Inhibition of ATM kinase upregulates levels of cell death induced by cannabidiol and γ-irradiation in human glioblastoma cells

Abstract

Despite advances in glioblastoma (GBM) therapy, prognosis of the disease remains poor with a low survival rate. Cannabidiol (CBD) can induce cell death and enhance radiosensitivity of GBM but not normal astrocytes. Inhibition of ATM kinase is an alternative mechanism for radiosensitization of cancer cells. In this study, we increased the cytotoxic effects of the combination of CBD and γ-irradiation in GBM cells through additional inhibition of ATM kinase with KU60019, a small molecule inhibitor of ATM kinase. We observed in GBM cells treated by CBD, γ-irradiation and KU60019 high levels of apoptosis together with strong upregulation of the percentage of G2/M-arrested cells, blockade of cell proliferation and a massive production of pro-inflammatory cytokines. Overall, these changes caused both apoptotic and non-apoptotic inflammation-linked cell death. Furthermore, via JNK-AP1 activation in concert with active NF-κB, CBD upregulated gene and protein expression of DR5/TRAIL-R2 and sensitize GBM cells to TRAIL-induced apoptosis. In contrast, CBD notably decreased in GBM surface levels of PD-L1, a critical immune checkpoint agent for T-lymphocytes. We also used in the present study TS543 human proneural glioma cells that were grown as spheroid culture. TS543 neurospheres exhibited dramatic sensitivity to CBD-mediated killing that was additionally increased in combination with γ-irradiation and KU60019. In conclusion, treatment of human GBM by the triple combination (CBD, γ-irradiation and KU60019) could significantly increase cell death levels in vitro and potentially improve the therapeutic ratio of GBM.

RETRACTED ARTICLE: Inhibition of autophagic flux differently modulates cannabidiol-induced death in 2D and 3D glioblastoma cell cultures

“…We additionally used ATM inhibitor (ATMi) KU60019 35 , alone or in combination with γ-irradiation and CBD, to investigate its downstream effects on autophagy. We previously confirmed specificity of ATM kinase inhibitor (ATMi) KU60019 (at concentration 1-2 μM) for suppression γ-irradiation-induced ATM activation in U87MG cells 31 . Interestingly, the presence of ATMi (1-2 μM) in non-irradiated or, especially, in γ-irradiated GBM cells was linked with upregulation of LC3-II levels 24-48 h after treatment ( Fig.…”

“…Additional investigations also confirmed a cytotoxic role of cannabinoids for several other types of cancer 12,30,51 . A number of studies demonstrated the efficiency of combined treatments of cannabinoids together with γ-irradiation in both cell culture and in animal experiments 17,18,28,31 . CBD treatment can induce apoptotic death in GBM using both the intrinsic and extrinsic death pathways 52 while γ-irradiation results in enhancing apoptotic and non-apoptotic cell death, especially in the presence of ATM inhibitor 31,53 .…”

“…A number of studies demonstrated the efficiency of combined treatments of cannabinoids together with γ-irradiation in both cell culture and in animal experiments 17,18,28,31 . CBD treatment can induce apoptotic death in GBM using both the intrinsic and extrinsic death pathways 52 while γ-irradiation results in enhancing apoptotic and non-apoptotic cell death, especially in the presence of ATM inhibitor 31,53 . However, CBD and THC could be involved in the regulation of additional cell signaling networks including autophagy.…”

“…To overcome the resistance of proneural TS543 GBM that was driven by a PDGFR-regulated pathway to targeted tyrosine kinase inhibitors, we used CBD treatment of TS543 3D culture. In our recent study, we demonstrated effective killing of proneural TS543 GBM cells grown as 3D spheroid culture by CBD 31 . Additional investigation of the main signaling pathways linked with the autophagy regulation in TS543 cells after relatively short www.nature.com/scientificreports www.nature.com/scientificreports/ (3 h) treatment by CBD (5-20 μM) revealed, as expected, a strong dose-dependent activation/phosphorylation of JNK1/2 and MAPK p38.…”

“…Some experiments were performed with adherent U87MG cells grown as 2D monolayer cultures in cell media supplemented with 5% FBS that could promote autophagy. GBM cells were treated by CBD (5-20 μM) and γ-irradiation (5-10 Gy), as previously described 28,31 . A conventional surrogate biomarker of radiation-induced DNA damage, active Ataxia Telangiectasia Mutated (ATM) kinase (Ser1981-P), which played a crucial role as a sensor of double-strand brakes in genomic DNA and as the initiator of DNA repair after nuclear ionizing irradiation 32 , was detected by Western blotting 6 h after treatment.…”