Musculoskeletal
Pericellular Matrix Formation Alters the Efficiency of Intracellular Uptake of Oligonucleotides in Osteosarcoma Cells

https://doi.org/10.1016/j.jss.2008.02.037Get rights and content

One of the crucial roles of tumor extracellular matrix is to act as a barrier to drug delivery. In this study, we analyzed the relationship between the formation of tumor extracellular matrix and the efficiency of intracellular uptake of oligonucleotides in human osteosarcoma cell lines, HOS, and MG-63. Oligonucleotides used in this study were nuclear factor-kappa B (NF-κB) decoy, which might be a therapeutic tool for neoplasms. Pericellular matrix formation was examined by particle exclusion assay. Cellular uptake of fluorescein isothiocyanate-labeled NF-κB decoy was evaluated by fluorescent microscopy and flow cytometry. Effects of NF-κB decoy on cell viability and cell cycle arrest in MG-63 cells were determined by MTT assay and flow cytometry, respectively. MG-63 cells exhibited abundant pericellular matrix with time compared with HOS cells. Uptake of fluorescein isothiocyanate-labeled NF-κB decoy decreased in MG-63 cells with time but not in HOS cells in both monolayer and three-dimensional culture using matrigel. However, after enzymatic removal of pericellular matrix, the uptake markedly recovered in MG-63 cells. NF-κB decoy inhibited cell proliferation and induced G0/G1 cell cycle arrest in MG-63 cells. These results suggest that abundant pericellular matrix might disturb the uptake of NF-κB decoy, and modification of pericellular matrix composition would increase the efficacy of exogenous oligonucleotides treatment for neoplasms.

Introduction

The extracellular matrix (ECM) has multiple functions in pathological conditions, i.e., offering substrate for cell migration, storage of growth factors, initiating signals toward gene expressions [1, 2], and forming a barrier for cancer cells against host immunocompetent cells [3]. Increased matrix deposition of hyaluronan, which is an abundant component of the ECM, could promote cell invasion by providing a suitable environment for cancer cells [4], stimulating cell motility via interactions with cell surface receptors of hyaluronan [5].

Other than these functions, the ECM is a potent barrier to the delivery and penetration of complex biopharmaceuticals such as monoclonal antibodies, therapeutic proteins, nanoparticles, or genes. The ECM acts as a dispersive filter, controlling the composition of extracellular fluid and the rate of molecular trafficking. Because of uniformly elevated interstitial fluid pressure in solid tumors, drug delivery through the ECM relies on passive diffusive transport [6]. Different ECM components including collagen, glycosaminoglycans, and proteoglycans form a complex structured gel. Resistance to interstitial flow has been strongly linked to glycosaminoglycans, especially hyaluronan (HA) [7, 8, 9]. It is thus crucial to identify the ECM constituents and characteristics that restrict diffusion and to determine how these are affected by tumor type.

Nuclear factor-kappa B (NF-κB) is a critical transcriptional factor. The activated NF-κB regulates various genes involved in growth control, apoptosis, invasion and metastasis of many cancer cells. Constitutive activation of NF-κB has been shown in some tumors and is positively related to tumorigenesis, including esophageal cancer, cervical cancer, renal cancer, and Ewing sarcoma [10, 11, 12, 13]. Thus, activated NF-κB has been suggested as a therapeutic target in malignant tumors. It has been demonstrated that NF-κB activation can be inhibited by decoy oligonucleotides [14, 15, 16, 17], which are a synthetic double-strand oligo-DNA containing a sequence corresponding to the consensus of the NF-κB binding sites [18]. Inhibition of inducible NF-κB activation using NF-κB decoy were demonstrated to reduce both TNF-α induced cell invasion and motility in osteosarcoma cell lines [19].

Osteosarcoma is the most common primary malignant tumor of bone [20, 21]. Despite aggressive chemotherapy and radical resection of the tumor, a significant proportion of patients eventually develop pulmonary metastasis and succumb to the disease. There is a pressing need to develop novel approaches to the current medical treatment of osteosarcoma. Osteosarcoma is a heterogeneous group of lesions with diverse morphology and clinical behavior. More understanding of the basic biology of this tumor, such as the role of tumor ECM, may provide additional approaches for treatment. Previous studies have demonstrated that the manipulation of ECM in osteosarcoma cells alters the cell behavior. Depletion of abundant HA-rich ECM by antisense hyaluronan synthase-2 oligonucleotides or hyaluronan oligosaccharides leads to suppression of tumorigenicity by the osteosarcoma cells [22, 23]. Recently, the role of ECM as a modulator of antitumor drugs has been suggested [24, 25, 26]. Several studies have provided evidence that doxorubicin, etoposide, and hydroxy-cyclophosphamide-induced cell death could be suppressed in the presence of ECM [24, 25, 26, 27]. Therefore, ECM may provide protection against antitumor drugs or host immunocompetent cells, and contribute to the repopulation of tumor cells following treatment. However, no study has been undertaken to clarify the roles of ECM in the efficiency of intracellular uptake of exogenous added pharmaceuticals, especially small molecules such as NF-κB decoy oligonucleotides.

In this study, we determined the roles of pericellular ECM in the intracellular uptake of NF-κB decoy oligonucleotides in the human osteosarcoma cell lines, MG-63 and HOS, with or without pretreatment with hyaluronidase, which abrogates the HA-rich pericellular ECM in MG-63 cells. Because our previous study demonstrated that MG-63 cells have HA-rich abundant pericellular matrix, whereas HOS has poor matrix pericellulary [22], these two cell lines were used for cellular uptake of oligonucleotides in this study. Additionally, the levels of NF-κB activation in these osteosarcoma cell lines, and the effects of NF-κB decoy on cell growth and cell cycle were also investigated.

Section snippets

Chemicals

Dulbecco's modified Eagles medium and trypsin ethylenediamine tetraacetic acid (EDTA) were obtained from Life Technologies, Inc. (Grand Island, NY). Fetal bovine serum was purchased from Hyclone (Logan, UT). Cell Proliferation Kit I was purchased from Roche (Mannheim, Germany). FuGene 6 was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Antihuman phospho-NF-κB p65 polyclonal antibody was purchased from Cell Signaling Technology (Beverly, MA). Matrigel was purchased from BD

Cell-Associated Matrix Assembly by MG-63 and HOS Cells

One of the functions of tumorous ECM might be the protection of tumor cells from antitumor drugs or host immunocompetent cells. The cell-associated matrix surrounding living cells is best visualized with particle exclusion assay. As can be seen in Fig. 1A and B, MG-63 cells exhibited more abundant ECM formation with time compared with HOS cells. As shown in Fig. 1D, morphometric analysis indicated a statistically significant difference in cell-associated matrix between HOS and MG-63 cells (P <

Discussion

The transport barrier composed of the ECM is particularly important in tumors because it may prevent the penetration of therapeutic agents. Previous studies reported the role of ECM composition in the penetration of high molecular weight therapeutic agents. Netti et al. reported that resistance to IgG penetration is related to tumor rigidity and collagen organization [30]. IgG diffusion is hindered both by the collagen network and by the hydrophilic gel of proteoglycans [31]. Hyaluronidase is

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