nu-7026 and Brain-Neoplasms

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a critical role in non-homologous end-joining repair of DNA double-strand breaks (DSB) induced by ionizing radiation (IR). Little is known, however, regarding the relationship between DNA-PKcs and IR-induced angiogenesis; thus, in this study we aimed to further elucidate this relationship. Our findings revealed that lack of DNA-PKcs expression or activity sensitized glioma cells to radiation due to the defective DNA DSB repairs and inhibition of phosphorylated Akt(Ser473) . Moreover, DNA-PKcs deficiency apparently mitigated IR-induced migration, invasion and tube formation of human microvascular endothelial cell (HMEC-1) in conditioned media derived from irradiated DNA-PKcs mutant M059J glioma cells or M059K glioma cells that have inhibited DNA-PKcs kinase activity due to the specific inhibitor NU7026 or siRNA knockdown. Moreover, IR-elevated vascular endothelial growth factor (VEGF) secretion was abrogated by DNA-PKcs suppression. Supplemental VEGF antibody to irradiated-conditioned media was negated enhanced cell motility with a concomitant decrease in phosphorylation of the FAK(Try925) and Src(Try416) . Furthermore, DNA-PKcs suppression was markedly abrogated in IR-induced transcription factor hypoxia inducible factor-1α (HIF-1α) accumulation, which is related to activation of VEGF transcription. These findings, taken together, demonstrate that depletion of DNA-PKcs in glioblastoma cells at least partly suppressed IR-inflicted migration, invasion, and tube formation of HMEC-1 cells, which may be associated with the reduced HIF-1α level and VEGF secretion. Inhibition of DNA-PKcs may be a promising therapeutic approach to enhance radio-therapeutic efficacy for glioblastoma by hindering its angiogenesis.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Movement; Chromones; Culture Media, Conditioned; DNA Breaks, Double-Stranded; DNA Repair; DNA-Activated Protein Kinase; Endothelial Cells; Glioblastoma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Morpholines; Neoplasm Invasiveness; Neovascularization, Pathologic; Neovascularization, Physiologic; Radiation Tolerance; Radiation, Ionizing; RNA, Small Interfering; Signal Transduction; Vascular Endothelial Growth Factor A

First line chemotherapeutics for brain tumors (malignant gliomas) are alkylating agents such as temozolomide and nimustine. Despite growing knowledge of how these agents work, patients suffering from this malignancy still face a dismal prognosis. Alkylating agents target DNA, forming the killing lesion O(6)-alkylguanine, which is converted into DNA double-strand breaks (DSBs) that trigger apoptosis. Here we assessed whether inhibiting repair of DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ) is a reasonable strategy for sensitizing glioma cells to alkylating agents. For down-regulation of HR in glioma cells, we used an interference RNA (iRNA) approach targeting Rad51 and BRCA2, and for NHEJ we employed the DNA-PK inhibitor NU7026. We also assessed whether inhibition of poly(ADP)ribosyltransferase (PARP) by olaparib would enhance the killing effect. The data show that knockdown of Rad51 or BRCA2 greatly sensitizes cells to DSBs and the induction of cell death following temozolomide and nimustine (ACNU). It did not sensitize to ionizing radiation (IR). The expression of O(6)-methylguanine-DNA methyltransferase (MGMT) abolished all these effects, indicating that O(6)-alkylguanine induced by these drugs is the primary lesion responsible for the formation of DSBs and increased sensitivity of glioma cells following knockdown of Rad51 and BRCA2. Inhibition of DNA-PK only slightly sensitized to temozolomide whereas a significant effect was observed with IR. A triple strategy including siRNA and the PARP inhibitor olaparib further improved the killing effect of temozolomide. The data provides evidence that down-regulation of Rad51 or BRCA2 is a reasonable strategy for sensitizing glioma cells to killing by O(6)-alkylating anti-cancer drugs. The data also provide proof of principle that a triple strategy involving down-regulation of HR, PARP inhibition and MGMT depletion may greatly enhance the therapeutic effect of temozolomide.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Base Sequence; Brain Neoplasms; BRCA2 Protein; Cell Line, Tumor; Chromones; DNA Damage; Flow Cytometry; Glioma; Homologous Recombination; Humans; Microscopy, Fluorescence; Morpholines; Protein Kinase Inhibitors; Rad51 Recombinase; RNA, Small Interfering