NO to cancer: The complex and multifaceted role of nitric oxide and the epigenetic nitric oxide donor, RRx-001

Abstract

The endogenous mediator of vasodilation, nitric oxide (NO), has been shown to be a potent radiosensitizer. However, the underlying mode of action for its role as a radiosensitizer - while not entirely understood - is believed to arise from increased tumor blood flow, effects on cellular respiration, on cell signaling, and on the production of reactive oxygen and nitrogen species (RONS), that can act as radiosensitizers in their own right. NO activity is surprisingly long-lived and more potent in comparison to oxygen. Reports of the effects of NO with radiation have often been contradictory leading to confusion about the true radiosensitizing nature of NO. Whether increasing or decreasing tumor blood flow, acting as radiosensitizer or radioprotector, the effects of NO have been controversial. Key to understanding the role of NO as a radiosensitizer is to recognize the importance of biological context. With a very short half-life and potent activity, the local effects of NO need to be carefully considered and understood when using NO as a radiosensitizer. The systemic effects of NO donors can cause extensive side effects, and also affect the local tumor microenvironment, both directly and indirectly. To minimize systemic effects and maximize effects on tumors, agents that deliver NO on demand selectively to tumors using hypoxia as a trigger may be of greater interest as radiosensitizers. Herein we discuss the multiple effects of NO and focus on the clinical molecule RRx-001, a hypoxia-activated NO donor currently being investigated as a radiosensitizer in the clinic.

Keywords: Cancer; Hypoxia; Nitric oxide; Radiosensitization; Radiotherapy.

Graphical abstract

Fig. 1

The nitric oxide–nitrite–nitrate pathway. Under normal oxygen parameters (normoxia), nitric oxide (NO) is produced by NOS from L-Arg. In the absence of oxygen (hypoxia), nitrite is reduced by a variety reductases, including deoxyhemoglobin to produce NO. Further reduction/oxidation of NO can lead to production of metabolites.

Fig. 2

RRx-001: structure and primary metabolites (Hb: hemoglobin, GSH: glutathione).

Fig. 3

Molecular model of RRx-001 bound to the beta-Cys-93 of deoxyhemoglobin showing proximity to heme.

Fig. 4

NO release from RRx-001 treated RBCs. Left: schematic of reduction of serum nitrite to nitric oxide by RRx-001-bound hemoglobin. Right: RRx-001 potentiates the nitrite reductase activity of hemoglobin. Rate of NO formation of nitric oxide from nitrite compared to controls (Fens et al. [66]).

Fig. 5

Combination of RRx-001 and radiation. XRT alone or in combination with RRx-001 in HT-29 cells (P<0.01) (left) and SCCVII cells (right) (P<0.01).

Fig. 6

Tumor growth curves in mice showing the effect of a combination of RRx-001 with radiation (P<0.05 between combination and single agent groups).

Fig. 7

Time and sequence dependence of the radiosensitizing effect of RRx-001 showing the relationship between 4× TGD and the dosing schedule of RRx-001 and radiation. RRx-001 was dosed at t=0, ±2, and ±24 h. Total body radiation (7 Gy) was administered at t=0 h.

Fig. 8

Microbubble ultrasound imaging of tumor blood flow in mouse. Perfusion rate reaches a maximum at 6 h with the effect persisting for at least 48 h.

Fig. 9

Radioprotection of intestinal crypt cells by RRx-001. Mean crypt cells per duodenum (left) and jejunum (right) sections compared to TBI only dose. ♦=TBI; □=TBI+RRx-001.