| Summary: | 博士 === 國立陽明大學 === 生物醫學影像暨放射科學系暨研究所 === 98 === Higher oxidative stress is one feature of highly proliferative cancer cells. In the light of the differences on antioxidant network between cancer and normal cells, targeting key molecules regulating redox homeostasis in cancer cells is the developing strategy for cancer therapy. Human glioblastoma multiform (GBM) is the most malignant brain tumor possessing highly resistance to clinical treatment. This study aims to investigate whether destructing redox balance in GBM cells could overcome the resistance and raise the efficiency of radio- or chemical therapy. Using microarray analysis, two radiation-inducible genes sestrin 2 (SESN2) and xeroderma pigmentosum group C (XPC) were selected for further investigation in this study. When the induction of XPC or SESN2 was silenced by siRNA technology, the cytotoxicity of U87 cells to IR was significantly increased. To investigate the modes of action of these 2 genes, this study was divided into two parts. Part I: SESN2 silencing induces oxidative stress via accumulation of platelet-derived growth factor receptor beta (PDGFRß) in glioma cells. SESN2 is an essential molecule in redox homeostasis as evident that cells deficient with SESN2 triggers oxidative stress and lead to cell growth delay without exogeneous stimuli. However, how SESN2 regulating redox homeostasis and cell proliferation remains unclear. This study found SESN2 silencing gradually increased ROS and growth inhibition accompanied with the induction of autophagy. At the initial stage of SESN2 silencing, the levels of PDGFRß protein were dramatically increased and globally distributed within cells. The accumulation of PDGFRß after SESN2 silencing was due to the attenuation of its degradation and ubiquitination. Immunoprecipitation analysis showed no direct binding between SESN2 and PDGFRß, suggesting that SESN2 silencing indirectly interfered with degradation pathway of PDGFRß protein. When SESN2 and PDGFRß were double silenced, SESN2-mediated effects including ROS induction, autophagy activation, growth inhibition were rescued indicating that PDGFRß is the mediator for SESN2 to administer its functions. Part II: XPC silencing sensitizes glioma cells to arsenic trioxide (ATO) via increased oxidative damage. XPC is known to be involved in nucleotide excision repair. XPC is different from other repair-related molecules, because it can be induced by irradiation and ATO treatment. XPC silencing substantially sensitized U87 cells to ATO resulting from the emergency of senescence and autophagy cell death. In regard to DNA repair regulation, XPC silencing did not inference the level of DNA strand breaks and oxidative DNA lesion, 8-OHdG after ATO exposure. However, ATO-induced ROS was higher in XPC-silencing cells and the amount of hyperoxidized peroxiredoxin and protein carbonylation were both increased. As to antioxidant systems, XPC silencing suppressed ATO-induced activation of glutathione and catalase. After treatment of antioxidant N-acetylcysteine, the effects mediated by XPC silencing such as ATO toxicity, induction of autophagy, increase of ROS, activation of AP-1 were rescued, indicating XPC silencing sensitized GBM cells to ATO via enhancement of oxidative damages. In conclusion, this study demonstrated that GBM cells could be sensitized to ROS generating agents by disturbing the intracellular redox balance.
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