2. Academic Validation
  3. Directly imaging the localisation and photosensitization properties of the pan-mTOR inhibitor, AZD2014, in living cancer cells

Directly imaging the localisation and photosensitization properties of the pan-mTOR inhibitor, AZD2014, in living cancer cells

  • J Photochem Photobiol B. 2020 Dec;213:112055. doi: 10.1016/j.jphotobiol.2020.112055.
Abdullah R Ahmed 1,  Alessia Candeo 2,  Sofia D'Abrantes 3,  Sarah R Needham 2,  Rahul B Yadav 4,  Stanley W Botchway 5,  Anthony W Parker 6
Affiliations

Affiliations

  • 1 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.
  • 2 Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK.
  • 3 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.
  • 4 Evotec (UK) Ltd, 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire OX14 4RZ, UK.
  • 5 Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK. Electronic address: stan.botchway@stfc.ac.uk.
  • 6 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.
PMID: 33142217 DOI: 10.1016/j.jphotobiol.2020.112055
Abstract

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 μM) 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.

Keywords

AZD2014; FLIM;light sheet fluorescence microscopy; Fluorescence microscopy; GFP; INK128, mTOR; Multiphoton; PDT; Photosensitizer.

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