Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells

AbstractOur previous studies have shown the role of radiation-induced urokinase plasminogen activator (uPA) expression in the progression of meningioma. In the present study, we investigated whether modulation of DNA methylation profiles could regulate uPA expression. Initially, radiation treatment was found to induce hypomethylation in meningioma cells with a decrease in DNA (cytosine-5)-methyltransferase 1 (DNMT1) and methyl-CpG binding domain protein (MBD) expression. However, oxidative damage by H2O2 or pretreatment of irradiated cells with N-acetyl cysteine (NAC) did not show any influence on these proteins, thereby indicating a radiation-specific change in the methylation patterns among meningioma cells. Further, we identified that hypomethylation is coupled to an increase in uPA expression in these cells. Azacytidine treatment induced a dose-dependent surge of uPA expression, whereas pre-treatment with sodium butyrate inhibited radiation-induced uPA expression, which complemented our prior results. Methylation-specific polymerase chain reaction on bisulfite-treated genomic DNA revealed a diminished methylation of uPA promoter in irradiated cells. Transfection with small hairpin RNA (shRNA)-expressing plasmids targeting CpG islands of the uPA promoter showed a marked decline in uPA expression with subsequent decrease in invasion and proliferation of meningioma cells. Further, radiation treatment was found to recruit SP1 transcription factor, which was abrogated by shRNA treatment. Analysis on signaling events demonstrated the activation of MAP kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) in radiation-treated cells, while U0126 (MEK/ERK inhibitor) blocked hypomethylation, recruitment of SP1, and uPA expression. In agreement with our in vitro data, low DNMT1 levels and high uPA were found in intracranial tumors treated with radiation compared to untreated tumors. In conclusion, our data suggest that radiation-mediated hypomethylation triggers uPA expression in meningioma cells

uPAR/cathepsin B overexpression reverse angiogenesis by rescuing FAK phosphorylation in uPAR/cathepsin B down regulated meningioma.

BACKGROUND:Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression. METHODOLOGY/PRINCIPAL FINDINGS:In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model. CONCLUSION/SIGNIFICANCE:These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma

Characterization of CC-531 as a Rat Model of Colorectal Liver Metastases.

PURPOSE:Surgical resection of colorectal liver metastases is not achievable in more than 70% of the cases. Although the liver directed therapies have become a part of the stand of care, lack of a preclinical model impedes the assessment of toxicity and therapeutic benefits attributed several candidate drugs or treatment regimens that can be designed. In the present study we aim develop and characterize a rat colorectal liver metastasis model. MATERIALS AND METHODS:Growth characteristics of CC-531 cells were determined in vitro followed by subcapsular liver implantation in syngeneic WAG/Rij rats. Tumor growth progression was followed over 3 weeks by ultrasound (US) and magnetic resonance imaging (MRI). Growth characteristics were also assessed by histopathology and immunohistochemistry in harvested tumor tissues. RESULTS:The doubling time of CC-531 cells was found be under 24hrs and all the implanted rats grew tumors. US imaging showed hypoechoic masses and MRI showed contrast enhancement representing complex tumor microenvironments. Hematoxylin and Eosin staining confirmed tumor growth and uniform CD31 staining in tumor confirmed even vessel density. CONCLUSION:CC-531 can be used as a metastatic rat tumor colorectal liver metastases model with well-defined characteristics that can be readily followed by imaging whilst having a therapeutic window for interventions

MR imaging.

<p>Coronal contrast enhanced MRI images for the same rat at 8 and 16 days post injection of tumor cells, demonstrating rapid increase in tumor volume.</p

Quantitative MRI.

<p>Shown in (a) and (b) are two adjacent coronal slices of a representative rat liver with advanced stage tumor. Tumor lobes are identifiable as hyperintense relative to adjacent normal liver tissue within T2-weighted anatomic images. Shown In panels (c)–(h) are the corresponding parametric maps produced from dynamic contrast enhanced (DCE) image series. (f)These depict the vascular volume and permeability of tissues within the tumor region. R2* difference map from gas-challenge BOLD MR studies in this tumor model are shown in panel; significant heterogeneity in both gas-challenge signal changes and quantitative DCE measurements suggest a heterogeneous tumor microenvironment with regional variations in perfusion, tissue oxygenation and blood volume.</p

Irreversible Electroportation Therapy.

<p>Representative coronal T2 weighted MR images from two Wistar rats prior to IRE (A.I) and 7 days post IRE (A.II). White arrows = control tumors, and dashed arrows = tumors that under went IRE.</p

Ultrasound and Gross Pathology.

<p>(a) Transverse ultrasound images demonstrates a well-defined hypoehoic tumor in the anterior aspect of the left hepatic lobe. (b) Explanted gross specimen reveals tumors corresponding to the lesions seen on US (arrow heads).</p

Tumor inoculation.

<p>Using a 29 G needle, 5*106 CC-531 cells in a cellular suspension were injected into the anterior subcapsular left hepatic lobe.</p

Vascular Interventional Radiology-Guided Photothermal Therapy of Colorectal Cancer Liver Metastasis with Theranostic Gold Nanorods

We report sub-100 nm optical/magnetic resonance (MR)/X-ray contrast-bearing theranostic nanoparticles (TNPs) for interventional image-guided photothermal therapy (PTT) of solid tumors. TNPs were composed of Au@Gd₂O₃:Ln (Ln = Yb/Er) with X-ray contrast (∼486 HU; 10¹⁴ NPs/mL, 0.167 nM) and MR contrast (∼1.1 × 10⁸ mM^–1 S^–1 at 9.4 T field strength). Although TNPs are deposited in tumors following systemic administration <i>via</i> enhanced permeation and retention effect, the delivered dose to tumors is typically low; this can adversely impact the efficacy of PTT. To overcome this limitation, we investigated the feasibility of site-selective hepatic image-guided delivery of TNPs in rats bearing colorectal liver metastasis (CRLM). The mesenteric vein of tumor-bearing rats was catheterized, and TNPs were infused into the liver by accessing the portal vein for site-selective delivery. The uptake of TNPs with hepatic delivery was compared with systemic administration. MR imaging confirmed that delivery <i>via</i> the hepatic portal vein can double the CRLM tumor-to-liver contrast compared with systemic administration. Photothermal ablation was performed by inserting a 100 μm fiber-optic carrying 808 nm light <i>via</i> a JB1, 3-French catheter for 3 min under DynaCT image guidance. Histological analysis revealed that the thermal damage was largely confined to the tumor region with minimal damage to the adjacent liver tissue. Transmission electron microscopy imaging validated the stability of core–shell structure of TNPs <i>in vivo</i> pre- and post-PTT. TNPs comprising Gd-shell-coated Au nanorods can be effectively employed for the site-directed PTT of CRLM by leveraging interventional radiology methods