Ion-Channel Activity and Mesenchymal Differentiation in Radiation Resistance in Glioblastoma Multiforme

摘要

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor Current standard therapy is surgery combined with radio-therapy and/or chemo-therapy Unfortunately it is still the most difficult-to-treat tumor due to the existence of resistant tumor cells According to the previous studies radio-therapy induced mesenchymal differentiation (MD) which is a process of epithelial-mesenchymal transition and MD may promote GBM to establish radiation resistance Transforming Growth Factor-beta (TGF-β) is a modifier of radiation response and plays an important role in cell transformation Therefore TGF-β pathway might involve in developing radiation resistance via regulating MD Also radiation treatment influences on calcium-activated potassium channel activity it might relate to radiation resistance Thus we explored the correlation of calcium-activated potassium channel activity and MD in the process of establishing radiation resistance in human GBM cell lines We first examined the effect of radiation doses on cell viability by trypan blue and observed cell morphology According to the observation the cells showed morphology change were called transformed cells Also we chose the radiation dose of 50% cell viability for the established of consecutive-irradiation-induced resistance The radiation dose of the cell viability greater than 50% was chosen to establish high-dose-irradiation-induced resistance as positive control When viability of cells recovered from consecutive-irradiation exposures which were higher than positive control the cells were defined as consecutive-irradiation-induced resistance To develop radiation resistance 3 5Gy exposure for 6 times were required for 1306MG 2Gy exposure for 4 times were needed for U87MG and 7Gy for 4 times were required for CNS-1 As establishing radiation resistance the numbers of transformed cells increased Moreover immunocytochemistry staining showed expression of certain MD markers on transformed were different from on non-transformed cells such as E-cadherin forming clusters reduced the density of the expression of β-catenin and pan-cytokeratins and increased the density of the expression of N-cadherin and Fibronectin in transformed cells In addition immunoblotting results showed mesenchymal marker N-cadherin increased and epithelial marker beta-catenin decreased in radiation resistance Electrophysiology results indicated consecutive irradiation reduced big potassium channel (BK channel) activity but high-dose irradiation increased BK channel activity However radiation does not impacted on intermediate potassium channel (IK channel) activity Finally we used TGF-β and TGF-β receptor inhibitor LY364947 to regulate the activity of TGF-β/Smad pathway The immunoblotting results revealed that inhibition of TGF-β pathway reversed MD in consecutive-irradiation-induced resistant cell line via increasing the expression of β-catenin and reducing the expression of N-cadherin and Fibronectin Therefore our results suggest that mesenchymal differentiation and decrement of BK channel activity may both lead to radiation resistance and TGF-β/Smad pathway regulated the expression of certain MD markers during developing radiation resistance Finally we concluded that the developing of radiation resistance is a continuous and dynamic processes and not only biochemical factors but also electrophysiological factors play important role in these processes

獎項日期	2015 7月 30
原文	English
監督員	Chun-I Sze (Supervisor)