Everolimus (RAD001) and anti-angiogenic cyclophosphamide show long-term control of gastric cancer growth in vivo

Cancer Biol Ther. 2008 Sep;7(9):1377-85. doi: 10.4161/cbt.7.9.6416. Epub 2008 Sep 2.

Abstract

Metronomic dosing of cytotoxic drugs such as cyclophosphamide has shown anti-angiogenic activity, most likely by inducing hypoxia in tumors. Hypoxia leads to activation of escape mechanisms allowing tumor cell survival. This potentially limits the activity of anti-angiogenic strategies. We hypothesized that mTORC1 inhibition by everolimus (RAD001) leads to suppression of HIF-1alpha and VEGF resulting in synergistic anti-tumor activity in combination with anti-angiogenically dosed cyclophosphamide. In vitro, effects of everolimus on mTORC1 signaling, proliferation, cell cycle, HIF-1alpha expression and VEGF secretion were evaluated in two gastric cancer cell lines. In vivo, anti-tumor activity of everolimus in combination with metronomic cyclophosphamide was studied in a NCI-N87 human gastric cancer SCID mouse xenograft model. Expression of Ki-67 and HIF-1alpha, activated caspase 3, microvascular density (MVD) and tumor necrotic area assessed. Everolimus decreased proliferation and attenuated production of HIF-1alpha as well as VEGF in gastric cancer cells in vitro. In vivo, everolimus significantly inhibited tumor growth. This anti-tumor activity was linked to a significant increase in tumor necrotic area (p < 0.02) and trends for decreased proliferation, increased apoptosis, decreased HIF-1alpha and lower tumor MVD (p = n.s.). The combination of everolimus and cyclophosphamide resulted in a striking and highly significant long-term tumor growth control compared to monotherapy (p < 0.001), which was associated with a sharp increase in central tumor necrosis (p < 0.001). In conclusion, the combination of everolimus and metronomic cyclophosphamide showed synergistic anti-tumor activity. Depriving cancer cells by everolimus of factors necessary for their survival under hypoxia induced by anti-angiogenic chemotherapy appears to be a promising approach for treatment of gastric cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiogenesis Inhibitors / pharmacology*
  • Angiogenesis Inhibitors / therapeutic use
  • Animals
  • Antineoplastic Agents / pharmacology*
  • Antineoplastic Agents / therapeutic use
  • Caspase 3 / metabolism
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cyclophosphamide / pharmacology*
  • Cyclophosphamide / therapeutic use
  • Drug Therapy, Combination
  • Enzyme Activation / drug effects
  • Everolimus
  • Female
  • Humans
  • Immunohistochemistry
  • Ki-67 Antigen / metabolism
  • Mice
  • Mice, SCID
  • Necrosis / pathology
  • Neovascularization, Pathologic / drug therapy*
  • Neovascularization, Pathologic / genetics
  • Neovascularization, Pathologic / metabolism
  • Random Allocation
  • Sirolimus / analogs & derivatives*
  • Sirolimus / pharmacology
  • Sirolimus / therapeutic use
  • Stomach Neoplasms / blood supply*
  • Stomach Neoplasms / drug therapy*
  • Stomach Neoplasms / genetics
  • Stomach Neoplasms / metabolism
  • Tumor Burden / drug effects
  • Xenograft Model Antitumor Assays / methods

Substances

  • Angiogenesis Inhibitors
  • Antineoplastic Agents
  • Ki-67 Antigen
  • Cyclophosphamide
  • Everolimus
  • Caspase 3
  • Sirolimus