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Research Article

Bortezomib induces autophagic death in proliferating humanendothelial cells

Daniela Bellonia, Lorenzo Veschinia, Chiara Foglienib, Giacomo Dell'Antonioc,Federico Caligaris-Cappioa,d, Marina Ferrarinia,1, Elisabetta Ferreroa,⁎,1

aMyeloma Unit, Department of Oncology, IRCCS H San Raffaele, Milan, ItalybDepartment of Cardiology, IRCCS H San Raffaele, Milan, ItalycDepartment of Pathology, IRCCS H San Raffaele, Milan, ItalydUniversità Vita-Salute IRCCS H San Raffaele, Milan, Italy

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Laboratory of LymphoiFax: +39 02 2643 2611.

E-mail address: elisabetta.ferrero@hsr.it (E.1 Contributed equally to the work.

0014-4827/$ – see front matter © 2009 Elseviedoi:10.1016/j.yexcr.2009.11.005

A B S T R A C T

Article Chronology:

Received 18 September 2009Revised version received9 November 2009Accepted 9 November 2009Available online 13 November 2009

The proteasome inhibitor Bortezomib has been approved for the treatment of relapsed/refractorymultiple myeloma (MM), thanks to its ability to induce MM cell apoptosis. Moreover, Bortezomibhas antiangiogenic properties. We report that endothelial cells (EC) exposed to Bortezomibundergo death to an extent that depends strictly on their activation state. Indeed, while quiescentEC are resistant to Bortezomib, the drug results maximally toxic in EC switched towardangiogenesis with FGF, and exerts a moderate effect on subconfluent HUVEC. Moreover, ECactivation state deeply influences the death pathway elicited by Bortezomib: after treatment,angiogenesis-triggered EC display typical features of apoptosis. Conversely, death of subconfluentEC is preceded by ROS generation and signs typical of autophagy, including intense cytoplasmicvacuolization with evidence of autophagosomes at electron microscopy, and conversion of thecytosolic MAP LC3 I form toward the autophagosome-associated LC3 II form. Treatment with the

specific autophagy inhibitor 3-MA prevents both LC3 I/LC3 II conversion and HUVEC cell death.Finally, early removal of Bortezomib is accompanied by the recovery of cell shape and viability.These findings strongly suggest that Bortezomib induces either apoptosis or autophagy in EC;interfering with the autophagic response may potentiate the antiangiogenic effect of the drug.

© 2009 Elsevier Inc. All rights reserved.

Keywords:

Endothelial cellsAntiangiogenesisAutophagyApoptosisBortezomib

Introduction

Multiple myeloma (MM), a tumor of fully differentiated plasmacells, remains a fatal disease despite all available therapies [1].Targeting the ubiquitin-proteasome system is emerging as asuccessful strategy in cancer [2], and the proteasome inhibitorBortezomib (Velcade™) has been approved for the treatment ofnewly diagnosed and relapsed/refractory MM, given its remark-

d Malignancies, Departmen

Ferrero).

r Inc. All rights reserved.

able efficacy in inducing MM plasma cell apoptosis [3,4]. Inaddition, growing evidence shows that Bortezomib acts as anantiangiogenic agent [5]. Bone marrow (BM) angiogenesis is thehallmark of MM progression and correlates with disease activity[6,7]. Bortezomib was found to exert an antiangiogenic effect onMM endothelial cells (MMEC) through the inhibition of severalangiogenesis-related functions, such as proliferation, chemotaxisand capillary formation on Matrigel [5]. We recently showed that

t of Oncology, IRCCS H San Raffaele, Via Olgettina 60 20131 Milan Italy.

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angiogenesis-driven HUVEC are particularly sensitive to Bortezo-mib-induced death [8]. However, the mechanisms of Bortezomib-induced EC death have not yet been fully characterized.

So far, the best-described pathway of EC death is that viaapoptosis. Morphologically, apoptosis is defined by cell shrinkage,blebbing of the plasma membrane and typical nuclear condensa-tion and fragmentation [9]. The process can be activated by twomajor pathways: the “extrinsic” pathway, which is triggered by adeath receptor belonging to the TNF-receptor superfamily [10,11],and the “intrinsic” pathway, where extracellular or internal insultslead to mitochondrial damage [12]. Apoptosis initiated by eitherpathway is executed through the activation of a cascade ofcaspases [13]. Recently, autophagy has emerged as an additionaltype of programmed EC death [14]. Autophagy is an evolutionaryconserved pathway that plays a major role in the bulk degradationof most long-lived proteins and defective or superfluous organelles[15,16]. The autophagic pathway is characterized at the ultra-structural level by the accumulation of cytoplasmic autophagicvacuoles, which subsequently fuse with lysosomes [17]. Notably,autophagy should be considered an adaptive response that mayprolong cell survival [18]. By nature, EC are highly resistant toinjury and death inflicted by a wide series of mechanical andbiochemical stimuli. This feature may limit the outcome ofantiangiogenic strategies. Natural inhibitors of angiogenesis, suchas angiostatin [19] or thrombospondin-1 [20], have been shown tostimulate EC apoptosis. However, recent studies have shown thatother antiangiogenic agents, including endostatin [21], oxidizedLDL [22,23] and human plasminogen fragments [14], specificallystimulate EC autophagy. Elucidating the biology of these otherforms of cell death may provide us with a new weapon for tumortherapy. Given the high dependence of MM growth and progres-sion on BM angiogenesis, and the potential of Bortezomib as anantiangiogenic agent, we aimed to investigate the mechanisms ofBortezomib-induced EC death.

To do so, we compared the effects of Bortezomib on quiescentand subconfluent HUVEC vs. HUVEC triggered by FGF, a pivotalfactor in promoting angiogenesis [24–26], which we used as amodel of MMEC.

Materials and methods

Proteasome inhibitor, reagents and antibodies

Bortezomib (from Millennium Pharmaceuticals, Inc., Cambridge,MA), was stored at −20 °C and diluted in culture mediumimmediately before use. Human rh-FGFb (PeproTech, Rocky Hill,NJ) was used at 10 ng/ml. Anti-LC3 polyclonal antibody and HRP-conjugated anti-rabbit IgG used for Western blot were from MBLMedical and Biological Laboratories CO. (Naka-ku Nagoya, Japan);anti-LC3 polyclonal antibody for immunofluorescence was fromSanta Cruz Biotechnology (Santa Cruz, CA). The autophagyinhibitor 3-methyl adenine (3MA) was purchased from Sigma-Aldrich Ltd.

Hypoxia treatment

HUVEC were incubated in a temperature and humidity controlledC-chamber (BioSpherix, Redfield, NY), in an atmosphere contain-ing 5% O2, 5% CO2, 90% N2.

HUVEC and cell lines

Human umbilical vein endothelial cells (HUVEC) were isolatedfrom human cord by collagenase treatment as described [27] andcultured in 1% gelatin-coated flasks using endotoxin-free Medium199 (BioWhittaker, Cambrex Bio Science Verviers, Belgium)supplemented with 20% FCS (complete medium, CM). All experi-ments were carried out with HUVEC during passages 1–4. Thehuman myeloma cell line LP1 was purchased from DSMZ(Braunschweig, Germany).

Detection of apoptosis

Apoptosis was assessed by staining with FITC-conjugated Annex-inV (BenderMedical Systems, Prodotti Gianni, Milan, Italy) and thevital dye propidium iodide (PI) [28]. Cells were run on a FACScaninstrument (Becton Dickinson, Mountain View, CA). Caspase-3activation was evaluated by the use of the cell permeant substrateRed DEVD-FMK (BioVision Research Products, Mountain View,CA), as in ref. [29]. Mitochondrial membrane potential wasmeasured using the lipophilic probe JC-1 (BIOMOL Research Lab,PlymouthMeeting, PA), which aggregates in healthymitochondriaand fluoresces red while diffusing in the cytoplasm and greenupon mitochondrial damage [8].

HUVEC proliferation assay

HUVEC proliferation was assessed as described [27]. Briefly,3.5×103 cells resuspended in CM were seeded onto gelatin-coatedwells and incubated in the presence or absence of FGF (10 ng/ml),either under normoxic (20%O2) or hypoxic (5%O2) conditions for 24h. HUVEC were fixed with 2.5% glutaraldehyde, then stained with0.1% crystal violet and solubilized with 10% acetic acid. Theabsorbance at 590 nm of each well was then measured using amicroplate reader (Bio-Rad, Milan, Italy). The number of cells ineachwellwas calculatedby interpolating the absorbance values on acalibration curve prepared in parallel with known amounts of cells.

Confocal and microscopic analyses

Confocal analysis to detect LC3 was performed on HUVEC plated ongelatin-coated coverslips, fixed (2% paraformaldehyde) and per-meabilizedwith 0.01% Triton X-100 and then incubated 1 h RTwiththe anti-LC3 antibody, followed by Alexa 488-conjugated IgG(Molecular Probes, Eugene, Oregon). Images were randomlyacquired and confocal microscopic analysis was performed using aBio-Rad Laser Scanning Microscope MRC-1024 (objective Plan-apo63×, NA 1:4), or a Leica TCS SP2 Laser Scanner ConfocalMicroscope,as described [8]. For H&E analysis we used an Eclipse55imicroscopeequipped with a DS-L1 camera (Nikon, Tokyo, Japan); for phasecontrast microscopy, images were recorded with a Coolpix 4500digital photo camera (Nikon, Tokyo, Japan).

TEM

Cell culture monolayers or cell pellets were fixed for 15 min with4% paraformaldehyde and 2.5% glutaraldehyde. Monolayers werethen detached with rubber and centrifuged at high speed. Pelletswere postfixed in 1% OsO4 in phosphate buffer for 1 h, washed,dehydrated in ethanol and 2-hydroxy-propyl-methacrylate

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(HPMA, Merck, Darmstadt, Germany) and embedded in Epon.Conventional thin sections were collected on uncoated grids,stained with uranyl acetate and lead citrate and examined with aLeo 912-Omega electron microscope (Zeiss, Germany) [30].

Western blotting

WB analysis was performed as described [8]. Briefly, HUVEC weregrown to reach the subconfluence on gelatin-coated flasks, treatedwith the indicated effectors, washed and lysed with ice-cold lysisbuffer (50 mM Tris–HCl, pH 7.2; 0,1% Nonidet P-40; 250 mM NaCl;2 mM EDTA; 10% glycerol) supplemented with complete proteaseinhibitors (“Complete” cocktail, Roche Diagnostics, Mannheim,Germany). Lysates were centrifuged at 13,000 rpm for 20 min at4 °C, and supernatants were collected. Samples (50 μg of protein)were electrophoresed on NuPAGE 4–12% acrylamide gel (Invitro-gen, CA) and transferred to nitrocellulose membranes (AmershamBiosciences, UK) by standard procedures.Membraneswere blockedwith 5% nonfat dry milk in PBS-Tween20 and were incubated withanti-LC3 antibody. After washing, membranes were incubatedwiththe appropriate horseradish peroxidase-conjugated secondaryantibodies and developed by chemiluminescence (Amersham,Bioscience). Protein quantificationwas performed by densitometryusing molecular analyst ImageJ software (NIH, USA).

ROS measurement

We assessed ROS production by incubating HUVEC with the cell-permeable fluorescent probe hydroethidine (HE; Invitrogen, S.r.l.,San Giuliano Milanese, Italy) as previously described [28]. Briefly,cells were treated with 100 nM Bortezomib for 20 h, then stainedwith 2 μmol/l HE for 1 h at 37 °C, washed and analyzed by flowcytometry. Hydrogen peroxide was used as a positive control(100 μmol/l). Only living cells, gated according to scatterparameters, were used for the analysis.

Statistical analysis

Data are presented as mean±SEM. Comparisons for each pair werecalculated using the ANOVA Test (JMP software, SAS, Cary, NC).When indicated, Student's ttestwas applied. p≤0.05was consideredstatistically significant (⁎); p≤ 0.001, highly significant (⁎⁎).

Results

Bortezomib induces HUVEC cell death in vitro, depending ontheir proliferation rate

We have shown that Bortezomib exerts a direct antiangiogenic effecton HUVEC, triggered by hypoxia and FGF [8]. On this line, we firstinvestigatedwhether theeffect of BortezomibonHUVECviabilitywasdependent upon their proliferation state. In particular, we consideredthree different conditions: HUVEC made quiescent by confluence,spontaneously proliferating HUVEC (herein defined as subconfluent)and HUVEC induced to proliferate by treatment with FGF or cultureunder hypoxic (5% O2) conditions. These culture conditions wereactually able to discriminate different proliferation rates as illustratedin Supplementary Fig. 1. As shown in Fig. 1, the ability of Bortezomibto induce HUVEC cell death strictly depended on their proliferative

state. In particular, treatment with Bortezomib 100 nM, a dosecomparable to those obtained in therapy, did not result in significanttoxicity in quiescent HUVEC, even after extended culture periods(Figs. 1A and B). Indeed, confluent HUVEC treated with Bortezomibneither detached nor could be stained with Annexin V/PI and onlyshowed shape modifications, substantially consisting in their elon-gation. Conversely, highly proliferating cells, i.e. HUVEC cultured inthepresence of FGF, substantially died after a 20h treatmentwith100nM of Bortezomib (Fig. 1B, middle panel). Similar results wereobtained when HUVEC were switched toward angiogenesis byculture under hypoxic conditions, as previously reported [8].Subconfluent HUVEC, albeit less sensitive to Bortezomib treatmentcompared to proliferating HUVEC, detached and diedwith prolonged48–72 h exposure (Fig. 1B, left panel). Compared to MM cell line LP1(Fig. 1B, right panel), irrespective of their proliferation rate, HUVECdied upon treatment with higher doses (ten times) of Bortezomib.

Bortezomib induces apoptosis in actively proliferating HUVEC

We then assessed hallmarks of apoptosis (Annexin V binding,caspase-3 activation and mitochondrial depolarization) in quies-cent vs. FGF-activated HUVEC. As shown in Fig. 1C, both Annexin Vbinding and caspase-3 activation, the latter evaluated with thespecific fluorogenic substrate (red-DVED-fmk), were negligible inquiescent HUVEC exposed to 100 nM Bortezomib for 20 h (Fig. 1C,left and middle panels). In addition, the percentage of damagedmitochondria, assessed in terms of mitochondria depolarization,was similar to that of untreated cells (Fig. 1C, right panels). On theother hand, when HUVEC proliferation was driven by FGF, asubstantial fraction of cells displayed Annexin V staining (Fig. 1C).Furthermore, FGF treated cells showed caspase-3 activation andmitochondrial depolarization to an extent comparable to that ofBortezomib-treated LP1 MM cells, which we used as positivecontrol of apoptosis (Fig. 1C) [31]. Overall, these data indicate thatactively proliferating HUVEC undergo apoptosis through themitochondrial pathway when treated with Bortezomib.

Bortezomib induces early morphological changes insubconfluent HUVEC, reversible upon drug removal

As shown in Fig. 1B, subconfluent HUVEC died only after prolongedexposure to Bortezomib. However, remarkable morphologicalchanges were evident in subconfluent HUVEC already followingshort treatment with 100 nM Bortezomib. Fig. 2A showshematoxylin and eosin staining of Bortezomib-treated HUVEC: at20 h cells uniformly acquired a spindle-like morphology andshowed important cytoplasmic vacuolization, while nuclear shapewas preserved; at the same time point, only a fraction of cells died,as assessed by Annexin V/PI staining (Fig. 1B). We then assessedwhether Bortezomib-induced morphological modifications werereversible (Fig. 2B). To do so, we treated subconfluent HUVEC with100 nM Bortezomib for 20 h and 48 h, thus obtaining a progressiveworsening of cell morphology and cell loss. In a parallel set ofexperiments, Bortezomib was removed after 20 h and cells werecultured for an additional 24 h. After removal of the drug, HUVECrecovered their morphology and the ability to proliferate (Fig. 2B).These data suggest that Bortezomib-induced HUVEC death ispreceded by a series of morphological changes that develop overan extended time period; during this “window”, removal of thedrug prevents Bortezomib-induced cell death.

Fig. 1 – Bortezomib induces apoptosis in vitro in actively proliferating HUVEC. (A) HUVEC either subconfluent or grown toconfluency were treated or not with Bortezomib (Btz) 100 nM for 20 h (OM 20×). (B) Left panel: Subconfluent (white bars) andconfluent (grey bars) HUVEC were treated with Bortezomib (100 nM) for the indicated time points. Middle panel: HUVEC, eitherconfluent or subconfluent or FGF-driven were treated with Bortezomib 100 nM for 20 h. Results are expressed as a percentage ofAnnexin/PI positive cells and are means±SEM of five independent experiments. Right panel: LP1 MM cells were treated withBortezomib 10 nM and analyzed as above. Results are expressed as means±SEM of three independent experiments. ⁎ indicatedsignificant and ⁎⁎ indicated highly significant. (C) HUVEC confluent or proliferating (plus FGF) were treated with Bortezomib 100nM for 20 h and analyzed for Annexin/PI staining, caspase-3 activation and mitochondrial depolarization by FACS analysis. Cellsshowing caspase-3 activation stained with the specific substrate Red DEVD-FMK while cells undergoing mitochondrialdepolarization were characterized by a loss of red fluorescence. LP1 cells treated with Bortezomib were used as control. Results arerepresentative of one out of three independent experiments.

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Bortezomib induces early ultrastructural modifications insubconfluent HUVEC, evocative of autophagy

To investigate deeper the nature of the morphological changesdescribed above, we assessed the ultrastructure of Bortezomib-treated subconfluent HUVEC with transmission electron micros-copy (Fig. 3). HUVEC treated with Bortezomib, but not the controls

(A), revealed a cytoplasm rich in large vacuoles,mainly perinuclear(B), with organelle inclusions (so-called myelin figures) (C). Thepresence of double-membrane vesicles, which are considered thehallmark of autophagy, is documented in G. Cell nuclei maintainedoval shape with sparse heterochromatin (B), as in the control (A).Pycnosis and apoptotic bodies, observed in LP1 cells followingBortezomib treatment (E, F), were absent in HUVEC.

Fig. 2 – Bortezomib induces dramatic morphologic changes in subconfluent HUVEC that can be reversed upon removal of the drug.(A) Subconfluent HUVEC treated with Bortezomib showed progressive cytoplasmic thinning accompanied by intensevacuolization, while nuclearmorphologywas unaffected (H&E). (B) Subconfluent HUVEC (5×104) were cultured for 20 h and 48 h inthe presence of Bortezomib. NT correspond to untreated cells. In a separate set of experiments, cells were treated for 20 h withBortezomib, washed and allowed to grow for an additional 24 h. Results are representative of four experiments.

Fig. 3 – Ultrastructural evidence of autophagy in Bortezomib-treated HUVEC. Untreated (NT) HUVEC and LP1 (A and D, respectively)and Bortezomib-treated cells were processed for TEM as described in Materials and methods. Images were recorded at differentmagnifications using a Leo 912-Omega transmission electronmicroscope. Note the presence of double-membrane autophagosomes(G, arrows) and single-membrane vacuoles that contained disintegrated materials (C) in HUVEC; the nucleus of HUVEC remainedintact (B). Conversely, LP1 treated cells showed nuclear chromatin condensation (E) and pycnosis (F).

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Bortezomib-induced autophagic response is blocked by 3-MA

To confirm the ultrastructural evidence of vesicular autophago-somes, the autophagosome-related MAP-LC3 protein was identi-fied in Western blots derived from subconfluent cultures treatedor not with Bortezomib for 20 h. Microtubule-associated LC3protein has been used extensively as a biomarker of autophagy

Fig. 4 – Bortezomib treatment determines autophagy-associated LCHUVEC were treated with 100 nM Bortezomib or not (NT). (A) WestLC3-I represents the cytoplasmic form (18 kDa), and LC3-II represenwere quantitated by densitometric analysis and the ratio betweenrepresentative of three is depicted. (B) LC3 expression was monitormicroscopy (OM 60×). Upon treatment, cells increased the total convacuoles-associated form. Representative cells are shown and LC3 eThe effects of 3-MA on Bortezomib-induced LC-3modificationswere(D and E) Subconfluent HUVEC were treated with Bortezomib 100 ntheir morphology and Ann V/PI staining and FACS analysis. Results

[32,33]. This protein normally exhibits diffuse cytosolic distribu-tion, but is processed and localized to autophagosomes duringautophagy. Western blot analysis allows identification of twoforms of LC3 protein, the cytosolic 18-kDa LC3-I molecule, and the16-kDa LC3 II protein, the latter representing the autophagosome-associated form. As shown in Fig. 4A, both forms were seen athigher levels in Bortezomib-treated cells than in controls.

3 modifications, which can be inhibited by 3-MA. Subconfluentern blot analysis of HUVEC using antibodies against LC3 protein.ts the vacuoles-associated form (16 kDa) of the protein. Proteinsthe 16 kDa and 18 kDa was calculated. One experimented in untreated and Bortezomib-treated HUVEC using confocaltent of LC3 protein and acquired a dotted staining typical of thexpression calculated by mean fluorescence intensity (MFI). (C)assessed byWB analysis. γ-Tubulinwas used as loading control.M in the presence or absence of 3-MA 10 nM and evaluated forare representative of three independent experiments.

Fig. 5 – Bortezomib induces ROS generation in subconfluentHUVEC. Intracellular content of ROS was determined inuntreated and Bortezomib-treated HUVEC by hydroethidine(HE) staining and FACS analysis. (A) HE staining (grayhistograms) in untreated and Bortezomib-treated HUVEC. H2O2

was used as positive control. Results depicted were obtained bygating on living cells and are representative of fourindependent experiments. Numbers indicate Δ meanfluorescence intensity (ΔMFI). (B) Delta mean fluorescenceintensity (ΔMFI) of HE staining in untreated, Bortezomib- orH2O2-treated, subconfluent HUVEC. Results are mean±SEM offour independent experiments. Statistical differences weredetermined using the unpaired two-tailed Student's t test.

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Moreover, quantification of the two forms in treated and controlcells demonstrated the conversion to the vacuole-associated formmainly in the former. Indeed, the 16 kDa/18 kDa ratio was 1.72 inuntreated cells and 2.44 in Bortezomib-treated HUVEC. Accord-ingly, compared to the control, Bortezomib-treated cells showedan increase in LC3 staining (MFI 15.1 vs. 34.2, respectively), with adot-like or punctate MAP LC3 pattern (Fig. 4B), compatible withthe localization of LC3 protein within vacuoles. It is known that 3-methyladenine (3-MA) is a specific inhibitor of early stages of theautophagic process [34]. To further confirm the induction of theautophagic pathway, we assessed the capability of 10 mM 3-MA toblock LC3 conversion. As shown in Fig. 4C, treatment with 3-MAindeed prevented LC3-II generation induced by Bortezomib asdemonstrated by the markedly reduced 16 kDa/18 kDa ratio (0.21compared to 1.03, with Bortezomib alone).

Finally, we found that blockade of the autophagic pathwayprevented Bortezomib-induced cell death. In fact, subconfluentHUVEC incubated for 20 h with Bortezomib in the presence of 3-MA, exhibited a more adherent phenotype (Fig. 4D) and also adecreased staining with Annexin V/PI (Fig. 4E), overall confirmingthe autophagic nature of Bortezomib-induced EC damage. Uponprolonged exposure (48 h) to Bortezomib and 3-MA, however,HUVEC died of necrosis, as indicated by double staining withAnnexin V/PI (not shown).

Bortezomib promotes ROS generation in subconfluent HUVEC

In order to dissect possible mechanisms underlying Bortezomib-induced cell damage in HUVEC, we measured ROS production, anindicator of oxidative stress which has been implicated ininduction of autophagy [35]. Indeed, we recently reported thatBortezomib treatment disrupts redox homeostasis in MM cell lines[28]. To do so, we treated subconfluent HUVEC with Bortezomib100 nM and then assessed ROS formation by staining the cells withHE. As shown in Fig. 5A, Bortezomib treatment induced an increasein HE staining, which was even more pronounced than thatobtained with H2O2 that we used as a positive control. Indeed,upon a 20 h treatment with Bortezomib or H2O2, HE stainingsignificantly increased compared to control (ΔMFI 311±46 vs.399±25 in Bortezomib-treated HUVEC, and 344±48 in H2O2

treated HUVEC, Fig. 5B). This finding suggests that Bortezomibinduces oxidative stress also in HUVEC.

Discussion

Antiangiogenic therapies are endowed with a stringent rationaleand have emerged as a promising approach for the treatment ofboth solid tumors and hematological malignancies [36–39]. Such astrategy can be successful only if the enhanced EC death isrestricted to proliferating cells of the nascent vasculature.Therefore, the identification of factors that influence EC resistanceto death may help to optimize the outcome of antiangiogenicapproaches. In addition to a direct effect on MM plasma cells, theproteasome inhibitor Bortezomib mediates antiangiogenesis,which can be further exploited for MM therapy [5]. In the presentstudy, we evaluated the effect of Bortezomib on HUVEC, expres-sing different activation states, and found that: i) the sensitivity ofEC to Bortezomib-induced cell death parallels their proliferationrate, being maximal in EC switched toward angiogenesis and

negligible in quiescent EC; ii) Bortezomib induces death either viaan apoptotic or an autophagic pattern, again depending on ECactivation.

We [8] and others [5], have previously shown that Bortezomibdisrupts the angiogenic potential of EC, both indirectly byinhibiting the release of angiogenic factors [5] and directly throughthe induction of cytotoxicity [8]. Notably, in our hands the latterwas prominent when HUVEC were switched toward angiogenesiswith VEGF and hypoxic conditions, which are features common toseveral tumors. We now extend this observation showing thatBortezomib-induced cell death paralleled the activation state ofHUVEC; indeed, quiescent HUVEC were completely resistant, andprogressively became more sensitive according to their prolifer-ation rate. In particular, Bortezomib-induced HUVEC death wasmaximal when the drug was added to cells cultured in thepresence of FGF, indicating that it may overcome the anti-apoptotic signature conveyed by angiogenic factors which arehighly expressed inside MM microenvironment [6,7]. It istherefore conceivable that inside a tumor, EC may be exposed todifferent gradients of pro-angiogenic stimuli which may affect theproliferation rate and ultimately their sensitivity to drugs. This

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notion may partially account for the failure of antiangiogenicstrategies.

As previously reported with other angiogenesis inhibitors,Bortezomib activates two different, while interconnected path-ways of cell death, autophagy and apoptosis [14,40], dependingon HUVEC activation state. Indeed, FGF-activated EC underwentclassical apoptosis, via the intrinsic mitochondrial pathway;conversely, subconfluent EC underwent morphological andultrastructural changes, including the presence of typicalautophagosomes reminiscent of an autophagic fate. Thesefeatures, together with increased expression of total MAP LC3protein and conversion to the vacuoles-associated LC3-II form,definitely prove this notion, further substantiated by the specificinhibition of both LC3-II generation and Bortezomib-induceddeath obtained with 3-MA. Autophagy is a process by whichlong-lived proteins and larger cytoplasmic components aredegraded [41]. Mechanisms activating the autophagic pathwayin subconfluent HUVEC-treated with Bortezomib are so farunclear. It is known that the inhibition of the 26S proteasomeby Bortezomib leads to the accumulation of misfolded proteins,resulting in endoplasmic reticulum stress followed by acoordinated reparative cellular response called unfolded proteinresponse (UPR) [31,42]. Endoplasmic reticulum stress is also apotent inducer of autophagy where it promotes cell survival[43]. On the other hand, our present finding of an alteration ofredox homeostasis as indicated by ROS production provides apossible explanation for the induction of autophagic cell death insubconfluent HUVEC. Indeed, ROS have been demonstrated totrigger autophagy through several distinct mechanisms involv-ing, among others, members of the Atg4 family [35].

Although autophagy is initiated as a protective response tostress, persistent autophagy can lead to cell death. Given therecovery of EC morphology and viability upon withdrawal ofBortezomib and the protective effect obtained with 3-MA, we canspeculate that also in subconfluent HUVEC autophagy is activatedas a pro-survival response to damage inflicted by Bortezomib.Similarly, autophagy was recently reported in mouse embryofibroblasts upon treatment with Bortezomib and proposed as amechanism to escape cell death, implying co-treatment withinhibitors of autophagy as a tool to potentiate anti-cancertreatments [44].

In conclusion, our data suggest that Bortezomib induces deathof HUVEC via alternative pathways depending on their activationstate, and implicate that EC may initiate autophagy as a survivalresponse to limit the effects of angiogenesis inhibitors. This findingdiscloses the possibility to optimize the antiangiogenic effect of thedrug, not only by targeting activated EC, but also by interferingwith their autophagic survival response.

Acknowledgments

We thank Carla Panzeri (Alembic Facility, HSR, Milan) for technicalassistance at TEM, Alessandro Fulgenzi (Department of Pathology,University of Milan) for the statistical analysis and Simone Cenciand Niccolò Pengo (Division of Genetics and Cell Biology, HSR,Milan) for discussion and suggestion. We also thank Michael John(Università Vita-Salute, San Raffaele, Milan) for the English-language editing of the manuscript. The work was partiallysupported by BOSH S.p.A.

Appendix A. Supplementary data

Supplementary Fig. 1. FGF and hypoxia are highly mitogenic inHUVEC. 3.5×103 HUVEC (dotted lines) were seeded in triplicateand assessed for their proliferative response to FGF and hypoxia(5% O2) by crystal violet densitometric assay. The proliferative ratewas determined 24 h after seeding and calculated as described inMaterials and methods.

Note: The Supplementary material accompanying this article isavailable at doi:10.1016/j.yexcr.2009.11.005.

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