title: Selective mode of action of plumbagin through BRCA1 deficient breast cancer stem cells.
abstract: Studies over the past decade and half have identified cancer stem cells (CSCs) to be responsible for tumorigenesis, invasion, sustenance of metastatic disease, radio- and chemo-resistance and tumor relapse. Recent reports have described the plasticity of breast CSCs (BCSCs) to shift between the epithelial and mesenchymal phenotypes via Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial Transition (MET) states as the reason for their invasive capabilities. Additionally, BRCA1 has been found to be a mammary stem cell fate determinant. However, it is not clear what would be the best marker that can be used for identifying CSCs in BRCA1 mutated cancers. Also, anticancer agents that can reduce CSC population in a BRCA1 defective condition have not been addressed so far.
  
  
  
  Putative BCSCs were identified based on Hoechst exclusion, CD44(+)/24(-/low) expression and Aldehyde Dehydrogenase 1 (ALDH1) positivity using flow cytometry. The 'stemness' of the isolated ALDH1+ cells were analysed by immunofluorescence, western blotting for stem cell and EMT markers as well as in vitro mammosphere assays. Induction of Reactive Oxygen Species (ROS) by Plumbagin (PB) in BCSCs was assayed by Dichloro-dihydro-fluorescein diacetate (DCF-DA) staining. Ovarian cancer xenografts treated with PB were subjected to immunohistochemical analysis to study the ability of PB to target CSCs.
  
  
  
  We have confirmed that ALDH1 positivity is the best marker for the identification of BCSCs in BRCA1-defective breast cancer cell lines when compared to the CD marker profile and Side Population (SP) analysis. BRCA1 status was observed to be a determinant of the abundance of epithelial-like (ALDH1+) or mesenchymal-like (CD44(+)/24(-/low)) BCSCs, and the reconstitution of a full length, wild type BRCA1 in HCC1937 breast cancer cells possessing a mutated BRCA1, transforms them from 'stem-like' to more 'mesenchymal'. For the first time we have shown that Plumbagin (PB), a naturally occurring naphthoquinone which is predominantly a ROS inducer, could reduce BCSCs specifically in BRCA1-defective, basal-like cancer cells.
  
  
  
  The best marker for identifying BCSCs in BRCA1 defective condition could be ALDH1 and that BRCA1 mutated BCSCs would be mostly 'stem like' than 'mesenchymal'. Also ROS inducers like PB could reduce BCSCs in BRCA1 defective cancers.
authors.affiliation: Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, 695014, India.|Present address: Center for Cancer Research, National Cancer Institute, Building 567, Room 254, Frederick, MD, 21702-1201, USA.
authors.forename: Veena
authors.initials: V
authors.lastname: Somasundaram
authors.affiliation: Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, 695014, India.
authors.forename: Sreelatha K
authors.initials: SK
authors.lastname: Hemalatha
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, 55905, USA.
authors.forename: Krishnendu
authors.initials: K
authors.lastname: Pal
authors.affiliation: Department of Hematology, Mayo Clinic College of Medicine, Rochester, MN, 55905, USA.
authors.forename: Sutapa
authors.initials: S
authors.lastname: Sinha
authors.affiliation: Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, 695014, India.
authors.forename: Asha S
authors.initials: AS
authors.lastname: Nair
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, 55905, USA.
authors.forename: Debabrata
authors.initials: D
authors.lastname: Mukhopadhyay
authors.affiliation: Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, 695014, India. priyasrinivas@rgcb.res.in.
authors.forename: Priya
authors.initials: P
authors.lastname: Srinivas
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology* / Q000627:therapeutic use; D019313:BRCA1 Protein / Q000235:genetics* / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology*; D016190:Carboplatin / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D016513:Mice, SCID; D009285:Naphthoquinones / Q000494:pharmacology* / Q000627:therapeutic use; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D014408:Biomarkers, Tumor; D009285:Naphthoquinones; D016190:Carboplatin; C014758:plumbagin
keywords: ALDH1; BRCA1; Cancer stem cells; Plumbagin