N EW S L E T T E RFounder’s Day Special Issue October 2017

Development of customized strategies for improving the efficacy of cancer radiotherapy

Sundarraj Jayakumar, Deepak Sharma and S. Santosh Kumar Radiation Biology and Health Sciences Division

Sundarraj Jayakumar is the recipient of the DAE Young Scientist

Award for the year 2015

Abstract

We have developed DNA damage and DNA damage based gene expression as predictive markers of radiosensitivity in cancer cells which would find application in customized radiotherapy. We have identified Nrf2 as a target for modulating radiosensitivity in cancer cells and normal cells. We have also developed Nrf2 centric drugs for mitigation of radiation injury.

Introduction Cancer is one of the major causes of death worldwide. Radiation therapy is an important treatment modality employed for cancer. But due to the inter-individual differences in radiosensitivity of cancer cells, the efficacy of the radiotherapy is affected. Further, normal tissue toxicity is also a major concern to the clinicians during radiotherapy. The efficacy can be improved by prediction of radiosensitivity of cancer cells and through modulation of radiation responses of normal and cancer cells. Clonogenic assay is considered as gold standard for predicting radiosensitivity.However, this assay cannot be used under clinical settings because the tumor samples derived from patients seldom grow in a petri dish to form colonies. Hence there is a need for development of rapid and reliable assay for predicting the radiosensitivity, which will help in designing personalized radiotherapeutic regimen. Apart from prediction, modulation of radiosensitivity is also important for improving the outcome of radiotherapy. For modulation of radiation response, understanding the molecular players governing radiosensitivity of cell is pivotal. Drugs which target the proteins that are contributing to radioresistance of cancer cells can be effective sensitizers and the drugs which activate survival proteins specifically in normal cells can be potential radioprotectors. When cells are exposed to gamma radiation, free radicals are formed as a result of water radiolysis and these free radicals in turn cause

damage to cellular macromolecules like protein and DNA. Damage to DNA (double strand breaks) is the most critical lesion, which if unrepaired can lead to cell death. Cells activate many signaling mechanisms and repair pathways which help in salvaging the DNA damage. These factors can be used as targets for predicting and modulating the radiosensitivity. One ubiquitous transcription factor which gets activated during the radiation exposure is Nrf2. Under normal conditions, this transcription factor is sequestered in the cytoplasm and during oxidative stress,Nrf2 is translocated to the nucleus and thereby transactivates the target genes which are involved in anti-oxidant response leading to the survival of the cell. Targeting Nrf2 can be a good strategy for radiosensitization of tumor cells and radioprotection of normal cells. Drugs whichtarget this molecule can be good radio-modulators. Materials and Methods Tumor cells were obtained from National Centre for Cell Science, Pune. Chemicals and reagents were procured from Sigma-Aldrich. Clonogenic assay Clonogenic assay determines the ability of a cell to divide multiple times and form a macroscopic colony. Tumor cells were cultured in the petri dish for 12 h and then they were exposed to radiation. They were further cultured for two weeks for the colony development. The colonies were fixed using

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N EW S L E T T E RFounder’s Day Special Issue October 2017

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N EW S L E T T E RFounder’s Day Special Issue October 2017

the presence of antioxidant response elements (ARE) in their promoter region. The expression of RAD51 family of genes was decreased in Nrf2 knockdown cells, suggesting that the Nrf2 is involved in DNA repair through the homologous recombination repair. Hence our results clearly indicated that apart from involvement in anti-oxidant response, Nrf2 is also modulates DNA repair and thereby contributes to radiation resistance in cancer cells (Fig.3). Development of new drugs for modulation of cellular radio-sensitivity Many phytochemicals and their derived compounds were screened to modulate the radiosensitivity of cancer cells and normal cells. We found that dimethoxy curcumin (DIMC), a synthetic analogue of curcumin, modulated radiosensitivity of cancer cells. The bio-availability and stability of DIMC is more as compared to curcumin.Our studies revealed that DIMC at 2.5 µM concentration increased the radiosensitivity of the cancer cells and cancer stem cells. This effect was due to inhibition of thioredoxin reductase- an Nrf2 dependent gene. We have also identified another drug which is a derivative of green plant pigment chlorophyll (BARC Radio Modifier-BRM). Preclinical studies using BRM demonstrated its radioprotective

efficacy against lethal doses of radiation in mice. Safety and toxicological studies in multiple species of rodents have been completed under GLP conditions. BRM is well tolerated up to 5g/Kg body weight. This technology has been incubated with Innovative Drug Research Solutions Ltd., Bangalore. The formulation has been optimized and 2 lakh tablets have been manufactured under GMP conditions for human clinical trials. Conclusion Our efforts have identified a simple, fast and efficient protocol for predicting the radio-sensitivity of patient derived cancer cells. This protocol will be useful for customized radiotherapy. Further, we have identified Nrf-2 and thioredoxin reductase as druggable targets for modulation of radiosensitivity. Based on these studies, we have developed new radio-modifying drugs for use in clinic. Acknowledgements We acknowledge the contributions of all our collaborators and co-authors in the publications. We also thank the Head RB&HSD and Director BSG for the constant encouragement and support.

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