Fig 2: Effects of cannabinoids and ?-irradiation on cell signaling pathways and regulation of apoptosis in glioblastoma cells(A) Dose-dependent effects of CBD alone, in combination with VitE (50 µM), or in combination with ?-irradiation (10 Gy) on apoptosis of human glioblastoma cells, which was determined by PI staining DNA and the flow cytometry. The pooled results of four independent experiments are presented on the upper panel. Error bars represent means + S.D. (p < 0.05, Student’s t-test). Stars, open arrows and black arrows indicate significant differences between CBD-treated and (CBD+10Gy) treated U87MG, U118MG and T98G cells, respectively. (B) CBD-induced cell death was partially suppressed in the presence of SR144528 (20 µM), a CB2 receptor antagonist, or Vitamin E. U87MG cells were immunostained using anti-ß-tubulin monoclonal Ab. Bar = 50 µm. (C) Regulation of signaling pathways and apoptosis in the presence of cannabinoids. ?THC and endocannabinoids (eCB) induce signaling cascades through CB1 and CB2 receptors. ?THC could also affect growth factor receptors (GFR) signaling. CB1/2 receptors coupling to Gi signaling assemble is associated with activation of RAF-MEK-ERK pathway in the normal neural and glial cells, as well as in glioma cells. Activation of the PI3K/Akt-mTORC1 pathway by GFR with tyrosine kinase activity (RTK) might be negatively affected by CB1 receptor-dependent activation of ERK. RTK signaling activates also JAK/STAT3 pathway. ?THC-induced activation of CB1 and CB2 receptors upregulates ceramide synthesis in the endoplasmic reticulum (ER) and induces ER-stress, which suppresses/ downregulates RTK-induced AKT and ERK activation in glioma cells. It further suppresses mammalian target of rapamycin complex-1 (mTORC1) activity accompanied by suppression of the general translation but upregulation of autophagy. In contrast, cannabidiol (CBD) may directly penetrate into the cells without binding CB1/2 receptors. In the cells, CBD dramatically upregulates ROS production and ROS-dependent network, including activation of JNK. MAPK p38 could be activated by CBD in both ROS-dependent and ROS-independent mechanisms. These signaling pathways further regulate mitophagy, autophagy and the mitochondrial apoptotic pathway in glioblastoma cells, synergizing with ?-irradiation-induced cell death. CB1/2 receptors still could control CBD-induced signaling. TNFa /TNFR1 signaling might play a dual role via regulation of both survival functions (via NF-?B) and apoptotic or necroptotic functions. CBD treatment in combination with ?-radiation strongly affects TNF and TNF-R1 expression with the subsequent induction of proinflammatory cytokine (IL6 and IL8) expression and secretion. CBD could upregulate production of TRAIL and surface expression of TRAIL-R2 restoring external apoptotic signaling in glioblastoma cells.

Fig 3: Effects of CBD on TNFa and TRAIL expression and secretion and surface expression of death receptors in glioblastoma cells(A) Effects of CBD (20 µM) on TNF-promoter-luciferase reporter activity in transiently transfected glioblastoma cells. “Minimal” -36TNFpr-Luc, -615TNFpr-Luc (wt) and two mutated constructs, -615TNFpr(mutCRE)-Luc and -615TNFpr(mutAP1)-Luc were used. The pooled results of three independent experiments are presented in panel A. Error bars represent means ± S.D. (p < 0.05, Student’s t-test). Stars and black circles indicate a significant difference in the TNF promoter activity after CBD treatment in U87MG and U118MG cells, respectively. (B) Effects of CBD (20 µM) for 6 h on TRAIL-promoter-luciferase activity in transiently transfected glioblastoma cells, 24 h after transfection. (C, D) Quantitative real-time PCR analysis of the kinetics of TNFa mRNA levels after treatments of U87MG and U118MG cells by CBD (20 µM) and ?-irradiation (5 Gy) alone or in combination. The graphs indicate the fold change of target gene mRNA levels against time-point control after normalized to reference gene (beta-Actin). The pooled results of four independent experiments are presented in panel B. Error bars represent means + S.D. (p < 0.05, Student’s t-test). Stars indicate a significant difference in mRNA levels between control and CBD-pretreated cells, black circles - between irradiated (5 Gy) and irradiated in the presence of CBD (20 µM) cells. (E) Western blot assay of active and total NF-?B p65, TNFa p17 and TRAIL p32 levels 6 h after treatment by CBD in glioma cells. (F) TNFa protein levels secreted into the media after indicated treatments of glioblastoma cells were detected by ELISA. (G) Effects of CBD treatment on surface expression of death receptors in U87MG and U118MG glioblastoma cells. Surface expression of the main death receptors DR5/TRAIL-R2, FAS, TNFR1 and TNFR2 in U87MG and U118MG cells was determined by immunostaining and the flow cytometry. Cells were treated overnight in presence of 0.1% DMSO (green lines) or 20 µM CBD (red lines); ns–non-specific staining.