Aldea Mihaela D, Petrushev Bobe, Soritau Olga, Tomuleasa Ciprian I, Berindan-Neagoe Ioana, Filip Adriana G, Chereches Gabriela, Cenariu Mihai, Craciun Lucian, Tatomir Corina, Florian Ioan-Stefan, Crivii Carmen B, Kacso Gabriel
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.
J BUON. 2014 Apr-Jun;19(2):502-11.
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.
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.
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).
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.
胶质母细胞瘤干细胞（GSCs）是导致传统治疗后疾病预后不佳的原因，其由过度活跃的信号通路驱动，如PI3K/AKT/mTOR和RAS/RAF/MAPK。我们研究的目的是通过将作为mTOR抑制剂的二甲双胍（Met）与作为RAF抑制剂的索拉非尼（Soraf）联合使用，来靶向体外培养的GSCs。
在基础条件下培养的GSCs分别用Met、替莫唑胺（TMZ）、Soraf、Met+TMZ和Met+Soraf处理；未处理的一组作为对照。在药物暴露4小时时，我们通过2',7'-二氯荧光素二乙酸酯（DCFDA）测定法测量活性氧（ROS）水平，通过碘化丙啶（PI）-V膜联蛋白染色测量细胞凋亡，并使用荧光染料罗丹明123测量外排泵活性。在24小时时，我们通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐（MTT）测定法测量细胞增殖、细胞凋亡和丙二醛（MDA）水平。将MTT结果与非干细胞性胶质母细胞瘤细胞和成骨细胞培养物的相应测量结果进行比较。
Met+Soraf对GSCs和非干细胞性胶质母细胞瘤细胞具有最高的抗增殖作用（p<0.001）。Met和Soraf单一疗法均对GSCs表现出选择性细胞毒性作用（p<0.001），而对非干细胞性胶质母细胞瘤细胞未检测到作用（p>0.05）。Soraf而非Met影响正常细胞的增殖。与单独使用任何一种药物相比，Soraf在产生高水平ROS、降低外排泵活性和产生最高凋亡率方面与Met表现出协同作用（p<0.001）。
GSCs对Met和Soraf的联合使用高度敏感，该联合用药可减少细胞增殖、增加氧化应激、抑制外排泵活性并最终杀死GSCs。我们坚信这些结果值得进一步进行体内研究。
