Biomedicine & Pharmacotherapy 176 (2024) 116829

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Review

Targeting Nrf2 signaling pathways in the role of bladder cancer: From

signal network to targeted therapy
Liang Wu a, b, Zhao Hu a, b, Xiao-fen Song a, b, Yu-jian Liao a, b, Jiang-huan Xiahou a, b, Yuan Li a, b,
Zhong-hua Zhang a, b, *
a
Department of Urinary Surgery, Xinyu People’s Hospital, 369 Xinxin North Road, Xinyu, Jiangxi Province 338000, PR China
b
Department of Urinary Surgery, The Affiliated Xinyu Hospital of Nanchang University, 369 Xinxin North Road, Xinyu, Jiangxi Province 338000, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Bladder cancer (BC) is the most common malignancy of the urinary system and often recurs after tumor removal
Nuclear factor erythrocyte 2-associated factor 2 and/or is resistant to chemotherapy. In cancer cells, the activity of the signaling pathway changes significantly,
(Nrf2) affecting a wide range of cell activities from growth and proliferation to apoptosis, invasion and metastasis. Nrf2
Bladder cancer
is a transcription factor that plays an important role in cellular defense responses to a variety of cellular stresses.
Therapeutic strategy
Drug resistance
There is increasing evidence that Nrf2 acts as a tumor driver and that it is involved in the maintenance of
Oxidative stress malignant cell phenotypes. Abnormal expression of Nrf2 has been found to be common in a variety of tumors,
including bladder cancer. Over-activation of Nrf2 can lead to DNA damage and the development of bladder
cancer, and is also associated with various pathological phenomena of bladder cancer, such as metastasis,
angiogenesis, and reduced toxicity and efficacy of therapeutic anticancer drugs to provide cell protection for
cancer cells. However, the above process can be effectively inhibited or reversed by inhibiting Nrf2. Therefore,
Nrf2 signaling may be a potential targeting pathway for bladder cancer. In this review, we will characterize this
signaling pathway and summarize the effects of Nrf2 and crosstalk with other signaling pathways on bladder
cancer progression. The focus will be on the impact of Nrf2 activation on bladder cancer progression and current
therapeutic strategies aimed at blocking the effects of Nrf2. To better determine how to promote new chemo­
therapy agents, develop new therapeutic agents, and potential therapeutic targets.

1. Introduction underlying molecular pathways involved in tumorigenesis [10,11].

Cancer is the greatest threat to public health worldwide and the
leading cause of human death [1,2]. The occurrence of cancer is due to
genome mutation and abnormal expression of genetic factors caused by
exogenous and endogenous factors, leading to malignant transformation
of normal cells into cancer cells [3,4]. Bladder cancer (BC) is the most
common malignancy of urinary system, with high morbidity and mor­
tality [5]. According to the World Health Organization (WHO) annual
report, approximately 81,400 patients in the United States will develop
bladder cancer by 2030, most of whom will be men (approximately 62,
100) [6]. Bladder cancer is mainly divided into non-muscle-invasive
bladder cancer and muscle-invasive bladder cancer [7]. The main treat­
ments for bladder cancer are surgery, chemotherapy and radiation.
However, these traditional therapies are not efficient enough in fighting
cancer, making tumor cells prone to recurrence and migration [8,9].
Therefore, gene therapy has been introduced as a new tool to regulate the
Multiple studies have shown that a variety of signaling pathways have
been reported to be abnormally expressed in bladder cancer and involved
in the transformation and phenotypic progression of bladder cancer [12,
13]. Among them, nuclear factor erythrocyte 2-associated factor 2 (Nrf2)
is an important signaling pathway for the occurrence of bladder cancer
[14]. Recent experimental studies have confirmed that DNA mutations or
methyl groups can induce changes in the expression of Nrf2 signaling
molecules, which may be related to bladder malignant tumors [15,16].
These findings highlight the potential value of Nrf2 in bladder cancer.
Nrf2 is an important transcriptional regulator, which plays a variety
of roles in REDOX homeostasis, protein degradation, DNA repair, and
regulation of exogenous metabolism and immune response [17,18].
Nrf2 has become a prime target for research in the fields of inflamma­
tion, cancer metabolism, cancer prevention and cancer therapy [19].
Normally, the activation of Nrf2 prevents the development of cancer.
However, in some types of cancer Nrf2 can increase Nrf2 activity

* Corresponding author at: Department of Urinary Surgery, Xinyu People’s Hospital, 369 Xinxin North Road, Xinyu, Jiangxi Province 338000, PR China.
E-mail addresses: 290163901@qq.com (L. Wu), 18807905594@139.com (Z.-h. Zhang).

https://doi.org/10.1016/j.biopha.2024.116829
Received 3 October 2023; Received in revised form 9 May 2024; Accepted 26 May 2024
Available online 30 May 2024
0753-3322/© 2024 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
L. Wu et al.
through gene mutation [20]. Studies have shown that Nrf2 can be
over-activated by various factors in bladder cancer [21]. Over-activation
of Nrf2 involves the transformation of normal cells into cancer cells
through changes in cell metabolism and functional adaptation [22]. It
can also cause cancer cell proliferation and protect tumor cells from
oxidative stress, chemotherapy drugs and radiation therapy, as well as
chemotherapy resistance and poor prognosis [23] (Fig. 1). Therefore,
Nrf2 targeting cancer progression could provide a new perspective for
designing more effective drugs [24]. In this review, we reviewed the
biological characteristics of Nrf2, the role and mechanism of Nrf2 in the
pathological process of bladder cancer, and some extracts targeting Nrf2
to improve bladder cancer research progress. The effective treatment
strategies for bladder cancer with abnormal Nrf2 expression were also
discussed. It may provide a new idea for the clinical treatment and
development of Nrf2 targeting drugs.
2. Nrf2 signaling pathway
Nrf2 is a ubiquitous transcription factor, belonging to the tran­
scription factor cap’-n’-collar (CNC) family, an alkaline leucine zipper
protein located in the chromosome 2q31.2 region and containing 605
amino acids [25]. The Nrf2 protein sequence is highly conserved across
species and is usually distributed in the cytoplasm of mammalian cells
and is essential for maintaining cell homeostasis [26,27]. The functional
components of the Nrf2 signaling system include: Nrf2 protein
(Kelch-like ECH associated protein 1 (Keap1)) and antioxidant response
elements (ARE). Keap1 is a cysteine-rich cytoplasmic protein of
Cullin3-dependent E3-ubiquitin ligase complex and an intrinsic inhibi­
tor of Nrf2 [28]. The ARE sequence is located in the regulatory region of
genes involved in cell growth, oxidation and detoxification responses,
metabolism, immune response, cell survival, signaling and cell cycle
[29]. Nrf2 forms a heterodimer with small Maf protein, which is
responsible for binding to ARE and inducing NQO1, HO-1 and GST[30].
Nrf2 contains seven highly conserved Neh domains (Neh1-Neh7), which
are thought to be critical for its action and inhibition [31]. Among them,
the Neh1 domain mediates the binding of Nrf2 to the ARE sequence, and
Fig. 1. Under normal circumstances, Nrf2 has anticancer effect on normal cells through its target gene, and the overexpression of Nrf2 has carcinogenic effect on
transformed malignant cells.
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Biomedicine & Pharmacotherapy 176 (2024) 116829
the Neh2 domain mediates the interaction with the negative Nrf2
regulator Keap1 [32]. The C-terminal Neh3 domain collaboratively ac­
tivates the transcription of Nrf2 target genes with the Neh4 and Neh5
domains through reverse transcriptional activity. The Neh6 domain
participates in the negative regulation of Nrf2 stability, while the Neh7
domain inhibits Nrf2 transcriptional activity [33]. Nrf2 protein has 3
nuclear localization sequences (NLS) and 2 nuclear export sequences
(NES), which can bind to import and export proteins respectively [34].
Under normal non-stress conditions, Nrf2 in the cytoplasm promotes
ubiquitination by binding to the repressor molecule Keap1 and via E3
ubiquitin ligase. It exists in the cytoplasm in the form of inactive com­
plexes to maintain cell homeostasis through ROS-dependent oxidation
process [35]. However, under abnormal conditions, the cysteine resi­
dues of Keap1 are modified by electrophilic agents, oxidants, and
pro-inflammatory cytokines, prompting a conformational change of
Keap1 and its dissociation from Nrf2, resulting in the translocation of
Nrf2 to the nucleus [36–38]. After nuclear translocation, Nrf2 plays an
important role in the cellular defense system by controlling the
expression of antioxidants and detoxification genes in normal cells to
control allobiotics and oxidative stress conditions [33] (Fig. 2). How­
ever, in cancer cells, the Nrf2 gene is mutated in the DLG and ETGE
motifs of the Neh2 domain [39]. It leads to increased Nrf2 activity in
cancer cells, providing cell protection and promoting the survival and
proliferation of cancer cells [20].
3. Nrf2 pathway in bladder cancer
Under physiological conditions, cells in the body can eliminate free
radicals and active metabolites produced by cell metabolism, such as
ROS and RNS. If the amount of these active substances exceeds the
antioxidant capacity of the cell, it will cause an imbalance [40]. Under
normal conditions without stress, low expression of Nrf2 in cytoplasm
can maintain cell homeostasis. However, in cancer cells, the metabolism
of cancer cells is not regulated by genes, resulting in continuous aerobic
glycolysis and pyruvate oxidation in mitochondria, resulting in exces­
sive ROS and over-activation of antioxidant defense, resulting in Nrf2
L. Wu et al. Biomedicine & Pharmacotherapy 176 (2024) 116829

Fig. 2. (A) Schematic diagram of the structure of Nrf2, which contains seven highly conserved domains called Neh domains. Neh1 is required for the formation of
complexes with the transcription factor s-Maf, DNA binding, and binding to ubcm2. Neh2 contains the ETGE and DLG sequences required for keap1 binding, as well
as seven ubiquitin lysine residues that target Nrf2 for proteasomal degradation. Neh3 is required for transcriptional activation (CHD6 binding). Neh4 and Neh5 are
trans-active domains that bind activators (CBP, RAC3) or repressors (GR, HRD1). Neh6 regulates Nrf2 stability by binding to β-TrCP. Finally, Neh7 can interact with
the Nrf2 repressor protein RXRa. (B) Schematic diagram of Nrf2/keap1 signaling pathway.

gene mutation and abnormal Nrf2 expression. The abnormal expression
of Nrf2 can lead to oxidative stress disorder and disorder, the imbalance
of cellular antioxidant capacity and cancer-promoting procedures, thus
stimulating the malignancy of cancer cells/tissues [41–43]. Nrf2 is
abnormally activated in bladder cancer, and high expression of Nrf2
leads to an increase in cancer cell progression and proliferation and a
decrease in the survival rate of bladder cancer patients [44]. Koza­
kowska M et al. [45] found that compared with healthy tissues, the
expression of Nrf2 in bladder cancer was up-regulated along with HO-1
and HIF. Nrf2 activation has also been shown to increase HIF-1α in
bladder cancer tissues, thereby enhancing the proliferation, migration
and invasion of bladder cancer cells [46,47]. In addition, elevated Nrf2
levels lead to decreased overall survival and disease-free survival in
patients with bladder cancer, and inhibition of Nrf2 can reverse these
results [48]. In addition, over-activation of Nrf2 can lead to bladder
cancer resistance by inhibiting ROS-dependent DNA damage [21]
(Fig. 3). Based on these results, it is not difficult to see that inhibition of
Nrf2 is beneficial to the inhibition of bladder cancer. However, more
experimental data are needed to definitively evaluate Nrf2 as a thera­
peutic target [15].
4. Crosstalk with other signal pathways
Studies have confirmed that a variety of signaling pathways are
involved in the regulation of the pathological process of bladder cancer.
Including NF-κB and YAP pathways [49–51]. There are interactions
between signaling pathways, and the enhancement of one signaling
pathway may enhance or inhibit another pathway. In bladder cancer,
Nrf2 pathway can interact with other cell signal transduction pathways.
4.1. NF-κB signaling pathway
Nuclear factor κ-B (NF-κB) is an important signaling pathway
involved in many biological processes, such as immune and inflamma­
tory responses, proliferation, apoptosis, and EMT [52]. Activation of the
NF-κB pathway has been shown to be critical to bladder cancer-related
mechanisms, and activation of NF-κB signaling can promote tumor
progression by stimulating cancer cell proliferation, inhibiting cell
death, and promoting mutation accumulation in the long term [53].
After phosphorylation of IκB, NF-κB is released, enabling nuclear
translocation and involvement in bladder cancer cell proliferation,
migration and angiogenesis [54]. There is growing evidence of crosstalk
between Nrf2 and NF-κB signaling pathways [55]. Related studies in
bladder cancer have also reported that the interaction between Nrf2 and
NF-κB signaling pathway can jointly participate in the regulation of
disease course [56,57]. HO-1 is an important NrF2-regulated antioxi­
dant enzyme, and the effect of HO-1 on the expression of
pro-inflammatory cytokines is related to the inactivation of the NF-κB
pathway [58]. HO-1 restricts the activity of NF-ĸB by inhibiting the
degradation of IĸB and HO-1 effectively inhibits the phosphorylation
and subsequent degradation of IkB, resulting in reduced transcription of
NF-ĸB translocation and its downstream inflammatory genes [59]. In
addition, it has been reported that NF-ĸB inhibits Nrf2 signaling at the
transcriptional level [60]. NF-ĸB competitive deprivation of Nrf2’s
CREB-binding protein (CBP) results in Nrf2 inactivation, and NF-ĸB
recruits histone deacetylase 3 (HDAC3), resulting in local hypo­
acetylation that blocks Nrf2 signaling [60]. Crosstalk between Nrf2 and
NF-κB signaling has also been found in bladder cancer studies.

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L. Wu et al.
Fig. 3. In cancer cells, excessive ROS production dissociates keap1 and Nrf2, resulting in the translocation of Nrf2 to the nucleus to bind to ARE and S-Maf, thus gene
transcription, leading to angiogenesis and migration. At the same time, the overexpression of Nrf2 can lead to resistance to chemotherapy drugs.
4.2. YAP signaling pathways
YAP is a key component of the Hippo tumor inhibition pathway and
is involved in multiple tumor cell proliferation and chemotherapy
resistance [61]. YAP activation can induce cancer stem cell prolifera­
tion, chemotherapy resistance and metastasis, and its inhibition can
increase sensitivity to chemotherapy [62]. In bladder cancer, YAP is
activated, and the activated cells are involved in bladder cancer cell
proliferation and migration. Some reports have shown that there is a
crosstalk between YAP and Nrf2 that jointly participate in the patho­
logical process of bladder cancer [63]. FOXM1 is the downstream target
of YAP [64]. Overexpression of FOXM1 is associated with bladder cancer
and plays a crucial role in regulating cell proliferation, differentiation,
transformation and drug resistance [65,66]. YAP directly induces
FOXM1 transcription in a TEAD-dependent manner, and the activated
FOXM1 acts as the upstream gene of Nrf2 to transcribe and activate Nrf2
[67]. Upregulation of YAP in bladder cancer cells was accompanied by
an increase in the GSH/GSSG ratio and Nrf2 expression, while elimi­
nation of YAP decreased the GSH/GSSG ratio and Nrf2 expression. It
also reduces cell viability, increases apoptosis induction and inhibits
migration of chemotherapy-resistant bladder cancer cells [68,69].
5. Role of Nrf2 pathway in the development of bladder cancer
5.1. Metastasis
Cancer metastasis is the spread of cancer cells from the primary
tumor to distant sites and is an important cause of cancer-related death.
Metastasis involves a variety of molecular mechanisms, such as
epithelial-mesenchymal transformation (EMT) and lost-nest apoptosis
[70]. EMT is a key process associated with cancer metastasis and
treatment resistance. Adhesion protein e-cadherin and matrix metal­
loproteinase (MMP) are important substances in cancer metastasis. The
expression of adhesion protein e-cadherin is conducive to the inhibition
of EMT, while the increase of MMPs can lead to the migration and
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Biomedicine & Pharmacotherapy 176 (2024) 116829
metastasis of cancer cells [71,72]. Overactivated Nrf2 in cancer cells can
down-regulate e-cadherin and thus increase EMT, thereby increasing the
ability of metastasis [73,74]. More and more evidence shows that Nrf2 is
involved in the process of tumor invasion and metastasis. In bladder
cancer, highly expressed Nrf2 participates in the ARE sequence binding
of the Notch1 promoter region, enhancing the metastasis and invasion
ability of cancer cells and the EMT process [22,75].
5.2. Angiogenesis
Angiogenesis (development of new blood vessels) is a key process in
the growth and progression of tumor cells, and tumor progression and
invasion are usually accompanied by the production of new blood ves­
sels [76]. Another important factor in angiogenesis is hypoxia, which
activates angiogenic mediators such as HIF and VEGF transcription
factors [77]. Hypoxia-inducible factor 1-α (HIF-1α), a major transcrip­
tion factor responsible for adaptive hypoxic conditions, is activated
under hypoxic conditions in tumor cells, thereby stimulating the tran­
scription of various growth factors and extracellular matrix (ECM)
remodelers to create vascularis [78]. Hypoxia triggers Nrf2/ARE
pathway and promotes tumor vascular development. Forceful blocking
of HIF-1 signaling in the absence of Nrf2 results in a decrease in capillary
density [79]. Meanwhile, activated Nrf2 induces angiogenesis by stim­
ulating HIF-1α-dependent vascular endothelial growth factor (VEGF)
expression in cancer cells and promoting cancer growth [80,81].
5.3. Drug resistance
Chemotherapy resistance is an important and serious problem for the
success of chemotherapy, as well as an important factor affecting tumor
prognosis [82]. However, down-regulation of Nrf2 by inhibiting the
expression of certain target genes can induce its differentiation and
make it sensitive to chemotherapy drugs [83,84]. The role of Nrf2 in
chemotherapy resistance has been demonstrated in several types of
cancer, including ovarian, lung, and cisplatin resistant bladder cancer
L. Wu et al. Biomedicine & Pharmacotherapy 176 (2024) 116829

[85–87]. For example, Nrf2 is involved in cisplatin resistance in ovarian inhibitors may be an effective therapeutic strategy.
cancer, and its knockdown makes ovarian cancer cells sensitive to
cisplatin treatment [88]. In addition, in lung cancer cells, forced acti­
6.1. Potential Nrf2 inhibitors in bladder cancer
vation of Nrf2 induces chemotherapy resistance, while deletion or
Keap1 overexpression enhances sensitivity [89,90]. In addition, studies
Studies have shown that a large number of related inhibitors can play
have found that stable overexpression of Nrf2 leads to increased resis­
a therapeutic role in bladder cancer by inhibiting Nrf2 pathway activity
tance of cancer cells to chemotherapy drugs such as cisplatin, adria­
(Fig. 4). Glutathione (GSH) maintenance is essential for the genesis,
mycin and etoposide [90]. Cisplatin is the core treatment for metastatic
progression, survival, proliferation and metastasis of cancer cells [97,
bladder cancer; however, cisplatin resistance is a common clinical
98]. Sang et al. have demonstrated that Jolkinolide B can significantly
problem [91]. Studies related to bladder cancer have also found that
enhance cisplatin sensitivity of cisplatin resistant bladder cancer cells by
drug resistance in bladder cancer cells is often accompanied by
inhibiting GSH synthesis in bladder cancer, increasing intracellular ROS
over-activation of Nrf2 [92]. Nrf2 promotes resistance to cisplatin by
levels and activating endoplasmic reticulum stress-induced apoptosis
inducing biosynthesis of the cell protective enzyme glutathione (GSH)
[99]. Mesulfamide (MTX-211) is an epidermal growth factor receptor
[87]. Taurine up-regulated gene 1 (TUG1) is an oncogene involved in
inhibitor and may be a potential antitumor agent. In bladder cancer,
tumorigenesis and is responsible for chemotherapy resistance [93].
MTX-211 can effectively inhibit cell proliferation and induce late cell
TUG1 is associated with doxorubicin resistance in bladder uroepithelial
apoptosis by promoting keap1-mediated Nrf2 ubiquitination degrada­
carcinoma (UCB) [94]. It was found that TUG1 was overexpressed in
tion, inhibiting Nrf2 pathway and reducing GSH levels [100]. Aila is a
UCB, and Nrf2 was also found to be positively correlated with TUG1
natural compound extracted from the Ailanthus plant and can inhibit the
expression in UCB tissues, and Nrf2 positively regulated the expression
proliferation of different cancer cell lines [101,102]. Reports have
of TUG1, thus leading to adriamycin resistance in UCB cells. while
shown that Aila is an effective Nrf2 inhibitor, which can inhibit the
knocking down Nrf2 re-sensitized adriamycin resistant UCB cells to
progression of bladder cancer and overcome the chemotherapy resis­
adriamycin therapy [92]. In addition, Ciamporcero E et al. [69] found
tance of bladder cancer [63,68]. Aila strongly reduces the expression of
that after induction of cisplatin resistance in bladder cancer cells, Nrf2
Nrf2, YAP and c-Myc in bladder cancer cells to inhibit the proliferation
expression increased significantly, while silencing Nrf2 could
and migration of cisplatin-resistant bladder cancer cells [68]. In addi­
re-sensitize chemotherapy-resistant cells to cisplatin treatment. Subse­
tion, some biomolecules have also been reported in bladder cancer to
quent studies also confirmed that inhibiting the expression of Nrf2 could
reverse cancer cell resistance to cisplatin. For example, neural precursor
effectively reverse the resistance of bladder cancer cells to cisplatin [63,
cell expressed developmentally down-regulated 4-like (NEDD4L) is an
95,96]. Therefore, Nrf2 may be a promising target for overcoming
E3 ubiquitin ligase, It plays an important role in various biological
chemotherapy resistance. Based on these findings, targeting Nrf2 and
processes such as development, tumorigenesis and tumor progression
reducing its function in cancer cells may be a functional strategy.
[103]. In bladder cancer, NEDD4L inhibits the functions of cell viability,
apoptosis, migration and invasion of bladder cancer cells and reverses
6. Treatment of bladder cancer targeting Nrf2 pathway
the resistance of cancer cells to cisplatin by inactivating the Nrf2
signaling pathway [95]. Tumor necrosis factor-associated apoptosi­
Based on the above description, the overactivation of Nrf2 in bladder
s-inducing ligand (TRAIL), a member of the TNF superfamily, can induce
cancer can promote the occurrence and development of cancer cells and
apoptosis in tumor or infected cells [104]. TRAIL can effectively inhibit
induce the resistance of cancer cells to chemotherapy drugs. Numerous
Nrf2 nuclear translocation, thus promoting apoptosis of cancer cells
studies have shown the efficacy of targeting Nrf2 in a variety of diseases.
[105]. GULP1 (phagocytic junction protein 1 containing PTB domain) is
Therefore, the down-regulation of Nrf2 in cancer cells by various
a human homolog of death of Caenorhabditis elegans 6 (CED6) [106]. In

Fig. 4. Related inhibitors can play a therapeutic role in bladder cancer by inhibiting Nrf2 pathway activity. Neural precursor cell expressed developmentally down-
regulated 4-like (NEDD4L), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), Phagocytic junction protein 1 containing PTB domain (GULP1).
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L. Wu et al. Biomedicine & Pharmacotherapy 176 (2024) 116829

bladder urothelial carcinoma (UCB), silencing of GULP1 promotes nu­
clear accumulation of Nrf2, leading to sustained activation of Nrf2
signaling and conferring cisplatin resistance. While overexpression of
GULP1 can effectively inhibit the expression of Nrf2, thereby reversing
cisplatin resistance in UCB cells [107]. In addition to reversing cisplatin
resistance, inhibitors can also inhibit cancer progression and play a
protective role in bladder cancer. For example, thymosin-alpha (PTMA)
is a small acidic nuclear protein, and loss of nuclear PTMA expression in
human bladder tumors is an adverse prognostic indicator of shorter
disease-free survival. Nuclear PTMA can play a tumor suppressive role in
bladder cancer by down-regulating the expression of Nrf2 in bladder
cancer [108]. In addition, abnormal expression of some lncrnas is
closely related to the progression and prognosis of bladder cancer [109].
For example, bladder cancer apoptosis-associated transcript (AATBC) is
a novel LncRNA that is overexpressed in bladder cancer tissues and
cancer cell lines [110]. AATBC expression was positively correlated with
tumor grade and pT stage of bladder cancer. While AATBC knockout
leads to increased proliferation inhibition and apoptosis in vitro and
inhibits tumor growth in vivo [111]. The mechanism is that the
knockout of AATBC plays a protective role in bladder cancer by
down-regulating Nrf2, thereby inhibiting the proliferation of cancer
cells and promoting apoptosis of cancer cells [112] (Table 1).
6.2. Potential strategies to overcome drug resistance in bladder cancer
6.2.1. Endogenous molecular inhibitors
Research has reported that various endogenous molecules, such as
tumor protein P53 [113], activating transcription factor-3 [114],
E-cadherin [115], caveolin-1 [116] and GSK3 [117] are usually neces­
sary for normal cellular homeostasis. The activation or activation of
these molecules can down-regulate Nrf2 to play a protective role by
disrupting the Nrf2 pathway and its expression levels through different
mechanisms. For example, E-cadherin enables Keap1 to recruit Nrf2 via
β-catenin to reduce endogenous Nrf2 levels, thus playing a role in
chemotherapy resistance. The activation of P53 can directly or indi­
rectly down-regulate Nrf2 and produce higher levels of ROS, thus pro­
moting apoptosis. It is important to note that the cell biology of Nrf2
associated with cancer should be thoroughly understood before using
these endogenous molecules for therapy.
6.2.2. Exogenous natural inhibitors
Due to the complexity of the tumor microenvironment, endogenous
inhibitors are largely limited. Therefore, some exogenous inhibitors
have gradually entered the research scope of scholars, such as exogenous
natural inhibitors. Some exogenous natural inhibitors not only inhibit
Nrf2, but also promote the sensitivity of resistant cancer cells to anti-
cancer drugs. For example, vitamin C plays an antioxidant role in the
tumor microenvironment to inhibit oxidative stress and combines with
ARE to inhibit nuclear translocation of Nrf2, thereby reducing the level
of GSH and reversing the resistance associated with imatinib treatment
[118]. It was observed that Nrf2 resistant cancer cells had high levels of
Nrf2 and other downstream ARE driver genes, such as NQO-1 and HO-1.
Some natural inhibitors, such as cryptotanshinone and luteolin, have
been found in relevant cancer studies to significantly inhibit the
expression of Nrf2 and other downstream ARE driver genes (NQO1 and
HO-1) in cancer cells, thereby improving the resistance of cancer cells to
chemotherapy drugs [119,120]. However, the feasibility and safety of
these natural inhibitors in bladder cancer need to be further studied.
6.2.3. Nanocarrier delivery system
Nanostructured delivery systems appear to be a potentially viable
strategy to overcome drug resistance [121]. Nanocarriers can protect
siRNA from degradation during systemic circulation and transport
siRNA to target cells to avoid non-specific delivery [122]. In addition,
through the EPR (Enhanced Permeability and retention) effect, these
particles can accumulate better at the tumor site over a longer period of
time, thus providing extended drug release [123,124]. In a recent study,
the use of specific small interfering RNA (siRNA) loaded into nano­
carriers that target the Nrf2 gene (siNrf2) is an attractive alternative
because it can improve specificity. Experimental results showed that
siNrf2 loaded on guanidine carbosilane dendrimers could rapidly enter
cells and inhibit the expression of Nrf2, thereby increasing the sensi­
tivity of cancer cells to cisplatin and reducing the migration of cisplatin
resistant bladder cancer cells caused by high Nrf2 levels [125]. Although
dendritic polymers loaded with Sirnas for fighting cancer disease have
not yet been approved for clinical use. But it’s not hard to find a
promising delivery system for in-vivo gene therapy drugs.
7. Combination treatment strategy
`Due to the complexity of the tumor microenvironment, it is difficult
to control the progression and recurrence of cancer with a single tradi­
tional treatment. Therefore, the combination treatment strategy has
gradually become an inevitable trend in cancer treatment [126]. Through
combination therapy, Nrf2 signaling pathway inhibitors can not only
restore the sensitivity of tumor cells to growth, but also increase the
sensitivity of tumors to chemotherapy, radiation, and hormone therapy.
7.1. Combination with chemotherapy

Table 1
Nrf2 signaling pathway inhibitors may improve the efficacy of
Related inhibitor that Inhibition Nrf2 signaling with potential protective effects
chemotherapy and induce apoptosis by destroying the protective barrier
on bladder cancer.
of drug resistance in cancer cells. Aila can inhibit drug resistance in
Inhibitors Model Mechanisms Effects and Outcome Refer- cancer cells by interfering with the interaction of p23 and heat shock
ences
protein 90 (Hsp90) [127]. In bladder cancer, the combination of Aila
MTX-211 Cell, Inhibit Nrf2 Inhibit proliferation and [100] and cisplatin can effectively reverse the tolerance of bladder cancer cells
Mouse signaling induce apoptosis of
to cisplatin, induce cell cycle stagnation in the G0/G1 phase and in­
cancer cells
Aila Cell, inhibits Nrf2, Inhibit proliferation, [63,68] crease cell apoptosis [128]. Curcumin is a compound with great po­
Mouse YAP and migration and cisplatin tential in inhibiting chemotherapy resistance, and combined with
c-Myc resistance cisplatin has a synergistic inhibitory effect on bladder cancer cells.
NEDD4L Cell, Inhibit Nrf2 Inhibit proliferation, [95] Curcumin reduces the tolerance of cancer cells to cisplatin by
Mouse signaling migration and reverse
cisplatin resistance
down-regulating Nrf2 and reducing the expression of NrF2-mediated
TRAIL Cell, Inhibit Nrf2 Promote apoptosis [105] typical phase I enzymes (GSTP1 and NQO1). It can also enhance the
Mouse signaling sensitivity of tumor cells to chemotherapy drugs [129]. The results of
GULP1 Cell, Overexpression Reverse resistance [107] these trials may further our understanding of this combination and
Mouse GULP1 inhibits
provide new insights into strategies to overcome resistance.
Nrf2 signaling
Thymosin- Cell, Inhibit Nrf2 Inhibit proliferation and [108]
α Mouse signaling migration 7.2. Combined with radiation therapy
• AATBC Cell, Down-regulate Inhibit proliferation and [112]
Mouse AATBC inhibits promote apoptosis Radiation therapy is an important treatment strategy for cancer,
Nrf2 signaling

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L. Wu et al.
which can improve the cure rate of cancer patients [130]. Despite ad­
vances in radiation therapy, cancer mortality remains high [131].
Studies have shown that overexpression of Nrf2 can promote radiation
resistance [132]. A recent study showed that the Nrf2 inhibitor diallyl
disulfide can inhibit the activation of Nrf2 signaling and can be safely
combined with radiotherapy to improve radiotherapy outcomes in
NSCLC patients [133]. In conclusion, the combination of Nrf2 signaling
pathway inhibitors with radiation therapy may be a promising cancer
treatment.
7.3. Combination with immunotherapy
Cancer is a genetic disease and a chronic immune disease [134].
Immunotherapy can improve the ability of immune cells to kill tumor
cells, so that the body can play a direct anti-tumor role. Previous studies
have shown that BCG is a significantly effective treatment for bladder
cancer due to its ability to effectively reduce tumor activity [135,136].
Arsenic trioxide (As 2 O 3) has been reported to inhibit bladder cancer
[135]. In bladder cancer, As2O3 combined with BCG can inhibit the
Nrf2 pathway to improve the production of dendritic cells and increase
the number of CD4 + T cells, which can promote the apoptosis of
bladder tumor cells and reduce the volume of tumors [137]. However,
these findings are preliminary and the underlying mechanisms remain to
be further elucidated.
8. Conclusion
Bladder cancer (BC) is one of the most common tumors of the urinary
system, and traditional treatment is currently the main treatment
method. Due to the complexity of the tumor microenvironment, multi­
ple signaling pathways acting on the cancer pathological process make
these traditional therapies not efficient enough in anti-cancer. Up-
regulation/down-regulation of various pathways in cancer cells can
directly regulate their ability to proliferate, angiogenesis, metastasis,
and multidrug resistance, all of which are important factors in cancer
development. Under normal conditions, Nrf2 is a key transcription
factor in cells, and its induced gene expression program plays a key role
in cellular defense responses to a variety of cellular stresses, most
importantly oxidative stress. While under abnormal conditions, over-
activation of Nrf2 can lead to DNA damage and tumor development.
Multiple studies have confirmed the abnormal expression of Nrf2 in
bladder cancer, and as a tumor driver, the abnormal expression of Nrf2
can initiate cancer-promoting procedures to induce cancer cell pro­
gression, metastasis, angiogenesis and chemotherapy resistance, as well
as reduce the survival rate of patients. While inhibiting Nrf2 activity in
bladder cancer can reverse this process. This suggests that Nrf2 is a
carcinogenic agent and the Nrf2 pathway may be a potential target or
mechanism for bladder cancer therapy.
Funding
This work is not supported by any institution and funding.
CRediT authorship contribution statement
Liang Wu: Writing – original draft, Writing – review & editing. Zhao
Hu: Software. Xiao-fen song: Formal analysis. Yu-juan liao: Method­
ology. Jiang-huan Xiahou: Software. Yuan Li: Formal analysis.
Zhonghua Zhang: Writing – original draft, Writing – review & editing.
Declaration of Competing Interest
Liang Wu, Zhao Hu and Zhong-Hua Zhang conceived and designed
the review. Liang Wu drafted the manuscript; Xiao-fen Song and Yu-juan
Liao assisted in the preparation of the charts; Jiang-huan Xiahou and
Yuan Li edited and revised the manuscript. All authors read and
7
Biomedicine & Pharmacotherapy 176 (2024) 116829
approved the final manuscript.
All authors have declared that they have not received any funds or
other support from any organization that may be interested in the sub­
mitted works, and that there are no other relationships or activities that
may affect the submitted works.
Data availability
No data was used for the research described in the article.
Acknowledgements
Not applicable.
Ethics Approval and Consent to Participate
Not applicable.
Consent for Publication
Not applicable.
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