Phenethyl caffeate benzo[kl]xanthene lignan with DNA interacting properties induces DNA damage and apoptosis in colon cancer cells Vinod Vijayakurup a , Spatafora Carmela b , Daquino Carmelo b , Tringali Corrado b, ⁎, Priya Srinivas c , Srinivas Gopala a, ⁎⁎ a Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, 695011, India b Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria 6, I-95125 Catania, Italy c Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695014, India abstract article info Article history: Received 8 June 2012 Accepted 12 October 2012 Keywords: Xanthene Lignans CAPE DNA damage Apoptosis Aims: Phenethyl caffeate benzoxanthene lignan (PCBL) is a synthetic compound with DNA interacting, antiangiogenic, antiproliferative and tumor cell death inducing abilities. Though PCBL exhibits the qualities of a prospective antitumor agent, the basic mechanism of PCBL induced cell death remains unknown. This study aims to analyze the molecular mechanisms of PCBL induced cell death in tumor cells to further substan- tiate its antitumor abilities. Main methods: MTT assay was used for finding cell proliferation inhibition, flow cytometric analysis for the detection of cell cycle arrest, comet assay for DNA break detection and immunofluorescence for analyzing H2AX phosphorylation. Western blot analysis was used to detect the activation of different proteins related to DNA damage response and apoptosis. Key findings: PCBL inhibited proliferation of WiDr cells more efficiently than its analog, MCBL. Comet analysis of PCBL treated WiDr cells and activity of various DNA damage response proteins such as γ-H2AX, BRCA1, ATR and Chk1 in PCBL treated cells demonstrated the DNA damaging property of PCBL. Effector molecules of apoptosis such as caspase-3, caspase-7 and caspase-9 were found activated along with PARP cleavage in PCBL treated cells, suggesting apoptosis as the main mode of cell death. PCBL induced cell death was found associated with the activation of MAPK signaling. Inhibition of ERK, one of the MAPKs, by U0126 improved the apoptosis inducing ability of PCBL. Significance: In vitro findings suggest that PCBL works by initiating DNA damage and inducing apoptosis in cancer cells and thus could be considered for further preclinical studies. © 2012 Elsevier Inc. All rights reserved. Introduction Lignans and related compounds (neolignans, oxyneolignans and mixed lignans) are a well-known class of plant secondary metabolites with wide structural variety. Many compounds among them have prom- ising biological properties, such as cytotoxic, antimitotic, antileishmanial, antiangiogenic, cardiovascular and antiviral activity (Apers et al., 2002, 2005; Bose et al., 2009; Ghisalberti, 1997; Prasad et al., 2005; Van Miert et al., 2005). These compounds are normally dimers constituted by two phenylpropanoid (C 6 C 3 ) units, which are joined through a radical pheno- lic oxidative coupling mechanism during the biosynthetic process. Within the family of lignans, benzo[kl]xanthene lignans are a group of compounds rarely encountered in nature, characterized by a benzo[kl]xanthene core: a few examples reported in the literature include rufescidride, mongolicumin A and yunnaneic acid H isolated respectively from Cordia rufescens, Taraxacum mongolicum and Salvia yunnanensis (Shi et al., 2008; Silva et al., 2004; Tanaka et al., 1997). Owing to the rarity of benzo[xl]xanthene lignans in nature and its limited availability due to ineffective synthetic methods, biological properties of these family of lignans have not been explored until recent times. Some of us have recently synthesized few intensively fluorescent benzo[kl]xanthene lignans by Mn-mediated oxidative coupling of caffeic acid phenethyl ester (CAPE) and methyl ester (Daquino et al., 2009). These novel synthetic compounds were analyzed by NMR methods and molecular docking approach, for their DNA interacting properties. Among them, bis (2-phenylethyl)-6,9,10-trihydroxybenzo [kl]xanthene-1,2-dicarboxylate (or, more shortly, phenethyl caffeate benzoxanthene lignan, PCBL), was the most promising compound with respect to the DNA interacting ability and tumor cell death inducing Life Sciences 91 (2012) 1336–1344 ⁎ Corresponding author. Tel.: +39 095 7385025; fax: +39 095 580138. ⁎⁎ Correspondence to: S. Gopala, Department of Biochemistry, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695011, Kerala, India. Tel.: +91 471 2524689; fax: +91 471 2446433. E-mail addresses: ctringali@unict.it (T. Corrado), srinivasg@sctimst.ac.in (S. Gopala). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.10.013 Contents lists available at SciVerse ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie