DMA, a Bisbenzimidazole, Offers Radioprotection by Promoting NFκB Transactivation through NIK/IKK in Human Glioma Cells

BACKGROUND: Ionizing radiation (IR) exposure often occurs for human beings through occupational, medical, environmental, accidental and/or other sources. Thus, the role of radioprotector is essential to overcome the complex series of overlapping responses to radiation induced DNA damage. METHODS AND RESULTS: Treatment of human glioma U87 cells with DMA (5- {4-methylpiperazin-1-yl}-2-[2'-(3, 4-dimethoxyphenyl)-5'-benzimidazolyl] in the presence or absence of radiation uncovered differential regulation of an array of genes and proteins using microarray and 2D PAGE techniques. Pathway construction followed by relative quantitation of gene expression of the identified proteins and their interacting partners led to the identification of MAP3K14 (NFκB inducing kinase, NIK) as the candidate gene affected in response to DMA. Subsequently, over expression and knock down of NIK suggested that DMA affects NFκB inducing kinase mediated phosphorylation of IKKα and IKKβ both alone and in the presence of ionizing radiation (IR). The TNF-α induced NFκB dependent luciferase reporter assay demonstrated 1.65, 2.26 and 3.62 fold increase in NFκB activation at 10, 25 and 50 µM DMA concentrations respectively, compared to control cells. This activation was further increased by 5.8 fold in drug + radiation (50 µM +8.5 Gy) treated cells in comparison to control. We observed 51% radioprotection in control siRNA transfected cells that attenuated to 15% in siRNA NIK treated U87 cells, irradiated in presence of DMA at 24 h. CONCLUSIONS: Our studies show that NIK/IKK mediated NFκB activation is more intensified in cells over expressing NIK and treated with DMA, alone or in combination with ionizing radiation, indicating that DMA promotes NIK mediated NFκB signaling. This subsequently leads to the radioprotective effect exhibited by DMA

Luciferase assays for NFκB activation in U87 cells.

<p>Cells were co-transfected with pNFκB-Luc and pRenilla. After 24 h, cells were irradiated (8.5 Gy) with or without DMA treatment (50 µM) and stimulated with TNF-α (20 ng/ml) for 4 h prior to cell lysis. Luciferase activity was determined to quantify NFκB transcription activity. Relative Luciferase activity (RLA) for NFκB activation was increased in DMA treated cells compared with irradiated or control cells. The triplicate samples were used and the data was presented as mean±SD from three independent experiments. (*indicates statistical significance by T-test, *p<0.00001).</p

Effect of DMA on survival of HDF, MCF10A and U87 cell lines.

<p>(A) Structure of the DNA minor groove binding ligand DMA –5-(4-methylpiperazin-1-yl)-2-[2′-(3,4-dimethoxyphenyl)-5′-benzimidazolyl] benzimidazole.(B) Effect of varying concentrations of DMA on metabolic viability studied by MTT assay in exponential growing HDF,MCF10A and U87 cells at 24 h, (C) At 48 h (D) At 72 h.Values are mean (±SD) of three independent experiments.Statistical significance by T-test p<0.05.</p

List of pathways and number of genes in each pathway identified in U87 cells in three different treatment conditions DMA (50 µM), radiation (8.5 Gy) and DMA+ radiation (50 µM +8.5 Gy) after 4 h by Microarray hybridization studies.

<p>List of pathways and number of genes in each pathway identified in U87 cells in three different treatment conditions DMA (50 µM), radiation (8.5 Gy) and DMA+ radiation (50 µM +8.5 Gy) after 4 h by Microarray hybridization studies.</p

Effect of transfection with siRNA-NIK on the phosphorylation of IKKα and IKKβ.

<p>U87, human glioma cells were transiently transfected with siRNA-NIK. Phosphorylation of IKKα, IKKβ was absent concurrent with equivalent expression of IκBα in response to both DMA and radiation treatment.</p

Pathway construction based on the proteins identified from 2D PAGE and ESI-MS/MS Analysis

<p>. Genes/proteins identified from proteomic data analyses were categorized into pathways based on NCBI, OMIM, KEGG and Gene Cards. Colour boxes represent the proteins identified by ESI-MS/MS- chaperones and folding catalysts (HSP70, CALR, TRX, PRX2, PDIA3); structural proteins (UBCEP80, H2B1, ACTG, TPM4, VIM, FABPE); Single stranded nucleic acid binding proteins NPM1,PCBP1); Glycolytic pathway enzymes (PGM, ENO1,TPI); PARK7,NACA. Blue boxes indicate interacting partners not identified by ES-MS/MS but are common between the identified proteins as interacting partners. Red arrows indicate that the proteins identified belonging to common categories and possessing common interacting partners converge onto MAPK signal transduction pathway.</p

Effect of NFκB inducing kinase overexpression on the phosphorylation of IKKα and IKKβ.

<p>U87, human glioma cells were transiently transfected with p3XFLAG CMV10 NIK. Phosphorylation of IKKα, IKKβ was advanced in NIK overexpressing cells as compared to siRNA transfected cells in response to DMA treatment, alone or in combination with IR.</p

Sequences of primers used for quantitation of gene expression.

<p>Sequences of primers used for quantitation of gene expression.</p

Relative quantitation of gene expression for MAPK pathway genes in U87 cells.

<p>U87 cells were treated with 50 µM DMA and/or 8.5 Gy ionizing radiation. RNA samples were prepared 4 h after treatments. YWHAZ (14-3-3 zeta), MAP3K3 (Mitogen activated protein kinase kinasekinase 3), MAP3K7 (Mitogen activated protein kinase kinasekinase 7), MAP3K14 (Mitogen activated protein kinase kinase 14), MAPK10 (Mitogen activated protein kinase 10) and NFκB (Nuclear Factor Kappa B/Rel A subunit) were quantitated by Real Time PCRusing RNA samples from U87 glioma cell line. Values represent Mean±S.D.(*indicates statistical significance by T-test, *p<0.05).</p