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We recently reported that Phenethyl caffeate benzoxanthene lignan (PCBL), a semisynthetic compound derived from Caffeic Acid Phenethyl Ester (CAPE), induces DNA damage and apoptosis in tumor cells. In this study, we further investigated whether PCBL induces autophagy in WiDr cells. We also analyzed the pathways regulating autophagy and the role of autophagy in PCBL-induced cell death. Our acridine orange staining and LC3 II expression results suggest that PCBL induces autophagosomes in WiDr cells. The levels of LC3 II expression we observed after co-treatment of PCBL with bafilomycin A1 and the reductions in p62 expression we observed after PCBL treatment in WiDr cells demonstrate increased autophagic flux, a reliable indicator of autophagic induction. The increased Beclin 1 expression in PCBL-treated cells and the incapacity of PCBL to induce LC3 II in 3-methyladenine (3-MA)-treated cells we observed suggests that PCBL-induced autophagy is class III PI3-kinase dependent. PCBL did not alter phosphorylation of the mTOR substrate p70 S6 kinase, indicating that PCBL-induced autophagy was not mTOR regulated. Two autophagy related proteins, Atg5 and Atg12, also remained uninduced during PCBL treatment. The increased caspase activity and expression levels of LC3 II and p62 we observed in response to PCBL treatment in primary glioma cells demonstrates that PCBL-induced apoptosis and autophagy were not cell line specific. Pharmacological inhibition of autophagy did not alter the antitumor efficacy of PCBL in WiDr cells. This attests to the bystander nature of PCBL-induced autophagy (in terms of cell death). In toto, these data suggest that PCBL induces a class III kinase dependent, but mTOR independent, bystander mode of autophagy in WiDr cells.

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 substantiate 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., (Copyright © 2012 Elsevier Inc. All rights reserved.)