|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2008) 32, 11991206 (Printed in Great Britain)
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
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.
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
2.2 Anoikis induction
To induce anoikis cells were seeded (5
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
2.4 Transmission electronic microscope
Cells were washed in PBS and pellets were fixed by incubating with 2.5% glutaraldehyde for 2
2.5 Flow cytometric analysis
To quantify apoptosis by flow cytometry, cells (5
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
2.7 Western blot analysis
Cells were harvested and suspended in 30
2.8 Statistical analysis
Collected data were expressed as mean
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.
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 72
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 24
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
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 24
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 24
When attachment was deprived transcription level of PI3K increased at 24
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 24
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 24
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 24
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 72
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 48th
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.
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.
Clemo NK, Arhel, NJ, Barnes, JD, Baker, J, Moorghen, M, Packham, GK. The role of the retinoblastoma protein (Rb) in the nuclear localization of BAG-1: implications for colorectal tumour cell survival. Biochem Soc Trans 2005:33:Pt 4:676-8
Diaz-Montero CM, McIntyre, BW. Acquisition of anoikis resistance in human osteosarcoma cells does not alter sensitivity to chemotherapeutic agents. BMC Cancer 2005:5:1:39
Douma S, Van Laar, T, Zevenhoven, J, Meuwissen, R, Van Garderen, E, Peeper, DS. Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB. Nature 2004:430:7003:1034-9
Hellwinkel OJ, Muller, J, Pollmann, A, Kabisch, H. Osteosarcoma cell lines display variable individual reactions on wildtype p53 and Rb tumour-suppressor transgenes. J Gene Med 2005:7:4:407-19
Osanai M, Murata, M, Chiba, H, Kojima, T, Sawada, N. Epigenetic silencing of claudin-6 promotes anchorage-independent growth of breast carcinoma cells. Cancer Sci 2007 Oct:98:10:1557-62
Zhang Y, Lu, H, Dazin, P, Kapila, Y. Squamous cell carcinoma cell aggregates escape suspension-induced, p53-mediated anoikis: fibronectin and integrin alphav mediate survival signals through focal adhesion kinase. J Biol Chem 2004:279:46:48342-9
Received 24 September 2007/9 April 2008; accepted 5 July 2008doi:10.1016/j.cellbi.2008.07.002