title: Chelidonine isolated from ethanolic extract of Chelidonium majus promotes apoptosis in HeLa cells through p38-p53 and PI3K/AKT signalling pathways.
abstract: OBJECTIVE
  To evaluate the role of chelidonine isolated from ethanolic extract of Chelidonium majus in inducing apoptosis in HeLa cells and to assess the main signalling pathways involved.
  
  
  METHODS
  Cells were initially treated with different concentrations of chelidonine for 48 h and the median lethal dose (LD50) value was selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological analysis of nuclear condensation and DNA damage and fragmentation were measured by 4',6-diamidino-2-phenylindole staining and comet assay. Further, reactive oxygen species (ROS) generation, cell cycle arrest and change in mitochondrial membrane potential were also examined and analyzed by flow cytometry. Evaluation of interaction of drug with CT DNA was investigated by circular dichroism (CD) spectral analysis to find any possible drug-CT DNA interaction. The mRNA and protein expressions of major signal proteins like p38, p53, protein kinase B (AKT), phosphatidylinositol 3-kinases (PI3K), Janus kinase 3 (JAK3), signal transducer and activator of transcription 3 (STAT3) and E6 and E7 oncoproteins as well as the pro-apoptotic genes and antiapoptotic genes were also estimated by reverse transcriptase-polymerase chain reaction and Western blotting.
  
  
  RESULTS
  Based on LD(50) value (30 μg/mL) of chelidonine, three doses were selected, namely, 22.5 μg/mL (D1), 30.0 μg/mL (D2) and 37.5 μg/mL (D3). Results showed that chelidonine inhibited proliferation and induced apoptosis in HeLa cells through generation of ROS, cell cycle arrest at sub-G1 and G0/G1 stage, change in mitochondrial membrane potential and fragmentation of DNA. Results of CD spectra showed effective interaction between chelidonine and calf thymus DNA. Studies of signalling pathway revealed that chelidonine could efficiently induce apoptosis through up-regulation of expressions of p38, p53 and other pro-apoptotic genes and down-regulation of expressions of AKT, PI3K, JAK3, STAT3, E6, E7 and other antiapoptotic genes.
  
  
  CONCLUSIONS
  Chelidonine isolated from Chelidonium majus efficiently induced apoptosis in HeLa cells through possible alteration of p38-p53 and AKT/PI3 kinase signalling pathways.
authors.affiliation: Department of Zoology, University of Kalyani, Kalyani, India.
authors.forename: Avijit
authors.initials: A
authors.lastname: Paul
authors.forename: Kausik
authors.initials: K
authors.lastname: Bishayee
authors.forename: Samrat
authors.initials: S
authors.lastname: Ghosh
authors.forename: Avinaba
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Sourav
authors.initials: S
authors.lastname: Sikdar
authors.forename: Debrup
authors.initials: D
authors.lastname: Chakraborty
authors.forename: Naoual
authors.initials: N
authors.lastname: Boujedaini
authors.forename: Anisur Rahman
authors.initials: AR
authors.lastname: Khuda-Bukhsh
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053119:Benzophenanthridines / Q000737:chemistry / Q000494:pharmacology*; D059447:Cell Cycle Checkpoints; D049109:Cell Proliferation / Q000187:drug effects; D028281:Chelidonium / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D053119:Benzophenanthridines; D019253:Proto-Oncogene Proteins c-bcl-2; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C062047:chelidonine; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D048051:p38 Mitogen-Activated Protein Kinases