Radiation-induced H3K9 tri-methylation in E-cadherin promoter during lung EMT: in vitro and in vivo approaches using vanillin

Abstract

Radiotherapy is an important treatment regime for lung cancer, worldwide. However, radiation-induced pneumonitis and fibrosis are the treatment-limiting toxicities among patients who have undergone radiotherapy. The epithelial cells via epithelial to mesenchymal transition [EMT] acquires mesenchymal phenotype, which ultimately leads to fibrosis. Many investigations are focussed on understanding the signalling pathways mediating in EMT, however, the role of histone methylation is less understood in radiation-induced lung EMT. In the present study, we analysed the effect of vanillin, an antioxidant, on histone methylation during radiation-induced EMT. The thoracic region of Wistar rats was irradiated with a fractionated dose of X-ray (3 Gy/day) for two weeks (total of 30 Gy). The irradiated animals were sacrificed at the 8th and 16th weeks and tissues were used for analyses. Our data showed that radiation decreased the level of antioxidant enzymes such as SOD, catalase and reduced glutathione that would ultimately enhance oxidative stress in the tissues. Histopathological analysis revealed that radiation increased the infiltration of inflammatory cells to the tissue injury site. Total global histone methylation was increased upon irradiation, which was effectively prevented by vanillin administration. Vanillin enhanced E-cadherin expression and decreased the mesenchymal markers N-cadherin and vimentin in the irradiated lung tissue. The ChIP-qPCR analysis suggested that snail expression in the nucleus might involve in the enrichment of suppressive marker H3K9me3 on the E-cadherin promoter. Finally, we suggested that vanillin administration decreased radiation-induced oxidative stress and EMT expression. Additionally, irradiation increased the H3K9 methylation status with nuclear translocation of snail during lung EMT.

Keywords:

• Histone methylation
• EMT
• vanillin
• radiation

Acknowledgment

The authors acknowledge Department of Biochemical Engineering FIST vide grant SR/FST/ETI-331/2013 for the use of upright fluorescence microscope (Nikon Eclipse NIU). We acknowledge Vishnu Cancer Hospital, Thanjavur, Tamil Nadu, India for providing an X-ray facility (LINAC). We also acknowledge Central animal facility, SASTRA Deemed University for their kind support in carrying out animal experiments.

Disclosure statement

The author declares that there is no conflict of interest.

Author contributions

Sunil Gowda SN, carried out most of the experiments, Raghavi R, Ilakya S, Dhayalan D, and Nirupama helped in the experimentation. Dr. David R helped in animal experiments and Dr Prabhu helped in pathological scoring and analysis. Devipriya N, helped in planning the complete concept, reviewing and correction of the manuscript.

Additional information

Funding

The present study was funded by the Science and Engineering Research Board [YSS/2014/000518] and Department of Science and Technology, New Delhi [SL/P-12/2016] Government of India.