Keywords: Bcl-2 proteins, pancreatic beta cells, antioxidative defense status, pro-inflammatory cytokines, apoptosis

Pro-inflammatory cytokines are key mediators in the selective and progressive destruction of insulinproducing beta cells during type 1 diabetes development. However, the mechanisms of cytokineinduced beta cell apoptosis are not fully understood.

This study demonstrates that pro-inflammatory cytokines strongly modified the expression of the antiapoptotic protein Bcl-2 and the pro-apoptotic BH3-only proteins Bad, Bim, and Bid in primary rat islets and insulin-producing RINm5F cells. Overexpression of mitochondrially located catalase (MitoCatalase) specifically increased basal Bcl-2 and decreased basal Bax expression, suppressed cytokine-mediated reduction of Bcl-2, and thereby prevented the release of cytochrome c, Smac/DIABLO and the activation of caspase-9 and -3. Thus, cytokine-mediated decrease of Bcl-2 expression and the sequentially changed Bax/Bcl-2 ratio are responsible for the release of proapoptotic mitochondrial factors, activation of caspase-9, and ultimately caspase-3.

These results indicate that activation of the intrinsic/mitochondrial apoptosis pathway is essential for cytokine-induced beta cell death and the mitochondrial generation of reactive oxygen species, in particular mitochondrial hydrogen peroxide, differentially regulates the Bax/Bcl-2 ratio.

Introduction

Pancreatic beta cell apoptosis plays a crucial role in the pathogenesis of Type 1 Diabetes mellitus (T1DM) [START_REF] Atkinson | ADA Outstanding Scientific Achievement Lecture 2004. Thirty years of investigating the autoimmune basis for type 1 diabetes: why can't we prevent or reverse this disease?[END_REF][START_REF] Eizirik | A choice of death--the signal-transduction of immune-mediated beta-cell apoptosis[END_REF][START_REF] Kim | Recent progress in research on beta-cell apoptosis by cytokines[END_REF]. This process is initiated by two main pathways: the "extrinsic" or death receptor and the "intrinsic" or mitochondrial apoptosis pathway [START_REF] Huerta | Screening and detection of apoptosis[END_REF][START_REF] Millan | Apoptosis-inducing factor and colon cancer[END_REF]. The intrinsic death pathway involves loss of mitochondrial homeostasis, particularly of the outer mitochondrial membrane integrity, and subsequently the release of mitochondrial pro-apoptotic factors including cytochrome c.

Once released, cytochrome c promotes the assembly of the so-called apoptosome, consisting of cytochrome c, apoptosis-protease activating factor-1 (Apaf-1), ATP/dATP, and pro-caspase-9. Upon formation of this complex, activated caspase-9 triggers the processing and activation of effector caspases, which ultimately culminates in apoptotic cell death [START_REF] Danial | Cell death: critical control points[END_REF][START_REF] Kroemer | Mitochondrial membrane permeabilization in cell death[END_REF][START_REF] Susnow | Bcl-2 family proteins as regulators of oxidative stress[END_REF]. Permeablization of the mitochondrial outer membrane and the consequent release of cytochrome c are tightly regulated by a group of proteins known as the Bcl-2 protein family [START_REF] Adams | The Bcl-2 protein family: arbiters of cell survival[END_REF]. This family is composed of pro-and anti-apoptotic proteins that share up to four conserved regions known as Bcl-2 homology (BH) domains. Anti-apoptotic members such as Bcl-2 and Bcl-X L contain all four subtypes of BH domains and promote cell survival by inhibiting the function of the pro-apoptotic Bcl-2 family members. The pro-apoptotic members can be further subdivided into two subfamilies: multidomain proteins (e.g. Bax and Bak), containing BH1-3, or the BH3-only proteins (e.g. Bad, Bim, Bid, Noxa, and Puma) that contain only the BH3 domain critical for the induction of apoptosis [START_REF] Danial | Cell death: critical control points[END_REF][START_REF] Strasser | The role of BH3-only proteins in the immune system[END_REF]. Although the precise mechanisms by which these proteins regulate cell death are not fully understood, it appears that the interaction and the ratio between anti-and pro-apoptotic Bcl-2 family proteins determine the fate of cells exposed to apoptotic stimuli [START_REF] Adams | The Bcl-2 protein family: arbiters of cell survival[END_REF][START_REF] Wong | Bcl-2 family proteins: the sentinels of the mitochondrial apoptosis pathway[END_REF].

Studies with non-insulin-secreting cells suggested that reactive oxygen species (ROS), in particular hydrogen peroxide (H 2 O 2 ), decrease the expression of Bcl-2 and increase that of proapoptotic proteins, e.g. Bax, thereby changing the Bax/Bcl-2 ratio which determines the susceptibility of cells to apoptosis [START_REF] Korsmeyer | Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell death[END_REF][START_REF] Oltvai | Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[END_REF][START_REF] Raisova | The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis[END_REF]. In addition, the pro-inflammatory cytokines interleukin 1 (IL-1), tumor necrosis factor  (TNF-), and interferon 

5

(IFN-) down-regulated the expression of the anti-apoptotic protein Bcl-2 in pancreatic islets [START_REF] Piro | Bovine islets are less susceptible than human islets to damage by human cytokines[END_REF][START_REF] Trincavelli | Upregulation of mitochondrial peripheral benzodiazepine receptor expression by cytokine-induced damage of human pancreatic islets[END_REF][START_REF] Van De Casteele | Specific expression of Bax-omega in pancreatic beta-cells is down-regulated by cytokines before the onset of apoptosis[END_REF]; whereas, the overexpression of Bcl-2 in beta cells protected against cytokine-induced cell death [START_REF] Barbu | Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential (Deltapsi(m)) in pancreatic RINm5F cells: prevention by Bcl-2[END_REF][START_REF] Rabinovitch | Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction[END_REF][START_REF] Tran | Discrete and complementary mechanisms of protection of beta-cells against cytokine-induced and oxidative damage achieved by bcl-2 overexpression and a cytokine selection strategy[END_REF].Furthermore, inhibition of Bax mitochondrial translocation by the potential Bax suppressor protein V5 (also known as Ku70) resulted in a higher resistance against apoptotic stimuli in HEK293T and HeLa cells [START_REF] Sawada | Ku70 suppresses the apoptotic translocation of Bax to mitochondria[END_REF] and also against cytokine-induced toxicity in human pancreatic beta cells [START_REF] Grunnet | Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells[END_REF] The relation between ROS and the pro-inflammatory beta cell toxic cytokines IL-1, TNF-, and IFN- in their influence on the expression of anti-(Bcl-2 and Bcl-X L ) and pro-apoptotic (Bax, Bad, Bim, and Bid) proteins is still unknown. Therefore, the expression pattern of these proteins and its relation to the toxicity of pro-inflammatory cytokines, as well as its dependence on the antioxidative defense status, was analyzed in primary rat islets and in insulin-producing RINm5F cells overexpressing antioxidative enzymes. Materials and Methods

Tissue culture of RINm5F cells

Insulin-producing RINm5F tissue culture cells were cultured as described earlier in RPMI-1640 medium supplemented with 10 mM glucose, 10% (v/v) fetal calf serum (FCS), penicillin, and streptomycin in a humidified atmosphere at 37 °C and 5% CO 2 [START_REF] Lortz | Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes[END_REF]. The RINm5F cells overexpressing catalase in the cytosol (CytoCatalase) or in mitochondria (MitoCatalase) were generated as described before in detail [START_REF] Gurgul | Mitochondrial catalase overexpression protects insulin-producing cells against toxicity of reactive oxygen species and proinflammatory cytokines[END_REF]. Briefly, for overexpression of catalase in cytosol or mitochondria, human catalase cDNA was subcloned into the pcDNA3 (cytosolic overexpression) or pCMV/myc/mito-plasmid (mitochondrial overexpression) and the stabile clones were selected through resistance against G418. Cells transfected with the pCMV/myc/mito vector lacking insert were used as control cells. Expression of antioxidative enzymes in the cells was analyzed by catalase enzyme activity measurement [START_REF] Gurgul | Mitochondrial catalase overexpression protects insulin-producing cells against toxicity of reactive oxygen species and proinflammatory cytokines[END_REF]. The catalase enzyme activities were as follows in the control cells with an empty vector and in cells overexpressing

CytoCatalase or MitoCatalase, respectively (in U/mg total protein): control cells, 31 ± 0.2;

CytoCatalase, 346 ± 2.8; MitoCatalase, 594 ± 18.8.

Rat islet isolation

Pancreatic islets were isolated from 250-300 g adult male Lewis rats by collagenase digestion, separated by Ficoll gradient, and handpicked under a stereo microscope [START_REF] Tiedge | Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells[END_REF]. Isolated islets were cultured overnight in RPMI-1640 medium supplemented with 5 mM glucose, 10% FCS, penicillin, and streptomycin at 37 °C in a humidified atmosphere of 5% CO 2 .

Exposure to cytokines

Control and transfected RINm5F cells were seeded at different concentrations depending on the further experimentation and allowed to attach for a period of 24 h. For islet incubations, 100-150 uniformly sized handpicked, precultured islets were used. Cells were exposed to 600 U/ml human IL-1 or a combination of cytokines (cytokine mixture) consisting of 60 U/ml IL-1, 185 U/ml human TNF-, and 14 U/ml IFN- (PromoCell, Heidelberg, Germany) for a subsequent 24 h incubation. The indicated cytokine concentrations were selected based upon the results of previous studies (Lortz et al., [START_REF] Nerup | On the pathogenesis of IDDM[END_REF][START_REF] Nielsen | Gene expression profiles during beta cell maturation and after IL-1beta exposure reveal important roles of Pdx-1 and Nkx6.1 for IL-1beta sensitivity[END_REF]. IL-1 is the most potent beta cell toxic cytokine and strongly contributes to beta cell death by activation of the NFB and MAPK transduction pathways.

TNF- and IFN- alone are significantly weaker in this respect; however in synergy with IL-1 they potentiate its deleterious action through induction of additional signaling pathways [START_REF] Eizirik | A choice of death--the signal-transduction of immune-mediated beta-cell apoptosis[END_REF]. Therefore, the used cytokine mixture contained 10-fold less IL-1 than the IL-1 solution alone, in order to achieve a comparable toxicity [START_REF] Souza | Interaction between proinflammatory and anti-inflammatory cytokines in insulin-producing cells[END_REF].

Real-time Quantitative RT-PCR

Total RNA from insulin-producing RINm5F cells was isolated using the Chomczynski protocol, while the RNA from incubated islets was isolated with NucleoSpin RNA/Protein columns (Macherey-Nagel, Düren, Germany). RNA was quantified and analyzed by the Experion automated electrophoresis system (BioRad Laboratories, Hercules, CA, USA). For cDNA synthesis, random hexamers were used to prime the reaction of the RevertAid H -M-MuLV reverse transcriptase (Fermentas, St. Leon-Rot, Germany). QuantiTect SYBR Green technology (Qiagen, Hilden, Germany), which uses a fluorescent dye that binds only double-stranded DNA, was employed. The reactions were performed using the DNA Engine Opticon Sequence Detection System (BioRad Laboratories, Hercules, CA, USA). Samples were first denatured at 94 °C for 3 min followed by 40

PCR cycles comprised of a melting step at 94 °C for 30 s, an annealing step at 60 °C for 30 s, and an extension step at 72 °C for 30 s. Primers for qRT-PCR were used at an annealing temperature of 60 °C and gave an amplicon of 79-216 base pairs (Table 1). The optimal parameters for the PCR reactions were empirically defined and the purity and specificity of the amplified PCR product in each experiment was verified by melting curves. All analyzed transcripts showed Ct-values, which were at least 10 Ct-values lower than the blank values. Each PCR amplification was performed in triplicate.

Data are expressed as relative gene expression after normalization to the beta actin housekeeping gene using the Qgene96 and LineRegPCR software.

Tissue fractionation

Trypsinized RINm5F cells were collected by centrifugation at 700 g for 5 min, washed twice Fractions were obtained through differential centrifugation. Cell homogenates were centrifuged at 500 g for 10 min at 4 °C to discard nuclei and unbroken cells. The mitochondrial fraction was obtained through centrifugation for 10 min at 10 000 g at 4 °C. The supernatant was then centrifuged for another 60 min at 100 000 g at 4 °C, to separate the microsomal and the cytoplasmic fractions [START_REF] Lenzen | Transamination of neutral amino acids and 2-keto acids in pancreatic B-cell mitochondria[END_REF].

Western blot analyses

RINm5F whole cell extracts (for detection of Bcl-2 family members) or subcellular fractions (for cytochrome c and Smac/DIABLO detection) were sonified in ice-cold PBS on ice for 15 s at 60 W with a Braun-Sonic 125 sonifier. Protein content was determined by the BCA assay (Pierce, Rockford, IL, USA) or the Bradford assay. Whole islet cell proteins were obtained through a combined RNA/protein isolation procedure (NucleoSpin RNA/Protein columns, Macherey-Nagel, Düren, Germany) and prepared for Western blot analyses in accordance with the manufacturer's protocol.

RINm5F cell protein (20-30 g) or the protein fraction of 100-150 islets per lane were separated by a 12.5% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Nonspecific binding sites of the membranes were blocked with 5% non-fat dry milk for either 1 h at room temperature or overnight at 4 °C. Then, the membranes were incubated with specific primary antibodies for either 4 h at room temperature or overnight at 4 °C. The following antibodies were used: Bcl-2 (sc-7382, diluted 1:500), Bax (sc-7480, 1:250), Smac/DIABLO (sc-12683, 1:250), cytochrome c (sc-7159, 1:250), and actin (sc-1615, 1:250) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Bcl-X L (610211, 1:1000) (BD Biosciences, Franklin Lakes, NJ, USA); phospho-Ser 136 Bad, (44-524, 1:500) (Biosource, Camarillo, CA, USA); Bid (1:500), (R&D Systems, Minneapolis, MN, USA); and Bim (ap-330, 1:1000) (Stressgen, Victoria, BC, Canada). The excess of primary antibody was removed by three washes with wash buffer (PBS, 0.1% Tween 20, 0.1% BSA). Subsequently, the membranes were incubated with peroxidase-labeled secondary antibodies at a dilution of 1:20 000 at room temperature for 1 h. The 9

protein bands were visualized by chemiluminescence using the ECL detection system (Amersham Bioscience, Freiburg, Germany). The intensity of the bands was quantified with respect to -actin bands through densitometry with the Gel-Pro Analyzer 6.0 program (Media Cybernetics, Silver Spring, MD, USA).

Flow cytometric quantification of caspase-9 and caspase-3 activity

Caspase-9 and -3 activities were determined with the CaspGLOW staining kits (Promocell, Heidelberg, Germany). Control and overexpressing RINm5F cells were seeded at a density of 1 x 10 6 cells per well and allowed to attach for a period of 24 h at 37 °C before incubation with the indicated cytokines. After 24 h exposure to IL-1 or the cytokine mixture, the cells were trypsinized and collected by centrifugation at 700 g for 5 min. Cell pellets were resuspended in 1 ml medium and 300 µl of each sample were transferred to Eppendorf tubes. According to the manufacturer's protocol, FITC-LEHD-FMK (caspase-9) or Red-DEVD-FMK (caspase-3) was added to these cells and incubated for 45 min at 37 °C followed by washing twice with wash buffer. The cells were resuspended in 1 ml of washing buffer and 20 000 cells of each sample were analyzed by flow cytometry (CyFlow ML, Partec, Münster, Germany) using the FL-1 channel (488 nm/527 nm) for caspase-9 or FL-2 channel (488 nm/575 nm) for caspase-3.

Data analysis

The data are expressed as means ± SEM. Statistical analyses were performed by ANOVA followed by Dunnett's test for multiple comparisons using the Prism analysis program (Graphpad, San Diego, CA, USA).

Expression of Bcl-2-related proteins in primary rat islets and insulin-producing cells

Mitochondrial integrity and stability are dependent upon the expression and interaction of proand anti-apoptotic Bcl-2 proteins. Therefore, the expression of anti-and pro-apoptotic Bcl-2 family members in primary rat islets and insulin-producing RINm5F tissue culture cells was characterized by qRT-PCR. Gene expression of the anti-apoptotic (Bcl-2 and Bcl-X L ) and pro-apoptotic (Bax, Bad, Bim, and Bid) Bcl-2 proteins could be detected in primary rat islets and RINm5F cells. The proapoptotic protein Bax showed the highest gene expression level of all investigated Bcl-2 proteins and its expression level was 5-fold higher in primary rat islets and 8.5-fold higher in RINm5F cells, compared with the anti-apoptotic protein Bcl-2. Moreover, Bcl-X L and Bad revealed a 2-fold higher gene expression than Bcl-2 in primary islets and 4-fold higher in RINm5F cells; whereas the relative gene expression of the BH3-only proteins Bim and Bid was comparable with that of Bcl-2 (Fig. 1a andb). Overall, the expression pattern of all investigated Bcl-2-related proteins was comparable in primary rat islets and RINm5F cells.

Effects of pro-inflammatory cytokines on the expression of Bcl-2-related proteins in primary rat islets and insulin-producing cells

Exposure of primary islets and insulin producing cells to IL-1 alone resulted in alteration of multiple genes, cellular function impairment, and apoptosis. In combination with TNF- and /or IFN- these phenomena could be synergistically potentiated [START_REF] Eizirik | A choice of death--the signal-transduction of immune-mediated beta-cell apoptosis[END_REF]. To characterize the influence of these beta cell toxic cytokines on the gene expression pattern of Bcl-2 related proteins, primary rat islets and RINm5F cells were incubated with IL-1 alone or with a proinflammatory cytokine mixture consisting of IL-1, TNF-, and IFN-. A 24 h exposure of primary rat islets to IL-1 or the cytokine mixture caused a significant reduction of Bcl-2 (P < 0.001) and Bad (IL-1: P < 0.05; cytokine mixture: P < 0.01) gene expression. In contrast, the gene expression of Bcl-X L (P < 0.01) and Bid (IL-1: P < 0.01; cytokine mixture: P < 0.001) was significantly increased under the same treatment conditions, whereas that of Bim was only slightly but not significantly 11 upregulated. The gene expression of Bax remained unchanged after cytokine treatment (Fig. 2a). The changes in expression patterns, induced by cytokines in primary rat islets, were also observed in RINm5F tissue culture cells (Fig. 2c). Again, expression of Bcl-2 was significantly reduced by IL-1 alone and the cytokine mixture (P < 0.001). The Bad gene expression was only marginally affected by exposure to IL-1 alone, while the cytokine mixture induced a significant 25% decrease of Bad (P < 0.05). The Bcl-X L gene expression was significantly increased by 38% only after cytokine mixture treatment (P < 0.01). However, in contrast to primary rat islets, the Bim gene expression in RINm5F culture cells was significantly induced by IL-1 and the cytokine mixture (IL-1: P < 0.01; cytokine mixture: P < 0.001), whereas the Bid gene expression was similarly enhanced as observed in primary islets (IL-1: P < 0.01; cytokine mixture: P < 0.001, Fig. 2c). As in primary rat islets, the gene expression of Bax was not influenced by IL-1 or the cytokine mixture.

To verify the gene expression data obtained by qRT-PCR, Western blot analyses were performed. As shown in Fig. 2b andd, the protein expression of all investigated Bcl-2-related proteins in rat islets and RINm5F cells showed a very high correlation with the gene expression data.

Nevertheless, the observed cytokine-induced increase of Bcl-X L gene expression in primary islets could not be verified on protein level, whereas the Bim protein expression was significantly induced after treatment with IL-1 alone or the cytokine mixture (P < 0.05). Since the BH3-only protein Bad remains under control conditions in an inactive phosphorylated state and is sequestered in the cytosol by a 14-3-3 molecule [START_REF] Zha | Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L)[END_REF], we examined not only the total Bad protein expression, but also the cytokine-mediated dephosphorylation of Bad at Ser 136 . In rat islets and in RINm5F cells, IL-1 alone and the cytokine mixture induced Bad Ser 136 dephosphorylation (Fig. 2b andd), without affecting total Bad expression (data not shown).

Taken together, these findings demonstrate that pro-inflammatory cytokines modify the expression of most of the investigated Bcl-2-related proteins, indicating a complex regulation and a possible participation of these proteins in cytokine-induced beta cell toxicity, both in primary rat islets and in insulin-producing RINm5F cells. Among the investigated Bcl-2 proteins, Bcl-2, Bim, and Bid expression revealed the highest alterations in response to pro-inflammatory cytokines. 12

Effects of antioxidative enzymes on the expression of Bcl-2-related proteins in insulinproducing cells

In beta cells and insulin-producing cell lines the H 2 O 2 inactivating capacity in comparison to other tissues is extremely low [START_REF] Lenzen | Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues[END_REF][START_REF] Tiedge | Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells[END_REF]. Catalase overexpression in insulin-producing RINm5F cells resulted in a higher resistance against different chemical ROS donors.

However, stable overexpression of catalase in the mitochondrial compartment protected these cells more efficiently against cytokine-induced toxicity than the overexpression of catalase in the cytosolic compartment [START_REF] Gurgul | Mitochondrial catalase overexpression protects insulin-producing cells against toxicity of reactive oxygen species and proinflammatory cytokines[END_REF][START_REF] Lortz | Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes[END_REF]. Therefore, these RINm5F cells stably overexpressing the H 2 O 2 detoxifying enzyme catalase in the cytosol (CytoCatalase) or mitochondria (MitoCatalase) were used to examine the influence of antioxidative enzymes on the expression of Bcl-2-related proteins.

RINm5F cells overexpressing CytoCatalase showed significantly lower Bcl-2 gene expression (P <0.01), whereas the expression level of Bax was only slightly increased (Bcl-2: 32% and Bax:

141% of the control cells). In accordance with this, protein expression analysis also revealed a significantly lower Bcl-2 (P <0.001) and up-regulated Bax expression (Bcl-2: 10% and Bax: 135% of the control cells). Thus, the calculated Bax to Bcl-2 ratio of the CytoCatalase cells increased 4.4-fold at the mRNA level and 13.5-fold at the protein level, compared with control cells (Fig. 3a andb).

In contrast, Bcl-2 gene expression in MitoCatalase cells was significantly higher and that of Bax significantly lower than in control cells (P <0.05, Bcl-2: 204% and Bax: 48% of the control cells).

Western blot analysis also revealed elevated Bcl-2 and significantly decreased Bax (P <0.05) protein levels (Bcl-2: 206% and Bax: 46% of the control cells, Fig. 3b). Based on this observation, the ratio of Bax to Bcl-2 decreased 4.3-fold at the mRNA level and 3.3-fold at the protein level, compared with control cells. The expression levels of Bcl-X L , Bad, Bim, and Bid did not differ significantly between control cells and cells overexpressing CytoCatalase or MitoCatalase (data not shown). Therefore, only the expression of Bcl-2 and Bax in response to cytokines was characterized in these catalase overexpressing RINm5F cells. In contrast, Bax gene expression was not affected by cytokine treatment in control cells and cells overexpressing CytoCatalase or MitoCatalase (Fig. 5a). At the protein level, similar expression profiles of Bax were observed in control and CytoCatalase cells. Conversely, Bax protein expression was surprisingly decreased in CytoCatalase cells after incubation with the cytokine mixture (48% of untreated cells, P <0.01, Fig. 5b). In MitoCatalase cells Bax protein expression was only moderately but not significantly increased after cytokine treatment (IL-1 128% and cytokine mixture 151% of untreated cells, Fig. 5b). (cytokine mixture: P <0.05, Fig. 6a). On the other hand, the overexpression of MitoCatalase fully prevented this cytokine-induced cytochrome c release into the cytosol (Fig. 6a). In addition to the cytochrome c release data, the subcellular distribution of the intramitochondrial pro-apoptotic protein, second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO), a protein that antagonizes the inhibitory effects of the inhibitor of apoptosis protein (IAP) family, in particular of X-linked IAP, was investigated (Fig. 6b). After cytokine treatment, Smac/DIABLO was released into the cytosol in control (P <0.01) and CytoCatalase overexpressing cells (IL-1: P <0.05; cytokine mixture: P <0.01). The extent of cytokine-mediated Smac/DIABLO release into the cytosol was comparable to the release of cytochrome c. However, in MitoCatalase overexpressing cells, no significant release of Smac/DIABLO from the mitochondria into the cytosol could be detected (Fig. 6b).

Cytokine-induced release of cytochrome c from mitochondria and activation of caspase-9 and -3 in insulin-producing cells

Since cytosolic cytochrome c is part of the apoptosome, executing the processing of procaspase-9 to activated caspase-9, the activity of this caspase was quantified in these cell clones.

Control cells treated with the indicated cytokines resulted in a significant activation of caspase-9 (IL-1: 190%, cytokine mixture: 208% compared with untreated cells, P <0.01). This cytokine-mediated activation of caspase-9 was also observed in CytoCatalase overexpressing cells (IL-1: 235% (P <0.05), cytokine mixture: 271% (P <0.01) compared with untreated cells). Nonetheless, the overexpression of MitoCatalase suppressed this cytokine-mediated caspase-9 activation nearly completely (IL-1: 127%, cytokine mixture: 120% compared with untreated cells, Fig. 7a). In addition, the cytokine-induced activation of the executioner caspase-3 was assessed as a characteristic hallmark of apoptotic cell death. As shown in Fig. 7b the exposure of control cells to IL-1 alone or the cytokine mixture resulted in a significant activation of caspase-3 (IL-1: 213%, cytokine mixture:

202% compared with untreated cells, P <0.05). CytoCatalase overexpressing cells showed also 16

Discussion

In the present study we show that the pro-apoptotic protein Bax is expressed at significantly higher levels than the anti-apoptotic protein Bcl-2, in both primary rat islets and insulin-producing Previously, it was suggested that Bcl-2 family members are key regulators of apoptosis and that the stoichiometry of pro-and anti-apoptotic molecules decides the fate of the cell [START_REF] Youle | The BCL-2 protein family: opposing activities that mediate cell death[END_REF]. High Bax activity, induced through various apoptotic stimuli, has been considered a critical event in the process of beta cell apoptosis [START_REF] Grunnet | Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells[END_REF][START_REF] Tonnesen | Inhibition of nuclear factor-kappaB or Bax prevents endoplasmic reticulum stress-but not nitric oxide-mediated apoptosis in INS-1E cells[END_REF].

Interestingly, in the present study the pro-apoptotic protein Bax showed the highest expression level of all investigated Bcl-2-related proteins in primary rat islets and insulin-producing RINm5F cells;

whereas its antagonist Bcl-2 showed the lowest expression. This imbalance between pro-and antiapoptotic proteins contributes to the particular vulnerability of insulin-producing cells to apoptosis.

These results are consistent with previous studies performed on isolated human islets, showing that Bax is hyperexpressed compared with Bcl-2; it was postulated that this high Bax expression was caused by the stressful islet isolation procedure [START_REF] Thomas | Proapoptotic Bax is hyperexpressed in isolated human islets compared with antiapoptotic Bcl-2[END_REF]. However, our findings in both insulin-producing tissue culture cells and isolated primary rat islets strongly suggest that this imbalance is a characteristic of pancreatic beta cells and can be further enhanced by pro-apoptotic stimuli.

Exposure of pancreatic beta cells to pro-inflammatory cytokines has been previously shown to modulate the expression of Bcl-2 family members. In particular, Bcl-2 expression was down-regulated in mouse and human pancreatic islets upon cytokine exposure [START_REF] Piro | Bovine islets are less susceptible than human islets to damage by human cytokines[END_REF][START_REF] Trincavelli | Upregulation of mitochondrial peripheral benzodiazepine receptor expression by cytokine-induced damage of human pancreatic islets[END_REF], 17

2002; [START_REF] Van De Casteele | Specific expression of Bax-omega in pancreatic beta-cells is down-regulated by cytokines before the onset of apoptosis[END_REF]; whereas, overexpression of Bcl-2 protected beta cells against cytokine-mediated cell death [START_REF] Barbu | Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential (Deltapsi(m)) in pancreatic RINm5F cells: prevention by Bcl-2[END_REF][START_REF] Rabinovitch | Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction[END_REF][START_REF] Tran | Discrete and complementary mechanisms of protection of beta-cells against cytokine-induced and oxidative damage achieved by bcl-2 overexpression and a cytokine selection strategy[END_REF]. In the present study with primary islets and insulin-producing RINm5F cells, Bcl-2 expression was also significantly decreased upon exposure to IL-1 and a cytokine mixture, whereas the expression of Bax remained unchanged. This cytokine-mediated modulation of Bcl-2 expression resulted in a markedly elevated Bax/Bcl-2 ratio, which may determine the apoptotic potential of primary islets and RINm5F cells under stress conditions.

Pro-inflammatory cytokines induced expression of the BH3-only proteins Bim and Bid and activated Bad, through Ser 136 dephosphorylation, in primary rat islets and insulin-producing RINm5F cells. Bim gene and protein expression were shown to be inducible especially by ER stress inducing compounds, such as thapsigargin. Interestingly, the Bim induction was more profound on the protein than on the mRNA level, possibly due to post-translational modifications resulting in higher protein stability [START_REF] Puthalakath | ER stress triggers apoptosis by activating BH3-only protein Bim[END_REF]. In agrement with this study, in primary rat islets and RINm5F cells, Bim protein expression was more pronounced than gene expression in response to cytokines, suggesting that ER stress is also triggered by the cytokines, as previously shown [START_REF] Eizirik | The role for endoplasmic reticulum stress in diabetes mellitus[END_REF].

Bid, proteolytically cleaved by death receptor-activated caspase-8, and calcineurin-mediated Bad Ser 136 dephosphorylation have been considered to be required for beta cell apoptosis [START_REF] Grunnet | Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells[END_REF][START_REF] Mckenzie | Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells[END_REF]. These BH3-only proteins antagonize not only the anti-apoptotic activity of Bcl-2 and Bcl-X L , but Bid and Bim can also bind Bax with high affinity, thereby inducing its conformational change and translocation to mitochondria [START_REF] Strasser | The role of BH3-only proteins in the immune system[END_REF][START_REF] Youle | The BCL-2 protein family: opposing activities that mediate cell death[END_REF].

This results in an amplification of the caspase cascade through engagement of the intrinsic apoptosis pathway. However, neither CytoCatalase nor MitoCatalase had significant effects on basal or cytokine-induced Bim, Bid, and Bad expression, indicating that the detoxification of H 2 O 2 in both cellular compartments is not involved in BH3-only protein regulation. Despite cytokine-mediated activation of BH3-only proteins in all cell clones, the MitoCatalase cells prevented the cytokineinduced beta cell apoptosis, most likely through decreasing Bax and increasing Bcl-2 expression.

The expression of Bcl-2-related proteins is precisely regulated on the transcriptional and post- 18 cells suggested that ROS, in particular H 2 O 2 , decrease the expression of Bcl-2 and increase that of proapoptotic proteins, e.g. Bax [START_REF] Korsmeyer | Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell death[END_REF][START_REF] Oltvai | Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death[END_REF][START_REF] Raisova | The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis[END_REF]. On the other hand, the reduction of oxidative stress by ROS scavengers and H 2 O 2 inactivating enzymes could prevent down-regulation of Bcl-2 expression, resulting in higher resistance to apoptotic stimuli in other cell types [START_REF] Faucher | Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells[END_REF][START_REF] Hildeman | Control of Bcl-2 expression by reactive oxygen species[END_REF][START_REF] Pugazhenthi | Oxidative stress-mediated downregulation of bcl-2 promoter in hippocampal neurons[END_REF][START_REF] Wang | Peroxide is a key mediator of Bcl-2 down-regulation and apoptosis induction by cisplatin in human lung cancer cells[END_REF]. While beta cells and insulin-producing cell lines show a moderate even though overall appropriate expression of the superoxide-radical-inactivating enzyme in mitochondria, the H 2 O 2 inactivating capacity is extremely low compared with other tissues [START_REF] Lenzen | Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues[END_REF][START_REF] Tiedge | Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells[END_REF][START_REF] Lortz | Importance of mitochondrial superoxide dismutase expression in insulin-producing cells for the toxicity of reactive oxygen species and proinflammatory cytokines[END_REF]. This imbalance is further accentuated by the cytokine-induced expression of MnSOD leading to an aggravation of an already limited antioxidative defense capacity. Thus, these cells highly efficiently dismutate superoxide radicals, but they are prone to accumulate H 2 O 2 and exhibit a high susceptibility to ROS, especially under inflammatory conditions [START_REF] Lenzen | Oxidative stress: the vulnerable beta-cell[END_REF].

Quantification of intracellular H 2 O 2 formation in insulin-producing RINm5F cells exposed to proinflammatory cytokines by using the specific H 2 O 2 sensor HyPer revealed a significant accumulation of H 2 O 2 within the mitochondria [START_REF] Lenzen | Nitrooxidative stress as a mechanism of cytokine-mediated toxicity in insulin-producing cells[END_REF]. ROS dissipation through overexpression of catalase resulted in an increased resistance against chemical ROS donors and also cytokine-induced toxicity of these cells. Importantly, catalase overexpression did not affect typical cytokine signaling events such as NFB activation and iNOS induction [START_REF] Gurgul | Mitochondrial catalase overexpression protects insulin-producing cells against toxicity of reactive oxygen species and proinflammatory cytokines[END_REF][START_REF] Lortz | Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes[END_REF]. ROS formation, induced by pro-inflammatory cytokines, primarily targets mitochondria, causing beta cell damage and death [START_REF] Lenzen | Oxidative stress: the vulnerable beta-cell[END_REF].

Overexpression of MitoCatalase prevented mitochondrial H 2 O 2 accumulation [START_REF] Lenzen | Nitrooxidative stress as a mechanism of cytokine-mediated toxicity in insulin-producing cells[END_REF] and completely abolished the cytokine-mediated down-regulation of Bcl-2, which resulted in increased basal Bcl-2 and decreased Bax expression. These results are consistent with previous reports showing that overexpression of GPX1 and treatment with the synthetic antioxidant MnTBAP, also mitochondrially located, increased Bcl-2 expression and protected against ROS-mediated Bcl-2 downregulation [START_REF] Faucher | Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells[END_REF][START_REF] Orrenius | Mitochondrial oxidative stress: implications for cell death[END_REF][START_REF] Wang | Peroxide is a key mediator of Bcl-2 down-regulation and apoptosis induction by cisplatin in human lung cancer cells[END_REF]. 19

caspase-9 and activation of caspase-3. On the other hand, the overexpression of cytosolically located catalase failed to prevent cytokine-induced Bcl-2 down-regulation, to reverse the imbalanced Bax/Bcl-2 ratio, and to suppress the release of mitochondrial apoptotic factors resulting in the caspase cascade activation. However, the cytokine-induced caspase-3 activation was slightly attenuated in these cells, suggesting a possible involvement of cytosolic H 2 O 2 in cytokine-mediated beta cell death. This is consistent with the earlier observed partial protection against pro-inflammatory cytokine toxicity in CytoCatalase overexpressing cells [START_REF] Gurgul | Mitochondrial catalase overexpression protects insulin-producing cells against toxicity of reactive oxygen species and proinflammatory cytokines[END_REF][START_REF] Lortz | Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes[END_REF]. These findings indicate that H 2 O 2 of mitochondrial origin induced by pro-inflammatory cytokines is the major oxidative species responsible for Bcl-2 down-regulation and activation of downstream apoptotic events in insulinproducing cells.

Pro-inflammatory cytokines, especially TNF-, are known to increase mitochondrial ROS production in many cell types [START_REF] Yang | Proinflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells[END_REF]. Thus, mitochondrial ROS are crucial for full toxicity of cytokines. The precise mechanism involving mitochondrial ROS signaling in cytokineinduced apoptosis is still unknown, but it has been postulated that the direct damaging effects of mitochondrial ROS include cardiolipin peroxidation, facilitation of mitochondrial permeability transition, and inhibition of mitochondrial metabolism [START_REF] Orrenius | Reactive oxygen species in mitochondria-mediated cell death[END_REF]. Besides these direct damaging effects of mitochondrial ROS, the effects of MitoCatalase overexpression on Bcl-2 gene and protein expression suggest a crosstalk between mitochondrial ROS generation and Bcl-2 expression.

The Bcl-2 expression is predominantly regulated by the phosphorylated transcription factor, cAMP response element-binding protein (CREB), which is up-regulated by the PI-3K/Akt pathway and down-regulated by Ca 2+ dependent phosphatases, including calcineurin [START_REF] Pugazhenthi | Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein[END_REF][START_REF] See | Oxidative stress induces neuronal death by recruiting a protease and phosphatase-gated mechanism[END_REF]. Bcl-2 promoter activity has been impaired through cytokine-induced downregulation of phosphorylated CREB in MIN6 cells, whereas the overexpression of wild-type CREB protected against cytokine-induced toxicity in these cells [START_REF] Jambal | Cytokine-mediated down-regulation of the transcription factor cAMP-response element-binding protein in pancreatic beta-cells[END_REF]. Indeed, we also observed a cytokine-induced reduction of CREB dependent Bcl-2 promoter activity in RINm5F cells, which was diminished through overexpression of MitoCatalase (unpublished observation). Thus, it is likely that CREB is the major link between mitochondrial H 2 O 2 and the decrease in Bcl-2 gene expression in beta cells. Alternatively, as recently reported, Bcl-2 promoter activity can be modulated

20

by the p53 pathway [START_REF] Bourgarel-Rey | Transcriptional down-regulation of Bcl-2 by vinorelbine: identification of a novel binding site of p53 on Bcl-2 promoter[END_REF], which can be initiated by mitochondrial ROS [START_REF] Niizuma | Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival[END_REF][START_REF] Soberanes | p53 mediates particulate matter-induced alveolar epithelial cell mitochondria-regulated apoptosis[END_REF]. with IL-1 (600 units/ml), or with a cytokine mixture (60 units/ml IL-1, 185 units/ml TNF-, and 14 units/ml IFN-). Total RNA was isolated after 24 h incubation, qRT-PCR was performed with a Bcl-2 specific primer set, and the relative expression levels were normalized to the housekeeping gene - with IL-1 (600 units/ml), or with a cytokine mixture (60 units/ml IL-1, 185 units/ml TNF-, and 14 units/ml IFN-). Total RNA was isolated after 24 h incubation, qRT-PCR was performed with a Bax specific primer set, and the relative expression levels were normalized to the housekeeping gene - after 24 h incubation with IL-1 (600 units/ml) or with a cytokine mixture (60 units/ml IL-1, 185 units/ml TNF-, and 14 units/ml IFN-). Thereafter, the cells were lysed and fractionated into

  cold PBS, and centrifuged at 700 g for 5 min at 4 °C. The cell pellets were resuspended in 500 l of ice-cold H-medium (70 mM sucrose, 210 mM mannitol, 20 mM HEPES, 150 mM KCl, and 0.5 mM EGTA, pH 7.4) supplemented with Complete protease inhibitor cocktail (Roche Diagnostics, Manheim, Germany), and homogenized on ice with 25-30 strokes in a Potter-Elvehjem homogenizer.

  s c r i p t I. Mehmeti et al.

  A c c e p t e d M a n u s c r i p t I. Mehmeti et al.

  of pro-inflammatory cytokines on the expression of Bcl-2 and Bax in insulin-producing cells overexpressing antioxidative enzymesNext, we examined the effects of pro-inflammatory cytokines on the expression of Bcl-2 and Bax in insulin-producing RINm5F cells stably overexpressing CytoCatalase or MitoCatalase. Control and CytoCatalase cells, incubated for 24 h with IL-1 alone or in combination with TNF- and IFN-, resulted in a significant reduction of Bcl-2 gene expression (control cells: IL-1 52% (P <0.001) and cytokine mixture 50% (P <0.001) of untreated cells; CytoCatalase cells: IL-1 67% (P <0.01) and cytokine mixture 55% (P <0.001) of untreated cells). On the other hand, MitoCatalase cells showed no significant reduction of Bcl-2 gene expression after cytokine treatment (Fig.4a). These gene expression results were confirmed by Western blot analyses. As shown in Fig.4b, Bcl-2 protein expression in control cells was reduced to a similar extent as the gene expression in response to cytokines (IL-1: 59% and cytokine mixture: 51% of untreated cells, P <0.001). However, the reduction of Bcl-2 protein expression in cells overexpressing CytoCatalase was markedly greater after cytokine incubation (IL-1: 28% and cytokine mixture: 23% of untreated cells, P <0.001); whereas, Bcl-2 protein expression remained unchanged in cells overexpressing MitoCatalase (IL-1 115% and cytokine mixture 109% of untreated cells). Thus, MitoCatalase overexpression completely prevented cytokine-induced Bcl-2 down-regulation.

  influence of the variant Bax/Bcl-2 ratios detected in the control, MitoCatalase, and CytoCatalase cells on mitochondrial stability after incubation with IL-1 or the cytokine mixture, the cytokine-induced release of cytochrome c from mitochondria into the cytosol was studied by Western blot analysis. A 24 h treatment of control and CytoCatalase overexpressing cells with IL-1 or the cytokine mixture induced an augmented cytochrome c release into the cytosol

  after cytokine treatment. However, only cells treated with the cytokine mixture exhibited a statistically significant activation of caspase-3 (IL-1: 158%, cytokine mixture: 179% (P <0.05) compared with untreated cells). In accordance with the prevention of caspase-9 activation in MitoCatalase cells, cytokine-induced caspase-3 activation was also completely abolished by MitoCatalase (IL-1: 98%, cytokine mixture: 104% compared with untreated cells, Fig.7b).Taken together, these results on cytochrome c release and caspase-9 activation indicate mitochondria are a major target of pro-inflammatory cytokines finally leading to caspase-3 activation and induction of apoptosis. Prevention of this intrinsic apoptosis pathway by MitoCatalase overexpression suggests mitochondrially generated H 2 O 2 is involved in this process.

  RINm5F cells. Exposure to cytotoxic cytokines strongly increased the Bax/Bcl-2 ratio in favor of the pro-apoptotic Bax protein. However, overexpression of mitochondrially located H 2 O 2 detoxifying catalase (MitoCatalase) decreased the Bax expression and increased expression of Bcl-2 in insulinproducing cells. In addition, cells overexpressing MitoCatalase prevented the cytokine-induced release of the mitochondrial pro-apoptotic factors cytochrome c and Smac/DIABLO and consequently the activation of caspase-9 and the downstream executioner caspase-3. Taken together, these data indicate that ROS-mediated loss of mitochondrial integrity and an increased Bax/Bcl-2 ratio are crucial for cytokine-induced beta cell apoptosis.

  translational level and modified by various stimuli including ROS. Studies with non-insulin-secreting A c c e p t e d M a n u s c r i p t I. Mehmeti et al.

Furthermore,

  MitoCatalase overexpression successfully suppressed cytokine-mediated release of cytochrome c and consequently the activation of the caspase cascade, as shown by the inhibition of A c c e p t e d M a n u s c r i p t I. Mehmeti et al.

  Further experiments are needed to distinguish the impact of CREB, p53, and possibly other transcription factors on the Bcl-2 promoter level and to determine the influence of mitochondrial ROS on their activity in beta cells.Overall, the present results show that ROS of mitochondrial origin are major mediators in the regulation of cytokine-induced beta cell apoptosis. Moreover, we showed that MitoCatalase overexpression can compensate for the cytokine-mediated increase of the Bax/Bcl-2 ratio and prevent cytochrome c release and the activation of caspase-9 and -3. The efficient detoxification of mitochondrial H 2 O 2 protects against cytokine-mediated beta cell apoptosis and provides insights into the action of mitochondrial ROS and their redox signaling during beta cell apoptosis.

Fig. 1 .

 1 Figure legends

Fig. 2 .

 2 Fig. 2. Effects of pro-inflammatory cytokines on gene and protein expression of Bcl-2-relatedproteins in primary rat islets and insulin-producing RINm5F cells. (a) and (c) Isolated rat islets

Fig. 3 .

 3 Fig. 3. Effects of cytoplasmic and mitochondrial catalase overexpression on the expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax in insulin-producing RINm5F cells. (a) Gene expression of Bcl-2 and Bax in RINm5F control cells and cells overexpressing catalase in the cytoplasm (CytoCatalase) or in mitochondria (MitoCatalase) under control conditions. Total RNA was isolated 24 h after cell seeding, qRT-PCR was performed with gene specific primer sets, and the relative expression levels were normalized to the housekeeping gene -actin. (b) Protein

Fig. 4 .

 4 Fig. 4. Effects of pro-inflammatory cytokines on Bcl-2 gene and protein expression in insulinproducing RINm5F control cells and cells overexpressing CytoCatalase or MitoCatalase. (a) Gene expression of Bcl-2 in RINm5F control cells and cells overexpressing catalase in the cytoplasm (CytoCatalase) or in mitochondria (MitoCatalase) after incubation under control conditions,

Fig. 5 .

 5 Fig. 5. Effects of pro-inflammatory cytokines on Bax gene and protein expression in insulinproducing RINm5F control cells and cells overexpressing CytoCatalase or MitoCatalase. (a) Gene expression of Bax in RINm5F control cells and cells overexpressing catalase in the cytoplasm (CytoCatalase) or in mitochondria (MitoCatalase) after incubation under control conditions,

  actin. (b) Cell clones were treated as in (a) and Bax protein expression was quantified by Western blotA c c e p t e d M a n u s c r i p t I. Mehmeti et al. 27 analysis and normalized to the housekeeping protein -actin. The Bax gene or protein expression in each cell clone under control conditions was set as 100%. A representative blot is shown for Bax protein expression. Depicted values are means ± SEM, n = 6 mRNA expression or n = 4 protein expression. **P < 0.01 vs. untreated cells.

Fig. 6 .

 6 Fig. 6. Cytokine-induced release of cytochrome c and Smac/DIABLO from mitochondria of insulin-producing RINm5F control cells and cells overexpressing CytoCatalase or MitoCatalase. Western blot analysis of cytochrome c (a) and Smac/DIABLO (b) release of RINm5F control cells and cells overexpressing catalase in the cytoplasm (CytoCatalase) or in mitochondria (MitoCatalase)

Fig. 7 .

 7 Fig. 7. Effects of pro-inflammatory cytokines on caspase-9 and caspase-3 activation in insulinproducing RINm5F control cells and cells overexpressing CytoCatalase or MitoCatalase. Quantification caspase-9 (a) and caspase-3 (b) activation of RINm5F control cells and cells overexpressing catalase in the cytoplasm (CytoCatalase) or in mitochondria (MitoCatalase) after 24 h

  Figure 1

  Figure 3

  Figure 5

Figure 7

 7 Figure 7

Acknowledgments 1.1.1.1.1 We are grateful to Martin Wirth for his skillful technical assistance. This work was supported by the European Union (STREP Savebeta, contract no 036903 in the Framework Program 6 [FP6] of the European Community).

Conflict of interest

The authors declare that they have no conflict of interest.

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


Daniela Roell

Aria Baniahmad

The natural compounds atraric acid and N-butylbenzenesulfonamide as antagonists of the human androgen receptor and inhibitors of prostate cancer cell growth

Keywords: Prunus africanus, Prunus africana, natural compound, antihormone, antagonist, co-repressor Content 1. Introduction 2. Pygeum africanum

Extracts from the plant Pygeum africanum are widely used in the therapy of benign prostate hyperplasia (BPH) and in combinational therapy for prostate cancer, the second leading cause of cancer death and the mostly diagnosed form of cancer in men. The androgen receptor (AR) plays a crucial role in the development of the prostate as well as in prostate diseases. Even though the extracts from Pygeum africanum are considered as beneficial for prostate diseases in clinical trials, and some active compounds for treatment of BPH could be identified, compounds responsible for AR inhibition and the molecular mechanism for inhibition of prostatitis need to be identified. Recently, atraric acid and N-butylbenzenesulfonamide were isolated from a selective dichlormethane extract of Pygeum africanum as two novel AR antagonistic compounds. The molecular mechanisms of AR inhibition were analyzed and are summarized here. Both compounds are the first known natural, complete and specific AR antagonist.

Introduction

Prostate cancer (PCa) is one of the major health problems in elderly men and one of the most often diagnosed malignancies. In western countries it even emerged as the second leading cause of cancer death for men [START_REF] Edwards | Annual Report to the Nation on the Status of Cancer, 1975-2006, Featuring Colorectal Cancer Trends and Impact of Interventions (Risk Factors, Screening, and Treatment) to Reduce Future Rates[END_REF]. As a non-malignant form of prostate diseases benign prostate hyperplasia (BPH) is also a very common disease affecting aging males with sometimes severe influence for quality of life [START_REF] Shah | Fingerprinting the diseased prostate, associations between BPH and prostate cancer[END_REF].

BPH is associated with bothersome lower urinary tract symptoms by interfering with normal daily activities and sleep patterns [START_REF] Skolarikos | Lower urinary tract symptoms and benign prostatic hyperplasia[END_REF]. Both prostatic diseases, BPH and PCa, are characterized by the progressive enlargement of the prostate.

The androgen receptor (AR) plays an important role in the development of the prostate and in the development and progression of prostatic diseases like BPH and PCa. The AR is a nuclear hormone receptor that can be activated by androgens like testosterone and dihydrotestosterone (DHT) [START_REF] Tenbaum | Nuclear receptors: structure, function and involvement in disease[END_REF]. The AR consists of an amino-terminal domain harbouring a potent transactivation function, followed by a DNA-binding domain and A c c e p t e d M a n u s c r i p t

Roell and Baniahmad 3 a carboxy-terminal ligand binding domain. After androgen binding a conformational change of the AR is induced, followed by the translocation into the nucleus and the binding to androgen response elements of AR target genes. Hence, the AR acts as a transcription factor to transactivate or to inhibit gene expression. The transcriptional properties of the AR are partly dependent on binding of co-regulators (co-activators and co-repressors) [START_REF] Brinkmann | Mechanisms of androgen receptor activation and function[END_REF][START_REF] Burke | Co-repressors[END_REF]Rosenfeld et al. 2001;[START_REF] Feldman | The development of androgen-independent prostate cancer[END_REF][START_REF] Papaioannou | Co-activator and co-repressor interplay on the human androgen receptor[END_REF][START_REF] Baniahmad | Nuclear hormone receptor co-repressors[END_REF].

In general, co-repressors (CoR) have been identified as cofactors required for targeting gene repression by those transcription factors having a silencing function [START_REF] Burke | Co-repressors[END_REF]. Although, many CoR for the AR have been identified [START_REF] Wang | Androgen receptor corepressors: an overview[END_REF], a silencing domain for AR has not yet been described. CoR including those that bind to the silencing domain of the thyroid hormone receptor such as SMRT (silencing mediator for RAR and TR), NCoR (nuclear receptor co-repressor) and Alien interact with the AR. In these studies the Nterminus of the AR was determined as the interacting region, the region of the receptor that also interacts with co-activators [START_REF] Dotzlaw | The amino terminus of the human AR is target for corepressor action and antihormone agonism[END_REF]2003;[START_REF] Hodgson | The androgen receptor recruits nuclear receptor CoRepressor (N-CoR) in the presence of mifepristone via its N and C termini revealing a novel molecular mechanism for androgen receptor antagonists[END_REF].

As the AR is the main effector of PCa cell proliferation and progression (Brinkman et al., 2000;[START_REF] Ntais | Molecular epidemiology of prostate cancer: androgens and polymorphisms in androgen-related genes[END_REF] the inhibition of the AR plays an important goal in PCa therapy. A common treatment of PCa is the combination of hormone-ablation and treatment with AR antagonists (antihormones). Interestingly, CoR inhibit AR-mediated transactivation in the presence of specific currently therapeutically used androgen agonists [START_REF] Dotzlaw | The amino terminus of the human AR is target for corepressor action and antihormone agonism[END_REF].

One can differentiate between pure antagonists and mixed agonists/antagonists also termed partial antagonists [START_REF] Migliari | Antiandrogens: a summary review of pharmacodynamic properties and tolerability in prostate cancer therapy[END_REF]. The pure AR-antagonists such as casodex or hydroxyflutamide (OH-Fl) are non-steroidal compounds that allow the recruitment of CoR to the AR and inhibit its activity as well as the PCa proliferation [START_REF] Dotzlaw | The amino terminus of the human AR is target for corepressor action and antihormone agonism[END_REF][START_REF] Song | Antiandrogen Effects of Mifepristone on Coactivator and Corepressor Interactions with the Androgen Receptor[END_REF][START_REF] Papaioannou | Co-activator and co-repressor interplay on the human androgen receptor[END_REF][START_REF] Baniahmad | Nuclear hormone receptor co-repressors[END_REF][START_REF] Powell | Mechanisms of androgen receptor repression in prostate cancer[END_REF]. Also the cellular distribution of the AR seems to be changed by these ligands [START_REF] Tyagi | Dynamics of IntracellularMovement and Nucleocytoplasmic Recycling of the Ligand-Activated Androgen Receptor in Living Cells[END_REF][START_REF] Farla | Antiandrogens prevent stable DNAbinding of the androgen receptor[END_REF][START_REF] Marcelli | Quantifying Effects of Ligands on Androgen ReceptorNuclear Translocation, Intranuclear Dynamics, and Solubility[END_REF][START_REF] Nakauchi | A Differential Ligand-mediated Response of Green Fluorescent Protein-tagged Androgen Receptor in Living Prostate Cancer and Non-prostate Cancer Cell Lines[END_REF]. The partial AR antagonist

4

cyproterone acetate (CPA), which is a steroidal compound, represses the AR-mediated transactivation but exhibits also partial agonistic activity by partially activating the ARmediated transactivation. The level of CPA-mediated repression of AR is strictly dependent on the presence of CoR [START_REF] Dotzlaw | The amino terminus of the human AR is target for corepressor action and antihormone agonism[END_REF]2003), indicating that antihormone therapy of PCa is successful in the presence of functional CoR.

Interestingly, pure and partial antagonists show a different recruitment pattern of CoR on androgen response elements (ARE). In the presence of casodex the AR CoR SMRT is recruited to the prostate specific antigen (PSA) promoter but not to the mouse mammary tumor virus promoter (MMTV), containing several AREs, whereas in the presence of CPA SMRT is recruited to the MMTV promoter [START_REF] Dotzlaw | The amino terminus of the human AR is target for corepressor action and antihormone agonism[END_REF][START_REF] Shang | Formation of the androgen receptor transcription complex[END_REF][START_REF] Kang | Coregulator recruitment and histone modifications in transcriptional regulation by the androgen receptor[END_REF]. Also differences of recruitment among CoR can be observed on various AREs (Papaioannou et al., 2005 and unpublished data). These results indicate that the CoR recruitment is on one hand dependent on the ARE of the target gene, suggesting a structural influence of the DNA and on the other hand on the type of ligand. A possible explanation is that a specific AR structure is imposed by both the type of ligand and the DNA sequence of a particular ARE that dictates binding specificity of CoR to the AR [START_REF] Papaioannou | Co-activator and co-repressor interplay on the human androgen receptor[END_REF].

A further complexity emerges by observations that the interaction of CoR with the AR in the presence of partial antagonists can also be regulated through competition with co-activators.

Using CPA a co-activator/CoR competition was observed and interestingly an increasing expression of either CoR or co-activator shifts the AR towards a more repressed or activated state, respectively [START_REF] Papaioannou | Co-activator and co-repressor interplay on the human androgen receptor[END_REF]. Thus, at present in PCa therapy only non-steroidal compounds are generally known as pure AR antagonists and CPA represents a steroidal compound that exhibits mixed agonist/antagonist activity. One major molecular mechanism seems to be that these compounds require CoR-mediated repression of AR target genes.

However, antihormone treatment is effective only for a limited period of about 16-24 months after which PCa becomes androgen-independent [START_REF] Feldman | The development of androgen-independent prostate cancer[END_REF][START_REF] Eisold | Inhibition of MAPKsignaling pathway promotes the interaction of the corepressor SMRT with the human androgen receptor and mediates repression of prostate cancer cells growth in the presence of antiandrogens[END_REF].

5

Different mechanisms seem to be involved in this process e.g. a shifted co-activator-CoR ratio [START_REF] Balk | Androgen receptor as a target in androgen-independent prostate cancer[END_REF]. (I) CoR may have lost the ability to repress the AR, possibly due to CoR modification e.g. phosphorylation by activation of mitotic signaling pathways [START_REF] Baniahmad | Nuclear hormone receptor co-repressors[END_REF], (II) an overexpression of the AR that leads to titration of CoR [START_REF] Chen | Molecular determinants of resistance to antiandrogen therapy[END_REF], or (III) mutation of the AR [START_REF] Denmeade | A history of prostate cancer treatment[END_REF]) that might induce an AR conformation leading to AR-mediated transactivation. Unfortunately, some of the occurring AR mutations during PCa progression render therapeutically used antiandrogens into AR agonists [START_REF] Farla | Antiandrogens prevent stable DNAbinding of the androgen receptor[END_REF]. In consequence those therapeutics then become useless even disadvantageous because they can promote PCa progression.

Radical prostatectomy or local irradiations are other option for PCa therapy if the carcinoma is limited to the prostatic tissue [START_REF] Cato | Rapid actions of steroid receptors in cellular signaling pathways[END_REF]. Nevertheless the surgery of the prostate often leads to side effects like incontinence, impotence and marks psychological problems for most men. Additionally men developing PCa are in most cases seniors for which a surgery and the anesthesia is buried with threat.

PCa as well as BPH are also treated with inhibitors (e.g. finasteride) of the 5α-reductase, the enzyme that converts testosterone into its more active metabolite DHT [START_REF] Levin | A scientific basis for the therapeutic effects of Pygeum africanum and Serenoa repens[END_REF]. A decrease of the total level of androgens and a shrinkage of the prostate are the consequence [START_REF] Levin | A scientific basis for the therapeutic effects of Pygeum africanum and Serenoa repens[END_REF]. Finasteride only targets one of the two isoforms of the 5α-reductase and therefore is not sufficient to block androgens levels, especially in advanced PCa [START_REF] Vis | Key target of hormonal treatment of prostate cancer. part 2: the androgen receptor and 5α-reductase[END_REF].

The luteinizing hormone releasing hormone (LHRH) is manly responsible for the synthesis of testosterone. Therefore LHRH analoga decrease the amount of LHRH receptors and in consequence lead to a down-regulation of the androgen-production (Corbin 1982). However, also the adrenal gland produces a remarkable amount of androgen that also acts in the prostate. In this case LHRH-analoga fail their function. Thus, LHRH-analoga are mostly combined with antiandrogens for the treatment of PCa to reach a complete blockage of hormone activity [START_REF] Fuhrman | Recent developments in molecular action of antihormones[END_REF][START_REF] Labrie | Stimulation of androgen -dependent gene expression by the adrenal precursors dehydroepi-androsterone and androstenedione in the rat ventral prostate[END_REF] A c c e p t e d M a n u s c r i p t

Roell and Baniahmad

Besides treatment of PCa and prostatic diseases with the above described therapies the treatment with plant extracts is a common therapy [START_REF] Dreikorn | The role of phytotherapy in treating lower urinary tract symptoms and benign prostatic hyperplasia[END_REF]Schleich et al., 2006b).

Phytotherapeutic agents represent nearly half of the medications dispensed for BPH in Italy, compared to 5% for alpha blockers and 5% for 5α-reductase inhibitors and in Germany and Austria phytotherapy is the first-line treatment for mild to moderate urinary obstructive symptoms and represent 29% of all drugs described for the treatment of BPH [START_REF] Wilt | Pygeum africanum for benign prostate hyperplasia[END_REF]. Even though those plant extracts are considered as beneficial for BPH and PCa only few of the active compounds have been characterised and none were shown to bind directly to the AR.

Pygeum africanum

The evergreen tree Pygeum africanum is distributed across the entire African continent in mountain regions. It is also known as Prunus africana or Prunus africanus, a member of the Rosaceae family. Its bark is traditionally used to treat chest pain, malaria and fevers [START_REF] Bombardelli | Prunus Africana[END_REF]. European travellers became interested in this tree when they learned in the 1700s from South African tribes how to treat bladder discomfort and the "old man´s disease" with the bark [START_REF] Simons | Passing problems: prostate and prunus[END_REF]. As this plant seemed to have many effects on different diseases some of its active components had been isolated and investigated. The active compounds include phytosterols (e.g. beta-sitosterol) that have antiinflammatory effects by inhibiting the production of pro-inflammatory prostaglandins and pentacyclic triterpenes (e.g. ursolic and oleanic acids) that have anti-endema properties [START_REF] Murray | The healing power of herbs[END_REF][START_REF] Simons | Passing problems: prostate and prunus[END_REF]. In addition ferucil acid n-esters (e.g. n-docosanol and tetraconsanol) have been isolated from extracts that reduce prolactin levels and block the accumulation of cholesterol in the prostate [START_REF] Murray | The healing power of herbs[END_REF][START_REF] Simons | Passing problems: prostate and prunus[END_REF]. Prolactin as well as cholesterol is involved in the AR-activating pathway, whereas prolactin increases the testosterone uptake in the prostate and cholesterol increases binding sites for DHT [START_REF] Murray | The healing power of herbs[END_REF][START_REF] Simons | Passing problems: prostate and prunus[END_REF]. [START_REF] Isaacs | Importance of the natural history of benign prostatic hyperplasia in the evaluation of pharmacologic intervention[END_REF]. In France Pygeum africanum is the most commonly used medicine for BPH [START_REF] Ishani | Pygeum africanum for the treatment of patients with benign prostatic hyperplasia: a systematic review and quantitative meta-analysis[END_REF][START_REF] Levin | A scientific basis for the therapeutic effects of Pygeum africanum and Serenoa repens[END_REF]. Herby, the pulverized bark is incorporated into capsules and commercially available as the remedy Tadenan ® and in other European countries under various different trade names [START_REF] Ishani | Pygeum africanum for the treatment of patients with benign prostatic hyperplasia: a systematic review and quantitative meta-analysis[END_REF]. In the USA phytotherapeutic agents like extracts of Pygeum africanum have been marketed for prostate health including PCa prevention and treatment [START_REF] Shenouda | Phytosterol Pygeum africanum regulates prostate cancer in vitro and in vivo[END_REF].

Several studies have confirmed the positive effect of the extracts of Pygeum africaum on prostatic diseases [START_REF] Breza | Efficacy and acceptability of tadenan (Pygeum africanum extract) in the treatment of benign prostatic hyperplasia (BPH): a multicentre trial in central Europe[END_REF][START_REF] Cristoni | Botanical derivatives for the prostate[END_REF][START_REF] Katz | Flavonoid and botanical approaches to prostate health[END_REF][START_REF] Gerber | Phytotherapy for benign prostatic hyperplasia[END_REF][START_REF] Wilt | Pygeum africanum for benign prostate hyperplasia[END_REF][START_REF] Buck | Is there a scientific basis for the therapeutic effects of serenoa repens in benign prostatic hyperplasia? Mechanisms of action[END_REF]Dedhia et al., 2008). Tadenan ® inhibited the proliferation of fibroblasts from human hyperplastic prostate and bladder [START_REF] Edgar | A critical review of the pharmacology of the plant extract of Pygeum africanum in the treatment of LUTS[END_REF]. Also, Tadenan ® has been shown to be a potent inhibitor of rat prostatic fibroblast proliferation in response to direct activators of protein kinase C. Moreover, the extract of Pygeum africaum is an inhibitor of a variety of growth factors including basic fibroblast growth factor, epidermal growth factor and insulin like growth factor [START_REF] Yablonsky | Antiproliferative effect of Pygeum africanum extract on rat prostatic firbroblasts[END_REF] and of G-protein coupled receptors [START_REF] Solano | Effects of pygeum africanum extract (Tadenan ® ) on vasoactive intestinal peptide receptors, G proteins , and adenylyl cyclase in rat ventral prostate[END_REF]. Other studies with male rats revealed that a co-treatment or a post-treatment of Tadenan ® together with DHT suppressed the effects of DHT on micturition [START_REF] Choo | Functional evaluation of Tadenan on micturition and experimental prostate growth induced with exogenous dihydrotestosterone[END_REF][START_REF] Yoshimura | Effect of Pygeum africanum Tadenan on micturition and prostate growth of the rat secondary to coadministered treatment and post-treatment with dihydrotestosterone[END_REF]. The co-treatment in addition decreased the prostatic weight [START_REF] Yoshimura | Effect of Pygeum africanum Tadenan on micturition and prostate growth of the rat secondary to coadministered treatment and post-treatment with dihydrotestosterone[END_REF]. In animal models extract of Pygeum africanum revealed to modulate bladder contractility by reducing the sensitivity of the bladder to electrical stimulation, phenylephrine, adenosine triphosphate and carbacol [START_REF] Andro | Pygeum africanum extract for the treatment of patients with benign prostatic hyperplasia: a review of 25 years of published experience[END_REF].

Recently, the ethanolic extracts of Pygeum africanum were shown to inhibit human PCa cell growth, induce apoptosis and alter cell kinetics [START_REF] Shenouda | Phytosterol Pygeum africanum regulates prostate cancer in vitro and in vivo[END_REF]. TRAMP mice, a mouse model to investigate PCa, exhibited a reduction of PCa incidence compared to the control group after feeding with Pygeum africanum extract [START_REF] Shenouda | Phytosterol Pygeum africanum regulates prostate cancer in vitro and in vivo[END_REF].

In a single phase II study with patients having prostate hypertrophy a regression of the prostate size after Tadenan ® administration was observed [START_REF] Mathé | A pygeum africanum extract with so-called phyto-estrogenic action markedly reduces the volume of true and large prostatic hypertrophy[END_REF]. In clinical [START_REF] Andro | Pygeum africanum extract for the treatment of patients with benign prostatic hyperplasia: a review of 25 years of published experience[END_REF][START_REF] Yablonsky | Antiproliferative effect of Pygeum africanum extract on rat prostatic firbroblasts[END_REF][START_REF] Chatelain | Comparison of once and twice daily dosage forms of Pygeum africanum extract in patients with benign prostatic hyperplasia: a randomized, double-blind study, with long term open label extension[END_REF]. These results were confirmed by a multicentral trial in Europe, in which also nocturia was tested with the result that 32% of the male patients exhibited less symptoms [START_REF] Breza | Efficacy and acceptability of tadenan (Pygeum africanum extract) in the treatment of benign prostatic hyperplasia (BPH): a multicentre trial in central Europe[END_REF].

Even though all these effects of Pygeum africanum have been proven in vitro, in animal models and in clinical trials, the possibility for active antiandrogenic compounds to inhibit the AR remained elusive. Therefore, we investigated diverse extracts (n-hexane, dichlormethane, methanol, methanol-water and water) from the stem bark of Pygeum africanum for their antiandrogenic potential using first reporter gene assays (Schleich et al., 2006b). The selective dichlormethane or ethanolic extract from Pygeum africanum bark material elicited the highest bioactivity. Hence, a bioassay-guided fractionation was performed resulting in the isolation of two novel antiandrogenic compounds with a novel chemical structure from Pygeum africanum: atraric acid (AA) and N-butylbenzenesulfonamide (NBBS) (Schleich et al., 2006a;2006b).

The natural compounds AA and NBBS

The benzolic methylester AA is compared to steroids a very small molecule (Fig. 1). AA occurs in lichens and has been isolated from higher plants like Newbouldia laevis [START_REF] Gormann | Furanonaphthoquinones, atraric acid and a benzofuran from the sterm barks of Newbouldia laevis[END_REF], Alseodaphne andersonii [START_REF] Lee | Chemical constituents from Alseodaphne andersonii[END_REF], Acer nikense [START_REF] Nagumo | Studies on the conctituents of Aceraceae plants. XIII. Diarylheptanoids and other phenolics from Acer nikoense[END_REF] and Frullania brasiliensis [START_REF] Bardón | Eremophilanolides and other constituents from the argentine liverwort Frullania brasiliensis[END_REF]. Previously it was found as a degradation product of various natural depsides. Even though AA is present in the extract of the bark from Pygeum africanum it is unknown whether it is a secondary metabolite of the plant biosynthesis itself or if it is produced by lichens colonizing the tree and producing polyketides through the acetate-polymalonate pathway. It is known that atranorin, a depside of hematommic acid, and AA are biosynthesized through the polyketide pathway by various lichens [START_REF] Burgeois | Atraric acid, a marker for epiphytic lichens in the wood used in cooperage : identifiaction and quantification by GC/MS/(MS)[END_REF].

9

NBBS was shown to be produced by the bacteria Pseudomonas sp. AB2 in greenhouse soil [START_REF] Kim | Isolation and identification of antifungal Nbutylbenzenesulfonamide produces by Pseudomonas sp AB2[END_REF] and is like AA compared to steroids a small molecule (Fig. 1). Commonly NBBS is used as a plasticizer in the polyamide and co-polyamide production. It is also applied in the synthesis of sulfonyl carbamate herbicides. Because of its stability and persistance in the environment relatively high concentration have been found in wastewater and snow (concentrations up to 100 µg/L) (Huppert et el. 1998, Poliakova et al., 2000).

These findings reveal the possibility for NBBS to accumulate in organisms, but since we could isolate NBBS from several independent bark extracts and in Tadenan ® it is likely that NBBS is present naturally in Pygeum africanum bark or could be produced by organisms living on the bark of the Pygeum africanum tree. Thus, even though NBBS can also be synthesized chemically it originates in nature.

Nevertheless, both compounds AA and NBBS are present in all investigated extracts of Pygeum africanum and therefore are more likely to be natural products.

According to WHO the P. africanum extracts are well tolerated in humans. Pre-studies with mice treated several times a week with AA did not indicate any side effects, assuming a good tolerance of AA (data not published). Concerning NBBS Even though it was found to be neurotoxic for rabbits [START_REF] Strong | Nbutylbenzenesulfonamide: a neurotoxic plasticizer inducing a spastic myelopathy in rabbits[END_REF] experiments with rats showed only slight effects at very high doses that were just of short duration [START_REF] Kumar | Brain uptake, pharmacokinetics, and tissue distribution in the rat of neurotoxic Nbutylbenzenesulfonamide[END_REF]. In vitro studies revealed neuronal cells to be more sensitive in growth to NBBS treatment than glioma cells [START_REF] Nerurkar | Preliminary observations on the in vitro toxicity of N-butylbenzenesulfonmide: a newly discovered neurotoxic[END_REF] indicating different responses of cell types to NBBS.

In reporter gene assays a concentration of 10 µM AA exhibited a repression of androgen ARmediated transactivation by about 90% (Schleich et al., 2006a). AA exhibits still 50% of inhibition at a concentration of 1 µM and failed to repress the AR-mediated transactvation at 0.1 µM (Schleich et al., 2006a). We synthesized ethyl, n-propyl and n-butyl derivatives of AA and found an even higher antiandrogenic activity of the ethyl and the n-propyl derivative, arguing that the length of the ester chain plays a crucial role in AR antagonism (Schleich et al., 2006a).

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

Roell and Baniahmad 10 Compared to AA, NBBS showed less efficacy on the androgen activated AR-mediated transactivation as 90% repression were obtained only with higher concentrations (100 µM), a concentration of 10 µM exhibited 50% repression and the antiandrogenic effect was lost at a dosage of 1µM of NBBS [START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. A battery of NBBS derivatives was chemically synthesized to modify the butyl side chain and the benzene ring by substitutions with the aim of achieving more potent antiandrogens [START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF].

Derivatives with a shortened butyl side chain or a substituted benzene ring at the paraposition caused a loss of the antiandrogenic potential. This strongly suggests the importance of the length of the side chain and an unsubstituted benzene ring at the para-position as antiandrogenic compound [START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. Replacing the butyl side chain with a pentyl or a geranyl group in turn enhanced the antiandrogenic activity compared to NBBS, indicating that a hydrophobic side chain is important for AR antagonism but further extension weakened the effect [START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. Notably, also substituents at the metaposition of the benzene ring enhanced the antiandrogenic activity.

In summary more potent derivatives of either AA or NBBS were synthesized. The antiandrogenic activity was mainly depending on the length of the alkyl side chain and in case of NBBS the position of the benzene substituents, revealing a crucial role of small changes in the structure of the natural compounds.

A marker for PCa is the serum level of the prostate specific antigen (PSA), an AR target gene. The endogenous PSA expression in the androgen-dependent human PCa cell line LNCaP harbouring an AR mutant, was significantly repressed by AA and NBBS (Papaioannou et al., 2009a;[START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. Similar results were observed, performing real-time PCR with the androgen-independent growing PCa cell line C4-2, also containing the AR-T877A mutant.

To allow a specific targeted inhibition a very important characteristic of antiandrogens should be the AR specificity. The effect of both AA and NBBS was analyzed for the transcriptional properties of close related nuclear hormone receptors such as the progesterone receptor

11

(PR), the glucocorticoid receptor (GR), the estrogen receptor α (ER) and ERβ as well as the thyroid hormone receptor β (TRβ). In case of PR both isoforms were tested, the PR-B form with a more potent transactivation function and the PR-A form, which is generated by an internal translation start site. For ER both the ERα and the ERβ were investigated. Neither AA nor NBBS had any measurable effect on the ligand-induced transactivation of GR, PR-A, TRβ, or one of the ER receptors in the concentrations used for 90% of AR inhibition (Papaioannou et al., 2009a;[START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. NBBS as well as AA showed an inhibitory effect at high concentrations for the PR-B form and AA additionally had estrogenic potential at high concentration of 100 µM (Papaioannou et al., 2009a;[START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. This slight repression and activation of other nuclear hormone receptors only at high employed concentrations suggest that AA and NBBS are AR specific antiandrogens.

Eventually PCa becomes therapy resistant after a certain time of treatment with hormone ablation therapy. This therapy resistance may be due to several reasons one of which is due to AR mutations like the often occurring AR point mutation T877A in castration resistant PCa [START_REF] Culig | Swith from antagonist to agonist of the androgen receptor bicalutamide is associated with prostate tumor progression in a new model system[END_REF]. This point mutation in the ligand binding domain (LBD) of the AR renders the antiandrogenic metabolite OH-Fl of the recently used antihormone flutamide into an AR agonist. Notably, AA and NBBS exhibit repression of the AR-T877A mutant and therefore seem to have another molecular mechanism to mediate inhibition of AR compared to OH-Fl (Papaioannou et al., 2009a;[START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF].

One of the major goals in PCa therapy is the growth inhibition of the cancer cells. AA and NBBS decreased the cell proliferation of several human prostate cancer cell lines either growing androgen dependently and containing the wild type AR or the AR-T877A mutant or growing androgen independently and containing the AR-T877A mutant (C4-2). In contrast, growth of PC3 cells lacking expression of the AR or non PCa cells also lacking AR expression were not effected by AA and NBBS, suggesting an AR-dependent growth inhibitory mechanism imposed by AA and NBBS (Papaioannou et al., 2009a;[START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. Also, neither toxicity nor apoptosis could be observed during the treatment. The synthetic AR agonist R1881 promotes beside PCa cell proliferation also the invasion through the extracellular matrix, in consequence invasion of cells, an important step for PCa metastasis [START_REF] Chuan | Androgen induction of prostate cancer cell invasion is mediated by ezrin[END_REF]. In line with this AA inhibited the androgen-induced invasion of human LNCaP cells (Papaioannou et al., 2009a) while AA alone had no influence on the cell invasion. Thus, AA inhibits the agonist-induced invasion of LNCaP cells through the extracellular matrix and therefore could be a potential inhibitor also for PCa metastasis.

Insights in the molecular mechanism of AA and NBBS

To identify the important AR sites for the inhibitory mechanism of AA and NBBS on ARmediated transactivation several AR deletion mutants were analyzed. The results suggest that the AR-LBD is targeted by AA and NBBS, indicating binding of the compounds to the AR-LBD that may lead to competition with androgen binding (Papaioannou et al., 2009a;[START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. This hypothesis was verified by whole cell competition binding analysis with AR agonists confirming the release of androgens by either AA and NBBS indicating that both compounds act as competitive inhibitors (Papaioannou et al., 2009a;[START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF].

One possible mechanism to inhibit AR could be to reduce its protein level such as by enhanced protein degradation. However, treatment of PCa cells with AA or NBBS did not reveal a decrease of the AR protein suggesting that AA and NBBS do not induce AR degradation and suggesting that AA and NBBS use other mechanisms to inhibit the AR.

Another hypothesis was that AA or NBBS might induce CoR binding to AR. However, testing the interaction of three CoR such as SMRT, NCoR and Alien did not indicate a binding of these CoR to either AA or NBBS bound AR.

A further possibility for NBBS and AA to inhibit AR-mediated transactivation would be the inhibition of the translocation of AR into the nucleus. Using GFP-AR as expected the staining was cytoplasmatic without androgens whereas the majority of staining was observed in the nucleus with androgen treatment of cells. Interestingly, the androgen-induced translocation of

13

fractionation experiments. Thus, the data suggest that both compounds inhibit the androgen induced AR translocation into the nucleus (Papaioannou et al., 2009a;[START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. In contrast to casodex also recently published diarylthiohydantions with antiandrogenic activity such as AA and NBBS do not themselves translocate the AR into the nucleus [START_REF] Tran | Development of a secondgeneration antiandrogen for treatment of advanced prostate cancer[END_REF]. However, it was not investigated whether this diarylthiohydantions also inhibit the nuclear translocation of the AR in the presence of androgens.

Taken together, two compounds from the bark of Pygeum africanum were isolated that exhibit a novel molecular mechanism to repress the AR-mediated transactivation by inhibiting its nuclear translocation.

Discussion

With AA and NBBS, to our knowledge the first natural AR antagonists were isolated and identified from Pygeum africanum, from which plant extracts are used for a long time in the treatment of prostatic diseases.

DHT as well as the synthetic androgen R1881 are very hydrophobic molecules and are thought to not require an active process to pass the cell membrane [START_REF] Fedoruk | Pglycoprotein increases the efflux of the androgen dihydrotestosterone and reduces androgen responsive gene activity in prostate tumor sells[END_REF].

AA and NBBS are more hydrophilic and therefore the mode of cellular uptake may not be comparable, as the lipophilic cell membrane is a barrier for those molecules. Hence, the used concentrations for cell treatment do not necessarily reflect the intracellular concentrations of both compounds, so the concentrations may be lower than those added in the experiments.

In addition it was shown for other natural compounds that they require a similar concentration range of action [START_REF] Zi | Silibinin decreases prostate-specific antigen with cell growth inhibition via G1 arrest, leading to differentiation of prostate carcinoma cells: implications for prostate cancer intervention[END_REF]2000;[START_REF] Xing | Quercetin inhibits the expression and function of the androgen receptor in LNCaP prostate cancer cells[END_REF][START_REF] Kiviharju | Antiproliferative and proapoptotic activities of triptolide (PG490), a natural product entering clinical trials, on primary cultures of human prostatic epithelial cells[END_REF][START_REF] Rosenberg Zand | Flavonoids can block PSA production by breast and prostate cancer cell lines[END_REF][START_REF] Thelen | Silibinin downregulates prostate epithelium-derived Ets transcription factor in LNCaP prostate cancer cells[END_REF]. It is also possible that AA and NBBS may act together with other compounds and therefore potentiating the overall effect on prostatic diseases. For instance extracts form Pygeum africanum were shown to inhibit membrane receptors that are involved in AR signaling and thus suppress AR activity (Yablonski et al., 1997;[START_REF] Szolnoki | The effect of Pygeum africanum on fibroblast growth factor (FGF) and transforming growth factor beta (TGF beta 1/LAP) expression in animal model[END_REF] , 2001;Santa Maria Margalef et al., 2003;[START_REF] Boulbès | Pygeum africanum extract inhibits proliferation of human cultured prostatic fibroblasts and myofibroblasts[END_REF][START_REF] Edgar | A critical review of the pharmacology of the plant extract of Pygeum africanum in the treatment of LUTS[END_REF][START_REF] Li | Nerve growth factor and substance P: expression in a rat model of diabetic bladder[END_REF][START_REF] Quiles | Antiproliferative and apoptotic effects of the herbal agent Pygeum africanum on cultured prostate stromal cells from patients with benign prostatic hyperplasia (BPH)[END_REF] (Fig. 2).

The molecular mechanisms of currently clinically used antiandrogens for PCa treatment is based on the recruitment of co-repressors or through changing chromatin accessibility [START_REF] Dotzlaw | The amino terminus of the human AR is target for corepressor action and antihormone agonism[END_REF][START_REF] Shang | Formation of the androgen receptor transcription complex[END_REF][START_REF] Kang | Coregulator recruitment and histone modifications in transcriptional regulation by the androgen receptor[END_REF][START_REF] Hodgson | The androgen receptor recruits nuclear receptor CoRepressor (N-CoR) in the presence of mifepristone via its N and C termini revealing a novel molecular mechanism for androgen receptor antagonists[END_REF][START_REF] Hong | Modulation of the expression and transactivation of androgen receptor by the basic helixloop-helix transcription factor Pod-1 through recruitment of histone deacetylase 1[END_REF][START_REF] Metzger | LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription[END_REF]. These antiandrogens permit AR translocation into the nucleus as well as the recruitment of the AR with CoR to its target genes [START_REF] Jenster | Nuclear import of the human androgen receptor[END_REF][START_REF] Masiello | Bicalutamide functions as an androgen receptor antagonist by assembly of a transcriptionally inactive receptor[END_REF][START_REF] Zhu | Macrophage/cancer cell interactions mediate hormone resistance by a nuclear receptor derepression pathway[END_REF]. One basis of resistance of PCa to the hormone therapy may be due to malfunctioning of AR inhibition by these CoR. Therefore, the different CoR-independent molecular mechanism of AA and NBBS to inhibit the AR nuclear translocation may be a useful tool for hormone treatment of those refractory PCa (Fig. 3).

Comparing the IC 50 of AA (3 µM) and NBBS (10 µM) with that of casodex ( 1 Furthermore, we synthesized more potent AA and NBBS derivatives with the same underlying molecular mechanism for the analyzed NBBS derivatives. The enhanced antiandrogenic potential of this derivatives may be due to their higher lipophilicity compared to AA and NBBS respectively, but structural modifications seem also to play a role as also more lipophilic derivatives of AA and NBBS were synthesized that showed less or even no anitandrogenic potential (Papaioannou et al., 2009b and data not published). Additional substitutions at the benzolic ring of NBBS also resulted in some cases in an increased inhibition of AR-mediated transactivation [START_REF] Papaioannou | NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth[END_REF]. Thus, NBBS and AA may serve as novel chemical platform molecules to inhibit the AR transactivation and PCa growth and may serve as lead structures for new AR antagonists. AA is benzolic methylester with two additional methyl groups on C-atoms at positions 3 and 6. As subtituents on position 2 and 4 the benzol ring carries hydroxyl groups. The n-propyl derivative is elongated at the alkyl side chain. The scaffold of NBBS is also a benzol ring, whereas the substituent is a sulfone amide group followed by a butyl chain. The indicated NBBS derivative carries an additional tri-fluride group at the meta position of the benzol ring and the amide an additional hydrogen. All four molecules are smaller compared to steroids such as the androgen DHT. Further derivatives of AA and NBBS have other changed side chains or substituents (see text for details).

7

  Since the late 1960´s bark extracts of Pygeum africanum are used in Europe to treat BPH

  A c c e p t e d M a n u s c r i p t Roell and Baniahmad 8 trials the application of Tadenan ® has been shown to improve the symptoms of BPH and is well tolerated

  µM) (Hadratta et al., 2005) both compounds exhibit only slightly weaker efficiencies. In comparison with the clinically used antiandrogen flutamide (IC 50 of 116 µM) (Hadratta et al., 2005) AA and NBBS are even more potent. Thus, assuming that this natural compounds could be quite potent PCa inhibitors in vivo.

Figure Legends Fig 1 .

 Legends1 Figure Legends

Fig 2 .Fig 3 .

 23 Fig 2. Inhibition pathways of the AR by extract of Pygeum africanum.

  Fig.1

  Figure

  Fig.3

Acknowledgement

This work was supported by the Deutsche Krebshilfe to A.B.