J. Cancer Mol. 4: 109-115, 2008

[Research Paper]

Eurycomanone Induces Apoptosis through the Up-Regulation of p53 in Human Cervical Carcinoma Cells

Nurkhasanah Mahfudh and Azimahtol Hawariah Lope Pihie

School of Biosciences and Biotechnology, National University of Malaysia, 43600 Bangi, Selangor, Malaysia [N. Mahfudh, A. H. Lope Pihie]; Faculty of Pharmacy Ahmad Dahlan University, Jl. Prof Soepomo, Janturan, Yogyakarta, Indonesia [N. Mahfudh]

Abstract:

    AIM: Eurycomanone is a cytotoxic compound found in Eurycoma longifolia Jack.  Previous studies had noted the cytotoxic effect against various cancer cell lines.  The aim of this study is to investigate the cytotoxicity against human cervical carcinoma cells and the mode of action.

METHODS: The cytotoxicity of eurycomanone was evaluated using methylene blue staining assay and the mode of cell death was detected by Hoechst 33258 nuclear staining, TUNEL assay and flow cytometry with Annexin-V/propidium iodide double staining.  The protein expression of p53, E6, E6-AP, Bax and Bcl-2 were studied by Western blotting.  Immunostaining assay was used to confirm the up-regulation of p53 and Bax in cancer cells.

RESULTS: The findings suggested that eurycomanone was cytotoxic on cancerous cells (CaOv-3, HeLa, HepG2, HM3KO, MCF-7) and less toxic on normal cells (MDBK, Vero).  Furthermore, various methods proved that apoptosis was the mode of death in eurycomanone-treated cervical cancer HeLa cells.  The characteristics of apoptosis including chromatin condensation, DNA fragmentation and apoptotic bodies were found following eurycomanone treatment.  This study also found that apoptotic process triggered by eurycomanone involved the up-regulation of p53 tumor suppressor protein.  The up-regulation of p53 was followed by the increasing of pro-apoptotic Bax and decreasing of anti-apoptotic Bcl-2.  However, eurycomanone did not affect the E6 and E6-AP protein expression.

CONCLUSION: The data suggest that eurycomanone was cytotoxic on HeLa cells by inducing apoptosis through the up-regulation of p53 and Bax, and down-regulation of Bcl-2, independently of functional E6 and E6-AP activity.

Received 5/26/08; Revised 8/5/08; Accepted 8/9/08.

¹Correspondence: Dr. Nurkhasanah Mahfudh, School of Biosciences and Biotechnology, National University of Malaysia, 43600 Bangi, Selangor, Malaysia. E-mail: nurkhas@gmail.com

²Abbreviations: HPV, human papillomavirus; TUNEL, TdT-mediated dUTP nick end labeling; FITC, fluorescein isothiocyanate; PI, propidium iodide; PS, phosphatidylserine.

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