title: Inhibition of breast cancer cell proliferation in repeated and non-repeated treatment with zoledronic acid.
abstract: BACKGROUND
  Zoledronic acid is used to treat bone metastases and has been shown to reduce skeletal-related events and exert antitumor activity. The present in vitro study investigates the mechanism of action of Zoledronic Acid on breast cancer cell lines with different hormonal and HER2 patterns. Furthermore, we investigated the efficacy of repeated versus non-repeated treatments.
  
  
  METHODS
  The study was performed on 4 breast cancer cell lines (BRC-230, SkBr3, MCF-7 and MDA-MB-231). Non-repeated treatment (single exposure of 168 hrs' duration) with zoledronic acid was compared with repeated treatment (separate exposures, each of 48 hrs' duration, for a total of 168 hrs) at different dosages. A dose-response profile was generated using sulforhodamine B assay. Apoptosis was evaluated by TUNEL assay and biomolecular characteristics were analyzed by western blot.
  
  
  RESULTS
  Zoledronic acid produced a dose-dependent inhibition of proliferation in all cell lines. Anti-proliferative activity was enhanced with the repeated treatment, proving to be statistically significant in the triple-negative lines. In these lines repeated treatment showed a cytocidal effect, with apoptotic cell death caused by caspase 3, 8 and 9 activation and decreased RAS and pMAPK expression. Apoptosis was not observed in estrogen receptor-positive line: p21 overexpression suggested a slowing down of cell cycle. A decrease in RAS and pMAPK expression was seen in HER2-overexpressing line after treatment.
  
  
  CONCLUSIONS
  The study suggests that zoledronic acid has an antitumor activity in breast cancer cell lines. Its mechanism of action involves the decrease of RAS and RHO, as in osteoclasts. Repeated treatment enhances antitumor activity compared to non-repeated treatment. Repeated treatment has a killing effect on triple-negative lines due to apoptosis activation. Further research is warranted especially in the treatment of triple-negative breast cancer.
authors.affiliation: Osteoncology Center, IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), via P, Maroncelli 40, 47014, Meldola, FC, Italy. t.ibrahim@irst.emr.it.
authors.forename: Toni
authors.initials: T
authors.lastname: Ibrahim
authors.forename: Laura
authors.initials: L
authors.lastname: Mercatali
authors.forename: Emanuele
authors.initials: E
authors.lastname: Sacanna
authors.forename: Anna
authors.initials: A
authors.lastname: Tesei
authors.forename: Silvia
authors.initials: S
authors.lastname: Carloni
authors.forename: Paola
authors.initials: P
authors.lastname: Ulivi
authors.forename: Chiara
authors.initials: C
authors.lastname: Liverani
authors.forename: Francesco
authors.initials: F
authors.lastname: Fabbri
authors.forename: Michele
authors.initials: M
authors.lastname: Zanoni
authors.forename: Wainer
authors.initials: W
authors.lastname: Zoli
authors.forename: Dino
authors.initials: D
authors.lastname: Amadori