Transfection of NF-κB decoy oligodeoxynucleotide suppresses pulmonary metastasis by murine osteosarcoma

Cancer Gene Therapy (2011) 18, 250–259 r 2011 Nature America, Inc. All rights reserved 0929-1903/11 www.nature.com/cgt ORIGINAL ARTICLE Transfection of NF-jB decoy oligodeoxynucleotide suppresses pulmonary metastasis by murine osteosarcoma A Nishimura1, K Akeda1, T Matsubara1, K Kusuzaki1, A Matsumine1, K Masuda2, T Gemba3, A Uchida1 and A Sudo1 1 Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, Mie, Japan; 2Department of Orthopaedic Surgery, University of California, San Diego, CA, USA and 3AnGes MG, Osaka, Japan Nuclear factor-kappa B (NF-kB) has a pivotal role in the progression and distant metastasis of cancers, including malignant bone tumors. To inhibit NF-kB activation, a new molecular therapy using synthetic double-stranded oligodeoxynucleotide (ODN) as a ‘decoy’ cis element against NF-kB has been developed. To determine whether pulmonary metastasis of osteosarcoma is reduced by inhibiting the action of NF-kB, NF-kB decoy ODN was transfected into the nuclei of murine osteosarcoma cells with high pulmonary metastatic potential, the LM8 cell line, using a three-dimensional alginate spheroid culture model. An in vitro study demonstrated the successful transfection of LM8 cells cultured in alginate beads by ‘naked’ NF-kB decoy ODN and that the activation of NF-kB signaling was significantly suppressed. Tumor growth was not affected by transfection of NF-kB decoy ODN, however, the expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) mRNA was markedly decreased. Furthermore, the transfection of ‘naked’ NF-kB decoy ODN effectively suppressed pulmonary metastasis in an in vivo alginate bead transplantation model. Our results suggest that NF-kB has a central and specific role in the regulation of tumor metastasis and could be a molecular target for development of anti-metastatic treatments for osteosarcoma. Cancer Gene Therapy (2011) 18, 250–259; doi:10.1038/cgt.2010.75; published online 24 December 2010 Keywords: osteosarcoma; nuclear factor-kappa B; synthetic double-stranded oligodeoxynucleotide Introduction Osteosarcoma is the most common primary malignant tumor of bone in children. Despite multidisciplinary treatments for this tumor, a significant proportion of patients developed pulmonary metastasis and eventually succumbed to the disease. Therefore, there is an urgent need to develop new approaches to suppress the progression to pulmonary metastasis.1 The transcription factor nuclear factor-kappa B (NFkB) is a heterodimeric DNA-binding protein that consists of two major polypeptides, p50 and p65.2 In resting cells, NF-kB is sequestered in the cytoplasm by IkB proteins. Stimulus-mediated phosphorylation and subsequent proteolytic degradation of IkB allows the release and nuclear translocation of NF-kB, where it transactivates several target genes, such as vascular endothelial growth factor (VEGF), inter-cellular adhesion molecule-1 (ICAM-1), interleukin-1 (IL-1) and matrix metalloproteinases (MMPs).3 As these NF-kB-related gene expressions Correspondence: Dr K Akeda, Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu city, Mie 514-8507, Japan. E-mail: Received 2 April 2010; revised 17 July 2010; accepted 26 September 2010; published online 24 December 2010 are considered to be involved in a series of sequential steps, including invasion, intravasation, survival in the circulation, adhesion and growth in distant organs, it is generally thought that NF-kB has an essential role in tumor progression and metastasis.4 Increased and aberrant NF-kB signaling activity has been extensively documented in cancer cells, with implications for cellular proliferation, antiapoptosis, promotion of angiogenesis and metastatic tumor spread.5–8 Moreover, blocking the NF-kB signaling pathway has been reported to inhibit bone metastasis of breast cancer,9 and the angiogenesis, invasion and metastasis of prostate cancer10 and melanoma.11 Importantly, an increased activation of NF-kB has also recently been identified in a human osteosarcoma cell line and is thought to contribute to the maintenance of a highly proliferative malignant phenotype.12–17 To inhibit NF-kB activation, a new molecular therapy using synthetic double-stranded oligodeoxynucleotide (ODN) as a ‘decoy’ cis element against NF-kB has been developed.18 When the NF-kB decoy ODN is transfected into cells, it binds competitively to activated NF-kB and prevents transactivation of the target genes. The NF-kB decoy ODN strategy has been applied to various diseases, such as re-stenosis after angioplasty or stenting, glomerulonephritis, rheumatoid arthritis and atopic dermatitis (see review19). In oncology, it has been reported that the intra-tumor injection of NF-kB decoy ODN inhibited Transfection of NF-jB decoy in murine osteosarcoma A Nishimura et al the cachexia induced by adenocarcinoma20 and that intravenous treatment with NF-kB decoy ODN inhibited the hepatic metastasis of M5076 reticulosarcoma in mice.21 Therefore, we hypothesized that transfection with NF-B decoy ODN would suppress the tumor growth and pulmonary metastases of osteosarcoma. Using murine osteosarcoma cells from two metastatic clones, the parental Dunn cell line and its derivative LM8 with greater metastatic potential to the lung,22 we have recently established a novel alginate-encapsulated tumor spheroid model23 to mimic the in vivo microenvironment. The purpose of this study was to examine the effects of NF-B decoy ODN on tumor progression and metastasis-related gene expression in vitro, as well as pulmonary metastasis in vivo, using the LM8 cell line in the alginateencapsulated tumor spheroid model. Materials and methods Three-dimensional alginate spheroid culture The LM8 murine osteosarcoma cell line, which has a high pulmonary metastatic potential, was used for this study. The LM8 cell line was derived from the original Dunn cell line24 by in vivo selection.22 In this study, LM8 cells were seeded at a density of 2.0  106 cells in 175 cm2 culture flasks. When 490% confluency was reached at day 2, the cells were digested using 0.05% trypsin and encapsulated in 1.2% low-viscosity alginate (Keltone LV; Kelco, Chicago, IL) in 0.15 M sodium chloride (NaCl) at a concentration of 4.0  106 cells per ml.23 Encapsulation was achieved by gently expressing drops of the cell suspension through a 21 gauge needle from a 10 ml syringe into 102 mM calcium chloride; each drop was instantly transformed into a semisolid microspheric bead. After 10 min of incubation to allow further polymerization, the newly formed beads were washed three times with normal saline, followed by one wash with Dulbecco’s modified Eagle medium (DMEM: Gibco BRL, Grand Island, NY) to remove excess calcium chloride. The beads were then cultured in complete medium: DMEM supplemented with 10% fetal bovine serum (FBS: Life Technologies, Gaithersburg, MD) and (...truncated)