title: Chloroform Extract of Solanum lyratum Induced G0/G1 Arrest via p21/p16 and Induced Apoptosis via Reactive Oxygen Species, Caspases and Mitochondrial Pathways in Human Oral Cancer Cell Lines.
abstract: Solanum lyratum (SLEC) Thunberg (Solanaceae) has been used as a traditional herbal medicine in China for centuries. Numerous studies have shown that SLEC Thunberg (Solanaceae) extract inhibited cancer cell growth in vitro. Herein, we investigated cell death-induced by EcoAc, water, chloroform, butanol extract of SLEC in human oral cancer cell lines (HSC-3, SAS, and CAL-27) in vitro. Different SLEC extract induced cytotoxic effects in human oral cancer cells were examined by contrast phase microscopy. We selected the chloroform extract of SLEC to examine the cytotoxic effects by using DAPI staining, comet assays, flow cytometric assay, Western blotting and examination of confocal laser microscopy. SLEC decreased the percentage of viable cells, induced G0/G1 arrest and apoptosis. These effects were concentration- and time-dependent manners. SLEC increased protein levels of p21, p16, CDK2, and cyclin D1 in HSC-3, SAS, and CAL-27 cells. Also, SLEC increased CDK6 in HSC-3 and CAL-27 cells, but inhibited CDK6 in SAS cells. Cyclin E in HSC-3 and SAS cells was increased by SLEC, but it was inhibited in CAL-27 cells. SLEC suppressed the anti-apoptotic proteins Bcl-2 and Bcl-xl, but increased the pro-apoptotic proteins Bax and Bad in HSC-3, SAS, and CAL-27 cells. SLEC promoted the production of reactive oxygen species (ROS) and Ca²⁺, decreased the mitochondrial membrane potential (Δψm) and stimulated NO production in HSC-3, SAS, and CAL-27 cells. Specific caspase inhibitors (caspase-8 inhibitor: Z-IETD-FMK; caspase-9 inhibitor: Z-LEHD-FMK and caspase-3 inhibitor: Z-DEVD-FMK) for caspase-8, -9, and -3 blocked SLE-activated caspase-8, -9, and -3 activities which were associated with an increase in the percentage of viable cells. Taken together, SLE induced G0/G1 arrest and apoptosis via extrinsic- and intrinsic-dependent pathways in HSC-3, SAS, and CAL-27 cells.
authors.affiliation: * Departments of Biological Science and Technology, China Medical University, Taichung 404, Taiwan.
authors.forename: Chiz-Hao
authors.initials: CH
authors.lastname: Chiu
authors.affiliation: ** Division of Neurosurgical Oncology, Neurological Institute, Taichung Veterans General Hospital, Taichung 407, Taiwan.|†† Institute of Medical Sciences, Tzu Chi University, Hualien 970, Taiwan.
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Chou
authors.affiliation: † School of Chinese Medicine, China Medical University, Taichung 404, Taiwan.
authors.forename: Jing-Pin
authors.initials: JP
authors.lastname: Lin
authors.affiliation: ‡ Departments of Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan.
authors.forename: Chao-Lin
authors.initials: CL
authors.lastname: Kuo
authors.affiliation: ‡‡ Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei, Taiwan.
authors.forename: Hsu-Feng
authors.initials: HF
authors.lastname: Lu
authors.affiliation: § Departments of Physiology, China Medical University, Taichung 404, Taiwan.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Huang
authors.affiliation: ¶ School of Pharmacy, China Medical University, Taichung 404, Taiwan.
authors.forename: Chien-Chih
authors.initials: CC
authors.lastname: Yu
authors.affiliation: ∥ Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan.
authors.forename: Meng-Liang
authors.initials: ML
authors.lastname: Lin
authors.affiliation: * Departments of Biological Science and Technology, China Medical University, Taichung 404, Taiwan.|§§ Department of Biotechnology, Asia University, Wufeng, Taichung 413, Taiwan.
authors.forename: Jing-Gung
authors.initials: JG
authors.lastname: Chung
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D002725:Chloroform; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism*; D009062:Mouth Neoplasms / Q000378:metabolism / Q000473:pathology*; D009363:Neoplasm Proteins / Q000378:metabolism*; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016192:Resting Phase, Cell Cycle / Q000187:drug effects* / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects*; D032322:Solanum / Q000737:chemistry*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; C493956:CDKN1A protein, human; C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D009363:Neoplasm Proteins; D010936:Plant Extracts; D017382:Reactive Oxygen Species; D002725:Chloroform; D020169:Caspases; D002118:Calcium
keywords: Apoptosis; Caspase Cascades; Chloroform Extract of Solanum lyratum (SLEC); G0/G1 Arrest; Oral Cancer Cells