Vulnerability of Antioxidant Drug Therapies on Targeting Nrf2-Trp53-Jdp2 Axis in Controlling the Tumorigenesis

1. Introduction

It has been known that the control of balance between reactive oxygen species (ROS) and redox status is an important way to address the occurrence and development of tumor cells without compromising normal cells [1]. This idea depends upon the theory that cancers have a homeostatic balance of oxidation, which is produced by phase I enzyme ligands, and antioxidation, which is mediated by phase II enzyme ligands and is arranged to favor the hallmark actions of normal cells or cancer cells, such as proliferation, survival, migration, and metastasis (Figure 1) [2]. Thus, the ROS balance is critical for regulating oxidative stress and antioxidation: mediated by the transcription factors such as to aryl hydrocarbon receptor (AHR) and nuclear factor erythroid 2-related factor 2 (NRF2), respectively. The extensive redox conditions damage biocomponents, and cancers are known to compensate for this damage by enhancing the expression of antioxidants. Thus, the development of cancers depends on these antioxidant proteins to regulate the oxidation and antioxidation status of macromolecules within cells to maintain the ROS balance. Thus, a switch in cancer redox activity produced by antioxidation or increased ROS production causes the threshold of the ROS balance of cancer cells to be altered, which causes cell cycle arrest or cell death [3]. Indeed, redox-modulating drugs that cause apoptosis are important in defining ROS-induced cell death, which is important for anticancer therapy [3]. Although drugs that control ROS are expected to have therapeutic applications in cancer, drugs targeting ROS have shown only limited success in preclinical trials compared with other anticancer drugs [4]. These results raise the possibility that tumor cells may not be more sensitive to ROS than normal cells. Thus, these trials offered less success than anticipated. The lack of biomarkers to measure endogenous redox levels might also prevent success [5]. Furthermore, the complexity and redundancy of drugs targeting ROS/redox are not well characterized. Recent reports have shown the heterogeneity of phase II enzyme reagents against cancer organoids [6]. Thus, the use of antioxidants for cancer treatment should be applied with caution. In this review, the heterogeneity of phase II enzyme agents is discussed in the context of cancer treatment and how to avoid these difficulties in preclinical trials of phase II drugs for cancer treatment.

2. ROS in Cancer Cells

Assuming a crucial role for ROS in cancer cells, controlling endogenous ROS by altering the function of mitochondrial constituents is a possible anticancer strategy. These strategies may inhibit ROS-mediated cancer occurrence and progression by causing oxidative damage, such as ROS-mediated apoptosis [7,8]. Thus, preclinical research on antioxidant production and weak pro-oxidants was conducted to understand their merits. Compared with normal cells, cancer cells generate excessive ROS, increasing their sensitivity to further increases in ROS-related cell damage and committing them to apoptosis. Pro-oxidants may thus have antitumor functions. Therapeutic trials of antioxidants targeting ROS control have included nonenzymatic drugs, including NRF2 agonists [9] and vitamins [10,11], or targeting ROS via the enzymatic production of antioxidants, such as nitrogen oxide inhibitors [12,13], superoxide dismutase mimetics [14], N-acetylcysteine, and glutathione (GSH) esters [15,16]. Many such clinical trials have been conducted (Table 1), and many such agents against ROS production have been developed, but their actions involve ROS homeostasis and antioxidation, which are not specific to cancer.

For example, the repression of glutathione peroxidase 2 (GPx2) in breast cancer may enhance cancer progression due to hypoxic signals, aberrant vascularization, and a metabolic switch to the aerobic glycolysis/oxidative phosphorylation (OXPHOS) axis. The complex expression of GPx2 depends on metabolic specific drives and microenvironments, such as hyperproliferation-derived hypoxia.

Another consideration is that ROS has a dual function in cancer occurrence, which might depend on endogenous ROS (Figure 1) [17]. This dichotomy further complicates the antioxidation response to the decomposition of H₂O₂ through a change in the glutathione-SH (GSH)–glutathione sulfide (GSSG) axis. Hypoxia-inducible factor-1β which was named differently as AHR nuclear translocator (ARNT), is a component of the complex of phase I enzymes with AHR. Thus, crosstalk between AHR signals and hypoxia signals might occur, leading to overlap. In addition, the development of cancers also involves contrasting levels of GPx2 expression, which are correlated with the progression of cancer and poor prognosis. Decreased GPx2 expression was detected in colon cancer, pancreatic cancer, cancers of the bladder and urinary tract, and esophageal colon carcinomas, whereas increased GPx2 expression was detected in several carcinoma [18]. This apparent contradiction of GPx2 levels might be due to differences in the levels of the intact endogenous ROS production machinery [18].

3. NRF2 Has Dual Roles in Tumorigenesis

NRF2 has dual and contradictory roles in cancer [19]. Abnormal increased expression of NRF2 can be related with a poor prognosis. The constitutive level of NRF2 in many cancer cells might induce the expression of pro-survival genes and promote the proliferation of tumor cells via metabolic alterations, inhibit cancer specific apoptosis, and increase the self-renewal function of cancer stem cells. Moreover, NRF2 contributed to chemoresistance, radio-resistance, and inflammation, inducing carcinogenesis. Many NRF2 inhibitors have been reported to treat cancers [20]. These trials targeted NRF2 might provide a new tool in cancer treatment.

3.1. Tumor Suppressive Actions of NRF2

The NRF2 protein is activated by tumor suppressor gene products such as breast cancer susceptibility gene I and p21^Cip1 by inhibiting KEAP1 (Kelch like ECH associated protein 1/NRF2 complex formation in microenvironmental cells in tumors [21,22]. The degradation of the oncogene tyrosine kinase Fyn blocks its activation [23]. Nrf2-deficient mice reduced the production of antioxidants and repressed the expression of markers specific for phase II enzyme encoded genes. The quinone induced the severe oxidative stress rendered Nrf2-deficient mice more prone to skin cancer, while the Nrf2-medited expressions of NAD(P)H quinone dehydrogenase 1 (Nqo1) and Gst were inhibited [24]. Similarly, Nrf2-deficient mice exposed to carcinogens developed more cancers included stomach [25], liver [26], and urinary bladder [27] as compared with wild type mice, suggesting that Nrf2 is the critical factor controlling inflammation.

Many compounds isolated from plants, including carnosol, curcumin, resveratrol, and sulforaphane, and synthetic chemicals like oltipraz and oleanane triterpenoids, exhibited the chemo-preventive functions by activating the Nrf2-antioxidant response element (ARE)-regulated genes. These Nrf2 activators mainly controlled the antioxidation reaction by changing the intermolecular disulfide (S–S) bonds between two cystine residues of Keap1 proteins at Cys273 and Cys288 to commit the accumulation of Nrf2 [28]. Sulforaphane (SFN) is committed to inducing the transcription of phase II enzyme-encoding genes while inhibiting phase I enzyme-encoding gene and facilitating the cell death of cancer cells through the TP53 mechanism [29]. In addition, SFN targeted nuclear factor kappa B (NF-κB) [30] and Activation protein-1 (AP-1) complexes with an anti-inflammatory effect [31]. Other synthetic oleanane triterpenoids inhibited tumorigenesis by inhibiting the transcription of oncogenes, including k-RAS, TP53, BRCA1, and Erb-B2 Receptor Tyrosine Kinase 2, in some cancers [32,33].

In fact, a lot of therapeutics that controlled the NRF2 activity have been currently applied in clinical trials; these drugs played a significant role in repressing the cancers and could be augmented further by chemo-preventive compounds. Paradoxically, NRF2-deficient cancer cells were more prone to death of cancerous cells by oxidative stress but more resistant to chemo-preventive compounds. Thus, the effective drugs targeting NRF2 pathways represented a crucial strategy to develop the useful chemo-preventive medications. However, we need accumulate the further pre-clinical data for convincing the efficiency of these drugs against cancer patients.

Nrf2-knockout mice showed that the level of nitric oxide synthase activated by cyclooxygenase 2 and the level of tumor necrosis factor (Tnf) are greater than those in normal mice, indicating that Nrf2 is able to suppress the proinflammatory molecules [34]. In addition, Nrf2-dependent activation of Nqo1 decreased the levels of Tnf and Interleukin 1 after incubation with lipopolysaccharide. Although ROS are the molecular targets of NRF2-mediated anti-inflammatory effects, NRF2 might function as an anti-inflammation mediator without inducing ROS, which were reported by regulating the expressions of axis encoding the macrophage receptor with collagenous structure and CD36, which are not concerned with the oxidative response [35]. Furthermore, NRF2 protected against cell damages induced by H₂O₂ exposure via the p38/MAPK pathway [36,37]. Similarly, NRF2 inhibited the NF-κB pathway by stabilizing the inhibitor of nuclear factor κB kinase subunit-α (IKK-α) to inhibit the degradation of IKK-β [38]. By contrast, the NF-κB p65 subunit was reported to compete with NRF2 through the CH-KIK domain of the cyclic AMP response element binding protein (CREB) binding protein CBP coactivator [39]. Thus, these different mechanistic inputs of NRF2 should be clarified to determine how it suppressed the oncogenicity by controlling the level of ROS within cells.

3.2. Oncogenic Functions of NRF2

NRF2 is usually constitutively activated in tumorigenic cells. For example, somatic mutations in KEAP1 and/or NRF2 genes, exon skipping in the NRF2 genome, methylation/demethylation of the KEAP1 promoter, accumulation of p62/sequestosome-1, and mutation of fumarate hydrolase were reported [40,41] Constitutively activated NRF2 promoted cell proliferation through metabolic changes; inhibiting apoptosis; promoting angiogenesis, and invasion/ metastasis; and promoting drug resistance in various cancers [40,41]. Forced expression of NRF2 promoted the transcriptions of the oncogenes like MYC, KRAS, and BRAF [42]. Moreover, NRF2 activated oncogenes by suppressing the activity of tumor suppressor gene products like phosphatase tensin homolog/glycogen synthetase kinase 3/β-transduction repeat-containing E3 ubiquitin-protein ligase [43]. Indeed, NRF2 increased the proliferation of tumor cells by increasing the expression of glycolytic enzymes included glucose-6-phosphate dehydrogenase, phosphoglucomutase dehydrogenase, transketolase and trans-aldolase [44], which controlled genes involved in fatty acid-lipid metabolism [45], growth-associated genes [46], and cell cycle regulators [47]. The activation of NRF2 via the glucose-regulated protein 78/phosphorylated protein kinase RNA-like ER kinase/NRF2 signaling pathway enhanced the transcriptions of glycolytic genes and simultaneously to induced the inhibition of the TCA cycle related genes, which enhanced the Warburg effect [48].

Increased levels of NRF2 suppressed the endogenous ROS level in cancer stem cells (CSCs) compared with noncancer stem cells, suggesting the enrichment of stemness phenotypes [49,50,51,52,53]. For example, the CSCs with reduced mitochondrial-derived ROS exhibited the cancer stemness-associated properties, such as epithelial-to-mesenchymal transition (EMT) [48,61]. Similarly, persistent activation of NRF2 improved the self-renew ability of CSCs, by maintaining the cell quiescence and reducing the intracellular levels of ROS [54,55]. Mesenchymal stem cells (MSCs) in the microenvironment cells promoted the spread of cancer cells by enhancing their motility and invasion ability [56,57]. Thus, NRF2 was required to maintain the stemness of MSCs and caused their cell deaths under the oxidative stress condition [58]. Furthermore, because the continuous expression of NRF2 significantly benefited the cancer cells, these cells frequently developed to be the NRF2 dependency [59,60].

In addition, NRF2 derived oncogenic activity also stimulated the angiogenesis capacity, mainly by activating heme oxygenase 1 (HO-1) [61], which in turn stimulated the vascular endothelial growth factor to enhance the angiogenesis [62]. siRNA against NRF2 downregulated HO-1 and sensitized the acute myeloid leukemia cells to TNF-induced cell death. This effect indicates that NRF2 inhibits the apoptosis of cancer cells by regulating HO-1 [63]. In addition, NRF2 upregulated the antiapoptotic protein B-cell lymphoma 2 (Bcl2), while it downregulated the activity of BAX and caspase 3 to protect against the etoposide/radiation-mediated cell death following drug resistance [64]. Furthermore, NRF2 inhibited the activation of proapoptotic JNKs [65] and induced selective autophagy reactions via Keap1 degradation [66,67]. However, the forced expression of NRF2 allowed the autophagy-dependent cancer cells to overcome the loss of autophagy function [68].

Increased accumulation of NRF2 in the nucleus was associated with proliferation capacity. For instance, phosphoinositide 3-kinase (PI3K)-Akt activation coupled with depletion of Keap1 resulted in Nrf2-dependent proliferation of hepatocytes and cholangiocytes [69,70]. However, because the ability of stable Nrf2 was not sufficient to alter from a cancer defender to a cancer driver, the additional mutations of oncogenes and tumor suppressor genes were absolutely required [71,72,73]. Keap1 mutations, such as Kras/Hras mutations and Trp53 loss of function, are required to produce the NRF2-dependent cancer models [74]. Furthermore, NRF2-dependent malignancies with somatic mutations of KEAP1 and NRF2 differed depending on the specific organs. Thus, tissue-specific variation is another issue for the study of NRF2-dependent cancer, which might be discussed further.

In resistance to medical therapy, NRF2-controled drug efflux transporters are predictors of resistance for drug inoculations. For example, multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1-5 (MRP1-5), and breast cancer-resistant protein (BCRP) are critical for controlling issues to regulate the therapy because they induced the abnormal NRF2 activation to induce the widespread chemoresistance [75,76,77,78]. These issues might be the complementary trials for drug delivery to the cancer cells.

4. NRF2-Tragetted Drugs in Cancer Prevention

In the initiation stage of cancer, NRF2 activation can suppress carcinogenesis. Under normal conditions, NRF2 maintains antioxidation homeostasis and exerts anti-inflammatory effects and antitumorigenic effects, thus supporting cell survival.

Heterogeneity of Antioxidation Drugs against Cancer

Wu et al. (2023) recently reported the heterogeneity of redox ligands controlling the growth and progression of human gastric cancer organoids [6]. They performed experiments to compare the efficiency of three drugs, perillaldehyde (PEA) and cinnamaldehyde (CE), and the regular antioxidative drug sulforaphane (1-isothiocyanato-4- (methane sulfinyl) butane; SFN), and measured cancer occurrence by assessing xenotransplantation in SCID mice. PEA extracted from Perilla frutescens [79] showed the antifungal, and antioxidant activities in addition to other biological functions [80,81,82]. PEA activates NRF2 and represses oxidative stress-induced innate immunity in human keratinocytes [83]. CIN is a β-unsaturated aldehyde from cinnamon [84]. In addition, the antioxidant activity of CIN was demonstrated in mice [85]. Exposure to CIN induced autophagy-dependent cell death through epigenetic alteration by the histone methylating enzyme G9a in the endoplasmic reticulum [86]. CIN inhibited the AHR axis and induced an NRF2-mediated antioxidation response [87]. Thus, PEA and CIN might function in NRF2-dependent antioxidation to inhibit ROS generation without affecting AHR-mediated oxidative stress pathway. SFN, as a dietary isothiocyanate synthesized from a precursor in Brassica, was also examined. It is one of the most typical ligands of phase II enzymes pathway including NQO1, GST α-1, and HO-1, which are required to eliminate chemically damaged DNA. SFN induced cell cycle arrest at S-phase and apoptosis in a TP53-dependent manner in gastric cancer (GC) cells [88]. In addition, SFN inhibited the metastasis of breast cancer [89].

Another issue is the differential outputs on function of AHR- and NRF2-mediated phase I and II pathways. Both PEA and CIN induced an NRF2-mediated phase II enzyme response, which was mediated by ARE-containing genes [86] but was not involved in the phase I pathway [87]. By contrast, the effects of SFN were reported to be involved in both enzymatic pathways; SFN produced NRF2-dependent phase II promoter activation and AHR-dependent activation of phase II promoters such as NQO1 [90,91]. This activation provides unambiguous evidence of the different actions of PEA/CIN and SFN in preventing cancer-inducing activity. It is surprising to observe the contrasting actions of PEA/CIN and SFN in inhibiting gastric cancer development, ROS generation, and cellular apoptosis. The TP53 mutation might explain these differential effects because 3-D organoids exhibited TP53 mutations, which might affect the alterations of phase I and phase II enzyme reactions (Figure 2).

5. Phase I Drugs in Clinical Trials

Phase I and II enzyme pathways are known to overlap and are regulated by the “ARF–NRF2” gene battery [92]. In addition, the AP1/ATF transcription factor and histone chaperone Jun dimerization protein 2 (Jdp2) played a role in regulating AHR promoter activity via the NRF2 complex [93]. This mechanism was called as the AHR–NRF2–JDP2 gene battery.

In the case of AHR therapeutics, the present application of AHR antagonists in cancer disorders is a reasonable strategy for preclinical trials. AHR antagonists inhibit immune suppression, and AHR agonists overcome chronic inflammation and autoimmune disorders in cancer patients. The AHR antagonist IK-175 is well known to be effective in an anticancer therapeutic trial when combined with anti-PD1 antibodies. A phase Ia/b open-label study of OK175, alone or in combining with nivolumab, which suppressed locally advanced solid tumors and urothelial carcinoma, has been reported (NCT04200963) [94]. The microbial metabolite indole-3-aldehyde (3-IAld) exhibited strong anti-inflammatory activity via AHR [95,96]. Compelling results with 3-IAld were obtained in a dextran sodium sulfate (DSS)-mediated inflammatory bowel disease of mouse model. 3-IAld repaired colon damage and improved epithelial barrier integrity through AHR, and IL22 cured the immune checkpoint inhibitor (ICI)-induced colitis but did not block the antitumor effect of ICI [97]. These data indicate that the AHR ligand of 3-IAld could be developed as an agent for treating human disease.

BAY 2416964 was a novel oral AhR inhibitor that prevent the AHR ligand-induced immunosuppressive effects and enhanced the proinflammatory activity of antigen-in-human phase I clinical trials [98]. Studies in vitro showed that BAY 2416964 restored immunological activity in human and mouse cells, stimulated antigen-specific cytotoxic T-cell responses, and killed tumors. Studies in vivo showed that its oral application was well tolerated and demonstrated antitumor activity in a syngeneic mouse cancer model.

6. Phase II Drugs in Clinical Trials

Clinical trials using phase II enzyme drugs have been reported. For example, SFN has achieved only limited success in prostate cancer patients [99]. However, SFN is not effective for treating breast cancer patients [100]. SFN inhibited the progression of GC [88,101,102,103,104]. Thus, further studies are required to determine the usefulness of SFN.

In addition, the mutation status of oncogenes and antioncogenes is also crucial for the efficacy of treatment with phase II enzyme drugs. The expressions of TP53, NRF2, and JDP2 were tremendously repressed in the growth of organoids exposed with PEA and CIN as compared with those in control normal organoids. However, a 1.5- to 1.75-fold increase in NRF2 and TP53 expression was found in SFN-treated organoids compared with in control organoids. Exposure to PEA or CIN repressed the expression of proteins of the TP53–NRF2 axis and a third factor histone chaperoneJDP2, but SFN increased the expression of TP53 and NRF2 proteins. In the case of a tumor, the p53 mutation itself did not induce the tumor formation, but tumors developing from areas with p53 mutation and loss of heterozygosity were larger and demonstrated extensive chromosomal instability compared with lesions arising in normal epithelium [105]. Most TP53 mutations in cancers are found as the missense mutations rather than truncations or deletions. Both dominant-negative effects and gain-of-function activities were observed in the case of mutant p53 [106]. Mutant TP53 interacts with NRF2, but positive or negative effects of NRF2 have been reported [107,108,109]. Non-small cell lung cancers carrying mutant TP53 but not mutant NRF2 or KEAPl displayed higher levels of NRF2 mRNA than wild-type TP53 tumors [110]. In similar, the oncogenes KRAS, BRAF, and MYC promoted the increased transcription of NRF2 gene and its target genes, which might induce a greater decrease in cellular molecules [42]. Mutant p53 prolonged TNFα-induced NF-κB signaling [111], and mutation of p53 can upregulate Nrf2 via the NF-κB axis. However, in 3-D gastric cancer organoids, PEA and CIN treatment reduced the protein expression of TP53 and Nrf2 and reduced ROS and caspase 3 activity [6].

By contrast, SFN exposure induced NRF2 and other redox functions to be dominant. Some reports have indicated that the levels of phase II enzymes, such as NQO1, are increased in cancer tissues compared with healthy tissues and that NQO1 stabilized the wild-type TP53, especially under oxidative stress [112]. Compared with those of wild-type TP53, the TP53 mutants exhibited increased binding to NQO1 [113]. By contrast, the function of NRF2 as an antioxidation response factor was blocked in the case of R273H in p53-expressing cancer cells upon oxidative stress, and the NRF2 antioxidant response was impaired because of the decreased expression of NQO1 and HO-1 [114].

Mutant p53 enhanced the Warburg effect by activating the glucose transporters GLUT5/6 and GLUT3 via NF-κB axis and GLUT1 by stimulating its transferring to the plasma membrane. Various glycolytic enzymes are also stimulated by mutant p53, which caused increased glucose uptake and glycolysis [115]. The effect of the microenvironment with p53 mutations can also affect cancer generation.

One TP53 mutant, APR-246, has been tested in clinical applications [116]. The APR-246 mutant inhibited thioredoxin reductase 1 (TrxR1) [117], thioredoxin (Trx), glutaredoxin, and ribonucleotide reductase [118] and depleted cellular GSH [116,119,120]. Recent studies revealed that APR-246 was not specific to cancer cells, but its effects might be cell-type specific [121].

In general, the expression of p53 counteracted the expression of ARE-containing antioxidant genes such as cystine/glutamate transporter (SLC7a11; x-CT), NQO1, and GST-α1, which are Nrf2 targets [122]. p53 deletion increased ROS, DNA oxidation, and mutations. The introduction of the dietary supplementation with the antioxidant N-acetylcysteine subsequently improved karyotype stability and prevented the early onset of mutations for tumorigenesis. Three p53 mutations, K117R, K161R, and K162R, were generated and resulted in impaired p53-mediated cell cycle arrest, senescence, and apoptosis. Unlike p53-knockout mice, this p53 mutant mice did not develop early-stage lymphoma [123,124]. KRAS depletion and the depletion of RalB and IκB-related TANK-binding kinase 2 (TBK1) induced to activate p53 in a ROS- and NRF2-dependent manner. Similarly, the IκB kinase inhibitor BAY 11-7085 and dominant-negative mutant IκB-αM inhibited the NF-κB activity and increased the levels of phosphorylated p53, p53, and p21^Cip1 in a ROS-dependent manner [125]. The p53–Mdm2 protooncogene (MDM2)–ARF network can lead to unconventional and unique innovative approaches for developing a new generation of genetically informed and clinically effective cancer therapies [126].

Wild-type TP53 repressed Jdp2 promoter activity, but TP53-knockout mutant cells did not, which was demonstrated using HCT116 p53^−/− cells [127]. JDP2 enhances the NRF2-dependent antioxidant response [128]. JDP2 functions not only as a transcription factor but also as a histone chaperone for targeting specific acetylated histones. In addition, it has inhibitory effects on p300-mediated histone acetylation [129]. Therefore, the expression of TP53 and JDP2 was reported to be coregulated during transcription and chromatin regulation. These alterations might influence the function of NRF2 in TP53-mutant cancer cells or organoids. Thus, the molecular interactions of mutated TP53, JDP2, and NRF2 should be clarified.

Therefore, the contradictory results with PEA or CIN and SFN should be studied at the molecular level to identify the distinct pathways involved in human GC.

7. Discussion

This review highlights the role of the transcriptional balance of key factors between cellular antioxidation and oxidation in cancer development. The dedicated interplay to control NRF2 and AHR master regulators of ROS homeostasis should be defined as the context-dependent nature of NRF2/AHR actions in each cancer case. Ingenuity pathway analysis (IPA) of the signal networks between AHR and NRF2 revealed that they connected to shared target genes, such as genes related to the cell cycle, and chromatin regulators, including cyclin-dependent kinase inhibitor 1 (CDKN1A; p21^CIP1) and EP300 (histone acetylase p300) (Figure 3). JDP2, such as CDKN1A, has been reported to play a role in cell cycle regulation by controlling cell growth via cyclin A2 [130] and p300/CBP acetyltransferase to function as a HAT on histones [129].

Given the complexity of antioxidation mechanisms in different cancer patients, the cancer types, and stages of cancer progression, manipulating redox status may be inappropriate. Thus, the precise regulation of redox balance is important and has become a key target in searching the next-generation of redox-regulating agents for cancer therapy. A further understanding of redox mechanisms in cancer initiation and progression is still challenging by repeating the examples to see the experimental validation.

A variety of responses to NRF2-mediated cancer development, which are obtained by 2-D cell culture, should be reexamined using 3-D organoids. These responses might be due to the intratumor heterogeneity of CSCs and to committed subpopulations with distinct microenvironments. The following questions should be addressed: (i) Do antioxidant exposures, as cancer treatments, demonstrate the positive and negative dual effects in cancer types, caner stages or the endogenous ROS dependent manner? (ii) Are there specific treatments with different doses in different tumor tissues that need treatment with antioxidant therapies for required clinical outcomes? (iii) Is JDP2–NRF2-induced metabolic reprogramming in response to phase II enzyme ligands are required for CSCs or niches which are dependent upon the status of TP53 mutation? Such questions are currently ongoing. The techniques used to measure endogenous ROS levels in vivo and in 3-D organoid tips might be the next targets for applying redox drug therapy for cancer treatment, in addition to further identifying markers for CSCs and microenvironments. These technologies might validate antioxidant therapy for tumors. Thus, we need more experiments of mechanistic analysis of phase II drugs against cancer development.