Raphaela ; Fritsche
email: raphaela.fritsche@charite.de

Aurelia Noske

Ute Ungethüm

Ralf-Jürgen Kuban

Per-Ulf Peter M Schlag

Janine Tunn

Karle

Jonas Veit

Manfred Krenn

Christine Dietel

Sers

Per-Ulf Tunn

Janine Karle

Veit Krenn

Manfred Dietel

Christine ; Sers

Raphaela Fritsche-Guenther

Dr. rer. nat Raphaela Fritsche

De novo expression of EphA2 in osteosarcoma modulates activation of the mitogenic signaling pathway

Keywords: EphA2, EFNA1, MAPK, Osteosarcoma EphA2, EFNA1, MAPK, osteosarcoma

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Introduction

Conventional osteosarcoma, also called classical osteosarcoma, is the most common malignant primary bone tumor in children and adolescents with a profound risk of metastasis to the lung [START_REF] Campanacci | Bone Tumors[END_REF] . This highly aggressive neoplasm mainly affects patients in the second decade of life when the growth spurt is highest, with 60% of patients under the age of 25 [START_REF] Gelberg | Growth and development and other risk factors for osteosarcoma in children and young adults[END_REF] . Before the use of effective chemotherapy, the 2-year overall survival rates for patients with osteosarcomas was 15% to 20% following surgical resection and/or radiotherapy [START_REF] Friedman | The therapy of osteogenic sarcoma: current status and thoughts for the future[END_REF] . Surgery and chemotherapy since then led to an improvement in long-term survival rates to 50% to 80% [START_REF] Arndt | Common musculoskeletal tumors of childhood and adolescence[END_REF] . Nevertheless, drug resistance and poor clinical outcome due to early metastasis are still a problem.

Using microarray analysis we recently detected several ephrins and their cognate receptors deregulated in human bone tumors such as giant cell tumors and chondrosarcomas [START_REF] Guenther | Giant cell tumors of the bone: molecular profiling and expression analysis of Ephrin A1 receptor, Claudin 7, CD52, FGFR3 and AMFR[END_REF] .

Ephrin receptors (Eph) are the largest group of receptor tyrosine kinases, however to date most information about ephrin receptors and ephrin ligands (EFN) was gained from melanoma and epithelial tumors [START_REF] Varelias | Human osteosarcoma expresses specific ephrin profiles: implications for tumorigenicity and prognosis[END_REF] . In mouse melanoma cells, expression of the EphA2 receptor was shown to stimulate a distinct invasion program resulting in metastasis formation [START_REF] Parri | EphA2 reexpression prompts invasion of melanoma cells shifting from mesenchymal to amoeboid-like motility style[END_REF] . In carcinomas, there is already ample information on the diverse roles of different EFN and Eph and their signaling [START_REF] Surawska | The role of ephrins and Eph receptors in cancer[END_REF] . In colorectal cancer, loss of the EphA1 protein has been correlated with invasion and metastasis and is therefore associated with a poor prognosis [START_REF] Dong | Downregulation of EphA1 in colorectal carcinomas correlates with invasion and metastasis[END_REF][START_REF] Herath | Epigenetic silencing of EphA1 expression in colorectal cancer is correlated with poor survival[END_REF] .

In contrast, EphA2 was found upregulated in many carcinomas and exhibits both a kinasedependent and kinase independent role in the metastasis of prostate cancer [START_REF] Taddei | Kinase-dependent and -independent roles of EphA2 in the regulation of prostate cancer invasion and metastasis[END_REF] . The research on ephrin involvement in tumor development and progression is complicated by the fact that ephrin receptors and their ligands display forward and reverse signaling, yet the impact of both EphA and B family members in angiogenesis is now well documented [START_REF] Oike | Regulation of vasculogenesis and angiogenesis by EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells[END_REF] . Based on current molecular knowledge, targeted and rational therapeutic strategies are developed and nowadays successfully improve survival of cancer patients. Nevertheless, there is a continued medical need to study the basic molecular mechanisms of osteosarcoma in order to develop bone tumor-specific therapeutic strategies. The current study aimed to identifying genes differentially expressed between normal human osteoblasts and osteosarcoma tissue and to define genes that may be involved in tumor progression. This analysis revealed a distinct ephrin expression pattern and an increased expression of EFNA1 ligand and EphA2 receptor in human osteosarcoma tissue.

Material and methods

Patients and tissues

Tumor samples were obtained from the Max-Delbrueck-Center, division of Pathology, Berlin-Buch, Germany. Patient characteristics are described in Tab. 1. The tissue specimens were fixed in 4% neutral buffered formaldehyde, bone containing tissue was EDTA-decalcified and embedded in paraffin. For each case, hematoxylin and eosin (H&E)-stained slides were carefully reviewed and the diagnosis of the tumor was confirmed according to WHO criteria.

Written informed consent was obtained from each patient. The median age of osteosarcoma patients is 37 and ages vary from 7 years to 74 years. This is unusual as osteosarcoma typically affects adolescents, but we were not able to use younger patients based on the fact, that our sample collection in the institute include only these patients. All of the osteosarcoma tumors are primary.

Cell culture

Primary osteoblast cells (HOBc) and the osteosarcoma cell line SaOS2 were purchased from Provitro GmbH, Berlin, Germany and from ATCC (American Type Culture Collection) and (Biochrome) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. All cells were incubated in a humidified atmosphere with 5% CO 2 at 37°C. For signaling pathway analyses 1µg/ml EFNA1/F c (R&D), 50µM U0126 (Promega) or 50µM DMSO (Sigma) were used for the time points indicated. For proliferation assays cells were directly plated on 96plates or 96-polyhema-plates and treated with 1µg/ml EFNA1/F c up to 96h. For anchorageindependent growth media containing EFNA1/F c was changed every 24h. Proliferation of cells was measured using the MTT cell proliferation kit (Roche). For wound healing assay, cells were grown on 6-Well dishes or glass slides as a confluent monolayer and wounded with a 10µl pipette tip. Afterwards, 1µg/ml EFNA1/F c in the presence of fetal calf serum was added and wound closure was observed up to 48 hours.

Microarray analysis

All tissues were cryopreserved in liquid nitrogen immediately after removal and stored at -80°C. RNA was isolated from frozen samples and fresh cells using Tri-Reagent (Sigma) as well as the Rneasy-mini-kit (Qiagen). cRNA was synthesized from 5µg of total RNA. Synthesis of biotin-labeled cRNA was performed using the BioArray High Yield RNA Transcription kit (Enzo Diagnostics, Farmingdale, N.Y.). The amplified cRNA was hybridized to HG U133A arrays (Affymetrix, Santa Clara, CA). After a scan of the array surface, the computer-generated image of the array was overlaid with a virtual grid, controlled by Microarray Suite 5.0 software (Affymetrix). The Data Mining Tool 3.0 (Affymetrix) and average results from different replicates and to perform statistical analysis to compare the sample groups. Differential gene expression between osteosarcoma and non-neoplastic osteoblasts was defined according to the following criteria: 1) upregulated genes are present in both replicates of osteosarcoma metastases and differential expression between conventional versus HOBc (>1.5) and metastases vs. HOBc (>1.5) is mandatory, 2) downregulated genes are present in both replicates of HOBc and a differential expression between conventional vs. HOBc (>1.5) and metastases vs. HOBc (>1.5) is mandatory.

Expression levels below 1,000 were defined as background. The data were submitted to the GEO (Gene Expression Omnibus) database (GEO accession number GSE14359, geo@ncbi.nlm.nih.gov).

Immunohistochemistry

Immunohistochemical analyses were performed on paraffin-embedded tissues. As a nonneoplastic control, a tissue microarray (TMA) containing n=4 fetal and n=6 adult bone samples (each in duplicates; Provitro GmbH, Berlin, Germany) was used. Antigen retrieval was performed by incubation in trypsine (0.5%, Zymed Digest-All Kit). To block endogenous peroxidase activity, peroxidase blocking reagent (DAKO) was used. The primary antibodies EFNA1, EFNA2, EFNB1, EFNB3 (all 1:10, Zymed) and EphA2 (1:10, Upstate) were incubated over night at 4°C. The chromogene was 3, 3'-diaminobenzidine (DAB, DAKO) and nuclear counter staining was performed with hemalaun. All samples were semi-quantitatively scored by two different observers using a score modified according to Remmele and Stegner [START_REF] Remmele | Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue[END_REF] . 1. A score to measure the percentage of positively stained cells (PP) was performed as follows: 0 = no staining, 1 = 1-25%, 2 = 26-50%, 3 = 51-75%, 4 = 76-100%.

Immunoblot analysis

Protein extracts were prepared by incubation with sodiumdodecylsulfate (SDS)-cell lysis buffer (10% SDS, 1M TrisHCl pH 7.5, EDTA 0.5M pH8). Reagents for SDS-PAGE and Western blotting were obtained from Bio-Rad Laboratories (Richmond, CA). Electrophoresis was performed and proteins were transferred to nitrocellulose membranes (Schleicher & Schüll). Membranes were blocked and specific proteins were detected by incubation with rabbit anti-human EFNA1 (V-18, 1:200 Santa Cruz), mouse anti-human EphA2 (Clone D7, 1:500 Upstate), mouse anti-human p-Tyr (1:1,000 Santa Cruz), rabbit anti-human P-Erk1/2 (p44/42 MAPK Thr202/Tyr204 1:1,000 Cell Signaling), mouse anti-human Erk1 (1:5,000 BD) and mouse anti-human actin (1:5,000 Chemicon) antibodies over night at 4°C following HRP-conjugated rabbit anti-mouse (1:5,000 Dianova) or goat anti-rabbit (1:2,000 Cell Signaling) secondary antibodies. Blots were developed using ECL system from Amersham.

The quantification of blots was done using ImageJ.

Immuofluorescence staining

Cells were grown on glass slides and fixed with acetone. Immunofluorescence staining was performed using mouse anti-human EphA2 (Clone D7, 1:10 Upstate) antibody and visualized with Alexa488 (1:300 Invitrogen). Nuclei were counterstained with DAPI. Images were recorded using a fluorescence microscope (Zeiss).

Results

Expression of ephrins in osteosarcomas

RNA derived from eight osteosarcoma samples (n=4 conventional, n=4 metastases) and from the human osteoblast primary culture HOBc were analyzed using Affymetrix HG U133A microarrays. 1,163 genes were found significantly upregulated in conventional and metastatic osteosarcomas as compared to HOBc. 31 genes were expressed >10 fold in osteosarcomas compared to the non-neoplastic osteoblasts. Among them, EFNA1, a ligand for EphA tyrosine kinase receptors, was expressed >10 fold higher in osteosarcoma samples compared to osteoblasts (Tab. 2A). In addition, 2,059 genes were significantly downregulated between the osteosarcomas and HOBc. 37 genes were expressed less than 10 fold in osteosarcoma samples compared to osteoblasts (Tab. 2B).

During a recent study we discovered several ephrin genes deregulated in bone-related tumors, such as giant cell tumor of the bone [START_REF] Guenther | Giant cell tumors of the bone: molecular profiling and expression analysis of Ephrin A1 receptor, Claudin 7, CD52, FGFR3 and AMFR[END_REF] . For example, EFNA1 and EphA3 were upregulated in recurrent as compared to primary giant cell tumors, while EphA1 and EphA4 were found downregulated in giant cell tumor recurrences. This prompted us to suggest that tissue-specific expression patterns of ephrins are associated with the development and progression of bone tumors. The observation that the EFNA1 ligand light up as highly upregulated in the osteosarcoma samples versus non-neoplastic osteoblasts focused our further survey to the ephrin family and their expression in osteosarcoma tumor samples (Tab.

3). Within the 22,283 Affymetrix probe sets are five EFNA ligand probe sets (EFNA1-5), six EphA receptor probe sets (EphA1-5, EphA7), three EFNB ligand probe sets (EFNB1-3) and five EphB receptor probe sets (EphB1-4, EphB6).

EFNA1, EphA2 and EphA3 were found upregulated in osteosarcoma tissue compared to HOBc. No significant differences between conventional and metastases could be observed. EFNA4, EphA1, EphA4 and EphA5 were found to be expressed above background only in 1/8 osteosarcoma samples but below background in HOBc. Expression levels for EFNA2,

Ephrin proteins are expressed at elevated levels in osteosarcomas

To confirm the differential expression of ephrins at the protein level, we stained osteosarcoma tissue samples (n=10 primary tumors and n=7 metastases) using anti-EFNA1, anti-EFNA2, anti-EFNB1, anti-EFNB3 and anti-EphA2-specific antibodies. For EFNA1, EFNB1 and EphA2 a tissue microarray harboring several non-neoplastic human bone samples (n=18) were stained in addition. The immunoreactive scores for all antigens are shown in Tab. 4.

EFNA1 protein levels are increased in osteosarcomas

As already suggested from the microarray analysis, a significant upregulation of EFNA1 was detected in osteosarcomas compared to non-neoplastic bone (p=0.0003). EFNA1 expression was observed in 7 out of 8 cases of both fetal and adult osteocytes, in osteoblasts and in osteoclasts of non-neoplastic bone (Fig. 1A), but no significant differences between fetal and normal adult bone were found (p=0.33). However, EFNA1 was detectable at higher intensities in all osteosarcoma samples, where nearly 80% of tumor cells stained positive (Fig. 1B), yet no significant differences between conventional osteosarcomas and metastases were found (p=1). The EFNA1 immunoreactivity was localized to the membrane and to the cytoplasm of the tumor cells. Interestingly, five osteosarcoma samples showed a nuclear EFNA1 expression (Fig. 1C).

EphA2 protein is expressed de novo in osteosarcomas

In contrast to the EFNA1 ligand, the EphA2 receptor was not present in fetal and adult nonneoplastic bone (Fig. 1D), but 13/17 osteosarcoma samples showed expression of the EphA2 receptor (Fig. 1E,F). The EphA2 immunoreactivity was largely confined to tumor cells around the bone formations (Fig. 1E arrow). Again, a significant upregulation of EphA2 in osteosarcomas compared to non-neoplastic bone could be confirmed at the protein level (p=0.000024), no significant differences between conventional osteosarcomas and metastases were found (p=1).

Differential expression of EFNB1, EFNA2 and EFNB3

4/8 fetal bone samples showed a cytoplasmic expression of EFNB1 (Fig. 2A), while no EFNB1 protein was found in the adult bone (Fig. 2B). The EFNB1 immunoreactivity was largely confined to the osteoblasts of fetal bone and to the tumor cells around the bone formations. In contrast to adult bone, all osteosarcoma tissue samples showed an EFNB1 protein expression (Fig. 2C,D). A significant upregulation of EFNB1 in osteosarcomas compared to non-neoplastic bone could be confirmed at the protein level (p=0.000054). Like EphA2 receptor, the EFNB1 ligand appeared de novo expressed in osteosarcoma primary tumors, but was significantly downregulated in the metastases as compared to conventional osteosarcomas (p=0.015). EFNA2 (Fig. 3A,B) and EFNB3 (Fig. 3C,D) showed no significant differences between conventional osteosarcomas and metastases (p=0.3 and p=1, respectively). The EFNA2, EFNB1 and EFNB3 immunoreactivity was localized to the cytoplasm of the tumor cells. Interestingly, an expression of EFNA2 and EFNB3 was also found in the endothelial cells of tumor blood vessels (Fig. 2B, D arrows).

EFNA1-mediated EphA2 receptor phosphorylation and internalization

The deregulation of EFNA1 or EphA2 and the EFNA1-EphA2 ligand-receptor signaling plays an important role in tumor development and metastasis in several carcinomas. To investigate receptor protein could be detected in the cell lines (Fig. 4A). SaOS2 expressed the highest level of EphA2, while only a weak EphA2 expression could be observed in HOBc. EFNA1 was detected at nearly similar levels in all cell lines, with slightly lower levels in ZK58, SaOS2 and HOBc.

EFNA1-mediated aggregation of EphA2 receptors stimulates receptor internalization and degradation in breast and prostate carcinomas [START_REF] Macrae | A conditional feedback loop regulates Ras activity through EphA2[END_REF][START_REF] Pratt | Activation of the EphA2 tyrosine kinase stimulates the MAP/ERK kinase signaling cascade[END_REF] . Because of the high EphA2-expression in SaOS2 and MNNG/HOS, these cell lines were used for further analysis. The EphA2 subcellular localization without addition of the soluble ligand revealed a membranous expression at the sites of SaOS2 cell-cell contact and enrichment within membrane ruffles (Fig. 4B). In MNNG/HOS cells, EphA2 was mainly concentrated at sites of cell-cell contact (Fig. 4C). Analysis of EphA2 localization following the addition of EFNA1/F c revealed a redistribution of the EphA2 receptor from the plasma membrane within 10min after treatment of SaOS2 and MNNG/HOS cells (Fig. 4B,C). EphA2 immunoreactivity as measured by Western blot decreased within one hour following EphrinA1/F c exposures and 70-90% of the EphA2-protein was lost after 2h in SaOS2 and MNNG/HOS cells (Fig. 5A,B).

EphA2 is enriched at cell-cell contacts in non-transformed epithelial cells where it binds the ligand and becomes phosphorylated [START_REF] Zantek | Ecadherin regulates the function of the EphA2 receptor tyrosine kinase[END_REF] . In carcinoma cells, EphA2 often remains non-phosphorylated because overexpression is accompanied by an altered EphA2 localization resulting in a failure to bind the EFNA1 ligand [START_REF] Zantek | Ecadherin regulates the function of the EphA2 receptor tyrosine kinase[END_REF] . However, unlike other tyrosine receptor kinases, EphA2 receptors have a kinase activity independent of phosphorylation and are even assumed to participate in prostate cancer metastasis [START_REF] Taddei | Kinase-dependent and -independent roles of EphA2 in the regulation of prostate cancer invasion and metastasis[END_REF][START_REF] Zantek | Ecadherin regulates the function of the EphA2 receptor tyrosine kinase[END_REF] . In SaOS2 and MNNG/HOS cells the EphA2 receptor is overexpressed and localized in membrane ruffles and at cell-cell-contacts. presence of the EFNA1/F c soluble ligand, because we showed that both ligand and receptor are present in osteosarcomas. In both osteosarcoma cell lines, an increase in phospho-tyrosine protein level was detectable 2min after binding of the soluble ligand (Fig. 5A,B). After two hours, only weak phosphorylation could be detected, which was consistent with the internalization of the EphA2 receptor following ligand binding. These results suggested that in osteosarcoma cells the interaction between EphA2 receptor and EFNA1 ligand, both overexpressed in tumor samples, might result in phosphorylation and activation of the EphA2 receptor.

EFNA1-ligand binding to the EphA2 receptor induces activation of MAPK signaling

Previous reports suggested that in breast and prostate carcinoma cells ephrin receptor-ligand interaction results in receptor phosphorylation and downregulation of phospho-Erk1/2 levels through a negative feedback-loop [START_REF] Macrae | A conditional feedback loop regulates Ras activity through EphA2[END_REF] . In contrast, Pratt et al. 2002 found an increased Erk1/2 activity after treatment with EFNA1/F c in MDA-MB-231 breast cancer cells [START_REF] Pratt | Activation of the EphA2 tyrosine kinase stimulates the MAP/ERK kinase signaling cascade[END_REF] . To determine the effects of EphA receptor activation following ligand application in osteosarcomas, we assessed Ras-MAPK-signaling through Western blot analysis following EFNA1/F c ligand binding. In SaOS2 and MNNG/HOS cells Erk1/2 phosphorylation increased within 2min after EFNA1 ligand treatment, peaked at 2h and 30min, respectively and then slowly declined (Fig. 5A,B). These data clearly demonstrate that elevated levels of both EFNA1 ligand and EphA2 receptor in osteosarcomas are likely to contribute to an increased mitogenic signaling via the Ras-MAPK pathway.

EFNA1-EphA2 signaling enhances cell migration in osteosarcoma cells

Previous reports have suggested an involvement of the EphA receptor class in breast cancer cell migration and invasion [START_REF] Fang | A kinase-dependent role for EphA2 receptor in promoting tumor growth and metastasis[END_REF] . Therefore we tested the effect of EphA2 activation by EFNA1/F c on anchorage-independent and anchorage-dependent growth and migration of EphA2-expressing cells. MNNG/HOS cells were treated with 1µg/ml EFNA1/F c for 96h and cell growth was analyzed. MNNG/HOS cells showed a small increase in growth on plastic 96h after EFNA1/F c -treatment (Fig. 6A). In addition, MNNG/HOS cells also showed a 20% increase in anchorage-independent growth, suggesting a limited growth stimulatory role of EFNA1-EphA2 signaling (Fig. 6B). For migration studies, MNNG/HOS cells were treated with 1µg/ml EFNA1/F c up to 48h (Fig. 6C). A slight increase in migration using the soluble ligand compared to untreated cells was observed after 24h and 48h.

EFNA1/F c mediated stimulation of EphA2 induces autoregulation of EphA2 and EFNA1

Earlier investigations had suggested that EphA2 expression can be stimulated via EGFR and the downstream Mek/Erk signaling in the human epidermal carcinoma cell line A431 and in the head and neck carcinoma cell line HN5 [START_REF] Larsen | Activation of the EGFR gene target EphA2 inhibits epidermal growth factor-induced cancer cell motility[END_REF] . Our investigations in osteosarcoma cells now revealed a role for the EphA2 receptor itself in the activation of this pathway. Therefore we investigated whether the EphA2 and EFNA1 genes might exert an autoregulation following EFNA1 ligand binding. Treatment of MNNG/HOS cells with the ligand induced EFNA1 mRNA after 5min and the elevated levels persisted up to 48h (Fig. 7A). EphA2 mRNA showed a slight upregulation after 1h in MNNG/HOS cells, however overall the induction was weak (Fig. 7A). These results indicate a potential autoregulatory loop, which increases mainly EFNA1 mRNA levels in osteosarcoma cells upon ligand binding. To define to what extent the EFNA1 and EphA2 autoregulation was MAPK-dependent we treated MNNG/HOS cells with the Mek-inhibitor U0126 and measured protein and mRNA expression of EphA2 and EFNA1 by Western blot and RT-PCR, respectively. Both EphA2 and EFNA1 protein and mRNA were reduced through treatment with U0126 (Fig. 7B).

Discussion

Today, a number of molecular markers have been identified in osteosarcomas, some of which have the potential to act as novel therapeutic targets [START_REF] Clark | A review of clinical and molecular prognostic factors in osteosarcoma[END_REF] . For example VEGF expression in osteosarcoma correlates with the level of microvascular density and the presence of pulmonary metastases [START_REF] Kaya | Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis[END_REF] . In addition, increased levels of MMP9 are associated with increased metastatic potential and the receptor tyrosine kinases ErbB2/Her-2 and c-Met are overexpressed and/or amplified in bone tumors [START_REF] Onda | ErbB-2 expression is correlated with poor prognosis for patients with osteosarcoma[END_REF][START_REF] Coltella | Role of the MET/HGF receptor in proliferation and invasive behavior of osteosarcoma[END_REF][START_REF] Kido | Overexpression of matrix metalloproteinase (MMP)-9 correlates with metastatic potency of spontaneous and 4-hydroxyaminoquinoline 1-oxide (4-HAQO)-induced transplantable osteosarcomas in rats[END_REF] . Current treatment procedures involve surgical resection and neoadjuvant chemotherapy involving methotrexate, cisplatin, and adriamycin and have resulted in tumor-type dependent response rates up to 80%.

Nevertheless, a significant fraction of patients (app. 30%) die because of metastases.

Therefore, an improved understanding of the molecular mechanisms driving osteosarcoma progression and metastasis and the establishment of novel diagnostic and therapeutic targets is urgently needed.

Using microarray analysis, we detected a distinct expression profile for several ephrins and their receptors in human osteosarcoma tissue samples when compared to non-neoplastic human bone. The Ephrin receptors and ligands are associated with tumor development, progression and metastasis in numerous human cancers. The EphA2 receptor is upregulated in colorectal, lung and breast cancer, as well as in melanomas and is often found to correlate with tumor stage [START_REF] Saito | Expression of EphA2 and E-cadherin in colorectal cancer: correlation with cancer metastasis[END_REF] . Several publications described a role in angiogenesis, migration and tumor compartimentalization [START_REF] Brantley | Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo[END_REF][START_REF] Brantley-Sieders | EphA2 receptor tyrosine kinase regulates endothelial cell migration and vascular assembly through phosphoinositide 3-kinase-mediated Rac1 GTPase activation[END_REF] . However, a functional diversity was observed within the ephrin family. For example, an oncogenic role was found for the EphA/EFNA ligand-receptor system but a tumor suppressive role for EphB receptors [START_REF] Batlle | EphB receptor activity suppresses colorectal cancer progression[END_REF][START_REF] Noren | Paradoxes of the EphB4 receptor in cancer[END_REF] . In osteosarcoma, ephrin expression was first described by Varelias et al. (2002), who found distinct expression profiles for several ephrins within tumor samples and osteosarcoma cell lines [START_REF] Varelias | Human osteosarcoma expresses specific ephrin profiles: implications for tumorigenicity and prognosis[END_REF] . Using microarray analysis and immunohistochemistry we extended these data and confirmed high overexpression of the EphA2 receptor and the EFNA1 ligand in the majority of human receptor interaction in osteosarcoma. Our microarray data also imply upregulation of the EphA3 receptor in osteosarcomas. This suggests that EFNA1 might also interact with this receptor, however at the moment an involvement of EphA3 in osteosarcoma is unknown.

In cells derived from breast cancer and other carcinomas, controversial results were also reported with respect to signaling cascade activation upon EFNA1-mediated EphA2 stimulation. Macrae et al. found a negative feedback loop involving transcriptional regulation of EphA2 and suppression of Ras function [START_REF] Macrae | A conditional feedback loop regulates Ras activity through EphA2[END_REF] , also Wykosky et al. recently suggested that soluble EFNA1 upon interaction with EphA2 suppresses Ras/MAPK signaling in cells derived from glioblastoma multiforme 30 . In contrast, others suggested activation of MAPK signaling through the EFNA1 ligand [START_REF] Pratt | Activation of the EphA2 tyrosine kinase stimulates the MAP/ERK kinase signaling cascade[END_REF] . Stimulation of osteosarcoma cells with the soluble EFNA1 ligand induced MAPK signaling as indicated by Erk1/2 phosphorylation. Activation of MAPK signaling and the control of downstream effectors such as Fos and Jun have key cellular functions such as proliferation and migration and are linked to osteosarcoma development [START_REF] Wagner | Fos/AP-1 proteins in bone and the immune system[END_REF] . In contrast to the data described in breast and also in prostate cancer cells, applications of the EFNA1 ligand to MNNG/HOS cells induced migration and to a lower extend also proliferation. This observation indicates that EFNA1 EphA2 upregulation in osteosarcoma might be positively involved in the known oncogenic pathways responsible for osteosarcoma formation and progression. Interestingly our data also suggest a positive feedback loop including the EFNA1 EphA2 ligand-receptor system and acting via 2005 extended these findings and demonstrated a repulsive response of PC3 prostate cancer cells following EFNA1 application; however in the absence of FAK dephosphorylation [START_REF] Parri | EphrinA1 repulsive response is regulated by an EphA2 tyrosine phosphatase[END_REF] .

These observations are in line with our data indicating a migratory response of osteosarcoma cells in vitro. We were however unable to detect FAK dephosphorylation following EFNA1 application (data not shown). Thus it remains to be determined to what extend FAK is involved in EFNA1-induced migration of osteosarcoma cells and suggests a totally different role in growth and migration control of EFNA1 ligand-EphA2 receptor interaction in bone tumors as compared to epithelial tumors. The strong upregulation of EFNA1 ligand and EphA2 receptor in osteosarcomas marks the ephrins as interesting potential therapeutic target. Noberini et al (2008) showed, that small molecules are able to selectively inhibit the interaction of EphA2 with its natural ligand [START_REF] Noberini | Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors[END_REF] . These compounds also inhibit ephrin-induced phosphorylation of EphA2 without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Thus, further analysis on the role of EFNA1 and EphA2 in osteosarcomas might determine whether EphA2 is a possible therapeutic target for the treatment with small inhibitory molecules in osteosarcoma patients. Table 1: Tissue from patients with osteosarcoma used for microarray and immunohistochemical analyses. * used for microarray and immunohistochemical analyses. +: not used for immunohistochemical staining. f = female. m = male.

Number

  growth medium (PromoCell, Heidelberg, Germany) and McCoys5A media (Gibco) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin, respectively. The osteosarcoma cell lines HOS, MNNG/HOS, OST, SJSA, MG63 and ZK58 were a gift from Prof. Dr. G. Gaedicke, Clinic for General Pediatrics and Experimental Oncology, Charité Universitätsmedizin Berlin, Berlin, Germany and cultured in RPMI1640

  2. A staining intensity score (SI) was established to measure the intensity of positively stained cells: 0 = no expression, 1 = weak expression, 2 = moderate expression, 3 = strong expression. The combined immunoreactivity score (IRS) value was calculated through PPxSI (maximum ). Statistical analyses were performed executing the Mann-Whitney-U-Test and Bonferroni correction p-values using SPSS software 13.0. Differences were considered significant at p < 0.05.

  and EphA7 were below background. Among the B-ligands, only EFNB2 showed a high expression in osteosarcomas and HOBc, whereas expression below background was observed for EFNB1, EFNB3 and EphB1. For the B-class receptors EphB2, EphB3 (6/8 osteosarcoma samples), EphB4 and EphB6 (5/8 osteosarcoma samples) an expression level above background in osteosarcomas, but no differential expression was detected. With the exception of EphB4, ephrinB receptor expression in HOBc was below background.

  potential of ephrin signaling in osteosarcomas, we further analyzed EFNA1 and EphA2 expression and regulation in vitro. Osteosarcoma cell lines were tested for their EFNA1 and EphA2 protein expression. Using Western blot, different levels of EFNA1 ligand and EphA2

  the ligand was also detected in fetal and adult normal tissue but strongly upregulated in tumor tissue, de novo expression was detected for the EphA2 receptor in tumor tissue. This indicates that during osteosarcoma development a potential change in ligand-receptor interaction within the A-class of ephrins might occur. The EFNA1 ligand is able to bind the EphA2 receptor with high affinity[START_REF] Bartley | B61 is a ligand for the ECK receptor protein-tyrosine kinase[END_REF] , yet binds also other members of the Aclass receptors. Absence of EphA2 from fetal and adult normal human bone, but a strong upregulation during tumor development suggests a specific role for the EFNA1 ligand-EphA2

  . In prostate cancer cells the EFNA1 ligand induces dephosphorylation of focal adhesion kinase (FAK) followed by inhibition of cell spreading and migration[START_REF] Miao | Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation[END_REF] . Parri et al.

Aknowledgement

  This study was supported by the national genome research network (Nationales Genomforschungsnetz, NGFN, SIPAGE) and by the German Federal Ministry of Education and Research (BMBF) through the program FORSYS Partner (grant 0315261).

Figure 1 :

 1 Figure 1: Expression of EFNA1 ligand and EphA2 receptor in non-neoplastic human

Figure 2 :

 2 Figure 2: Expression of EFNB1 ligand in non-neoplastic human bone and in

Figure 3 :

 3 Figure 3: Expression of EFNA2 ligand and EFNB3 ligand in conventional and

Figure 4 :

 4 Figure 4: Internalization of EphA2 receptor following EFNA1 ligand application. (A) left

Figure 5 :

 5 Figure 5: Activation of P-Erk1/2 via EFNA1-stimulated receptor activation. SaOS2 (A)

Figure 6 :

 6 Figure 6: Receptor stimulation via EFNA1 ligand has a limited effect onto cell growth.

Figure 7 :

 7 Figure 7: Autoregulation of EFNA1 ligand and EphA2 receptor through EFNA1/F c

Table 2 :

 2 Genes with at least 10 fold overexpression (A) or suppression (B) in osteosarcoma compared to HOBc. Fc: fold change. ID: Affymetrix identity.

		Type	Sex Age Localization
	OS1*	conventional	f	15	Femur
	OS2	conventional	m	16	Tibia
	OS3	conventional	m	74	Humerus
	OS4*	metastasis	m	40	Lung
	OS5	metastasis	m	59	Lung
	OS6*	metastasis	f	21	Lung
	OS7	metastasis	f	20	Lung
	OS9*	conventional	m	23	Tibia
	OS10	metastasis	m	24	Lung
	OS11	metastasis	f	45	Lung
	OS12*	metastasis	m	67	Lung
	OS14	conventional	m	56	Scapula
	OS15*	conventional	f	74	Femur
	OS16* +	conventional	f	37	Lung
	OS17	conventional	m	54	Tibia
	OS18*	conventional	m	7	Femur
	OS20	conventional	m	33	Femur
	OS21	conventional	m	59	Femur
	OS24*	conventional	m	17	Femur

A:

Genes with at least 10 fold overexpression in osteosarcomas compared to HOBc

	209087_x_at	MCAM	12.2 melanoma cell adhesion molecule
	219478_at	WFDC1	12.1 WAP four-disulfide core domain 1
	266_s_at	CD24	12.0 CD24 molecule
	ID 202746_at	Symbol ITM2A	fc 11.4 integral membrane protein 2A Gene
	207370_at 208146_s_at	IBSP CPVL	41.7 integrin-binding sialoprotein 11.1 carboxypeptidase, vitellogenic-like
	201721_s_at 219607_s_at	LAPTM5 29.8 lysosomal multispanning membrane protein 5 MS4A4A 11.0 membrane-spanning 4-domains, subfamily A, member 4
	218678_at 211343_s_at COL13A1 10.7 collagen, type XIII, alpha 1 NES 28.7 nestin
	217028_at 201720_s_at 204712_at 202747_s_at 214574_x_at 209301_at	CXCR4 LAPTM5 23.4 lysosomal multispanning membrane protein 5 25.7 chemokine (C-X-C motif) receptor 4 WIF1 22.2 Wnt inhibitory factor-1 F o r ITM2A 10.6 integral membrane protein 2A LST1 10.2 leukocyte specific transcript 1 CA2 10.1 carbonic anhydrase II F o r
	203305_at 213068_at 215049_x_at	F13A1 DPT CD163	P P 20.6 coagulation factor XIII, A1 polypeptide e e 19.6 dermatopontin 19.0 CD163 molecule e r e r
	201117_s_at	CPE	18.6 carboxypeptidase E R R
	204379_s_at	FGFR3	17.8 fibroblast growth factor receptor 3 e e
	201116_s_at 202878_s_at	CPE CD93	17.5 carboxypeptidase E 15.9 Complement component 1, q subcomponent, receptor 1 v i e v i e
	207977_s_at	DPT	15.8 dermatopontin	w w
	215783_s_at	ALPL	14.5 alkaline phosphatase, liver/bone/kidney
	217897_at	FXYD6	14.5 FXYD domain containing ion transport regulator 6
	206488_s_at	CD36	13.7 CD36 molecule (thrombospondin receptor)
	203645_s_at	CD163	13.6 CD163 molecule
	221558_s_at	LEF1	13.5 lymphoid enhancer-binding factor 1
	218002_s_at	CXCL14 13.4 chemokine (C-X-C motif) ligand 14
	202023_at	EFNA1	13.0 ephrin-A1

B:

Genes with at least 10 fold suppression in osteosarcomas compared to HOBc

	201109_s_at THBS1	12.7		thrombospondin 1
	ID 212344_at	Symbol SULF1	fc 12.6		Gene sulfatase 1
	209395_at 214767_s_at HSPB6 CHI3L1	123.9 chitinase 3-like 1 (cartilage glycoprotein-39) 12.3 heat shock protein, alpha-crystallin-related, B6
	209396_s_at CHI3L1 205397_x_at SMAD3	87.0 12.2		chitinase 3-like 1 (cartilage glycoprotein-39) SMAD family member 3
	205792_at 212230_at	WISP2 PPAP2B	65.4 12.1		WNT1 inducible signaling pathway protein 2 phosphatidic acid phosphatase type 2B
	203851_at 202275_at	IGFBP6 G6PD	37.6 12.0		insulin-like growth factor binding protein 6 glucose-6-phosphate dehydrogenase
	218468_s_at GREM1 208502_s_at PITX1	31.8 11.7		gremlin 1, cysteine knot superfamily, homolog paired-like homeodomain 1
	204948_s_at FST 212143_s_at IGFBP3 F 27.2 23.3 o r 221111_at IL26 11.4 202434_s_at CYP1B1 11.3 202436_s_at CYP1B1 11.3 F o r	(Xenopus laevis) follistatin insulin-like growth factor binding protein 3 interleukin 26 cytochrome P450, family 1, subfamily B, polypeptide 1 cytochrome P450, family 1, subfamily B, polypeptide 1
	206157_at 212992_at	PTX3 AHNAK2 11.2 22.6 P pentraxin-related gene, rapidly induced by IL-1 beta AHNAK nucleoprotein 2 P
	201107_s_at THBS1 204037_at LPAR1	21.6 11.0	thrombospondin 1 e lysophosphatidic acid receptor 1 e
	209355_s_at PPAP2B 207345_at FST	20.3 10.9		phosphatidic acid phosphatase type 2B e r follistatin e r
	203963_at 202949_s_at FHL2 CA12 202765_s_at FBN1 204036_at LPAR1	20.0 19.4 10.5 10.4		carbonic anhydrase XII four and a half LIM domains 2 R e fibrillin 1 R lysophosphatidic acid receptor 1 e
	204508_s_at CA12 202912_at ADM 205924_at RAB3B	18.2 18.0 10.3		carbonic anhydrase XII adrenomedullin v i e RAB3B, member RAS oncogene family v i e
	209687_at	CXCL12	17.6		chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) w w
	205475_at	SCRG1	16.6		scrapie responsive protein 1
	206172_at	IL13RA2 16.0		interleukin 13 receptor, alpha 2
	201108_s_at THBS1	15.2		thrombospondin 1
	213112_s_at SQSTM1 14.2		sequestosome 1
	203939_at	NT5E	13.4		5'-nucleotidase, ecto (CD73)
	212226_s_at PPAP2B	12.9		phosphatidic acid phosphatase type 2B

Table 3 : Ephrin expression profile in osteosarcoma tissue samples compared to the non neoplastic osteoblast primary culture HOBc.

 3 Relative expression levels below 1,000 were defined as background. OS: osteosarcoma.

	Relative	A-Ligand	A-Receptor	B-Ligand	B-Receptor
	Expression
	significant	EFNA1	EphA1	EFNB2	EphB2
	(>1,000)	(HOBc below 1,000, 8/8	(1/8 OS above 1000,	(all probes above 1,000)	(HOBc below 1,000, 8/8
		OS above 1,000)	HOBc below 1,000)		OS above 1,000)
		EFNA4	EphA2		EphB3
		(1/8 OS above 1,000,	(all probes above 1,000)		(HOBc and 2/8 OS below
		HOBc below 1,000)	EphA3		1000)
			(HOBc and 1/8 OS below		EphB4
			1,000)		(all probes above 1,000)
			EphA4		EphB6
			(1/8 OS above 1, 000,		(5/8 OS above 1,000)
			HOBc below 1,000)
			EphA5
			(1/8 OS above 1000,
			HOBc below 1,000)
	not	EFNA2	EphA7	EFNB1	EphB1
	significant	(all probes below 1,000)	(all probes below 1,000)	(all probes below 1,000)	(all probes below 1,000)
	(<1,000)	EFNA3		EFNB3
		(all probes below 1,000)		(all probes below 1,000)
		EFNA5
		(all probes below 1,000)

Table 4 : Immunohistochemical analyses of EFNA1, EFNA2, EFNB1, EFNB3 and EphA2 in human osteosarcoma and non-neoplastic bone tissue samples.

 4 The immunohistochemical staining was calculated through the immunoreactive score (IRS) of the whole tissue sample. PP: percentage of positively stained cells. SI: intensity of positively stained cells.

		Osteosarcoma			Bone
		Conventional	Metastases Total	Fetal	Adult	Total
	SI	2.6	2.6	2.6	0.9	1.9	1.5
	EFNA1
	PP	3.8	3.3	3.6	1.3	2.4	1.9
	ligand
	IRS	10.1	9.1	9.7	2.6	4.7	3.8
	SI	2.1	2.1	2.1
	EFNA2
	PP	3.3	2.9	3.1
	ligand
	IRS	7.9	6.1	7.2
	SI	2.7	1.9	2.4	1.6	0.0	0.7
	EFNB1
	PP	3.0	2.1	2.6	1.0	0.0	0.4
	ligand
	IRS	8.1	4.3	6.5	3.3	0.0	1.4
	SI	2.0	2.0	2.0
	EFNB3
	PP	3.0	2.4	2.8
	ligand
	IRS	6.9	5.7	6.4
	SI	1.0	1.3	1.1	0.0	0.0	0.0
	EphA2
	PP	1.8	1.3	1.6	0.0	0.0	0.0
	receptor
	IRS	2.4	2.6	2.5	0.0	0.0	0.0

Published on behalf of the British Division of the International Academy of Pathology Histopathology


Marco Schumacher

Anja Hautzinger

Annette Rossmann

Susanne Holzhauser

Daniela Popovic

Anke Hertrampf

Sabine Kuntz

Michael Boll

Uwe Wenzel
email: uwe.wenzel@ernaehrung.uni-giessen.de

Chrysin blocks topotecan-induced apoptosis in

Keywords: ABC-transporters, apoptosis, caspases, topotecan, chrysin, ß-catenin

teaching and research institutions in France or abroad, or from public or private research centers.

MRP, multidrug resistance-associated protein; NEAA, nonessential amino acids; Pgp, P-phosphoglycoprotein; pH i , intracellular pH; TBS-T, Tris-buffered saline-Tween; Z-DEVD-AMC, carboxybenzoxy-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarine.

Introduction

Phosphoglycoprotein-170 (P-gp), multidrug resistance-associated protein-2

(MRP-2), and breast cancer resistance protein (BCRP) are ATP-binding cassette (ABC) transporters located at the apical membrane of enterocytes that may drive their substrates from inside the cell back into the intestinal lumen [START_REF] Dietrich | ABC of oral bioavailability: transporters as gatekeepers in the gut[END_REF][START_REF] Chan | The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability[END_REF]. Those substrates encompass various synthetic and natural lipophilic compounds [START_REF] Chan | The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability[END_REF][START_REF] Suzuki | Single nucleotide polymorphisms in multidrug resistance associated protein 2 (MRP2/ABCC2): its impact on drug disposition[END_REF][START_REF] Doyle | Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2)[END_REF].

Moreover, the inhibitor specificities of P-gp, BCRP and MRP-2 were shown to be highly overlapping, and general inhibitors of ABC transporters were more lipophilic and aromatic than specific inhibitors and non-inhibitors [START_REF] Matsson | Identification of novel specific and general inhibitors of the three major human ATP-binding cassette transporters P-gp, BCRP and MRP2 among registered drugs[END_REF]. Whereas the physiological role of ABC transporters might be to reduce the body load of potentially harmful compounds [START_REF] Chan | The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability[END_REF] their overexpression in tumors confers a multi-drug resistance (MDR) phenotype to cancer cells [START_REF] Murakami | Intestinal efflux transporters and drug absorption[END_REF][START_REF] Zhou | Structure, function and regulation of P-glycoprotein and its clinical relevance in drug disposition[END_REF]. In vitro screening has identified a number of pharmaceuticals which can selectively inhibit pumps such as P-gp, by virtue of their being substrates for these pumps [START_REF] O'connor | The pharmacology of cancer resistance[END_REF]. However, several generations of pharmaceutical inhibitors of P-gp have been examined in preclinical and clinical studies and these circumvention trials have largely failed to demonstrate the anticipated increase in therapeutic efficacy [START_REF] O'connor | The pharmacology of cancer resistance[END_REF]. At least in part this may be due to the redundancy of efflux pumps in resistant cancer cells but transport independent mechanisms could also account for this "multifactorial multidrug resistance" [START_REF] Su | Inhibition of efflux transporter ABCG2/BCRP does not restore mitoxantrone sensitivity in irinotecan-selected human leukemia CPT-K5 cells: evidence for multifactorial multidrug resistance[END_REF].

Besides the affection of ABC transporters by drugs they can be modulated by ingredients of plant foods or herbal products such as the flavonoids [START_REF] Alvarez | Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response[END_REF]. In that context, influences of several flavonoids have been demonstrated to be exerted at the level of expression, posttranslational modification, or transport function [START_REF] Alvarez | Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response[END_REF]. The latter can be influenced through an effect on the transporters´ATPase activity. Both, stimulation, e.g. by glabridin, and inhibition, e.g. by genistein and quercetin, have been described [START_REF] Cao | Role of P-glycoprotein in the intestinal absorption of glabridin, A c c e p t e d M a n u s c r i p t 23 an active flavonoid from the root of Glycyrrhiza glabra[END_REF][START_REF] Pulido | Interaction of enrofloxacin with breast cancer resistance protein (BCRP/ABCG2): Influence of flavonoids and role in milk secretion in sheep[END_REF]. Another possible mechanism for flavonoid-mediated inhibition of ABC-transporters is by competitive inhibition when flavonoids act as substrates [START_REF] Alvarez | Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response[END_REF][START_REF] Wang | Involvement of Pglycoprotein in regulating cellular levels of Ginkgo flavonols: Quercetin, kaempferol, and isorhamnetin[END_REF]. Whereas the free aglycones appear to be mainly transported by P-gp, intracellularly formed glucuronidated and sulfated conjugates can be transported by MRP-2 and BCRP [START_REF] Alvarez | Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response[END_REF][START_REF] Vaidyanathan | Transport and metabolism of the tea flavonoid (-)epicatechin by the human intestinal cell line Caco-2[END_REF][START_REF] Brand | Metabolism and transport of the citrus flavonoid hesperetin in Caco-2 cell monolayers[END_REF].

In the present study, we tested in Caco-2 human colon carcinoma cells the influence of the flavonoid chrysin, contained in honey, propolis and fruit skin [START_REF] Alvarez | Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response[END_REF], on the transport of fluorescent substrates for P-gp, MRP-2, and BCRP, respectively. By Western Blotting we determined the impact of chrysin on the expression level of the transporters. The net effect on transport activity was estimated then by measuring the accumulation of the anti-tumor drug topotecan. DNA-fragmentation was quantified as a measure of topotecan-induced apoptosis and estimations of caspase-activities and pH i served the discovery of the mechanisms underlying alterations in the apoptosis rate. Immunocytochemistry detecting ß-catenin in fixed Caco-2 cells was finally performed in order to associate the effects of chrysin on apoptosis and differentiation with cellular localization of this transcription factor whose deregulation is intimately linked to tumor formation [START_REF] Näthke | Cytoskeleton out of the cupboard: colon cancer and cytoskeletal changes induced by loss of APC[END_REF].

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

Materials and methods

Materials

Chrysin, fluorescent substrates (except CMFDA) and inhibitors of ABC transporters were purchased from Sigma (Deisenhofen, Germany). CMFDA was from Invitrogen (Karlsruhe, Germany). Cell culture plates were from Renner (Dannstadt, Germany). All other materials needed for cell culture, SYTOX-Green, BCECF-AM, and EnzChek Kit for measuring caspase activities were from Invitrogen (Karlsruhe, Germany). Topotecan was from Molekula (Lisses, France). Quadriperm wells were from Greiner Bio-one (Frickenhausen, Germany). DNase free RNase was from MBI Fermentas (Heidelberg, Germany). Caspase-8 substrate Ac-IETD-AFC, caspase-8

inhibitor Ac-IETD-CHO, and mouse-anti-ß-catenin were from BD Biosciences (Heidelberg, Germany). Rabbit-anti-P-gp, mouse-anti-MRP-2, mouse-anti-BCRP, mouse-anti-actin, and horseradish peroxidase labeled goat-anti-mouse-IgG and goatanti-rabbit-IgG antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). Biotinylated anti-mouse-IgG was obtained from Vector Laboratories (Munich, Germany) and FluoroLink Cy5 labeled streptavidin by Amersham Biosciences (Freiburg, Germany). The Bio-Rad-Protein-Assay was purchased from BioRad (Munich, Germany). All other chemicals were obtained from Sigma (Deisenhofen, Germany).

Cell culture

Caco-2 cells (HTB 37, passage 31) were provided by American Type Culture Collection (ATCC) and were used between passages 40 and 65. Cells were cultured and passaged in DMEM supplemented with 10% FCS, 2 mM glutamine, 1% MEM non essential amino acids and 70 µg/ml gentamycin in a humidified incubator at 37°C under an atmosphere of 5% CO 2 . Cells were passaged at preconfluent densities by the use of a solution containing 0.05 % trypsin and 0.5 mM EDTA.

P-gp, MRP-2 and BCRP mediated transport of topotecan

For estimation of the ABC-transporters activities the fluorescent dyes rhodamine 123, CMFDA, and Hoechst 33342 were used at 1 µM concentrations as substrates for P-gp [START_REF] Van Der Sandt | Specificity of doxorubicin versus rhodamine-123 in assessing P-glycoprotein functionality in the LLC-PK1, LLC-PK1:MDR1 and Caco-2 cell lines[END_REF], MRP-2 [START_REF] Förster | Compound profiling for ABCC2 (MRP2) using a fluorescent microplate assay system[END_REF], and BCRP [START_REF] Hooijberg | Online fluorescent method to assess BCRP/ABCG2 activity in suspension cells[END_REF], respectively. Contributions of the respective transporters for overall transport were verified by using the selective inhibitors verapamil, MK-571, and fumitremorgin C [START_REF] Li | Involvement of Pglycoprotein, multidrug resistance protein 2 and breast cancer resistance protein in the transport of belotecan and topotecan in Caco-2 and MDCKII cells[END_REF]. Cells were exposed to substrates in Krebs buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl 2 , 1 mM MgSO 4 , 10 mM glucose, 10 mM HEPES, pH 7.4) for 3 h over which transport of the substrates was linear. Thereafter cells were washed twice with ice-cold Krebs buffer and fluorescence of rhodamine 123 and CMFDA was measured at emission of 538 nm after excitation at 485 m, whereas emission of Hoechst 33342 was estimated at 460 nm after excitation at 355 nm using a microtiter plate reader (Fluoroskan Ascent, Thermo Electron, Dreieich, Germany). The accumulation of topotecan was assessed according to its fluorescent properties with excitation at 355 nm and emission at 538 nm [START_REF] Gryczynski | Fluorescence spectral properties of the anticancer drug topotecan by steady-state and frequency domain fluorometry with one-photon and multi-photon excitation[END_REF].

Apoptosis assays

Caspase-3 and -8 activities were determined using the EnzChek activity assay Kit according to the manufacturer´s instructions. In brief, 0.5x10 6 cells per well were A c c e p t e d M a n u s c r i p t 8 seeded onto 6-well plates and allowed to adhere for 48 h. Cells were then exposed for 24 h to the test compounds. Subsequently, cells were trypsinized, cell numbers were determined and then the cells were pelleted for 5 min at 300 x g. The pellets were suspended in 50 µl of lysis buffer and centrifuged at 45.000 x g for 5 min.

Cleavage of Z-DEVD-AMC (final concentration 100 µM) was followed in the fluorometer by excitation at 355 nm and emission of 460 nm each 20 min over 5 h.

For inhibition of caspase-3 Ac-DEVD-CHO was added. For determination of caspase-8 activities Ac-IETD-AFC (final concentration 100 µM) was used. Cleavage of the substrate was followed by measuring emission at 538 nm after excitation at 355 nm.

Nuclear fragmentation as a late marker of apoptosis was determined by staining of DNA with SYTOX-Green nucleic acid stain. Cells (1 x 10 6 ) were seeded onto glass slides and incubated in Quadriperm wells with the test compounds for 48 h. Thereafter glass slides were washed with PBS and cells were fixed with 2% paraformaldehyde. DNA was stained with 0.25 µM SYTOX-Green solution and visualized under an epifluorescence microscope (Zeiss Axioskope 2) equipped with a band path excitation filter of 470 nm and a long path emission filter of 520 nm.

Photographs were made from at least three independent cell batches and apoptotic cells were determined according to the number of cells displaying chromatin condensation and nuclear fragmentation versus total cell counts.

FACS-analysis was performed in order to quantify the amount of cells with significantly lower amounts of intact DNA which appear as a subG1-peak as a characteristic of apoptosis. Therefore, Caco-2 cells were seeded at a density of 1 x 10 6 onto 25 cm 2 culture flasks and were incubated for 48 h in the presence or the absence of the test compounds after having reached 40% confluency. Cells were trypsinized, pelleted by centrifugation at 500 x g for 3 min, washed twice with PBS and adjusted to 1 x 10 6 cells/ml. Cells were then fixed by suspending them at 4°C in

pH i -measurements

For pH i -measurements cells were loaded with BCECF by preincubating the cells with 5 µM of the lipophilic acetoxymethyl-ester (BCECF-AM) at 37°C for 45 min.

Subsequently the monolayers were washed with Krebs buffer, pH 7.4. Prior to pH imeasurements the medium was taken off from the cells and was reapplied directly after measurements. This proceeding was chosen in order to simulate the conditions used for apoptosis measurements and moreover, to neglect influences on pH i -measurements by

Western blotting

Caco-2 cells were incubated in 75 cm 2 flasks and scraped off in ice-cold PBS containing 0.2% EDTA. Cells were pelleted subsequently by centrifugation for 5 min at 300 g. The pelleted cells were dissolved subsequently in lysis buffer (10 mM HEPES, 1 mM EDTA, 1 mM DTT, 0.2% protease inhibitor cocktail, pH 7.5) and frozen overnight at -80°C. Thereafter, cells were homogenized by 20 strokes with a Teflon homogenizer. Samples were centrifuged for 20 min at 380 g to remove intact cells and the supernatant was centrifuged at 40.000 x g for 20 min in order to enrich the plasma membrane fraction. The final pellet was resuspended in membrane buffer (10 mM HEPES, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.2% protease inhibitor cocktail) and protein content was determined by the Bradford reaction.

The proteins of the membrane fractions were resolved by SDS-PAGE according to the method described by Schagger and von Jagow [START_REF] Schagger | Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa[END_REF] and were electroblotted onto PVDF membranes. Control of protein transfer and identification of the molecular weight marker proteins was achieved by Ponceau Red staining.

Thereafter, the blotting membranes were blocked for 1 h with TBS-T containing 1% milk powder and then incubated for 1 h with the primary antibodies recognizing P-gp times with TBS-T and bound antibodies were detected after 1 h incubation with horseradish peroxidase conjugated secondary antibodies (1:1000) using an ECL system according to the manufacturer´s instructions.

Immunocytochemistry

For immunofluorescent detection of ß-catenin, cells grown on poly-L-lysine coated glass slides were fixed by 2% paraformaldehyde for 10 min on ice. Thereafter, cells were incubated for 2 h in a humidity chamber with a 1:500 dilution of anti-ßcatenin. After washing four times with TBS-T, adding for 1 h biotinylated anti-mouse-IgG in a 1:200 dilution and washing twice with TBS-T and TBS, respectively, bound antibodies were detected using FluoroLinkCy5 labeled streptavidin in a 1:500 dilution. Cell nuclei were stained by 0.25 µM SYTOX-Green solution during a 15 min incubation. Antibody staining was detected at an emission of 645-720 nm after excitation at 633 nm, and SYTOX-Green nucleic acid stain at emissions of 500-535 nm after excitation at 488 nm by confocal laser scanning microscopy (DMIRBE Leica, Wetzlar, Germany).

Calculations and statistics

All calculations were performed by using Prism 4.01 (Graph PAD, Los Angeles, CA). For each variable 4-8 independent experiments were carried out. Data are given as the mean  S.E.M. Variance analysis between groups was performed by One-way ANOVA and significance of differences between control and treated cells were determined by a Tukey´s test.

Chrysin inhibits P-gp, MRP-2, and BCRP in Caco-2 cells and increases the accumulation of topotecan

To test the influence of chrysin on ABC-transporters its impact on the accumulation of P-gp substrate rhodamine 123, the MRP-2 substrate CMFDA, and the BCRP-substrate Hoechst 33342 were estimated. Chrysin dose-dependently increased the accumulation of the P-gp substrate rhodamine 123, the MRP-2 substrate CMFDA, and the BCRP-substrate Hoechst 33342 with very similar EC 50values around 50 µM (Fig. 1A). Maximum accumulations of the substrates in the presence of 250 µM chrysin, moreover, were very similar to those achieved by the specific transport-inhibitors verapamil for P-gp, MK-571 for MRP-2, and fumitremorgin for BCRP, respectively (Fig. 1B). Moreover, in the presence of specific inhibitors 250 µM chrysin was not able to enhance the accumulation of the fluorescent dyes significantly (data not shown), suggesting that transport of the substrates by the respective transporters under the conditions used is rather specific.

None of the three inhibitors was able to increase the accumulation of the topoisomerase-I inhibitor topotecan as potently as did chrysin (Fig. 2A). However, equal accumulation potency could be achieved when all three inhibitors were coadministered (Fig. 2A). Moreover, the dose-dependent accumulation increase of topotecan by chrysin revealed an EC 50 -value of 68.6  0.8 µM which is in the range of the EC 50 -values observed for chrysin with regard to the inhibition of P-gp, MRP-2, and BCRP (Fig. 2B).

A c c e p t e d M a n u s c r i p t 13

Chrysin increases the expression of ABC-transporters in Caco-2 cells

The increased accumulation of topotecan observed in the presence of chrysin suggests the flavonoid to be a MDR-reversing agent. In this regard, however, effects on the expression rates must be considered as well especially concerning longer periods of treatment. Western blotting experiments revealed that chrysin enhanced significantly the expression of all three ABC-transporters as did topotecan (Fig. 3A-C). More importantly, chrysin was able to stimulate transporter expression even in the presence of topotecan (Fig. 3A-C). Over a time period of 48 h, however, the inhibition of transporter function prevails to the enhanced expression as accumulation of topotecan was still increased by chrysin at this time point of incubation (Fig. 3D).

Chrysin inhibits potently topotecan-induced apoptosis

Surprisingly, chrysin almost completely abolished topotecan-induced chromatin condensation and DNA-fragmentation as markers of proceeded apoptosis (Fig. 4A). On the basis of these apoptosis markers dose-response relationships revealed a half maximal inhibition of topotecan-mediated apoptosis at 128 µM (Fig. 4B). Moreover, FACS analysis showed that 48% of the cell population were present in a sub-G 1 -peak caused by fragmented DNA in apoptotic cells after topotecan treatment (Fig. 4C). Accordingly, the FACS analysis verified the results obtained by microscopic assessment of apoptotic nuclei. Besides the fact that chrysin almost completely inhibited topotecan-induced apoptosis when assessed by FACS, it could be shown that the flavonoid arrests cells in G 2 /M as this population increases from 8% in the control to 25% in chrysin treated cells (Fig. 4C). Finally, not only apoptosis as a cancer preventive parameter was inhibited but also differentiation. Whereas efficiently inhibited differentiation when triggered by butyrate (Fig. 4D).

Chrysin inhibits the topotecan-mediated activation of caspases-3 and -8

According to data showing that transport-independent effects of P-gp exist on caspase-activities we tested whether up-stream caspase-8 and effector-caspase-3

were modulated by chrysin. Indeed chrysin was able to potently inhibit topotecanmediated caspase-8 and caspase-3 activation (Fig. 5A). One of the postulated mechanisms through which this inhibition could be achieved is via increasing pH i .

However, chrysin did not significantly affect topotecan-induced intracellular acidification, nor did the base imidazole inhibit caspase-activation and apoptosis although it prevented the topotecan-induced pH i -decline (Fig. 5B).

Chrysin stabilizes cytosolic ß-catenin

Since both caspase-8 and -3 were shown in previous studies to be involved in processing of cytosolic ß-catenin, processed ß-catenin was detected by Western blotting (Fig. 6A) and localization of this cancer promoting transcription factor was studied by immunocytochemistry (Fig. 6B). It could be shown that topotecan cleaves ß-catenin whereas chrysin stabilizes ß-catenin in the cytosol (Fig. 6A). Moreover, chrysin caused a removal of ß-catenin from the plasma membrane into the cytosol as did topotecan and the same was visible when both compounds were combined (Fig. 6B). These results suggest that stabilized cytosolic ß-catenin might transmit the effects of chrysin on inhibition of apoptosis. In this context it was demonstrated that A c c e p t e d M a n u s c r i p t 16

Discussion

Multi-drug resistance is a phenomenon based on various adaptations of cancer cells to escape the killing actions of chemotherapeutics [START_REF] Pajak | Overview how adenocarcinoma cancer cells avoid immuneand chemotherapy-induced apoptosis[END_REF]. On a molecular level those adaptations are exemplified by immune-escape mechanisms [START_REF] Begley | Targeted therapies to improve tumor immunotherapy[END_REF], the overexpression of detoxifying enzymes of phase-I or -II metabolism [START_REF] Moscow | Multidrug resistance[END_REF], or the enhanced expression of ABC-transporters [START_REF] Stavrovskaya | Transport proteins of the ABC family and multidrug resistance of tumor cells[END_REF]. The overexpression of ABCtransporters promotes the outward-transport of most commonly used cancer drugs in spite of their different chemical structures and thereby prevents the achievement of effective drug concentrations in the target cells. This causes an immense clinical problem and consequently there has been intense search for compounds which can act to reverse MDR phenotype in cultured cells, in animal models, and ultimately in patients [START_REF] Lee | Reversing agents for ATP-binding cassette drug transporters[END_REF]. Within this search a number of natural compounds were identified that interact with ABC-transporters, and in contrast to the first generation MDR-reversing agents, which were usually drugs, have been regarded to possess fewer side effects [START_REF] Lee | Reversing agents for ATP-binding cassette drug transporters[END_REF]. Amongst those compounds, many flavonoids appear to serve as substrates of the ABC-transporters P-gp, MRP-2, and BCRP and thus may reverse MDR by competitive inhibition [START_REF] Alvarez | Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response[END_REF]. On the other hand, we have shown in a previous study that selected flavonoids were able to increase P-gp expression in human colorectal cancer cells in vitro and in the mouse intestine in vivo [START_REF] Lohner | Flavonoids alter P-gp expression in intestinal epithelial cells in vitro and in vivo[END_REF]. Whereas in normal cells induced expression of ABC-transporters might serve the excretion of the inducing xenobiotic, i.e. the flavonoid, in cancer cells MDR could derive.

Using human Caco-2 cells as an in vitro model for colorectal cancer we demonstrate in the present study that the flavonoid chrysin, occurring abundantly in fruit skins [START_REF] Alvarez | Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response[END_REF], is able to inhibit three apical ABC-transporters, i.e. P-gp, MRP-2, and BCRP, dose-dependently. The chemotherapeutic topotecan appears to be extruded and BCRP, respectively, resulted in similar increases in the accumulation of the drug, whereas the higher rate of accumulation caused by chrysin was achieved only when all three inhibitors were applied simultaneously. Chrysin consequently appeared to meet the criteria of an effective MDR-reversing agent by efficient blockade of topotecan efflux. Moreover, those effects can be expected to occur under in vivo conditions as well since concentrations of polyphenols in the low mM range may be considered as plausible in the gut lumen after ingestion of a normal diet rich in fruits and vegetables [START_REF] Serra | Bioavailability of procyanidin dimers and trimers and matrix food effects in in vitro and in vivo models[END_REF][START_REF] Sergent | Differential modulation of ochratoxin A absorption across Caco-2 cells by dietary polyphenols, used at realistic intestinal concentrations[END_REF]. Obviously the inhibition of transport by chrysin prevails versus the also observed increased expression of all three efflux-pumps since increased accumulation of topotecan could still be detected after 48 h of incubation when transporters had already been up-regulated. Therefore it was quite astonishing that in spite of increased topotecan accumulation the apoptosis-inducing activities of topotecan, quantified after 48 h, were completely inhibited by chrysin. Since beyond apoptosis, differentiation is another crucial parameter which has been shown to be diminished in cancer cells [START_REF] Van Der Flier | Stem cells, self-renewal, and differentiation in the intestinal epithelium[END_REF], we also tested whether chrysin has influence on differentiation of Caco-2 cells. Certain dietary compounds, such as butyrate from fiber, have been shown in culture selectively to promote and restore differentiation in neoplastic cells and, thereby, have the potential to eliminate aberrant cells at an early stage of tumor formation [START_REF] Wong | Colonic health: fermentation and short chain fatty acids[END_REF]. As shown in many studies before, butyrate efficiently induced differentiation in Caco-2 cells. Notably, beyond blocking topotecan-induced apoptosis chrysin greatly diminished differentiation as caused by butyrate.

The apoptosis inhibiting activities of chrysin might be due to the enhanced expression of ABC-transporters. This assumption is derived from experiments in cells which were transfected with mutant P-gp. It was demonstrated that the transportdeficient P-gp potently reduced caspase activities [START_REF] Tainton | Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux[END_REF] in accordance with our including that mediated by ligation of Fas [START_REF] Smyth | The drug efflux protein, Pglycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis[END_REF]. Since caspase-activation requires intracellular acidification [START_REF] Matsuyama | Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis[END_REF] and moreover, P-gp was suggested to act anti-apoptotic by prevention of a decline in pH i [START_REF] Robinson | Human MDR 1 protein overexpression delays the apoptotic cascade in Chinese hamster ovary fibroblasts[END_REF][START_REF] Johnstone | Multiple physiological functions for multidrug transporter P-glycoprotein?[END_REF], we first tested whether chrysin has an impact on pH i . Chrysin did not affect the diminished pH i which was caused by topotecan thus excluding that the inhibition of caspase-3 and -8 by chrysin was due to a stabilizaton of pH i . Moreover, preventing the topotecan-induced pH i -decline by addition of the base imidazole did not inhibit the ability of topotecan to activate caspases-8 and -3, supporting that a low pH i is not necessary for caspase activation in Caco-2 cells.

Inhibition of apoptosis has been described to be mediated by ß-catenin, a transcription factor of the Wnt-signaling pathway which is disregulated in colon cancer development [START_REF] Shitashige | Wnt signaling inside the nucleus[END_REF][START_REF] Gavert | beta-Catenin signaling in biological control and cancer[END_REF]. Since ß-catenin can be degraded by the downstream effector caspase-3 [START_REF] Steinhusen | Apoptosis-induced cleavage of beta-catenin by caspase-3 results in proteolytic fragments with reduced transactivation potential[END_REF]42] we finally assessed the impact of chrysin on ß-catenin stability but also on its intracellular localization. Degradation of this tumor-promoting factor, as caused by topotecan, was clearly inhibited by chrysin. On the other hand, cellular localization of ß-catenin, which was found predominantly in the cytosol and nuclei of topotecan-treated cells, was not affected by chrysin. Since a caspase-3inhibitor prevented the degradation of ß-catenin and also apoptosis it must be suggested that stabilization of ß-catenin is the prime mechanism underlying the enhancing effects of chrysin on cancer cell survival. This is in agreement with studies concluding that an important function of ß-catenin cleavage during apoptosis is the removal of its transcription activation [START_REF] Steinhusen | Apoptosis-induced cleavage of beta-catenin by caspase-3 results in proteolytic fragments with reduced transactivation potential[END_REF]. The stabilization of ß-catenin might finally was shown for P-gp in brain endothelial cells that its promoter can be activated by ßcatenin signaling [START_REF] Lim | Activation of beta-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells[END_REF]. Since expression of BCRP and MRP-2 increased under activation of canonical Wnt/ß-catenin signaling as well [START_REF] Lim | Activation of beta-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells[END_REF] it can be suggested that stabilized ß-catenin activates a number of ABC-transporters by promoter activation.

In addition to apoptosis, ß-catenin was shown in Caco-2 cells to influence differentiation [START_REF] Mariadason | Down-regulation of beta-catenin TCF signaling is linked to colonic epithelial cell differentiation[END_REF][START_REF] Kirkland | Type I collagen inhibits differentiation and promotes a stem cell-like phenotype in human colorectal carcinoma cells[END_REF]. In this regard the enhanced localization of ß-catenin in the plasma membrane was associated with increased differentiation [START_REF] Mariadason | Down-regulation of beta-catenin TCF signaling is linked to colonic epithelial cell differentiation[END_REF][START_REF] Kirkland | Type I collagen inhibits differentiation and promotes a stem cell-like phenotype in human colorectal carcinoma cells[END_REF]. Butyrate was shown in colon cancer cells to cause sequestration of ß-catenin to the cell membrane as well, suggesting that a possible role of butyrate is to promote differentiation via increasing the levels of ß-catenin available for cell adhesion [START_REF] Vincan | Sodium butyrateinduced differentiation of human LIM2537 colon cancer cells decreases GSK-3beta activity and increases levels of both membrane-bound and Apc/axin/GSK-3beta complex-associated pools of beta-catenin[END_REF].

However, that ß-catenin is directly involved in differentiation of Caco-2 cells is rather unlikely since butyrate in our experiments resulted in identical localization and fragmentation patterns of ß-catenin as topotecan and whereas both compounds are potent apoptosis inducers (not shown for butyrate), only butyrate led to an increase in differentiation. On the other hand it can not be excluded that chrysin exerts its differentiation inhibiting activities via ß-catenin stabilization and a concomitant increase in ABC-transporter expression since indeed a role for P-gp in regulating cellular differentiation has been postulated given the down-regulation of endogenous P-gp expression during differentiation of pluripotent stem cells [START_REF] Chaudhary | Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells[END_REF].

In conclusion, our studies show that the enhanced expression of three ABCtransporters in human colorectal cancer cells by the flavonoid chrysin is associated with prevention of topotecan-induced apoptosis. This inhibition of apoptosis occurs in spite of increased drug accumulation that is caused by the inhibition of transport activities. The most plausible explanation for the observed effects is provided by the inhibition of caspases which protects ß-catenin from caspase-mediated cleavage.

*Graphical Abstract

A c c e p t e d M a n u s c r i p t 2 Abbreviations:

 2 Abbreviations: ABC, ATP binding cassette; Ac-DEVD-CHO, acetyl-aspartyl-glutamylvalyl-aspartyl-aldehyde; Ac-IETD-AFC, acetyl-isoleucyl-glutamyl-threonyl-aspartyl-7amino-4-trifluoromethylcoumarin, AP, alkaline phosphatase; BCECF-AM, 2´,7´-bis-(2carboxyethyl)-5-(and 6)-carboxyfluorescein acetoxymethylester; BCRP, Breast cancer resistance protein; CMFDA, 5-chloromethylfluorescein diacetate; DMEM, Dulbecco's Modified Eagle Medium; ECL, Enhanced chemiluminescence; DTT, dithiothreitol; FACS, fluorescence activated cell sorting; FCS, Fetal calf serum; HEPES, N-hydroxyethylpiperazine-N′-2-ethanesulfonate; MDR, Multi drug resistance;

A c c e p t e d M a n u s c r i p t 5

 5

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

7

 7

A c c e p t e d M a n u s c r i p t 9 ethanol for 24

 924 h. Thereafter, the suspension was centrifuged for 3 min at 500 x g, and the pellet was washed twice in 38 mM sodium citrate buffer (pH 7.4). After washing and centrifugation, cells were incubated with the dye solution containing 7-amino actin (1 mg/ml), 1U of DNase free RNase in sodium citrate buffer. Cell cycle analysis was performed using a FACScan (Becton Dickinson, San Jose, CA, U.S.A.) and the Lysis II Ver. 1.1 software.2.5. Differentiation assay0.5 x 10 6 Caco-2 cells were seeded onto 25 cm 2 -culture flasks and thereafter incubated for 48 h with the test compounds. Cells were washed twice with PBS and trypsinized and the harvested cells were pelleted for 10 min at 1500 g. The pellets were resuspended in 1 M diethanolamine buffer, pH 9.8 with 0.5 mM MgCl 2 and homogenized. 50 µl of the homogenate were mixed with 940 µl diethanolamine buffer and 10 µl AP substrate 4-nitrophenol phosphate (1.25 mM stock solution). AP-activity was calculated according to UV-absorption of the product 4-nitrophenol which absorbs light at 405 nm.

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

 10 diffusing out of the cells during these long periods. Intracellular H + -activity was determined by measuring the intensity of emission at 538 nm after excitation of the fluorophore at 444 nm (isosbestic point) and 485 nm (pH-sensitive wavelength), respectively, using the microtiter plate reader. The 485/444 fluorescence ratio was converted to pH i from a calibration curve generated by estimation of the fluorescence ratio in buffers of different pH[START_REF] Boyarsky | Inadequacy of high K+/nigericin for calibrating BCECF. II. Intracellular pH dependence of the correction[END_REF].

( 1 :

 1 500), MRP-2 (1:50), and BCRP (1:50), respectively. Blots were washed three A c c e p t e d M a n u s c r i p t 11

A c c e p t e d M a n u s c r i p t 14 neither

 14 topotecan nor chrysin affected differentiation in Caco-2 cells, chrysin

A c c e p t e d M a n u s c r i p t 15 inhibition

 15 of the downstream caspase-3 not only prevented topotecan-induced cleavage of ß-catenin (Fig.6C) but also apoptosis as caused by topotecan (Fig.6D).

A c c e p t e d M a n u s c r i p t 17 from

 17 Caco-2 cells by all three transporters since specific inhibitors for P-gp, MRP-2,

A c c e p t e d M a n u s c r i p t 18 observations

 18 that apoptosis was inhibited by chrysin instead of inhibition of transport activities and the concomitant enhanced topotecan levels observed inside Caco-2 cells. Moreover, classical multidrug resistant human tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death,

A c c e p t e d M a n u s c r i p t 19 be

 19 transmitted into enhanced expression of the investigated ABC-transporters as it

A c c e p t e d M a n u s c r i p t 21 A

 21

Figure legends:Fig. 1 .

 1 Figure legends:

Fig. 2 .

 2 Fig. 2. Chrysin enhances the accumulation of topotecan. Caco-2 cells were incubated for 3 h with 25 µM topotecan in the absence (control) or presence of 40 µM verapamil, 50 µM MK-571, 10 µM fumitremorgin, or 250 µM chrysin, respectively.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Chrysin increases the expression of ABC-transporters in the presence of topotecan. Cells were exposed for 48 h to medium alone (control) or in addition to 250 µM chrysin, 25 µM topotecan, or a combination of chrysin and topotecan.Thereafter, membrane fractions were enriched, proteins extracted and separated by SDS-PAGE before blotted proteins were probed with specific antibodies for P-gp (A), MRP-2 (B), or BCRP (C) (left panels). Intensities of bands were assessed as

Fig. 5 .

 5 Fig. 5. Chrysin inhibits topotecan-induced activation of caspase-3 and -8. Activities of caspase-3 and caspase-8 in extracts generated from cells that were exposed to medium alone (control), to 25 µM topotecan, or a combination of topotecan and 250 µM chrysin or 10 mM imidazole were estimated after 24 h by measuring the fluorescence generated by cleavage of Z-DEVD-AMC and Ac-IETD-AFC,

Acknowledgements

This study was supported by the Deutsche Forschungsgemeinschaft; Grant number: WE 2684/7-1 and 7-2. The authors greatly acknowledge the expert technical assistance of Mrs. Eva Czynski. We are indebted to Mr. Alan McDonley for critically reading the manuscript.