Human NQO1 as a Selective Target for Anticancer Therapeutics and Tumor Imaging

Abstract

Human NAD(P)H-quinone oxidoreductase1 (HNQO1) is a two-electron reductase antioxidant enzyme whose expression is driven by the NRF2 transcription factor highly active in the prooxidant milieu found in human malignancies. The resulting abundance of NQO1 expression (up to 200-fold) in cancers and a barely detectable expression in body tissues makes it a selective marker of neoplasms. NQO1 can catalyze the repeated futile redox cycling of certain natural and synthetic quinones to their hydroxyquinones, consuming NADPH and generating rapid bursts of cytotoxic reactive oxygen species (ROS) and H₂O₂. A greater level of this quinone bioactivation due to elevated NQO1 content has been recognized as a tumor-specific therapeutic strategy, which, however, has not been clinically exploited. We review here the natural and new quinones activated by NQO1, the catalytic inhibitors, and the ensuing cell death mechanisms. Further, the cancer-selective expression of NQO1 has opened excellent opportunities for distinguishing cancer cells/tissues from their normal counterparts. Given this diagnostic, prognostic, and therapeutic importance, we and others have engineered a large number of specific NQO1 turn-on small molecule probes that remain latent but release intense fluorescence groups at near-infrared and other wavelengths, following enzymatic cleavage in cancer cells and tumor masses. This sensitive visualization/quantitation and powerful imaging technology based on NQO1 expression offers promise for guided cancer surgery, and the reagents suggest a theranostic potential for NQO1-targeted chemotherapy.

Keywords: NQO1 turn-on probes; antioxidant enzymes; futile substrates; near-infrared fluorophores; targeted therapy; theranostic drugs; tumor imaging; tumor-selective therapies; β-lapachone.

Figure 1

The location and composition of the human NQO1 gene and its expression controlled by the ARE-NRF2 pathway are shown. E = exon, In = intron.

Figure 2

Functions and roles of NQO1 in quinone metabolism and cellular defense against oxidative stress and other stresses, and impact on p53 steady-state levels are represented. Linkage of increased NQO1 expression with carcinogenesis and opportunities for selective therapeutic strategies through futile substrates is displayed.

Figure 3

Catalysis of a futile cycle substrate by NQO1 and the linkage of the resulting redox imbalance to different routes of cell death are shown. ER, endoplasmic reticulum; DAMPS, damage-associated molecular patterns.

Figure 4

(A). NQO1 FANTOM Tag expression as reported in different human tissues. The tissue data for RNA expression obtained through a Cap Analysis of Gene Expression (CAGE) generated by the FANTOM5 project are shown. The values represent scaled Tags Per Million (data adapted from https://www.proteinatlas.org/ENSG00000181019-NQO1/tissue, accessed on 27 July 2024). (B). Relative NQO1 protein expression in different human cancer types. Most cancers display strong cytoplasmic positivity in a fraction of cells, greater in lung cancer, followed by colorectal, endometrial, stomach, and pancreatic cancers (Data adapted from https://www.proteinatlas.org/ENSG00000181019-NQO1, accessed on 24 July 2024).

Figure 4

(A). NQO1 FANTOM Tag expression as reported in different human tissues. The tissue data for RNA expression obtained through a Cap Analysis of Gene Expression (CAGE) generated by the FANTOM5 project are shown. The values represent scaled Tags Per Million (data adapted from https://www.proteinatlas.org/ENSG00000181019-NQO1/tissue, accessed on 27 July 2024). (B). Relative NQO1 protein expression in different human cancer types. Most cancers display strong cytoplasmic positivity in a fraction of cells, greater in lung cancer, followed by colorectal, endometrial, stomach, and pancreatic cancers (Data adapted from https://www.proteinatlas.org/ENSG00000181019-NQO1, accessed on 24 July 2024).

Figure 5

Chemical structures of compounds reported to inhibit the catalytic activity of NQO1.

Figure 6

The mechanism of action of β-lapachone. The futile catalysis of the substrate to its hydroxyquinone and semiquinone forms and the consequent generation of ROS, increased production of H₂O₂, consequent PARP-1 activation, and metabolic perturbations are shown.

Figure 7

Details of cellular mechanisms leading to cell death, necroptosis, and release of DAMPS by β-lapachone are represented. Inhibition of radiation resistance and DNA repair as reported in the literature are also shown.

Figure 8

Futile catalysis of deoxynyboquinone by NQO1 and the resulting changes in cellular redox and physiology are shown.

Figure 9

Design strategies of NIR fluorescent probes for detection and quantitation of NQO1 activity in cells and theranostic applications thereof in human cancers. A general design and sensing mechanism of canonical activatable NQO1 probes and self-immolative linker-based activatable NQO1 probes is shown.

Figure 10

The structure of an NQO1-activatable NIR fluorescent probe called NIR-ASM (shown in part (B)). (A) Synthesis of NIR-ASM by coupling ASM with quinone propionic acid (QPA) and properties of NIR-ASM. (C) NIR-ASM activation and fluorescence generation in NQO1expressing cancer cells.

Figure 11

(A) Diagrammatic summary of techniques for NQO1 imaging using NIR-ASM and resulting fluorescence in NQO1-positive cancer cells and lack of it in NQO1-negative (normal) cells after incubation of 10 µM NIR-ASM for 1 h is shown. (B) Flow cytometry assays to determine NIR-ASM activation by NQO1-positive and -negative cell lines (C) Application of live in vivo fluorescence imaging of NQO1-positive tumors developed in nude mice.

Figure 12

(A): Confocal red fluorescence images of NQO1-positive cancer cells and NQO1-negative normal cells after incubation of 10 µM NIR-ASM for 1 h. The cell nuclei were counterstained with Hoechst 33342 (reproduced from ref. [168] with permission). (B): Results of live animal fluorescence imaging of endogenous NQO1 activity in A549 tumor xenografts after intravenous administration of NIR-ASM (5 mg/kg) to tumor-bearing nude mice (reproduced from ref. [168] with permission). The upper panel shows mice with NQO1-proficient A549 tumors and the lower panel shows the same animal with both A549 and NQO1-deficient MDA-MB-231 tumors on the left and right flanks respectively. Tumors and organs were harvested from the A549 tumor-bearing mice after NIR-ASM injections and imaged for ex vivo fluorescence as well. Fluorescence only in the tumor but not in the spleen (Sp), pancreas (Pa), lungs (Lu), brain (B), heart (H), kidneys (Ki), liver (Li), and intestine is evident, showing specificity.