Effect of Glut-1 and HIF-1α double knockout by CRISPR/CAS9 on radiosensitivity in laryngeal carcinoma via the PI3K/Akt/mTOR pathway

Hypoxic resistance is the main obstacle to radiotherapy for laryngeal carcinoma. Our previous study indicated that hypoxia-inducible factor 1 alpha (HIF-1 alpha) and glucose transporter 1 (Glut-1) double knockout reduced tumour biological behaviour in laryngeal carcinoma cells. However, their radioresistance mechanism remains unclear. In this study, cell viability was determined by CCK8 assay. Glucose uptake capability was evaluated by measurement of F-18-fluorodeoxyglucose radioactivity. A tumour xenograft model was established by subcutaneous injection of Tu212 cells. Tumour histopathology was determined by haematoxylin and eosin staining, immunohistochemical staining, and TUNEL assays. Signalling transduction was evaluated by Western blotting. We found that hypoxia induced radioresistance in Tu212 cells accompanied by increased glucose uptake capability and activation of the PI3K/Akt/mTOR pathway. Inhibition of PI3K/Akt/mTOR activity abolished hypoxia-induced radioresistance and glucose absorption. Mechanistic analysis revealed that hypoxia promoted higher expressions of HIF-1 alpha and Glut-1. Moreover, the PI3K/Akt/mTOR pathway was a positive mediator of HIF-1a and/or Glut-1 in the presence of irradiation. HIF-1 alpha and/or Glut-1 knockout significantly reduced cell viability, glucose uptake and PI3K/Akt/mTOR activity, all of which were induced by hypoxia in the presence of irradiation. In vivo analysis showed that knockout of HIF-1 alpha and/or Glut-1 also inhibited tumour growth by promoting cell apoptosis, more robustly compared with the P13K inhibitor wortmannin, particularly in tumours with knockout of both HIF-1 alpha and Glut-1. HIF-1 alpha and/or Glut-1 knockout also abrogated PI3K/Akt/mTOR signalling transduction in tumour tissues, in a manner similar to wortmannin. HIF-1 alpha and/or Glut-1 knockout facilitated radiosensitivity in laryngeal carcinoma Tu212 cells by regulation of the PI3K/Akt/mTOR pathway.