Cell Biology International (2009) 33, 119-124 - Xinjun Liang and others - Effects of Survivin on cell proliferation and apoptosis in MG-63 cells in vitro

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Cell Biology International (2009) 33, 119–124 (Printed in Great Britain)

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Effects of Survivin on cell proliferation and apoptosis in MG-63 cells in vitro

Xinjun Liang^a¹, Mingxu Da^b¹, Zhiqiang Zhuang^a, Weiguang Wu^a, Zhuang Wu^a, Yanfeng Wu^a and Huiyong Shen^a^*

^aDepartment of Orthopaedics, Sun-Yat-Sen Memorial Hospital, Sun-Yat-Sen University, 107 West Yanjiang Road, Guangzhou 510120, PR China
^bDepartment of General Surgery, Gansu Provincial Hospital, 160 Donggang West Road, Lanzhou 730000, PR China

Abstract

Osteosarcoma is the most frequent primary malignant tumor of the skeleton and occurs mainly in children and adolescent. The prognosis of osteosarcoma is very poor due to its aggressive and no effective treatment. This study is the first to investigate the anti-cancer effects of antisense pEGFP-C1-Survivin on human osteosarcoma cells. It was shown in our results that Survivin blockaded could significantly induce apoptosis and inhibit the invasive of osteosarcoma cells line MG-63. The effects were probably produced by the decreased expression of Survivin induced by antisense pEGFP-C1-Survivin which was examined by RT-PCR and western blotting. All these suggested that Survivin should be very important in the development of osteosarcoma and Survivin blockaded by using antisense pEGFP-C1-Survivin could markedly inhibit the proliferation and invasion of osteosarcoma cells line MG-63, partially reversed their malignant phenotype. Targeting Survivin might be a promising option in the treatment of osteosarcoma.

Keywords: Osteosarcoma, Survivin, Apoptosis.

¹These authors contributed equally to this manuscript.

^*Corresponding author.

1 Introduction

Osteosarcoma is the most frequent primary malignant bone tumor and occurs mainly in children and adolescent (Klein and Siegal, 2006). It is an extremely destructive malignancy that most commonly arises in the long bones (DuBois and Demetri, 2007). Limited improvements had been got by using conventional methods including surgery, radiotherapy and chemotherapy in the past two decades (Lee et al., 2008). Despite the successful use of neoadjuvant chemotherapy in the treatment of osteosarcoma, many patients who have got osteosarcoma remain resistant to treatment due to rapid progression, metastasis, and tendency to chemotherapy resistance (Chou et al., 2005; Lee et al., 2004; Bacci et al., 2007). Gene therapy as a new method to treat tumors has been introduced to osteosarcoma for a few years and has got great achievements (Kishida et al., 2007; Cho et al., 2007). Therapeutic strategies using targeted block the expression of some oncogene may be an effective way to the treatment of osteosarcoma (Lamoureux et al., 2007; Ganjavi et al., 2006; Kersting et al., 2007).

Survivin is a member of the inhibitors of apoptosis (IAP) gene family which is recently described and plays an important role in the inhibition of apoptosis and control of cell division (Ambrosini et al., 1997; Troeger et al., 2007; Stauber et al., 2007; Carelli et al., 2007). It deserves attention as a selective target for cancer therapy because it is highly expressed in most malignancies and embryonic tissues but is undetectable or expressed at a very low level in normal adult tissues (Tanabe et al., 2004; Yamashita et al., 2007; Gazzaniga et al., 2003; Cohen et al., 2003; Gordon et al., 2007). Some studies have shown that Survivin is up regulated in osteosarcoma which indicates a poor prognosis and increased risk of metastasis (Wang et al., 2006; Trieb et al., 2003; Osaka et al., 2007). Thus, Survivin may involve in the development of osteosarcoma and therefore may be a useful target for the therapy of osteosarcoma.

As discussed before, we suppose that Survivin blockade may reduce the proliferation and invasion of osteosarcoma cells. Several studies reported that Survivin knockdown by using antisense RNA, shRNA or other methods could increase the apoptosis rate of many cancer cells (Paduano et al., 2006; Ma et al., 2005; Pratt et al., 2006; Ai et al., 2006; Zhou et al., 2004; Chao et al., 2004). But there is still little report that whether Survivin down regulation could induce the apoptosis of osteosarcoma cells. Therefore, the purpose of this study was designed to observe the effects of antisense Survivin plasmid on the proliferation, apoptosis, migration and invasion of osteosarcoma cells in vitro by using MTT, Flow cytometry and Transwell invasion assay. Furthermore, we detected the changes of the expression of Survivin in osteosarcoma cells by RT-PCR and western blot to investigate the effects of antisense Survivin plasmid on gene expression.

2 Materials and methods

2.1 Cell line and culture conditions

Human osteosarcoma cell line MG-63 was obtained from American Type Culture Collection (ATCC). Cells were cultured in the DMEM complete medium supplemented with 2mM l-glutamine and 10% heat-inactivated fetal bovine serum (FBS) at 37°C humidified atmosphere containing with 5% CO2.

2.2 Construction of antisense Survivin plasmids

Survivin cDNA was cloned from a cDNA pool derived from the human osteosarcoma cell lines MG-63 by reverse transcription polymerase chain reaction (RT-PCR), which was performed by using the following primers: sense, 5′-GGA TCC GCA TGG GTG CCC CGA CGT TG-3′, and antisense, 5′-GAA TTC GCT CCG GCC AGA GGC CTC AA-3′, containing BamHI and EcoRI restriction sites, respectively (underlined sequences). The 459bp PCR product was subsequently extracted from the agarose gel and cloned to the eukaryotic expression vector pEGFP-C1 vector (Clontech, USA) and the final production (termed antisense pEGFP-C1-Survivin) was confirmed by restriction enzyme digestion and sequencing.

2.3 Transfection of cells with antisense pEGFP-C1-Survivin

Twenty-four hours before transfection, 1.5–2×10⁵ osteosarcoma cell line, MG-63, was plated into 6-well tissue culture plates. Cells were rinsed with 2ml of PBS before transfection. When everything was ready, MG-63 cells were transfected with antisense pEGFP-C1-Survivin by using Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer's protocol. For each well, 3–5μg purified plasmid mixed with 8–10μl Lipofectamine in 2ml serum-free DMEM was used. Six hours later, the medium was replaced by complete medium. The cells were further incubated in 5% CO2 at 37°C and harvested for analysis at different time points. Untreated cells, cells treated with liposome alone, and cells treated with control vector pEGFP were used for comparison.

2.4 Measurement of cell growth by MTT

The growth inhibition rate of MG-63 cells was measured by Methyl thiazolyl tetrazolium (MTT) assay. Cells were plated onto 96-well plates at a density of 5000 cells per well and divided into different groups as described before. After transfected by antisense pEGFP-C1-Survivin for 6h, the serum-free medium was replaced by complete medium. The cells were further incubated in 5% CO2 at 37°C for another 18h, 42h, and 66h. Subsequently, 20μl of MTT reagent (Sigma; 5mg/ml) and 180μl DMEM were changed and reacted for 4h at 37°C. Then 150μl Dimethyl Sulphoxide (DMSO) was added to each well. The plate was shaken on a rotary platform for 10min, and then the absorbance at wavelength 490nm was measured by use of microplate reader. The ratio of the absorbance of treated cells relative to that of the control cells was calculated and expressed as a percentage of cell death.

2.5 Apoptosis assay

MG-63 cells were trypsinized and collected after pre-treatment for 48h. Cells were rinsed twice in PBS and fixed with ice-cold 70% ethanol and stored at −4°C until use. The cells were centrifuged again and resuspended in 200μl PBS, then 100μl of RNase I (1mg/ml) and 100μl of PI (400μg/ml) were added to the cell suspensions and incubated at 37°C for 30min and analyzed by flow cytometry. Experiments were conducted in at least triplicate.

2.6 Detecting the mRNA expression of Survivin by reverse transcription-PCR

To detect the change of the mRNA expression of Survivin after the treatment of Survivin blockage by antisense pEGFP-C1-Survivin, RT-PCR was performed using a one-step RT-PCR kit (Life Technologies, USA) according to the manufacturer's instructions. After 48h, MG-63 cells were collected and washed twice in PBS, centrifuged and collected again. Total RNA was isolated by Trizol reagent (Invitrogen, USA). The primer pairs were designed according to the sequences in the GenBank as follows. Survivin (447bp): sense 5′-CGA CGA CTT CTC CCG CCG CTA CCG C-3′; and antisense 5′-CCG CAT GCT GGG GCC GTA CAG TTC C-3′. A 230bp fragment of GAPDH was used as the internal control: sense 5′-ACG GAT TTG GTC GTA TTG GG-3; antisense 5′-TGA TTT TGG AGG GAT GTC GC-3′. The cDNA synthesis and pre-denaturation were performed as follows: 1 cycle of 94°C for 5min. PCR amplification was continued for 35 cycles of 94°C for 15s, 60°C for 30s, and 72°C for 1min. A final extension was performed for 10min at 72°C. PCR products were identified by 1.5% agarose gels electrophoresis containing 0.5% ethidium bromide (EB). All experiments were repeated at least three times.

2.7 Measurement of Survivin protein by western blotting

About 1×107 MG-63 cells were gathered after pre-treatment for 48h as described previously. After medium was removed, cells were rinsed twice with PBS solution at room temperature. Plasmosin was extracted. Subsequently, cell lysates were centrifuged at 12,000rpm for 10min at 4°C. Supernantant was collected and protein concentrations were determined. Primary antibodies for Survivin and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Horseradish peroxidase conjugated goat anti-rabbit was used as secondary antibodies. Proteins were visualized with chemiluminescence's luminal reagents. To check for protein loading, the blots were also incubated with an anti β-actin polyclonal antibody.

2.8 Migration and invasion assay

A transwell invasion assay was used to detect the invasive property of MG-63 cells' migration. After pre-treatment for 48h, MG-63 cells were divided into four groups as described above. For invasion studies, matrigel was added to the upper surface of the transwell membrane and allowed to dry in a sterile environment overnight. 1×10⁵ MG-63 cells were suspended in medium without serum and then the cells were plated onto the upper surface of the transwell membrane in a total volume of 100μl. The lower chambers were filled with 0.4ml of media containing 10% FBS. The cells were incubated at 37°C humidified atmosphere containing with 5% CO2. 24h later, cells on the upper surface of the membrane were gently scrubbed with a cotton bud, and the cells migrating to the lower compartment of the filters were fixed in methanol and stained with hematoxylin eosin (HE). The number of cells that had invaded was then counted using a hemocytometer. Each sample was assayed in triplicate.

2.9 Statistical analysis

All experiments were repeated at least three times. Results were expressed as means±SD. Significance was defined at the level of P<0.05.

3 Results

3.1 Effects of antisense pEGFP-C1-Survivin on MG-63 cells' proliferation

MG-63 cells' proliferation was detected by Methyl thiazolyl tetrazolium (MTT) after pre-treatment as described before. As shown in Fig. 1A, notable changes were observed 48h after treatment with transfection of antisense pEGFP-C1-Survivin, and another 24h later, the inhibition rate reached the peak (P<0.05). The inhibition rates were significantly lower in the other three groups (P>0.05).

Fig. 1

Effects of antisense pEGFP-C1-Survivin on cell proliferation and apoptosis. A. MG-63 cells were transfected by antisense pEGFP-C1-Survivin for 24h, 48h, and 72h. The inhibition rate of MG-63 cells was measured by MTT assay. Results were show in the value of absorbance at 490nm. Untreated cells, cells treated with liposome alone, and cells treated with control vector pEGFP were used for comparison. B Apoptosis of osteosarcoma cells line MG-63 induced by antisense pEGFP-C1-Survivin was observed by flow cytometry. Cells were divided into four groups as described before. After pre-treatment for 48h, cells were collected and the apoptosis rate of cells was examined by flow cytometry.

3.2 Effects of antisense pEGFP-C1-Survivin on MG-63 cells' apoptosis

It was reported that Survivin involved in cell proliferation, growth, anti-apoptosis, and tumorigenesis. Survivin has been shown highly expression in osteosarcoma (Wang et al., 2006; Trieb et al., 2003; Osaka et al., 2007). To further verify the effects of antisense pEGFP-C1-Survivin on cell apoptosis, flow cytometry analysis was used. As shown in Fig. 1B, we found that apoptosis was induced in MG-63 cells after transfected with antisense pEGFP-C1-Survivin (P<0.05). No significant changes were observed in untreated cells, the cells treated with liposome alone, and cells treated with control vector pEGFP (P<0.05).

3.3 Down-regulating effects of antisense pEGFP-C1-Survivin on Survivin mRNA and protein

To observe the Survivin blockaded by antisense pEGFP-C1-Survivin, the mRNA and protein expression of Survivin were detected by using RT-PCR and western blotting. In our results, we found in Fig. 2 that the mRNA and protein expression of Survivin were decreased significantly in the MG-63 cells treated with antisense pEGFP-C1-Survivin than controls, which suggested that the inhibition effects of MG-63 cells may be induced by the Survivin blockaded.

Fig. 2

Expression of Survivin mRNA and protein in human osteosarcoma cells line MG-63 after transfected with antisense pEGFP-C1-Survivin was detected by RT-PCR and western blot. Cells were treated with antisense pEGFP-C1-Survivin for 48h in the experiment group. The other three groups as described before were used as comparison. A PCR products were identified by 1.5% agarose gels electrophoresis containing 0.5% ethidium bromide. Relative sizes of RT-PCR products are as follows: Survivin, 447bp; and GAPDH, 230bp. Molecular size markers are the 100bp ladders. B Immunochemical detection of expression of Survivin protein in MG-63 cells by mouse anti-human monoclonal antibodies. The expression of β-actin protein was used as control.

3.4 Effects of antisense EGFP-C1-Survivin on the cell migration and invasion

Migration and invasion are important features of tumor cells. To study the effects of Survivin knocked down by antisense pEGFP-C1-Survivin on the invasive property of MG-63 cells, transwell invasion assay was used. Cells in control groups showed high invasive activity in vitro, whereas the invasive activity of cells in the experiment group treated with antisense pEGFP-C1-Survivin decreased notablely. This result suggested that Survivin might play an important role in the invasion of osteosarcoma cells and targeted inhibition of Survivin by using antisense pEGFP-C1-Survivin could reverse this malignant feature. It means that Survivin might be a useful marker in the therapy of osteosarcoma.

4 Discussion

Osteosarcoma is the most common primary malignant neoplasm of the skeleton in children and adolescent. It occurs frequently in the long bones, such as, the distal of femur and the proximal humerus (Kim et al., 2008). Its natural history is characterized by a rapidly progressive course with early metastasis to the lungs. The prognosis for patients with osteosarcoma is very poor due to the early pulmonary metastasis (Graat et al., 2008). The survival rate of patients who got osteosarcoma five years is only 20% by treated with traditionary therapy methods such as surgery, radiotherapy and chemotherapy (Okada et al., 2004).

During the past few years, the survival rate and quality of life of the patients who had osteosarcoma were improved notablely with the development of new-adjuvant chemotherapy (Bacci et al., 2003). But still many patients with osteosarcoma remained resistant to treatment due to rapid progression and metastasis (Kager et al., 2003). How to postpone the progression and metastasis was the criticality for the therapy of osteosarcoma. Gene therapy as a new method to treat tumors has been found to be an effective way to treat cancers by regulating the expression of some oncogenes and factors relative to the development of tumors.

Several studies have reported that Survivin is a novel member of inhibitors of apoptosis (IAPs) family and is expressed during fetal development and in many tumor tissues, but it lacks expression in terminally differentiated adult tissues (LaCasse et al., 1998; Rahman et al., 2006; Ryan et al., 2006). Over expression of Survivin correlates with resistance to chemotherapy and poor prognosis in patients. Recently studies have found that Survivin may play an important role in the development of osteosarcoma (Osaka et al., 2006). Therefore, we suppose that target blocked the expression of Survivin of osteosarcoma may induce the apoptosis of osteosarcoma cells and improve the chemotherapy sensitivity.

Therapeutic strategy using antisense plasmid has been found to be an effective way to down regulate the expression of oncogene and reduce the apoptotic threshold in tumor cells. Several experiments have been done to observe the effects of antisense Survivin plasmid on tumors and have got great achievements (Tu et al., 2003; Kappler et al., 2007). But there is still little research about the effects of antisense Survivin plasmid on osteosarcoma. In this study, we constructed an antisense Survivin eukaryotic expression vector, which was named antisense pEGFP-C1-Survivin. Targeting Survivin by this antisense plasmid was supposed to inhibit the growth and induce the apoptosis of osteosarcoma cells. To confirm this, MTT and flow cytometry assays were used in our study. Cells were divided into four groups, which were cells treated with antisense pEGFP-C1-Survivin, untreated cells, cells treated with liposome alone, and cells treated with control vector pEGFP. As shown in Fig. 1, we found that the inhibition rate and apoptosis rate increased significantly in the MG-63 cells transfected by the antisense pEGFP-C1-Survivin 48h later and the effects reached the peak for another 24h. There were no such changes being observed in the other three groups. All these suggested that Survivin blockaded could decrease the proliferation and could induce the apoptosis of osteosarcoma cells.

We have known that high levels of Survivin mRNA and protein expression were significantly associated with clinical resistance to chemotherapy and due to the development and progression of osteosarcoma. RT-PCR and western blotting were used to survey whether the mRNA and protein expression of Survivin in osteosarcoma cells could be down regulated by antisense pEGFP-C1-Survivin. As observed in Fig. 2, the Survivin mRNA and protein expression degraded conspicuously when transfected with antisense pEGFP-C1-Survivin than that in the other three groups, which suggested that antisense pEGFP-C1-Survivin produce its killing effects by inhibiting the expression of Survivin.

It was reported that highly expression of Survivin represented the ability of migration and invasion of tumor cells. In the present study, transwell invasion assay was used to detect the changes of invasive property in MG-63 cells after treatment with antisense pEGFP-C1-Survivin. We could see in Fig. 3 that the invasive activity of osteosarcoma cells decreased notablely when the Survivin expression was blockaded. But there were no significantly changes observed in the other groups.

Fig. 3

The effect of antisense pEGFP-C1-Survivin on the migrating activity of MG-63 cells was detected by a transwell invasion assay. MG-63 cells were divided into four groups as described. After pre-treatment for 48h, the number of cells that had invaded was then counted using a hemocytometer. Each sample was assayed in triplicate.

These results revealed that targeting Survivin expression using antisense pEGFP-C1-Survivin might be an effective therapy for osteosarcoma. As reported, Survivin knocked down might increase the sensitivity to chemotherapy. Several studies are now under way to test the efficacy of antisense pEGFP-C1-Survivin in combination with cytotoxic chemotherapy on tumor cells in vitro. It is expected that Survivin blockaded may be a useful gene therapy method in the treatment of osteosarcoma.

In conclusion, we suggested that Survivin play an important role in the development of osteosarcoma. The antisense pEGFP-C1-Survivin could efficiently down regulate the expression level of Survivin and induce cell apoptosis in vitro. We conceive that Survivin might be considered as a therapeutic target for the treatment of osteosarcoma, Survivin inhibition in combination with chemotherapy may increase clinical effectiveness of the treatment of osteosarcoma.

Acknowledgements

We thank Dr Renyun Xia for the help of the experiment. A part of this work was done in the department of orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China.

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Received 28 June 2008/22 August 2008; accepted 13 October 2008

doi:10.1016/j.cellbi.2008.10.013

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