Abstract
Background: Glioblastoma (GBM) highly expresses Src tyrosine kinase involved in survival, proliferation, angiogenesis and invasiveness of tumor cells. Src activation also reduces reactive oxygen species (ROS) generation, whereas Src inhibitors are able to increase cellular ROS levels. Methods: Pro-oxidative effects of two pyrazolo[3,4-d]pyrimidine derivatives—Src tyrosine kinase inhibitors, Si306 and its prodrug pro-Si306—were investigated in human GBM cells U87 and patient-derived GBM-6. ROS production and changes in mitochondrial membrane potential were assessed by flow cytometry. The expression levels of superoxide dismutase 1 (SOD1) and 2 (SOD2) were studied by Western blot. DNA damage, cell death induction and senescence were also examined in GBM-6 cells. Results: Si306 and pro-Si306 more prominently triggered ROS production and expression of antioxidant enzymes in primary GBM cells. These effects were followed by mitochondrial membrane potential disruption, double-strand DNA breaks and senescence that eventually led to necrosis. Conclusion: Src kinase inhibitors, Si306 and pro-Si306, showed significant pro-oxidative potential in patient-derived GBM cells. This feature contributes to the already demonstrated anti-glioblastoma properties of these compounds in vitro and in vivo and encourages clinical investigations.
Highlights
Brain tumors represent 90% of all tumors of the central nervous system (CNS) [1]
The potential of Src tyrosine kinase inhibitors to generate reactive oxygen species (ROS) and reactive nitrogen species (RNS) was analyzed by flTohwecpyototemnettirayl.oTfhSerpcrteysreonscieneofksiunpaesreoixnidheibaitnoirosn,tohygdernoegreantepRerOoxSidaendanRdNpSerwoxaysnaitnrait-e lyanzeiodnbwyafsloinwvecsyttiogmateedtryin
The intensity of dihydrorhodamine 123 (DHR) fluorescence, which is proportional to the H2O2 and ONOO− levels, increased in Si306-treated GBM-6 cells, resulting in 8.02% of DHR-positive cells compared to the control, which had 5.78% (Figure 3b)
Summary
Brain tumors represent 90% of all tumors of the central nervous system (CNS) [1]. Glioblastoma (GBM) is the most frequent and aggressive (WHO grade IV) type of malignant CNS tumor and is associated with poor prognosis, with a median patient survival of 12 to 15 months [2,3]. Glioblastoma (GBM) highly expresses Src tyrosine kinase involved in survival, proliferation, angiogenesis and invasiveness of tumor cells. Results: Si306 and pro-Si306 more prominently triggered ROS production and expression of antioxidant enzymes in primary GBM cells. These effects were followed by mitochondrial membrane potential disruption, double-strand DNA breaks and senescence that eventually led to necrosis. Conclusion: Src kinase inhibitors, Si306 and pro-Si306, showed significant pro-oxidative potential in patient-derived GBM cells. This feature contributes to the already demonstrated anti-glioblastoma properties of these compounds in vitro and in vivo and encourages clinical investigations
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