Abstract P1-05-05: Hyperthermia regulates transporter expression via ROS production and enhances the cytotoxicity of doxorubicin
Authors: Terasaki A , Kurokawa H , Terasaki M , Ito H , Matsui H , Ichioka E , Tsushima Y , Manaka-Iguchi A , Bando H , Hara H 
Published: February 2018

Abstract
Introduction: Hyperthermia (HT) is a non-invasive cancer therapy. Treatment temperature between 41°C to 44°C has no cytotoxic damage in normal cells, however shows cytotoxicity in cancer cells because of the underdeveloped vascular system. HT often used with other cancer therapy such as radiation therapy and chemotherapy. However mechanism of synergistic effect using these therapies remains unclear. Compared to 37°C, 42°C is mild heat stress for cells, thus superoxide anion is released from tissue. Superoxide anion is produced by mitochondrial electron transport chain. Reactive oxygen species (ROS), produced by mild heat stress, can be released from mitochondria. We have previously reported that ATP-binding cassette sub-family G member 2 (ABCG2) expression was suppressed by increasing mitochondrial ROS, and induction of the cancer specific porphyrin accumulation. ABCG2 is a transporter of doxorubicin (DOX), therefore we hypothesized that synergistic effect of HT and chemotherapy would be induced by down-regulation of ABCG2 expression via intracellular ROS increase. In this study, we investigated if cytotoxic effect of breast cancer cell using DOX can be enhance by HT via intracellular ROS increase.
Materials and methods: The murine breast cancer cell line, 4T1E was incubated at 37°C or 42°C for 1h. Intracellular ROS generation after HT treatment was detected by electron spin resonance (ESR). Twenty four hours after HT treatment, cells were incubated in medium containing 0, 0.1 and 1 μM DOX for 24 h. Cell viability was measured using the Cell Counting Kit 8, a water-soluble tetrazolium-8 based colorimetric assay. ABCG2 expression in whole cells was analyzed by Western blotting.
Results and discussion: ESR signal peak with HT treatment became high as compared to without HT treatment, indicating intracellular ROS level was increased by HT treatment. Cell viability and ABCG2 expression were decreased by DOX exposure and by HT treatment. The enhancement of HT treatment effect by DOX is considered to be result of down-regulation of ABCG2 expression by ROS. When cells were exposed to DOX with 5-aminolevulinic acid (ALA), cell viability reduced further. Since it is known that porphyrin is introduced by ALA and is transported by ABCG2, we speculate that ALA worked as a competitive inhibitor of DOX excretion transporter to enhance cell death. ESR signal peak in ALA treatment cells was higher than that in non-ALA treatment cells. Significant increase in cellular damage by HT treatment was shown by adding ALA, but not without ALA. Moreover, cell death induced by HT and ALA treatment was suppressed by adding N-acetylcysteine (NAC), which is an antioxidant. These results suggest that cellular damage of HT treatment is due to ROS production induced by ALA.
Conclusion: HT treatment involved intracellular ROS production and down-regulated the expression of ABCG2 protein. HT treatment also enhanced the cell damage by DOX. Cell death by DOX was enhanced by combination with HT and ALA treatment, possibly via intracellular ROS generation, and was suppressed by additing antioxidant.
Citation Format: Terasaki A, Kurokawa H, Terasaki M, Ito H, Matsui H, Ichioka E, Tsushima Y, Manaka-Iguchi A, Bando H, Hara H. Hyperthermia regulates transporter expression via ROS production and enhances the cytotoxicity of doxorubicin [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-05.