PMID- 33142217 OWN - NLM STAT- MEDLINE DCOM- 20210602 LR - 20231110 IS - 1873-2682 (Electronic) IS - 1011-1344 (Print) IS - 1011-1344 (Linking) VI - 213 DP - 2020 Dec TI - Directly imaging the localisation and photosensitization properties of the pan-mTOR inhibitor, AZD2014, in living cancer cells. PG - 112055 LID - S1011-1344(20)30505-4 [pii] LID - 10.1016/j.jphotobiol.2020.112055 [doi] LID - 112055 AB - The range of cellular functions the mechanistic target of rapamycin (mTOR) protein performs makes it an attractive drug target for cancer therapy. However, the cellular localisation and mode of action of second generation inhibitors of mTOR is poorly understood despite the level of attention there is in targeting the mTOR protein. We have therefore studied the properties of the pan-mTOR inhibitor AZD2014, an ideal candidate to study because it is naturally fluorescent, characterising its photochemical properties in solution phase (DMSO, PBS and BSA) and within living cells, where it localises within both the nucleus and the cytoplasm but with different excited state lifetimes of 4.8 (+/- 0.5) and 3.9 (+/- 0.4) ns respectively. We measure the uptake of the inhibitor AZD2014 (7 muM) in monolayer HEK293 cells occurring with a half-life of 1 min but observe complex behaviour for 3D spheroids with the core of the spheroid showing a slower uptake and a slow biphasic behaviour at longer times. From a cellular perspective using fluorescence lifetime imaging microscopy AZD2014 was found to interact directly with GFP-tagged mTORC1 proteins including the downstream target, S6K1. We observe light sensitive behaviour of the cells containing AZD2014 which leads to cell death, in both monolayer and spheroids cells, demonstrating the potential of AZD2014 to act as a possible photodynamic drug under both single photon and multiphoton excitation and discuss its use as a photosensitizer. We also briefly characterise another pan-mTOR inhibitor, INK128. CI - Crown Copyright (c) 2020. Published by Elsevier B.V. All rights reserved. FAU - Ahmed, Abdullah R AU - Ahmed AR AD - Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK; Larch House, Woodlands Business Park, Breckland, Linford Wood, Milton Keynes MK14 6FG, UK. FAU - Candeo, Alessia AU - Candeo A AD - Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK. FAU - D'Abrantes, Sofia AU - D'Abrantes S AD - Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK; CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Gray Laboratories, ORCRB Roosevelt Drive, Oxford OX3 7DQ, UK. FAU - Needham, Sarah R AU - Needham SR AD - Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK. FAU - Yadav, Rahul B AU - Yadav RB AD - Evotec (UK) Ltd, 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire OX14 4RZ, UK. FAU - Botchway, Stanley W AU - Botchway SW AD - Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK. Electronic address: stan.botchway@stfc.ac.uk. FAU - Parker, Anthony W AU - Parker AW AD - Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK. Electronic address: a.w.parker@stfc.ac.uk. LA - eng PT - Journal Article DEP - 20201015 PL - Switzerland TA - J Photochem Photobiol B JT - Journal of photochemistry and photobiology. B, Biology JID - 8804966 RN - 0 (Antineoplastic Agents) RN - 0 (Benzamides) RN - 0 (Benzoxazoles) RN - 0 (Fluorescent Dyes) RN - 0 (Morpholines) RN - 0 (Protein Kinase Inhibitors) RN - 0 (Pyrimidines) RN - 0BSC3P4H5X (vistusertib) RN - EC 2.7.1.1 (MTOR protein, human) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) RN - JGH0DF1U03 (sapanisertib) SB - IM MH - Animals MH - Antineoplastic Agents/*chemistry/pharmacology MH - Apoptosis MH - Benzamides/*chemistry/pharmacology MH - Benzoxazoles/chemistry/pharmacology MH - Cell Line, Tumor MH - Cell Membrane Permeability MH - Cell Proliferation MH - Cricetulus MH - Fluorescent Dyes/*chemistry MH - Humans MH - Kinetics MH - Microscopy, Fluorescence, Multiphoton MH - Morpholines/*chemistry/pharmacology MH - Optical Imaging MH - Photochemotherapy MH - Protein Kinase Inhibitors/*chemistry/pharmacology MH - Pyrimidines/*chemistry/pharmacology MH - TOR Serine-Threonine Kinases/*antagonists & inhibitors PMC - PMC7762844 OTO - NOTNLM OT - AZD2014 OT - FLIM;light sheet fluorescence microscopy OT - Fluorescence microscopy OT - GFP OT - INK128, mTOR OT - Multiphoton OT - PDT OT - Photosensitizer COIS- The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. EDAT- 2020/11/04 06:00 MHDA- 2021/06/03 06:00 PMCR- 2020/12/01 CRDT- 2020/11/03 20:13 PHST- 2020/05/27 00:00 [received] PHST- 2020/09/24 00:00 [revised] PHST- 2020/10/12 00:00 [accepted] PHST- 2020/11/04 06:00 [pubmed] PHST- 2021/06/03 06:00 [medline] PHST- 2020/11/03 20:13 [entrez] PHST- 2020/12/01 00:00 [pmc-release] AID - S1011-1344(20)30505-4 [pii] AID - 112055 [pii] AID - 10.1016/j.jphotobiol.2020.112055 [doi] PST - ppublish SO - J Photochem Photobiol B. 2020 Dec;213:112055. doi: 10.1016/j.jphotobiol.2020.112055. Epub 2020 Oct 15.