Potential Glioblastoma cell survival mechanisms in Temozolomide-induced resistance

• Main Authors: Chia-WeiHsu, 許家瑋
• Other Authors: Chun-I Sze
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/5k9va7

碩士 === 國立成功大學 === 細胞生物與解剖學研究所 === 103 === WHO defines the Glioblastoma multiforme (GBM) as a grade IV glioma, which is the most common and hostile malignant primary brain tumor in human population. Previous studies showed that the GBM median survival is less than 15 months. The 5 year survival rate is less than 4%. Temozolomide (TMZ) is one of the most widely used drugs to treat GBM. Recently, it has been showed that TMZ treatment can induce apoptosis, autophagy, cell senescence and cell cycle arrest depend on MGMT expression level. However, the TMZ-induced resistance formation mechanism remains unclear. This study intends to establish two TMZ-induced resistance cell lines and test the role of apoptosis, cell senescence, autophagy and cell cycle arrest in their survival and death. The 1306-MG (human GBM), CNS-1 (rat GBM), and human astrocyte cell lines were used for establish TMZ-induced resistance (TIR) model. All of these cell lines were checked for MGMT expression level by immunoblotting. Trypan Blue Exclusion Assay (TBEA) method and cell counting were used to assess cell viability. Furthermore, TUNEL was used to observe apoptosis, LC3 staining was used to observe autophagy, SA-βGal was used to observe cell senescence, and zymography was used to observe MMP-9 activity, and cell flow-cytometry was used to determine cell cycle change. The MGMT expression levels of TIR GBM were not changed through generation of resistance. The cell viability increased through establishment of TIR accumulation under same concentrations of TMZ treatment; TIR significantly reduced the numbers of senescence cells in 1306-MG and CNS-1 TIR cell; the autophagy level and MMP-9 activity of TIR-GBM were elevated in 1306-MG and CNS-1 cells with generation of TIR cell; TIR cells showed significantly decreased SubG1 phase of cell cycle through generation of TIR cell, while both 1306-MG and CNS-1 showed decreased cell cycle distribution of S and G2/M phases. We had established 1306-MG and CNS-1 TMZ-induced resistance model. We also found that TIR formation accompanied with decreased apoptosis, diminished cellular senescence, arising autophagy, cell cycle arrest at G0/G1 phase, and increased MMP-9 activity. We conclude apoptosis, autophagy, cellular senescence, and cell cycle arrest all participate in the development of TMZ-induced resistance.