Combination of Zoledronic Acid and serine/threonine Phosphatase Inhibitors Induces Synergistic Cytotoxicity and Apoptosis in Human Breast Cancer Cells via Inhibition of PI3K/Akt Pathway

Tumour Biol. 2016 Mar;37(3):3665-73. doi: 10.1007/s13277-015-3265-x. Epub 2015 Oct 13.


The aim of this study was to investigate the cytotoxic and apoptotic effects of zoledronic acid (ZA) in combination with serine/threonine protein phosphatase inhibitors, calyculin-A (CA) and okadaic acid (OA), in human MCF-7 and MDA-MB-231 breast cancer cells. XTT cell viability assay was used to evaluate cytotoxicity. DNA fragmentation and caspase-3/7 activity assays were performed to evaluate apoptosis. Activities of phosphatase 1 (PP1) and phosphatase 2A (PP2A) were measured by serine/threonine phosphatase ELISA kit. Expression levels of PI3K, p-PI3K, Akt, p-Akt, Bcl-2, p-Bcl-2, Bad, and p-Bad proteins were evaluated by Western blot analysis. Combination of ZA with either CA or OA showed synergistic cytotoxicity and apoptosis as compared to any agent alone in both MCF-7 and MDA-MB-231 breast cancer cells. Combination treatment also resulted in inhibition of both PP1 and PP2A activities. Both agents used alone or in combination did not induce significant changes in total PI3K, Akt, Bcl-2, and Bad expressions, while p-PI3K, p-Akt, p-Bcl-2, and p-Bad levels were reduced by the combination treatment as compared to agents alone. Moreover, apoptotic effect of combination treatment was significantly inhibited in the presence of LY294002, a specific PI3K inhibitor, in both breast cancer cell lines. In conclusion, synergistic apoptotic effect of the combination treatment is correlated with the block of the PI3K/Akt signal pathway in breast cancer cells.

Keywords: Calyculin-A; Combination; Okadaic acid; PI3K/Akt pathway; Synergism; Zoledronic acid.

MeSH terms

  • Apoptosis / drug effects*
  • Blotting, Western
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Diphosphonates / pharmacology*
  • Dose-Response Relationship, Drug
  • Drug Synergism
  • Enzyme Inhibitors / pharmacology*
  • Female
  • Humans
  • Imidazoles / pharmacology*
  • MCF-7 Cells
  • Okadaic Acid / pharmacology
  • Oxazoles / pharmacology
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Signal Transduction / drug effects*
  • Zoledronic Acid


  • Diphosphonates
  • Enzyme Inhibitors
  • Imidazoles
  • Oxazoles
  • Okadaic Acid
  • Zoledronic Acid
  • calyculin A
  • Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins c-akt
  • Phosphoprotein Phosphatases