title: Clausenidin from Clausena excavata induces apoptosis in hepG2 cells via the mitochondrial pathway.
abstract: BACKGROUND
  Clausena excavata Burm.f. is used locally in folk medicine for the treatment of cancer in South East Asia.
  
  
  OBJECTIVE
  To determine the mechanism of action of pure clausenidin crystals in the induction of hepatocellular carcinoma (hepG2) cells apoptosis.
  
  
  METHODS
  Pure clausenidin was isolated from Clausena excavata Burm.f. and characterized using1H and13C NMR spectra. Clausenidin-induced cytotoxicity was determined by MTT assay. The morphology of hepG2 after treatment with clausenidin was determined by fluorescence and Scanning Electron Microscopy. The effect of clausenidin on the apoptotic genes and proteins were determined by real-time qPCR and protein array profiling, respectively. The involvement of the mitochondria in clausenidin-induced apoptosis was investigated using MMP, caspase 3 and 9 assays.
  
  
  RESULTS
  Clausenidin induced significant (p<0.05) and dose-dependent apoptosis of hepG2 cells. Cell cycle assay showed that clausenidin induced a G2/M phase arrest, caused mitochondrial membrane depolarization and significantly (p<0.05) increased expression of caspases 3 and 9, which suggest the involvement of the mitochondria in the apoptotic signals. In addition, clausenidin caused decreased expression of the anti-apoptotic protein, Bcl 2 and increased expression of the pro-apoptotic protein, Bax. This finding was confirmed by the downregulation of Bcl-2 gene and upregulation of the Bax gene in the treated hepG2 cells.
  
  
  CONCLUSIONS
  Clausenidin extracted from Clausena excavata Burm.f. is an anti-hepG2 cell compound as shown by its ability to induce apoptosis through the mitochondrial pathway of apoptosis. Clausenidin can potentially be developed into an anticancer compound.
authors.affiliation: MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, Serdang, Selangor, Malaysia; Department of Biochemistry, Kaduna State University, Main Campus, PMB 2336 Kaduna, Nigeria. Electronic address: petermwaziri@gmail.com.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Waziri
authors.affiliation: Department of Veterinary Pathology and Microbiology, Faculty of Veterinary, University Putra Malaysia, Serdang, Selangor, Malaysia. Electronic address: rasedee@gmail.com.
authors.forename: Rasedee
authors.initials: R
authors.lastname: Abdullah
authors.affiliation: Laboratory of Vaccine and Therapeutics, Institute of Bioscience, University Putra Malaysia, Serdang, Selangor, Malaysia. Electronic address: skyeap2005@gmail.com.
authors.forename: Swee Keong
authors.initials: SK
authors.lastname: Yeap
authors.affiliation: Laboratory of Vaccine and Therapeutics, Institute of Bioscience, University Putra Malaysia, Serdang, Selangor, Malaysia. Electronic address: aro675@gmail.com.
authors.forename: Abdul Rahman
authors.initials: AR
authors.lastname: Omar
authors.affiliation: MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, Serdang, Selangor, Malaysia. Electronic address: ahmadbstmm@yahoo.com.
authors.forename: Ahmad Bustamam
authors.initials: AB
authors.lastname: Abdul
authors.affiliation: Department of Chemistry, Faculty of Science, University Putra Malaysia, Serdang, Selangor, Malaysia. Electronic address: kartinee@upm.edu.my.
authors.forename: Nur Kartinee
authors.initials: NK
authors.lastname: Kassim
authors.affiliation: MAKNA Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, Serdang, Selangor, Malaysia. Electronic address: keepibinformed@yahoo.co.uk.
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Malami
authors.affiliation: Department of Chemistry, Faculty of Science, University Putra Malaysia, Serdang, Selangor, Malaysia. Electronic address: thiruventhan_90@yahoo.com.
authors.forename: Thiruventhan
authors.initials: T
authors.lastname: Karunakaran
authors.affiliation: School of Public Health, Zhengzhou University, Zhengzhou City, Henan Province, PR China. Electronic address: mustyimam@gmail.com.
authors.forename: Mustapha Umar
authors.initials: MU
authors.lastname: Imam
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D054700:Clausena / Q000737:chemistry*; D015870:Gene Expression; D056945:Hep G2 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008855:Microscopy, Electron, Scanning; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D010936:Plant Extracts / Q000494:pharmacology*
chemical_list: D010936:Plant Extracts; D053148:Caspase 3; D053453:Caspase 9
keywords: Apoptosis; Bax; Bcl 2; Clausenidin; Liver cancer; MMP