Abstract B76: Acquired resistance of pancreatic cancer cells to 2-methoxyestradiol is associated with the upregulation of manganese superoxide dismutase in the mitochondria.

Background/Objectives: Pancreatic cancer is an exceptional aggressive cancer, and acquired drug resistance in this type of cancer is common. Reactive oxygen species (ROS) play an essential role in cell apoptosis, which is a key mechanism by which radio- or chemotherapy induces cell killing. Mitochondria are the major source of ROS in cells. Thus, alterations in the expression of mitochondrial proteins involved in ROS production or scavenging may be closely linked to the resistance of cancer cells to radio- or chemotherapy. The objective of this study was to use a comparative proteomic method to screen the mitochondrial proteins that confer the resistance of pancreatic cancer cells to ROS-inducing anticancer compounds. Methodology/Principal Findings: In order to eliminate the complications caused by genetic background variations, we generated a pancreatic cancer isogenic stable cell line that differs in sensitivity to 2-methoxyestradiol (2-ME), a ROS-inducing anticancer compound. The isogenic stable cell line was generated by exposing the pancreatic cancer MIA PaCa-2 cells (an undifferentiated pancreatic cancer cell line) to increasing concentrations of 2-ME (0.5-2.5 µM) over a three month period. One clone that survived from the long-term exposure to 2-ME showed strong 2-ME resistance, which was gauged by both cell apoptotic assay and the long-term clonogenic survival assay. We measured cellular ROS levels with flow cytometry using a fluorescent probe DCFH-DA, and it was shown that the resistant cells contained an elevated level of ROS, almost 3-fold that of the parental cells. We then used a two-dimensional gel electrophoresis based comparative proteomics method to compare the expressions of mitochondrial proteins between the parental and the resistant cells, followed by mass spectrometry analysis of the differentially expressed proteins. One protein identified to be upregulated in the resistant cells was manganese superoxide dismutase (SOD2), a mitochondrial protein that converts superoxide radicals to hydrogen peroxides (H 2 O 2 ). To examine whether the upregulated SOD2 was related to the 2-ME resistance of the resistant cells, we silenced or ectopically overexpressed SOD2 gene in the cells. The results demonstrated that silencing of SOD2 with small hairpin RNA resensitized the resistant cells to 2-ME, and stable overexpression of SOD2 led the parental cells to 2-ME resistance. In addition, the 2-ME-resistant cells also demonstrated resistance to gamma-ray radiation, suggesting the acquired 2-ME resistance of pancreatic cancer cells is not restricted to ROS-inducing compound 2-ME but is also linked to the radio-resistance of the cells. Because H 2 O 2 , the product of SOD2, is also toxic to cells, we examined the expression or activity of several important mitochondrial proteins that are involved in detoxifying H 2 O 2 in the mitochondria. The results demonstrated that the expression or activity of those proteins was not enhanced, suggesting they may not positively contribute to the enhanced 2-ME-resistance of the resistant cells. Conclusions: Our results suggest that pancreatic cancer cells acquire resistance to ROS-inducing anticancer agents through increasing the threshold of ROS tolerance of the cells (hence, the resistance to the oxidative agent-mediated cell killing and apoptosis). Upregulation of SOD2 expression in the mitochondria plays a key role in this enhanced tolerance to ROS-inducing anticancer drugs, and potentially also to ionizing radiation. Citation Format: Jianhong Zhou, Yuchun Du. Acquired resistance of pancreatic cancer cells to 2-methoxyestradiol is associated with the upregulation of manganese superoxide dismutase in the mitochondria. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B76.