title: Induction of intrinsic apoptosis in leukaemia stem cells and in vivo zebrafish model by betulonic acid isolated from Walsura pinnata Hassk (Meliaceae).
abstract: BACKGROUND
  Leukaemia stem cells (LSC) have been associated with disease relapse and chemotherapy resistance. Betulonic acid (BA), a pentacyclic lupane-type triterpenoid, was reported to exhibit cytotoxicity toward various cancer cells and to be capable of inducing intrinsic apoptosis in solid tumours. However, the in vitro and in vivo apoptotic effects of BA against LSC remain unknown.
  
  
  OBJECTIVE
  We aimed to determine whether BA isolated from bark of Walsura pinnata Hassk (Meliaceae) has pro-apoptotic effects on LSC in in vitro and in vivo models.
  
  
  METHODS
  The population of high purity LSC was isolated from the Kasumi-1 cell line using magnetic sorting and characterised by flow cytometry. Cell viability was assessed using the MTS assay to examine dose- and time-dependent effects. The colony formation assay was performed in MethoCult® H4435 enriched media. Apoptosis was analysed using Annexin-V and propidium iodide staining, mitochondrial transmembrane potential was studied using JC-1 staining, and expression of apoptosis related genes (BAX, Bcl-2 and survivin) was evaluated by real time-polymerase chain reaction (RT-PCR). Caspase 3/7 and 9 activities were monitored through Promega Caspase-Glo® over a period of 24h. The in vivo antileukaemia activity was evaluated using LSC xenotransplanted zebrafish, observed for DNA fragmentation from apoptosis by TUNEL assay.
  
  
  RESULTS
  BA maintained its potency against the LSC population in comparison to parental Kasumi-1 cells (fold differences ≤ 1.94) over various treatment time points and significantly inhibited the formation of colonies by LSC. Apoptosis was triggered by BA through the upregulation of BAX and suppression of Bcl-2 and survivin genes with the loss of mitochondrial transmembrane potential, leading to the activation of caspase 9 followed by downstream caspase 3/7. BA was able to suppressed leukaemia formation and induced apoptosis in LSC xenotransplanted zebrafish.
  
  
  CONCLUSIONS
  The results demonstrate that BA inhibited the proliferative and colonogenic properties of LSC. BA induced apoptosis in LSC through the mitochondria pathway and was effective in the in vivo zebrafish model. Therefore, BA could be a lead compound for further development into a chemotherapy agent against LSC.
authors.affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia; Centre of Natural Products and Drug Discovery (CENAR), University of Malaya, 50603 Kuala Lumpur, Malaysia. Electronic address: leongkh@um.edu.my.
authors.forename: Kok Hoong
authors.initials: KH
authors.lastname: Leong
authors.affiliation: Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Mohamad Azrul
authors.initials: MA
authors.lastname: Mahdzir
authors.affiliation: Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Mohd Fadzli Md
authors.initials: MF
authors.lastname: Din
authors.affiliation: Centre of Natural Products and Drug Discovery (CENAR), University of Malaya, 50603 Kuala Lumpur, Malaysia; Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Khalijah
authors.initials: K
authors.lastname: Awang
authors.affiliation: Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, 812-8582, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Department of Research and Development of Next Generation Medicine, Faculty of Medical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Kasem
authors.initials: K
authors.lastname: Kulkeaw
authors.affiliation: Division of Cell Regulation Systems, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Ishitani
authors.affiliation: Department of Clinical Study, Center for Advanced Medical Innovation, Kyushu University, Fukuoka, 812-8582, Japan; Center for Clinical and Translational Research, Kyushu University, Fukuoka, 812-84582, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sugiyama
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008296:Malaysia; D029582:Meliaceae / Q000737:chemistry*; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D024301:Plant Bark / Q000737:chemistry; D013234:Stem Cells / Q000187:drug effects*; D015027:Zebrafish / Q000378:metabolism*; D000094062:Betulinic Acid
chemical_list: D009828:Oleanolic Acid; C118308:betulonic acid; D000094062:Betulinic Acid
keywords: Apoptosis; Betulonic acid; Leukaemia stem cells; Meliaceae; Zebrafish