Abstract
OBJECTIVE Over-expression of transforming growth factor β1 (TGF-β1) has been observed in many advanced cancers. The present study was aimed at developing potential antisense oligonucleotides (ASONs) to repress TGF-β1 expression in rhabdomyosarcoma (RMS) RD cells, and to examine their effect on tumorigenicity of RD cells in vivo.
METHODS ASONs targeting the region surrounding the start codon of TGF-β1 were synthesized and transferred into cells in the form of complexes with Lipofectamine 2000. The TGF-β1 protein was determined by immunofluorescence and ELISA. The cell viability and cell cycle were examined by MTT and flow cytometry. The RD cells, with or without TGF-β1 ASON, in 50 μl of serum-free EMDM medium were injected subcutaneously into the right flank of nude mice. The tumors were then measured and weighed.
RESULTS The ASON sequence targeting the first start site at bases 841-855 of the human TGF-β1 gene had the greatest effect on attenuating the expression of TGF-β1 (P < 0.05). The ASONs induced a decrease in OD values after 6 d (P < 0.05). Analysis of the cell cycle revealed that the ASON induced a significant decrease in cells in the S phase and an increase in cells in the G1 phase (P < 0.05). In the nude mice model, the mean tumor volume, after 2 weeks of treatment with Lipofectamine or ASON, decreased to 88.5% or 55% respectively, compared to the control tumor size, resulting in a significant difference (P < 0.01).
CONCLUSION The sequence of the ASON, which targeted the start condon at the bases 841-855 of the human TGF-β1 gene, was demonstrated to be a useful agent for studying the regulation of TGF-β1 over-expression in RD cells, and has important therapeutic potential for suppressing the tumorigenicity of human RMS in vivo.
keywords
Introduction
TGF-β1 has emerged as a potent inhibitor of the progression of early tumor stages. However, in later stages of tumorigenesis, it contributes to tumor progression by inducing an epithelial-to-mesenchymal transition (EMT), and cell invasion and migration of epithelial tumor cells[1]. Our previous studies demonstrated, using human embryonal RD rhabdomyosarcoma (RMS) cells, that high exogenous levels of the TGF-β1, and TGF-β1 autocrines inhibited growth of RD cells[2,3]. However, under physiological conditions the function of autocirneTGF-β1 is not concentration dependent.
RMS is the most common soft-tissue malignant tumor in children. Multimodality therapy involving surgery, chemotherapy, and radiotherapy is necessary in RMS cases, but the outcome of patients with metastatic RMS remains poor[4]. Therefore, the development of more effective therapeutic agents and strategies is much needed. Since RMS cells are thought to arise as a consequence of regulatory disruption of cellular growth and differentiation[5], an understanding of the process may open new avenues for the treatment of RMS through the development of targeted therapies. The present study was aimed at developing a potential antisense oligonucleotide (ASON) to repress TGF-β1 expression in rhabdomyosarcoma RD cells, and to examine their effect on tumorigenicity of these cells in vivo.
Materials and Methods
Cell culture
RD cells were obtained from the American Type Culture Collection (ATCC). Normal human primary skeletal myoblasts (Mb) were maintained in culture from previous studies[6]. The cells were grown in Dulbecco’s modified Eagles’s medium (DMEM) containing 10% fetal calf serum (FCS) in 5% CO2 at 37°C. The human SMMC-7721hepatocarcinoma cell line, obtained from the Cell Bank of Chinese Academic of Science, was used as a positive control which expressed TGF-β1[7].
Antisense oligodeoxynucleotides and transfection
Antisense phosphorothioate oligodeoxynucleotides were synthesized by an Applied Biosystems Model 3900 DNA synthesizer, and labeled with caroboxyfluorescein-5-succimidyl ester (FAM) (Shenggong, Shanghai, China). The sequence of the TGF-β1 ASON was 5’-CGG CAT GGG GGA GGG-3’, which was complementary to the 841-855 bases of TGF-β1. This site had been shown to be the most successful for inhibition[8,9]. SON, which is complimentary to antisense oligos, was chosen as a control sequence. MSON is similar to ASON, but contains two nucleotide changes in the middle. The oligos were delivered into cells in the form of complexes with Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacture’s instructions, as previously described[10].
Enzyme-linked immunosorbent assay (ELISA)
RD cells were plated at 4 × 104 cells per well in 96-well plates in the absence or presence of TGF-β1 ASON for 1, 2, 4, and 6 days. The TGF-β1 protein content in the medium was determined by ELISA (Jingmei Co., Guangzhou, China), according to the manufacturer’s instructions. Briefly, the TGF-β1 antibody was coated inside the wells of 96-well plates and the TGF-β1 measured using biotinylated detection secondary antibodies and streptavidin horseradish peroxidases. Absorbance was determined with an ELISA reader (Model 550, BIO-RAD Hercules, CA, USA).
Immunofluorescence staining
RD cells, after cultivating on coverslips for 24 h in DMEM containing 10% FCS, were treated with TGF-β1 ASON for 6 days. Coverslips were then washed with iced PBS and fixed with 4% paraformaldehyde for 15 min at 4°C. After washing with PBS, the cells were blocked with 5% goat serum in PBS containing 1% bovine serum albumin (BSA) at 37°C for 30 min. Then the cells were incubated at 4°C overnight with mouse anti-TGF-β1 (Dako, Glostrup, Denmark) in PBS containing 1% BSA. After the cells were washed with PBS, a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG was added for 1 h at room temperature. The slides were then washed, mounted, and photographed with a fluorescence microscope (BX2-FLB3-000 OLYMPUS, Japan). At least 200 cells/slide in random fields were counted at 20 × to determine the percentage of positive cells. Each experiment was repeated at least 3 times.
MTT assay
The effect of TGF-β1 ASON on cell growth was evaluated by a viability assay and growth inhibition. The RD cells were plated at 4 × 104 cells per well in 96-well plates in the absence or presence of TGF-β1 ASON for 1~6 days. During the last 4 h of each treatment, each well was plused with 10 μl MTT (Sigma, USA), followed by incubation at 37°C for 4 h to allow MTT formazan formation. The medium and MTT were replaced by 100 μl of dimethylsulfoxide to dissolve the formazan crystals. After 30 min, the optical density (OD) at 557 nm was determined using a microplate reader (Model 550, BIORAD Hercules, CA, USA).
Flow cytometric cell cycle analysis
After treatment with TGF-β1 ASON, the RD cells were harvested on day 6, fixed with 70% ice-cold ethanol, and stored at 4°C for 2 h. The cells then were centrifuged at 1,000 r/min for 10 min. The cell pellet then was gently resuspended in 1 ml of hypotonic fluorochrome solution and analyzed using a FACS can flow cytometer (COULTESR, Elite, ESP, USA).
Xenografts in nude mice
Sixty male BALB/c nude mice (5 weeks old, mean weight 18 g) were purchased from the SLAC Laboratory Animal Co. Ltd, Shanghai, China. The mice were divided into groups I~VI, using 10 mice per treatment group. RD cells, with or without TGF-β1 ASON, were injected subcutaneously into the left flank in 50 μl of serum-free medium. DMEM was injected subcutaneously into the right flank of the mice. The mice were injected as follows: each mouse from group I was injected with the RD cell suspension, group II with Lipofectamine (20 μl/ml), group III with TGF-β1SON, and group VI with TGF-β1 ASON. Tumor growth was monitored using calipers on alternate days. After establishing the model for 2 weeks, the mice were sacrificed and the tumors removed. The tumors were then weighed and measured. Tumor volume (V, mm3) was calculated as (L × W2)/2, where L = length (mm) and W = width (mm). The percentage in tumor volume was determined by V (treated RD)/V (RD control).
Statistical analysis
Data were presented as the mean ± standard deviation. Analysis was carried out using SPSS 10.0 (SPSS Inc, Chicago, L, USA) Differences between the control samples and oligo-treated samples were compared using the paired sample t-test. P values < 0.05 were considered significant.
Results
Effects of TGF-β1 ASON on TGF-β1 protein content
To assess whether the oligonucleotides were able to modify the expression of TGF-β1, we investigated their effects on cellular TGF-β1 protein content by ELISA. The TGF-β1 protein levels were increased in the RD groups and SMMC-7721 control group with increasing time compared to the normal myoblasts. However, the increase of TGF-β1 in RD was attenuated by TGF-β1 ASON (Fig.1). Immunofluorescence staining was performed at the same time to analyze the expression of TGF-β1 (Fig.2 and Table 1). Control RD cells showed 85% TGF-β1-positive cells. With addition of TGF-β1 ASON for 6 days, 42% TGF-β1-positive cells developed. No changes were observed in RD cells with addition of TGF-β1MSON and SON. These results indicate that the sequence of TGF-β1 ASON could be a useful agent to study over-secretion of TGF-β1 in RMS cells.
TGF-β1 protein expression was measured by ELISA. Mb (normal myoblasts) control group; SMMC-7721 (hepatocarcinoma cell line) control group. Each experiment was performed three times.
Immunofluorescence analysis of RD cells with Lipofectamine, ASON, MSON, and SON. Positive cells were detected and quantified as described in materials and methods.
Comparison of TGF-β1-positive cells (%).
Inhibitory effect of TGF-β1 ASON on the growth of RD cells
To investigate the effect of TGF-β1 ASON on growth of RD cells, they were treated with TGF-β1 ASON for 1~6 days. TGF-β1 ASON induced a decrease in OD values in time-dependent manner compared with control cells (Fig.3). Significant differences in viability were observed between untreated and TGF-β1 ASON-treated cells after 6 days (P < 0.05). A histogram of DNA content revealed that TGF-β1 ASON induced a significant decrease in cells in the S phase (22.4 %) and an increase in cells in the G1 phase (69.7%) (P < 0.05) (Table 2). The results indicated that growth was arrested in the G0/G1 transition of the RD cells, and that cell death did not occur.
Cells were seeded and treated with TGF-β1 ASON (Mean ± SD, n = 3, P < 0.05 vs. the control group).
Analysis of the cell cycle (%).
Anti-tumorigenicity effect of TGF-β1 ASON on RD cells in vivo
A model using male nude mice was used to evaluate antitumor activity of TGF-β1 ASON against RD cells; tumor volume percentage was determined by V (Treated RD)/V (RD control). In order to exclude the toxic effect of media, it was used as a control in group II. The mean tumor volume, after 2 weeks of treatment with Lipofectamine, SON, and ASON decreased to 88.5% and 55% of the control tumor size respectively (Fig.4). There was a significant difference between group VI (TGF-β1 ASON) and group II (Lip) (P < 0.01). No significant differences were observed between groups II (Lip) and III (TGF-β1 SON) (P >0.05).
Antitumor effect of TGF-β1 ASON in vivo (Mean ± SD, n = 10, aP > 0.05, bP < 0.01 vs. the control group).
Discussion
ASONs have been widely used to evaluate gene function in vivo, and several antisense therapeutics are in clinical trials[11,12]. In our study, several potential antisense oligodeoxynucleotides were synthesized in an attempt to identify one that would specifically reduce TGF-β1 expression, and modify cellular growth properties. The region surrounding the first start condon was targeted by ASON (described in materials and methods), as this is the most successful inhibition site[8,9]. For the cellular applications and our in vivo studies, chemically modified oligonucleotides were used. The majority of the above studies employed 2′-O-methyl (2′-O-Me) substituted RNA oligonucleotides[13,14]. ELISA and immunofluorenscence were applied in our research to assess effects of these modified oligos on TGF-β1 expression. As shown in Fig.1, the TGF-β1 protein levels were increased in the RD groups and SMMC-7721 control group with increasing time compared to the Mb cells. However, this increase was attenuated by ASON (P < 0.05). Taken together, our data suggest that the sequence of TGF-β1 ASON targeting the transcriptional start site (bases 841-855) could be useful in studies aimed at understanding the regulation of TGF-β1 expression in RMS cells.
Previous studies have demonstrated that the human embryonal RMS RD cell line over-expresses TGF-β1, receptors, downstream molecules Smads 2, 3, and 4[2]. Therefore the RD cell line is a suitable model to study the TGF-β1 autocrine loop involved in growth and differentiation of RMS. Preliminary research has shown that exposure of RD cells at a 2 and 5 ng/ml concentration of TGF-β1 will suppress growth of RD cells[3].
In our study, the relationship between TGF-β1 and growth of RD cells was investigated. The cells were treated with ASON targeting the transcriptional start site (bases 841-855) for 1~6 days showing that TGF-β1 ASON decreased in growth in a time-dependent manner compared to control cells. Significant differences in viability were observed in TGF-β1 ASON-treated cells after 6 days (P < 0.05). Analysis of the cell cycle revealed that TGF-β1 ASON induced a significant decrease in the number of cells in the S phase and enhanced the number of cells in the G1 phase. Thus, the RD cells were growth arrested at the G0/G1 transition, rather than dying. It is possible that previous studies on the effect of growth of RD cells using high extraneous concentrations of TGF-β1 produced artfactual results[3], and that in the physiological state, TGF-β1 actually promotes the growth of RD cells. TGF-β1 inhibits muscle-specific gene expression in G0/G1 when there is a stimulation of cell proliferation. RMS represents a class of myoblasticderived tumors, expressing some muscle-specific markers and myogenic promoting transcription factors. However, RMS cells fail to undergo terminal differentiation into skeletal muscle[5,15]. In related studies, we found that TGF-β1 ASON-induced myogenic differentiation correlated with increased expression of myogenin. This protein is more sensitive to repression by growth factor signals compared to myf5 and MyoD. Myogenin is expressed in proliferating myoblasts prior to differentiation (data not shown). These data suggest that TGF-β1-mediated inhibition of myogenic differentiation could be a secondary consequence of cell proliferation.
One of the enigmas of TGF-β1 biology relating to cancer is why tumors over-secrete bioactive TGF-β1, when it acts as a growth inhibitor? In a conditional mouse skin model, expression of the TGF-β1 transgene was switched on at specific stages of carcinogenesis[16]. When TGF-β1 was induced early during tumor formtion, it suppressed tumorigenesis, while induction at a later time, accelerated metastasis. In a sarcoma model, over-expression of TGF-β1 leads to growth inhibitory effects in vitro (tumor suppression action). However, when xenografts of sarcoma cells are placed in syngeneic rats, the TGF-β1-expressing tumor cells established tumors much faster. The final tumor size was also larger, an effect that is fully inhibited by anti-TGF-β1 neutralizing antibodies against TGF-β1[17]. In our present study, nude mice were used to evaluate the antitumor activity of TGF-β1 ASON against RD cells. Results showed that the tumor volumes decreased with TGF-β1 ASON treatment compared to controls, demonstrating that TGF-β1 is capable of directly inducing the necessary steps toward tumorigenicity.
Different growth mechanisms in RMS have been demonstrated by treatment of RD cells with the TPA phorbol ester, which inhibits the activation of secreted latent TGF-β1 and induces growth arrest and myogenic differentiation in these cells[18]. Alterations of certain signaling pathways, for example IGFs and myostatin, produce similar effects of differentiation in RMS cells [19,20]. These findings highlight the complexity of tumorigenicity in RMS. Recent studies have demonstrated that the signaling of TGF-β1 from cell surface to the nucleus can also be directed through the MAPK (ERK2) pathway besides the TGF-β1/Smad signaling pathway[21]. Thus, TGF-β1 promotes tumorigenicity by a mechanism that does not depend upon over-expressed receptors (Tβ RI and Tβ RII) and involves activation of intracellular MAPK and Rho GTPase activities.
When treated with TGF-β1 ASON, the cells were decreased in the S phase in vitro, and tumor volumes decreased in vivo. These data indicate that there is a role for elevated TGF-β1 gene expression in the regulation of malignant cell proliferation and tumor dissemination, and that these pathways are important components of RMS progression.
Footnotes
This work was supported by grants from the Jiangsu Provincial Higher Institution Natural Science Foundation (No.2134605) and the Jiangsu Provincial Post-Doctoral Foundation (No.51208).
- Received November 22, 2007.
- Accepted March 27, 2008.
- Copyright © 2008 by Tianjin Medical University Cancer Institute & Hospital and Springer
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