title: Rottlerin-induced autophagy leads to the apoptosis in breast cancer stem cells: molecular mechanisms.
abstract: BACKGROUND
  Autophagy is an indispensable lysosomal self-digestion process involved in the degradation of aggregated proteins and damaged organelles. Autophagy is associated with the several pathological processes, including cancer. Cancer stem cells (CSCs) play significant roles in cancer initiation, progression and drug resistance. Recent studies have demonstrated the antitumor activities of plant-derived chemopreventive agent rottlerin (Rott). However, the molecular mechanism by which Rott induces autophagy in breast CSCs has not been investigated.
  
  
  RESULTS
  The objectives of this study were to examine the molecular mechanism by which Rott induces autophagy which leads to apoptosis in breast CSCs. Treatment of breast CSCs with Rott for 24 h resulted in a concentration dependent induction of autophagy, followed by apoptosis as measured by flow cytometry. Electron microscopy confirmed the presence of autophagosomes in Rott treated breast CSCs. Western blot analysis showed that Rott treatment increased the expression of LC3, Beclin-1 and Atg12 that are accumulated during autophagy. Prolonged exposure of breast CSCs to Rott caused apoptosis which was associated with the suppression of phosphorylated Akt and mTOR, upregulation of phosphorylated AMPK, and downregulation of anti-apoptosis Bcl-2, Bcl-X(L), XIAP and cIAP-1. Knock-down of Atg7 or Beclin-1 by shRNA inhibited Rott-induced autophagy at 24 h. Our study also demonstrates that pre-treatment of breast CSCs with autophagosome inhibitors 3-methyladenine and Bafilomycin, as well as protein synthesis inhibitor cycloheximide inhibited Rott-induced autophagy and apoptosis. Rott induces autophagy via extensive cytoplasmic vacuolization in breast CSCs. Molecular docking results between C2-domain of protein kinase C-delta and Rott indicated that both hydrogen bonding and hydrophobic interactions contributed significantly for ligand binding with minimum binding affinity of ≈ 7.5 Kcal/mol. Although, autophagy inhibitors suppress the formation of cytoplasmic vacuolization and autophagy in breast CSCs, the potency of Rott to induce autophagy and apoptosis might be based on its capability to activate several pathways such as AMPK and proteasome inhibition.
  
  
  CONCLUSIONS
  A better understanding of the relationship between autophagy and apoptosis would eventually allow us to discover novel drugs for the treatment of breast cancer by eliminating CSCs.
authors.forename: Dhruv
authors.initials: D
authors.lastname: Kumar
authors.forename: Sharmila
authors.initials: S
authors.lastname: Shankar
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, and Internal Medicine, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA. rsrivastava@kumc.edu.
authors.forename: Rakesh K
authors.initials: RK
authors.lastname: Srivastava
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000098:Acetophenones / Q000737:chemistry / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis; D001343:Autophagy; D001578:Benzopyrans / Q000737:chemistry / Q000494:pharmacology*; D001943:Breast Neoplasms; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D062105:Molecular Docking Simulation; D014411:Neoplastic Stem Cells / Q000502:physiology*; D051745:Protein Kinase C-delta / Q000737:chemistry / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014617:Vacuoles / Q000378:metabolism
chemical_list: D000098:Acetophenones; D000972:Antineoplastic Agents, Phytogenic; D001578:Benzopyrans; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; C085746:rottlerin; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D051745:Protein Kinase C-delta; D055372:AMP-Activated Protein Kinases