title: The mTOR inhibitor RAD001 sensitizes tumor cells to the cytotoxic effect of carboplatin in breast cancer in vitro.
abstract: OBJECTIVE
  The phosphatidylinositol 3-kinase (PI3K)/protein kinase B(AKT)/mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in many types of cancer, including breast cancer. It is recognized that breast cancer cells develop resistance to a variety of standard therapies through the activation of this pathway. We hypothesized that targeting this signaling by the mTOR inhibitor RAD001 may potentiate the cytotoxicity of a conventional chemotherapeutic drug, carboplatin, and enhance the treatment efficacy for breast cancer.
  
  
  METHODS
  Cell proliferation was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; cell apoptosis with enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used for the analysis of cell cycle distribution and mitochondrial membrane function. Gene expression at the protein level was determined by Western blot.
  
  
  RESULTS
  MTOR inhibitor RAD001 enhanced the sensitivity of breast cancer cells to carboplatin. RAD001 in combination with carboplatin resulted in synergistic inhibition of cell proliferation and caspase-independent apoptosis in these cells. Moreover, in MCF-7 and BT-474 cells, synergistic effects of this combination on G₂/M cell cycle arrest and regulation of different molecules responsible for cell cycle transition and apoptosis were observed. The p53 pathway was involved in the synergism of RAD001 and carboplatin on breast cancer cell proliferation and apoptosis, since the synergistic effect was demonstrated in all tested breast cancer cell lines with wild-type p53 and the use of p53 inhibitor partially antagonized the effect of RAD001 and carboplatin on p53 and p21 expression, as well as their inhibitory effect on cell proliferation. However, a synergistic effect of the combination of the two drugs on cell proliferation was observed in two p53-mutated cell lines with high AKT expression, suggesting that an alternative mechanism underlying the observed synergism exists.
  
  
  CONCLUSIONS
  Our results suggest that the combination of RAD001 and carboplatin is a promising treatment approach for breast cancer. On the basis of these results, we have initiated a phase I/II clinical trial with the combination of carboplatin and RAD001 in patients with metastatic breast cancer.
authors.affiliation: Division of Oncology and Hematology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. jan.eucker@charite.de and hongyu.liu@charite.de.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Liu
authors.forename: Chuanbing
authors.initials: C
authors.lastname: Zang
authors.forename: Jan-Hendrik
authors.initials: JH
authors.lastname: Schefe
authors.forename: Sandra
authors.initials: S
authors.lastname: Schwarzlose-Schwarck
authors.forename: Anne-Constanze
authors.initials: AC
authors.lastname: Regierer
authors.forename: Elena
authors.initials: E
authors.lastname: Elstner
authors.forename: Carsten-Oliver
authors.initials: CO
authors.lastname: Schulz
authors.forename: Christian
authors.initials: C
authors.lastname: Scholz
authors.forename: Kurt
authors.initials: K
authors.lastname: Possinger
authors.forename: Jan
authors.initials: J
authors.lastname: Eucker
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D000068338:Everolimus; D005260:Female; D005434:Flow Cytometry; D016158:Genes, p53; D006801:Humans; D008564:Membrane Potentials; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000970:Antineoplastic Agents; D000068338:Everolimus; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus