Ethyl acetate extracts of Nepenthes ventricosa x sibuyanensis leaves cause growth inhibition against oral cancer cells via oxidative stress

Abstract

Jen-Yang Tang,1,2 Tzu-Jung Yu,3 Li-Ching Lin,4–6 Sheng-Yao Peng,3 Chun-Lin Wang,7 Fu Ou-Yang,8,9 Yuan-Bin Cheng,10 Hsueh-Wei Chang9,11–131Department of Radiation Oncology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; 2Department of Radiation Oncology, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan; 3Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; 4Department of Radiation Oncology, Chi-Mei Foundation Medical Center, Tainan 71004, Taiwan; 5School of Medicine, Taipei Medical University, Taipei 11031, Taiwan; 6Department of Optometry, Chung Hwa University of Medical Technology, Tainan 71703, Taiwan; 7Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu 30062, Taiwan; 8Division of Breast Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan; 9Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan; 10Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; 11Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan; 12Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; 13Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung 80708, TaiwanIntroduction: The genus Nepenthes of the pitcher plants contains several natural and hybrid species that are commonly used in herbal medicine in several countries, but its possible use in cancer applications remains unknown as yet.Methods: In this study, we investigated the antioral cancer properties using ethyl acetate extracts of the Nepenthes hybrid (Nepenthes ventricosa x sibuyanensis), namely EANS. The bioactivity was detected by a MTS-based cell proliferation assay and flow cytometric or Western blot analysis for apoptosis, oxidative stress, and DNA damage.Results: Treatment for 24 hrs of EANS inhibited all three types of oral cancer cells that were tested (Ca9-22, CAL 27, and SCC9), with just a small difference to normal oral cells (HGF-1). This antiproliferation was inhibited by pretreatments with the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC), and the apoptosis inhibitor (Z-VAD). EANS treatment increased the subG1 population and it also dose- and time-dependently induced annexin V- and pancaspase-detected apoptosis as well as cleaved caspases 3 and 9 overexpressions in the oral cancer cells (Ca9-22). After EANS treatment of Ca9-22 cells, intracellular ROS and mitochondrial superoxide (MitoSOX) were overexpressed and mitochondrial membrane potential (MMP) was disrupted. Moreover, DNA damages such as γH2AX and 8-oxo-2ʹ-deoxyguanosine (8-oxodG) were increased after EANS treatment to Ca9-22 cells. The EANS-induced effects (namely, oxidative stress, apoptosis, and DNA damage) were suppressed by ROS scavenger.Conclusion: Our findings demonstrated that EANS inhibits ROS-mediated proliferation against oral cancer cells.Keywords: Nepenthes, oral cancer, oxidative stress, apoptosis, DNA damage

Keywords

• Nepenthes
• oral cancer
• oxidative stress
• apoptosis
• DNA damage.