QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 6 June 2006
doi: 10.1242/jcs.03011

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cat, B. Articles by Brenneisen, P. Search for Related Content PubMed PubMed Citation Articles by Cat, B. Articles by Brenneisen, P.

Enhancement of tumor invasion depends on transdifferentiation of skin fibroblasts mediated by reactive oxygen species

¹ Institute for Biochemistry and Molecular Biology I, Heinrich-Heine-University, 40225 Düsseldorf, Germany
² Henkel KGaA, 40191 Düsseldorf, Germany

View larger version (44K): [in a new window]   Fig. 1. TGFß1-mediated transition of fibroblasts to myofibroblasts. (A) Subconfluent HDFs were cultured in control conditioned medium (CMHDF), in co-culture with SCL-1 tumor cells (co-cultureHDF,SCL), treated with 10 ng/ml rTGFß1 in CMHDF (CMHDF,TGF), treated with conditioned medium of SCL-1 cells (CMSCL) or treated with CMSCL containing 5.0 µg TGFß1-neutralizing antibody (CMSCL,anti TGF) for different time periods and then immunostained for SMA. Representative images are shown. Dotted line highlights the tumor cell cluster, the arrows indicate the nuclei of the tumor cells. Bar, 20 µm. (B) Subconfluent HDFs were treated with 10 ng rTGFß1/ml CMHDF for 24 hours. The level of SMA protein was determined by western blot. The densitometric values represent the fold increase over control, which was set at 1.0. The data represent means ± s.e.m. of three independent experiments.

View larger version (21K): [in a new window]   Fig. 2. Antioxidants downregulate TGFß1-mediated expression of SMA. (A) Subconfluent HDFs were cultured in CMHDF and either untreated or pretreated for 4 hours with 5.0 mM NAC or for 24 hours with 0.5 µM Na2SeO3 before addition of 10 ng/ml rTGFß1. TGFß1 and the antioxidants were present for an additional 48 hours. The level of SMA protein was determined by western blot. Three independent experiments were performed. (B) HDF monolayer cultures were cultured in CMHDF either containing 0.6 nM SeP for 48 hours before treatment with 10 ng/ml TGFß1 for a further 48 hours or containing SeP for the complete time of 96 hours. The level of SMA protein was determined by western blot. The densitometric analysis describes protein expression as a percentage, setting rTGFß1-treated controls at 100%. The experiments were performed in duplicate. (C,D) Subconfluent HDFs were cultured in CMHDF and either untreated or pretreated for 24 hours with 0.5 µM Na2SeO3 (C) or ebselen (D) before addition of 10 ng/ml rTGFß1. TGFß1 and selenite or ebselen were present for an additional 48 hours. The levels of cytosolic glutathione peroxidase (GPx) (C) and of SMA (D) were detected by western blot. -tubulin was used as loading control. Quantitative data were standardized to -tubulin and densitometric values represent fold increase over control, which was set at 1.0. Data are representative of two independent experiments.

View larger version (14K): [in a new window]   Fig. 3. Antioxidants lower TGFß1-initiated lipid peroxidation. (A) Subconfluent HDFs were cultured in CMHDF and either untreated or pretreated for 24 hours with 50 µM Trolox or 10 µM BHT before addition of 10 ng/ml rTGFß1. TGFß1 and the antioxidants were present for an additional 48 hours. The cell lysates were assayed by western blotting for SMA. The experiments were performed in triplicate. (B) Time-course analysis of rTGFß1-treated HDFs. At the indicated time points, cell lysates were prepared and the content of intracellular LOOH determined. FeSO4 and Asc2P were used as a positive control. The data represent the mean ± s.e.m. of four independent experiments. **P<0.01 versus mock-treated controls (C) (ANOVA, Dunnett's test). (C,D) Subconfluent HDFs were incubated as described in Fig. 2A and Fig. 3A before treatment with 10 ng/ml rTGFß1 for an additional 1 hour (see C) or 2 hours (see D) in the presence of the antioxidants. Cell lysates were prepared and subjected to LOOH measurements (see C) or detection of conjugated dienes (see D). Three independent experiments were performed. **P<0.01 (C) and *P<0.05 (D) versus rTGFß1-treated cells (ANOVA, Dunnett's test).

View larger version (29K): [in a new window]   Fig. 4. ROS-mediated signaling is independent of Smad2 phosphorylation. (A) Time-course analysis of TGFß1-initiated phosphorylation of Smad2. Subconfluent HDFs were untreated (C) or treated with 10 ng/ml rTGFß1 CMHDF for the indicated times. The cell lysates were subjected to western blot analysis for phospho-Smad2. The densitometric data (in arbitrary units, a.u.) represent means ± s.e.m. of three independent experiments. (B) Subconfluent HDFs were preincubated with the antioxidants as described (see Fig. 2A, Fig. 3A) before addition of 10 ng/ml rTGFß1 for a further 10 minutes. The cell lysates were subjected to western blotting analysis for phospho-Smad (P-Smad2) and total Smad2/3. Total Smad2/3 was also used as a loading control. Data shown are representative of three independent experiments.

View larger version (17K): [in a new window]   Fig. 5. Involvement of PKC in TGFß1/ROS-dependent expression of SMA. (A) Subconfluent fibroblasts in CMHDF were preincubated with the PKC inhibitors Ro 31-8220 and Ro 32-0432 for 1 hour before treatment with 10 ng/ml rTGFß1 in combination with each inhibitor. Expression of SMA was detected by western blots. The densitometric analysis describes protein expression as fold increase over control, which was set at 1.0. The data represent the mean ± s.e.m. of three independent experiments. (B) Time-course analysis for activation of PKC was performed using subconfluent HDFs either untreated (C) or treated with 10 ng/ml rTGFß1 CMHDF for the indicated time periods. The cell lysates were subjected to western blot analysis for phospho-PKC. The image is representative of two independent experiments. The densitometric analysis represents fold increase over control (C) which was set at 1.0. (C) Subconfluent HDFs were preincubated with the antioxidants as described prior to addition of 10 ng/ml rTGFß1 for a further 1 minute. Western blot analysis was performed for phospho-PKC and total PKC. Total PKC bands indicate the loading control. Data shown are representative of three independent experiments. (D) Subconfluent HDFs were preincubated with PKC inhibitor RO 31-8220 for 1 hour (closed circles) before treatment with rTGFß1 in CMHDF or 1 mM H2O2 for the indicated time. Increase of DCF fluorescence was followed over 15 minutes versus untreated controls (open circle). The experiments were performed in duplicate. Arrows indicate addition of rTGFß1 or H2O2.

View larger version (22K): [in a new window]   Fig. 6. Antioxidants inhibit TGFß1-mediated transdifferentiation in dermal and skin equivalents. (A) Fibroblasts seeded for 2 days in the dermal equivalent (DE) were untreated or treated with NAC, selenite or Trolox before stimulation with rTGFß1. After a further 48 hours, the collagen gel was dissolved with collagenase, the cells lysed and subjected to western blot analysis for SMA. Detection of -tubulin confirmed equal loading. The diameter (in cm) of the contracted or non-contracted collagen lattices was used as a measure of the contractile force of the cells. Three independent experiments were performed. Bar, 1 cm. (B) Histological structure of a skin equivalent (HE staining). cc-GAG, collagen-chitosan-glycosaminoglycan; d, dermis; e, epidermis; f, fibroblasts; k, normal human epidermal keratinocytes; sc, stratum corneum. Bar, 25 µm. (C) Skin equivalents were incubated for 3 days with 10 ng/ml rTGFß1 keratinocyte-SFM medium (without supplements) or in combination with 5 mM NAC. After dispase II treatment, the dermis was homogenized and 50 µl clear lysate or sample subjected to western blot analysis. -tubulin was used as a loading control. Quantitative data were standardized to -tubulin and densitometric values represent fold increase over the control, which was set at 1.0. The image is representative of two independent experiments.

View larger version (35K): [in a new window]   Fig. 7. Prevention of transdifferentiation lowers the invasive capacity of tumor cells. (A) Conditioned media of HDFs (CMHDF), myofibroblasts (CMMF), SCL-1 cells (CMSCL) and HDF, which were cultured in CMHDF and treated with rTGFß1 and antioxidants (CMMF(antioxidant)) were used for the invasion assays based on matrigel-coated transwells as described in the Materials and Methods. The total number of tumor cells migrating towards the chemoattractive media over a 72 hour time period is a measure of the invasive capacity. The data represent the mean ± s.e.m. of five independent experiments. The insets show Coomassie Blue-stained tumor cells on the lower side of the cell culture insert, migrating either as a cluster of cells (a, dotted lines) or as single cells (b, arrow head). ***P<0.001 versus CMMF (ANOVA, Dunnett's test). (B) Human cytokine antibody arrays were used to compare the pattern of secreted growth factors and cytokines in the conditioned media (CM) of human dermal fibroblasts (HDF) and myofibroblasts (MF), which were collected after a 48 hour period of secretion. The most prominent signals representing interleukin-6 (IL-6), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are shown. Tissue inhibitor of matrix metalloproteinase (TIMP)-1 represents a negative control. The image is representative of two independent experiments, each experiment is a comparison of CMHDF with CMMF. (C) Subconfluent HDFs were cultured in CMHDF and either untreated or treated with antioxidants before addition of 10 ng/ml rTGFß1 for an additional 24 hours. Thereafter, the medium was replaced by fresh medium (without TGFß1 and antioxidants) and after a further 48 hours, the conditioned medium was collected and subjected to VEGF, HGF and IL-6 ELISA. ***P<0.001 versus untreated CMMF (ANOVA, Dunnett's test). (D) Conditioned medium from myofibroblasts (CMMF) was untreated or treated with 5 µg/ml neutralizing antibody medium, and the total number of migrating SCL-1 tumor cells is a measure of the chemoattractive capacity of antibody-treated conditioned medium compared with untreated CMMF, which was set at 100%. Three independent experiments (mean ± s.e.m.) were performed. **P<0.01 versus untreated CMMF (ANOVA, Dunnett's test).

View larger version (34K): [in a new window]   Fig. 8. Novel concept of chemoprevention of tumor progression. Tumor cells release growth factors and cytokines, e.g. TGFß1, which initiate ROS-dependent changes of gene expression in stromal cells through receptors, resulting in transdifferentiation and release of proinvasive signals. Proinvasive signals stimulate the invasive capacity of cancer cells. These signals are lowered or prevented by treatment of stromal cells with antioxidants and/or micronutrients, finally preventing the generation of tumor-educated myofibroblasts and leading to attenuation of tumor invasion.