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

A comparison of folate status in women of child-bearing age in Korea and in the United States

Taisun Hyun,1 Suguna Badiga,2 Han Byul Jang,1 Young-Hee Han,1 Chandrika J Piyathilake21Department of Food and Nutrition, Chungbuk National University, Cheongju, Korea; 2Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL, USABackground: Even though several studies have demonstrated that periconceptional supplementation with folic acid (FA) reduces the occurrence of neural tube defects, FA fortification has been a topic of intense debate due to the possible adverse effects of higher folate status on several health conditions. Several countries, including Korea, have been indecisive as to whether fortification is warranted or not. It is therefore helpful for these countries to compare folate concentrations in their populations with populations exposed to mandatory FA fortification.Purpose: To evaluate the differences in the distribution of circulating concentrations of folate in Korea and the United States (US) at different time points.Methods: The Korean study populations consisted of women of child-bearing age recruited in 1999 and in 2009. The US study populations consisted of women of child-bearing age recruited in the post FA fortification era (2005 and 2009). Plasma and red blood cell (RBC) folate concentrations were measured using the Lactobacillus casei microbiological assay.Results: The percentage of US women with neural tube defect-protective levels of RBC folate was significantly higher compared to Korean women in 1999 and 2009. However, in 2009, when FA supplements became readily available for Koreans, 50% of Korean women in the study achieved the neural tube defect-protective level of RBC folate; 11% of them demonstrating supraphysiologic concentrations of plasma folate. Even though FA fortification in the US resulted in more than 80of women achieving >400 ng/mL of RBC folate by 2009, nearly 50% also demonstrated having supraphysiologic concentrations of plasma folate, which prompted some researchers to raise concerns about possible adverse effects of higher folate status on several health conditions.Conclusion: Encouraging Korean women of reproductive age to take FA supplements and evaluating the outcome of such efforts would be worthwhile prior to implementing a population-wide mandatory FA fortification in Korea.Keywords: folate, fortification, child-bearing ag

Long-term results of pancreatic stents in chronic pancreatitis

Pancreatic stenting is a new nonsurgical treatment for patients with chronic pancreatitis and pain. We studied the long-term safety and efficacy of pancreatic stenting. Between 1982 and 1993, 51 patients with chronic pancreatitis and persistent pain with dominant strictures in the pancreatic duct were treated with plastic pancreatic stents. Stent insertion was successful in 49 of 51 patients. Early complications occurred in 9 of the 51 patients (18%). Patients were followed for a median of 34 months (range 6 to 128). Nine of the 49 patients (82%) had clinical improvement. Sixteen of these 40 patients still had their stents in place. Stents were removed in 22 of the 40 patients with persistent beneficial response in all (median follow-up 28.5 months). The long-term effect of stenting could not be evaluated in the remaining 2 patients because they had a double bypass operation. Stent dysfunction occurred in 27 of the 49 patients (55%) and was successfully treated by exchanging the stent. Pancreatic stenting was associated with minimal early complications, but stent dysfunction remained a frequent late complication. Pancreatic drainage resulted in clinical improvement in 40 of the 49 patients (82%). Twenty-two of these 40 patients maintained the beneficial response after stent removal (28.5 months

A higher degree of expression of DNA methyl transferase 1 in cervical cancer is associated with poor survival outcome

Chandrika J Piyathilake,1 Suguna Badiga,1 Samuel G Borak,2 Janaka Weragoda,1 Sejong Bae,3 Roland Matthews,4 Walter C Bell,2 Edward E Partridge5 1Department of Nutrition Sciences, 2Department of Pathology, 3Department of Medicine, The University of Alabama at Birmingham, Birmingham, AL, 4Department of Obstetrics and Gynecology, Morehouse School of Medicine, Atlanta, GA, 5Comprehensive Cancer Center, The University of Alabama at Birmingham, Birmingham, AL, USA Background: Even though novel therapies based on aberrant DNA methylation could be of particular importance for the treatment of cervical cancer (CC) because the oncoproteins E6/E7 of high-risk human papillomaviruses, the causative agents for developing CC, have the capacity to bind and upregulate DNA methyltransferases (DNMTs), to our knowledge, no previous studies have evaluated the expression of this enzyme in CC in relation to survival outcomes. The purpose of the study was to evaluate the expression of DNMT1 in CC and its association with survival outcomes.Methods: The study population consisted of 76 women treated for primary CC and followed up by the University of Alabama at Birmingham (UAB) cancer registry. The expression of DNMT1 was examined using immunohistochemistry, and the degree of expression of DNMT1 was expressed as a percentage of cells positive for DNMT1 and its intensity. Cox proportional hazards model was used to assess the relationship between the degree of expression of DNMT1 and overall survival after adjusting for relevant covariates.Results: The expression of DNMT1 was significantly higher in CC cells compared to that in the normal cervical epithelium. A higher percentage of cells positive for DNMT1 and a higher intensity score for DNMT1 were significantly associated with poor survival outcome (hazard ratio [HR] =4.3, P=0.03 and HR =4.9, P=0.02, respectively).Conclusion: Our findings suggested that the degree of expression of DNMT1 could be considered as a target in the epigenetic treatment of CC. Replication of our results in other study populations with CC could create the opportunity of using DNMT inhibitors to treat CC. Keywords: DNMT1, cervical cancer, survival outcome&nbsp

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