title: Metformin and the mTOR inhibitor everolimus (RAD001) sensitize breast cancer cells to the cytotoxic effect of chemotherapeutic drugs in vitro.
abstract: OBJECTIVE
  Metformin appears to interfere directly with cell proliferation and apoptosis in cancer cells in a non-insulin-mediated manner. One of the key mechanisms of metformin's action is the activation of adenosine monophosphate activated protein kinase (AMPK). AMPK is linked with the phosphatidylinositol 3-kinase (PI3K)/ phosphatase and tensin homolog (PTEN)/protein kinase B (AKT) pathway and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) cascades--all known for being frequently dysregulated in breast cancer. Therefore, simultaneously targeting AMPK through metformin and the PI3K/AKT/mTOR pathway by an mTOR inhibitor could become a therapeutic approach. The aim of this study was to evaluate the anticancer effect of metformin alone and in combination with chemotherapeutic drugs and the mTOR inhibitor RAD001.
  
  
  METHODS
  The proliferation of breast cancer cells was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; and the cell apoptosis with enzyme-linked immunosorbent assay (ELISA). Gene expression at the protein level was determined by western blot.
  
  
  RESULTS
  We tested metformin alone and in combination with RAD001 and/or chemotherapeutic agents (carboplatin, paclitaxel and doxorubicin, respectively) on several human breast cancer cell lines with respect to cell proliferation, apoptosis and autophagy. Metformin alone inhibited cell proliferation and induced apoptosis in different breast cancer cell lines (ERα-positive, HER2-positive, and triple-negative). The cytotoxic effect of metformin was more remarkable in triple-negative breast cancer cell lines than in other cell lines. The cell apoptosis induced by metformin is, at least partly, caspase-dependent and apoptosis inducing factor (AIF)-dependent. Interestingly, we demonstrated that metformin induced cell autophagy. Inhibiting autophagy with chloroquine, enhanced the treatment efficacy of metformin, indicating that autophagy induced by metformin may protect breast cancer cells from apoptosis. We further demonstrated that co-administration of metformin with chemotherapeutic agents and RAD001 intensified the inhibition of cell proliferation. The analysis of cell cycle-regulating proteins cyclin D, cyclin E and p27 by western blot indicated that the synergistic inhibition of G1 phase of the cell cycle by the combination treatment of metformin, chemotherapeutic drugs and/or RAD001 contributed to the synergistic inhibition of cell proliferation.
  
  
  CONCLUSIONS
  Our investigation provides a rationale for the clinical application of metformin within treatment regimens for breast cancer.
authors.affiliation: Department of Oncology and Hematology, University Hospital of Berlin, Berlin, Germany. hongyu.liu@charite.de
authors.forename: Hongyu
authors.initials: H
authors.lastname: Liu
authors.forename: Christian
authors.initials: C
authors.lastname: Scholz
authors.forename: Chuanbing
authors.initials: C
authors.lastname: Zang
authors.forename: Jan H
authors.initials: JH
authors.lastname: Schefe
authors.forename: Piet
authors.initials: P
authors.lastname: Habbel
authors.forename: Anne-Constanze
authors.initials: AC
authors.lastname: Regierer
authors.forename: Carsten-Oliver
authors.initials: CO
authors.lastname: Schulz
authors.forename: Kurt
authors.initials: K
authors.lastname: Possinger
authors.forename: Jan
authors.initials: J
authors.lastname: Eucker
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D016190:Carboplatin / Q000494:pharmacology; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000068338:Everolimus; D005260:Female; D006801:Humans; D008687:Metformin / Q000494:pharmacology*; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D008687:Metformin; D000068338:Everolimus; D016190:Carboplatin; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus