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Cell Biology International (2011) 35, 607–615 (Printed in Great Britain)

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Apoptotic effect of eugenol in human colon cancer cell lines

Saravana Kumar Jaganathan*, Abhijit Mazumdar†, Dilip Mondhe‡ and Mahitosh Mandal§¹

*PSNA College of Engineering and Technology, Kothhandaraman Nagar, Dindugal624 622, Tamil Nadu, India, †Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, U.S.A., ‡Indian Institute of Integrative Medicine, Jammu180 001, Jammu and Kashmir, India, and §School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721 302, West Bengal, India

Eugenol, a natural compound available in honey and various plants extracts including cloves and Magnoliae flos, is exploited for various medicinal applications. Since most of the drugs used in the cancer are apoptotic inducers, the apoptotic effect and anticancer mechanism of eugenol were investigated against colon cancer cells. Antiproliferative effect was estimated using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay]. Earlier events like MMP (mitochondrial membrane potential), thiol depletion and lipid layer break were measured by using flow cytometry. Apoptosis was evaluated using PI (propidium iodide) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay and DNA fragmentation assay. MTT assay signified the antiproliferative nature of eugenol against the tested colon cancer cells. PI staining indicated increasing accumulation of cells at sub-G₁-phase. Eugenol treatment resulted in reduction of intracellular non-protein thiols and increase in the earlier lipid layer break. Further events like dissipation of MMP and generation of ROS (reactive oxygen species) were accompanied in the eugenol-induced apoptosis. Augmented ROS generation resulted in the DNA fragmentation of treated cells as shown by DNA fragmentation and TUNEL assay. Further activation of PARP (polyadenosine diphosphate-ribose polymerase), p53 and caspase-3 were observed in Western blot analyses. Our results demonstrated molecular mechanism of eugenol-induced apoptosis in human colon cancer cells. This research will further enhance eugenol as a potential chemopreventive agent against colon cancer.

Key words: apoptosis, cell cycle, colon cancer, eugenol, honey, ROS

Abbreviations: DCFH-DA, 2′,7′-dichlorofluorescin diacetate, DMEM, Dulbecco's modified Eagle's medium, IC₅₀, percentage at which 50% cells were dead approximately, MMP, mitochondrial membrane potential, MO, Mercury Orange, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, NAC, N-acetyl-l-cysteine, PARP, polyadenosine diphosphate-ribose polymerase, PI, propidium iodide, ROS, reactive oxygen species, RPMI, Roswell Park Memorial Institute, TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling

¹To whom correspondence should be addressed (email mahitosh@smst.iitkgp.ernet.in).

Received 21 February 2010/18 August 2010; accepted 2 November 2010

Published as Cell Biology International Immediate Publication 2 November 2010, doi:10.1042/CBI20100118

© The Author(s) Journal compilation © 2011 Portland Press Limited