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

Paolillo,Mayra; Galiazzo,Marisa   C.; Daga,Antonio; Ciusani,Emilio; Serra,Massimo; Colombo,Lino; Schinelli,Sergio

doi:10.3892/ijo.2018.4583

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

Authors:
- Mayra Paolillo
- Marisa C. Galiazzo
- Antonio Daga
- Emilio Ciusani
- Massimo Serra
- Lino Colombo
- Sergio Schinelli
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Affiliations: Department of Drug Sciences, University of Pavia, I-27100 Pavia, Italy, Institute for Research, Hospitalization and Care-University Hospital (IRCCS-AOU) San Martino-Cancer Research Institute (IST), I-16132 Genoa, Italy, Fondazione IRCCS Neurological Institute C. Besta, I-20133 Milan, Italy
Published online on: October 3, 2018 https://doi.org/10.3892/ijo.2018.4583
Pages: 2683-2694

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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.

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