Nrf2 activity as a potential biomarker for the pan-epigenetic anticancer agent, RRx-001

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulatory transcription factor that plays an important role in the antioxidant response pathway against anticancer drug-induced cytotoxic effects. RRx-001 is a new anticancer agent that generates reactive oxygen and nitrogen species, and leads to epigenetic alterations in cancer cells. Here we report the RRx-001 mediated nuclear translocation of Nrf2 and the activation of expression of its downstream enzymes HO-1 and NQO1 in tumor cells. Inhibition of intrinsic Nrf2 expression by Nrf2-specific siRNA increased cell sensitivity to RRx-001. Molecular imaging of tumor cells co-expressing pARE-Firefly luciferase and pCMV-Renilla luciferase-mRFP in vitro and in vivo in mice revealed that RRx-001 significantly increased ARE-FLUC signal in cells in a dose- and time-dependent manner, suggesting that RRx-001 is an effective activator of the Nrf2-ARE signaling pathway. The pre-treatment level of ARE-FLUC signal in cells, reflecting basal activity of Nrf2, negatively correlated with the tumor response to RRx-001. The results support the concept that RRx-001 activates Nrf2-ARE antioxidant signaling pathways in tumor cells. Hence measurement of Nrf2-mediated activation of downstream target genes through ARE signaling may constitute a useful molecular biomarker for the early prediction of response to RRx-001 treatment, and thereby guide therapeutic decision-making.

A novel hypoxia-selective epigenetic agent RRx-001 triggers apoptosis and overcomes drug resistance in multiple myeloma cells

“…Our finding in MM cells is consistent with similar observations using RRx-001 in other cell systems 53, 54 .…”

Hypoxic conditions increases H2S-induced ER stress in A2870 cells

“…We observed elevated levels of this protein in cells subjected to hypoxia combined with GYY4137. These results are in agreement with results obtained in study of Zhang and coworkers, who found that in human breast cancer the high NR2F2 transcript level was associated with favorable clinical outcome, which might be due to NR2F2 0 s inhibitory effect on TGF-b-dependent EMT and its role in inhibiting chemoresistance [35]. However, controversial data obtained by other experiments (for example, NR2F2 expression) was found both a positive (ovarian) and negative (prostate and breast) prognostic factor for cancer patients [36].…”

RRx-001: a systemically non-toxic M2-to-M1 macrophage stimulating and prosensitizing agent in Phase II clinical trials

“…Multiple lines of evidence indicate that metabolites of RRx-001 cross the blood-brain barrier: (1) in an ADME study with 14 C-labeled RRx-001, radioactivity was detected in the CNS [8]; (2) in an ongoing Phase I/II trial acronymed BRAINSTORM, a significant reduction in the Dynamic contrast-enhancedmagnetic resonance imaging (DCE-MRI)-derived volume transfer constant, K trans , has been demonstrated in multiple patients after one dose of single agent RRx-001, indicative of reduced vascular permeability; and (3) the combination of RRx-001 with artemisinin, in an experimental cerebral malaria model, synergistically decreased animal mortality, neurocognitive sequelae, cerebral vascular changes, and inflammatory cell accumulation in brain microvessels [10].…”

“…The protective effects of RRx-001 on normal tissue may be mediated through the induction of the Nrf2-regulated antioxidant cell response, which neutralizes free radical damage [2]. In addition to radioprotective properties, RRx-001 is also associated with chemoprotection.…”

“…In addition to radiosensitization, RRx-001 demonstrated potent anticancer activity as a single agent [1]; significant cytotoxic enhancement was also present in combination with standard chemotherapy and immunotherapy through depletion of cancer stem cells (CSCs) and activation of p53 and Nrf2 pathways [1][2][3]. Moreover, RRx-001 has shown evidence of in vivo radio-and chemoprotection; in fact, the Armed Forces Radiobiology Research Institute is currently researching RRx-001 as a radioprotectant in case of a nuclear emergency.…”