ku-55933 and Lung-Neoplasms

The role of Ataxia-telangiectasia mutated (ATM) in response to DNA damage has previously been studied, but its underlying mechanisms specific to ionizing radiation (IR) have remained to be elucidated. In this study, function of ATM on radiation-induced cell death in lung cancer H1299 cells was analysed.. Human lung cancer cells, H1299, were used, and cell models with ATM(-/-) and MAPK14(-/-) were established by genetic engineering. Radiosensitivity was analysed using colony formation assays. Western blotting and co-immunoprecipitation were implemented to detect protein expression and interaction. MDC staining and GFP-LC3 relocalization were used to detect autophagy.. Autophagy as well as phosphorylation of ATM was activated by ionizing radiation. Both the inhibitor of ATM, KU55933 and ATM silencing reduced phosphorylation of ATM and MAPKAPK2 expression. Both ATM(-/-) and MAPK14(-/-) cells displayed hypersensitivity. IR increased autophagy level by more than 129% in DMSO-treated cells, while only by 47% and 27% in KU55933-treated and ATM(-/-) cells respectively. MAPK14 knock-down alone gave rise to the basal autophagy level, but decreased notably after IR. KU55933 and ATM knock-down inhibited IR-induced autophagy by activating mTOR pathways. Both Beclin1-PI3KIII and Beclin1-MAPKAPK2 interactions as were remarkably affected by silencing either ATM or MAPK14.. ATM promoted IR-induced autophagy via the MAPK14 pathway, mTOR pathway and Beclin1/PI3KIII complexes. MAPK14 contributed to radiosensitization of H1299 cells.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Ataxia Telangiectasia Mutated Proteins; Autophagy; Beclin-1; Cell Line, Tumor; Cell Survival; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Membrane Proteins; Mitogen-Activated Protein Kinase 14; Morpholines; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Pyrones; Radiation, Ionizing; RNA Interference; RNA, Small Interfering; Signal Transduction; TOR Serine-Threonine Kinases

Radiotherapy is a key component of cancer treatment. Because of its importance, there has been high interest in developing agents and strategies to further improve the therapeutic index of radiotherapy. DNA double-strand repair inhibitors (DSBRIs) are among the most promising agents to improve radiotherapy. However, their clinical translation has been limited by their potential toxicity to normal tissue. Recent advances in nanomedicine offer an opportunity to overcome this limitation. In this study, we aim to demonstrate the proof of principle by developing and evaluating nanoparticle (NP) formulations of KU55933, a DSBRI. We engineered a NP formulation of KU55933 using nanoprecipitation method with different lipid polymer nanoparticle formulation. NP KU55933 using PLGA formulation has the best loading efficacy as well as prolonged drug release profile. We demonstrated that NP KU55933 is a potent radiosensitizer in vitro using clonogenic assay and is more effective as a radiosensitizer than free KU55933 in vivo using mouse xenograft models of non-small cell lung cancer (NSCLC). Western blots and immunofluorescence showed NP KU55933 exhibited more prolonged inhibition of DNA repair pathway. In addition, NP KU55933 leads to lower skin toxicity than KU55933. Our study supports further investigations using NP to deliver DSBRIs to improve cancer radiotherapy treatment.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; DNA Breaks, Double-Stranded; Drug Carriers; Drug Delivery Systems; Humans; Lactic Acid; Lipids; Lung Neoplasms; Male; Mice; Mice, Nude; Mice, SCID; Microscopy, Fluorescence; Morpholines; Nanomedicine; Nanoparticles; Neoplasm Transplantation; Neoplasms; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Pyrones; Radiation-Sensitizing Agents; Radiotherapy

Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells.. Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis.. IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK alpha subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21(waf/cip) as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further.. We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21(waf/cip) and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21(waf/cip) pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

Topics: AMP-Activated Protein Kinases; Ataxia Telangiectasia Mutated Proteins; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Enzyme Activation; Female; G2 Phase; Humans; Lung Neoplasms; Male; Metformin; Morpholines; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Pyrazoles; Pyrimidines; Pyrones; Radiation Tolerance; RNA, Small Interfering; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Premature or stress-induced senescence is a major cellular response to chemotherapy in solid tumors and contributes to successful treatment. However, senescent tumor cells are resistant to apoptosis and may also reenter the cell cycle. We set out to find a means to specifically induce senescent tumor cells to undergo cell death and not to reenter the cell cycle that may have general application in cancer therapy.. We investigated the mechanisms regulating cell survival in drug-induced senescent tumor cells. Using immunofluorescence and flow cytometry-based techniques, we established the status of the ataxia telangiectasia mutated (ATM) signaling pathway in these cells. We assayed the requirement of ATM signaling and p21(CIP1) expression for survival in premature senescent tumor cells using pharmacologic inhibitors and antisense oligonucleotides.. The ATM/ATR (ATM- and Rad3-related) signaling pathway was found to be constitutively active in drug-induced senescent tumor cells. We found that blocking ATM/ATR signaling with pharmacologic inhibitors, including the novel ATM inhibitors KU55933 and CGK733, induced senescent breast, lung, and colon carcinoma cells to undergo cell death. We show that the mechanism of action of this effect is directly via p21(CIP1), which acts downstream of ATM. This is in contrast to the effects of ATM inhibitors on normal, untransformed senescent cells.. Blocking ATM and/or p21(CIP1) following initial treatment with a low dose of senescence-inducing chemotherapy is a potentially less toxic and highly specific treatment for carcinomas.

Topics: Ataxia Telangiectasia Mutated Proteins; Benzeneacetamides; Breast Neoplasms; Carcinoma; Cell Cycle; Cell Cycle Proteins; Cell Survival; Cellular Senescence; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; DNA-Binding Proteins; Drug Evaluation, Preclinical; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Lung Neoplasms; Morpholines; Protein Serine-Threonine Kinases; Pyrones; Thiourea; Tumor Cells, Cultured; Tumor Suppressor Proteins