Effects of NFκB decoy oligonucleotides released from biodegradable polymer microparticles on a glioblastoma cell line
Introduction
Minimal progress has been made in our ability to treat glioblastoma (GBM) over the past century. While surgery, chemotherapy and radiation may remain primary treatment modalities for glioblastomas, they have significant deleterious side effects and drawbacks. The tumor is usually invasive through the normal brain. Incomplete removal of the tumor and development of drug-resistance by the remaining tumor cells ultimately lead to tumor progression [1]. Although many innovative techniques have improved the quality of survival for many patients, the median survival following diagnosis and adjuvant treatment still remains only approximately one-year [2]. Previous studies have focused on identifying the distinct biological features of high-grade human GBM [3], [4], [5], [6], [7], [8], [9], [10], [11].
Nuclear factor-kappa B (NFκB) activation has recently been shown to be important in regulating cell proliferation in a variety of tumor cells [12], [13], [14], [15], [16]. Activation of NFκB involves phosphorylation and dissociation of the inhibitory IκB protein from a cytoplasmic complex with NFκB [17], [18], [19]. After serine phosphorylation of IκB, the inhibitory protein is ubiquinated and degraded by the proteasome pathway [20], [21]. Liberated NFκB is translocated into the nucleus where it induces transcription of responsive genes by binding to DNA κB motifs. Activation of NFκB is correlated with changes in cyclin D1 protein expression and enzymatic activity of cyclin-dependent kinase 4 (Cdk-4). Cyclin D1 is a cell cycle regulatory protein that is expressed in proliferating cells during G1 phase of the cell cycle. It is not expressed in quiescent cells, such as neurons or mature glial cells, which are in the G0 phase. Cyclin D1 complexes with Cdk-4 to form an active kinase, which phosphorylates the retinoblastoma gene product [22]. Phosphorylation of Rb promotes progression through the cell cycle [12], [22].
If errant NFκB activation in GBM is related to a disregulation in the cell cycle yielding uncontrolled cell growth, blocking the translocation pathway of NFκB might inhibit tumor cell proliferation. We have recently shown that specific inhibition of NFκB can be achieved by using a molecular decoy oligonucleotide (ODN) strategy in dorsal root ganglion neurons [23]. The molecular strategy designed to inhibit NFκB activity required the generation of cis decoy ODNs against NFκB binding sites. Binding of liberated NFκB to phosphorothioated decoy deca-nucleotide elements (5′-GGGATTTCCC-3′) within the cytoplasm prevents DNA binding and transactivation by the transcription factor. Therefore, such “decoy” ODN strategies may inhibit the NFκB activity in GBM cell lines and reduce tumor cell growth. Generation and efficiency of NFκB decoy elements have been previously determined [23], [24].
It is probable that such molecular therapeutic strategies will involve local delivery of NFκB decoy ODNs. Even if a potential therapy is effective, it may not work because appropriate delivery has not been achieved. In this regard, incorporation of NFκB decoy ODNs into a biodegradable microparticle delivery scheme would be particularly attractive. Incorporation of biological molecules into synthetic matrices such as microparticles provides a method for sustained and controlled local delivery [25], [26], [27], [28], [29], [30], [31], [32]. This approach is an alternative to cell-based delivery systems during the phase of drug development. A major advantage of biodegradable synthetic microparticles is the ability to control the rate of drug release to obtain the desired local therapeutic concentrations by changing the structure and biodegradation rate of the polymers. Using such a delivery system may also offer other advantages such as site-specific delivery and protection of ODNs from degradation prior to release [33].
In the present study, experiments were designed to determine whether NFκB activation would cause disregulation of GBM cell growth, whether decoy ODNs could inhibit NFκB activity, whether NFκB decoy ODNs would inhibit GBM cell line growth, whether ODNs could be released from PLGA microparticles in a controlled fashion, and whether NFκB decoy ODNs released from PLGA microparticles would have inhibitory effects on GBM cell line growth.
Section snippets
Raw materials
NFκB phosphorothioated decoy ODNs 5′-GGGATTTCCC-3′ and mutated ODNs 5′-GGGCTTTCCC-3′ (underlined base indicates mutation) were purchased from Oligos Etc., Inc. (Wilsonville, OR) on a 50 μm scale, which were purified to ⩾90% with gel electrophoresis and HPLC purification. NFκB decoy and mutant ODNs for electrophoretic mobility gel shift (EMSA) were generated by the Mayo Clinic Molecular Biology Core Facility as follows: 5′-CCTTGAAGGGATTTCCCTCC-3′ and 3′-GGAACTTCCCTAAAGGGAGG-5′ (consensus
NFκB activity in GBM cell lines
In the first line of experiments, the level of NFκB activity in three GBM cell lines was compared to that in primary glial cultures. EMSA revealed NFκB activity was detected in all GBM cell lines (arrow) but not in primary glial cultures (Fig. 1). It suggests that the disregulation of tumor cell growth could be caused by NFκB activation. The NFκB activity was most significant on the U87 cell line. Therefore, we selected this cell line for the evaluation of the inhibition of NFκB activity.
Inhibition of NFκB activity by decoy ODNS
Discussion
This study was conducted to answer the following five questions: (1) Could NFκB activation be the reason for disregulation of GBM cell growth? (2) Could the decoy ODNs inhibit the NFκB activity? (3) Do the decoy ODNs have inhibitory effects on GBM cell line growth? (4) Could the ODNs be released from PLGA microparticles in a controlled manner? (5) Do the ODNs released from PLGA microparticles also have inhibitory effects on GBM cell line growth?
We have demonstrated that NFκB activation is
Acknowledgements
This work was supported by NIH Grant NS39764 (AJW), Lindse-Bok Neuro-oncology Fellowship (JSG), John Smith Foundation (MJY), and AirCast Foundation (MJY). The GBM cell lines were a generous gift from Dr. C. David James (Mayo Foundation).
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