title: Evaluation of the cytotoxicity, cell-cycle arrest, and apoptotic induction by Euphorbia hirta in MCF-7 breast cancer cells.
abstract: BACKGROUND
  Euphorbia hirta L. (Euphorbiaceae) has been used as a folk remedy in Southeast Asia for the treatment of various ailments.
  
  
  OBJECTIVE
  The current study evaluates the cytotoxicity, cell-cycle arrest, and apoptotic induction by E. hirta in MCF-7 breast cancer cells.
  
  
  METHODS
  Cytotoxic activity of methanol extract of whole part of E. hirta was determined by the MTT assay at various concentrations ranging from 1.96 to 250.00 µg/mL in MCF-7 cells. Cell morphology was assessed by light and fluorescence microscopy. Apoptosis and cell-cycle distribution were determined by annexin V staining and flow cytometry. DNA fragmentation, caspase activity, and reactive oxygen species (ROS) assays were performed using the commercially available kits. To identify the cytotoxic fraction, E. hirta extract was subjected to bioassay-guided fractionation.
  
  
  RESULTS
  Euphorbia hirta exhibited significant inhibition of the survival of MCF-7 cells and the half inhibitory concentration (IC50) values was 25.26 µg/mL at 24 h. Microscopic studies showed that E. hirta-treated cells exhibited marked morphological features characteristic of apoptosis. Euphorbia hirta extract also had an ignorable influence on the LDH leakage and generating intracellular ROS. The flow cytometry study confirmed that E. hirta extract induced apoptosis in MCF-7 cells. Euphorbia hirta also resulted in DNA fragmentation in MCF-7 cells. Moreover, E. hirta treatment resulted in the accumulation of cells at the S and G2/M phases as well as apoptosis. The caspase activity study revealed that E. hirta extract induced apoptosis through the caspase-3-independent pathway by the activation of caspase-2, 6, 8, and 9. Euphorbia hirta hexane fraction, namely HFsub4 fraction, demonstrated highest activity among all the fractions tested with an IC50 value of 10.01 µg/mL at 24 h.
  
  
  CONCLUSIONS
  This study revealed that E. hirta induced apoptotic cell death and suggests that E. hirta could be used as an apoptosis-inducing anticancer agent for breast cancer treatment with further detailed studies.
authors.affiliation: a Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, USM , Penang , Malaysia .
authors.forename: Yuet Ping
authors.initials: YP
authors.lastname: Kwan
authors.affiliation: b RIKEN-USM Joint Research Unit , RIKEN , Wako , Saitama , Japan .
authors.forename: Tamio
authors.initials: T
authors.lastname: Saito
authors.affiliation: c School of Biological Sciences, Universiti Sains Malaysia , Penang , Malaysia .
authors.forename: Darah
authors.initials: D
authors.lastname: Ibrahim
authors.affiliation: d Advanced Medical and Dental Institute, Universiti Sains Malaysia , Penang , Malaysia .
authors.forename: Faisal Muti Saleh
authors.initials: FM
authors.lastname: Al-Hassan
authors.affiliation: a Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, USM , Penang , Malaysia .
authors.forename: Chern
authors.initials: C
authors.lastname: Ein Oon
authors.affiliation: e Faculty of Dentistry , Dental Research & Training Unit, and Oral Cancer Research and Coordinating Centre (OCRCC), University of Malaya , Kuala Lumpur , Malaysia , and.
authors.forename: Yeng
authors.initials: Y
authors.lastname: Chen
authors.affiliation: a Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, USM , Penang , Malaysia .
authors.forename: Subramanion L
authors.initials: SL
authors.lastname: Jothy
authors.affiliation: f Faculty of Health, Nanomedicine - Laboratory of Immunology and Molecular Biomedical Research (LIMBR) , School of Medicine (SoM), Institute for Frontier Materials (IFM), Deakin University , Waurn Ponds , Victoria , Australia.
authors.forename: Jagat R
authors.initials: JR
authors.lastname: Kanwar
authors.affiliation: a Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, USM , Penang , Malaysia .
authors.forename: Sreenivasan
authors.initials: S
authors.lastname: Sasidharan
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D028481:Euphorbia / Q000737:chemistry; D005260:Female; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D061986:MCF-7 Cells; D018384:Oxidative Stress / Q000187:drug effects; D008517:Phytotherapy; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D010946:Plants, Medicinal; D017382:Reactive Oxygen Species / Q000378:metabolism; D059807:S Phase Cell Cycle Checkpoints / Q000187:drug effects*; D013997:Time Factors; D014709:Vero Cells
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D010936:Plant Extracts; D017382:Reactive Oxygen Species; D020169:Caspases
keywords: Anticancer; G2/M phase arrest; S phase arrest; caspase