Analysis of oxidative stress response in clinically relevant radioresistant cells

Radiation therapy is one of the choices to treat malignant tumors. Although radiation therapy has been established as an excellent local treatment for malignant tumors, existence of radiation resistant cell is a major problem to overcome. To reveal radioresistant mechanism, we investigated using clinically relevant radioresistant (CRR) cells that had been obtained by exposing to 2 Gy/day X-rays for more than 30 days. CRR cells show not only radioresistance but also resistant to anticancer drug docetaxel. CRR cells also show low frequency of DNA double strand breaks, low mitochondrial membrane potential and produce little amount of reactive oxygen species (ROS). However, the molecular mechanism to obtain radio-resistant is not clear. So, we try to reveal the resistant ability to other oxidative stress such as hydrogen peroxide (H2O2). First, we investigated the resistance of CRR cells against H2O2 using WST assay. As a result, these cells showed resistant to H2O2. Next, we examined gene expression and enzyme activity of catalase that is H2O2 catabolic enzyme. Catalase expression was up-regulated in CRR cells. However, catalase enzyme activity was down-regulated. We also investigated mitochondrial DNA (mtDNA) copy number. It was shown that the mtDNA copy number was decreased in CRR cells. In addition, the amount of ATP, ATPase gene expressions and plasma membrane potential were investigated in CRR cells. It was shown that the amount of ATP decreased, ATPase gene expressions were up regulated and plasma membrane potential were low in CRR cells. Furthermore, increase of internal H2O2 amount and lipid peroxidation was investigated. As a result, increase of internal H2O2 amount and lipid peroxidation were seen in parental cells 2h after H2O2 administration. On the other hand, increase of internal H2O2 amount and lipid peroxidation were not seen in CRR cells 2h after H2O2 administration. These results suggest that the decrease of cell response through plasma membrane component is the main factor rather than internal oxidative stress scavenging enzyme activity to obtain resistance against oxidative stress in CRR cells.