MD Krisztina Rusai
email: rusai@gyer1.sote.hu

Ágnes Prókai

Beáta Szebeni

Andrea Fekete

András Treszl

Ádám Vannay

Veronika Müller

György Reusz

Uwe Heemann

Jens Lutz

Tivadar Tulassay

Attila J Szabó

Role of Serum and Glucocorticoid-Regulated Kinase-1 in the protective effects of erythropoietin during renal ischemia/reperfusion injury

In vitro, cultures of HEK293 cells were exposed to 16 h hypoxia. Incubation with EPO at a dosis of 400 U/ml exerted a protective effect on cell death assessed by LDH release and Annexin-V FACS analysis. This was paralleled by up-regulation of SGK1 expression, as well as phosphorylation. Downregulation of SGK1 expression by small interfering RNA technique ameliorated the antiapoptotic effect of EPO treatment.

In an in vivo rat model of unilateral renal I/R injury, rats were treated with 500 U/kg EPO 24 h prior to ischemia. EPO resulted in less severe tissue injury and ameliorated the elevation in creatinine and urea nitrogen levels 24 h after reperfusion. Furthermore, SGK1 expression and phosphorylation were higher in EPO compared to vehicle treated rats as demonstrated by real-time PCR, Western blot and immunofluorescence technique.

We conclude that EPO protects from renal I/R injury and SGK1 might contribute to the mediation of EPO effects under ischemic conditions. Keywords: EPO, apoptosis, hypoxia, ischemia/reperfusion, kidney, SGK1

A c c e p t e d M a n u s c r i p t INTRODUCTION Erythropoietin (EPO) is a glycoprotein hormone primarily responsible for the regulation of red blood cell production [START_REF] Krantz | Erythropoietin[END_REF]. However, increasing evidence suggests that independently of its erythropoietic effects, it also exerts protective actions against ischemic injury in various organs [START_REF] Lacombe | Biology of erythropoietin[END_REF].

EPO is up-regulated in brain, spinal cord and kidney after hypoxic stimuli and it protects the cells from apoptosis [START_REF] Brines | Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury[END_REF][START_REF] Sirén | Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress[END_REF][START_REF] Sharples | Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia-reperfusion[END_REF]. Furthermore, exogenous EPO administration after renal ischemia/reperfusion (I/R) injury has also been demonstrated to ameliorate tissue injury mainly by preventing apoptotic cell death [START_REF] Sharples | Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia-reperfusion[END_REF][START_REF] Bahlmann | Erythropoietin regulates endothelial progenitor cells[END_REF][START_REF] Yang | Preconditioning with erythropoietin protects against subsequent ischemia-reperfusion injury in rat kidney[END_REF]. I/R injury is among the leading causes of morbidity and mortality in various diseases including acute renal failure, heart failure, stroke and post-transplant graft dysfunction. The balance between the activation of pro-and anti-apoptotic pathways in the post-ischemic tissue plays a critical role in ischemia-induced organ dysfunction as apoptotic cell death has been recognized as a major mode of cell demise in I/R injury [START_REF] Broe | Apoptosis in acute renal failure[END_REF].

EPO exerts its immediate anti-apoptotic effects via an activation of multiple signalling pathways of which phosphorylation and activation of the phosphatidylinositol 3-kinase (PI3-kinase) [START_REF] Cai | Phosphatidylinositol-3-kinase signaling is required for erythropoietinmediated acute protection against myocardial ischemia/reperfusion injury[END_REF] and signal transducer and activator of transcription 3 (STAT3) kinase [START_REF] Zhang | Signal transducers and activators of transcription 5 contributes to erythropoietin-mediated neuroprotection against hippocampal neuronal death after transient global cerebral ischemia[END_REF] seem to play pivotal roles. However, the underlying mechanisms have still not been fully described and possibly, additional pathways are involved in the protective effects of EPO.

Activation of the PI3-kinase induces to the phosphorylation of protein kinase B (Akt). Akt has multiple effects on cell survival by directly maintaining mitochondrial integrity and through the inhibition of several pro-apoptotic mediators [START_REF] Chong | Erythropoietin is a novel vascular protectant through activation of Akt1 and mitochondrial modulation of cysteine proteases[END_REF]. The serum-and glucocorticoid-inducible protein kinase 1 (SGK1) is a serine-threonine kinase which is 50-55% homologue to Akt. SGK1 is also directly phosphorylated and, thus activated by the phosphoinositide-dependent kinase 1 (PDK1) which acts in a PI3-kinase-dependent manner [START_REF] Alessi | 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase[END_REF] and share a number of common target molecules [START_REF] Leong | Expression of the serum-and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells[END_REF].

The expression and activation of SGK1 is increased by various cell stress stimuli such as hyperosmotic stress, ultraviolet radiation, heat shock [START_REF] Webster | Immediate-early transcriptional regulation and rapid mRNA turnover of a putative serine/threonine protein kinase[END_REF], oxidative stress [START_REF] Leong | Expression of the serum-and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells[END_REF] and hypoxia [START_REF] Aoyama | Serum and glucocorticoid-responsive kinase-1 regulates cardiomyocyte survival and hypertrophic response[END_REF].

Under these conditions SGK1 acts as a strong anti-apoptotic kinase mainly by phosphorylating FKHRL1 and IKKβ [18].

Recently, we have shown that SGK1 is up-regulated and exerts anti-apoptotic effects after renal I/R injury in kidney-derived tubular cells and in the kidney tissue (manuscript accepted in Cell Physiol Biochem).

Since the protective effects of EPO are mediated mainly by the PI3-kinase pathway and SGK1 seems to be one of the main downstream targets of this pathway, we hypothesized that the anti-apoptotic effects of EPO during ischemic renal injury might be mediated by the PI3-kinase regulated SGK1.

Cell culture

The human embryonic kidney (HEK) 293 cell line derived from human embryonic kidney cells was purchased from American Type Culture Collection (ATCC, Rockville, MD). All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) (PAN Biotech GmbH, Aidenbach, Germany) and were propagated at 37 °C in a humidified air containing 5% CO 2 incubator.

In vitro hypoxia model

HEK 293 cells were seeded out and plated on 6-well plates at 5x10 5 /ml 24 h prior to the induction of hypoxia.

For hypoxia the medium was changed to serum-and glucose-free DMEM saturated with 95% N 2 /5% CO 2 and cells were placed in a 37 °C airtight box for 16 h. For normoxic conditions, culture medium was changed to serum-and glucose-free DMEM and cells were placed in a 37 °C/5% CO 2 incubator for 16 h. After 16 h of incubation time cells were analyzed.

Human recombinant EPO epoetin beta was obtained from Roche Hungary (Budapest, Hungary) and was added to the cell culture medium in a final concentration of 400 U/ml 2 h prior to the induction of hypoxia or to control normoxic cells. Placebo solution, also obtained from Roche Hungary served as vehicle control.

Annexin V Analysis

Annexin V-propidium-iodid (PI) apoptosis kit was used according to the manufacturer's protocol to detect apoptosis in vitro (Caltag Laboratories, Karlsruhe, Germany). In brief, cells were washed with phosphate buffered saline (PBS) and stained with Fluorescein labelled Annexin V and PI and then analyzed by flow cytometry. Annexin V and PI positive cells were considered as apoptotic cells.

SGK-1 gene silencing

For sgk-1 gene silencing small interfering RNA (siRNA) technique was used. Sgk-1-specific target sequence (5′-AAUGUGAAGCACCCUUUCCUG -3′), was annealed and cloned into pSilencer-4.1-CMV-Neo (Ambion) at BamHI-HindIII sites according to the manufacturer's instructions.

In vivo renal I/R model

Animals and surgery

Male Wistar rats (weighing 250-275 g) were subjected to 50 mg/kg pentobarbital sodium anaesthesia (Nembutal, Abbott Laboratories, Budapest, Hungary) and the abdomen was opened through a midline incision left renal artery and vein were clipped by an atraumatic vascular clamp for 50 min during which right nephrectomy was performed. After 50 min of ischemia, the clamp was removed and the animals were allowed to recover. After 2 h and 24 h of reperfusion, rats were reanaesthetized and the remnant kidney was removed.

EPO treated rats were given 500 U/kg EPO Roche Hungary (Budapest, Hungary) intraperiotenally, 24 h prior to ischemia and animals were randomized into the following groups (1) sham + vehicle, (2) sham + EPO, (3) I/R 2h + vehicle, (4) I/R 2h + EPO, (5) I/R 24h + vehicle and (6) I/R 24h + EPO. Sham-operated rats (groups 1 and 2) underwent identical surgical procedures without unilateral renal clamping and were maintained under anaesthesia for the duration of the experiment (N=8/each group).

All animal experiments were approved by the Ethical Committee of the Semmelweis University.

Lactate dehydrogenase (LDH) and renal function parameters

LDH release into the cell supernatant, serum creatinine and blood urea nitrogen (BUN) levels were analyzed by an automatic laboratory analyzer (Synchron CX5, Beckman Coulter, Krefeld, Germany). LDH results are expressed as a percentage of total cellular LDH per well measured from cells lysed in 1% Triton-X 100 and corrected with appropriate media controls and total protein.

Renal histopathology

Paraffin sections of kidneys fixed in 4% neutral buffered formalin were stained with hematoxylin and eosin (H&E) and with periodic acid-Schiff (PAS) reagent. Histological changes typical to I/R injury such as tubular architecture, tubular dilation, swelling and necrosis, luminal congestion with loss of brush border, interstitial edema and infiltration of polymorphonuclear neutrophils were semiquantitatively evaluated and scored. Two independent observers examined the slides by light microscopy in a blinded fashion.

A c c e p t e d M a n u s c r i p t

Reverse transcription-polymerase chain reaction (RT-PCR)

Human SGK1 mRNA expression in HEK293 cells were determined by RT-PCR analysis. Total RNA was isolated from the cells by RNeasy Total RNA Isolations Kit (Qiagen GmbH, Hilden, Germany) according to the instructions of the manufacturer. The quality and quantity of the RNA were confirmed photometrically. [START_REF] Heemann | Lipopolysaccharide pretreatment protects from renal ischemia/reperfusion injury. possible connection to an interleukin-6dependent pathway[END_REF] After initial denaturation at 94ºC, 35 cycles of amplification at the accurate annealing temperature for human SGK1 and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were performed. The primer sequences and annealing temperatures are shown in Table 1. PCR products were separated on ethidium-bromide stained 2.5% agarose gels, then visualized and photographed under ultraviolet light.

Signals were quantified by densitometry and corrected for the GAPDH signal using an image analysis software program (Gel-Pro Analyser 3.1 Software).

Real-time polymerase chain reaction

Rat SGK1 mRNA expression in the renal tissues was assessed by SYBR Green real-time PCR on a Light Cycler system (Roche Diagnostics, Mannheim, Germany). PCRs were performed in glass capillaries, in a final volume of 20 μl containing 3 mM MgCl 2 , 0.2 μM of each primer, 2 μl LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany) and 1 μl of cDNA. The conditions of PCRs were as follows: one cycle at 95°C for 8 min, followed by 55 cycles at 95°C for 3 s, 59°C for 5 s and 72 °C for 28 s with a single fluorescence detection point at the end of the extension. mRNA expression of GAPDH in tissue samples was determined by real-time PCR quantification using fluorescence resonance energy transfer (FRET) hybridization probes on a Light Cycler system (Roche Diagnostics, Mannheim, Germany) in a final volume of 20 μl containing 2 mM MgCl 2 , 0.17 nM of each hybridization probe, 0.5 nM of each primer, 2 μl of the Light-Cycler FastStart DNA Master HybProbe (Roche Diagnostics) and 1 μl of cDNA. The PCR reaction was carried out under the following conditions: one cycle at 95°C for 8 min, followed by 50 cycles at 95°C for 4 s, 55°C for 8 s and 72°C for 22 s with a single fluorescence detection point at the end of the annealing segment.

Western blotting

Cells and kidney tissues were homogenized by a cell disrupter in chilled lysis buffer containing 1 mg/mL aprotinine, 5 mg/mL leupeptin, 1 M Tris, 0.5 M EGTA, 0.25 M NaF, 0.5 M PMSF, 0.5 M A c c e p t e d M a n u s c r i p t 8 Na 3 VO 4 and 0.1% Triton X-100. The lysate was centrifuged at 13.000 g for 10 min to remove insoluble debris. Protein concentrations in the supernatant were determined by the Bradford method using BSA as standard. All reagents for PAGE and Western blot were purchased from Sigma Aldrich (Munich, Germany). Lysates were solubilized in a buffer of 12.5 mM Tris-HCl, pH 6.7, containing 4.0% SDS, 1 mM EDTA, 15% glycerol and 0.01% bromphenolblue. 50 g protein of the samples was separated on 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Amersham, GE Healthcare Europe GmbH, Munich, Germany). The membranes were blocked with 5% non-fat dry milk (NFDM) for 1 h at room temperature and then probed with rabbit polyclonal anti-SGK1 antibody (1:400, Upstate, Germany) in 5% NFDM overnight at 4 C. The membranes were washed with Tween-phosphate-buffered saline, and incubated with anti-rabbit immunoglobuline antibody, a horseradish peroxidase conjugated from goat (1:5000, Amersham) in 5% NFDM for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence (ECL, Amersham) by Kodak Image Station 400 D. Prestained standards were used as molecular weight markers (Biorad Laboratories, Munich, Germany).

Signals on Western blots were quantified by densitometry using Gel-Pro Analyser 3.1 Software.

Double labeling using immunofluorescence microscopy

Kidney sections were immediately snap-frozen in 30% sucrose for immunohistochemical analysis.

They were embedded in Shandon cryomatrix (ThermoElectron Co.), cut into 5-10 μm thick slices with a cryostat, and stored at -80°C. After rinsing in PBS, they were treated in methanolic H 2 O 2 for 30 min. Tissue sections were permeabilized in 0.2% Triton X-100 in PBS and then blocked in 0.5% NFDM for 30 min at room temperature. The samples were incubated overnight at 4°C with primary antibodies (anti-total SGK 1:100, Santa Cruz, anti-phospho-SGK1 1:100, Cell Signaling), washed with PBS, and then incubated with secondary antibodies (Alexa Fluor 488 1:100, Alexa Fluor 546 1:100, Invitrogen, Budapest, Hungary) for 30 min at room temperature. DNA was stained with Hoechst 33342 (1:1000 Sigma, Hungary) for 10 min at room temperature. Appropriate controls were performed omitting the primary antibodies to ensure their specificity and to avoid auto-fluorescence.

Stained slides were analysed in a blinded fashion, localisation and expression of SGK1 was detected.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). Data were tested using the Chi-Square-or Mann-Whitney U test using the SPSS statistical software package (v. 13.0, SPSS GmbH, Munich, Germany). Data were analyzed using one-way ANOVA followed by Dunnett post hoc test or Kruskal-Wallis ANOVA for nonparametric data. P<0.05 was considered significant.

In vitro experiments

EPO prevents apoptosis after hypoxia in HEK 293 cells

Exposure of HEK293 cells to 24 h hypoxia caused significant cell death when compared to normoxic cells detected by LDH release into the supernatant (P<0.01). In particular, apoptosis of cells caused by hypoxia was demonstrated by Annexin V FACS analysis and proved to be elevated in comparison to normoxic cells (P<0.01) (Figure 1A-B.). Hypoxia-induced overall cell death and also specifically, apoptosis were reduced by incubation with EPO (400 U/ml) compared to vehicle treated cells (P<0.01) (Figure 1A-B.).

EPO stimulates the expression and phosphorylation of SGK1

Incubation of HEK293 cells with EPO increased SGK1 mRNA and protein expression both under normoxic and hypoxic conditions (P<0.01 vs. vehicle treated cells). Hypoxia stimulated phosphorylation of SGK1 (P<0.01 vs. vehicle treated cells, respectively) which was further increased by EPO (P<0.01 vs. vehicle treated cells) (Figure 2A-B.).

The effect of SGK1 siRNA on the anti-apoptotic action of EPO in hypoxia

To further examine the role of SGK1 in the mediation of EPO actions, we inhibited SGK1 expression by plasmid-mediated siRNA technique. Cells in which SGK1 expression was inhibited showed reduced protection after EPO treatment in hypoxic injury detected by LDH release and Annexin V FACS analysis (both P<0.01) (Figure 1A-B.).

In vivo experiments

Effect of EPO on renal dysfunction and post-ischemic structural damage caused by I/R

Administration of EPO to sham-operated animals did not have any effect on renal function (Figure 3A-B.).

Renal I/R injury increased post-ischemic serum creatinine and BUN levels 24 h after repefusion (both P<0.001 vs. sham) (Figure 3A-B., Table 2.). In the earlier time-point, 2 h after reperfusion, no significant elevation in serum renal function parameters were detected. A single bolus of EPO (500 U/kg) resulted in lower levels of serum creatinine and BUN at 24 h after reperfusion compared to vehicle treated animals (P<0.01) (Figure 3A-B.).

Renal I/R injury caused marked alterations in renal histology compared with kidneys taken from sham-operated animals (Figure 4A.). At 2 h of reperfusion, in contrast to the renal function parameters, there were already changes in the kidney tissue compared to sham operated animals:

Effect of EPO on SGK1 expression and phosphorylation in renal I/R injury

Administration of EPO resulted in a higher SGK1 mRNA expression at all time-points compared to vehicle treated animals (P<0.01 vs. shams, 2h, 24h, respectively) (Figure 5A.). EPO treatment led to the highest elevation in SGK1 mRNA expression in sham-operated animals. SGK1 mRNA expression was temporarily elevated 2 h after reperfusion (P<0.05 vs. sham) (Figure 5A.), but at 24 h of reperfusion, it returned to the level of sham-operated rats. Similarly to the vehicle-treated rats, in EPO-treated animals SGK1 mRNA expression decreased 24 h after reperfusion compared to the 2h+EPO rats (P<0.05) (Figure 5A.).

Parallel to the changes in mRNA expression, EPO treatment resulted in markedly elevated SGK1 protein levels compared to the vehicle-treated rats both in sham-operated and in IR 2h animals (P<0.01 respectively), however, 24 h after reperfusion, no difference was detected (P>0.05) (Figure

5B.).

The activated phosphorylated form of SGK1 was detectable only 2 h after reperfusion in the vehicletreated animals. On the contrary, in the EPO treated rats, SGK1 phoshorylation was detectable already in the sham-operated animals and it was higher in the 2h IR group (P<0.01 vs. vehicletreated rat) (Figure 5B.).

To show the location of SGK1 and phosphorylated SGK1, we performed immunofluorescent (IF) staining of the kidneys. IF demonstrated that SGK1 was expressed in the tubular cells. In accordance with the Western blot measurements EPO treatment up-regulated total and phosphorylated SGK1 in The glycoprotein hormone EPO, which is mainly responsible for the regulation of erythropoesis, seems to also have strong anti-apoptotic effects under hypoxic and oxidative stress, both main features of an in vivo I/R injury. In the present study, we investigated the role of SGK1 in the renoprotective effects of EPO during renal I/R injury.

In our experiments, EPO protected both cultured kidney cells and also the kidney tissue against hypoxic and ischemic injury. HEK293 cells treated with 400 U/mL recombinant EPO were protected from cell death caused by 16 h of hypoxia. Furthermore, in a rat model of renal I/R injury, EPO treatment (500 U/kg) 24 h prior to the induction of ischemia improved post-ischemic renal function and diminished renal structural damage.

These results are well in accordance with earlier observations providing convincing evidence for a protective role of EPO. Yang et al. were the first to demonstrate that a high-dose of EPO (3000 U/kg) 24 h before bilateral renal artery clamping protected the kidneys from ischemic injury. At 24 h post-reperfusion, both the rise in creatinine level and the apoptosis of proximal tubular epithelial cells were attenuated in EPO treated animals [18]. Moreover, Sharples et al. presented that EPO in a lower dose (300 U/kg) protected the kidney against unilateral I/R injury when even given as late as 30 min after the onset of reperfusion [START_REF] Sharples | Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia-reperfusion[END_REF]. As they did not detect any renal hemodynamic effects, and the EPO receptor is known to be expressed in renal tubular epithelial cells they also proposed a direct of EPO on proximal tubular epithelial cells and protects them from hypoxia-induced apoptosis. These findings are backed up by Huang et al. who found no effect on cortical and papillary perfusion in normal rats after acute EPO administration either [START_REF] Huang | Study of the actions of human recombinant erythropoietin on rat renal haemodynamics[END_REF]. Furthermore, in a model of unilateral ureteral obstruction leading to tubular apoptosis and tubulointerstitial fibrosis, EPO administration directly attenuated the apoptosis of tubular cells and the extent of fibrotic expansion [START_REF] Kitamura | Nonerythropoietic derivative of erythropoietin protects against tubulointerstitial injury in a unilateral ureteral obstruction model[END_REF].

All these data further support our findings, that EPO in vitro could directly reduce the rate of apoptosis of HEK293 cells after a hypoxic insult, however the exact molecular mechanisms has not been clearly elucidated until recently. SGK1 which is ubiquitously expressed and induced by stress and hormones (including gluco-and mineralocorticoids, insulin) [18] activates ion channels, carriers and also the Na+-K+-ATPase. It regulates the activity of enzymes and transcription factors (e.g., FKHRL1, NF-κB) and thus, participates mainly in the regulation of cell proliferation, apoptosis, ion transport and hormone release [START_REF] Lang | Patho)physiological significance of the serum-and glucocorticoid-inducible kinase isoforms[END_REF].

In our experiments, hypoxia induced mRNA expression of SGK1 which was paralleled by elevated protein levels and phosphorylation. The same tendency could be also detected in vivo during renal A c c e p t e d M a n u s c r i p t 13 I/R injury. mRNA expression reached its highest level 2 h after reperfusion, whereas protein levels peaked 24 h after reperfusion. Interestingly, marked phosphorylation of SGK1 was detected only 2 h after reperfusion.

Binding of EPO to its receptor leads to the phosphorylation of janus kinase 2 (JAK2) which subsequently activates multiple cascades recruiting the PI3-kinase, STAT5 and MAP Kinase [START_REF] Bahlmann | Erythropoietin and renoprotection[END_REF]. Furthermore, it induces the transcription of several hypoxia-related genes via the activation and nuclear translocation of the transcription factor NF-κB. EPO can cause increased expression of Bcl-2 and heat shock protein-70 [START_REF] Heemann | Lipopolysaccharide pretreatment protects from renal ischemia/reperfusion injury. possible connection to an interleukin-6dependent pathway[END_REF], and also induces hypoxia inducible factor-1 α synthesis in renal I/R injury [START_REF] Imamura | Erythropoietin protects the kidneys against ischemia reperfusion injury by activating hypoxia inducible factor-1alpha[END_REF].

Here, we demonstrate the novel finding that EPO induces the expression and phosphorylation of SGK1. Treatment of cells with recombinant EPO resulted in a significant up-regulation of SGK1 expression both under normoxic and hypoxic conditions. Furthermore, EPO treatment in vivo led to increased SGK1 mRNA and protein expression in the kidney tissue both in sham-operated and ischemized rats. The ratio of phosphorylated and un-phosphorylated SGK1 was also higher in the EPO treated groups both under in vitro hypoxic and in vivo ischemic conditions. In vivo, we not only measured the expression of SGK1, but also performed IF staining to show the localization of the kinase and its possible relevance to the site of EPO action in the kidney. We could demonstrate that SGK1 was present and upregulated in the tubular cells which are described to be the most sensitive to ischemic injury. This finding indicates that EPO not only regulates the expression, but also triggers the phosphorylation, thereby the activation of SGK1. Based on previous literary data, it is conceivable that activation of SGK1 by EPO might be mediated through the PI3-kinase signalling pathway.

To investigate if the EPO-induced induction and activation of SGK1 has a significant impact on the protective effect of EPO, we inhibited SGK1 expression in cells by plasmid-mediated siRNA. We showed that in those cells in which SGK1 expression was decreased, the anti-apoptotic effect of EPO was attenuated. There was an elevated release of LDH and a higher rate of apoptotic Annexin V-PI positive cells compared to EPO treated cells. All these data confirm the direct effect of EPO on SGK1 mediated protection against apoptosis. The elevation in serum BUN levels is abrogated by EPO (500 U/kg) administered intraperitoneally 24 h before induction of ischemia (*P<0.001 vs. I/R 24+veh). There was no difference in the BUN levels either between the sham-operated groups or between the animals 2 h after reperfusion.

  ) protects the kidneys from ischemia/reperfusion (I/R) injury; however, the exact signalling mechanisms are not fully understood. The serum and glucocorticoid-regulated kinase 1 (SGK1) is an anti-apoptotic protein kinase regulated through the phosphatidylinositol 3kinase (PI3-kinase) pathway by cellular stimuli, hormones and growth factors. The objective of the present study was to examine the role of SGK1 in the renoprotective effects of EPO in renal I/R injury.

  investigated the effects of EPO administration on SGK1 expression and activation during renal I/R injury both under in vitro and in vivo conditions. Furthermore, we inhibited SGK1 through small interfering technique to further study the direct impact of SGK1 on the EPO action.A c c e p t e d M a n u s c r i p t5 METHODS AND MATERIALSReagentsHuman recombinant EPO epoetin beta and placebo were obtained from Roche Hungary (Budaörs, Hungary).

A c c e p t e d M a n u s c r i p t 6 To

 6 knock down Sgk1 expression, HEK293 cells were transfected with Sgk1-specific RNAi constructs and were examined 48 h after transfection. The pSilencer-4.1-CMV-Neo negative control construct was transfected similarly and used as a negative control.

  oedemic, swelling and blebbing of tubular cells were detected, but no difference was demonstrated between the vehicle and EPO group. Parallel to the marked elevation in renal function parameters, 24 h after reperfusion, widespread degeneration of tubular architecture was detected. Both apoptosis and necrosis of tubular cells were observed. Apoptotic tubular cells had chromatin condensation, pyknosis, and karyorrhexis in the nucleus leading to cytoplasmic condensation triggering the cell to shrink and form numerous vacuoles within the cytoplasm. Necrosis of tubular cells were characterized by chromatin flocculation, swelling and degeneration of the entire cytoplasm and the mitochondrial matrix, blebbing of the plasma membrane, and eventual shedding of the cytoplasmic contents into the extracellular space. Necrosis and apoptosis of tubular cells result in tubular atrophy and loss of brush borders (Figure 4A.). 24 h after reperfusion renal sections of EPO treated animals demonstrated a reduction of the histological features of renal injury such as markedly less tubular necrosis and apoptosis resulting in significantly less atrophy and infiltration of leukocytes compared to vehicletreated rats (P<0.01) (Figure 4A-B.).

  and in 2h IR animals, whereas 24 h after reperfusion, no major difference was seen as compared to the vehicle-treated rats. A c c e p t e d M a n u s c r i p t 12 DISCUSSION

  Our data report a new signaling molecule involved in EPO cytoprotective actions, since the present study identified for the first time an important role of SGK1 in the anti-apoptotic and possibly, renoprotective effect of EPO during renal I/R injury. The EPO induced up-regulation of SGK1 was observed both under in vitro and in vivo circumstances. Moreover, in vitro, inhibition of SGK1 blocked the protective effect of EPO under hypoxia confirming a direct role of SGK1 in the EPO effect.

A c c e p t e d M a n u s c r i p t 14 These

 14 observations provide insights into a novel signaling mechanism by which EPO partly exerts its potent tissue protective actions. Given our results along with previous reports, the clinical use of EPO possibly leading to reduced cellular damage due to ischemic events should be considered, however, further investigations are needed to establish the feasibility and efficacy of EPO in clinical settings. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 16 TITLES AND LEGENDS TO FIGURESTable 1. Primer pairs for RT-PCR analysis Table 2. Serum levels of creatinine and blood urea nitrogen (BUN) +P<0.001 vs. Shams *P<0.001 vs. I/R 24+veh.

Figure 1 .

 1 Figure 1. Erythropoietin (EPO) prevents hypoxia-induced cell death. (A) Human embryonic kidney (HEK) cultures undergo cell death when exposed to hypoxia for 24 h. EPO significantly abrogated cell death measured by Lactate dehydrogenase (LDH) release in a dose-dependent manner (*P<0.01 vs. Hypo+veh). Inhibition of SGK1 expression by siRNA technique reduced the protective effect of EPO (*P<0.01 vs. Hypo+EPO). (B) HEK cells undergo apoptotic cell death after exposure to 24 hhypoxia detected by Annexin V-Propidium iodid FACS analysis. Annexin V-PI -positive cells located in Q2 were considered to be apoptotic. EPO significantly ameliorated apoptotic cell death after hypoxic injury (*P<0.01 vs. Hypo+EPO), whereas inhibition of SGK1 expression by siRNA abolished the anti-apoptotic effect of EPO (*P<0.01 vs. Hypo+EPO)

Figure 2 .

 2 Figure 2. Erythropoietin (EPO) leads to induction and activation of SGK1 both in normoxic and hypoxic Human embryonic kidney (HEK) cultures. (A) SGK1 mRNA expression was measured in normoxic and hypoxic cells with and without EPO treatment by RT-PCR. EPO treatment led to significant up-regulation of SGK1 mRNA expression both in normoxic and hypoxic cells (*P<0.01 vs. vehicle treated cells, respectively). (B) SGK1 protein expression was measured in normoxic and hypoxic cells with and without EPO treatment. EPO treatment led to significant up-regulation of SGK1 both in normoxic and hypoxic cells (*P<0.05 vs. vehicle treated cells, respectively). EPO caused also increased phosphorylation of SGK1 in hypoxic cells, but not in normoxic cells (*P<0.01 vs. vehicle treated cells). PCRs and immunoblots are shown are representative of three independent experiments.

Figure 3 .

 3 Figure 3. Effect of erythropoietin (EPO) on renal dysfunction caused by ischemia/reperfusion (I/R) injury. Serum creatinine levels (A) and blood urea nitrogen (BUN) levels (B) were measured as respective biochemical markers of glomerular dysfunction. Data represent mean ± SD. (A) Renal I/R led to significant elevation in serum creatinine levels 24 h after reperfusion (+P<0.001 vs. Shams).The increase in serum creatinine is abrogated by EPO (500 U/kg) administered intraperitoneally 24 h

Figure 4 .

 4 Figure 4. Erythropoietin (EPO) attenuates the morphologic changes of ischemia/reperfusion (I/R) injury. (A) PAS-stained kidney sections are shown from each animal group at a magnification of 200x. I/R led to widespread degeneration of tubular architecture, tubular dilation, swelling, necrosis and apoptosis, loss of brush borders especially 24 h after reperfusion. (B) Figures are representative of n = 8 for all groups. I/R led to morphologic changes in the kidney tissue (+P<0.001 vs. shams) EPO treatment ameliorated I/R caused histological changes 24 h after reperfusion (*P<0.001 vs. I/R 24h+veh).

Figure 5 .

 5 Figure 5. Erythropoietin (EPO) induced SGK1 mRNA and protein expression. (A) SGK1 mRNA expression was assessed by real-time PCR analysis. I/R caused elevated SGK1 mRNA expression 2 h after reperfusion (%P<0.05 vs. shams). EPO induced SGK1 mRNA expression in all animal groups (*P<0.01 vs. vehicle treated animals). 24 h after reperfusion, SGK1 mRNA expression was decreased compared to the 2 h-animals in the EPO treated animals (+P<0.01 vs. I/R 2h+EPO). (B) SGK1 protein expression was assessed by Western blot analysis. I/R caused elevated SGK1 protein expression 24 h after reperfusion (%P<0.05 vs. Shams). EPO induced SGK1 protein expression in sham-operated and 2 h I/R groups (*P<0.01 vs. vehicle treated animals). Two and 24 h after reperfusion, SGK1 protein expression was decreased compared to the sham-animals in the EPO treated groups (+P<0.01 vs. Sham+EPO). (C) Immunofluorescent staining of the kidneys showed marked SGK1 expression located in the tubuli. Up-regulated total SGK1 (green fluorescence) (left panel) and phosphorylated-SGK1 (SGK1-P, red fluorescence) were stained in EPO treated animals in the sham-operated group and 2h after ischemia in the tubular cells. 24 h after reperfusion, no marked difference was demonstrated between the vehicle and EPO treated animals. Cell nuclei were labeled with Hoechst (blue staining).

  Figure 1A.

  Figure 3B.

Table 1 .

 1

		Creatinine	BUN
		( M)	(mM)
	sham+veh	53.0 ± 2.6	4.6 ± 1.2
	sham+EPO	48.8 ± 4.1	3.7 ± 0.5
	IR 2h+veh	59.5 ± 31.8	4.6 ± 1.2
	IR 2h+EPO	86.8 ± 11.6	6.3 ± 1.2
	IR 24h+veh	353.2 ± 36.1 +	39.3 ± 0.4+
	IR 24h+EPO	261.6 ± 50.9*,+	24.9 ± 4.5*,+

Table 2 .

 2

Acknowledgements

This study was supported by the NNF 78846 (OTKA), TAMOP-4.2.2-08/1/KMR-2008-0004, OTKA T071730, ETT 435/2006 grants. A. F., V. M. and AJ. Sz. are recipients of the Bolyai Scholarship. The technical assistance of Mária Bernáth is gratefully acknowledged.


V Badireenath Konkimalla

Dr Thomas Efferth
email: efferth@uni-mainz.de

Inhibition of epidermal growth factor receptor over-expressing cancer cells by the aphorphine-type isoquinoline alkaloid, dicentrine

Keywords: microarrays, natural product, oncogenes, pharmacology, pharmacogenomics, pharmacognosy, signal transduction, systems biology

The extraordinary relevance of EGFR in tumour biology makes it an exquisite molecular target for tumor therapy. Despite considerable success with these EGFR tyrosine kinase inhibitors in cancer therapy, resistance against these chemical compounds develops owing to the selection of point-mutated variants of EGFR. Therefore, there is an urgent need for the identification of novel EGFR tyrosine kinase inhibitors for treating tumours caused by such EGFR mutants. We found a preferential cytotoxicity of dicentrine towards U87MG.EGFR transduced with a constitutively deletion-activated EGFR expression vector as compared to non-transduced wild-type U87MG cells. As determined by microarray-based mRNA expression profiling, this preferential cytotoxicity was accompanied with an activation of BRCA1-mediated DNA damage response, p53 signalling, G1/S and G2/M cell cycle regulation, and aryl hydrocarbon receptor pathways. The activation of these signalling routes might be explained by the fact that dicentrine intercalates DNA and induces DNA strand break by inhibition of DNA topoisomerases. The cell cycle might be arrested by dicentrineinduced DNA lesions.

Introduction

During the past three decades, the members of the epidermal growth factor receptor (EGFR) gene family evolved in cancer biology as important factors affecting prognosis of cancer patients [START_REF] Volm | Expression of resistance factors (P-glycoprotein, glutathione S-transferase-pi, and topoisomerase II) and their interrelationship to protooncogene products in renal cell carcinomas[END_REF][START_REF] Konkimalla | Diagnosis and therapy of oral squamous cell carcinoma[END_REF][START_REF] Hirsch | Predctive value of EGFR and HER2 overexpression in advanced non-small cell lung cancer[END_REF]. The EGFR family consists of four members (HER1-HER4), which of HER1 (EGFR, erbB1) and HER2 (erbB2, c-neu) are best characterized. They represent a class of glycoproteins, which serve as receptors for EGF and other ligands. They activate signal transduction pathways involved in basic biological processes, such as cell proliferation, apoptosis, metastasis, and angiogenesis. HER1 and HER2 are frequently over-expressed in tumours and are associated with unfavourable prognosis [START_REF] Lemaire | Differential expression profiling of head and neck squamous cell carcinoma (HNSCC)[END_REF].

EGFR molecules are inactive monomers that are activated by binding of specific ligands (e.g., EGF and TGF-). Activation is associated with either homodimerisation with a second EGFR molecule or with another HER member (i.e., HER2). The dimerisation stimulates the intrinsic tyrosine kinase activity of EGFR, which regulates specific signal transduction cascades. Constitutive EGFR activation as a consequence of mutations or gene amplification causes deregulated cellular processes, such as proliferation, invasion, angiogenesis, cell motility, cell adhesion, inhibition of apoptosis, and DNA synthesis. The kinase activity is also associated with autophosphorylation of five tyrosine residues in the Cterminal EGFR domain. Mutations affecting EGFR expression foster carcinogenesis.

The extraordinary relevance of EGFR in tumour biology makes it an exquisite molecular target for tumour therapy. Apart from therapeutic antibodies, several small molecules have been developed as EGFR inhibitors [START_REF] Oliveira | Molecular biology of epidermal growth factor receptor inhibition for cancer therapy[END_REF]. For example, gefitinib (Iressa ® , Astra Zeneca, DE, USA) and erlotinib (Tarveca ® , Genentech Inc., CA, USA) are used for the treatment of non-small cell lung cancer and other tumour types [START_REF] Astsaturov | Targeting epidermal growth factor receptor signaling in the treatment of head and neck cancer[END_REF]. Both compounds belong to the class of quinazolinamines and exhibit their inhibitory activity on EGFR tyrosine kinases by competing with ATP for the ATP-binding pocket.

Despite considerable success with these EGFR tyrosine kinase inhibitors,,resistance develops owing to the selection of point-mutated variants of EGFR [START_REF] Perea | Predictors of sensitivity and resistance to epidermal growth factor receptor inhibitors[END_REF]. Therefore, there is an urgent need for the identification of novel EGFR tyrosine kinase inhibitors for treating tumours caused by such EGFR mutants.

Dicentrine is an aporphine-type isoquinoline alkaloid isolated from several medicinal plants, some of which have been used in traditional medicines to treat cancers and other diseases [START_REF] Lalezari | Major alkaloids of Glaucium flavum Grantz, population Ghom[END_REF][START_REF] Shafiee | Alkaloids of papaver genus IX. Alkaloids of Glaucium vitellinum Boiss and Buhse, population Seerjan and Glaucium pulchrum Stapf, population Elika[END_REF][START_REF] Shafiee | Alkaloids of Glaucium flavum Grantz, populations Isfahan and Kazerun[END_REF][START_REF] Vecchietti | Alkaloids of Ocotea brachybotra[END_REF][START_REF] Vecchietti | New aporphine alkaloids of Ocotea minarum[END_REF][START_REF] Chen | Bioactive principles from the roots of Lindera megaphylla[END_REF][START_REF] Stévigny | Cytotoxic aporphine alkaloids from Cassytha filiformis[END_REF][15]. Dicentrine has been shown to exert cytotoxic activity towards cancer cells in vitro and in vivo [START_REF] Tzeng | Inhibitory effects of isoquinoline-type alkaloids on leukemic cell growth and macromolecule biosynthesis[END_REF][START_REF] Kondo | Suppression of tumor cell growth and mitogen response by aporphine alkaloids, dicentrine, glaucine, corydine, and apomorphine[END_REF][START_REF] Huang | Anti-tumor effects of d-dicentrine from the root of Lindera megaphylla[END_REF]. Dicentrine binds to and intercalate DNA and inhibits the catalytic activity of DNA topoisomerases [START_REF] Woo | Topoisomerase II inhibition by aporphine alkaloids[END_REF][START_REF] Stévigny | Cytotoxic and antitumor potentialities of aporphinoid alkaloids[END_REF]. Recently, we found that dicentrine binds with high A c c e p t e d M a n u s c r i p t 4 affinity to the erlotinib pharmacophore of EGFR by means of an in silico modelling and virtual screening approach [START_REF] Konkimalla | Diagnosis and therapy of oral squamous cell carcinoma[END_REF]. This indicates that EGFR and related signalling pathways may play a role in mediating dicentrine's cytotoxic activity towards cancer cells.

The aim of the present investigation was to investigate, whether dicentrine may exert preferential cytotoxicity towards cancer cells over-expressing EGFR. To this end, we determined the cytotoxicity of dicentrine in non-transduced human U87MG glioblastoma cells and U87MG cells transduced with expression vectors harbouring either wild-type EGFR (U87MG.wtEGFR), deletion-activated EGFR (U87MG.EGFR), or EGFR with a point mutation in the tyrosine kinase domain (U87MG.PK). Furthermore, transcriptome-wide mRNA expression profiling of these cell lines with and without dicentrine treatment has been performed to elucidate signalling pathways involved in the cytotoxicity of dicentrine in transduced and non-transduced cell lines. Interestingly, a connection of BRCA1-mediated DNA damage response and other DNA damage response pathways has been unravelled, indicating a relationship of EGFR and DNA damage signalling upon challenge of cancer cells with dicentrine.

Material and Methods

Drugs: Dicentrine was kindly provided by the Drug Synthesis and Chemistry Branch, Chemotherapeutic Agents Repository, National Cancer Institute, U.S.A. The chemical structure of dicentrine is shown in Supplementary Figure 1.

Cell lines:

The establishment of the parental human glioblastoma cell line, U87MG and its derivatives, which over-express exogenous wild-type EGFR (U87MG.wtEGFR-2N), tyrosine kinase-deficient EGFR (U87MG.DK-2N), or constitutively active EGFR with a genomic deletion of exons 2-7 (U87MG.EGFR) has been described elsewhere [START_REF] Huang | The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling[END_REF]. The cell lines were kindly provided by Dr. W.K. Cavenee (Ludwig Institute for Cancer Research, San Diego, CA, USA). Cell culture conditions of these cell lines were as described [START_REF] Nagane | A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis[END_REF].

XTT proliferation assay: The toxicity of compounds was determined by means of the Cell Proliferation Kit II (Roche Diagnostics, Mannheim, Germany). This test is based on the cleavage of the yellow 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5carboxanilide inner salt (XTT) by ubiquitous dehydrogenases leading to the formation of an orange formazan dye. [START_REF] Scudiero | Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity using human and other tumor cell lines[END_REF]. The amount of dye is commensurate to the number of metabolic active cells. Fresh stock solutions of each compound were prepared in DMSO at a A c c e p t e d M a n u s c r i p t 5 concentration of 100 mM. A dilution series ranging from 10 -9 M to 10 -3 M was prepared using DMEM medium to perform the XTT test. Cells were diluted to a final concentration of 1×10 5 cells/mL. One hundred micro-litres of the cell suspension were sowed into the wells of a 96well culture plate (Costar, Corning, USA). Marginal wells were filled with 100 L of pure medium, in order to minimize effects of evaporation. Besides, wells filled with medium were required to determine the background absorbance caused by non-metabolized XTT. A row of wells containing cells was left untreated, and another row of wells containing cells was treated with 1 L DMSO. This served as solvent control. The other rows of wells containing cells were supplemented with different concentrations of compound. Each concentration was tested in at least two independent plates containing different batches of cells.

After incubation with compounds at 37 °C, 5 % CO 2 in humidified atmosphere, XTT reagent was freshly prepared and added to each well as specified by the manufacturer: XTTlabelling reagent and electron-coupling reagent were mixed in a ratio of 50:1, and 50 L of this mixture were added to each well of the 96-well plate. The plates were incubated for about 3 h at 37 °C, 5 % CO 2 in humidified atmosphere and read out after incubation. Quantification of cell cytotoxicity was performed in an ELISA plate reader (Bio-Rad, München, Germany) at 490 nm with a reference wavelength of 655 nm. Absorbance values at both wavelengths were subtracted. The cytotoxic effect of the treatment was determined as percentage of viability and compared to untreated cells [START_REF] Konkimalla | Effect of artemisinins and other endoperoxides on nitric oxide-related signaling pathway in RAW 264.7 mouse macrophage cells[END_REF]. The toxicity of compounds was determined by means of the formula Implemented in the simple ligand binding module of Sigma plot software (version 10.0).

RNA isolation:

Total RNA of EGFR-transduced and non-transduced U87MG human glioblastoma cells was extracted from the test samples using RNeasy ® Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions to obtain highly pure RNA. Isolated total RNA was re-suspended in sample buffer provided by the manufacturer. The concentration and quality of total RNA was verified by electrophoresis using the total RNA Nano chip assay on an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany). Only samples with RNA index values greater than 8.5 were selected for expression profiling. RNA concentrations were determined using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). All of the RNA samples were stored at -80 °C until used for microarray analyses. Diego, CA, USA) were prepared according to Illumina's recommended sample labelling procedure based on a previously published protocol [START_REF] Konkimalla | Effect of artemisinins and other endoperoxides on nitric oxide-related signaling pathway in RAW 264.7 mouse macrophage cells[END_REF][START_REF] Eberwine | Analysis of gene expression in single live neurons[END_REF]. In brief, 250 ng total RNA were used for complementary DNA (cDNA) synthesis, followed by an amplification/labelling step (in vitro transcription) to synthesize biotin-labelled cRNA according to the MessageAmpII aRNA Amplification kit (Ambion, Inc., Austin, TX). Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was column purified according to TotalPrep RNA Amplification Kit, and eluted in 60 µL of water. Quality of cRNA was controlled using the RNA Nano Chip Assay on an Agilent 2100 Bioanalyzer and spectrophotometrically quantified (NanoDrop).

Hybridization was performed at 58 °C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 50 ng cRNA/L, unsealed in a wet chamber for 20 h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed twice in E1BC buffer (Illumina Inc.) at room temperature for 5 min. After blocking for 5 min in 4 mL of 1 % (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10 min incubation in 2 mL of 1 g/mL Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1 % blocking solution. After a final wash in E1BC, the arrays are dried and scanned.

Scanning and data analysis: Microarray scanning was done using a bead station array scanner, setting adjusted to a scaling factor of 1 and PMT settings at 430. Data extraction was done for all beads individually, and outliers are removed when >2.5 MAD (median absolute deviation). All remaining data points were used for the calculation of the mean average signal for a given probe, and standard deviation for each probe was calculated.

Data analysis was done by normalisation of the signals using the cubic spline algorithm after background subtraction, and differentially regulated genes are defined by calculating the standard deviation differences of a given probe in a one-by-one comparison of samples or groups. Pathway analysis was done by using the Ingenuity Pathways Analysis software (version 5.5) from Ingenuity Systems (Redwood City, CA, USA).

Real-time RT-PCR:

The experiment was done on the Roche LC480 using ABgene Mastermix ''CM 215-A" and Roche Universal Probe Library. Oligos were designed using Roche Total reaction volume was 10 L. We used 400 nM final oligo concentration each, 100 nM final probe concentration and 6.3 ng RNA (transcribed in cDNA using Superscript) per reaction. We used transcribed cDNA from RNA obtained from untreated cells or cells treated with dicentrine at the 50 % inhibition dose (IC 50 ). No DNAse digest was done. Genes identified by microarray analyses were exemplarily analysed for validation by means of realtime RT-PCR (CDKN1A, E2F1). The housekeeping genes, HPRT1, G6PD, ALAS1, and HMBS, served as reference. The primer sequences are shown in Table 1.

All measurements are done in triplicates to calculate mean values and standard deviations. To calculate normalised mRNA expression values, we measured the sample's crossing point (expressed as a cycle number), the efficiency of the reaction, and the number of cycles completed to determine how much the DNA concentration must have increased for each sample by the end of the amplification. The analysis used these calculations to compare samples and to generate ratios as indicated by the manufacturer (Roche, Penzberg, Germany).

The final ratio resulting from the calibrator-normalised relative quantification was a function of PCR efficiency and of the determined crossing points. The ''concentration ratio" was determined as ratio of the target and the reference genes in the specific sample. To calculate the change of expression between untreated (calibrator) and dicentrine-treated samples, the concentration ratios of the sample was divided by the concentration ratio of the calibrator. This value is the ''normalised ratio". I . While the majority of differentially regulated genes were different between the different cell lines, 8 genes were commonly regulated in U87MG and U87MG.wtEGFR-2N cells, 10 genes in U87MG and U87MG.EGFR, and 37 genes in U87MG and U87MG.DK-2N cells.

Results

Cytotoxicity

The non-intersected or the uniquely regulated genes for dicentrine were considered as those molecular players that are responsible as mechanism of action for this compound. The variability in the number of such uniquely regulated genes in different EGFR-transduced cell lines suggested the diverse pathways activated by individual compounds to elicit their cellular effects.

In order to see, whether dicentrine elicits its activity through one or several pathways, we investigated which signalling pathways were affected by the action of dicentrine in U87MG.EGFR cells. For this reason, we analyzed all genes included in the Illumina biochip by means of the Ingenuity Pathway Analysis software. Out of >80 canonical pathways, five were significantly regulated upon dicentrine treatment (P<0.05). The 10 functional groups of genes with the lowest P-values are shown in Figure 3. Of them, genes involved in BRCA1mediated DNA damage response were the most prominent. Based on this result, we then asked which genes were affected upon dicentrine treatment. The genes associated with BRCA1-mediated DNA damage response are shown in Figure 4.

In addition to BRCA1-mediated DNA damage response, other pathways, which appeared in dicentrine-treated U87MG.EGFR but not in non-transduced U87MG cells, were the aryl hydrocarbon receptor (AHR) signalling route, the G1/S as well as the G2/M cell cycle checkpoint regulation routes, and the p53 signalling pathway (Figure 3). All of these pathways are related to DNA damage.

Validation of microarray data by real-time RT-PCR:

The results of DNA microarray hybridisations were exemplarily validated by real-time RT-PCR. For this purpose, two genes were chosen. One gene (CDKN1A) was found to be up-regulated, another one (E2F1) was down-regulated upon dicentrine treatment in microarray analyses. As can be seen in Table II, corresponding up-or down-regulation of these genes after dicentrine treatment was also observed by real-time RT-PCR. III, the IC 50 values for dicentrine significantly correlated with the cell doubling times of the cell lines. The mRNA expression of CCNE1 and CCNF and the proliferation-related genes, PAK4 and VEGFB were also significantly associated with the IC 50 values for dicentrine. Other cyclins did not correlate with dicentrine. Furthermore, we analyzed apoptosis-related genes and found significant relationships between the expression of CASP-7, NFKB1, PDCD4, and DAPK1 to the IC 50 values for dicentrine (Table III). An inverse correlation with borderline significance (P=0.07) was observed for the NFB-inhibitor, IKB. Other caspases as well as bcl-2 family members (bcl-2, bax, bcl-x, bxl-xL, and bad) did not correlate with response of tumor cell lines towards dicentrine.

Discussion

In the present investigation, we found a preferential cytotoxicity of dicentrine towards U87MG.EGFR transduced with a constitutively deletion-activated EGFR expression vector as compared to non-transduced wild-type U87MG cells. A possible explanation for preferential cytotoxicity of dicentrine towards EGFR-overexpressing cells is that dicentrine binds to EGFR receptor. Hence, EGFR-expressing cells are killed, while non-EGFRexpressing cells survive. A mean energy of docking of -8.1 kcal/mole and a pKi of 4.25 nM have been calculated in silico by 3D molecular docking of dicentrine to the three-dimensional structure of the binding site of the EGFR tyrosine kinase, erlotinib, at the ATP-binding site of EGFR [START_REF] Konkimalla | Diagnosis and therapy of oral squamous cell carcinoma[END_REF]. Based on this result, we have started the present investigation. The preferential cytotoxicity of dicentrine in EGFR-transfected cells found by XTT assays support the view that dicentrine inhibits EGFR-expressing cells by binding to EGFR.

The preferential activity of dicentrine towards U87MG.EGFR cells was indeed EGFR-specific, since we and others found that this cell line exhibited resistance rather than sensitivity towards other DNA damaging agents such as cisplatin or artesunate [START_REF] Nagane | Drug resistance of human glioblastoma cells conferred by tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-XL and caspase-3 like proteases[END_REF][START_REF] Efferth | Molecular modes of action of artesunate in tumor cell lines[END_REF]. Anticancer with other modes of action (paclitaxel, vincristine, homoharringtonine, L-alanosine) were also less active in U87MG.EGFR cells [START_REF] Efferth | Identification of gene expression profiles predicting tumor cell response to L-alanosine[END_REF][START_REF] Efferth | Molecular modes of action of cephalotaxine and homoharringtonine from the coniferous tree Cephalotaxus hainanensis in human tumor cell lines[END_REF]. Comparable data have been resported by other authors in other EGFR-expressing cells [START_REF] Wosikowski | Altered gene expression in drug-resistant human breast cancer cells[END_REF].

This preferential cytotoxicity was accompanied with an activation of BRCA1mediated DNA damage response, p53 signalling, G1/S and G2/M cell cycle regulation, and aryl hydrocarbon receptor pathways. The activation of these signalling routes might be explained by the fact that dicentrine intercalates DNA and induces DNA strand break by inhibition of DNA topoisomerases. The cell cycle might be arrested by dicentrine-induced DNA lesions.

Indeed, an association of dicentrine with DNA damage has previously been reported.

Dicentrine intercalates DNA and inhibits DNA topoisomerases I and II [START_REF] Woo | Topoisomerase II inhibition by aporphine alkaloids[END_REF][START_REF] Zhou | Isolation and biochemical characterization of a new topoisomerase I inhibitor from Ocotea leucoxylon[END_REF]. In this respect, dicentrine may be compared with classical anti-cancer drugs such as anthracyclines.

The interrelationship of EGFR and BCRA1 has not well been investigated [START_REF] Marks | EGFR mutant lung adenocarcinomas in patients with germline BRCA mutations[END_REF].

A c c e p t e d M a n u s c r i p t 10

There are hints that EGFR inhibits the catalytic and DNA-cleaving activity of DNA topoisomerase II [START_REF] Markovits | Inhibition of DNA topoisomerases I and II and induction of apoptosis by erbstatin and tyrphostin derivatives[END_REF][START_REF] Allen | Epidermal growth factor regulates topoisomerase II activity and drug sensitivity in human KB cells[END_REF]. Furthermore, inhibition of EGFR by small molecule inhibitors such as gefitinib resulted in synergistic interaction with the DNA topoisomerase inhibitor, irinotecan [START_REF] Koizumi | Synergistic interaction between the EGFR tyrosine kinase inhibitor gefitinib ("Iressa") and the DNA topoisomerase I inhibitor CPT-11 (irinotecan) in human colorectal cancer cells[END_REF]. The sensitivity of BRCA1-and BRCA2-deficient etoposide-induced DNA double strand breaks was mediated by DNA topoisomerase II [START_REF] Treszezamsky | BRCA1-and BRCA2-deficient cells are sensitive to etoposide-induced DNA double-strand breaks via topoisomerase II[END_REF], indicating a mechanistic relationship between both proteins. Although incompletely understood at present, it is worth speculating that EGFR cross-talks with DNA toposiomerases and BRCA1-mediated DNA damage response pathways. This cross-talk may be important for intercalation and DNA topoisomerase-mediated DNA damage of dicentrine.

Apart from the BRCA1-mediated DNA damage response pathway, other signalling routes were also differentially regulated between U87MG.EGFR and parental U87MG cells.

Among them were two signalling routes for arrest in G1/S or G2/M cell cycle phases. There is a plethora of investigations pointing to the eminent role of cell cycle arrest for cancer therapy.

Cancer cells arresting in one of these phases can resist the detrimental effects of cancer chemotherapy [START_REF] Efferth | Apoptosis and resistance to daunorubicin in human leukemic cells[END_REF][START_REF] Johansson | Cancer therapy: targeting cell cycle regulators[END_REF]. On the other side, compounds capable to release arrested cells such as caffeine lead to a re-sensitization of cancer cells towards chemotherapy [START_REF] Efferth | Expression of apoptosis-related oncoproteins and modulation of apoptosis by caffeine in human leukemic cells[END_REF]. The fact that dicentrine differentially affects signalling routes for G1/S or G2/M arrest in U87MG and U87MG.EGFR cells indicates that dicentrine interferes with cell cycle of cancer cells, possibly via the induction of DNA lesions.

The p53 signalling pathway was also activated in U87MG.EGFR cells upon challenge with dicentrine. It is well known that p53 is activated by DNA lesions, either spontaneously occurring due to the tumour suppressor function of p53 or upon chemotherapeutic treatment. In both cases, p53 can mediate cell cycle arrest, DNA repair, and/or apoptosis. We conclude that the results obtained by microarray hybridisations may point to dicentrine-induced DNA lesions, as already been found by others [START_REF] Woo | Topoisomerase II inhibition by aporphine alkaloids[END_REF][START_REF] Zhou | Isolation and biochemical characterization of a new topoisomerase I inhibitor from Ocotea leucoxylon[END_REF][START_REF] Tadaki | Clastogenicity of aporphine alkaloids in vitro[END_REF]. This point of view is also conceivable with the finding that the aryl hydrocarbon receptor (AhR) signalling pathway was involved in the action of dicentrine. This signalling pathway is of enormous relevance in carcinogenesis, but also for resistance towards CDKN1A (p21, Cip1) appeared as one of the differentially expressed genes upon dicentrine treatment in our microarray experiments. This gene encodes a cyclin-dependent kinase inhibitor, which blocks cell cycle progression at the G1 phase by interacting with cyclin E. Interestingly, CCNE1 (cyclin E1) was one of the genes which significantly correlated with response of the N.C.I. cell line panel to dicentrine. This indicates that cell cycle progression may be inhibited by CDKN1A-cyclin E interaction upon dicentrineinducesd DNA damage.

It should be taken into account that dicentrine as many other natural compounds alike targets many cellular structures in the organism and is not cancer-specific in its bioactivity spectrum. Besides its cytotoxic activity, dicentrine inhibited trypanosomes [15]. Dicentrine is a known inhibitor of -adrenoreceptors [START_REF] Teng | Dicentrine, a natural vascular alpha 1-adrenoceptor antagonist, isolated from Lindera megaphylla[END_REF][START_REF] Yu | Haemodynamic effects of dicentrine, a novel alpha 1-adrenoceptor antagonist: comparison with prazosin in spontaneously hypertensive and normotensive Wistar-Kyoto rats[END_REF][START_REF] Yu | Effects of dicentrine, a novel alpha 1-adrenoceptor antagonist, on human hyperplastic prostates[END_REF][START_REF] Mustafa | Dicentrine is preferentially antagonistic to rat aortic than splenic alpha 1-adrenoreceptor stimulation[END_REF]. It reduced hypertension and hyperlipidemia, two of the major risk factors for cardiovascular disease [START_REF] Yu | Effects of dicentrine on haemodynamic, plasma lipid, lipoprotein level and vascular reactivity in hyperlipidaemic rats[END_REF]. Electrophysiological examination of dicentrine on the conduction system of rabbit hearts showed that dicentrine exerted anti-arrhythmic action [START_REF] Young | The electrophysiological effects of dicentrine on the conduction system of rabbit heart[END_REF]. Dicentrine had beneficial effects on the rigidity of head and body circulation. It improved tube distensibility and wave transmission time [START_REF] Chang | Effects of dicentrine on the mechanical properties of systemic arterial trees in dogs[END_REF].

Furthermore, dicentrine inhibited sodium and potassium channels [START_REF] Su | Dicentrine, an alpha-adrenoceptor antagonist with sodium and potassium channel blocking activities[END_REF] and thromboxane formation during platelet aggregation [START_REF] Yu | Dicentrine, a novel antiplatelet agent inhibiting thromboxane formation and increasing the cyclic AMP level of rabbit platelets[END_REF]. It also exerted chromosomal damaging clastrogenic effects [START_REF] Tadaki | Clastogenicity of aporphine alkaloids in vitro[END_REF].

If dicentrine will be further developed for cancer treatment, derivatives with preferential activity towards cancer cells and fewer side effects on other tissues and cellular targets should be generated. Dicentrine represents an example of a natural product with multitarget activity. It is valuable as lead compound for the derivatisation of second generation compounds with improved pharmacological features.

In conclusion, the data of the present investigation imply that otherwise drug-resistant tumours with constitutive activation and/or over-expression of EGFR may be preferentially susceptible towards dicentrine due to inhibition of EGFR and regulation of EGFR-related downstream signalling pathways. Genes were selected for real-time RT-PCR analysis from the list of differentially expressed genes.

A c c e p t e d M a n u s c r i p t 6

 6 Probe labelling and Illumina Sentrix BeadChip array hybridization: Biotin-labelled cRNA samples for hybridisation on Illumina Mouse Sentrix-8 BeadChip arrays (Illumina Inc., San

  A c c e p t e d M a n u s c r i p t 7 ProbeFinder Webservice (https://qpcr2.probefinder.com/organism.jsp) and were synthesised by MWG. Two versions were designed per gene. All variants analysed span an intron to prevent cross reactions with possible genomic DNA contaminations.

  of dicentrine: Transduced and non-transduced U87MG glioblastoma cell lines were treated with different concentrations of dicentrine and subjected to the XTT assay. The dose response curves are shown in Figure 1 and the 50 % inhibition concentrations (IC 50 ) calculated from the dose response curves in Table I. The IC 50 value for dicentrine was 4.13to 9.09-fold higher in non-transduced U87MG cells (43.0 ± 4.44 µM) as compared to the three transduced cell lines. U87MG.EGFR cells transduced with a deletion-activated EGFR expression vector were most sensitive towards dicentrine (4.73 ± 0.294 µM). The IC 50 value of U87MG.DK-2N cells carrying a point mutation in the tyrosine kinase domain of EGFR was 8.69 ± 0.09 µM, and the IC 50 for U87MG.wtEGFR-2N was 10.4 ± 0.48 µM.

A c c e p t e d M a n u s c r i p t 8

 8 Differential gene regulation upon dicentrine treatment: Next, we were interested, whether specific mRNA expression profiles underlie the different IC 50 values. Therefore, we performed microarray-based mRNA hybridisations. An overview of the number of up-or down-regulated genes upon treatment of the different U87MG cell lines with dicentrine is given in Figure 2 and Supplementary Table

A c c e p t e d M a n u s c r i p t 9

 9 Cell cycle and apoptosis parameters:To analyze the role of cell cycle and apoptosis for response of tumor cells towards dicentrine in a general context, we correlated the IC 50 values for dicentrine of the tumor cell line panel of the Developmental Therapeutics Program of the National Cancer Institute, U.S.A., with the cell doubling times of the cell lines and the microarray-based mRNA expression of genes involved in cell cycle and apoptosis (http://dtp.nci.nih.gov). As shown in Table

  chemotherapy. Induction of the mdr1 gene, which is well-known to induce multidrug resistance, requires the AhR nuclear translocator [42]. Down-regulation of AhR repressor conferred resistance to apoptotic signals [43] and 17--estradiol-induced AhR overexpression with the characteristic phenotype of increased proliferation and resistance to apoptosis [44]. Given the general role of AhR for drug resistance, it is worth hypothesising A c c e p t e d M a n u s c r i p t 11 that AhR may also be involved in response of tumour cells to dicentrine, as indicated by the results of the present investigation.

Figure 1 :

 1 Figure legends:

Figure 2 :

 2 Figure 2: Genes uniquely and commonly regulated in transduced and non-transduced U87MG cell lines. Venn diagram representation was chosen to compare overlaps of differentially expressed genes after dicentrine treatment.

Figure 3 :

 3 Figure 3: Comparison of canonical pathway analysis for dicentrine-treated non-transduced U87MG and U87MG.EGFR transduced with a deletion-activated EGFR expression vector. Each bar represents the ratio of the number of genes that are regulated in a particular pathway upon treatment with dicentrine (IC 50 ) for 72 h. Only the top 10 canonical pathways are shown of U87MG (dark grey bars) and U87MG.EGFR (light grey bars) as analyzed by the Ingenuity Pathway Analysis software.

Figure 4 :Supplementary Figure 1 :

 41 Figure 4: BRCA1-mediated DNA damage response pathway. This pathway was identified as top canonical pathway differentially regulated by dicentrine in U87MG and U87MG.EGFR cells (see Figure 4). Genes in red colour indicate up-regulation, whereas genes in green colour indicate down-regulation of mRNA expression upon dicentrine treatment.

13 *

 13 Mean SEM of two independent experiments with each four-fold determination ** IC 50 of the corresponding cell line divided by IC 50 of U87MG

  a) Homo sapiens cyclin-dependent kinase inhibitor 1a (p21, Cip1) (CDKN1A), transcript variant 2 b) Homo sapiens E2F transcription factor (E2F1) * average hybridization signal (±SEM). ** Conc ratio (± Conc ratio STD).

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 2 Figure 2

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 3 Figure 3

Figure 4

 4 Figure 4

Table II :

 II Validation of mRNA microarray results

							Dicentrine-	Fold
	Gene Cell line	Method	Untreated cells	treated cells	change
	CDKN1A	a)		U87MG	microarray 10,604.6 ± 310.6* 20,263.1 ± 596.2	1.7
					RT-PCR	0.137 ± 0.0832**	0.533 ± 0.0465	3.89
				U87MG.EGFR microarray 7,846.0 ± 197.1	21,471.0 ± 472.6	2.7
					RT-PCR	0.025 ± 0.0013	0.268 ± 0.0094	10.74
	E2F1	b)	U87MG	microarray 330.0 ± 14.8	150.0 ± 5.1	-2.2
					RT-PCR	0.003 ± 0.0012	0.001 ± 0.0004	-3.21
				U87MG.EGFR microarray 505.3 ± 18.1	135.2 ± 5.8	-3.7
					RT-PCR	0.001 ± 0.0001	0.000 ± 0.000	-3.77

Acknowledgement: This work has been supported by a grant of the Dietmar Hopp-Foundation (St. Leon-Rot, Germany).

*Graphical Abstract