MusculoskeletalPericellular Matrix Formation Alters the Efficiency of Intracellular Uptake of Oligonucleotides in Osteosarcoma Cells
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
References (44)
- et al.
Invasive growth and topoisomerase-switch induced by tumorous extracellular matrix in osteosarcoma cell culture
Cell Biol Int
(2005) - et al.
Identification of two hyaluronan-binding domains in the hyaluronan receptor RHAMM
J Biol Chem
(1993) - et al.
Inhibition of inducible NF-κB activity reduces chemoresistance to 5-fluorouracil in human stomach cancer cell line
Exp Cell Res
(2003) - et al.
Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity
Exp Cell Res
(2005) - et al.
Hyaluronan oligosaccharides inhibit tumorigenicity of osteosarcoma cell lines MG-63 and LM-8 in vitro and in vivo via perturbation of hyaluronan-rich pericellular matrix of the cells
Am J Pathol
(2007) - et al.
Cellular uptake and intracellular fate of antisense oligonucleotides
Trends in Cell Biol
(1992) - et al.
Indole-3-carbinol suppresses NF-κB and IκB α kinase activation, causing inhibition of expression of NF-κ regulated antiapoptotic and metastatic gene products and enhancement of apoptosis in myeloid and leukemia cells
Blood
(2005) - et al.
Hyaluronidase enhances the therapeutic effect of vinblastine in intralesional treatment of Kaposi's sarcoma
J Am Acad Dermatol
(1997) - et al.
The microenvironment of the tumor–host interface
Nature
(2001) - et al.
Hyaluronidase-sensitive halos around adherent cellsTheir role in blocking lymphocyte-mediated cytolysis
J Exp Med
(1979)
Hyaluronan in Malignancies
Enhancement of fluid filtration across tumor vessels: Implication for delivery of macromolecules
Proc Natl Acad Sci USA
Water and solute transport in the extracellular matrix: Physical principles and macromolecular determinants
Flow through interstitium and other fibrous matrices
Q J Exp Psychol
Physiological function of connective tissue polysaccharides
Physical Rev
NF-κB activation in esophageal adenocarcinoma: Relationship to Barrett's metaplasia, survival, and response to neoadjuvant chemoradiotherapy
Ann Surg
NF-κB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix
Oncogene
Increased nuclear factor-κ B activation is related to the tumor development of renal cell carcinoma
Carcinogenesis
Inhibition of constitutive NF-κB activity suppresses tumorigenicity of Ewing sarcoma EW7 cells
Int J Cancer
In vivo transfection of cis element “decoy” against nuclear factor-κB binding site prevents myocardial infarction
Nat Med
Decoy oligodeoxynucleotides as novel cardiovascular drugs for cardiovascular disease
Ann NY Acad Sci
Normalizing mitochondrial superoxide production blocks three pathways of hyperglycemic damage
Nature
Cited by (17)
Tissue-engineered 3D models for elucidating primary and metastatic bone cancer progression
2019, Acta BiomaterialiaCitation Excerpt :Hydrogels have been widely used to study cancer cell invasion, matrix remodeling, angiogenesis, and responses to matrix properties such as stiffness and composition in soft tissue tumors [3]. Hydrogel-based models of primary bone cancer have been developed using naturally-derived polymers such as collagen type I [60,61], hyaluronic acid [61], alginate [62], and Matrigel [63,64] and synthetic materials such as poly(ethylene glycol diacrylate) (PEGDA) [65]. Although hydrogel models do not mimic the stiffness range of bone tissue, these have the advantage that they can be designed to mimic bone composition.
Osteomimetic matrix components alter cell migration and drug response in a 3D tumour-engineered osteosarcoma model
2019, Acta BiomaterialiaCitation Excerpt :Appreciation of the difference in characteristics of the bone and bone marrow is essential when aiming to replicate in vivo biology with an in vitro model of the disease. Indeed, bone density [6,7], platelet-assisted intravasation [8–10], hypoxia [11,12] and matrix composition [13,14] are all mediators of osteosarcoma development. The research presented herein focuses on the role of the matrix composition in the osteosarcoma niche on cancer cell behaviour.
Tissue engineered models of healthy and malignant human bone marrow
2019, Advanced Drug Delivery ReviewsCitation Excerpt :Biocompatible scaffolding approaches enabled OS cells to interact with biomechanical and biochemical cues that impact tumor formation and migration [181,193,195] and even enable culture of cancer stem cells (Fig. 5D) [196]. For example, nuclear factor-kappa B and the pericellular ECM were found to play critical roles in affecting pulmonary metastatic potential [197,198]; downregulation of the microRNA miR-29b-1 was identified as a novel therapeutic agent [199]; secreted frizzled related protein 2 was found irrelevant to proliferation but important to migratory and invasive potential [200]; the role of vascular endothelial cadherin in transdifferentiation of OS cells to endothelial-like cells, and the ability of the bisphosphonate zoledronic acid to mitigate this process [201,202]. Quite surprisingly given the native tumor biology, very few OS models are heterotypic [181].
Morphological, cytotoxic and apoptotic effects of different concentrations of nisin ZP on MG63 cells in a 3-dimensional culture system
2024, Asia-Pacific Journal of Molecular Biology and BiotechnologyEngineered bone marrow as a clinically relevant ex vivo model for primary bone cancer research and drug screening
2023, Proceedings of the National Academy of Sciences of the United States of America