Cancer Research The Future of Cancer Research: Science and Patient Impact Cancer Health Disparities Conference 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 3708-3715, April 15, 2007. doi: 10.1158/0008-5472.CAN-06-4164
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Orosco, A.
Right arrow Articles by Modrowski, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Orosco, A.
Right arrow Articles by Modrowski, D.

Cell, Tumor, and Stem Cell Biology

Syndecan-2 Affects the Basal and Chemotherapy-Induced Apoptosis in Osteosarcoma

Armelle Orosco1,2, Olivia Fromigué1,2, Céline Bazille1,2, Natacha Entz-Werle3, Pierre Levillain4, Pierre J. Marie1,2 and Dominique Modrowski1,2

1 Inserm, U606 and 2 Université Paris 7, Paris, France; 3 Biochemistry and Molecular Biology Department, CHRU Hautepierre, Strasbourg, France; and 4 Department of Anatomy and Pathologic Cytology, Medical University of Poitiers, Poitiers, France

Requests for reprints: Dominique Modrowski, Unit 606, Institut National de la Sante et de la Recherche Medicale, Hôpital Lariboisière, 2 rue Ambroise Paré, 75475 cedex 10, Paris, France. Phone: 33-1-49-95-63-58; Fax: 33-1-49-95-84-52; E-mail: dominique.modrowski{at}larib.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Syndecans are transmembrane heparan sulfate proteoglycans controlling cell adhesion, migration, and proliferation. We previously showed that syndecan-2 is involved in the control of apoptosis in cultured osteosarcoma cells. These data led us to the hypothesis that syndecan-2 may play a role in the apoptotic signaling in bone tumors. We immunohistochemically analyzed tissue sections from biopsies from 21 patients with well-characterized osteosarcoma. These tissues expressed low levels of syndecan-2 compared with osteoblasts and osteocytes in normal bone. Cultured human osteosarcoma cells also produced lower mRNA levels of syndecan-2 than normal osteoblastic cells. Moreover, the presence of syndecan-2 correlated with spontaneous apoptosis in osteosarcoma tissues as assessed by detection of DNA fragmentation in situ. Overexpression of syndecan-2 resulted in decreased number of migrating and invading U2OS osteosarcoma cells in Matrigel. In addition, overexpression of syndecan-2 sensitized human osteosarcoma cells to chemotherapy-induced apoptosis, increasing the response to methotrexate, doxorubicin, and cisplatin. Consistently, knockdown of the proteoglycan using stable transfection with a plasmid coding small interfering RNA resulted in inhibition of chemotherapy-induced apoptosis. Analysis of syndecan-2 expression both in biopsies and in corresponding postchemotherapy-resected tumors, as well as in cells treated with methotrexate or doxorubicin, showed that the cytotoxic action of chemotherapy can be associated with an increase in syndecan-2. These results provide support for a tumor-suppressor function for syndecan-2 and suggest that dysregulation of apoptosis may be related to abnormal syndecan-2 expression or induction in osteosarcoma. Moreover, our data identify syndecan-2 as a new factor mediating the antioncogenic effect of chemotherapeutic drugs. [Cancer Res 2007;67(8):3708–14]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Osteosarcoma is the most common primary bone cancer occurring mainly in children (1, 2). Aggressive chemotherapies and surgical treatment are currently done to achieve a disease-free state. However, overall 2-year survival rate in patients with osteosarcoma remains below 66% because a significant portion of patients respond poorly to chemotherapeutic protocols and because of frequent metastases occurring in lung and brain (2, 3). In cancer cells, dysregulation of apoptotic processes are believed to contribute to metastatic potential and to drug resistance (4, 5). Although such dysregulation may represent a potent source of new therapeutic targets, the molecular mechanisms that control osteosarcoma cell fate are largely unknown.

Syndecans are transmembrane heparan sulfate proteoglycans that serve as low-affinity receptors for various extracellular soluble and matrix components (6). They modulate the binding and availability of factors, thereby inducing downstream signaling pathways that control cell adhesion, migration, differentiation, and proliferation. Both tumor suppressor and tumor growth promoter activities have been reported for syndecans depending on the type of cancer (7–9). Syndecan-2 cooperates with integrins in lung carcinoma cells (10) to induce adhesion and was found associated with an antimetastatic effect (11). In contrast, syndecan-2, which is highly expressed in several colon cancer cell lines, is also involved in the adhesion and proliferation of these cells and promotes their tumorigenic activity (12, 13). It was shown that syndecan-2 expression occurs during mouse embryonic skeletal development and persists in differentiated bone structures (14). Moreover, human adult normal osteoblastic cells express high level of this proteoglycan (15). In contrast, some osteosarcoma cell lines were found to produce very low level of syndecan-2 (16). We recently reported that syndecan-2 is involved in the control of apoptosis in osteosarcoma cells. Indeed, overexpression of syndecan-2 in osteosarcoma cells induced signaling modifications such as inhibition of protein kinase C{delta} and extracellular signal-regulated kinase as well as activation of c-Jun-NH2-kinase, resulting in cell death (16, 17). These data led us to hypothesize that syndecan-2 may play a role in the apoptotic signaling in bone tumors and may control osteosarcoma cell fate in vivo. Here, we examined the expression and role of syndecan-2 in osteosarcoma cells in vivo. We show that syndecan-2 expression is low in bone tumors and this correlates with spontaneous apoptosis. To determine whether syndecan-2 could modulate the apoptotic response to chemotherapeutic drugs, we used methotrexate, doxorubicin, and cisplatin, drugs that are currently used in chemotherapeutic protocols to treat osteosarcoma. We show here that syndecan-2 sensitizes osteosarcoma cells to chemotherapy-induced apoptosis and that chemotherapeutic drugs can promote syndecan-2 expression. These results indicate that syndecan-2 may contribute to the antioncogenic effect of chemotherapeutic drugs in osteosarcoma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical samples. Slices of formalin-fixed and paraffin-embedded osteosarcoma tissues were obtained from biopsies in 21 patients. These biopsies have been histologically characterized by pathologists. For five patients, samples were also obtained from surgically resected tumors after preoperative chemotherapy. Informed consent was obtained from each patient or the patient's guardian.

Immunohistochemistry. Tissue sections were deparaffinized with xylene and rehydrated in decreasing concentrations of ethanol to distilled water. The sections were then washed in PBS containing 0.1% Tween 20 (pH 7.6). Background labeling was reduced by heparitinase III and chondroitinase ABC digestion (Sigma-Aldrich, Saint Louis, MO). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol. After multiple rinses in PBS and antigoat blocking serum, 2 µg of rabbit anti–syndecan-2 antibody that react with the cytoplasmic domain of syndecan-2 were added (Zymed, San Francisco, CA). Sections were then sequentially incubated in secondary biotinylated antirabbit antibody, in biotin-avidin amplification system, and finally with diaminobenzidine (DAB) substrate. The sections were counterstained with 0.1% methylene blue for tissue morphology, dehydrated with xylene, and mounted using Entelan mounting medium (Microscopy, Karlsruhe, Germany). Stained sections were examined independently by three persons, under a microscope, at high and low magnifications (x4, x20, and x40 objective lens) to select only the cellular signal. Syndecan-2 staining was scored by +/– and + system according the quantity of cells displaying a clear labeling and the intensity of staining. These qualitative data were then transformed to numerical ones.

In situ labeling of apoptotic cells. Detection of apoptosis assessed by DNA fragmentation was done using ApopTag technology (Intergen Company, New York, NY). Osteosarcoma tissue sections were deparaffinized, digested with heparitinase III, chondroitinase ABC, and proteinase K (Sigma-Aldrich). The slides were then incubated in 3% hydrogen peroxide in PBS. Following two rinses in PBS, sections were sequentially incubated in equilibrium buffer, with terminal deoxynucleotidyl transferase (TdT) and digoxigenin-labeled nucleotides. Then, a peroxidase-conjugated antidigoxigenin antibody was added to the tissue sections and the product was developed in DAB. Sections were counterstained with 0.1% methylene blue for tissue morphology and mounted using Entelan. Tissue sections treated with DNase for 30 min served as positive controls for detecting DNA fragmentation and negative controls included tissue sections processed without TdT enzyme to ensure that the staining was not due to nonspecific incorporation of nucleotides.

Osteosarcoma and osteoblastic cells. Human osteosarcoma cell lines were MG63, U2OS, SaOS2, and OHS4. Immortalized osteoblastic cells (18) or primary cultures of osteoblasts isolated from nonpathologic human bone (19) served as normal controls. All cells were maintained in DMEM supplemented with 10% FCS and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin).

Cell transfection and transduction. TRIP-GFP, a gift from Pierre Charneau, is a lentiviral HIV-1–derived vector expressing enhanced green fluorescent protein (GFP) from the cytomegalovirus immediate-early promoter (20). Syndecan-2 sequence was subcloned into this vector, replacing the GFP sequence between the BamH1-Kpn1 sites, to create TRIP-SYND2. Virion particles containing the TRIP-GFP, TRIP-SYND2, or the control empty vector (TRIP-EV) were produced by transient calcium phosphate cotransfection of 293T cells with the vector plasmid, an encapsidation plasmid (R8.2), and a vesicular stomatitis virus G protein envelope expression plasmid (pHCMV-G). Osteosarcoma cells were transduced with lentiviral vector particles in the presence of polybrene (10 µg/mL) for 48 h. The optimal virion concentration was previously determined using different concentrations of viral supernatant containing TRIP-GFP.

Stable expression of small interfering RNA. To inhibit syndecan-2 expression, U2OS cells were stably transfected with psiRNA plasmid (Invivogen, San Diego, CA) containing a sequence coding for small interfering RNA (siRNA) against syndecan-2 or a scrambled sequence as control. These siRNA sequences were selected with use of computer-assisted programs: for syndecan-2, 5'-GCTGACATCTGATAAAGACAT-3'; for the scrambled sequence, 5'-GGCCGTCACCAACATGTA-3'. The oligonucleotides containing the sequence for siRNA were annealed and cloned into psiRNA. U2OS cells were transfected with these constructs using the Transfast system (Promega, San Luis Obispo, CA) and selected in G418 antibiotic. The whole-cell population that survived antibiotic selection was amplified and used for the experiments.

Western blot analysis. Cells were washed with cold PBS and then lysed on ice in 50 mmol/L Tris-HCl (pH 8), containing 150 mmol/L NaCl, 5 mmol/L EDTA, a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IL), and 1% NP40. Protein content was determined using the DC Protein Assay (Bio-Rad, Hercules, CA). Protein lysates were subjected to SDS-PAGE and electrotransferred onto polyvinylidene difluoride membrane. The membrane was then blocked in 20 mmol/L Tris-HCl (pH 7.5), 137 mmol/L NaCl, 0.1% Tween 20, 1% bovine serum albumin (TBSTA), incubated with primary antibodies diluted in TBSTA, and then with appropriate horseradish peroxidase–conjugated antibodies. The signals were visualized using a chemiluminescent detection system (Pierce Biotechnology, Rockford, IL). Syndecan-2 protein overexpression was shown by the increased low molecular weight band level that corresponds to the monomeric nonglycosylated form of syndecan-2. We also checked as previously described (17) that transduction induced a strong increase in the glycosylated protein present in the membrane corresponding to functional syndecan-2 (data not shown).

RNA extraction and quantitative real-time PCR. Total RNA was isolated from cultured cells using a ready-to-use monophasic solution of phenol and guanidine isothiocyanate. Two micrograms were subjected to cDNA synthesis for 1 h at 37°C using 200 units M-MLRV reverse transcriptase (Life Technologies, Gaithersburg, MD). Syndecan-2 expression was analyzed by quantitative real-time PCR (qRT-PCR) using Sybr green (Abgene, Epsom, United Kingdom) and the Light Cycler instrument (Roche Molecular Biochemicals). Primers for syndecan-2 were as follows: 5'-TTGGCTTTCTCTTTGCAATTTT-3' and 5'-CCTTCATCCTTCTTTCTCATGC-3' and for reference genes; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-ATCACCATCTTCCAGGAGCG-3' and 5'-CCTGCTTCACCACCTTCTTG-3'; and 18S, 5'-CGGCTACCACATCCAAGGAA-3' and 5'-GCTGGAATTACCGCGGCT-3'. Fold changes for syndecan-2 was normalized using the {Delta}Ct formula.

Motility and Matrigel invasion assay. Cell invasion and motility assays were done using 24-well chambers (BD Biosciences, Bedford, MA) with or without Matrigel, respectively. Cells were trypsinized, counted, and then seeded in the upper chamber at 5 x 104 per well in serum-free medium. DMEM with 10% FCS was added in the bottom well to serve as chemoattractant. Cells were cultured for 24 h. Cells that did not migrate through the membrane or the Matrigel were discarded. Cells that had migrated were then fixed with 4% paraformaldehyde, stained with hematoxylin, and counted. Each experiment was done in triplicate.

Drug-induced apoptosis assay. MG63 and U2OS cells were seeded in 96-well culture plates (104 per well), cultured overnight, and treated with increasing doses of methotrexate, doxorubicin, or cisplatin for 24 h. Apoptosis was then detected using the Apopercentage apoptosis assay (Biocolor, Belfast, Northern Ireland) according to the manufacturer's protocol. Briefly, 20 µL of dye were added to the medium (final dilution 1/100). After 30 min, the cells were washed twice with PBS. Staining was analyzed by microscopy or cells were lysed in DMSO, and absorbance at 550 nm was then determined. In some wells, H2O2 (5 mmol/L) was added to the medium for 2 h before harvesting of the cells to induce apoptosis thereby serving as positive control.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. Cell-mediated reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in formazan was used as a cell viability assay. MG63 and U2OS cells were seeded and treated with drugs as for the apoptosis assay. After 24-h treatment, 10 µL of 3 mg/mL MTT were added to each well at room temperature. After 30 min, the insoluble formazan salt that was produced was solubilized in DMSO and absorbance measured at 540 nm.

Statistical analysis. Each experiment was done at least thrice each time and in triplicates. Two-tailed Student's t test and two-way ANOVA were done.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Osteosarcoma cells express lower syndecan-2 levels than normal osteoblasts in vivo. To determine whether syndecan-2 is expressed in osteosarcomas, we immunohistochemically analyzed tissue sections from biopsies from 21 patients with well-characterized osteosarcoma. In normal bone structures present in biopsies and in sections of nontransformed adult bone, both osteoblasts and osteocytes displayed a strong staining for syndecan-2 (Fig. 1A, a, b ). In contrast, in most osteosarcoma tissues, only a faint or no syndecan-2 labeling was present (Fig. 1A, c, d). When expressed at all, syndecan-2 was detected only in localized spotted areas. The proteoglycan appeared both in osteogenic and chondrogenic osteosarcomas. Syndecan-2 expression level was graded from 0.5 to 4, combining percentage and staining intensity of positive cells in cancerous bone and in normal bone. Results presented in Fig. 1B show that in 18 osteosarcomas out of 21, syndecan-2 expression was lower than in normal bone. Low levels of syndecan-2 was found both in high- and low-grade tumors (Fig. 1B). Consistent with these observations, osteosarcoma cells expressed from 3- to 20-fold less syndecan-2 than normal human osteoblastic cells as measured by qRT-PCR (Fig. 1C).


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		Figure 1. Osteosarcoma cells express lower syndecan-2 levels than normal osteoblasts in vivo. A, syndecan-2 expression was determined by immunohistochemistry on paraffin-embedded tissue sections. Representative slides from adjacent normal bone (a, b) and osteosarcoma (c, d) tissues. Immunoreactive syndecan-2 appears as a strong brown staining in osteoblasts and osteocytes in normal tissues (arrows). Magnifications, x250 (a and c) and x125 (b and d). B, syndecan-2 expression was graded according to the number of positive cells and the intensity of the immunoreactivity in normal bone tissues and in osteosarcoma tissues from 21 patients classified according to the grade of the tumor. C, syndecan-2 mRNA expression was determined in human osteosarcoma cell lines and in nontransformed human osteoblastic cells. Columns, means; bars, SE. *, P < 0.001, significant difference between osteosarcoma and normal bone cells or tissues.

 
Increased syndecan-2 expression results in decreased cell motility. A weak expression of syndecan-2 was consistently found in all subsets of analyzed osteosarcomas irrespective of classification according to the grade of the tumor (Fig. 1B), their osteogenic or chondrogenic phenotype, age of the patients, localization of the tumor, and survival status. Because syndecan-2 was previously shown to be involved in tumorigenic activity in sarcomas (12), we checked whether modulation of the expression of syndecan-2 in osteosarcoma cells affects their motility and their invasive capacity in vitro. To modify syndecan-2, we used a lentiviral vector coding the proteoglycan (TRIP-SYND2) or the control empty vector (TRIP-EV). TRIP-SYND2 allowed increasing syndecan-2 expression by 3- to 5-fold in MG63 cells and U2OS cells (Fig. 2A–C ). Then, we determined the capacity of transduced MG63 and U2OS cells to migrate, in response to chemoattractant medium containing 10% FCS, through a microporous membrane or a Matrigel-coated membrane. U2OS and MG63 cells exhibited very different motility and invasive capacities. U2OS cells migrated at a high rate through the membranes. In U2OS cells, overexpressing syndecan-2 resulted in a significant decrease in the number of migrating and invading cells (Fig. 2D). Although the same trend toward decreased motility was found, TRIP-SYND2 did not significantly modify this variable in MG63 cells. This was probably due to the very low basal capacity of migration of these cells. Overexpression of syndecan-2 did not render MG63 cells competent to invade Matrigel (Fig. 2D).


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Figure 2. Syndecan-2 overexpression in osteosarcoma cells results in decreased cell motility. MG63 and U2OS cells were transduced with empty vector, TRIP-EV (a and c) or TRIP-SYND2 (b and d), and syndecan-2 level was analyzed by Western blot (A and B) and qRT-PCR (C). TRIP-SYND2 induced a significant increase in syndecan-2 expression (P < 0.001). D, the effects of syndecan-2 overexpression on cell motility and invasive capacity were analyzed using transwell assays, counting the cells that migrate across a microporous membrane or across Matrigel, respectively. Columns, means; bars, SE. *, P < 0.01, significant difference between cells transduced with TRIP-SYND2 and TRIP-EV.

 
Syndecan-2 expression in osteosarcoma tissues is related to the level of basal apoptosis. To determine whether expression of syndecan-2 in vivo in osteosarcoma tissues is associated with apoptosis, we detected apoptotic cells in tissue slides consecutive to those used for the syndecan-2 immunostaining. We observed that most regions of the osteosarcoma tumors that exhibited no syndecan-2 staining also displayed a low number of apoptotic cells with fragmented DNA (Fig. 3A, a, b; arrowheads ). In contrast, areas where syndecan-2 was detected contained a significantly higher number of apoptotic cells (Fig. 3A, c, d; asterisks). The number of apoptotic cells and the number of cells with immunoreactive syndecan-2 in 18 fields within different osteosarcoma tissues were enumerated. The Pearson coefficient between syndecan-2 expression and the spontaneous apoptosis that occurs in osteosarcoma was r = 0.7, indicating a link between syndecan-2 and occurrence of apoptosis in vivo (Fig. 3B).


Figure 3
View larger version (57K):
[in this window]
[in a new window]

  		Figure 3. Syndecan-2 expression in osteosarcoma tissues is related to the level of basal apoptosis. A, apoptotic cells were stained using an in situ TdT-mediated nick-end labeling assay in paraffin-embedded tissue sections of osteosarcoma tumors. Fragmented DNA in nuclei of apoptotic cells appears as a brown staining. The corresponding fields were located in adjacent tissue sections stained for syndecan-2 by immunochemistry. Representative fields with (*) or without (arrowheads) syndecan-2 expression (b and d) and the corresponding fields stained for apoptotic cells (a and c). B, the number of apoptotic cells and syndecan-2–positive cells in the corresponding fields of adjacent tissue slides was counted. r is the Pearson correlation coefficient.

 
Syndecan-2 sensitizes osteosarcoma cells to cytotoxic drugs. Together, the finding that syndecan-2 is associated with apoptosis in osteosarcoma tissues and our previous results showing that syndecan-2 is able to modify osteosarcoma cell signaling to induce cell death suggest that the proteoglycan could modulate responsiveness to cytotoxic drugs. We therefore investigated the effect of syndecan-2 overexpression on the induction of apoptosis by methotrexate, doxorubicin, and cisplatin in MG63 cells. These cells that express very low basal level of the proteoglycan were transduced with TRIP-EV or TRIP-SYND2 as described (Fig. 2A and B) and then treated with different concentrations of the drugs for 24 h. Induction of apoptosis was measured by the Apopercentage assay. As expected, overexpression of syndecan-2 increased basal apoptosis level in MG63 and U2OS cells (Supplementary Data 1A and 1B). The addition of 5 mmol/L H2O2 for 2 h in the culture medium induced a high increase in dye accumulation in the cells (Supplementary Data 1C) and served as a positive control for apoptosis induction. Control MG63 cells were not sensitive to methotrexate even at high dosage (Fig. 4A ), whereas doxorubicin and cisplatin induced only weak apoptotic changes in these cells (Fig. 4B and C). Transduction with TRIP-SYND2 sensitized MG63 cells to methotrexate at dosage as low as 10 µmol/L (Fig. 4A). Moreover, the increased syndecan-2 expression also resulted in a stronger response to doxorubicin and cisplatin (Fig. 4B and C).


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Figure 4. Syndecan-2 sensitizes osteosarcoma cells to cytotoxic drugs. MG63 and U2OS cells were transduced with TRIP-SYND2 or control vector. Apoptosis induced by cytotoxic drugs was measured by colorimetric detection of Apopercentage dye uptake in MG63 cells treated with the indicated doses of methotrexate (A), doxorubicin (B), or cisplatin (C). Points, means; bars, SE. The slopes of the regression lines for the two conditions (TRIP-EV and TRIP-SYND2) differ significantly (P < 0.001).

 
Knockdown of syndecan-2 results in inhibition of apoptosis induction by drugs in osteosarcoma cells. To further document the role of syndecan-2 in the apoptotic response to chemotherapeutic drugs, we used U2OS cells, which express higher level of the proteoglycan and which are more sensitive to doxorubicin and to cisplatin than MG63 cells. Indeed, very low dosages of the two drugs induced apoptosis in U2OS (Fig. 5C and D ). We analyzed U2OS cell population expressing a siRNA sequence targeted against syndecan-2 (psiSYND2). In these cells, syndecan-2 level was reduced by 7- to 10-fold compared with the control cell population stably transfected with a scramble siRNA sequence (psiscramble; Fig. 5A and B). Inhibition of syndecan-2 expression resulted in a clear decrease in U2OS sensitivity to doxorubicin and cisplatin (Fig. 5C and D). These results suggest that syndecan-2 contributes to drug-induced apoptosis.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Figure 5. Syndecan-2 silencing results in inhibition of apoptosis induction by drugs in osteosarcoma cells. U2OS cells were transfected with psiRNA plasmid containing a sequence coding for siRNA against syndecan-2 (psi SYND2) or a scrambled sequence as control (psi Scramble) and selected for stable expression. The level of syndecan-2 in transfected cells was determined by Western blot (A) and qRT-PCR (B). C and D, apoptosis induced by cisplatin or doxorubicin was measured by colorimetric detection of Apopercentage dye uptake in psi Scramble– and psi SYND2–expressing cells. Points, mean; bars, SE. The slopes of the regression lines for the two conditions (psi Scramble and psiSYND2) differ significantly (P < 0.001).

 
The cytotoxic action of chemotherapeutic drugs is associated with increased syndecan-2 expression. Although syndecan-2 seems to be crucial for the cytotoxic activity of doxorubicin and cisplatin in U2OS cells as well as rendering MG63 cells sensitive to methotrexate, there was no clear correlation between the level of syndecan-2 found in tumors and the histologic response to chemotherapy as evaluated in situ by the pathologists (Fig. 1B). We therefore investigated whether chemotherapeutic drugs could modify syndecan-2 expression. We examined the presence of immunoreactive syndecan-2 in biopsies and in corresponding osteosarcoma tissues obtained from the surgical resection after a preoperative chemotherapy in five patients. We found that the level of syndecan-2 was higher in two samples of postchemotherapy-resected tumors compared with biopsies obtained pretreatment from the same patient (Fig. 6A ). In three other patients, a very weak or no difference in the syndecan-2 level was found in tissues obtained before and after treatment (data not shown). A striking result was that the increased syndecan-2 expression after induction chemotherapy was associated with the strongest histologic responses (50% and 42% of necrotic cells present after treatment in tumors) compared with the three patients characterized as very poor responders (20%, <10%, and <10% of necrotic cells, respectively) and who did not display any change in syndecan-2 expression postchemotherapy. These results suggest that the cytotoxic activity of the chemotherapeutic drugs may be associated with an induction of syndecan-2 in osteosarcoma. To confirm this finding, we measured syndecan-2 mRNA expression in osteosarcoma cells after 24-h exposure to various concentrations of methotrexate or doxorubicin. In MG63 and U2OS cells, methotrexate did not alter syndecan-2 expression. In contrast, doxorubicin induced a significant increase in syndecan-2 RNA (Fig. 6B). Syndecan-2 induction by doxorubicin was dose dependent in both cell lines and was of a greater magnitude in U2OS cells compared with MG63 cells. Experiments to evaluate the effect of the two drugs on cell viability in the same conditions indicated that the induction of syndecan-2 was related to the cytotoxic activity of the drugs in both MG63 and U2OS cells. Although methotrexate did not modify the viability of the two cell lines, doxorubicin induced a dose-dependent decrease in cell viability that was proportionately greater in U2OS than in MG63 cells (Fig. 6C). Overall, these results show that chemotherapeutic drugs are able to induce syndecan-2 expression in vivo and in vitro and that the induction of syndecan-2 may correlate with the cytotoxic effect of the drugs.


Figure 6
View larger version (44K):
[in this window]
[in a new window]

  		Figure 6. Syndecan-2 expression can be induced in osteosarcoma tissues and cells after chemotherapy. A, syndecan-2 level was determined by immunohistochemistry in osteosarcoma tissue sections obtained before and after preoperative chemotherapy in two patients. Syndecan-2 staining was increased after chemotherapy. B, syndecan-2 mRNA level was determined by qRT-PCR in MG63 and U2OS cells treated by grading doses of methotrexate or doxorubicin (top). GAPDH served as reference gene. Columns, mean of fold changes against nontreated cells; bars, SE. C, the effects of methotrexate and doxorubicin on cell viability were measured using a MTT assay. Results are expressed as the mean level for each condition relative to the mean value obtained for untreated cells. *, P < 0.05, significant difference between treated and untreated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Osteosarcoma is the most common malignant bone tumor in children (1, 2). Recent improvements in its treatment have resulted in long-term disease-free survival rates approaching 70% in patients with localized disease. However, a large portion of patients do not respond to chemotherapy protocols and go on to develop metastasis. These patients are at a higher risk of relapse and poor outcome. Intensified chemotherapies failed to overcome the tumor resistance in poor responders (21). Thus, it is crucial to understand the mechanisms that support this resistance to chemotherapy and to identify new targets to improve the management of osteosarcomas. In this study, we identified syndecan-2 as a new factor mediating basal and drug-induced apoptosis in osteosarcoma tissues and cells.

We show here that osteosarcoma tissues express very low levels of syndecan-2 compared with osteoblasts and osteocytes in normal bone. Consistent with this observation, we also found that cultured osteosarcoma cells produce low levels of syndecan-2 compared with normal human osteoblastic cells. Although the exact function of syndecan-2 in osteoblastic cells is not yet elucidated, reduced level of the proteoglycan could reflect a relatively undifferentiated phenotype in osteosarcomas. In support of this hypothesis, undifferentiated human bone marrow stromal cells (22) express lower syndecan-2 levels compared with more differentiated osteoblastic cells (data not shown). Moreover, syndecan-2 expression increases during conversion of myoblasts to osteoblasts in response to bone morphogenetic protein-2, suggesting that syndecan-2 expression is induced during osteogenic differentiation (23). Thus, the low expression of syndecan-2 may be an important event in osteosarcoma biology because terminal osteoblast differentiation is inhibited in osteosarcoma with decreased RUNX-2 and Osterix expression (24, 25) and because there is an inverse relationship between osteoblastic differentiation and tumorigenesis (24). Alteration of syndecan-1 expression was described in tumoral tissues and associated with loss of an antitumoral activity. For example, syndecan-1 was found to be down-regulated during epithelial cell transformation, resulting in the loss of the epithelial phenotype and acquisition of an invasive and metastatic phenotype (26, 27). We report here that overexpression of syndecan-2 results in decreased number of migrating and invading U2OS cells and does not render MG63 cells competent to invade Matrigel. These data support an antitumoral role for syndecan-2 in osteosarcoma. This is in contrast with the tumor promoter function that was described for the proteoglycan in colon cancer cells (28), but is in agreement with its ability to mediate cell binding to collagen and to inhibit cell invasion in other types of cancer cells (29).

Our finding that syndecan-2 levels correlate with spontaneous apoptosis in osteosarcoma tissues provides further support for a tumor-suppressor function for syndecan-2. This strengthens our previous in vitro data showing that syndecan-2 expression results in modifications of different signaling pathways that promote cell death (16, 17). Altogether, these data indicate that syndecan-2 is a key regulator of apoptosis in osteosarcoma cells and that low level of syndecan-2 may activate a default pathway resulting in apoptosis in these cancer cells.

Dysregulation of apoptosis due to alteration of p53 or phosphatidylinositol 3-kinase/AKT pathways or Bcl-2 overexpression have been previously shown to contribute to chemotherapeutic drug resistance, allowing cancer cells to escape stress effects induced by therapeutic agents (3). Here, we show that syndecan-2 level affects chemotherapeutic-induced apoptosis using methotrexate, doxorubicin, and cisplatin, drugs that are currently used in chemotherapy protocols to treat osteosarcoma. In MG63 cells that produce very low basal level of syndecan-2 and display none or only minimum response to the three drugs, increasing the expression of the proteoglycan results in a clear improvement in the apoptotic response to chemotherapeutic drugs. Moreover, in U2OS cells that express syndecan-2 at a higher basal level than MG63 cells, doxorubicin and cisplatin induced a much stronger apoptotic response than in MG63 cells. Silencing syndecan-2 expression in U2OS cells resulted in a clear inhibition of drug-induced apoptosis. Together, these results indicate that syndecan-2 is able to sensitize osteosarcoma cells to chemotherapy and contributes to apoptotic signaling propagation.

The level of syndecan-2 found in biopsies at diagnostic does not seem to be predictive of the histologic response to chemotherapy. However, we show here that syndecan-2 expression can be induced in osteosarcoma tissues after chemotherapy in some patients. Moreover, doxorubicin at dosages effective to reduce osteosarcoma cell viability also induced syndecan-2 expression in MG63 and U2OS cells, whereas methotrexate did not modify cell viability and syndecan-2 expression. Thus, syndecan-2 may represent a new target gene to improve chemotherapy. These data also suggest that induction of syndecan-2 is a key event mediating cytotoxic effects of chemotherapy. Consistent with our results, a positive relationship between syndecan-1 induction after chemotherapy and favorable prognosis was previously described in oral cancer (30). Others have also shown that syndecan-2 expression is decreased by interleukin-6, a cytokine that promotes osteoblastic cell survival and resistance to paclitaxel, an antimitotic agent, in osteosarcoma cells (31–33). Although larger studies are required to correlate syndecan-2 induction with various variables in osteosarcomas (grade, survival, metastasis), our data suggest that syndecan-2 may represent a new marker to evaluate the apoptotic response to preoperative chemotherapy.

In conclusion, our analysis of syndecan-2 expression in osteosarcoma tissues, supported by in vitro analysis in human osteosarcoma cell lines, clearly shows that this proteoglycan is involved in the control of cell death and affects the apoptotic response to chemotherapeutic agents in osteosarcoma.


   Acknowledgments
 
Grant support: A Ph.D. studentship from ARC (JR/MLD/MDV-A05/3) and a grant from Cancer and Solidarity Foundation, Geneva, Switzerland (A. Orosco).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. P. Charneau and E. Laplantine (Institut Pasteur, Paris, France) for the gift of the TRIP vector, Dr. B. Oyajobi (San Antonio, TX) for critical reading of the manuscript, and Prof. F. Debiais (CHR Poitiers, Poitiers, France) for her help.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 11/10/06. Revised 2/ 2/07. Accepted 2/14/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gibbs CP, Jr., Weber K, Scarborough MT. Malignant bone tumors. Instr Course Lect 2002;51:413–28.[Medline]
  2. Meyers PA, Gorlick R. Osteosarcoma. Pediatr Clin North Am 1997;44:973–89.[CrossRef][Medline]
  3. Link MP, Goorin AM, Miser AW, et al. The effect of adjuvant chemotherapy on relapse free survival in patients with osteosarcoma of the extremity. N Engl J Med 1986;314:1600–6.[Abstract]
  4. Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer 2002;2:277–88.[CrossRef][Medline]
  5. Lafleur EA, Koshkina NV, Stewart J, et al. Increased Fas expression reduces the metastatic potential of human osteosarcoma cells. Clin Cancer Res 2004;10:8114–9.[Abstract/Free Full Text]
  6. Bernfield M, Götte M, Park PW, et al. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 1999;68:729–77.[CrossRef][Medline]
  7. Inki P, Joensuu H, Grenman R, Klemi P, Jalkanen M. Association between syndecan-1 expression and clinical outcome in squamous cell carcinoma of the head and neck. Br J Cancer 1994;70:319–23.[Medline]
  8. Aref S, Goda T, El-Sherbiny M. Syndecan-1 in multiple myeloma: relationship to conventional prognostic factors. Hematology 2003;8:221–8.[CrossRef][Medline]
  9. Barbareschi M, Maisonneuve P, Aldovini D, et al. High syndecan-1 expression in breast carcinoma is related to an aggressive phenotype and to poorer prognosis. Cancer 2003;98:474–83.[CrossRef][Medline]
  10. Kusano Y, Oguri K, Nagayasu Y, et al. Participation of syndecan 2 in the induction of stress fiber formation in cooperation with integrin {alpha}5ß1: structural characteristics of heparan sulfate chains with avidity to COOH-terminal heparin-binding domain of fibronectin. Exp Cell Res 2000;256:434–44.[CrossRef][Medline]
  11. Munesue S, Kusano Y, Oguri K, et al. The role of syndecan-2 in regulation of cytoskeletal organization of Lewis lung carcinoma-derived metastatic clones. Biochem J 2002;363:201–9.[CrossRef][Medline]
  12. Park H, Kim Y, Lim Y, Han I, Oh ES. Syndecan-2 mediates adhesion and proliferation of colon carcinoma cells. J Biol Chem 2002;277:29730–6.[Abstract/Free Full Text]
  13. Kim Y, Park H, Lim Y, Kwon HJ, Woods A, Oh ES. Decreased syndecan-2 expression correlates with trichostatin-A induced-morphological changes and reduced tumorigenic activity in colon carcinoma cells. Oncogene 2003;22:826–30.[CrossRef][Medline]
  14. David G, Bai XM, Van der Schueren B, Marynen P, Cassiman JJ, Van den Berghe HS. Partial and temporal changes in the expression of fibroglycan (syndecan-2) during mouse embryonic development. Development 1993;119:841–54.[Abstract]
  15. Modrowski D, Baslé M, Lomri A, Marie PJ. Syndecan-2 is involved in the mitogenic activity and signaling of granulocyte-macrophage colony-stimulating factor in osteoblasts. J Biol Chem 2000;275:9178–85.[Abstract/Free Full Text]
  16. Modrowski D, Orosco A, Thevenard J, Fromigue O, Marie PJ. Syndecan-2 overexpression induces osteosarcoma cell apoptosis: implication of syndecan-2 cytoplasmic domain and JNK signaling. Bone 2005;37:180–9.[Medline]
  17. Orosco A, Thévenard J, Fromigué O, Marie PJ, Modrowski D. Dual involvment of PKC{delta} in the apototic signaling induced by syndecan-2 in osteoblastic cells. J Cell Biochem 2006;98:838–50.[CrossRef][Medline]
  18. Hay E, Hott M, Graulet AM, Lomri A, Marie PJ. Effects of bone morphogenetic protein-2 on human neonatal calvaria cell differentiation. J Cell Biochem 1999;71:81–93.
  19. de Pollack C, Arnaud E, Renier D, Marie PJ. Age-related changes in bone formation, osteoblastic cell proliferation, and differentiation during postnatal osteogenesis in human calvaria. J Cell Biochem 1997;64:128–39.[CrossRef][Medline]
  20. Dardalhon V, Herpers B, Noraz N, et al. Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap. Gene Ther 2001;8:190–8.[CrossRef][Medline]
  21. Bacci G, Forni C, Ferrari S. Neoadjuvant chemotherapy of osteosarcoma of the extremity: intensification of preoperative treatment does not in crease the rate of good histologic response to the primary tumor or improve the final outcome. J Pediatr Hematol Oncol 2003;25:845–53.[CrossRef][Medline]
  22. Ahdjoudj S, Lasmoles F, Oyajobi BO, Lomri A, Delannoy P, Marie PJ. Reciprocal control of osteoblast/chondroblast and osteoblast/adipocyte differentiation of multipotential clonal human marrow stromal F/STRO-1(+) cells. J Cell Biochem 2001;81:23–38.[CrossRef][Medline]
  23. Gutierrez J, Osses N, Brandan E. Changes in secreted and cell associated proteoglycan synthesis during conversion of myoblasts to osteoblasts in response to bone morphogenetic protein-2. J Cell Physiol 2006;206:58–7.[CrossRef][Medline]
  24. Thomas DM, Johnson SA, Sims NA, et al. Terminal osteoblast differentiation, mediated by runx2 and p27Kip1, is disrupted in osteosarcoma. J Cell Biol 2004;167:925–34.[Abstract/Free Full Text]
  25. Cao Y, Zhou Z, de Crombrugghe B, et al. Osterix, a transcription factor for osteoblast differentiation, mediates antitumor activity in murine osteosarcoma. Cancer Res 2005;65:1124–8.[Abstract/Free Full Text]
  26. Leppa S, Vleminckx K, Van Roy F, Jalkanen M. Syndecan-1 expression in mammary epithelial tumor cells is E-cadherin-dependent. J Cell Sci 1996;109:1393–403.[Abstract]
  27. Soukka T, Pohjola J, Inki P, Happonen RP. Reduction of syndecan-1 expression is associated with dysplastic oral epithelium. J Oral Pathol Med 2000;29:308–13.[CrossRef][Medline]
  28. Park H, Han I, Kwon HJ, Oh ES. Focal adhesion kinase regulates syndecan-2-mediated tumorigenic activity of HT1080 fibrosarcoma cells. Cancer Res 2005;65:9899–905.[Abstract/Free Full Text]
  29. Liu W, Litwack ED, Stanley MJ, Langford JK, Lander AD, Sanderson RD. Heparan sulfate proteoglycans as adhesive and anti-invasive molecules. Syndecans and glypican have distinct functions. J Biol Chem 1998;273:22825–32.[Abstract/Free Full Text]
  30. Nemeth Z, Szigeti K, Mathe M, Szabo G, Velich N, Suba Z. Effect of induction chemotherapy on changes of laminin and syndecan expression in oral squamous cell carcinomas: a prospective, randomized, clinicopathologic and immunohistochemical study. J Craniofac Surg 2005;16:205–12.[CrossRef][Medline]
  31. Bellido T, O'Brien CA, Roberson PK, Manolagas SC. Transcriptional activation of the p21(WAF1,CIP1,SDI1) gene by interleukin-6 type cytokines. A prerequisite for their pro-differentiating and anti-apoptotic effects on human osteoblastic cells. J Biol Chem 1998;273:21137–44.[Abstract/Free Full Text]
  32. Duan Z, Lamendola DE, Penson RT, Kronish KM, Seiden MV. Overexpression of IL-6 but not IL-8 increases paclitaxel resistance of U-2OS human osteosarcoma cells. Cytokine 2002;17:234–42.[CrossRef][Medline]
  33. Birch MA, Skerry TM. Differential regulation of syndecan expression by osteosarcoma cell lines in response to cytokines but not osteotropic hormones. Bone 1999;24:571–8.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Orosco, A.
Right arrow Articles by Modrowski, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Orosco, A.
Right arrow Articles by Modrowski, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online