ref. 35.Dennis Jr., G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H.C. Lempicki R.A. Genome Biol. 2003; 4: P3Crossref PubMed Google Scholar). The analysis identified clusters significantly enriched in specific functional categories (Table 1). In particular, five clusters shown in Fig. 2a (1.Brown J.M. Attardi L.D. Nat. Rev. Cancer. 2005; 5: 231-237Crossref PubMed Google Scholar, 5.Sperandio S. de Belle I. Bredesen D.E. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 14376-14381Crossref PubMed Scopus (780) Google Scholar, 6.Schneider D. Gerhardt E. Bock J. Müller M.M. Wolburg H. Lang F. Schulz J.B. Cell Death Differ. 2004; 11: 760-770Crossref PubMed Scopus (46) Google Scholar, 10.Abraham M.C. Lu Y. Shaham S. Dev. Cell. 2007; 12: 73-86Abstract Full Text Full Text PDF PubMed Scopus (88) Google Schola
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