ku-55933 and Brain-Neoplasms

Autophagy is a cytoprotective process, which occurs following temozolomide (TMZ) treatment, and contributes to glioma chemoresistance and TMZ treatment failure. However, the molecular mechanisms by which TMZ induces autophagy are largely unknown. In the current study, the ataxia‑telangiectasia mutated (ATM) inhibitor KU‑55933, adenosine monophosphate‑activated protein kinase (AMPK) inhibitor compound C, and U87MG and U251 cell lines were employed to investigate the molecular mechanisms of TMZ‑induced autophagy in glioma, and to evaluate the effects of autophagy inhibition on TMZ cytotoxicity. KU‑55933 and compound C were observed to inhibit the activation of autophagy‑initiating kinase ULK1 and result in a significant decrease of autophagy as indicated by depressed LC3B cleavage and acidic vesicular organelle formation. The activation of AMPK‑ULK1 was ATM dependent. Autophagy inhibition via the AMPK inhibitor compound C augmented TMZ cytotoxicity as observed by depressed cell viability, increased γH2AX‑marked double‑strand breaks (DSBs) and elevated numbers of apoptotic glioma cells. In conclusion, TMZ induced autophagy via ATM‑AMPK‑ULK1 pathways. TMZ chemoresistance may therefore be overwhelmed by targeting AMPK, particularly for the treatment of O6‑methylguanine DNA methyltransferase‑negative gliomas.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents, Alkylating; Ataxia Telangiectasia Mutated Proteins; Autophagy; Autophagy-Related Protein-1 Homolog; Brain Neoplasms; Cell Line, Tumor; Dacarbazine; Glioma; Humans; Intracellular Signaling Peptides and Proteins; Microtubule-Associated Proteins; Morpholines; Protein Serine-Threonine Kinases; Pyrones; Signal Transduction; Temozolomide

Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sensitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p < 0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p < 0.001), respectively] and also elevated the fraction of cells arrested in G2/M [U251 G2/M fraction: 61.8 ± 1.1 % vs. 35 ± 0.8 % (p < 0.001), respectively, and U87 G2/M fraction 25 ± 0.2 % vs.18.6 ± 0.4 % (p < 0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/M arrest. While KU-55933 did not enhance TMZ induced Chk1/Chk2 activation, it increased TMZ-induced residual γ-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These findings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Brain Neoplasms; Cell Cycle Proteins; Cell Division; Cell Proliferation; Dacarbazine; DNA Damage; DNA-Binding Proteins; Drug Resistance, Neoplasm; Flow Cytometry; G2 Phase; Glioblastoma; Humans; Immunoenzyme Techniques; Mice; Morpholines; Neurons; Protein Serine-Threonine Kinases; Pyrones; Temozolomide; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tumor Suppressor Proteins