Combining TMZ and SB225002 induces changes of CXCR2 and VEGFR signalling in primary human endothelial cells

Standard of care therapy for glioblastoma (GBM) consisting of surgical removal, temozolomide (TMZ) and radiotherapy fails to cure the disease and median survival is limited to 15 months. Therapeutic approaches targeting vascular endothelial growth factor (VEGF)‑mediated angiogenesis, one of the major drivers of tumour growth, have not prolonged patient survival as reported in clinical studies. Apart from VEGFR signalling, proangiogenic C‑X‑C motif chemokine receptor 2 (CXCR2) is of special interest as its ligands C‑X‑C motif chemokine ligand 2 (CXCL2) and interleukin‑8 (IL8) are upregulated and associated with reduced survival in GBM patients. As CXCR2 is also expressed by endothelial cells, the aim of the present study was to elucidate the effect of combination therapy on gene and protein expression of primary human endothelial cells (HUVECs). To mimic the GBM specific CXCL2/IL8 oversupply environment [referred to as stimulation (STIM)], HUVECs were treated with a cocktail of CXCL2/IL8 and/or TMZ and/or CXCR2‑antagonist SB225002 (SB). In brief, six treatment conditions were utilized: i) Control, ii) STIM (CXCL2/IL8), iii) TMZ + SB, iv) STIM + TMZ, v) STIM + SB, vi) STIM + TMZ + SB followed by either RNA‑isolation and RT‑qPCR for BAX, BCL2, vascular endothelial growth receptor (VEGFR)1/2, VEGF, CXCR1/2, CXCL2 and IL8 or immunofluorescence staining for VEGFR2 and CXCR2. SB and TMZ led to morphological changes of HUVECs and downregulated antiapoptotic BCL2