title: Dentatin isolated from Clausena excavata induces apoptosis in MCF-7 cells through the intrinsic pathway with involvement of NF-κB signalling and G0/G1 cell cycle arrest: a bioassay-guided approach.
abstract: BACKGROUND
  Clausena excavata Burm. f. has been used in folk medicines in eastern Thailand for the treatment of cancer.
  
  
  METHODS
  To investigate the apoptosis mechanism, we isolated dentatin (DTN) from this plant using a bioassay-guided approach. DTN-induced cytotoxicity was observed with the MTT assay. Acridine orange/propidium iodide staining was used to detect cells in early apoptosis and high content screening (HCS) to observe nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Apoptosis was confirmed with a clonogenic assay, DNA laddering and caspase 3/7 and 9 assays. Reactive oxygen species (ROS) formation, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. The involvement of nuclear factor-kappa B (NF-κB) was analysed with the HCS assay.
  
  
  RESULTS
  A significant increase in chromatin condensation in the cell nucleus was observed by fluorescence analysis. Apoptosis was confirmed by the reduced number of colonies in the clonogenic assay and the increased number of cellular DNA breaks in treated cells observed as a DNA ladder. Treatment of MCF-7 cells with DTN encouraged apoptosis with cell death-transducing signals that reduced MMP by down-regulation of Bcl-2 and up-regulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase 9 followed by the executioner caspase 3/7. DTN treatment significantly arrested MCF-7 cells at the G0/G1 phase (p<0.05) and ROS was significantly elevated. Moreover, DTN significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus.
  
  
  CONCLUSIONS
  Together, the results demonstrated that the DTN isolated from Clausena excavata inhibited the proliferation of MCF-7 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway with involvement of the NF-κB signalling pathway.
authors.affiliation: UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, Serdang, Selangor, Malaysia.
authors.forename: Ismail Adam
authors.initials: IA
authors.lastname: Arbab
authors.forename: Ahmad Bustamam
authors.initials: AB
authors.lastname: Abdul
authors.forename: Mohd Aspollah
authors.initials: MA
authors.lastname: Sukari
authors.forename: Rasedee
authors.initials: R
authors.lastname: Abdullah
authors.forename: Suvitha
authors.initials: S
authors.lastname: Syam
authors.forename: Behnam
authors.initials: B
authors.lastname: Kamalidehghan
authors.forename: Mohamed Yousif
authors.initials: MY
authors.lastname: Ibrahim
authors.forename: Manal Mohamed Elhassan
authors.initials: MM
authors.lastname: Taha
authors.forename: Siddig Ibrahim
authors.initials: SI
authors.lastname: Abdelwahab
authors.forename: Hapipah Mohd
authors.initials: HM
authors.lastname: Ali
authors.forename: Syam
authors.initials: S
authors.lastname: Mohan
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification* / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001681:Biological Assay / Q000379:methods*; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D054700:Clausena / Q000737:chemistry; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D015870:Gene Expression / Q000187:drug effects; D006575:Heterocyclic Compounds, 3-Ring / Q000302:isolation & purification* / Q000494:pharmacology*; D006801:Humans; D061986:MCF-7 Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016328:NF-kappa B / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D006575:Heterocyclic Compounds, 3-Ring; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; C581591:dentatin; D045304:Cytochromes c; D020169:Caspases