Contents lists available at ScienceDirect Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed Original article Modulation of Caspase-3 activity using a redox active vitamin K3 analogue, plumbagin, as a novel strategy for radioprotection Rahul Checker a,c,1 , Debojyoti Pal a,1 , Raghavendra S. Patwardhan a , Bhakti Basu b,c , Deepak Sharma a,c,∗ , Santosh K. Sandur a,c a Radiation Biology & Health Sciences Division, Bio-science Group, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India b Molecular Biology Division, Bio-science Group, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India c Homi Bhabha National Institute, Anushaktinagar, Mumbai, 400094, India ARTICLE INFO Keywords: Lymphocytes Thiol Radioprotector Apoptosis Protein interaction network Naphthoquinone ABSTRACT Radiation induced damage to normal cells is a major shortcoming of conventional radiotherapy, which ne- cessitates the development of novel radio-protective drugs. An ideal radio-modulator would protect normal cells while having cytotoxic efects on cancer cells. Plumbagin is a potent anti-tumour agent and has been shown to sensitize tumour cells to radiation-induced damage. In the present study, we have evaluated the radio-protective potential of plumbagin and found that it protected normal lymphocytes against radiation-induced apoptosis, but did not protect cancer cells against radiation. Plumbagin ofered radioprotection even when it was added to cells after irradiation. The ability of only thiol based antioxidants to abrogate the radio-protective efects of plum- bagin suggested a pivotal role of thiol groups in the radio-protective activity of plumbagin. Further, protein interaction network (PIN) analysis was used to predict the molecular targets of plumbagin. Based on the inputs from plumbagin's PIN and in light of its well-documented ability to modulate thiol groups, we proposed that plumbagin may act via modulation of caspase enzyme which harbours a critical catalytic cysteine. Indeed, plumbagin suppressed radiation-induced increase in homogenous caspase and caspase-3 activity in lymphocytes. Plumbagin also inhibited the activity of recombinant caspase-3 and mass spectrometric analysis revealed that plumbagin covalently interacts with caspase-3. Further, the invivo radioprotective efcacy of plumbagin (single dose of 2mg/kg body weight) was demonstrated by its ability to rescue mice against radiation (7.5 Gy; Whole Body Irradiation) induced mortality. These results indicate that plumbagin prevents radiation induced apoptosis specifcally in normal cells by inhibition of caspase-3 activity. 1. Introduction Radiotherapy, the principal modality for treatment of cancer, which is given to nearly half of all cancer patients, continues to be a double edged sword [1,2]. While there is no scepticism in the efcacy of radiotherapy in controlling cancer progression, the collateral damage to healthy normal cells remains a cause of immense concern [3]. Exposure of normal tissues to radiation during the course of radiation therapy often results in multiple side efects including acute and chronic tissue toxicities, which can sometimes result in irreversible changes like lung fbrosis, kidney failure and xerostomia [4]. Radiation induced damage to normal tissues during radiotherapy signifcantly reduces its scope for efective usage in cancer therapy and has directed researchers and clinicians to work towards technological improvements in radiation delivery as well as towards development of chemical modifers of ra- diation injury [5,6]. However, even with improved methods for tar- geted delivery of radiation to tumour site, normal tissue toxicity con- tinues to remain a major concern [7]. Hence, identifcation and development of compounds that protect normal tissues or minimize post-irradiation toxicity may help in reducing the unwanted side-efects in patients receiving radiotherapy [8,9]. Research on the development of suitable radiation countermeasures began in 1959 at Walter Reed Army Institute of Research (WRAIR) where 4000 compounds were screened for their radio-protective po- tential and WR-2721, also known as Ethyol or amifostine, emerged as the most potent molecule [10]. Amifostine was approved by the FDA for reduction of the radiation induced xerostomia in head and neck cancer patients [11]. Since amifostine has low to moderate efcacy and https://doi.org/10.1016/j.freeradbiomed.2019.09.001 Received 22 May 2019; Received in revised form 30 August 2019; Accepted 1 September 2019 ∗ Corresponding authors. Radiation Biology & Health Sciences Division, Bio-science Group, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India. E-mail addresses: dsharma@barc.gov.in (D. Sharma), sskumar@barc.gov.in (S.K. Sandur). 1 Both the authors have contributed equally. Free Radical Biology and Medicine 143 (2019) 560–572 Available online 04 September 2019 0891-5849/ © 2019 Published by Elsevier Inc. T