siRNA-Mediated Downregulation of MMP-9 and uPAR in Combination with Radiation Induces G2/M Cell-Cycle Arrest in Medulloblastoma

Abstract

Our previous work and that of other investigators strongly suggest a relationship between the upregulation of metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR) in tumor angiogenesis and metastasis. In this study, we evaluated the role of MMP-9 and uPAR in medulloblastoma cancer cell resistance to ionizing irradiation (IR) and tested the antitumor efficacy of siRNA (short interfering RNA) against MMP-9 [plasmid siRNA vector for MMP-9 (pM)] and uPAR [plasmid vector for uPAR (pU)] either alone or in combination [plasmid siRNA vector for both uPAR and MMP-9 (pUM)]. Cell proliferation (BrdU assay), apoptosis (in situ TUNEL for DNA fragmentation), and cell-cycle (FACS) analyses were carried out to determine the effect of siRNA either alone or in combination with IR on G 2 /M cell-cycle arrest in medulloblastoma cells. IR upregulated MMP-9 and uPAR expression in medulloblastoma cells; pM, pU, and pUM in combination with IR effectively reduced both MMP-9 and uPAR expression, thereby leading to increased radiosensitivity of medulloblastoma cells. siRNA treatments (pM, pU, and pUM) also promoted IR-induced apoptosis and enhanced IR-induced G 2 /M arrest during cell-cycle progression. While IR induces G 2 /M cell-cycle arrest through inhibition of the pCdc2-and cyclin B-regulated signaling pathways involving p53, p21/WAF1, and Chk2 gene expression, siRNA (pM, pU, and pUM) alone or in combination with IR induced G 2 /M arrest mediated through inhibition of the pCdc2-and cyclin B1-regulated signaling pathways involving Chk1 and Cdc25A gene expression. Taken together, our data suggest that downregulation of MMP-9 and uPAR induces Chk1-mediated G 2 /M cell-cycle arrest, whereas the disruption caused by IR alone is dependent on p53-and Chk2-mediated G 2 /M cell-cycle arrest. Mol Cancer Res; 9(1); 51-66. Ó2010 AACR.

RHOB influences lung adenocarcinoma metastasis and resistance in a host‐sensitive manner

“…This would explain why RHOB levels did not affect chemosensitivity to gemcitabine and cisplatin. Thus, RHOB could confer treatment resistance by cell‐autonomous effects related to its actions on microtubule regulation, or/and by increasing tumor‐host dependent MMPs secretion, which modulates radiosensitivity (Ganji et al., 2010). Furthermore, radio‐ and chemoresistance by RHOB could be mediated by tumor‐host interactions through changes in stromal angiogenesis (Kazerounian et al., 2013), and/or changes in host‐derived MMP secretion (Luis‐Ravelo et al., 2011).…”

“…In agreement with these findings, RHOB regulates migration and is a key regulator of uPAR signaling in cell adhesion, migration and invasion (Alfano et al., 2012; Vega et al., 2012). These cell functions may also account for the overt osseous colonization and participate in resistance to ionizing radiation, since MMP secretion has been shown to modulate radiosensitivity (Ganji et al., 2010). In our model, tumor–stroma interactions induced an increase in RHOB expression levels under co‐culture conditions; an event that could be relevant in the bone compartment.…”

Curcumin Induced Human Gastric Cancer BGC-823 Cells Apoptosis by ROS-Mediated ASK1-MKK4-JNK Stress Signaling Pathway

“…Enhanced chemiluminescence system was used to detect immunoreactive proteins with horseradish peroxidase (HRP)-conjugated secondary antibody. HRP-conjugated secondary antibody was measured by enhanced chemiluminescence (ECL, Thermo Scientific, Waltham, MA, USA) and were exposed using film exposure [ 49 ].…”

Role of MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with α5β1 integrin in glioma

“…Briefly, cells were plated in 96-microwell plates, incubated overnight and subjected to different treatments as described above. Colorimetric estimation of BrDU incorporation was carried out by quantification of antibody-peroxidase reaction product at dual wavelengths of 450/540 nm using multiwell spectrophotometer as described by Ganji et al ( 58 ). Percentage of BrDU incorporation was compared to mock-treated cells (100%) and represented as mean±SE values obtained in at least three independent experiments.…”