title: Induction of cell cycle arrest and apoptosis by betulinic acid-rich fraction from Dillenia suffruticosa root in MCF-7 cells involved p53/p21 and mitochondrial signalling pathway.
abstract: BACKGROUND
  Dillenia suffruticosa (Family: Dilleniaceae) or commonly known as "Simpoh air" in Malaysia, is traditionally used for treatment of cancerous growth including breast cancer.
  
  
  OBJECTIVE
  D. suffruticosa root dichloromethane extract (DCM-DS) has been reported to induce G0/G1 phase cell cycle arrest and apoptosis in caspase-3 deficient MCF-7 breast cancer cells. The present study was designed to investigate the involvement of p53/p21 and mitochondrial pathway in DCM-DS-treated MCF-7 cells as well as to identify the bioactive compounds responsible for the cytotoxicity of DCM-DS.
  
  
  METHODS
  Extraction of D. suffruticosa root was performed by the use of sequential solvent procedure. GeXP-based multiplex system was employed to investigate the expression of p53, p21, Bax and Bcl-2 genes in MCF-7 cells treated with DCM-DS. The protein expression was then determined using Western blot analysis. The bioactive compounds present in DCM-DS were isolated by using column chromatography. The structure of the compounds was elucidated by using nuclear magnetic resonance spectroscopy. The cytotoxicity of the isolated compounds towards MCF-7 cells was evaluated by using MTT assay. The percentage of betulinic acid (BA) in DCM-DS was determined by HPLC analysis.
  
  
  RESULTS
  The expression of p53 was significantly up-regulated at protein level. The expression of p21 at both gene and protein levels was significantly up-regulated upon treatment with DCM-DS, suggesting that the induction of G0/G1 phase cell cycle arrest in MCF-7 cells was via p53/p21 pathway. Bcl-2 protein was down-regulated with no change at the mRNA level, postulating that post-translational modification has occurred resulting in the degradation of Bcl-2 protein. Overall, treatment with DCM-DS increased the ratio of Bax/Bcl-2 that drove the cells to undergo apoptosis. A total of 3 triterpene compounds were isolated from DCM-DS. Betulinic acid appears to be the most major and most cytotoxic compound in DCM-DS.
  
  
  CONCLUSIONS
  DCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via p53/p21 pathway. In addition, DCM-DS induced apoptosis by increasing the ratio of Bax/Bcl-2. Betulinic acid, which is one of the major compounds, is responsible for the cytotoxicity of the DCM-DS. Therefore, BA can be used as a marker for standardisation of herbal product from D. suffruticosa. DCM-DS can also be employed as BA-rich extract from roots of D. suffruticosa for the management of breast cancer.
authors: Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Electronic address: foojhibiau@gmail.com.
authors: Jhi Biau
authors: JB
authors: Foo
authors: Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Electronic address: latifahsy@upm.edu.my.
authors: Latifah
authors: L
authors: Saiful Yazan
authors: Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors: Yin Sim
authors: YS
authors: Tor
authors: Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors: Agustono
authors: A
authors: Wibowo
authors: Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors: Norsharina
authors: N
authors: Ismail
authors: Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors: Chee Wun
authors: CW
authors: How
authors: Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors: Nurdin
authors: N
authors: Armania
authors: Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors: Su Peng
authors: SP
authors: Loh
authors: Laboratory of Natural Product, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors: Intan Safinar
authors: IS
authors: Ismail
authors: Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors: Yoke Kqueen
authors: YK
authors: Cheah
authors: Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
authors: Rasedee
authors: R
authors: Abdullah
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D045466:Dilleniaceae / Q000737:chemistry*; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects*; D006801:Humans; D061986:MCF-7 Cells; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D053978:Pentacyclic Triterpenes; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology; D018517:Plant Roots / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D014315:Triterpenes / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000187:drug effects; D000094062:Betulinic Acid
chemical_list: D051017:Apoptosis Regulatory Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D053978:Pentacyclic Triterpenes; D010936:Plant Extracts; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D014315:Triterpenes; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D000094062:Betulinic Acid
keywords: Apoptosis; Betulinic acid; Breast cancer; Dillenia suffruticosa; Triterpene