Cell Biology International (2008) 32, 1199-1206 - Dingsheng Lin and others - Bcl-2 and caspase-8 related anoikis resistance in human osteosarcoma MG-63 cells

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Cell Biology International (2008) 32, 1199–1206 (Printed in Great Britain)

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Bcl-2 and caspase-8 related anoikis resistance in human osteosarcoma MG-63 cells

Dingsheng Lin, Jie Feng and Weishan Chen^*

Department of Orthopedics, Institute of Orthopaedic Research, 2nd Affiliated Hospital, Medical College, Zhejiang University, #88 Jiefang Road, Hangzhou 310009, PR China

Abstract

Detachment of adherent cells from extracellular matrix results in apoptosis, a process termed “anoikis”. Resistance to anoikis is implicated in the progression of many malignancies by facilitating the migration and eventual colonization of distant sites. Human kidney epithelial cells 293T, human osteoblast cells hFOB 1.19 and human osteosarcoma cells Saos-2 significantly underwent anoikis when adherence was prevented. But human osteosarcoma MG-63 cells were distinctly anoikis resistant when detached. They formed large aggregates and showed little apoptosis compared to the other cells. When MG-63 cells were in suspension, caspase-8, physically associated with death receptor was activated by cell–matrix detachment, whereas. Caspase-3 and caspase-9 were not activated. Translational level of Bcl-2 significantly increased in a time-dependent manner, but the level of β-catenin and PI3K did not. Caspase-8 participates in an anoikis-inducing process in MG-63 cells at an early time, and overexpression of Bcl-2 blocks activation of caspase-8 making MG-63 cells anoikis resistant.

Keywords: Anoikis, Osteosarcoma, Bcl-2, Caspase-8, PI3K, β-catenin.

^*Corresponding author. Tel.: +86 571 8776 7023.

1 Introduction

Cell–matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation and aspects of cell growth control (Liotta and Kohn, 2004). When detached from the extracellular matrix (ECM), normal adherent cells undergo a form of apoptotic death known as anoikis (Frisch and Francis, 1994). Cancer cells do not become fatally undergo anoikis if separated from home turf. Resistance to anoikis is known as one of the hallmarks of malignant transformation. This plays an important role during tumor progression by affording increased survival time in the absence of matrix attachment, facilitating migration and re-attachment, and colonization of secondary sites (Diaz-Montero and McIntyre, 2003, 2005; Bretland et al., 2001).

Osteosarcoma is the most common malignant primary bone tumor. Up to 80% of patients show macroscopic pulmonary metastases at present. Little is known about its role during the progression of osteosarcoma, although resistance to anoikis has been described in many types of human malignancies including gastric cancer, mammary cancer, colon cancer, lung cancer (Zhang et al., 2004) and Diaz-Montero et al. (Diaz-Montero and McIntyre, 2003, 2005; Diaz-Montero et al., 2006) found that Saos-2 human osteosarcoma cells were sensitive to anoikis. They generated an anoikis resistant variant of the Saos-2 cell line by sequential culturing cells under adhered and suspended conditions. We compared cell apoptosis rate of human kidney epithelial cells 293T cells and human fetal osteoblasts hFOB 1.19 cells with that of osteosarcoma cell line MG-63 cell and Saos-2 cell in the detachment condition, and sought to understand the molecular mechanisms underlying resistance to anoikis in human osteosarcoma cells. This may provide alternative treatments that may prevent or reduce osteosarcoma cell migration.

2 Materials and methods

2.1 Cell culture

Human osteosarcoma cell lines MG-63, Saos-2 cells, human fetal osteoblasts hFOB 1.19 cells and human kidney epithelial cells 293T cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air in Dulbecco's modified Eagle's medium (D-MEM), McCoy's 5A medium, 1:1 mixture of Ham's F12 medium, Dulbecco's modified Eagle's medium and modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 2.0mM l-glutamine. For the subculture, the cell monolayer was washed twice with phosphate buffered solution (PBS) and incubated with trypsin-EDTA solution (0.05% trypsin, 0.25% EDTA) for 10min at 37°C to detach the cells. The effect of trypsin was inhibited by adding the FBS at 37°C. Cells were resuspended in culture medium for reseeding.

2.2 Anoikis induction

To induce anoikis cells were seeded (5×10⁵cells/well) on 6-well culture plate coated with poly(2-hydroxyethyl methacrylate) (poly-HEMA; Sigma, St. Louis, MO, USA). Poly-HEMA was dissolved in 95% ethanol (v/v) at 50mg/ml and added to 6-well cell culture wells at a density of 5mg/cm². The wells were allowed to dry overnight under sterile conditions followed by extensive PBS washes.

2.3 Cell morphology

MG-63 cells Saos-2 cells, 293T cells and hFOB 1.19 cells seeded on poly-HEMA treated and untreated wells, cells (5×10⁵cells/well) growing in suspension and adherent conditions were examined under a light microscope.

2.4 Transmission electronic microscope

Cells were washed in PBS and pellets were fixed by incubating with 2.5% glutaraldehyde for 2h at 4°C followed by 1% osmium tetroxide for 1h at 4°C. After dehydration in a graded series of ethanol and infiltration in propylene oxide, cells were embedded in Epon 812. Ultrathin 60–100nm sections were stained with uranyl acetate and lead citrate and the cell was observed by transmission electronic microscope (TEM) (Philips TECNAI 10) at 80kV.

2.5 Flow cytometric analysis

To quantify apoptosis by flow cytometry, cells (5×10⁵/well) were cultured on poly-HEMA treated and untreated 6-well tissue culture plates for 24h, 48h, 72h and 7days at 37°C in a humidified atmosphere of 5% CO2 in air. Adherent cells were detached with trypsin (0.05% trypsin, 0.25% EDTA) in PBS. Suspended cells were harvested in their culture medium and centrifuged at 500×g for 10min. Pellets were washed with PBS and fixed with ice-cold 75% ethanol (v/v) overnight at 4°C. After fixation, the cells were washed with PBS and stained with 500μl of PI solution (50μg/ml in PBS) containing 25μg/ml of RNase. Cells were incubated at 37°C for 30min and analysed by flow cytometry.

2.6 RT-PCR analysis

A reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to determine the expression of mRNA level. Total RNA was extracted from cells using Trizol reagent (Invotrogen). After extraction with chloroform (Sigma) and precipitation with isopropanol (Sigma), the RNA pellet was stored at −20°C in 80% ethanol. Before first strand synthesis, the RNA pellet was dried, dissolved in nuclease-free water and the RNA concentration was determined spectrophotometrically at A260. First strand cDNA was reverse transcribed from 2μg of total cellular RNA using random hexamers and the Superscript™ First Strand Synthesis System for RT-PCR (Invitrogen Life Technologies, USA). Targeted genes were amplified by PCR with primers: Human β-catenin sense 5′-gatttgatggagttggacatgg-3′, anti-sense 5′-tgttcttgagtgaaggactgag-3′; human phosphoinositide-3-kinase sense 5′-attgtgatacaccctccgtgga-3′, anti-sense 5′-tgatgaggtatgctaggcgacc-3′; human GAPDH sense 5′-acctgacctgccgtctagaa-3′ and anti-sense 5′-tccaccaccctgttgctgta-3′. The reaction profiles were used for the primer sets: denaturation step at 94°C for 5min, 94°C for 30s, 55°C for 30s and 72°C for 60s, for 30 cycles. All PCR reactions ended with a 10min incubation period at 72°C. The 247bp amplified product of GAPDH was used as an internal control. Ten microliters of aliquots of PCR products were run on 2% agarose gel electrophoresis followed by ethidium bromide staining, UV transillumination and image capture.

2.7 Western blot analysis

Cells were harvested and suspended in 30μl Western blot lysis buffer containing 50mmol/L Tris–HCl (pH 7.5), 150mmol/L NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1mmol/L PMSF, 100μmol/L leupeptin and 2μg/ml aprotinin at 0–4°C for 15min. After centrifugation at 1500×g for 10min at 0°C, the supernatants were collected and the proteins separated on 12% SDS-PAGE. After electrophoresis, equal amounts of protein from each sample were transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in TBST and incubated with rabbit anti-human β-catenin, Bcl-2, PI3K, caspase-3, caspase-8 and caspase-9 and β-actin (1:1000) antibody (Calbiochem) separately overnight at 4°C. After washing three times with TBST, the membrane was incubated at room temperature for 1h with horseradish peroxidase-conjugated secondary antibody diluted with TBST (1:10,000). The protein signals were visualized by an enhanced chemiluminescence reaction system (Amersham, Arlington Heights, IL, USA).

2.8 Statistical analysis

Collected data were expressed as mean±S.D. Statistical analysis was performed by the one-way ANOVA to express the difference within groups.

3 Result

3.1 Cell morphology under light microscopy

MG-63 cells in poly-HEMA treated wells formed large and compact aggregates and Saos-2 cells formed small and compact aggregate, whereas hFOB 1.19 cells and 293T cells did not form even very small and loose aggregates (Fig. 1). Cell–cell aggregation is an important phenotype during anoikis resistance which can provide a significant advantage in the processes of invasion and metastasis for tumor cells in vivo. MG-63 cells formed large aggregates, suggesting a strong ability to resist anoikis.

Fig. 1

Cell morphology of MG-63, Saos-2 293T and hFOB 1.19 cells in poly-HEMA treated (in suspension condition) wells (photographed at ×40 magnification) and poly-HEMA untreated (in adherent condition) wells (photographed at ×100 magnification) under light microscope. The background of the suspended cell culture wells is poly-HEMA.

3.2 Cell morphology and cell junction by TEM

After cells were seeded on poly-HEMA treated wells for 72h, 293T, hFOB 1.19 and Saos-2 cells showed apoptotic features compared to MG-63 cells under TEM. Nuclear membranes were irregular, crescent nucleus became condensed and fragmented and vacuoles in the cytoplasm were observed (Fig. 2). MG-63 cells were normal in appearance. Cell detachment induced anoikis in 293T, hFOB 1.19 and Saos-2 cells, but not MG-63 cells. MG-63 cells were anoikis resistant, but the cells established cell junctions including gap junction and desmosome.

Fig. 2

Cell morphology and cell junction of MG-63, Saos-2, 293T and hFOB 1.19 cells in poly-HEMA treated wells under transmission electronic microscope. Cells were seeded on poly-HEMA untreated (in adherent condition) wells for 24h and poly-HEMA treated (in suspension condition) wells for 72h respectively and were collected for TEM observation. Cells morphology were photographed at ×3700 magnification, cell junctions (adherent 24h) were photographed at ×30,000 magnification and cell junctions (suspended 72h) were photographed at ×65,000 magnification.

3.3 Frequency of apoptosis rate

To confirm the anoikis resistance of MG-63 cells, apoptosis was quantified. Apoptosis in hFOB 1.19 cells, 293T cells and Saos-2 cells cultured in poly-HEMA treated wells was significantly higher than in MG-63 cells (p<0.05). Apoptosis frequency of MG-63 cells cultured in poly-HEMA treated wells for 24h, 48h, 72h and 7days was 6.3%, 13.9%, 20.3% and 30.3%, respectively. Apoptosis in hFOB 1.19, 293 T and Saos-2 cells cultured in poly-HEMA treated wells for 7days was 89.2%, 95.9% and 82.1% separately. So MG-63 cells were distinctly anoikis resistant when growing in poly-HEMA treated wells for 24h, 48h, 72h and 7days (Fig. 3).

Fig. 3

The apoptosis rates of MG-63, Saos-2, 293T and hFOB 1.19 cells cultured in the suspension and adherent condition for the different time. Cells were seeded on poly-HEMA treated or poly-HEMA untreated wells for 24h, 48h, 72h, 7days. Then cells were collected for flow cytometric analysis. Results are expressed as means±S.D. of four observations. *p<0.05 compared with apoptosis rate of hFOB 1.19 cells.

3.4 Transcriptional and translational level of β-catenin and PI3K

β-catenin and PI3K are two important molecules in the process of anoikis resistant. Their transcriptional level and translational level in MG-63 cells were measured. The transcriptional level of β-catenin decreased when anoikis was induced for 24h compared to adherent condition, then increased in a time-dependent manner. It adjusted itself to a normal and active condition after the stimulation of detachment, but the translational level of β-catenin did not increase or decrease compared to adherent condition.

When attachment was deprived transcription level of PI3K increased at 24h and at 48h compared to adherent condition (Fig. 4). PI3K is composed of 85kDa (p85) and 110kDa (p110) subunits. p85 lacks PI3K activity and acts as an adapter coupling p110 to activated protein tyrosine kinase (Hiles et al., 1992). p85 and p110 translational level of PI3K were measured. The protein level of p85 increased after cells were detached from matrix for 72h. The translational level of p110 was the same no matter cells were in suspension or adherent.

Fig. 4

The transcriptional and translational level of β-catenin and PI3K in MG-63 cells seeded on poly-HEMA treated wells. (1: MG-63 cell cultured in poly-HEMA untreated wells for 24h as control 2, 3, 4: MG-63 cell cultured in poly-HEMA treated wells for 24h, 48h and 72h respectively) Results shown are representative of three independent experiments. *p<0.05 compared with control.

3.5 Protein level of Bcl-2 and caspase-3, caspase-8 and caspase-9

Caspases are the cysteine proteases that cleave at conserved aspartic acids and are critical to apoptosis. Caspase-3, caspase-8 and caspase-9 are the three key caspases during apoptosis and apoptosis can be inhibited by overexpression of Bcl-2. So activation of caspase-3, caspase-9 and caspase-8 and the protein level of Bcl-2 in MG-63 cells were detected. Caspase-3 and caspase-9 were not activated in MG-63 cells cultured in poly-HEMA treated wells for 24h, 48h, and 72h. Caspase-8 was activated and the maximal activation happened at 48h. The protein level of Bcl-2 increased in a time-dependent manner (Fig. 5).

Fig. 5

The translational level of caspase-3, caspase-8 and caspase-9 and Bcl-2 in MG-63 cells seeded on poly-HEMA treated wells. (1: MG-63 cell cultured in poly-HEMA untreated wells for 24h. 2, 3, 4: MG-63 cell cultured in poly-HEMA treated wells for 24h, 48h and 72h respectively). Results shown are representative of three independent experiments.

4 Discussion

This study shows that 293T cells and hFOB 1.19 cells undergo anoikis when adherence to ECM was denied. They are normal cells which were used as control cells in the experiment. The Saos-2 cells and MG-63 cells showed different results. Saos-2 cells developed a time-dependent anoikis in suspension in agreement with Diaz-Montero and McIntyre (2003, 2005). The anoikis of suspended Saos-2 increased in a time-dependent manner reaching 72.2% cell death at 72h of culturing. But MG-63 cells showed anoikis resistance with low apoptosis rates. MG-63 cells have a wild-type Rb gene while Saos-2 cells have no active RB gene (Hellwinkel et al., 2005). The role of Rb as tumor suppressor is firmly established, but it also has the ability to block apoptosis which is generally thought to be a property of oncogenes (Delston and Harbour, 2006). Anti-apoptotic functions of Rb contribute significantly to the genesis and progression of tumors (Dasgupta et al., 2006) though the mechanisms by which Rb regulates apoptosis are yet to be fully elucidated (Clemo et al., 2005).

Many phenotypic changes can contribute to the resistance to anoikis (Frisch and Ruoslahti, 1997; Frisch and Screaton, 2001). Fibroblasts grown at high density will form cell–cell contacts which confer anoikis resistance when cell–matrix anchorage are disrupted (Grossmann, 2002). Zhang et al. (2004) reported that the human oral squamous cell carcinoma (HSC-3) in suspension that formed multicellular aggregates had significantly lower levels of apoptosis than single cells. MG-63 cells in poly-HEMA treated wells formed large and compact spheroids and underwent anoikis resistance. But 293T, hFOB 1.19 and Saos-2 cells formed no aggregates or very small and loose aggregates and then underwent anoikis. Perhaps cell–cell contacts promote cell survival in the absence of interactions with the exrracellular matrix and create a permissive environment for cell proliferation (Shen and Kramer, 2004).

Cell junction includes tight junction, adhesion junction and gap junction. One paper reported (Osanai et al., 2007) that tight junctions are intercellular structures in epithelial and endothelial cells that primarily play a critical role in cell–cell adhesion. Recent evidence revealed that tight junctions are directly involved in the regulation of cellular functions such as proliferation, differentiation and apoptosis, due to the ability of tight proteins to recruit various signaling molecules that have proliferative and differentiative capacities, including transcription factors, lipid phosphatases and cell-cycle regulations. Cancer cells often exhibit loss of functional tight junctions and disruption of the tight junction structure is associated with cancer development. Transmission electronic microscope observation found that cell–cell junction formed when cells were deprived of attachment. Gap junction exists mostly of tissues and cells and is the normal cell junction of 293T, hFOB 1.19, Saos-2 and MG-63 cells. Adhesion junction (for example desmosome) exists between epithelial cells and is the normal cell junction of 293T cells but not hFOB 1.19, Saos-2 and MG-63 cells. It suggests that stimulation of detachment induces the formation of desmosome between these cells and it is the cadherins that exist in the middle of the desmosome, so cadherins may transduct survival or death signals between cells of detachment.

The molecular mechanisms involved in anoikis resistance are not well understood and there are many different views. TrkB protected cells against anoikis and allowed cells to proliferate as large spheroid aggregates in suspension in different epithelial cell lines and across species, by mechanisms that required activation of phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB) pathway (Liotta and Kohn, 2004; Douma et al., 2004; Desmet and Peeper, 2006; Geiger and Peeper, 2005). E-cadherin binding of renal epithelial cells promotes survival in a PI3K dependent fashion and cadherins form intracellular complexes with catenins which link the cadherin to the actin cytoskeleton or to the so-called Wnt/Wingless pathway of signal transduction through association with β-catenin (Grossmann, 2002). Overexpression of β-catenin renders epithelial cells anoikis resistant in vitro and promotes progression through the cell cycle (Fuchs et al., 2005). So β-catenin and PI3K are the two crucial molecules which have critical roles in anoikis resistance.

Diaz-Montero et al. demonstrated a crucial role for Src in anoikis resistant human SAOS-2 osteosarcoma cells and the survival pathway was mediated by the Src-dependent activation of the PI3K/Akt pathway in a manner independent of FAK activity. We found β-catenin and PI3K protein level did not increase in MG-63 cells growing in suspension, except PI3K p85, which has inhibitory effect on p110. So they don't participate in anoikis resistance in MG-63 cells. The difference between transcriptional level and translational level may be related to post-transcriptional modification and post-translational modification. As a negatively regulatory subunit for p110 phosphoinositide 3-kinases (PI3Ks), p85 can independently stimulate signaling pathways involved in actin cytoskeletal rearrangements, so maybe the increasement of p85 changed cytoskeletal of MG-63 cells resulted in anoikis resistant.

Caspases as the executioner of apoptosis plays an important role in the process of apoptosis. There are two pathways in caspase cascade (Grossmann, 2002; Reddig and Juliano, 2005): the cell surface death receptor pathway and the mitochondria-initiated pathway. In the death receptor pathway activation of caspase-8 following its recruitment to the death-inducing signaling complex is the critical event. In the mitochondrial-initiated pathway caspase-9 is activated. They cleave and activate downstream caspases and executioner caspases such as caspase-3, but these cascades can be inhibited by anti-apoptotic members of Bcl-2 (Frisch and Francis, 1994; Grossmann, 2002; Reddig and Juliano, 2005). Caspase-3 and caspase-9 were not activated all the time though the translational level of caspase-3 increased. The activation of caspase-3 not the protein level of caspase-3 is the important thing in the process of apoptosis. Caspase-3 has no effect on apoptosis if it is not activated so caspase-3 did not participate in the anoikis. Caspase-8 was activated and reached the maximal activation at the 48thh in MG-63 cells after detachment. Our results are coincident with Grossmann's point that caspase-8 can be activated by cell–matrix detachment (Grossmann, 2002). Frisch also demonstrated that caspase-8 was activated in a time-dependent fashion following suspension of MDCK cells (Frisch, 1999). The findings indicate a role for death receptor or proteins with related death domains in triggering anoikis. Overexpression of Bcl-2 has been reported to protect apoptosis induced by inhibition of contact with ECM in MDCK cells (Frisch and Francis, 1994). But Bcl-2 protein level did not correlate with cell survival in mammary epithelium (Park et al., 1999). The protein level of Bcl-2 increased in a time-dependent manner after MG-63 cells were deprived of attachment, so overexpression of Bcl-2 can protect osteosarcoma cells against anoikis.

It is possible that caspase-8 was activated to start the caspase cascade of cell death when MG-63 cells were detached from extracellular matrix, but the activation of caspase-8 was inhibited later by Bcl-2. The increased expression of Bcl-2 may tip the “life and death” reach a new balance. As expected caspase activation leads to proteolytic cleavage of numerous intracellular targets such as PKB/AKT, FAK and β-catenin (Grossmann, 2002). So PI3K and β-catenin may be cleaved by caspase-8 activation or other unknown proteases and their translational levels are not in agreement with their transcriptional level.

In summary, the initiation and execution of anoikis is mediated by different pathways and anoikis is a particularly useful tool to study the complex balance of life and death, homeostasis or neoplastic growth. Cell detachment from extracellular matrix may activate the initiation and execution of anoikis by activation of caspase-8 but overexpression of Bcl-2 may inhibit anoikis. Anoikis resistance of MG-63 cells is not related to protein level of β-catenin and PI3K, though it remains to be seen which of the complex web of intracellular signaling cascades is critical to the development of adhesion-independent survival during tumorigenesis in vivo. Such pathways would provide a powerful target for drug therapy against tumor invasion and metastasis.

Acknowledgements

The authors wish to express their thanks to Xiaobo Yan for the assistance in the RT-PCR and Western-Blot studies. The authors wish to thank also to the Electronic Microscope Staff in Medical College of Zhejiang University.

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Received 24 September 2007/9 April 2008; accepted 5 July 2008

doi:10.1016/j.cellbi.2008.07.002

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