Background
First developed for its cytoprotective properties against radiation, amifostine is now approved by the US Food and Drug Administration for clinical use as a cytoprotector in several anti-cancer therapies [
1]. Amifostine, also called Ethyol
®, is a phosphorylated aminothiol (WR-2721). Its intracellular activity relies on its dephosphorylation by membrane bound alkaline phosphatase [
2], thus producing the free thiol WR-1065. WR-1065 acts as a free radical scavenger and is considered to be the effective cytoprotector, affording protection against the toxic side effects of both chemotherapeutic agents and ionizing radiations [
3‐
5]. This cytoprotection has been shown to be mostly effective on normal cells and does not interfere with the efficacy of anticancer treatment in tumours [
6]. This selective effect on normal cells was attributed, in part, to the acidic microenvironment found in numerous tumour tissues, which decreases the rate of prodrug activation by the alkaline phosphatase and also to a lower expression of the enzyme in the tumour endothelium [
2]. Abnormal tumour vasculature is also thought to lower the drug's access to tumour tissues [
4]. Nevertheless, the drug does penetrate in cancer cells, even at a lower rate, and has often been described as enhancing the antitumour effects of chemotherapeutic agents and ionizing radiations [
4,
7,
8]. This latter property, albeit not clearly understood, may depend on the activation of key regulatory proteins in cancer cells, such as the tumour-suppressive p53 protein, which is activated by amifostine and can block cell proliferation in p53 WT tumour cells, or enhance apoptosis due to chemotherapeutic agents in p53 deficient-tumour cells [
9‐
12]. Amifostine has also been reported to act as a hypoxia-mimetic compound able to promote hypoxia-inducible factor (HIF)-1α accumulation both
in vitro and
in vivo [
13]. HIF-1α is a major stress regulator induced by cancer cells in response to ischaemia during tumour development. Stabilized by hypoxia, HIF-1α heterodimerizes with the HIF-1β subunit and activates the transcription of different sets of genes involved in numerous cellular processes including metabolism, apoptosis and angiogenesis [
14]. The most prominent pro-angiogenic factor activated by HIF in response to hypoxia is the vascular endothelial growth factor (VEGF-A). Cancer cells secrete VEGF-A as different isoforms that diffuse through the tumour microenvironment and bind to the specific transmembrane receptors VEGFR1, VEGFR-2 and neuropilin-1, mainly located on vascular endothelial cells (ECs) [
15,
16]. VEGFR-2 is believed to mediate most of the paracrine effects of VEGF-A known to promote angiogenesis: vascular permeability, EC proliferation, migration, survival and association in micro-capillary structures [
17]. Moreover, VEGFR-2 has been recently implicated in VEGF-A autocrine loops in EC, that seem to be essential to their survival and to the maintenance of the differentiated state of blood vessels [
18].
Induction of VEGF-A expression also occurs under different cellular stresses through HIF-1- independent pathways [
19‐
22]. As an example, VEGF-A expression can be activated by the inositol requiring enzyme 1 (IRE1)-dependent pathway, a branch of the unfolded protein response (UPR), in response to hypoxia or glucose deprivation [
21]. Reductive agents such as dithiothreitol (DTT) and homocysteine are also potent inducers of the UPR and of an activating transcription factor 4 (ATF4)-mediated transcription of the VEGF-A gene [
23‐
25]. Besides their effects on VEGF-A expression, thiolic-reducing agents may impair VEGF-A signalling on EC via their free radical scavenging properties. Indeed, these compounds may interfere with the production of reactive oxygen species (ROS) which is necessary to the phosphorylation of extracellular signal-regulated kinase (ERK) downstream of VEGFR-2, and to the mitotic response of EC [
26,
27]. These effects may significantly impact tumour growth and metastasis [
28‐
31].
Despite some reports on HIF expression, the effects of amifostine on angiogenesis have been poorly studied and the results found in the literature are often controversial. Using the developmental angiogenesis model of the chorio-allantoic membrane (CAM), both pro- and anti-angiogenic effects have been reported [
32,
33]. The anti-angiogenic effect was attributed to the ability of amifostine to modify the redox status of EC
in vitro. However, a proliferating effect of the drug was concomitantly observed [
34]. Moreover, Grdina
et al. described an anti-metastatic effect of amifostine in mice developing Sa-NH sarcoma, in association with high serum levels of the angiogenesis inhibitor angiostatin. This observation suggests that amifostine could have anti-angiogenic properties [
35,
36]. Little is written about the effects of amifostine on VEGF-A expression in these studies and nothing in relation to VEGF-A expression in tumour cells.
In order to better characterize the potential effects of amifostine on tumour angiogenesis, we undertook a systematic study to analyse its effect on VEGF-A expression, production and activity on human cancer and EC.
Methods
Reagents
We used culture media obtained from Invitrogen (CA, USA) and Cambrex (MD, USA). Fetal bovine serum (FBS) was obtained from Biowest (Nuaillé, France) and was heat-inactivated before use. Growth Factor Reduced Matrigel was obtained from Becton Dickinson Biosciences (NJ, USA). Bovine serum albumine (BSA), cell-culture grade gelatin, fibronectin, DTT, protamine sulphate, aminoguanidine and amifostine (WR-2721) were purchased from the Sigma Chemical Co (MO, USA). The metabolically active form of amifostine (WR-1065) was synthesized by Dr M. Zanda (CNR, Milan, Italy). The VEGF-A neutralizing polyclonal antibody was obtained from R&D Systems (Minneapolis, USA). Primary antibodies against human HIF-2α and β-actin were obtained from Santa Cruz Biotechnology (CA, USA); anti-human Ku80 from Serotec (Oxford, UK); anti- human HIF-1α from BD Transduction Laboratories (Le Pont de Claix, France); anti-VEGFR-2 from ReliaTech (Braunschweig, Germany); anti- phospho-eIF2α (Ser51), anti-eIF2α and antibodies to p42/44 and their phosphorylated counterparts from Cell Signaling Technology (MA, USA). Primers for ATF4 were as described by Namba
et al. [
37]. Primers for GADD34, CHOP, EDEM and BIP were as previously described [
21]. Other primer used for mRNA expression analysis, were reported in Additional File
1 (Table S1). All primers were obtained from Proligo (Paris, France).
Cell culture
Human mammary carcinoma cells MCF7 and colon carcinoma HCT116 cells were a gift from Dr P Hainaut (International Agency for Research on Cancer, Lyon, France). MCF7 cells and U87 human glioma cells (ATCC, HTB-14) were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% FBS. HCT116 cells were grown in McCoy medium, 10% FBS. Human umbilical EC (HUVEC; Clonetics, CA, USA) were propagated up to eight passages on a 0.2% gelatine matrix in endothelial growth medium (EGM-2; Bulletkit, Walkersville, USA). Treatments of tumour cells with amifostine were performed in the presence of 4 mM aminoguanidine (AG), as previously described [
10]. AG prevents the catabolization of amifostine by FBS Cu-dependant amine-oxydases into cytotoxic compounds [
38]. Amifostine treatments on HUVEC were performed without AG. Hypoxic conditions were obtained at 3% O
2 in a Heraeus incubator BB-6060.
Lentiviral vector constructs and MCF7 transduction
MCF7 were transduced using lentivectors expressing the fluorescent marker E-GFP and containing short hairpin RNA sequences against HIF-1α [HIF-1α.small hairpin (sh)RNA] or red fluorescent protein (RFP.shRNA) as described [
39]. For transduction, MCF7 cells (5.10
4 cells per well in a 24-multiwell plate) were incubated for 24 h in complete medium. Cells were then incubated with viral supernatants from 293T cells for 24 h at 37°C in the presence of protamine sulphate. Transduced cells were sorted out 5 days post-transduction by cytofluorimetry. Enhanced green fluorescent protein (E-GFP) positive cells were used for the experiments.
ATF4 RNA interference and transient transfection of MCF7
Small interfering RNAs (siRNAs) were purchased from Eurogentec (Liège, Belgium). The sequence of the ATF4-targeting SiRNA (SiATF4) was as previously described [
40]. A SiATF4 mutated in three nucleotides served as control (SiMUT, 51). Cells at a 50% density were transfected with 250 nM of SiRNA in OptiMEM using Lipofectamine Plus (Invitrogen, CA, USA). After 24 h, cells were either treated or not treated with amifostine; cells and supernatants were then collected for RNA isolation and protein analysis.
VEGF-A enzyme-linked immunoadsorbent assay (ELISA)
Cells at 60% confluence were grown in 5-cm diameter dishes for the indicated period of time. VEGF-A concentration was measured in cell-conditioned media using a commercial VEGF-A ELISA kit (R&D Systems, Minneapolis, USA). Assays were performed in triplicate and calibration curves were obtained using human recombinant VEGF-A. The results were analysed using the Softmax Pro4.0 software (Molecular Devices Corporation, CA, USA). Cells were counted using a cell counter (Coulter, Becton Dickinson, NJ, USA).
The paracrine effect of amifostine was assayed as follows. MCF7 cells were first incubated for 48 h, either in the presence of AG alone or with both AG- and amifostine. Conditioned media (CM) in these conditions were dialysed at 4°C in order to remove the two chemicals. HUVEC grown in 96-multiwell plates to 80% confluence were then starved for 4 h in DMEM without fetal calf serum (FCS) and incubated for 16 h in the presence of CM. Wst-1 assays (Roche Applied Science, IN, USA) were then performed as follow: Wst-1 reagent was added to the cell medium after a 12 h incubation with CM and absorbance was read 4 h later at 440 nm using a spectrophotometer (Molecular Devices Corporation). Results were analysed using the Softmax Pro 4.0 software (Molecular Devices Corporation). The assay was performed in triplicates. In similar experiments, CM were pre-incubated for 45 min at 37°C with a VEGF-A neutralizing antibody or with an irrelevant anti-ß-actin antibody, prior to addition to HUVEC.
Polymerase chain reaction analysis (PCR)
Reverse transcription (RT) were performed as previously described [
39]. A semi-quantitative analysis of VEGF-A was performed by co-amplifying VEGF-A and β-actin in a Thermal Cycler (Eppendorf AG, Hamburg, Germany) at 94°C for 40 s, 59°C for 30 s, 72°C for 50 s, throughout 35 cycles, with a final elongation step of 3 min at 72°C. Real time quantitative PCR (Q-PCR) analyses were performed using the MX3000p thermocycler (Stratagene, CA, USA) and the SYBRgreen dye (ABgene, Epsom, UK) methodology. The relative abundance of transcripts was calculated by using α-tubulin or β-actin as standards.
Immunoblot analysis
MCF7 cells and HUVEC were grown up to the subconfluence in 10 cm diameter dishes. HUVEC were starved for 24 h in EBM-2 medium, 2% FCS prior to treatments. Treatments were then performed in fresh medium (EBM-2 for HUVEC, complete culture medium for MCF7) and total protein extracts were collected on ice at different time points with an electrophoretic mobility shift assay buffer-B [
11] for HIF and mitogen activated protein kinase analysis or as described by Drogat
et al. for eIF-2α analysis [
22]. Forty to fifty micrograms of proteins were separated in SDS-PAGE gels and transferred to a 0.4 μm nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). Proteins of interest were detected using specific primary antibodies (see figure legends) and secondary antibodies were coupled to horseradish peroxidase (DAKO SA, Glostrup, Denmark). Blots were revealed using the enhanced chemiluminescence +reagent (Amersham Pharma Biotech, NJ, USA) followed by radioautography or direct quantification of chemiluminescence (Fujifilm LAS-3000).
VEGF-A radioligand binding studies
VEGF-A
165 was radio-labelled as previously described [
41] to a specific activity ranging from 20,000 to 100,000 cpm/ng. HUVEC were seeded at 7000 cells/cm2 in six-well plates, cultured for 2 days in EGM-2 and starved for 24 h in 2% FBS-containing EBM-2 prior to a 12 h-incubation in the presence, or not, of 1 mM WR-1065. The plates were washed once with phosphate buffered saline (PBS) and the cells were incubated for 2 h at 4°C with 125I-VEGF-A in DMEM, 20 mM HEPES, 0.2% gelatine. They were rinsed three times in PBS and finally solubilized at room temperature in a solution containing 2% Triton X-100, 10% glycerol and 1 mg/mL BSA. Total cell lysates were counted for radioactivity using a Kontron MR 250 gamma-counter. Each value reported in the graph was the mean of duplicate determinations. Unspecific binding was defined as the amount of radioactivity bound in the presence of 20 μg/mL protamine sulphate, which gave results similar to the use of a 100-fold excess of VEGF-A. The experiments were repeated three times with similar results. Binding data were analysed using the GraphPad Prism 4.0 software.
Transwell cell migration assays
HUVEC cell migration was measured using 24-well-Transwell plates (Becton Dickinson Labware, Le Pont de Claix, France). Prior to cell seeding, inserts (8-μm pore size) were precoated overnight at 4°C on the lower side using 10 μg/mL fibronectin in PBS. Membranes were then blocked 1 h at room temperature with 1% heat-inactivated BSA. HUVEC (105 cells per well) were seeded in the upper compartment and allowed to migrate for 7 h throughout the membranes in DMEM containing 0.5% FBS and 0.1% BSA and in the presence of different concentrations of WR-1065. VEGF-A (10 ng/mL) was added in the lower compartment as chemo-attractant. After migration, cells that had migrated were fixed and stained in a solution containing 30% methanol, 10% acetic acid and 0.1% Coomassie Blue. The extent of cell migration was analysed by counting cells in at least six random fields per Transwell filter using the LUCIA image analysis software (Laboratory Imaging, Praha, Czech Republic). Results are from three independent experiments.
Amifostine was tested for its ability to modulate endothelial cell differentiation into tube-like structures using a Matrigel surface assay. Cells (2 × 105 cells/mL of Growth Factor Reduced Matrigel, BD Biosciences, MA, USA) were seeded in EBM-2 medium supplemented with 0.2% FBS and 1% L-glutamine and allowed to adhere for 1 h. Media were then replaced by EBM-2 enriched with 0.5% FBS, containing increasing concentrations of WR-1065 with or without 10 mg/mL of VEGF-A. Cells were then incubated for 3 h at 37°C. Total length of tube-like structures was assessed using the LUCIA image analysis software. Results shown are representative of three independent experiments. Each data point is the mean of six randomly chosen fields.
Statistics
Statistical analysis of data was performed using the paired Student's t test P value (*P < 0.05; **P < 0.01).
Discussion
Our investigations showed that the radio- and chemo-protective drug amifostine is a potent inducer of VEGF-A expression, acting both at the mRNA and protein levels in several human cancer cell lines. Accumulation of VEGF-A mRNA was detected at concentrations of the drug (0.5 - 2 mM) that correspond to those measured in most tissues of treated rats and monkeys 10 to 30 min after an injection of a single protective dose [
8,
42]. This suggests that VEGF-A up-regulation may also occur along clinical cancer treatments of patients with the cytoprotective drug and questions about the possible angiogenic side effects of such treatments.
To date, the effects of amifostine on angiogenesis were still poorly studied and results were found somewhat controversial [
32]. In addition, little is known of the effects of amifostine on VEGF-A expression. Therefore, the potential impact of amifostine on angiogenesis has to be clarified, in particular in relation to the VEGF-A expression. A study from Koukourakis showed that HIF-1α was stabilized both
in vitro and
in vivo by high concentrations of amifostine (8 mM) [
13]. Since HIF-1α acts as a pro-angiogenic transcription factor and increases VEGF-A expression [
14], we first thought that it was responsible for VEGF-A up-regulation in cancer cells exposed to amifostine in our experiments. However, using concentrations of amifostine consistent with the clinical approach, we failed to observe in our cell models any accumulation of either HIF-1α or HIF-2α proteins in response to the drug treatment. Our data clearly show that HIF-1α activity is not involved in VEGF-A up-regulation under amifostine treatment at clinical doses, which led us to consider alternative VEGF-A stimulatory pathways.
Amifostine is a disulphide reducing agent that can generate redox stress in cells [
12] and other thiol-containing compounds are known to affect protein folding and to activate the UPR [
24]. We therefore investigated whether UPR was activated in response to amifostine and whether this could lead to VEGF-A mRNA expression as previously reported [
21]. The increased expression of several genes considered to be hallmarks of the UPR pathways (
BIP, EDEM, CHOP and
GADD34) following amifostine treatment suggests that amifostine did activate the UPR signalling pathways. However, the low increase in
BIP and
EDEM mRNA expression, which usually relies upon IRE1- and ATF6-dependant pathways [
44,
45], suggests that these pathways were not fully activated in response to amifostine. In addition, there was no detectable XBP1 splicing in treated cells, suggesting that IRE1 is not induced by amifostine and thus does not significantly contribute to VEGF-A up-regulation.
The strong increase in
CHOP and
GADD34 mRNA expression could be linked to either PERK activation or to the GCN2-dependant pathway, both leading to eIF2α phosphorylation, inhibition of cap-dependant translation and activation of the ATF4 transcription factor [
46,
47]. Interestingly, homocysteine, a compound related to amifostine by the presence of its thiol function, has been shown to induce ATF4 transcriptional activity, leading to increased expression of VEGF-A [
24]. Consistently, we observed a transient eIF2α phosphorylation in response to amifostine treatment, which suggests the involvement of ATF4 for VEGF-A expression. Indeed, abolition of ATF4 expression (~95%) using siRNA strategy clearly demonstrated that VEGF-A up-regulation upon exposure to amifostine strongly relies on ATF4. However, other pathways may also contribute to the amifostine-mediated effects on VEGF-A expression such as the c-jun N terminal kinase pathway (JNK). JNK is a known intermediate of VEGF-A mRNA and protein up-regulation [
48,
49] and we previously showed that this pathway is also activated by amifostine [
11]. Consistently, the chemical JNK inhibitor SP-600125 did reduce the extent of amifostine-induced VEGF-A activation by 1.4 fold (not shown). Interestingly, JNK activation is also linked to the UPR downstream of the ER-stress-induced activation of IRE1α, independently of its endoribonuclease activity [
50], and IRE1α is itself involved in the up-regulation of VEGF-A in cancer cells undergoing ischemia [
21]. Collectively, these data suggest that JNK might also synergize with ATF4 to up-regulate VEGF-A expression in response to amifostine.
The potential pro-angiogenic effect of amifostine in cancer cells may not be limited to the activation of VEGF-A expression. Experiments using VEGF-A blocking antibodies indicated that, in addition to this growth factor, treated tumour cells may also secrete other endothelial cell growth promoting factors. EGF or IL8 are possible candidates for this effect stand, and their expressions were up-regulated (up to 13- and 5.2-fold, respectively) in MCF7 cells upon stimulation by amifostine, as determined using transcriptome analysis (not shown). Amifostine is currently used as a cytoprotector in therapeutic treatment, either as a single dose treatment for cancer therapies or in long-term chronic delivery in the treatment of myelodysplasic syndromes [
4,
7,
51]. Side-induction of VEGF-A, or of any other pro-angiogenic factors, by cancer cells during these treatments may have detrimental effects on the therapeutical purposes. Therefore, it was necessary to investigate whether amifostine could develop secondary effects when applied directly on EC.
EC are subjected to the highest concentrations of amifostine following its intravenous administration. Comparatively, cancer cells accumulate lower concentrations of the drug, as it is known to hardly penetrate tumour masses [
4,
8]. Since we had previously found an anti-proliferative effect of amifostine on normal and cancer cells ([
10,
52] and unpublished data), we considered the possibility that amifostine also impedes EC proliferation, which could overcome the tumour-mediated pro-angiogenic effect. In fact, as reported here, each of the endothelial cell responses to VEGF-A was strongly inhibited by amifostine treatment, therefore suggesting that EC were indeed made unresponsive to tumour-mediated VEGF-A stimulation by clinical doses of the drug.
The origin of EC desensitization in response to amifostine treatment was then considered. The EC proliferation arrest should be considered as the consequence of a p53 dependent-cell cycle arrest in G1, as already observed in various cell types ([
10,
52] and unpublished data). In addition, we showed that amifostine plays a role in EC capacity to respond to VEGF-A. An immediate impairment of intracellular signalling was observed downstream to this interaction, as indicated by the 50% decrease of the VEGF-A-induced phosphorylation of the ERK kinases in the presence of amifostine. As ROS are essential second messengers in EC [
26,
27], this inhibitory effect may depends on the ROS scavenging properties of the drug, leading to the blockade of the downstream signalling of VEGFR-2.
In addition to this immediate effect, we observed a long-term effect of amifostine on the capacity of EC to bind VEGF-A. This was shown by a significant decrease in VEGF-A binding to its specific cell-surface receptors after 12 h of amifostine treatment. This long-term effect depends on the down-regulation of VEGFR-2 protein expression at the plasma membrane. This down regulation probably results from a combination of complex intra- and extra-cellular redox effects due to the free thiol group of WR-1065. Part of these effects may occur at the transcriptional level by inactivating redox sensitive transcription factors involved in VEGF-R2 expression as it does for p53 and NFKappa B [
10,
12,
53]. An additional effect may occur at the translational level through the global inhibition of translation due to eIF2a phosphorylation in response to treatment [
46,
47]. In addition, although no modulation of VEGF-A affinity to its receptors was observed in our experiments, we cannot completely rule out the possibility of an additional direct effect of the thiol group of amifostine on VEGF-A binding to its receptors. Indeed, VEGF-A exists as a disulphide-bond functional dimer. VEGF-A dimerization, which is essential for its biological activity, may be impaired under reducing conditions [
54]. Moreover, VEGFR-2 ligand binding sites are located within Ig-like extracellular domains that may also be sensitive to redox conditions [
17]. By potentially modifying the structure of the two partners, amifostine may inhibit the interaction between VEGF-A and VEGF-R2, therefore inducing a down-regulation of VEGF-R2 expression [
55]. Finally, activation of the stress induced kinase JNK by amifostine may trigger specific phosphorylation of VEGF-R2 receptor that could lead to its internalization and degradation as it has already been described for epidermal growth factor receptor [
56]. Thus, several characteristics of amifostine likely contribute to render endothelial cell unresponsive to external VEGF-A stimuli.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SD performed most of the technical part of the study, cell culture and treatment, PCR, ELISA and in vitro angiogenesis tests. XC performed VEGF-A binding studies on HUVECs. HR and FM performed viral constructs and viral transduction on MCF7 cells to block HIF1-alpha expression. MB performed WB on Eif2alpha protein and helped in the UPR pathway study. RS and MZ synthesized WR1065 necessary for the study. MM and AB provided scientific support and advised on UPR stress and angiogenesis studies. SN is the principal investigator of this study and performed the cell cycle analysis, the ATF4 part of the experiment, a part of the quantitative RT PCR, the writing and revision of the paper.