Abstract 906: Molecular imaging of RRx-001-induced oxidative stress in Nrf2-luciferase expressing SCC VII tumors in mice
Authors: Ning Shoucheng , Sekar Thillai Veerapazham , Paulmurugan Ramasamy , Scicinski Jan , Oronsky Bryan , Peehl Donna , Knox Susan J. 
Published: May 2015

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive master regulatory transcription factor that plays an important role in the antioxidant response pathway against anticancer drug-induced cytotoxic effects. Under normal conditions, Nrf2 is retained in the cytoplasm and degraded by Keap1. Under oxidative stress, the Nrf2-Keap1 interaction is disrupted and Nrf2 is released and translocated from cytoplasm to nucleus, where Nrf2 recruits sMaf, binds to the antioxidant response element and initiates transcription of antioxidant genes. Studies have reported that Nrf2 and its downstream target genes are overexpressed in cancer cells, giving cancer cells an advantage for survival and growth.

RRx-001 (also known as ABDNAZ) is a new promising anticancer agent (Cancer Res. 72:2600-08, 2012). RRx-001 generates reactive oxygen species (ROS) and nitric oxide (NO), modulates intracellular redox status, and triggers caspase-independent apoptosis in cancer cells. It was well tolerated and demonstrated anticancer efficacy in a recent phase I clinical trial. Here, we describe a series of molecular imaging studies showing that activation of Nrf2 may be a useful biomarker of response to RRx-001 therapy in vivo. SCC VII tumor cells were stably co-transfected with pcDNA-Nrf2-FLUC and pcDNA-RLUC-RFP plasmids. Single clones of cells expressing both the reporter genes were selected and evaluated by dual-luciferase reporter assay for reporter activities. Then, cells were tested for Nrf2-FLUC response to RRx-001 and to known Nrf2 activators. The results showed that RRx-001 induced a significant level of activation in SCC VII cells in a dose- and time-dependent manner, suggesting that RRx-001 is an effective activator of the Nrf2 pathway. Next, mice bearing Nrf2-firefly luciferase-Rluc-RFP-expressing SCC VII tumors were treated with RRx-001 and imaged for Nrf2 reporter gene expression using Bruker Xtreme and Xenogen IVIS spectrum in vivo imaging systems. We found that treatment with RRx-001 significantly upregulated expression of the Nrf2-FLUC reporter gene in SCC VII tumors as early as 6 hours following RRx-001 administration, that was maintained at high levels until 24 hours, while no activation was seen in the solvent-treated control mice. In separate experiments, we found that RRx-001 treatment activated endogenous Nrf2 expression and nuclear translocation in SCC VII tumor cells in vitro and in vivo in mice.

These new findings support the concept that RRx-001 generates ROS in cancer cells, which in turn activate Nrf2 and downstream antioxidant signaling pathways. These encouraging data indicate that Nrf2 might be useful as a molecular biomarker to monitor the response of targeted cancer cells to RRx-001 treatment, and for selection of cancer patients who would be most likely to respond and benefit from RRx-001 therapy.

Citation Format: Shoucheng Ning, Thillai Veerapazham Sekar, Ramasamy Paulmurugan, Jan Scicinski, Bryan Oronsky, Donna Peehl, Susan J. Knox. Molecular imaging of RRx-001-induced oxidative stress in Nrf2-luciferase expressing SCC VII tumors in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 906. doi:10.1158/1538-7445.AM2014-906