title: Securinine from Phyllanthus glaucus Induces Cell Cycle Arrest and Apoptosis in Human Cervical Cancer HeLa Cells.
abstract: BACKGROUND
  The Securinega-type alkaloids occur in plants belonging to Euphorbiaceae family. One of the most widely distributed alkaloid of this group is securinine, which was identified next to allosecurinine in Phyllanthus glaucus (leafflower). Recently, some Securinega-type alkaloids have paid attention to its antiproliferative potency towards different cancer cells. However, the cytotoxic properties of allosecurinine have not yet been evaluated.
  
  
  METHODS
  The cytotoxicity of the extract, alkaloid fraction obtained from P. glaucus, isolated securinine and allosecurinine against HeLa cells was evaluated by real-time xCELLigence system and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and 7-amino-actinomycin (7-AAD) staining and confirmed with fluorescent Hoechst 33342 dye. The assessment of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, the level of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), caspase-3/7 activity and cell cycle analysis were measured by flow cytometry. The enzymatic activity of caspase-9 was assessed by a luminometric assay. The expression of apoptosis associated genes was analyzed by real-time PCR.
  
  
  RESULTS
  The experimental data revealed that securinine and the alkaloid fraction were significantly potent on HeLa cells growth inhibition with IC50 values of 7.02 ± 0.52 μg/ml (32.3 μM) and 25.46 ± 1.79 μg/ml, respectively. The activity of allosecurinine and Phyllanthus extract were much lower. Furthermore, our study showed that the most active securinine induced apoptosis in a dose-dependent manner in the tested cells, increased the percentage of ROS positive cells and depolarized cells as well as stimulated the activity of ERK1/2, caspase-9 and -3/7. Securinine also induced cell cycle arrest in S phase. Real-time PCR analysis showed high expression of TNFRSF genes in the cells stimulated with securinine.
  
  
  CONCLUSIONS
  Securinine induces apoptosis and activates cell cycle checkpoints in HeLa cells which is associated with oxidative stress. The results indicate that the mitochondrial pathway is involved in the programmed cell death.
authors: Department of Biology and Pharmaceutical Botany, Medical University of Gdańsk, Gdańsk, Poland.
authors: Justyna
authors: J
authors: Stefanowicz-Hajduk
authors: Department of Pharmacognosy with Medicinal Plant Garden, Medical University of Gdańsk, Gdańsk, Poland.
authors: Barbara
authors: B
authors: Sparzak-Stefanowska
authors: Department of Pharmacognosy with Medicinal Plant Garden, Medical University of Gdańsk, Gdańsk, Poland.
authors: Mirosława
authors: M
authors: Krauze-Baranowska
authors: Department of Biology and Pharmaceutical Botany, Medical University of Gdańsk, Gdańsk, Poland.
authors: J Renata
authors: JR
authors: Ochocka
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001381:Azepines / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D006367:HeLa Cells / Q000187:drug effects*; D006572:Heterocyclic Compounds, Bridged-Ring / Q000494:pharmacology*; D006801:Humans; D007783:Lactones / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D029803:Phyllanthus / Q000737:chemistry; D010880:Piperidines / Q000494:pharmacology*; D010936:Plant Extracts / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D001381:Azepines; D006572:Heterocyclic Compounds, Bridged-Ring; D007783:Lactones; D010880:Piperidines; D010936:Plant Extracts; C000785:securinine