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Carcinogenesis Advance Access originally published online on January 3, 2008
Carcinogenesis 2008 29(2):404-410; doi:10.1093/carcin/bgm296
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Stromal resistance of fibroblasts against oxidative damage: involvement of tumor cell-secreted platelet-derived growth factor (PDGF) and phosphoinositide 3-kinase (PI3K) activation

Christel Werth, Dominik Stuhlmann, Bahar Cat, Holger Steinbrenner, Lirija Alili, Helmut Sies and Peter Brenneisen*

Institute of Biochemistry and Molecular Biology I, Heinrich-Heine-University, D-40225 Düsseldorf, Germany

* To whom correspondence should be addressed. Tel: +49 211 811 2715; Fax: +49 211 811 3029; Email: peterbrenneisen{at}web.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
A critical step in tumor progression is the interaction of malignant and stromal cells via paracrine mechanisms. Stromal cells, particularly fibroblasts, support cancer cells in invasion of the surrounding tissue for access to the vascular system. Here, the question is addressed of whether tumor cells induce ‘stromal resistance’, i.e. protect the microenvironment from oxidative damage. The supernatant of cultured skin-derived tumor cells was added to fibroblasts and was shown to protect the fibroblasts from hydrogen peroxide-mediated cell toxicity. The platelet-derived growth factor secreted from the cancer cells was identified as trigger of this protection in fibroblasts via the phosphoinositide 3-kinase pathway. These data suggest that prosurvival signals in stromal fibroblasts as initiated by tumor cells constitute a strategy of ‘stromal resistance’, illustrating a novel biological role of fibroblasts for the tumor microenvironment.

Abbreviations: cGPx, cytosolic glutathione peroxidase; CM, conditioned medium; EGF, epidermal growth factor; HDF, human dermal fibroblasts; NHEK, normal human epidermal keratinocytes; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI3K, phosphoinositide 3-kinase; ROS, reactive oxygen species; SCL, squamous cell line; SFM, serum-free medium; TGF, transforming growth factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Interaction between tumor cells and the stromal compartment is a crucial factor in tumor progression. Disturbance in the stroma, composed of endothelial, fibroblastic and myofibroblastic cells as well as macrophages and other inflammatory cells, is suggested to be essential during the invasion process (1). Tumor–stroma interaction is characterized by both local accumulation of extracellular matrix components and tumor cell-initiated modulation of the metabolism of resident cells, mainly surrounding the tumor cluster. This results in the generation of a stroma which is convenient for the cancer cells (2,3). As tumors can be considered as non-healing wounds, particularly cells such as fibroblasts have a prominent role in the progression, growth and spread of cancers. Fibroblasts are associated with cancer cells at all stages of tumor progression, and their functional contribution to that process is emerging (4,5).

In melanoma and carcinoma, a wide variety of cytokines [e.g. interleukin-1, interleukin-6, tumor necrosis factor-{alpha}] and growth factors [e.g. transforming growth factor (TGF)-β1, epidermal growth factor (EGF), platelet-derived growth factor (PDGF)] are expressed by both tumor and stromal cells which via autocrine and/or paracrine mechanisms promote neovascularization and tumor growth as well as migration during tumor invasion (3,6). For example, TGF-β1 and PDGF play important roles in a variety of cancers. Progression of human hepatocellular carcinomas was decreased by disturbance of both the PDGF and TGF-β1 signaling, resulting in the prevention of the epithelial-to-mesenchymal transition (7). Recently, tumor cell-derived TGF-β1 was shown to be responsible for both decrease in gap junctional intercellular communication between stromal fibroblasts (8) and mesenchymal-to-mesenchymal transition, resulting in myofibroblast formation which is associated with an increase in the invasive capacity of adjacent tumor cells in tumor–stroma interaction (4,9). Signaling through an autocrine PDGF/PDGF receptor (PDGFR) loop is an early oncogenic event in gliomagenesis and the increased expression of PDGF-AA and -BB correlates with the degree of malignancy (10).

Mitogenic signals initiated by binding of soluble factors such as TGF-β1, EGF and PDGF to the adequate receptor kinases are transduced by cytosolic serine/threonine signaling cascades, among them the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase pathways. The serine/threonine kinase Akt, also termed protein kinase B and a major PI3K target, has been implicated in antiapoptotic survival strategies of tumor cells (11). Recently, it was shown that the PDGFR inhibitor ST571 (Imatinib Mesylate) sensitizes chemoresistant glioma cells to cisplatin toxicity, depending on Akt inactivation (12). An EGF-initiated and Akt and extracellular signal-regulated kinase 1/2 mediated production of vascular endothelial growth factor in hepatocellular carcinoma cell lines was markedly inhibited by the EGF receptor tyrosine kinase inhibitor gefitinib (Iressa) (13). Furthermore, the TGF-β1-triggered involvement of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in the induction of epithelial-to-mesenchymal transition in human malignant keratinocytes (14) emphasizes the relevance of TGF-β1 and the mitogen-activated protein kinase pathway in carcinogenesis.

In that context, the transdifferentiation of fibroblasts to ‘tumor-educated’ fibroblasts, namely myofibroblasts, by tumor cell-derived TGF-β1 was recently shown to be a prerequisite for an increase in the aggressive behavior of the tumor cell during invasion (4). As these data underline the hypothesis for the essential role of tumor-associated fibroblasts and myofibroblasts in the progression, growth and spread of tumors (5,15), the objective in this study was to evaluate whether tumor cells may protect surrounding fibroblasts from extrinsically generated damage, especially oxidative damage. Herein, we show that tumor cell-derived PDGF mediates a prosurvival mechanism in hydrogen peroxide-challenged fibroblasts via a PI3K–Akt-mediated pathway. To our knowledge, this is the first report linking a tumor cell-mediated paracrine protective mechanism to tumor–stroma interaction.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Materials
Cell culture media [DMEM, keratinocyte serum-free medium (SFM) + supplements] were purchased from Invitrogen GmbH (Karlsruhe, Germany) and the defined fetal calf serum (FCS gold) was from PAA Laboratories (Linz, Austria). All chemicals were obtained from Sigma (Taufkirchen, Germany) unless otherwise stated. The Vivaspin 15R concentrator columns were delivered by Vivascience (Hannover, Germany). The protein assay kit (Bio-Rad DC, detergent compatible) was from Bio-Rad Laboratories GmbH (München, Germany). The RayBio® Human Cytokine Antibody Array V kit was purchased from Hölzl Diagnostics (Cologne, Germany). The enhanced chemiluminescence system (SuperSignal West Pico Maximum Sensitivity Substrate) was supplied by Pierce (Bonn, Germany). Recombinant human PDGF-BB was delivered from R&D Systems GmbH (Wiesbaden, Germany). Polyclonal rabbit antibodies raised against human phospho-Akt (Ser473), total Akt and phospho-PDGFR beta (Tyr751) were supplied by Cell Signaling Technology (New England Biolabs, Frankfurt a.M., Germany). The following secondary antibodies were used: polyclonal horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Dianova, Hamburg, Germany). Polyclonal rabbit anti-human PDGF-BB and goat anti-human PDGF-AA neutralizing antibodies were delivered by Acris Antibodies GmbH (Hiddenhausen, Germany) and R&D Systems, respectively.

Cell culture
Human dermal fibroblasts (HDF) were established by outgrowth from foreskin biopsies of healthy human donors with an age of 4–6 years. Cells were used in passages 4–12, corresponding to cumulative population doubling levels of 6–26 (16). The squamous cell line (SCL) A431, originally derived from a 85-year-old female with epidermal carcinoma, and the malignant melanoma line A375 from a 54-year-old female (17) were delivered by the American Type Culture Collection (LGC Promochem, Wesel, Germany). The cell lines A431 and A375, the dermal fibroblasts and the squamous carcinoma cell line SCL-1, originally derived from the face of a 74-year-old woman (18) (generously provided by N. Fusenig, DKFZ Heidelberg, Germany), were maintained in DMEM supplemented with glutamine (2 mM), penicillin (400 U/ml), streptomycin (50 µg/ml) and 10% fetal calf serum. Normal human epidermal keratinocytes (NHEK) were prepared from foreskin biopsies as described (19). The cells were grown in keratinocyte-SFM medium containing supplements (human EGF, bovine pituitary extract) and gentamycin (5 mg/ml) and were used for experiments between passages 2 and 6. Cells were grown in a humidified atmosphere of 5% CO2 and 95% air at 37°C.

Preparation of conditioned medium
Conditioned medium (CM) was obtained from SCL-1 (CMSCL), A431 (CMA431), A375 (CMA375), NHEK (CMNHEK) and HDF (CMHDF). For that, seeded 0.75 x 106 tumor cells were grown to subconfluence (~70% confluence), 2.0 x 106 NHEK to 80% confluence and 1.5 x 106 HDF cells to confluence in 175 cm2 culture flasks to get finally identical cell numbers. The serum/supplement-containing medium was removed, and the cells were incubated for further 48 h in 15 ml serum-free DMEM or supplement-free SFM medium before collection of the CM. CM were clarified by centrifugation at 1250g, and CM were used freshly or stored at –20°C for at most 1 week before use.

Fractionation of CM by ultrafiltration
Prior to ultrafiltration, precooled (4°C) cell culture supernatants were filtered through a 0.2-µm syringe filter to remove any cellular debris. Ultrafiltration of CM of SCL-1 tumor cells with Centriprep YM-10 (10 kDa molecular weight-cut off) at 1500g (4°C) resulted in a <10 kDa filtrate and >10 kDa retentate. This concentrated retentate was brought to the original volume with serum-free medium, and thereafter filtrated through Centriprep YM-30 (30 kDa molecular weight-cut off) at 1500g (4°C) resulting in a >10 to <30 kDa filtrate and a >30-kDa fraction which was brought to the original volume as described above. All fractions were tested for biological activity concerning phosphorylation of protein kinase Akt.

Cell viability
HDF were cultured in 24-well plates (Greiner Bio-One, Frickenhausen, Germany) to 90% confluency. Thereafter, the cells were washed and grown for 24 h in CMHDF, CMSCL, CMA431 or CMA375. Oxidative damage was caused by exposure to H2O2 (0.25, 1.0 mM) for additional 24 h. The protective capacity was examined by comparing cell viability with or without CMSCL, A431 or A375 preincubation. Cell viability was determined by measurement of the ability of HDF to metabolize MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) via mitochondrial dehydrogenases to a purple formazan dye as described (20). Cytotoxicity was calculated as the percentage of formazan formation in H2O2-treated cells compared with mock-treated cells.

SDS-PAGE and western blot
SDS-PAGE was performed according to the standard protocols published elsewhere (21) with minor modifications. Briefly, HDF were lysed after incubation with CMHDF or CMSCL in 2x SDS-PAGE buffer (125 mM Tris–HCl, 4% wt/vol glycerol, 100 mM dithiothreitol, pH 6.8). The protein amount of the samples was determined by using a modified Lowry method (Bio-Rad DC) and 5 µg total protein/lane were applied to 10% (wt/vol) SDS-polyacrylamide gels. After electroblotting onto nitrocellulose membrane (Hybond-C Extra, GE Healthcare, Freiburg, Germany), immunodetection was carried out using a 1:1000 dilution of primary phospho-Akt, total Akt and phospho-PDGFRβ antibodies, and a 1:10 000 dilution of goat anti-rabbit secondary antibody conjugated to horseradish peroxidase.

Antigen–antibody complexes were visualized by an enhanced chemiluminescence system on BioMax Light Film (Kodak, Rochester, NY). Equal loading was checked by Coomassie Blue staining. Molecular sizes of the bands were calculated by comparison with a prestained protein marker (Biomol, Hamburg, Germany). For quantification of the bands, the developed films were scanned by an image analysis system and analyzed with the AIDA image software (Raytest, Straubenhardt, Germany).

Human cytokine antibody array
A human protein cytokine array was performed according to the manufacturer’s protocol. Briefly, the membranes with the spotted cytokine antibodies were blocked with a blocking buffer, and thereafter incubated with 1 ml of CMSCL, CMNHEK or CMHDF at room temperature for 2 h. After washing, the membranes were treated with 1 ml of a cocktail of primary biotin-conjugated antibodies for additional 2 h at room temperature. Thereafter, the membranes were incubated with 2 ml of horseradish peroxidase-conjugated streptavidin/membrane at room temperature for 2 h. The membranes were developed by enhanced chemiluminescence system on BioMax Light Film (see supplementary Figure 1, available at Carcinogenesis Online).

Statistical analysis
All means were calculated at least from three independent experiments, and the error bars represent the standard deviation. Analysis of statistical significance was done by analysis of variance or Student’s t-test with, herein, #P > 0.05, *P < 0.01, **P < 0.001 and ***P < 0.0001 as levels of significance.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Tumor cell supernatant lowers toxicity of hydrogen peroxide
As tumor cells need tumor-associated fibroblasts/myofibroblasts to increase their invasive capacity (4), we addressed the question of whether the tumor cells might protect the fibroblasts from oxidative damage. Therefore, subconfluent fibroblast monolayer cultures (~80% confluence) were treated with CMSCL, CMA431, CMA375 and CMHDF, respectively, for 24 h prior to treatment with different concentrations of H2O2 for further 24 h. H2O2 was used as model substance to generate oxidative stress (22,23) as it was demonstrated that macrophages and neutrophils produce reactive oxygen species (ROS) such as singlet oxygen and H2O2 during oxidative burst in tumor–stroma interaction (24), and that H2O2 was used to generate oxystress in cancer therapy (25).

At concentrations of 0.25 and 1.0 mM H2O2, the viability of the fibroblasts was significantly lowered, showing a significant cytotoxicity of H2O2 on the fibroblasts which were maintained in CMHDF. The mean values ranged between 22 and 27% (1 mM H2O2) and from 69 to 73% (0.25 mM H2O2), respectively, compared with mock-treated cells (no H2O2) which were set at 100% (Figure 1). Unlike the CMHDF, preincubation of the cells with CMSCL, CMA431 or CMA375 resulted in protection against H2O2-mediated cytotoxicity. On average, the viability of the fibroblasts ranged from 48 to 65% and 86 to 92%, depending on the used H2O2 concentrations and the CM of the different tumor cell lines (Figure 1). These findings in correlation with the measured P values indicated a higher survival rate of HDF in CMSCL, CMA431 and CMA375 in comparison with CMHDF. As it was shown earlier that CMSCL triggers a disruption of gap junctional communication between fibroblasts (26) as well as a tumor invasion-promoting transition of fibroblasts to myofibroblasts (4), further studies were performed with the CM of the SCL-1.


Figure 1
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  		Fig. 1. Effect of tumor cell-derived supernatant on viability of HDF. Subconfluent fibroblast monolayer cultures were treated with serum-free supernatants of tumor cell lines SCL-1, A431, A375 (CMtumor cell) or HDF (CMHDF) for 24 h prior to treatment with different concentrations of H2O2 for additional 24 h. Cell viability was measured. At least three independent experiments were performed for each tumor cell line and the fibroblasts. #P > 0.05, *P < 0.01, **P < 0.001 and ***P < 0.0001 versus mock-treated cells (analysis of variance, Dunnett’s test).

 
Higher survival of fibroblasts in CMSCL depends on Akt/PKB
Active protein kinase Akt is a strong promoter for cell survival, as it inhibits several proapoptotic signaling components such as Bad, caspase-9 or transcription factors of the Forkhead family (27,28). In order to study the involvement of Akt in tumor cell-assisted and soluble factor-triggered protection of (stromal) fibroblasts against oxidative damage, a time course analysis of Akt phosphorylation, which was used as proof for Akt activation, was performed after treatment of subconfluent fibroblasts with either CMHDF or CMSCL (Figure 2A). A significant increase in phosphorylated Akt was detected at 15 min and peaked at 30 min after treatment with CMSCL. Subsequently, a progressive dephosphorylation of the activated Akt was observed between 45 min and 3 h. The total amount of Akt did not change significantly, excluding a CMSCL-dependent gene expression. After treatment with CMHDF, no significant change in phosphorylation of Akt was observed (data not shown). The subsequent experiments regarding Akt phosphorylation were performed at 30 min after treatment.


Figure 2
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  		Fig. 2. Effect of Akt/PKB on survival of fibroblasts. (A) Subconfluent fibroblast cultures were treated with either serum-free CMHDF (data not shown) or CMSCL, cell lysates were prepared after different time points, and subjected to western blot analysis. (B and C) Fibroblasts were preincubated for 2 h with the PI3K inhibitors wortmannin (WO, 100 nM) or LY294002 (25 µM) prior to lysis of the cells after 30 min after treatment with CMHDF or CMSCL. Lysates were analyzed by western blots. (D) Subconfluent fibroblasts were treated with serum-free CMHDF or CMSCL with or without wortmannin for 24 h prior to treatment with 1 mM H2O2 for additional 24 h. Cell viability was measured. #P > 0.05, **P < 0.001 (Student’s t-test). (A–D) Three independent experiments were performed.

 
As other kinases (29) have been discussed to be involved in phosphorylation of Akt apart from the classical PI3K and 3-phosphoinositide-dependent protein kinase 1 pathway (28), the PI3K inhibitors wortmannin and LY294002 (30) were used to check the upstream regulator of Akt phosphorylation. Subconfluent fibroblast monolayer cultures were incubated for 2 h with non-toxic concentrations of the inhibitors prior to treatment with CMSCL or CMHDF for 30 min. Wortmannin (Figure 2B) as well as LY294002 (Figure 2C) almost completely abrogated the CMSCL-dependent increase in phosphorylation of Akt. These data indicate the involvement of PI3K in activation of Akt, initiated by soluble factors of CMSCL.

To show that the higher survival of fibroblasts in CMSCL after oxidative stress is actually mediated by the involvement of the kinase Akt, subconfluent fibroblasts were treated with CMHDF or CMSCL with or without wortmannin for 24 h prior to treatment with H2O2 for further 24 h. Unlike the CMHDF and CMHDF in combination with wortmannin (CMHDF + wortmannin), preincubation of the cells with CMSCL alone resulted in a better protection against H2O2-mediated cytotoxicity. Here, the viability was lowered to only 52 ± 3% at a concentration of 1 mM H2O2 compared with the viability in CMHDF. Interestingly, the CMSCL-triggered protection against H2O2 toxicity was abolished by the inhibitor wortmannin, showing a survival rate of 24 ± 2% which is similar to that of H2O2-treated fibroblasts preincubated in CMHDF or CMHDF+wortmannin (Figure 2D).

Identification of the paracrine factor mediating Akt phosphorylation
In order to characterize the tumor cell-derived soluble factors responsible for protection of fibroblasts against ROS-mediated toxicity, a membrane filter fractionation assay was performed. The molecular masses of the assumed paracrine acting factors were identified to be ≥30 kDa, herein called the 30-kDa fraction, showing a significant increase in Akt phosphorylation. In comparison with the other fractions (<10 kDa, 10–30 kDa), the ‘active’ 30-kDa fraction resulted in a similar intensity of the Akt signal as the total supernatant of the tumor cells (CMSCL) at 30 min after treatment (Figure 3A).


Figure 3
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  		Fig. 3. Identification of the soluble factor derived from tumor cell supernatants. (A) At subconfluence, the serum-containing medium was replaced by SFM. Thereafter, the supernatants of fibroblast (CMHDF) and SCL-1 tumor cell (CMSCL) monolayer cultures were collected after 2 days and fractionated. Fresh fibroblast cultures were incubated for 30 min with the fractions, lysed and subjected to western blot analysis for phosphorylated Akt. Experiments were performed in duplicate. (B) Subconfluent fibroblasts were treated for 3 h with 1 mM suramin prior to incubation with serum-free CMSCL or CMHDF for 30 min. Experiments were performed in triplicate. (C) A human protein cytokine/growth factor array was performed as described in Material and methods. The membranes were developed by enhanced chemiluminescence. The array was performed in duplicate.

 
The polysulfonated naphthylurea suramin is a potent inhibitor of growth factor receptors such as EGF, TGF-β, PDGF and basic fibroblast growth factor (31,32), as well as a suppressor of signaling by protein kinases such as phosphoinositide kinases (33). Therefore, that agent was used to narrow down the range of potential soluble factors effective in protection of tumor-associated fibroblasts from ROS-mediated toxicity. Subconfluent fibroblasts were treated for 3 h with a non-toxic concentration of suramin in CMHDF prior to incubation with fresh CMSCL or CMHDF for 30 min. Figure 3B shows that suramin completely prevented the CMSCL-mediated phosphorylation of Akt. CMHDF did not increase its phosphorylation. Again, the total amount of Akt was not altered (Figure 3B).

As the previous data suggested that the tumor cell-derived soluble factor is a growth factor with a molecular weight ≥30 kDa, peptide arrays for detection of secreted proteins were performed. Using peptide arrays for CMHDF, CMNHEK and CMSCL, PDGF-BB was identified as a prominent protein spot overexpressed in CMSCL compared with CMNHEK and CMHDF, and fulfilling all the criteria described (Figure 3C, supplementary Figure 1, available at Carcinogenesis Online). TGF-β1, another growth factor whose expression and secretion were published earlier to be upregulated in the squamous carcinoma cell line SCL-1 (8), and interleukin-6, significantly upregulated in CMSCL as well (supplementary Figure 1, available at Carcinogenesis Online), had no effect on both Akt phosphorylation and protection against oxidative damage (data not shown).

PDGF-BB mediates Akt phosphorylation via its receptor PDGFRβ
To investigate whether CMSCL mediates Akt phosphorylation in fibroblasts via PDGF-BB and its receptor PDGFRβ, a time course analysis for receptor phosphorylation was performed. Subconfluent fibroblasts were treated for different times with the CM of the tumor cells. H2O2 at 1 mM and incubated for 5 min was used as positive control (34). The cells were harvested, lysed and subjected to western blot analysis. A rapid and significant increase in CMSCL-mediated phosphorylation of PDGFRβ was detected after 2.5 min. The signal peaked at 10 min, whereas at 30 min after treatment the phosphorylation signal was significantly lowered compared with both the signal at maximum intensity and the positive control (Figure 4A).


Figure 4
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  		Fig. 4. Phosphorylation of PDGFR and protein kinase Akt. (A) Subconfluent fibroblast cultures were treated with serum-free CMSCL, cell lysates were prepared after different time points, and subjected to western blot analysis for PDGF beta receptor. H2O2 (1 mM, 5 min) was used as positive control. (B) Fibroblasts were preincubated for 1 h with the inhibitor AG1295 (25 µM). After a 30 min treatment with serum-free CMHDF or CMSCL, the cells were lysed and lysates analyzed by western blot analysis. (C) Subconfluent fibroblasts were treated with serum-free CMHDF or CMSCL for 30 min. CMSCL was preincubated with 0.1 or 1.0 µg neutralizing PDGF-BB (nAb) per millimeter overnight at 4°C or untreated. Western blots were performed. (A–C) Three independent experiments were performed.

 
These data are in line with the time course analysis of Akt phosphorylation in CMSCL-treated cells (Figure 2A), suggesting that a PDGF-BB initiated PDGFRβ phosphorylation may affect the activation of Akt. To check that assumption, subconfluent fibroblasts were incubated for 1 h with a non-toxic concentration of AG1295 (6,7-dimethyl-2-phenylquinoxaline), a tyrphostin (35) and selective inhibitor of the PDGFR tyrosine kinases with higher affinity to PDGFRβ (36,37), prior to treatment with CMSCL. A combination of CMSCL and AG1295 completely prevented the CMSCL-induced phosphorylation of Akt. Both the inhibitor and CMSCL had no effect on the overall expression of the protein kinase (Figure 4B).

Furthermore, subconfluent fibroblasts were incubated in parallel with CMHDF, CMSCL and CMSCL in combination with two different concentrations of a PDGF-BB neutralizing antibody. Again, the CM of SCL-1 tumor cells increased the phosphorylation of Akt, whereas the PDGF-BB neutralizing antibody significantly decreased that phosphorylation intensity by 78%. Interestingly, the neutralizing antibody showed a concentration-independent effect (Figure 4C). To exclude an involvement of PDGF-AA in phosphorylation of Akt, a PDGF-AA neutralizing antibody was applied which had no effect on the CMSCL-dependent Akt phosphorylation (data not shown).

Tumor cell-derived PDGF-BB protects fibroblasts from oxidative damage
As the present data suggest that PDGF-BB secreted from tumor cells is the primary soluble factor protecting stromal fibroblasts from H2O2 toxicity (see Figure 1), we addressed the question of whether the PDGF-BB neutralizing antibody and the tyrphostin AG1295, respectively, lower the tumor cell supernatant-triggered survival of these cells. Subconfluent fibroblast monolayer cultures were treated with CMSCL or CMHDF for 24 h prior to treatment with different concentrations of H2O2. CMHDF had no protective effect. At concentrations of 0.25 and 1.0 mM H2O2, the viability of the fibroblasts was lowered by 26–36% and 75–83%, respectively, compared with mock-treated cells. In contrast to CMHDF, preincubation of the cells with CMSCL maintained cell viability or only marginally lowered the viability to ~71% at a concentration of 1 mM H2O2, indicating increased survival of HDF in CMSCL in comparison with CMHDF (Figure 5A). A combination of CMSCL and PDGF-BB neutralizing antibody or AG1295 counteracted the protective effect of CMSCL after H2O2 treatment. The survival rate dramatically decreased to a similar extent as described above for CMHDF- and H2O2-treated HDF.


Figure 5
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  		Fig. 5. Effect of tumor cell-derived supernatant on viability of HDF. (A) Subconfluent fibroblast monolayer cultures were treated with serum-free CMSCL or CMHDF for 24 h prior to treatment with different concentrations of H2O2 for additional 24 h. CMSCL was incubated in combination with 25 µM AG1295 and neutralizing PDGF-BB (1.0 µg/ml, nAb), respectively, or mock treated. Cell viability was measured. Four independent experiments were performed. #P > 0.05, **P < 0.001 and ***P < 0.0001 versus mock-treated cells (analysis of variance, Dunnett’s test). (B) Subconfluent fibroblast monolayer cultures were treated with CMHDF or CMHDF + PDGF–BB (25 ng/ml) for 1 h prior to treatment with different concentrations of H2O2 for additional 24 h. The survival rate was measured. Experiments were performed in triplicate. **P < 0.001 (Student’s t-test).

 
In order to show that PDGF-BB is the major soluble factor in mediating protection against H2O2 toxicity, subconfluent fibroblast monolayer cultures were treated with CMHDF or CMHDF containing human recombinant PDGF-BB (25 ng/ml) for 24 h prior to treatment with different concentrations of H2O2. Again, CMHDF had no protective effect. At concentrations of 0.25 and 1.0 mM H2O2, the viability of the fibroblasts was decreased on average to 66 and 18%, respectively, compared with mock-treated cells. In contrast to CMHDF, preincubation of the cells with CMHDF + PDGF lowered the viability to only 90 ± 2% at a concentration of 0.25 mM H2O2 and to 42 ± 5% at a concentration of 1 mM H2O2, indicating a similar protective effect of CMHDF + PDGF as seen with CMSCL (Figure 5B).

To exclude that H2O2 per se or PDGF-BB induce the major regulable antioxidant enzyme cytosolic glutathione peroxidase (cGPx) (4,38) which might mediate protection against H2O2-triggered oxidative damage, subconfluent fibroblasts were treated with CMSCL or CMHDF for 24 h prior to treatment with H2O2 alone or in combination with 0.1 mM mercaptosuccinate, described to be a cGPx-specific inhibitor (38,39). At a concentration of 0.25 mM H2O2, the viability of the fibroblasts maintained in CMHDF was lowered to 49 ± 5%, compared with mock-treated cells (no H2O2). Unlike CMHDF, preincubation of the cells with CMSCL or CMSCL in combination with mercaptosucccinate resulted in protection against H2O2-mediated cytotoxicity. The viability of the fibroblasts was decreased to only 93 ± 7% (CMSCL) and 92 ± 3% (CMSCL + mercaptosuccinate), respectively, indicating a cGPx-independent protection against oxidative damage.

In conclusion, our data show that protection of stromal fibroblasts from exogenous ROS-mediated cell death depends on tumor cell-derived soluble factors, here the platelet-derived growth factor-BB, initiating the PI3K/Akt survival pathway.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
ROS, oxidative stress and stromal resistance
The function of ROS is a ‘double-edged sword’. On the one hand, ROS are involved in redox signaling controlling proliferation, differentiation and cellular homeostasis (40,41). On the other hand, if the generation of ROS exceeds the system’s ability to neutralize and eliminate them, the situation of ‘oxidative stress’ is attained (22,23), going along with disruption of redox signaling and control (42). Because of this, oxidative stress has been implicated in a growing list of human diseases such as cardiovascular diseases, neurodegenerative diseases and cancer (43).

ROS play a central role in biological effects initiated by radio- and chemotherapeutic approaches as well. In that context, the generation of oxidative stress, comprising, for example, an overproduction of the highly reactive hydroxyl radicals (HO.), superoxide anions (Formula) and H2O2, is often preferred in radio- and chemotherapy. It takes advantage of the finding that neoplastic cells function with a heightened basal level of ROS-mediating signaling, therefore making them more susceptible to exogenously induced oxidative stress than their normal counterparts (25,44,45). Even though a higher susceptibility for ROS-initiated apoptosis or necrosis is true for several cancer types (46–48), it often looks different in practice. In addition to serious side-effects such as hair loss, vomiting, mucositis, rash, diarrhea and pneumonitis (49,50), on a molecular level chemo- and radiotherapeutic treatment of tumor cells is not rarely accompanied by escape of those cells from cell death: since the antioxidant system in the cells plays an essential role in elimination of ROS, the upregulation of antioxidant molecules in cancer cells is thought to contribute to radiation resistance (51,52). Furthermore, anticancer agent-induced oxidative stress was shown to activate antiapoptotic signaling pathways, such as Akt, Erk and nuclear factor-{kappa}B in breast cancer and pancreatic carcinoma cells resulting in poor prognosis (53,54). An increase in oxidative stress may contribute to cancer progression by amplifying genomic instability reflected in both the heterogeneity of the cell population within a tumor tissue and the problem of successful treatment of diverse cancers (55). In conclusion, both the detail molecular events in ROS-mediated malignant transformation and resistance against ROS-producing anticancer drugs are not fully understood. Furthermore, many studies dealing with the unraveling of the mystery of cancer resistance disregard the influence of the stromal cells in mediating that resistance.

As stromal cells, such as fibroblasts and endothelial cells, reveal genomic stability within the tumor–host microenvironment, stromal therapy (3) could emerge as a new strategy to combat invasion and metastatic spread. Recently, the ROS-mediated generation of tumor-educated fibroblasts was prevented by the use of antioxidants and micronutrients which contributed to the lowered invasive capacity of the adjacent tumor cells (4). In that context, the question was addressed herein of whether tumor cells may protect (stromal) fibroblasts from extrinsically generated oxidative damage, as tumor cells invade the tissue by means of those stromal cells. Therefore, tumor-educated fibroblasts were exposed to toxic concentrations of exogenously added H2O2, which was used as model substance in simulation of a chemotherapeutical approach This kind of oxidative stress increased cell death, which was counteracted by preincubation of the fibroblasts with tumor cell-derived supernatants. We hypothesize, that this ‘stromal resistance’ has a synergistic and supporting effect on the known molecular mechanisms resulting in resistance of neoplastic cells against anticancer agent and ionizing radiation-initiated oxidative stress.

PDGF and PI3K in tumor progression
In our study, resistance against H2O2-induced oxidative damage and cell death of dermal fibroblasts was initiated by the growth factor PDGF-BB that activated the PI3K-dependent survival pathway. PDGF, a homodimeric or heterodimeric molecule consisting mainly of PDGF-A and/or PDGF-B subunits, is known to be an important regulator of chemotaxis, proliferation and survival for a variety of cells (56). Aberrant signaling by the overexpressed PDGF-B subunit is involved in neoplastic transformation and tumor progression in a variety of cancers. For example, the degree of malignancy in gliomagenesis correlates with the establishment of an autocrine loop by overexpressed PDGF-B, and blockade of the PDGF/PDGFR pathway by dominant-negative mutants reverted the malignant phenotype of brain cells (10,57). Furthermore, overexpression of autocrine PDGF-A and PDGF-B was found in human malignant melanoma in vivo (58). Unlike, by paracrine stimulation of stromal fibroblasts and molecular ‘feedback’ mechanisms, PDGF-BB induced hyperproliferation of immortalized keratinocytes, thereby promoting tumorigenicity (59). These data focusing on paracrine mechanisms of PDGF-BB in tumor–stroma interaction are in line with the finding herein, that tumor cell-derived PDGF-BB initiates protection of stromal fibroblasts against oxidative damage.

Downstream of the PDGF/PDGFR-initiated signaling, the kinases PI3K and Akt were identified as key players in context of cell survival that comprises counteraction of apoptotic mechanisms (27,60). The involvement of PI3K/Akt in survival, migration and invasion of cancer cells was already demonstrated. For example, increased PI3K activity and higher levels of PI3K phosphorylated Akt stand for poor prognosis in primary meningiomas (61). Further on, a PI3K controlled overexpression and activation of members of the Akt family occur in more than two-thirds of human melanomas (62). In murine NIH3T3 fibroblasts overexpressing the human platelet-derived growth factor B-chain under the control of an SV40 promoter (NIH/sis), intact PI3K activity was required for the morphological alterations and the enhanced migratory response which are hallmarks for PDGF-BB-induced autocrine transformation (63). In addition, the PI3K/Akt pathway is involved in mediating survival signals which rescue Ewing tumor cells from induced cell death (64). Apparently, the functions of PI3K and Akt on cell migration and invasion must be tumor cell and tissue specific, a recurring theme in tumorigenesis which is based at least in part on the genomic instability of tumor cells.

The present study focused on the tumor microenvironment, represented by dermal fibroblasts. The protection of these fibroblasts from H2O2-dependent oxidative damage, correlating with increased cell survival, is mediated by the tumor cell-initiated and PI3K-triggered phosphorylation of the antiapoptotic Akt/PKB, and does not depend on both the H2O2-triggered activation of Akt as described earlier (65) and the upregulation of the H2O2 detoxifying cGPx. Our data are in line with the finding that PDGF-BB-initiated and PI3K-triggered phosphorylation of Akt protects against H2O2-induced apoptosis and promotes the migratory response of fibroblasts in a mouse model for wound healing (66), pointing out the importance of the PDGF–PI3K pathway for cell survival of fibroblasts. Furthermore, green tea polyphenols protect human skin fibroblasts against peroxynitrite-induced cytotoxicity via the PI3K pathway (67).

Taken together, the tumor cell initiated protection of (stromal) fibroblasts against increased ROS levels, which depends on the PI3K-controlled activation of its downstream target Akt, may contribute to the problems in chemo- and radiotherapy. The concept of stromal resistance emphasizes the importance of stromal cells in the development of rational clinical strategies. Fibroblasts are therefore a key determinant and important target for cancer therapies.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figure 1 can be found at http://carcin.oxfordjournals.org/.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Deutsche Krebshilfe e.V. (107160, 10-2223).


   Acknowledgments
 
We thank C. Wyrich for excellent technical assistance. H.S. is a Fellow of the National Foundation for Cancer Research, Bethesda, MD, USA.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received September 4, 2007; revised December 17, 2007; accepted December 17, 2007.


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