MMP-2 siRNA Inhibits Radiation-Enhanced Invasiveness in Glioma Cells

Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma.We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls.MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients

p-MMP-2 combined with radiation enhances apoptosis <i>in vivo</i>.

<p><b>A,</b> Immunohistochemical analysis of brain sections using anti-MMP-2, anti-VEGF and anti-pFAK antibodies. Sections were photographed (60×). Also shown is the negative control where the primary antibody was replaced by non-specific IgG (insets). <b>B,</b> Tissue sections of mice were evaluated with the TUNEL assay according to manufacturer's instructions and photographed under fluorescent microscopy (60×). For the negative control, samples were incubated with label solution (without terminal transferase) instead of TUNEL reaction mixture (insets). <b>C,</b> siRNA against MMP-2 inhibits U251 tumor cell invasion <i>in vivo</i>. H&E staining was performed according to standard protocol, and representative pictures of tumor sections from mock, pSV, p-MMP-2-treated mice are shown (20× and 60×). <b>D</b>, Immunohistochemical analysis of brain sections using anti-human nuclei (HuNu) antibody, a histological marker for identification of human cells (a specific human nuclear antigen). Entire brain sections were photographed (4×; middle row); shown on the top row is a non-tumor region (40×; top row); and shown on the bottom row is tumor and non-tumor overlapping region (40×; bottom row). Also shown is the negative control where the primary antibody was replaced by non-specific IgG (inset).</p

p-MMP-2 transfection inhibits radiation-enhanced glioma cell invasion.

<p>U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2, and irradiated as described earlier. Cells were trypsinized and counted, and 5×10⁵ cells from each treatment condition were allowed to invade transwell inserts containing 12-µm-pore polycarbonate membranes pre-coated with Matrigel for 24 h at 37°C. Afterwards, cells were fixed and stained with Hema-3. Cells that had migrated to the lower side of the membrane were photographed under a light microscope at 20× magnification. Percentages of invading cells were quantified by counting five fields from each treatment condition. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock or irradiated controls.</p

p-MMP-2 inhibits radiation-enhanced tumor culture medium-induced microtubule network formation in endothelial cells and downregulates expression of angiogenesis-associated molecules.

<p><b>A,</b> Human microvascular endothelial cells (5×10⁴) were seeded in 96-well plates and cultured with conditioned medium collected from U-251 and U-87 glioma cells transfected with mock, p-SV, and p-MMP-2, and irradiated as described earlier. 24 h after radiation treatment, the cells were washed, fixed and stained with Hema-3 and photographed. Percentages of branches were quantified by counting five fields in each condition. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock or irradiated controls. <b>B,</b> U-251 and U-87 transfection and radiation was carried out as described earlier. 24 h after radiation, whole cell lysates were prepared and analyzed by Western blotting for the angiogenic molecules VEGF, VEGFR-2 and p-VEGFR-2 as well as p-FAK, FAK, p-p38 and p38. GAPDH served as a loading control.</p

p-MMP-2 transfection in combination with radiation inhibits glioma cell migration.

<p>U-251 and U-87 cells were cultured for formation of spheroids as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020614#s4" target="_blank">Materials and Methods</a>. Spheroids were then transfected with mock, p-SV or p-MMP-2, and followed by irradiation as described earlier. At the end of the migration assay, spheroids were fixed and stained with Hema-3. Migration of cells from spheroids to monolayers was measured using a microscope calibrated with a stage and ocular micrometer. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock, p-SV or irradiated controls.</p

p-MMP-2 transfection inhibits radiation-enhanced MMP-2 activity and expression levels as well as cell viability.

<p><b>A,</b> U-251 and U-87 cells were transfected with mock (PBS), p-SV or p-MMP-2 (2 µg), and after 72 h of incubation, cells were irradiated with 0, 2, 4, 6 or 8 Gy and incubated for a further 24 h. Conditioned media was used to determine MMP-2 activity by gelatin zymography, and total cell lysates were used to determine MMP-2 levels by Western blotting. <b>B,</b> Total RNA was used to determine MMP-2 mRNA transcription levels by RT-PCR with gene-specific primers. GAPDH served as a loading control. <b>C,</b> U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2 and irradiated as described above. 24 h after radiation, the cells were fixed and processed to visualize MMP-2 expression. The cells were mounted using mounting media with DAPI to visualize the nucleus. <b>D,</b> U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2, and irradiated for 72 h after transfection. After a another 24 h of incubation, cell viability was analyzed by MTT assay (absorbance read at 550 nm). <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, significant difference from mock, p-SV or irradiated controls.</p

Radiation enhances MMP-2 and p-MMP-2 inhibits MMP-2 activity and expression in glioma cell lines.

<p><b>A,</b> U-251 and U-87 cells were irradiated with 0–12 Gy X-ray, incubated for 24 h, and conditioned medium collected. MMP-2 activity was determined by gelatin zymography. The band intensities of MMP-2 activity were quantified by densitometry. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from non-irradiated (0 Gy) conditioned medium. <b>B,</b> U-251 and U-87 cells were transfected with mock (PBS), p-SV or p-MMP-2 (1, 2 or 3 µg). After 72 h of incubation, conditioned media was used to determine MMP-2 activity by gelatin zymography, and total cell lysates were used to determine MMP-2 and MMP-9 levels by Western blotting. The band intensities of MMP-2 activity as well as MMP-2 and MMP-9 protein levels were quantified by densitometry and normalized with the intensity of the mock bands. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. <i>Columns</i>: mean of triplicate experiments; <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock or p-SV.</p

p-MMP-2 combined with radiation inhibits tumor growth <i>in vivo</i>.

<p>U-251 (1×10⁶) cells were injected intracerebrally into athymic mice. After ten days, animals were separated into five groups and were treated on alternate days with intracerebral injections of p-SV or p-MMP-2 for a total of 4 doses (60 µg per dose) and 2 doses of radiation (4 Gy per dose) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020614#s4" target="_blank">Materials and Methods</a>. Six weeks after the experiment was initiated, mice were euthanized with intracardiac perfusion of PBS, followed by formaldehyde. The brains were then removed. <b>A,</b> Six weeks after the experiment was initiated, an intraperitonal injection of 2.5 mg D-luciferin sodium salt diluted in 50 µL of PBS was given, and animals were photographed under the IVIS camera for fluorescent light emission. The brains were removed and fixed in 10% phosphate-buffered formaldehyde, and the fixed tissue samples were then processed into paraffin blocks. Brain sections (5 µM thick) were stained with hematoxylin and eosin (H&E), and photographed under a light microscope (4× and 40×). <b>B,</b> Every fifth or sixth brain section (5 µM thick) was stained with H&E solution, and the tumor masses (H&E-stained) were manually traced on the microscope attached computer screen. Areas were calculated using Image Pro Discovery Program software (Media Cybernetics, Inc., Silver Spring, MD). The total tumor volume was calculated as the summed area on all slices, multiplied by the slice separation. <i>Columns</i>: mean of area of tumor portion of all mice in the group (n = 8); <i>bars</i>: SD; *<i>p</i><0.01, **<i>p</i><0.001, significant difference from mock, p-SV or irradiated controls.</p