An RGD small-molecule integrin antagonist induces detachment-mediated anoikis in glioma cancer stem cells

Int J Oncol. 2018 Dec;53(6):2683-2694. doi: 10.3892/ijo.2018.4583. Epub 2018 Oct 3.

Abstract

The malignancy of glioblastoma (GB) is primarily due to the ability of glioma cancer stem cells (GSC) to disseminate into surrounding brain tissues, despite surgery and chemotherapy, and to form new tumoral masses. Members of the RGD-binding integrin family, which recognize the arginine-glycine-aspartic acid (RGD) sequence present in components of the extracellular matrix, and which serve a crucial function in the dissemination of GCS, are overexpressed in GB. Small-molecule integrin antagonists (SMIAs) designed to recognize RGD-integrins may therefore be an effective tool for decreasing GB infiltration and recurrence. In the present study, in vitro pro-apoptotic and infiltrative effects elicited by the SMIA 1a‑RGD in human GSC were investigated. Reverse transcription-quantitative polymerase chain reaction analysis revealed that, compared with normal human astrocytes, GSC grown on laminin-coated dishes overexpressed stemness markers as well as αvβ3 and αvβ5 integrins. In addition, dissociated GSC were identified to exhibit tumorigenic capacity when injected into immunodeficient mice. Using annexin/fluorescence-activated cell sorting analysis and ELISA nucleosome assays, it was identified that treatment of GSC with 25 µM 1a‑RGD for 48 h elicited detachment‑dependent anoikis not accompanied by necrosis-dependent cell death. A colorimetric proliferation assay indicated that 1a‑RGD did not affect cell viability, but that, instead, it markedly inhibited GSC migration as assessed using a Transwell assay. Western blot experiments revealed a decrease in focal adhesion kinase and protein kinase B phosphorylation with a concomitant increase in caspase-9 and -3/7 activity following 1a‑RGD treatment, suggesting that the pro-anoikis effects of 1a‑RGD may be mediated by these molecular mechanisms. Western blot analysis revealed no changes in specific markers of autophagy, suggesting further that 1a‑RGD-induced cell death is primarily sustained by anoikis-associated mechanisms. In conclusion, the results of the present study indicate that SMIA have potential as a therapeutic tool for decreasing GSC dissemination.

MeSH terms

  • Aged
  • Animals
  • Anoikis
  • Brain Neoplasms / drug therapy*
  • Brain Neoplasms / metabolism
  • Cell Differentiation / drug effects
  • Cell Movement / drug effects
  • Female
  • Gene Expression Regulation, Neoplastic / drug effects
  • Glioma / drug therapy*
  • Glioma / genetics
  • Glioma / metabolism
  • Humans
  • Integrin alphaVbeta3 / antagonists & inhibitors
  • Integrin alphaVbeta3 / genetics
  • Integrins / antagonists & inhibitors*
  • Male
  • Mice
  • Middle Aged
  • Neoplastic Stem Cells / drug effects*
  • Neoplastic Stem Cells / metabolism
  • Receptors, Vitronectin / antagonists & inhibitors
  • Receptors, Vitronectin / genetics
  • Signal Transduction / drug effects
  • Small Molecule Libraries / administration & dosage*
  • Small Molecule Libraries / chemical synthesis
  • Small Molecule Libraries / pharmacology

Substances

  • Integrin alphaVbeta3
  • Integrins
  • Receptors, Vitronectin
  • Small Molecule Libraries
  • integrin alphaVbeta5