Arsenic trioxide sensitizes promonocytic leukemia cells to TNFα-induced apoptosis via p38-MAPK-regulated activation of both receptor-mediated and mitochondrial pathways

Available online at www.sciencedirect.com

1773 (2007) 1653–1663www.elsevier.com/locate/bbamcr

Biochimica et Biophysica Acta

Arsenic trioxide sensitizes promonocytic leukemia cells to TNFα-inducedapoptosis via p38-MAPK-regulated activation of both receptor-mediated

and mitochondrial pathways

Donna Amrán a,b, Yolanda Sánchez a, Carlos Fernández a, Adrián M. Ramos a, Elena de Blas a,Jacqueline Bréard c, Consuelo Calle b, Patricio Aller a,⁎

a Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040, Madrid, Spainb Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Madrid, Spain

c Institut National de la Santé et de la Recherche Médicale, Faculté de Pharmacie, Chatenay-Malabry, France

Received 15 March 2007; received in revised form 14 June 2007; accepted 15 June 2007Available online 22 June 2007

Abstract

Treatment with the anti-leukemic drug arsenic trioxide (As2O3, 1–4 μM) sensitizes U937 promonocytes and other human myeloid leukemia celllines (HL60, NB4) to apoptosis induction by TNFα. As2O3 plus TNFα increases TNF receptor type 1 (TNF-R1) expression, decreases c-FLIPLexpression, and causes caspase-8 and Bid activation, and apoptosis is reduced by anti-TNF-R1 neutralizing antibody and caspase-8 inhibitor. Thetreatment also causes Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release from mitochondria, XIAP down-regulation, andcaspase-9 and caspase-3 activation. Bcl-2 over-expression inhibits cytochrome c release and apoptosis, and also prevents c-FLIPL down-regulationand caspase-8 activation, but not TNF-R1 over-expression. As2O3 does not affect Akt phosphorylation/activation or intracellular GSH content, norprevents the TNFα-provoked stimulation of p65-NF-κB translocation to the nucleus and the increase in NF-κB binding activity. Treatments withTNFα alone or with As2O3 plus TNFα cause TNF-R1-mediated p38-MAPK phosphorylation/activation. P38-MAPK-specific inhibitors attenuatethe As2O3 plus TNFα-provoked activation of caspase-8/Bid, Bax translocation, cytochrome c release, and apoptosis induction. In conclusion, thesensitization by As2O3 to TNFα-induced apoptosis in promonocytic leukemia cells is an Akt/NF-κB-independent, p38-MAPK-regulated process,which involves the interplay of both the receptor-mediated and mitochondrial executioner pathways.© 2007 Elsevier B.V. All rights reserved.

Keywords: Arsenic trioxide; TNFα; Apoptosis; Receptor-mediated pathway; Mitochondrial pathway; Myeloid leukemia cell

Abbreviations: Ac-DEVD-pNA, N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide;Ac-LEHD-pNA, N-acetyl-Leu-Glu-His-Asp-p-nitroanilide; APL, acute pro-myelocytic leukemia; DAPI, 4,6-diamino-2-phenylindole; FITC, fluoresceinisothiocyanate; FLIP, Flice inhibitory protein; GSH, reduced glutathione; JNK,Jun N-terminal kinase; mAb, monoclonal antibody; MAPK, mitogen-activatedprotein kinase; pAb, polyclonal antibody; MEK/ERK, mitogen-inducedextracellular kinase/extracellular signal-regulated kinase; PBS, phosphate-buffered saline; PI, propidium iodide; TNFα, tumour necrosis factor alpha;TNF-R, tumour necrosis factor receptor; TRAIL, tumour necrosis factor-relatedapoptosis-inducing ligand; Z-IETD-Fmk, Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F; Z-VAD-Fmk, Z-Val-Ala-Asp(OMe)-CH2F⁎ Corresponding author. Tel.: +34 918373112x4247; fax: +34 915360432.E-mail address: [email protected] (P. Aller).

0167-4889/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbamcr.2007.06.003

1. Introduction

Arsenic trioxide (As2O3, Trisenox™) is a clinically effectivedrug in the cure of acute promyelocytic leukemia (APL). At lowconcentrations (≤4 μM in plasma) As2O3 causes APL cyto-reduction by promoting cell differentiation and/or apoptosis.This property was originally attributed to the capacity of the drugto disrupt the promyelocytic leukemia-retinoic acid receptor α(PML-RARα) fusion protein, characteristic of most APLs [1].Nonetheless, As2O3 exhibits multiple effects which are alsoimportant for apoptosis [1,2]. This offers the possibility ofextending the therapeutic use of the drug to malignancies otherthan APLs, and actually promising pre-clinical assays have beencarried out using different hematological and solid tumour cell

1654 D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

types [3]. Nevertheless, the relatively low sensitivity of most celltypes to As2O3 would require generating strategies to increasethe apoptotic efficacy of the drug, and decrease the dosage toclinically affordable concentrations.

As2O3 is a mitochondria-targeting drug, which binds theadenosine nucleotide translocator [4] and interferes withmitochondrial respiration [5]. As most chemotherapeuticagents, As2O3 may induce apoptosis by activating the“intrinsic” (mitochondrial) pathway. This pathway is char-acterized by the release of cytochrome c and other mito-chondrial proteins to the cytosol, assembly and activation ofthe apoptosome and inhibition of IAPs (“inhibitor of apoptosisproteins”), and sequential activation of caspase-9 and effectorcaspases [6]. Hence, a manner of potentiating As2O3 toxicitymay be the combination with cytokines of the tumour necrosisfactor alpha (TNFα) family, which activate the “extrinsic”(receptor-mediated) pathway. This pathway is initiated bycytokine binding and activation of death receptors (TNF-Rs),followed by DISC (“death-inducing signalling complex”)formation and activation of initiator caspase-8 (or -10) andeffector caspases [6]. Among these cytokines, tumour necrosisfactor-related apoptosis-inducing ligand (Apo2L/TRAIL) isconsidered a promising anticancer agent because of itscapacity to preferentially induce apoptosis in tumour cellswith lower toxicity to normal cells [7]. The use of TNFαitself has been questioned because of the low sensitivity ofmany tumour cells and the inflammatory response induced byhigh doses of this cytokine [8]. Nevertheless, it has beenargued that combined treatments allowing a reduction indosage may still render TNFα effective as an anticanceragent [9,10].

Earlier reports indicated that As2O3 cooperate with TRAIL toinduce apoptosis in human leukemia cell lines [11,12]. As2O3

stimulated cytokine receptor expression and down-regulated c-FLIP (“FLICE-inhibitory protein”) expression, and these effectswere in turn mediated by Akt dephosphorylation [12]. On theground of these observations, we wanted to investigate thepossible cooperation between As2O3 and TNFα to induceapoptosis in leukemia cells. We selected the U937 promonocyticcell line as an appropriate model, since it exhibits low intrinsicsensitivity to both As2O3 [13] and TNFα [14]. The obtainedresults indicate that As2O3 sensitizes U937 and other myeloidleukemia cells to apoptosis induction by TNFα. Apoptosisbehaves as an Akt/NF-κB-independent, p38-MAPK-regulatedprocess, which involves TNF-R1 over-expression, c-FLIPLdown-regulation, and caspase-8 activation. Nonetheless, As2O3

plus TNFα also activates the mitochondrial pathway, whichregulates the activation of the receptor-mediated pathway.

2. Materials and methods

2.1. Reagents and antibodies

All components for cell culture were obtained from Invitrogen, Inc.(Carlsbad, CA, USA). 4,6-diamino-2-phenylindole (DAPI) was obtained fromServa (Heidelberg, Germany). The kinase inhibitors LY294002, PD98059,U0126, SB203580, SB220025, and SP600125, the caspase substrates N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA, specific for caspase-3) and

N-acetyl-Leu-Glu-His-Asp-p-nitroanilide (Ac-LEHD-pNA, specific for cas-pase-9), and the caspase inhibitors Z-Val-Ala-Asp(OMe)-CH2F (z-VAD-fmk,non-specific) and Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (z-IETD-fmk, specificfor caspase-8), were obtained from Calbiochem (Darmstadt, Germany).Recombinant TNFα and recombinant human TRAIL/Apo2L were obtainedfrom Strathmann Biotech AG (Hamburg, Germany). Monochlorobimane wasobtained from Molecular Probes (Eugene, OR, USA). Rabbit polyclonalantibodies (pAb) against human Akt, phospho-Akt (Ser471), p38-MAPK, andphospho-p38-MAPK (Thr180/Tyr182), and mouse anti-caspase-8 monoclonalantibody (mAb) (1C12), were obtained from Cell Signaling Technology(Beverly, MA, USA). Mouse anti-pigeon cytochrome c mAb clone 7H8.2C12and mouse anti-Bax mAb clone 6A7 were obtained from BD PharMingen (SanDiego, CA, USA). Mouse anti-human Bcl-2 (100) mAb, rabbit anti-human Bax(N-20) pAb, goat anti-human Bid (C-20) pAb, rabbit anti-human NF-κB p65(sc-109) pAb, and mouse anti-human tumour necrosis factor receptor 1 (TNF-R1) (H-5) mAb, were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA,USA). Mouse anti-XIAP (MIHA/ILP-a) mAb was obtained from MBLInternational Corporation (Woburn, MA, USA). Mouse anti-human CD95(Apo1/Fas) (DX2) mAb was obtained from eBioscience (San Diego, CA, USA).Rabbit anti-human FLIPL (the long isoform of the FLICE inhibitory protein)pAb was obtained from Upstate Cell Signalling Solutions (Lake Placid, LY,USA). Mouse anti-human TNF-RI/TNFRSF1A (16803) mAb, which specifi-cally neutralizes TNF-R1, was obtained from R&D Systems (Minneapolis, MN,USA). All peroxidase- and fluorescein isothiocyanate (FITC)-conjugatedimmunoglobulin G antibodies were obtained from DAKO Diagnósticos, S.A.(Barcelona, Spain). All other reagents were from Sigma (Madrid, Spain).

2.2. Cells and treatments

Human U937 (promonocytic), HL60 (myelomonocytic) and NB4 (acutepromyelocytic) leukemia cells, and stably Bcl-2-transfected U937 cells (U4clone) [15], were grown as earlier indicated [16]. Stock solutions ofcamptothecin and SB220025 (10 mM), LY294002, PD98059, SB203580 andSP600125 (20 mM), U0126 (2.63 mM), Ac-DEVD-pNA and Ac-LEHD-pNA(5 mM), z-VAD-fmk and z-IETD-fmk (25 mM), lonidamine (100 mM), andmonochlorobimane (200 mM), were prepared in dimethyl sulfoxide. Stocksolutions of cis-platinum(II)-diammine dichloride (cisplatin, 3.3 mM), TNFαand TRAIL/Apo2L (100 μg/ml), were prepared in distilled water. All thesesolutions were stored at −20 °C. Stock solutions of DAPI (100 μg/ml) andpropidium iodide (PI, 1 mg/ml) were prepared in phosphate-buffered saline(PBS). A stock solution of As2O3 (100 mM) was prepared in distilled water.These solutions were stored at 4 °C.

2.3. Determination of apoptosis, and measurement of caspaseactivities

Distinctive characteristics of apoptotic cells were chromatin condensation/fragmentation, as measured by DAPI staining and microscopy examination,reduced (sub-G1) DNA content, as measured by PI staining and flow cytometryexamination, and phosphatidylserine translocation from the inner to the outerlayer of the plasma membrane, as indicated by cell surface binding of FITC-labeled annexin V using flow cytometry assays. As a routine, we also examinedfree trypan blue or PI penetration into the cells, as an indication of plasmamembrane integrity. Caspase-9 and caspase-3 activities were determined in invitro assays, using as substrates Ac-LEHD-pNA and Ac-DEVD-pNA,respectively. Caspase-8 activation was evidenced by the appearance of pro-caspase-derived cleavage fragments, as determined by immunoblot assays.Annexin V binding was determined using a rh Annexin V/FITC Kit (BenderMedSystems GmbH, Vienna, Austria), following the procedure described by themanufacturer. A detailed description of all other procedures was presented inpreceding publications [16,17], and hence is omitted here.

2.4. Measurement of intracellular GSH content

The intracellular GSH content was determined by fluorometry after cellloading with monochlorobimane, following the previously described procedure[18].

1655D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

2.5. Cell fractionation and immunoblot assays

To obtain total cellular protein extracts, cells were collected by centrifuga-tion, washed with PBS, and lysed by 5-min heating at 100 °C followed bysonication in Laemmli's buffer containing a protease inhibitor cocktail, 10 mMsodium fluoride, and 1 mM sodium orthovanadate. To obtain cytosolic extracts,aimed at determining cytochrome c and Omi/HtrA2 release from mitochondria,cells were collected for centrifugation, re-suspended in 100 μl of ice-cold PBScontaining 80 mM KCl, 250 mM sucrose, and 200 μg/ml digitonin, and kept on

Fig. 1. Apoptosis induction by As2O3 and TNFα in U937 cells. The figure shows the fand D), annexin V binding to the cell surface (panel B), and reduction in DNA concombination. (A) Cells were permeabilized and stained with DAPI, and the chromstained with annexin V, and the fluorescence determined by flow cytometry. Numbersvertical dotted line. (C) Cells were permeabilized and stained with PI, and the fluorNumbers in parentheses indicate the fraction of cells with sub-G1 DNA content. (D) Cfor 24 h in the absence of the drug (TNF, As); or treated firstly for 24 h with TNFα alo(As–TNF). Except when otherwise is indicated, As2O3 was used at 4 μM; TNFαwas uwere simultaneously applied. Z-VAD-fmk (50 μM, panel A) was applied 30 min beforused. The bars in panels A and D represent the mean±S.D. of at least three determdeterminations with similar results.

ice for 5 min. After centrifugation (10000×g for 15 min at 4 °C) the pellet wasdiscarded. To obtain mitochondrial extracts, aimed at determining Baxtranslocation to mitochondria, cells were homogenized by repeatedly passingthem throughout a 25-gauge needle. The homogenate was first centrifuged at1000×g for 10 min, and the supernatant centrifuged again at 10000×g for 20 minto obtain the mitochondrial fraction, following the previously describedprocedure [17]. Nuclear extracts, aimed at determining p65 NF-κB transloca-tion, were obtained using the method of Schreibert et al. [19]. Fractions of total,mitochondrial, cytosolic or nuclear extracts, containing equal protein amounts,

requency of apoptotic cells, as determined by chromatin fragmentation (panels Atent (panel C), in U937 cell cultures treated with As2O3 and TNFα, alone or inatin examined by fluorescence microscopy. (B) Non-permeabilized cells werein parentheses indicate the fraction of annexin V-positive cells, delimited by theescence (as an indication of DNA content) was determined by flow cytometry.ells were treated for 24 h with As2O3 or TNFα alone, and then allowed to recoverne, washed, and then treated for 24 h with As2O3 alone (TNF–As); or vice versased at 40 ng/ml; the treatments lasted 24 h; and the drugs in combined treatmentse treatment with As2O3 plus TNFα. As controls (Cont), drug-untreated cells wereinations, and the profiles in panels B and C are representative of one of two

1656 D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

were analyzed by SDS-polyacrylamide gel electrophoresis, blotted ontomembranes, and immunodetected, as previously described [16].

2.6. Electrophoretic mobility gel shift assays

Nuclear extracts were obtained as indicated by Schreibert et al. [19]. Adouble-strand oligonucleotide containing the consensus binding site for NF-κB(5′-AGTTGAGGGGACTTTCCCAGGC-3′) was prepared. The conditions ofoligoprobe radioactive labeling, binding reaction, and electrophoretic separa-tion, were exactly as described by López-Rodríguez et al. [20]. For competitionexperiments, nuclear extracts were pre-incubated with 50-fold molar excess ofunlabelled oligonucleotide for 30 min at 4 °C before adding the labeled probe.

3. Results

3.1. Death induction

We firstly examined the capacity of As2O3 and TNFα tocause apoptosis in U937 promonocytic cells, as determined bychromatin condensation. As indicated in Fig. 1A, As2O3 wasalmost innocuous at 1–2 μM, and moderately toxic at 4 μM,while TNFα was almost innocuous at all assayed concentra-tions (from 20 to 80 ng/ml). When they were simultaneouslyapplied, TNFα and As2O3 synergistically induced apoptosis in

Fig. 2. Apoptosis induction in other myeloid cells lines, or using different cytokinesalone, and with the indicated concentrations of As2O3, alone (−) or in combination wiagonistic anti-CD95 antibody (αFas) or TRAIL, alone or in combination with As2O3.(CDDP), or 10 nM camptothecin (CPT), alone (−) or in combination with TNFα. Thewhen otherwise is indicated, As2O3 was used at 4 μM, TNFα at 40 ng/ml, and TRAIL

a concentration-dependent (from 1 to 4 μM As2O3) and time-dependent (from 8 to 24 h) manner. Allowing for quantitativedifferences inherent to the sensitivity of the used techniques,this response was corroborated by measuring the fraction ofcells exhibiting surface binding of annexin V (Fig. 1B), as wellas the accumulation of cells with sub-G1 DNA content (Fig.1C). Treatment with As2O3 for periods longer than 24 h alsoslightly caused cell accumulation at the G2/M phase of thegrowth cycle (result not shown). The pan-caspase inhibitor z-VAD-fmk greatly decreased the As2O3 plus TNFα-provokedcell death (Fig. 1A), indicating that it is bona fide caspase-dependent apoptosis. Under the used conditions, the frequencyof cells permeable to PI or trypan blue, which may indicateprimary or secondary (apoptosis-derived) necrosis, was below12% (results not shown).

The cooperation between antitumour drugs and cytokines isgenerally explained by the capacity of the chemotherapeuticdrug to sensitize to cytokine-induced apoptosis. Neverthelessthe opposite effect, namely sensitization by TNFα tochemotherapeutic drug-provoked apoptosis, was also reported[21, and references therein]. For this reason, new determina-tions were carried out in which As2O3 and TNFα weresequentially applied. Pre-treatment with As2O3 followed by

and antitumour drugs. (A) HL60 and NB4 cell cultures were treated with TNFαth TNFα. (B) U937 cell cultures were treated with the indicated concentrations of(C) U937 cell cultures were treated with 50 μM lonidamine (Lon), 4 μM cisplatinfrequency of apoptotic cells was determined by chromatin fragmentation. Exceptat 100 ng/ml, and the treatments lasted 24 h. All other conditions were as in Fig. 1.

1657D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

post-treatment with TNFα still allowed apoptosis potentiation,while no potentiation was obtained using the opposite sequence(Fig. 1D), indicating sensitization by As2O3 to TNFα-mediatedapoptosis.

For comparison, experiments were carried out using HL60myelomonocytic and NB4 acute promyelocytic leukemia cellsinstead of U937 cells, agonistic CD95 antibody and TRAILinstead of TNFα, and the antitumour drugs lonidamine,cisplatin and camptothecin, instead of As2O3. Determinationsof chromatin fragmentation indicated that: (i) As2O3 and TNFαalso cooperated to induce apoptosis in both HL60 and NB4 cells(Fig. 2A). (ii) Treatment with CD95 antibody was innocuous,and the apoptotic effect in combination with As2O3 was nothigher than that caused by As2O3 alone. On the other hand,TRAIL caused per se a moderate, concentration-dependenttoxicity at 50 to 150 ng/ml. This cytokine cooperated withAs2O3 to induce apoptosis, but the effect was approximatelyadditive (Fig. 2B). Finally, no sensitization to TNFα-inducedapoptosis could be obtained using lonidamine, cisplatin orcamptothecin (Fig. 2C). These effects were qualitativelycorroborated by measuring cell surface annexin-V bindingand accumulation of cells with sub-G1 DNA content (results notshown).

Fig. 3. Activation of the extrinsic apoptotic pathway. In panels A, C, D and F, total cepanels B and E apoptosis was determined at 24 h of treatment. (A) Cleavage-activatiothe appearance of 43 kDa and 18 kDa apoptosis-related fragments. (B) Frequency ofthe absence or in the presence of the caspase-8-specific inhibitor z-IETD-fmk (50 μMevidenced by the decrease in the 23-kDa Bid pro-form content. (D) TNF-R1 expressiocells in cultures treated with As2O3, alone or with TNFα, either in the absence or theexpression levels, in cells subjected to the indicated treatments. Z-IETD-fmk and n-represent the mean±S.D. of at least three determinations (*Pb0.01, Student's t testdeterminations with similar results. In these experiments, the levels of α-tubulin (C,conditions were as in Figs. 1 and 2.

3.2. Activation of the receptor-mediated pathway

Then, we wanted to determine caspase-8 activation, whichrepresents a critical event in the execution of the extrinsicapoptotic pathway. As indicated in Fig. 3A, treatment withAs2O3 plus TNFα caused pro-caspase-8 cleavage/activation,while As2O3 alone and TNFα alone were almost ineffective.Control determinations indicated caspase-8 activation bytreatment with TRAIL alone, which as indicated above sufficedto cause apoptosis. By contrast no activation was observed upontreatment with As2O3 plus CD95 antibody or cisplatin plusTNFα, which failed to cause or to potentiate apoptosis,respectively (see Fig. 2A and C). The caspase-8 inhibitor z-IETD-fmk reduced the toxic action of As2O3 plus TNFα toa level similar to that produced by As2O3 alone (Fig. 3B),corroborating the importance of this caspase for apoptosis.Finally, As2O3 plus TNFα also caused cleavage/activation ofBid, a substrate of caspase-8, as revealed by the decrease inthe amount of the 23-kDa Bid pro-form (Fig. 3C).

Treatment with As2O3 alone and with As2O3 plus TNFαactivated TNF-R1 expression, as determined by immunoblotassays, while no activation was obtained with TNFα alone or withcisplatin plus TNFα (included as a control) (Fig. 3D). Flow

llular protein extracts were assayed by immunoblot at 16 h of treatment, while inn of pro-caspase-8 in cells subjected to the indicated treatments, as evidenced byapoptotic cells in cultures treated with As2O3, alone or and with TNFα, either in). (C) Cleavage/activation of Bid in cells subjected to the indicated treatments, asn levels, in cells subjected to the indicated treatments. (E) Frequency of apoptoticpresence of 20 μg/ml neutralizing TNF-R1 antibody (n-αTNFR1). (F) C-FLIPLαTNFR1 were applied 30 min before treatments. The results in panels B and E). The results in panels A, C, D and F are representative of one of at least twoF) and total p38-MAPK (D) were also measured as loading controls. All other

Fig. 4. Activation of the intrinsic (mitochondrial) apoptotic pathway. (A, B) Thepresence of cytochrome c (Cyt c) and Omi/HtrA2 (Omi) in the cytosol (cyto),and of Bax in cytosolic and mitochondrial fractions (mito) (A), and the totalcellular levels of XIAP, Bcl-2 and Bax (B), were determined by immunoblotusing cytosolic, mitochondrial, and total cellular protein extracts, respectively.The level of α-tubulin was also measured as a loading control. (C) Caspase-9and caspase-3 activities were determined in in vitro assays using as substratesAc-LEHD-pNA and Ac-DEVD-pNA, respectively. The results (mean±S.D. ofthree determinations) are indicated in relation to untreated cells (Cont), whichreceived the arbitrary value of one. The values in panels A and B arerepresentative of one of two determinations. Except when otherwise indicated,the measurements were carried out at 16 h of treatment. All other conditionswere as in Fig. 1.

1658 D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

cytometry determinations were not sensitive enough to clearlydetect cell surface receptor expression (results not shown).Incubation with anti-TNF-R1 neutralizing antibody decreasedthe As2O3 plus TNFα-provoked cell death (Fig. 3E), whichcorroborates the importance of this receptor in mediating apop-tosis potentiation. Finally, treatment with As2O3 plus TNFαdecreased the expression of the long isoform of the caspase-8antagonist c-FLIP (c-FLIPL) (Fig. 3F). Control determinationsindicated c-FLIPL decrease upon treatment with TRAIL alone,which as indicated above caused caspase-8 activation, but not bytreatments with As2O3 plus anti-CD95 antibody or cisplatin plusTNFα, which failed to activate caspase-8. The short isoform ofc-FLIP (c-FLIPS) could not be detected in U937 cells [22].

While treatment with As2O3 alone activated TNF-R1 expres-sion, As2O3 toxicity was not decreased by incubation with anti-TNF-R1 neutralizing antibody (Fig. 3E) or with caspase-8inhibitor (Fig. 3B). Moreover, incubation with an anti-TNFα-blocking antibody was equally ineffective, and ELISA assaysfailed to detect TNFα increase in the supernatant of As2O3-treatedcells (results not shown). This excludes a possible intervention ofTNFα/TNF-R1 in mediating the apoptotic action of As2O3.

3.3. Activation of the mitochondrial pathway

As indicated above, As2O3 may induce apoptosis throughdirect binding to mitochondria. In addition, the cleavage/activation of Bid (see Fig. 3C) suggests the engagement of themitochondrial pathway in As2O3 plus TNFα-provoked apopto-sis. For these reasons, we examined the behaviour of factorscritical for the stimulation of this pathway, such as the release ofthe mitochondria-localized proteins cytochrome c and Omi/HtrA2, the expression and/or localization of XIAP, Bcl-2, andBax, and the activation of caspases -9 and -3. The obtainedresults are summarized in Fig. 4. While changes in cytochromec, Omi/HtrA2 and XIAP were very low in cells treated withAs2O3 alone, As2O3 plus TNFα clearly elicited cytochrome cand Omi/HtrA2 release from mitochondria, as revealed byimmunoblot assays using cytosolic extracts (Fig. 4A). Thistreatment also caused a decrease in the total XIAP levels (Fig.4B). Total Bcl-2 and Bax expression remained almost unaffected(Fig. 4B). However As2O3 induced Bax translocation tomitochondria, and this effect was potentiated by co-treatmentwith TNFα, as demonstrated by immunoblot using mitochon-drial and cytosolic extracts (Fig. 4A). This observation wasqualitatively corroborated by flow cytometry assays using ananti-Bax antibody (6A7 clone), which specifically recognizes aconformationally active Bax isoform [16] (result not shown).Finally, As2O3 induced LEHDase (indicative of caspase-9) andDEVDase (indicative of caspase-3) activities, and these effectswere increased by co-treatment with TNFα (Fig. 4C). Theseresults indicate that co-treatment with As2O3 plus TNFα in factpotentiates the activation of the intrinsic executioner pathway.

3.4. Effects of Bcl-2 over-expression

To investigate the relationship between the extrinsic andintrinsic pathways, experiments were carried out using Bcl-2-

transfected U937 cells (U4 clone), since Bcl-2 is known to blockthe apoptotic machinery at the mitochondrial level. Preliminarydeterminations indicated an approximately eight-fold increase inBcl-2 content in the U4 cells, in relation to the non-transfectedcells (results not shown). It was found that U4 cells not only wereunable to undergo cytochrome c release (Fig. 5A), but also failedto undergo apoptosis induction (Fig. 5B), caspase-8 activation(Fig. 5C), and c-FLIPL down-regulation (Fig. 5D), in response toAs2O3 plus TNFα. However, TNF-R1 expression was stillincreased in the U4 cells (Fig. 5D). This indicates that theexecution of the extrinsic pathway is under mitochondrialcontrol, which acts at some step(s) between receptor expressionand caspase-8 activation.

3.5. Akt phosphorylation, intracellular GSH, and NF-κBactivation

Earlier studies indicated that As2O3 may affect the phosphor-ylation/activation of Akt [12,23,24], an that this kinase protects

Fig. 5. Effects of Bcl-2 over-expression. The experiments were carried out using Bcl-2-transfected U937 cells (U4 clone). Non-transfected cells (U937) wereoccasionally included for comparison. (A) Release of cytochrome c to the cytosol, (C) cleavage/activation of caspase-8, and (D) expression levels of TNF-R1 and c-FLIPL, at the indicated times of treatment with As2O3 plus TNFα. (B) Frequency of apoptotic cells, at 24 h of treatment. The results in panels A, C and D arerepresentative of one of at least two determinations. The results in panel B represent the mean±S.D. of three determinations. All other experimental conditions were asin Figs. 1, 3 and 4.

1659D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

against As2O3 toxicity [25]. In some of these studies, thealteration in Akt phosphorylation was interpreted as a responseto the generation of reactive oxygen species elicited by As2O3

[23,24]. In addition, we recently reported that PI3K/Aktinhibitors decrease the intracellular level of reduced glutathione(GSH), which is a strict determinant of As2O3 toxicity [26, andreferences therein]. For these reasons, experiments were carriedout to analyze Akt phosphorylation and intracellular GSH inU937 cells treated with As2O3, alone and in combination withTNFα. As indicated in Fig. 6A, B, the treatments did not causesignificant alterations in Akt phosphorylation, nor altered theGSH content. This contrasts with the decrease in bothparameters provoked by the PI3K inhibitor LY294005, whichwas included as a control. Moreover, in our experiments As2O3

did not cause detectable alterations in intracellular reactiveoxygen species content, as determined using the peroxide-sensitive probe dichlorodihydrofluorescein diacetate (results notshown).

It is known that TNFα activates NF-κB, a transcription factorwith anti-apoptotic action [27]. In addition, it was reported thatAs2O3 may down-regulate NF-κB activation in myeloid cells[28]. For these reasons, we wanted to measure NF-κB activation

in cells treatedwithTNFα, alone and in combinationwithAs2O3.It was observed that treatment with TNFα alone stimulatedNF-κB binding to its DNA consensus sequence, but the bindingwas not reduced by co-treatment with As2O3 (Fig. 6C). Inagreement with this, As2O3 failed to prevent the TNFα-provokedstimulation of p65-NF-κB translocation to the nucleus (Fig. 6D).Collectively, these results indicate that the sensitization byAs2O3

to TNFα-provoked apoptosis may not be explained by altera-tions in Akt phosphorylation, GSH level, or NF-κB activity.

3.6. MAP kinase signalling

MAP kinases are essential factors in apoptosis signalling, andTNFαwas reported to stimulate JNK and p38-MAPK activation[27]. To investigate the possible involvement of MAPKs onapoptosis induction by As2O3 plus TNFα, experiments werecarried out using the p38 inhibitors SB203580 (10 μM) andSB220025 (1 μM), the JNK inhibitor SP600125 (10 μM), andthe MEK/ERK inhibitors PD98059 (20 μM) and U0126(2.5 μM). These concentrations were selected from precedingstudies, which indicated that they effectively prevented kinaseactivation without causing per se significant cell death [16,29].

Fig. 6. Akt phosphorylation, GSH content, and NF-κB activation. (A) Relativelevels of total and phosphorylated Akt (T-Akt and P-Akt, respectively), asrevealed by immunoblot assays, at the indicated times of treatment with As2O3,alone or with TNFα, and at 24 h of treatment with 30 μM LY294002 (LY, usedas a control). (B) Intracellular GSH content at 24 h of treatment with As2O3,alone or with TNFα, or with LY294002 (used as a control). The values arerepresented in relation to untreated cell cultures (approximate GSH content,9.0 nmol/106 cells), which received the arbitrary value of one. (C) NF-κBbinding activity, as determined by gel shift assays at the indicated times oftreatment with TNFα, alone or with As2O3. The positions of bound (NF-κB) andfree (arrow) oligoprobe are indicated. (D) Nuclear translocation of p65 NF-κB,as determined at 16 h of treatment by immunoblot using nuclear protein extracts.The corresponding cytosolic fractions were used as a control. The experimentswere repeated twice in panels A and D, and three times in panels B and C. Allother conditions were as in Fig. 1.

1660 D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

As indicated in Fig. 7A, the p38-MAPK inhibitors did not affectapoptosis induction by As2O3 alone, but they attenuatedapoptosis potentiation by As2O3 plus TNFα. By contrast, no

attenuation was observed using the JNK and MEK/ERKinhibitors. P38-MAPK inhibitor also attenuated cytochrome cand Omi/HtrA2 release, caspase-8 and Bid cleavage/activation,and Bax translocation (Fig. 7B). Immunoblot assays indicatedthat treatments with As2O3 caused p38-MAPK activation, asrevealed by the increase in protein phosphorylation, and theactivation was higher in combination with TNFα (Fig. 7C). Ofnote, treatment with TNFα alone, which as indicated abovefailed to cause apoptosis, sufficed to activate p38-MAPK (Fig.7C), and this effect was blocked by anti-TNF-R1 neutralizingantibody (Fig. 7D). Thus, p38-MAPK activation is a necessary,but not sufficient event for TNF-R1-mediated apoptosis.

4. Discussion

The results in this work indicate that the anti-leukemic drugAs2O3 cooperates with TNFα in inducing apoptosis in U937promonocytes and other human myeloid leukemia cell lines.This response is attributable to the capacity of As2O3 tosensitize the cells to the action of TNFα, to which they arenormally refractory. As2O3 and TRAIL also cooperate ininducing apoptosis. Nevertheless U937 cells are moderatelysensitive to TRAIL, as measured by caspase-8 activation andapoptosis induction, and the potentiation by As2O3 of TRAILtoxicity is relatively lower (additive effect) than in the case ofTNFα. Sensitization by As2O3 appears as a drug-specificresponse, since lonidamine, cisplatin and camptothecin failed tocooperate with TNFα in inducing apoptosis and, at least in thecase of cisplatin, in activating the receptor-mediated pathway.This may be surprising, since campthocecin and other chemo-therapeutic drugs stimulated TNFα-provoked apoptosis in othercell types [10,30]. The reason for such discrepancy remains tobe determined.

The present results also indicate that sensitization by As2O3 toTNFα-induced apoptosis involve the activation of the receptor-mediated pathway and the potentiation of the mitochondrialpathway, and that both pathways contribute to the final apoptoticresponse. Although As2O3 is a mitochondria-targeting agent,there are reports proving that this drug may activate themitochondrial or the receptor-mediated pathway, depending onthe used cell model. An example of this was given by thefunctional status of p53 in multiple myeloma cells. Thus, inmyeloma cells bearing wild type p53 As2O3 increased TRAILreceptor expression, but failed to activate caspase-8, andapoptosis was primarily executed throughout the mitochondrialpathway. By contrast, in p53-null myeloma cells, cells withmutated p53, or cells with siRNA-silenced p53, which are moresensitive to As2O3, the drug increased TRAIL and TRAILreceptor expression, caused c-FLIPL down-regulation, andelicited caspase-8 and Bid activation, with later engagement ofthe mitochondrial pathway [11,31,32]. Of note, U937 promono-cytic cells are p53-null cells [33], and in our experimentstreatment with As2O3 alone sufficed to increase TNF-R1expression, but in spite of it the drug failed to activate caspase-8/Bid. However the combination of As2O3 plus TNFα, whichincreased TNF-R1 expression and also caused c-FLIPL down-regulation, elicited caspase-8/Bid activation. The potentiation of

Fig. 7. p38-MAPK activation and effect of MAPK inhibitors. (A) Frequency of apoptotic cells in cell cultures treated for 24 h with As2O3, alone or with As2O3, eitherin the absence (−) or the presence of the p38-MAPK inhibitors SB203580 (SB1, 10 μM) and SB220025 (SB2, 1 μM), the JNK inhibitor SP600125 (SP, 10 μM), andthe MEK/ERK inhibitors PD98059 (PD, 20 μM) and U0126 (U, 2.5 μM). The results indicate the mean±S.D. of at least three determinations (*Pb0.005, Student'st test). (B) Release of cytochrome c and Omi/HtrA2, activation of caspase-8 and Bid, and translocation of Bax to mitochondria, in cells treated for 16 h with As2O3

plus TNFα, alone or with SB203580, as determined by immunoblot using cytosolic (cytochrome c, Omi/HtrA2), cytosolic and mitochondrial (Bax), and totalcellular (caspase-8, Bid) protein extracts. (C) Relative levels of total and phosphorylated p38-MAPK (p38-T and p38-P, respectively) upon treatment for theindicated time periods with As2O3 alone, TNFα alone, or As2O3 plus TNFα, as determined by immunoblot. (D) Relative levels of phosphorylated p38-MAPK, upontreatment for 16 h with TNFα, alone or in the presence of neutralizing TNF-R1 antibody (n-αTNFR1). The results in panels B–D are representative of one of at leasttwo determinations. The kinase inhibitors and n-αTNFR1 were applied 30 min before the treatments. All other conditions are as in Figs. 1, 3 and 4.

1661D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

the mitochondrial pathway by As2O3 plus TNFα involves theconcurrence of Bid and Bax activation, which favours proteinrelease from mitochondria, and XIAP down-regulation, whichrelieves caspases from the inhibitory action of that protein [6]. Asindicated above, Bid activation serves to engage the mitochon-dria in cytokine-initiated apoptosis. Bax is a substrate of p38-MAPK, and this kinase was demonstrated to regulate Baxphosphorylation/activation and translocation to mitochondria[34]. Hence, Bax activation may represent a key factor to explainthe regulation by p38-MAPK of As2O3 plus TNFα-provokedapoptosis. Finally, recent reports characterized XIAP expressionas a factor which determines cell resistance against cytokine-mediated apoptosis, and XIAP down-regulation as a central eventexplaining the synergism between TNFα family cytokines andchemotherapeutic drugs [35,36].

Akt and NF-κB regulates the expression and activity ofseveral pro-apoptotic or anti-apoptotic proteins, including c-FLIPand IAPs [12,28,37]. Moreover, Akt may regulate the synthesisand activity of γ-glutamylcysteine synthetase, the key-limitingenzyme of GSH biosynthesis [16,38]. The effect of As2O3 on Aktis controversial, since there are reports indicating that the drugdecreases [12,23] or increases [24] Akt phosphorylation inleukemia cells. However in our experiments As2O3 did not causesignificant alterations in Akt phosphorylation or intracellularGSH content in U937 cells treated with As2O3, alone and withTNFα. In the same manner, the behaviour of NF-κB does notsatisfactorily explain the sensitizing action of As2O3, since the

drug failed to prevent the TNFα-provoked NF-κB activation inU937 promonocytic cells. As an alternative explanation, c-FLIPand XIAP down-regulation might be the consequence ofincreased ubiquitinization/proteasome-mediated protein degrada-tion, as indicated by some authors [39,40]. Actually, earlierstudies reported an increase in ubiquitin-conjugated proteins inarsenic-treated cells [41]. Moreover, Omi/HtrA2 is released frommitochondria upon treatment with As2O3 plus TNFα, and thisprotein has been characterized as a serine protease which targetsand degrades IAPs [42,43].

Enforced expression of Bcl-2 and Bcl-XL is frequently used asa tool to block the mitochondrial apoptotic cascade, and to checkthe contribution of mitochondria to the final apoptotic effect. Inthis regard, earlier reports indicated that Bcl-2 over-expression didnot prevent or had minimal effects on apoptosis generation byTRAIL in HL60 cells [37], by As2O3 in multiple myeloma cellswith mutated p53 [31], by mytramycin A plus TNFα in TF-1human erythroleukemia cells [10], and by flavopiridol plusTRAIL in U937 cells [35]. Thus, in those experimental models themitochondrial pathway was dispensable for apoptosis execution.By contrast, in our experiments mitochondrial activation playedan important role, since Bcl-2 over-expressing cells wereprevented from undergoing caspase-8 activation and apoptosisin response to As2O3 plus TNFα. The blockade of caspase-8could be explained by the inability of the Bcl-2-over-expressingcells to down-regulate c-FLIP levels, but additional regulation atother steps of the DISC machinery may not be excluded.

1662 D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

In summary, co-treatment with low As2O3 concentrationssensitizes myeloid leukemia cells to apoptosis induction byTNFα, following a process which involves the interplay of theextrinsic (receptor-mediated) and mitochondrial pathways. Onthe one hand, the increase in TNF-R1 expression and down-regulation of c-FLIP expression lead to caspase-8 activation,and caspase-8-mediated Bid cleavage together with Baxactivation and XIAP down-regulation facilitate mitochondrial-dependent caspase-9 and -3 activation. In turn, mitochondrialpathway activation regulates caspase-8 activation and executionof the extrinsic pathway. By potentiating the apoptotic response,combined treatments may offer new opportunities for the use ofAs2O3 and TNFα as therapeutic agents.

Acknowledgements

Supported by grants SAF2004-01250 from the PlanNacional de Investigación Científica, Desarrollo e InnovaciónTecnológica, Ministerio de Educación y Ciencia, Spain; GR/SAL/0639/2004 from the Dirección General de Universidades eInvestigación, Consejería de Educación, Comunidad de Madrid,Spain; and grants from the Ligue National contre la Cancer(France). D.A., Y.S. and C.F. were recipients of predoctoralfellowships from the Universidad Complutense de Madrid(D.A.) and Plan de Formación de Personal Investigador,Ministerio de Educación y Ciencia, Spain (Y.S., C.F.).

References

[1] W.M. Miller Jr., H.M. Schipper, J.S. Lee, J. Singer, S. Waxman,Mechanisms of action of arsenic trioxide, Cancer Res. 62 (2002)3893–3903.

[2] H. Shi, X. Shi, K.J. Liu, Oxidative mechanisms of arsenic toxicity andcarcinogenesis, Mol. Cell. Biochem. 255 (2004) 67–78.

[3] A.M. Evens, M.S. Tallman, R.B. Gartenhaus, The potential role of arsenictrioxide in the treatment of malignant disease: past, present and future,Leuk. Res. 28 (2004) 891–900.

[4] N. Larochette, D. Decaudin, E. Jacotot, C. Brenner, I. Marzo, S.A. Susin,N. Zamzami, Z. Xie, J. Reed, G. Kroemer, Arsenite induces apoptosis viadirect effect on the mitochondrial permeability transition pore, Exp. CellRes. 249 (1999) 413–421.

[5] H. Pelicano, L. Feng, Y. Zhou, J.S. Carew, E.O. Hileman,W. Plunkett, M.J.Keating, P. Heng, Inhibition of mitochondrial respiration. A novelstrategy to enhance drug-induced apoptosis in human leukaemia cells byreactive oxygen species-mediated mechanism, J. Biol. Chem. 278 (2003)37832–37839.

[6] S. Fulda, K.M. Debatin, Extrinsic versus intrinsic apoptosis pathways inanticancer chemotherapy, Oncogene 25 (2006) 4798–4811.

[7] F.C. Kimberley, G.R. Screaton, Following a TRAIL: update on a ligandand its five receptors, Cell Res. 14 (2004) 359–372.

[8] I.C. Kettelhut, W. Fiers, A.L. Godberg, The toxic effect of tumour necrosisfactor in vivo and their prevention by cyclooxygenase inhibitors, Proc.Natl. Acad. Sci. U. S. A. 84 (1987) 4273–4277.

[9] P. Schotte, G. Van Loo, I. Carpentier, P. Vandenabeele, R. Beyaert, Lithiumsensitizes tumor cells in a NF-κB-independent way to caspase activationand apoptosis induced by tumor Necrosis Factor (TNF). Evidence for arole of the TNF receptor-associated death domain protein, J. Biol. Chem.276 (2001) 25939–25945.

[10] V. Duverger, A.M. Murphy, D. Sheehan, K. England, T.G. Cotter, I. Hayes,F.J. Muphy, The anticancer drug mithramycin A sensitizes tumour cells toapoptosis induced by tumour necrosis factor (TNF), Br. J. Cancer 90(2004) 2025–2031.

[11] Q. Liu, S. Hilsenbeck, Y. Gazitt, Arsenic trioxide-induced apoptosis inmyeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation ofcaspase-8 or caspase-9, and synergy with APO2/TRAIL, Blood 101 (2003)4078–4087.

[12] E. Szegezdi, S. Cahill, M. Meyer, M. O'Dwyer, A. Samali, TRAILsensitization by arsenic trioxide is caspase-8 dependent and involvesmodulation of death receptor components and Akt, Br. J. Cancer 94 (2006)398–406.

[13] Y. Jing, J. Dai, R.M. Chalmers-Redman, W.G. Tatton, S. Waxman, Arsenictrioxide selectively induces acute promyelocytic leukaemia cell apop-tosis via a hydrogen peroxide-dependent pathway, Blood 94 (1999)2102–2111.

[14] A. Cossariza, C. Franceschi, D.Monti, S. Salvioli, E. Bellesia, R. Rivabene,L. Biondo, G. Rinaldi, A. Tinari, W. Malorni, Protective effect ofN-acetylcysteine in tumor necrosis factor-alpha-induced apoptosis inU937 cells: the role of mitochondria, Exp. Cell Res. 220 (1995)232–240.

[15] Renvoize, R. Roger, N. Moulian, J. Bertoglio, J. Bréard, Bcl-2 expressionin target cells leads to functional inhibition of caspase-3 protease family inhuman NK and lymphokine-activated killer cell granule-mediatedapoptosis, J. Immunol. 159 (1997) 126–134.

[16] A.M. Ramos, C. Fernández, D. Amrán, D. Esteban, E. de Blas, M.A.Palacios, P. Aller, Pharmacologic inhibitors of extracellular signal-regulated kinase (ERKs) and c-Jun NH2-terminal kinase (JNK)decrease glutathione content and sensitize human promonocytic leukemiacells to arsenic trioxide-induced apoptosis, J. Cell. Physiol. 209 (2006)1006–1015.

[17] A. Troyano, P. Sancho, C. Fernández, E. de Blas, P. Bernardi, P. Aller, Theselection between apoptosis and necrosis is differentially regulated inhydrogen peroxide-treated and glutathione-depleted human promonocyticcells, Cell Death Differ. 10 (2003) 889–898.

[18] A. Troyano, C. Fernández, P. Sancho, E. de Blas, P. Aller, Effect ofglutathione depletion on antitumor drug toxicity (apoptosis and necrosis)in U-937 human promonocytic cells, J. Biol. Chem. 276 (2001)14115–47107.

[19] E.P. Schreiber, P. Matthias, M.M. Müller, W. Schaffner, Identificationof a novel lymphoid-specific octamer binding protein (OTF-2B) byproteolytic clipping bandshift assay (PCBA), EMBO J. 7 (1988)4221–4229.

[20] C. López-Rodríguez, M. Zubiaur, J. Sancho, A. Concha, A.L. Corbí, Anoctamer element functions as a regulatory element in the differentiation-responsive CD11c integrin gene promoter: OCT-2 inducibility duringmyelomononcytic differentiation, J. Immunol. 158 (1997) 5833–5840.

[21] K. Schmelz, T. Wieder, I. Tamm, A. Müller, F. Essmann, C.C. Geilen,K. Schulze-Osthoff, B. Dörken, P.T. Daniel, Tumor necrosis factor αsensitizes malignant cells to chemotherapeutic drugs via the mitochondrialapoptosis pathway independently of caspase-8 and NF-κB, Oncogene 23(2004) 6743–6759.

[22] C. Muñoz-Pinedo, C. Ruiz-Ruiz, C. Ruiz de Almodóvar, C. Palacios,A. López-Rivas, Inhibition of glucose metabolism sensitizes tumor cells todeath receptor-triggered apoptosis through enhancement of death-inducingsignalling complex formation and apical procaspase-8 processing, J. Biol.Chem. 278 (2003) 12759–12768.

[23] Y.J. Choi, J.W. Park, S.I. Suh, K.C. Mun, J.H. Bar, D.K. Song, S.P. Kim,T.K. Kwon, Arsenic trioxide-induced apoptosis in U937 cells involvegeneration of reactive oxygen species and inhibition of Akt, Int. J. Oncol.21 (2002) 603–610.

[24] H. Pelicano, J.S. Carew, T.J. McQueen, M. Andreeff, W. Plukett, M.J.Keating, P. Huang, Targeting Hsp90 by 17-AGG in leukemia cells:mechanisms for synergistic and antagonistic drug combinations witharsenic trioxide and Ara-C, Leukemia 20 (2006) 610–619.

[25] G. Tabellini, A. Cappellini, P.L. Tazzari, F. Falà, A.M. Billi, L. Manzoli,L. Cocco, A.M. Martelli, Phosphinositide 3-kinase/Akt involvement inarsenic trioxide resistance of human leukemia cells, J. Cell. Physiol. 202(2005) 623–634.

[26] A.M. Ramos, A. Fernández, D. Amrán, P. Sancho, E. de Blas, P. Aller,Pharmacologic inhibitors of PI3K/Akt potentiate the apoptotic action ofthe antileukemic drug arsenic trioxide via glutathione depletion and

1663D. Amrán et al. / Biochimica et Biophysica Acta 1773 (2007) 1653–1663

increased peroxide accumulation in myeloid leukemia cells, Blood 105(2005) 4013–4025.

[27] H. Wajant, K. Pfizenmaier, P. Scheurich, Tumor necrosis factor signalling,Cell Death Differ. 10 (2003) 45–65.

[28] S. Mathas, A. Lietz, M. Janz, H.M. Hinz, F. Jundt, C. Scheidereit,K. Bommert, B. Dorken, Inhibition of NF-κB essentially contributesto arsenic-induced apoptosis, Blood 102 (2003) 1028–1034.

[29] C. Fernández, A.M. Ramos, P. Sancho, D. Amrán, E. de Blas, P. Aller,12-O-tetradecanoylphorbol-13-acetate may both potentiate and decreasethe generation of apoptosis by the antileukemic agent arsenic trioxide inhuman promonocytic cells. Regulation by extracellular signal-regulatedprotein kinases and glutathione, J. Biol. Chem. 279 (2004) 3877–3884.

[30] S. Bergeron, M. Beauchmin, R. Bertrand, Camptothecin- and etoposide-induced apoptosis in human leukemia cells is independent of cell deathreceptor-3 and -4 aggregation but accelerates tumour necrosis factor-related apoptosis-inducing ligand-mediated cell death, Mol. Cancer Ther.3 (2004) 1659–1669.

[31] C. Akay, Y. Gazitt, Arsenic trioxide selectively induces early andextensive apoptosis via the APO2/caspase-8 pathway engaging themitochondrial pathway in myeloma cells with mutant p53, Cell Cycle 2(2003) 358–368.

[32] F. Kircelly, C. Akay, Y. Gazitt, Arsenic trioxide induces p53-dependentapoptotic signals in myeloma cells with SiRNA-silenced p53: MAP kinasepathway is preferentially activated in cells expressing inactivated p53, Int.J. Oncol. 30 (2007) 993–1001.

[33] S. Kanno, A. Higurashi, Y. Watanabe, A. Shouji, K. Asou, M. Ishikawa,Susceptibility to cytosine arabinoside (Ara-C)-induced cytotoxicity inhuman leukaemia cells, Toxicol. Lett. 152 (2004) 149–158.

[34] B.J. Kim, S.W. Ryu, B.J. Song, JNK- and p38 kinase-mediated phos-phorylation of Bax leads to its activation, and mitochondrial translocationand apoptosis of human hepatoma HepG2 cells, J. Biol. Chem. 281 (2006)21256–21265.

[35] R.R. Rosato, Y. Dai, J.A. Almenara, S.C. Maggio, S. Grant, Potent anti-leukemic interactions between flavopiridol and TRAIL/Apo2L involve

flavopiridol-mediated XIAP downregulation, Leukaemia 18 (2004)1780–1788.

[36] A.P. Kater, F. Dicker, M. Mangiola, K. Welsh, R. Houghtne, J. Ostresh,A. Nefzi, J.C. Reed, C. Pinilla, T.J. Kipps, Inhibitors of XIAP sensitizeCD40-activated chronic lymphocytic leukaemia cells to CD-95 mediatedapoptosis, Blood 106 (2005) 1742–1748.

[37] R. Bortul, P.L. Tazzari, A. Cappellini, G. Tabellini, A.M. Billi, R. Bareggi,L. Manzoli, L. Cocco, A.M. Martelli, Constitutively active Akt1 protectsHL60 leukaemia cells from TRAIL-induced apoptosis through amechanisms involving NF-kappaB activation and cFLIP(L) up-regulation,Leukaemia 17 (2003) 379–389.

[38] S.K. Kim, K.J. Woodcroft, S.S. Khodadadeh, R.F. Novak, Insulinsignaling regulates gamma-glutamyl-cysteine ligase catalytic subunitexpression in primary cultured rat hepatocytes, J. Pharmacol. Exp. Ther.311 (2004) 99–108.

[39] H.G. Zhang, J. Wang, X. Yang, H.C. Hsu, J.D. Mountz, Regulation ofapoptosis proteins in cancer cells by ubiquitin, Oncogene 23 (2004)2009–2015.

[40] Y. Yang, S. Fang, J.P. Jensen, A.M. Weissman, J.D. Ashwell, Ubiquitinprotein ligase activity of IAPs and their degradation in proteasomes inresponse to apoptotic stimuli, Science 288 (2000) 874–877.

[41] T. Bredfeldt, M.J. Kopplin, A.J. Gandolfi, Effects of arsenite on UROtsacells: low-level arsenite causes accumulation of ubiquitinated proteins thatis enhanced by reduction in cellular glutathione levels, Toxicol. Appl.Pharmacol. 198 (2004) 412–418.

[42] G. Van Loo,M. Van Gurp, B. Depuydt, S.M. Srinivasula, I. Rodríguez, E.S.Alnemri, K. Gevaert, J. Vandekerckhove, W. Declercq, P. Vandenabeele,The serine protease Omi/HtrA2 is released from mitochondria duringapoptosis. Omi interacts with caspase-inhibitor XIAP and inducesenhanced caspase activity, Cell Death Differ. 9 (2002) 20–26.

[43] S.M. Srinivasula, S. Gupta, P. Datta, Z. Zhang, R. Hedge, N. Cheong,T. Fernandes-Alnemri, E.S. Alnemri, Inhibitor of apoptosis proteins aresubstrates for the mitochondrial serine protease Omi/HtrA2, J. Biol.Chem. 278 (2003) 31469–31472.