title: Koenimbin, a natural dietary compound of Murraya koenigii (L) Spreng: inhibition of MCF7 breast cancer cells and targeting of derived MCF7 breast cancer stem cells (CD44(+)/CD24(-/low)): an in vitro study.
abstract: BACKGROUND
  Inhibition of breast cancer stem cells has been shown to be an effective therapeutic strategy for cancer prevention. The aims of this work were to evaluate the efficacy of koenimbin, isolated from Murraya koenigii (L) Spreng, in the inhibition of MCF7 breast cancer cells and to target MCF7 breast cancer stem cells through apoptosis in vitro.
  
  
  METHODS
  Koenimbin-induced cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Nuclear condensation, cell permeability, mitochondrial membrane potential, and cytochrome c release were observed using high-content screening. Cell cycle arrest was examined using flow cytometry, while human apoptosis proteome profiler assays were used to investigate the mechanism of apoptosis. Protein expression levels of Bax, Bcl2, and heat shock protein 70 were confirmed using Western blotting. Caspase-7, caspase-8, and caspase-9 levels were measured, and nuclear factor kappa B (NF-κB) activity was assessed using a high-content screening assay. Aldefluor™ and mammosphere formation assays were used to evaluate the effect of koenimbin on MCF7 breast cancer stem cells in vitro. The Wnt/β-catenin signaling pathway was investigated using Western blotting.
  
  
  RESULTS
  Koenimbin-induced apoptosis in MCF7 cells was mediated by cell death-transducing signals regulating the mitochondrial membrane potential by downregulating Bcl2 and upregulating Bax, due to cytochrome c release from the mitochondria to the cytosol. Koenimbin induced significant (P<0.05) sub-G0 phase arrest in breast cancer cells. Cytochrome c release triggered caspase-9 activation, which then activated caspase-7, leading to apoptotic changes. This form of apoptosis is closely associated with the intrinsic pathway and inhibition of NF-κB translocation from the cytoplasm to the nucleus. Koenimbin significantly (P<0.05) decreased the aldehyde dehydrogenase-positive cell population in MCF7 cancer stem cells and significantly (P<0.01) decreased the size and number of MCF7 cancer stem cells in primary, secondary, and tertiary mammospheres in vitro. Koenimbin also significantly (P<0.05) downregulated the Wnt/β-catenin self-renewal pathway.
  
  
  CONCLUSIONS
  Koenimbin has potential for future chemoprevention studies, and may lead to the discovery of further cancer management strategies by reducing cancer resistance and recurrence and improving patient survival.
authors.affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Ahmadipour
authors.affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Mohamed Ibrahim
authors.initials: MI
authors.lastname: Noordin
authors.affiliation: Medical Research Center, Jazan University, Jazan, Kingdom of Saudi Arabia.
authors.forename: Syam
authors.initials: S
authors.lastname: Mohan
authors.affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Aditya
authors.initials: A
authors.lastname: Arya
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Mohammadjavad
authors.initials: M
authors.lastname: Paydar
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Chung Yeng
authors.initials: CY
authors.lastname: Looi
authors.affiliation: UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia.
authors.forename: Yeap Swee
authors.initials: YS
authors.lastname: Keong
authors.affiliation: UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Malaysia.
authors.forename: Ebrahimi Nigjeh
authors.initials: EN
authors.lastname: Siyamak
authors.affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Somayeh
authors.initials: S
authors.lastname: Fani
authors.affiliation: Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Firoozi
authors.affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Chung Lip
authors.initials: CL
authors.lastname: Yong
authors.affiliation: Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, Serdang, Malaysia.
authors.forename: Mohamed Aspollah
authors.initials: MA
authors.lastname: Sukari
authors.affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Behnam
authors.initials: B
authors.lastname: Kamalidehghan
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001688:Biological Products / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000032:analysis / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D051927:CD24 Antigen / Q000032:analysis / Q000378:metabolism*; D002227:Carbazoles / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis / Q000378:metabolism*; D061986:MCF-7 Cells; D032101:Murraya / Q000737:chemistry*; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D001688:Biological Products; D014408:Biomarkers, Tumor; D051927:CD24 Antigen; D002227:Carbazoles; D018960:Hyaluronan Receptors; C000609614:koenimbin
keywords: MCF7 breast cancer stem cells; Murraya koenigii (L) Spreng; Wnt/β-catenin; glycogen synthase kinase 3β; koenimbin; nuclear factor kappa B