2-Deoxy-D-glucose and 6-aminonicotinamide-mediated Nrf2 down regulation leads to radiosensitization of malignant cells via abrogation of GSH-mediated defense

Abstract

Enhanced level of nuclear erythroid-related factor-2 (Nrf2) has been associated with cancer chemo/radioresistance. Therefore, the role of Nrf2 in radiosensitization of malignant cells induced by a combination of 2-deoxy-D-Glucose (2-DG) and 6-aminonicotinamide (6-AN) was investigated. Two established human malignant cells lines namely KB (head and neck squamous carcinoma) and BMG-1 (cerebral glioma) were used. Following treatment with a combination of 2-DG (5 mM) and 6-AN (5 μM), irradiated (2Gy) KB and BMG-1 cells were assessed for protein level of Nrf2, Keap1 and γ-glutamylcysteine synthetase (γ-GCS) by western blotting and mRNA expression of γ-GCS, glutathione reductase (GR) and glutathione peroxidase (GPx1) by RT-PCR at 24 hours post treatment. A significant decrease in the level of Nrf2 with a concomitant increase in Keap1 was observed in both the irradiated malignant cells at 24 hours following treatment with combination (2-DG + 6-AN). Down regulation of γ-GCS, GR and GPx1 at 24 hours following treatment with combination (2-DG + 6-AN) resulted in abrogation of glutathione (GSH)-mediated defense in both the irradiated malignant cells. Eventual accumulation of ROS led to radiosensitization of both the malignant cells. These results indicate that deregulated Nrf2-Keap1 signalling leads to the radiosensitization of malignant cells due to abrogated glutathione defense. Metabolic modification-mediated down regulation of Nfr2 and its downstream signalling may have a potential of improving tumour radiotherapy.

Keywords::

• Nrf2
• Radiosensitization
• 2-deoxy-D-glucose
• 6-aminonicotinamide
• Glutathione

Acknowledgement

We thank Dr. N. Raghuram, Associate Prof. School of Biotechnology, Guru Gobind Singh Indraprastha University, Delhi for his technical comments. We thank Mrs. Namita kalra and Mrs. Richa Bhardwaj for their technical help in flow cytometry experiments. We thank Dr. B S Dwarakanath and Dr. Sudhir Chandana for extending lab facilities. Mr. Pradeep Kumar Sharma received fellowship from ICMR, India. We also thank Dr. R P Tripathi, Director INMAS for his constant support.

Conflicts of interest

We confirm that all authors fulfil all conditions required for authorship. We also confirm that there is no potential conflict of interest as described in the Instructions for Authors. All authors have read and approved the manuscript.