title: Metformin plus sorafenib highly impacts temozolomide resistant glioblastoma stem-like cells.
abstract: OBJECTIVE
  Glioblastoma stem cells (GSCs), responsible for the dismal disease prognosis after conventional treatments, are driven by overactive signaling pathways, such as PI3K/ AKT/mTOR and RAS/RAF/MAPK. The objective of our study was to target in vitro-GSCs by combining metformin (Met) as a mTOR inhibitor, with sorafenib (Soraf) as a RAF inhibitor.
  
  
  METHODS
  GSCs cultured under basal conditions were treated with Met, temozolomide (TMZ), Soraf, Met+TMZ and Met+Soraf; as untreated arm served as control. At 4 hrs of drug exposure, we measured the level of reactive oxygen species (ROS) by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, apoptosis by prodium iodide (PI)-V Annexin staining and efflux pump activity by using the fluorescent dye rhodamine 123. At 24 hrs, we measured cell proliferation by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis and malondialdehyde (MDA) levels. MTT results were compared with corresponding measurements on cultures of non-stem glioblastoma cells and osteoblasts.
  
  
  RESULTS
  Met+Soraf exerted the highest antiproliferative effects in GSCs and non-stem glioblastoma cells (p<0.001). Both Met and Soraf monotherapy exhibited a selective cytotoxic effect on GSCs (p<0.001), while no effect was detected on non-stem glioblastoma cells (p>0.05). Soraf, but not Met, impacted the proliferation of normal cells. Soraf displayed synergism with Met in producing high levels of ROS, decreasing efflux pump activity and generating the highest apoptotic rates when compared to either drug alone (p<0.001).
  
  
  CONCLUSIONS
  GSCs were highly sensitive to the combination of Met and Soraf which reduced cell proliferation, increased oxidative stress, inhibited efflux pump activity and ultimately killed GSCs. We strongly believe that these results warrant further in vivo exploration.
authors: Research Center for Functional Genomics, Biomedicine and Translational Medicine, University of Medicine and Pharmacy Iuliu Hatieganu and Department of Functional Genomics, the Oncology Institute Prof. Dr. Ion Chiricuta, Cluj Napoca, Romania.
authors: Mihaela D
authors: MD
authors: Aldea
authors: Bobe
authors: B
authors: Petrushev
authors: Olga
authors: O
authors: Soritau
authors: Ciprian I
authors: CI
authors: Tomuleasa
authors: Ioana
authors: I
authors: Berindan-Neagoe
authors: Adriana G
authors: AG
authors: Filip
authors: Gabriela
authors: G
authors: Chereches
authors: Mihai
authors: M
authors: Cenariu
authors: Lucian
authors: L
authors: Craciun
authors: Corina
authors: C
authors: Tatomir
authors: Ioan-Stefan
authors: IS
authors: Florian
authors: Carmen B
authors: CB
authors: Crivii
authors: Gabriel
authors: G
authors: Kacso
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000473:pathology; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm; D005909:Glioblastoma / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D015227:Lipid Peroxidation; D008687:Metformin / Q000008:administration & dosage*; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000378:metabolism; D009536:Niacinamide / Q000008:administration & dosage / Q000031:analogs & derivatives*; D018384:Oxidative Stress; D010671:Phenylurea Compounds / Q000008:administration & dosage*; D020112:Rhodamine 123 / Q000378:metabolism; D000077157:Sorafenib; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D000077204:Temozolomide; D048490:raf Kinases / Q000037:antagonists & inhibitors*
chemical_list: D010671:Phenylurea Compounds; D020112:Rhodamine 123; D009536:Niacinamide; D003606:Dacarbazine; D008687:Metformin; D000077157:Sorafenib; D058570:TOR Serine-Threonine Kinases; D048490:raf Kinases; D000077204:Temozolomide