title: The indole-3-carbinol cyclic tetrameric derivative CTet inhibits cell proliferation via overexpression of p21/CDKN1A in both estrogen receptor-positive and triple-negative breast cancer cell lines.
abstract: BACKGROUND
  Indole-3-carbinol (I3C), an autolysis product of glucosinolates present in cruciferous vegetables, and its dimeric derivative (3,3'-DIM) have been indicated as promising agents in preventing the development and progression of breast cancer. We have recently shown that I3C cyclic tetrameric derivative CTet formulated in γ-cyclodextrin (γ-CD) efficiently inhibited cellular proliferation in breast cancer cell lines. This study aims to analyze the mechanisms involved in the in vitro inhibition of cell proliferation and to evaluate the in vivo antitumor activity of CTet in a xenograft study.
  
  
  METHODS
  Estrogen receptor-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines were exposed to CTet to evaluate cell cycle perturbation (propidium iodide staining and cytofluorimetric acquisition), induction of autophagic morphological features (co-localization of LC3b autophagosome marker and LAMP2a lysosome marker by immunofluorescence) and changes in protein expression (immunoblot and microarray-based gene expression analyses). To test the in vivo efficacy of CTet, female athymic nude mice inoculated with MCF-7 cells were i.p. treated with 5 mg/kg/day of CTet for five days/week for two weeks and the tumor mass was externally monitored.
  
  
  RESULTS
  CTet induced accumulation in G2/M phase without evidence of apoptotic response induction in both cell lines tested. In triple-negative MDA-MB-231 the autophagic lysosomal activity was significantly up-regulated after exposure to 4 μM of CTet for 8 hours, while the highest CTet concentration was necessary to observe autophagic features in MCF-7 cells. The inhibition of Akt activity and p53-independent p21/CDKN1A and GADD45A overexpression were identified as the main molecular events responsible for CTet activity in MCF-7 and p53-mutant MDA-MB-231 cells. In vivo, CTet administration was able to significantly inhibit the growth of MCF-7 xenotransplanted into nude mice, without adverse effect on body weight or on haematological parameters.
  
  
  CONCLUSIONS
  Our data support CTet formulated with γ-CD as a promising and injectable anticancer agent for both hormone-responsive and triple-negative breast tumors.
authors: Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via Saffi 2, 61029 Urbino, Italy.
authors: Mauro
authors: M
authors: De Santi
authors: Luca
authors: L
authors: Galluzzi
authors: Simone
authors: S
authors: Lucarini
authors: Maria Filomena
authors: MF
authors: Paoletti
authors: Alessandra
authors: A
authors: Fraternale
authors: Andrea
authors: A
authors: Duranti
authors: Cinzia
authors: C
authors: De Marco
authors: Mirco
authors: M
authors: Fanelli
authors: Nadia
authors: N
authors: Zaffaroni
authors: Giorgio
authors: G
authors: Brandi
authors: Mauro
authors: M
authors: Magnani
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016158:Genes, p53; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009687:Nuclear Proteins / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism; D047408:gamma-Cyclodextrins; D000097642:GADD45 Proteins
chemical_list: D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D007211:Indoles; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D009687:Nuclear Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D047408:gamma-Cyclodextrins; C572341:5,6,11,12,17,18,23,24-octahydrocyclododeca(1,2-b-4,5-b'-7,8-''-10,11-b''')tetraindole; C493956:CDKN1A protein, human; C439070:GADD45A protein, human; D000097642:GADD45 Proteins; C497811:LAMP2 protein, human