Activation of p38/JNK Pathway Is Responsible for Embelin Induced Apoptosis in Lung Cancer Cells: Transitional Role of Reactive Oxygen Species Deepa R. Avisetti 1,2 , K. Suresh Babu 3 , Shasi V. Kalivendi 1,2 * 1 Centre for Academy of Scientific & Innovative Research, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, Andhra Pradesh, India, 2 Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, Andhra Pradesh, India, 3 Natural Products Chemistry, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, Andhra Pradesh, India Abstract The natural product embelin has been demonstrated to possess a wide range of therapeutic properties, however, the mechanisms by which it exerts anticancer effects are not yet clear. By monitoring the molecular changes associated during early apoptotic phase, we have identified the crucial role of oxidative stress induced MAP kinase signalling as a predominant mechanism for its anticancer effects. Treatment of A549 lung cancer cells with embelin resulted in the enhancement of phospho-p38 and phospho-JNK levels as early as 4h. Pretreatment of cells with specific inhibitors of p38 (PD169316) and JNK (SP600125) abrogated embelin-induced caspase-3 activation. Studies employing embelin in the presence or absence of specific MAP kinase inhibitors indicated that the observed changes in phosphorylation levels of p38, JNK and ERK 1/2 are solely due to embelin and not because of cross-talk between MAP kinases. Reactive oxygen species (ROS) play a crucial role in embelin induced alterations in MAP kinase phosphorylation and apoptosis as pretreatment of cells with FeTMPyP mitigated this effect. The observed changes are not due to the inhibitory effect of embelin on XIAP as cells treated with SMAC-N7-Ant peptide, a specific inhibitor of XIAP’s BIR3 domain did not mimic embelin induced apoptotic effects. The findings of the present study clearly indicate the crucial role of p38 and JNK pathways in embelin induced apoptosis and provide us with new clues for improving its therapeutic efficacy. Citation: Avisetti DR, Babu KS, Kalivendi SV (2014) Activation of p38/JNK Pathway Is Responsible for Embelin Induced Apoptosis in Lung Cancer Cells: Transitional Role of Reactive Oxygen Species. PLoS ONE 9(1): e87050. doi:10.1371/journal.pone.0087050 Editor: Shrikant Anant, University of Kansas School of Medicine, United States of America Received September 24, 2013; Accepted December 17, 2013; Published January 22, 2014 Copyright: ß 2014 Avisetti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by SMiLE project from CSIR, India. Senior Research Fellowship to DRA from UGC, India, is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing Interests: The authors have declared that no competing interests exist. * E-mail: kalivendi@iict.res.in Introduction Embelin, an active component of fruits of Embelia ribes, has been demonstrated to possess a broad-spectrum of therapeutic proper- ties such as anticancer, anti-inflammation, anti-diabetes, anti- obesity, analgesic, anti-fertility and anti-helminthic [1–5]. The initial discovery of embelin as an inhibitor of XIAP by virtue of its interaction with the BIR3 domain and its observed selectivity towards cancer cells as compared to the normal cells inspired us to consider it as a lead compound for further studies against cancer [6]. As many of the cancers express elevated levels of XIAP and become refractory to apoptosis, treatment with embelin or in combination with other known anticancer drugs was found to sensitize them towards apoptosis [6,7]. Mechanism based studies indicate that embelin inactivates NF-kB by inhibiting nuclear transportation of p65 and also shown to inhibit STAT3 phosphorylation by inducing the expression of PTEN [8,9]. Specific efforts to identify the precise molecular target of embelin resulted in the identification of embelin as an inhibitor against XIAP’s BIR3 domain [6]. In addition, embelin was also demonstrated to be an inhibitor of 5-lipoxigenase and microsomal prostaglandin E2 synthase-1 (mPGES)-1; plasminogen activator inhibitor-1 (PAI-1) and P300/CBP associated factor (PCAF) [10– 12]. Moreover, embelin has been shown to interfere with the oxidative phosphorylation of mitochondria and can undergo both redox and non-redox mediated mechanisms [13,14]. Though the affinity of embelin against some of the molecular targets and cell signalling mechanisms have been identified, the primary intracellular target responsible for its anti-cancer property is not yet clear as many of the earlier studies have been carried out at later time points where the signal transduction cascade becomes complex due to the cross-talk between multiple cell signalling mechanisms [8,15,16,17]. Hence, in the present study, we sought to identify the alterations in signalling pathways responsible for the anticancer property of embelin during the early apoptotic phase. The present study identified for the first time the pivotal role of MAP kinase pathway, especially p38 and JNK, in embelin induced apoptosis. Materials and Methods Materials Embelin was purified from the fruits of Embelia ribes as described previously [18,19]. Minimal essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buff- ered saline (DPBS), penicillin, streptomycin, sulphorhodamine B (SRB), Ac-DEVD-7-AFC, Ac-LEHD-7-AFC, PD169316, SP600125, N-acetyl-L-cysteine (NAC), radioimmune precipitation PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e87050