Mechanistic review of sulforaphane as a chemoprotective agent in bladder cancer

Abstract

Regular consumption of cruciferous vegetables has numerous health benefits, including reduced cancer risk and improved patient outcomes. Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with a chemoprotective role against epithelial cancers, particularly of the bladder. Epithelial cells have several functions, including secretion, absorption, filtration, and protection from environmental insults. The specialized stratified epithelium of the bladder has direct and frequent contact with carcinogenic agents, increasing the likelihood of cancer initiation at this site. Carcinogen exposure, particularly from cigarette smoke or occupational exposure to aromatic amines, are the most significant risk factors for bladder cancer due to their ability to activate inflammatory pathways, induce free radicals, and damage DNA. SFN acts as an antioxidant by activating phase II enzymes involved in carcinogen detoxification to prevent DNA damage and inhibit tumor initiation, modulates multiple signaling pathways to inhibit tumor growth and progression, and has anti-inflammatory and immune-modulating properties to help protect against cancer. Due to these chemoprotective mechanisms, SFN has been studied as both mono- and adjuvant therapy in several bladder cancer models. Here we present a review of the effects of SFN on carcinogen-induced bladder cancer to support the inclusion of cruciferous vegetables as a chemoprotective strategy.

Introduction

Bladder cancer incidence and risk factors

Bladder cancer is the sixth most common cancer in the US and is more prevalent in males than in females [1-3]. The estimated incidence of bladder cancer and related cancer deaths in the US in 2023 are 82,290 and 16,710, respectively [2]. Clinically, patients often present with painless hematuria but may also experience urinary urgency, frequency, or dysuria. Patients with a late-stage disease may present with lymphadenopathy, flank pain, and weight loss [4,5]. Annually, 90% of bladder cancer diagnoses are urothelial carcinoma. Most cases (70%) will not be muscle-invasive at the time of diagnosis, but there is a 50-80% chance of bladder cancer recurrence within five years of diagnosis in these patients. Approximately 33% of newly diagnosed bladder cancers are muscle-invasive, and 4% present as distant metastatic disease [5,6]. The five-year overall survival rate for non-muscle invasive bladder cancer is 77%. However, patients with distant metastases have a five-year survival rate as low as 6% [7].

The most significant risk factor for the development of bladder cancer is a history of cigarette smoking, which is responsible for approximately 50% of bladder cancer cases in both men and women. Those who smoke have a three-times greater chance of being diagnosed with bladder cancer than those who do not smoke [8,9]. In older male populations, a 65+ pack-year history of smoking leads to a relative risk of 3.6 for bladder cancer compared to never-smokers (95% confidence interval [CI] = 2.2-4.6) [10]. The increased risk may, in part, be explained by increased DNA damage caused by smoking. For instance, DNA adducts, particularly 4-aminobiphenyl (cigarette smoke carcinogen) DNA adducts, are higher in bladder cancers of smokers than in non-smokers [11]. Other well-documented risk factors for bladder cancer include schistosomiasis, prior pelvic radiation, chronic bladder inflammation, and exposure to certain medications and occupational toxins, including arsenic, aromatic amines, and aniline dyes [8,12,13].

Isothiocyanate metabolism and bioavailability

Phytochemicals are plant-produced compounds that often serve as plant defense mechanisms and have been used for medicinal purposes for hundreds of years [14-17]. In recent decades, certain phytochemicals have emerged as potential or active anti-cancer therapies [18-21].

Isothiocyanates are active plant phytochemicals derived from inactive precursors, glucosinolates, which are uniquely present in cruciferous vegetables such as cabbage, kale, brussels sprouts, and broccoli [22,23]. Cruciferous vegetables of the Brassicaceae family are believed to have originated in Europe several thousand years ago [24]. They are rich in vitamins and minerals, including folic acid, vitamin A, iron, calcium, and zinc [25]. This family of vegetables harbor varying levels of glucosinolates, which contain sulfur groups that give cruciferous vegetables their characteristic pungent taste and smell [26]. The metabolism of glucosinolates begins with plant damage, such as chewing or chopping, which activates plant-derived myrosinase enzymes that convert inactive glucosinolates into isothiocyanates, thiocyanates, nitriles, goitrin, and epithionitriles. Myrosinase enzymes can also be produced in varied quantities by gut microbiota, which can assist in glucosinolate metabolism in the colon [26-28]. Isothiocyanates are believed to be important inducers of phase II detoxification enzymes and may play a role in chemoprevention [29]. Their overall metabolism and bioavailability depend on multiple factors, including mode of consumption, stomach acidity, gut microbiome, and genetic makeup [30,31].

Once produced, isothiocyanates undergo a series of metabolic reactions, including conjugation with glutathione by glutathione-S-transferases (GSTs) [32,33]. GSTs are phase II detoxification enzymes that neutralize endogenous and exogenous compounds and help to reduce oxidative stress. In humans, there are two super-families of GSTs, microsomal and cytosolic. Cytosolic GSTs are coded for by at least 16 genes [34]. GSTs, particularly the cytoplasmic enzymes GSTM1 and GSTP1, play an important role in isothiocyanate metabolism. GSTM1 is highly expressed in cells of the liver, which may further support its role in metabolism of SFN after oral consumption [30,35].

A variety of polymorphisms have been described in the GST enzymes, particularly of GSTM1 and GSTT1 [36,37]. While the overall frequencies of homozygous GSTM1- and GSTT1-null genotypes vary depending on racial and/or ethnic group, polymorphisms in GST enzymes exist across all populations [37]. For instance, GSTM1-null mutations affect up to 53% of all racial/ethnic groups while GSTT1-null mutations affect up to 21% of Caucasian, 64% of Asian populations, and 45% of African populations [35,38]. These mutations lead to a lack of a functional protein product, which may alter the metabolism and efficacy of isothiocyanates, particularly SFN, as chemopreventive agents [35,39,40].

GSTM1 mutations are perhaps the most well studied of the GST enzyme polymorphisms. In mice, knocking out GSTM1 leads to increased inflammation and oxidative stress [41]. In humans, epidemiologic studies suggest that individuals in the United States with active GSTM1 may gain a greater degree of protection from the consumption of cruciferous vegetables than those who are GSTM1-null [35]. However, epidemiologic studies in Asia suggest that GSTM1- or GSTT1-null individuals derive a higher degree of cancer protection from crucifer consumption than those who are GSTM1- and GSTT1-positive [42,43]. The differences in the predominant type of cruciferous vegetables consumed by Asian populations (Chinese cabbage) versus American populations (broccoli) may be one reason for these conflicting results [43]. Alternatively, GSTM1 metabolism of SFN may increase its secretion, and those with GSTM1-null mutations may ultimately have reduced SFN secretion, and therefore increased circulating levels of the anti-carcinogenic compound to combat cancer [44]. Finally, additional enzymes of the mercapturic acid pathway are required for isothiocyanate metabolism and differing levels of enzyme activity among individuals may also play a role in SFN activity and cancer prevention [44].

Individuals with GSTM1-null mutations may also have compensatory mechanisms for the loss of GSTM1 activity. Work done by Gasper, et al. indicated that GSTM1-null individuals metabolize SFN more rapidly within the first six hours after consumption, and excrete more urinary SFN metabolites within a 24-hour period, compared to individuals who have functional GSTM1 proteins [35]. Additional investigations indicated that the half-life of SFN is greater in those with GSTM1-null mutations, but that the type and quantity of urinary SFN metabolites were similar to those who were GSTM1-positive [45]. In respiratory epithelial cells with GSTM1-null mutations, SFN treatment led to a dose-dependent increase in overall GST activity, believed to be a compensatory mechanism of SFN metabolism [46]. Furthermore, in vitro analysis of GSTM1-null human lymphocytes showed that GSTM2 expression nearly doubled with SFN treatment, and GSTM2 also metabolized SFN at a comparable rate to function GSTM1 protein [47].

Anticancer mechanisms of SFN

SFN is a bioactive compound with both antioxidant and anti-inflammatory properties. SFN induces cancer cell apoptosis and cell cycle arrest while also activating enzymes responsible for detoxifying carcinogenic compounds [48,49]. SFN also improves the efficacy of certain traditional chemotherapeutic regimens [50,51]. SFN acts as a chemoprotective agent through various mechanisms, including the activation of phase II enzymes involved in carcinogen detoxification to prevent DNA damage and inhibit tumor initiation, as well as the regulation of multiple signaling pathways to inhibit tumor growth and progression. SFN also possesses immune-modulating properties that help to protect against cancer [48,49,52]. Such functions, along with the selective toxicity towards transformed cells, make SFN a potential candidate for the treatment and prevention of several types of cancer, especially epithelial cancers associated with high carcinogen exposure, like bladder cancer. Each of the anti-cancer mechanisms of SFN are reviewed below.

Antioxidant properties of SFN

Many of the most notable effects of SFN are mediated by its electrophilic properties and ability to activate phase II detoxification enzymes. The best-established mechanism is via protein nuclear factor erythroid 2-related factor-2 (Nrf2) activation. Under normal conditions, Nrf2 is bound by its inhibitor, Kelch-like ECH-associated protein 1 (Keap1). SFN inactivates Keap1, allowing Nrf2 to translocate to the nucleus and activate specific antioxidant response elements (ARE) [53-55]. A lesser established mechanism proposed by Li, et al. is that SFN induces mild increases in reactive oxygen species (ROS), leading to transcription factor EB (TFEB) activation. TFEB plays a role in activating antioxidant response elements and regulating cellular autophagy, ultimately reducing overall oxidative stress. TFEB also acts as a potential arm of protein Nrf2 activation [55].

Nrf2 is an important leucine zipper transcription factor that assists in drug metabolism by inducing antioxidant response element-mediated expression of phase II detoxification enzymes, including NAD(P)H: quinone oxidoreductase and GSTs [56-58]. Under homeostatic conditions, Keap1 binds to and inhibits Nrf2, which is eventually ubiquitinated and degraded [59,60]. Accumulation of ROS and electrophiles, due to SFN or other mechanisms, activates Nrf2 by modification of cysteine residues on Keap1, which weakens the association between Keap1 and Nrf2, allowing Nrf2 to disassociate from Keap1 and translocate to the cell nucleus [60,61]. Once in the nucleus, Nrf2 proteins bind ARE to stimulate the expression of phase II enzymes (N-acetyltransferase and glutathione S-transferase), which play crucial roles in detoxifying xenobiotics [61,62].

While phase II enzymes typically detoxify carcinogenic compounds, phase I enzymes generally activate xenobiotic compounds into their carcinogenic metabolites rather than inactivate them into neutral compounds [63-65]. In addition to activating phase II enzymes, SFN also decreases the expression of phase I enzymes, especially cytochrome P450 isoenzymes 2E1 (CYP2E1) and 3A4 in human hepatocytes [66,67]. Both phase I and phase II xenobiotic metabolism occur predominantly in the liver [68].

Activating Nrf2 with a subsequent increase of phase II enzyme expression is an essential chemoprotective mechanism of SFN in the bladder (Figure 1A). An example of this was previously recognized in human hepatocyte and hepatoma (HepG2) cell lines wherein SFN treatment increased Nrf2 and, therefore, glutathione levels [69]. Low doses of SFN (less than or equal to 5 μM) assisted in protecting both cell lines against hydrogen-peroxide-induced damage by activating the Nrf2/glutathione detoxification system. Cancer cells treated with SFN showed higher catalase levels, heme oxygenase 1, and NAD(P): quinone oxidoreductase-1 detoxification enzymes [69]. In cultured prostate cancer cells and prostatic adenocarcinomas in murine models, SFN treatment led to increased Nrf2 levels, with a subsequent decrease in ROS caused by androgen deprivation. Furthermore, Liu, et al. revealed that SFN treatment sensitized prostate cancer cells to radiation therapy via Nrf2 upregulation [70]. By reducing oxidative stress, SFN may play a role in preventing the DNA damage, mutation, and inflammation that contribute to carcinogenesis.

Figure 1.

Chemoprotective effects of SFN. A. SFN mediates antioxidant response by Nrf2 activation of phase II enzymes with subsequent repair of tobacco carcinogen-induced DNA damage in the transitional epithelium. B. SFN induces anti-inflammatory and immune responses by inhibiting the NF-κB pathway in epithelial cells. C. SFN induces epigenetic changes, cell cycle arrest, and apoptosis of transformed epithelial cells. Figure was created with BioRender.

Anti-inflammatory properties of SFN

Chronic inflammation is a contributing factor to the emergence of various cancer types. The relationships between pro- and anti-inflammatory cytokines, as well as inflammatory cells and their cytokine and chemokine milieus, play significant roles in the induction and progression of cancer [71,72]. High levels of ROS also create favorable environments for inflammation, tumor initiation, and proliferation through the oxidation of critical molecular structures, including lipids, proteins, and nucleic acids [73]. Through its action on the Keap1/Nrf2 pathway and induction of ROS detoxifying enzymes, SFN may mitigate inflammation and inhibit tumor cell progression (Figure 1B) [73-75].

ROS also generate inflammation by increasing the levels of tumor necrosis factor-alpha (TNF-α), which in turn increases the expression of the transcription factor Nuclear Factor Kappa B (NF-κβ) [76]. NF-κβ promotes oxidative stress and can induce inflammation if homeostatic conditions are not restored [73]. The inflammatory molecules released in response to NF-κβ include interleukin-6 (IL-6), interleukin-1β (IL-1β), and interferon-gamma (INF-γ), in addition to cyclooxygenase-2 (COX-2), vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1), all of which favor inflammation [77-80]. Alternatively, SFN treatment inhibits the release of NF-κβ from the inhibitory molecule, I-kB, and can prevent NF-κβ from binding to DNA, thereby reducing the expression of COX-2 and other inflammatory mediators [81-84]. Similarly, SFN treatment has also been found to decrease the cellular release of IL-1β, IL-2, IL-6, and IL-10, reduce the expression of VCAM-1 and ICAM-1 on endothelial cells exposed to lipopolysaccharide via downregulation of NF-κβ, as well as increase cell autophagy and apoptosis of transformed cells [76,79,85,86].

In addition to altering cytokine expression, SFN treatment modulates the function of several innate and adaptive immune cell types. For instance, SFN was previously found to regulate the antibody-dependent cellular cytotoxicity response of natural killer cells, leading to the destruction of recognized cancer cells [87]. SFN also plays an immunomodulatory role in certain inflammatory conditions by polarizing macrophages to an alternatively activated/M2 phenotype rather than a classically activated/M1 phenotype [88-90]. The M1 subtype is associated with a pro-inflammatory response characterized by increased inflammatory cytokines and ROS expression. Conversely, macrophages of the M2 subtype are associated with the production of anti-inflammatory cytokines and the activation of pathways that lead to antioxidant production [89,91,92].

Furthermore, SFN can reduce inflammation by inhibiting activated, non-transformed human T cells, especially TH17 cells, through increasing intracellular ROS levels, decreasing glutathione levels, and reducing the expression of RORgt, IL-17, and IL-22. The T cell activation markers CD25 and CD69 and the activation factor IL-2 are also decreased with SFN treatment compared to untreated controls [75]. Recently, Shen et al. discovered that SFN treatment potentiates the activities of chimeric antigen receptor T (CAR-T) cells in vitro and in vivo. CAR-T cells treated with SFN secreted more IFN-γ, perforin, and granzymes, and reduced CAR-T expression of programmed cell death 1 (PD-1), which binds programmed cell death ligand 1 (PD-L1) on tumor cells and promotes CAR-T exhaustion. In xenograft models, CAR-T cells in mice given SFN showed reduced tumor progression and prolonged survival [93]. Through the modulation of T cell activity, SFN may decrease T cell-mediated inflammation, prevent autoimmunity and potential carcinogenesis, and promote the death of transformed cells.

DNA modulation by SFN

In addition to its effects on antioxidant responses and inflammation, SFN alters cellular epigenetics by regulating histone deacetyltransferases (HDACs), which remove acetyl groups from histones, causing the repression of gene transcription (Figure 1C). The effects of SFN on HDACs have been shown in multiple models, including human colon and prostate cell lines, prostate cancer xenografts, and murine peripheral mononuclear cells [94]. Increased HDAC expression is a mechanism used by several cancer types to repress the expression of tumor suppressor genes and allow continued cell cycling and proliferation [95,96]. HDACs also play a role in the DNA damage response at cell cycle checkpoints and during the processing of double-stranded DNA breaks [97]. SFN was previously shown to inhibit HDACs competitively and protect against carcinogenesis [98-101]. For instance, in LNCaP and PC3 prostate cancer cell lines, SFN-induced HDAC inhibition led to increased levels of the tumor suppressor p21 with subsequent cell cycle arrest and increased Bax levels, which induced cancer cell apoptosis [94,102]. Furthermore, cell cycle analyses have shown that SFN inhibits cell cycle progression, particularly in the sub-G1 and G2/M phases, with associated reductions in cyclins B1 and D1, along with increased tumor suppressor proteins p21 and p53 activity [103-105].

The mild increase in ROS generated by SFN may also help disrupt mitochondrial membranes and cause the release of cytochrome c with subsequent apoptosis of cancer cells [106,107]. In HeLa cells and HepG2 cells, SFN induces the formation of apoptotic bodies and causes cell cycle arrest in the sub-G1 phase. SFN also increases Bax expression and downregulates Bcl-2 and Bcl-XL, further evidence that SFN induces apoptosis in transformed cells [108]. Additionally, in a study on SFN-induced apoptosis in prostate cancer cells, SFN led to the activation of caspase-3 and caspase-9, triggering apoptosis in tumor cells [107].

By inhibiting phase I enzymes and activating phase II detoxification enzymes, SFN may reduce the activation of carcinogens and ROS that interact with cellular DNA, thus preventing DNA mutations or reducing the mutation burden [109,110]. Some research suggests that SFN increases DNA damage and prevents double-stranded DNA break repair, leading to cell cycle arrest and apoptosis [111-113]. However, others report a decrease in DNA mutation burdens. For example, in HepG2 hepatoma cells, SFN exposure for three hours reduced DNA adduct levels by 66% [114]. Collectively, these mechanisms help prevent tumor initiation and progression (see Table 1 for a summary of mechanisms and corresponding references).

Table 1.

Anti-cancer mechanisms of sulforaphane and corresponding references

Mechanism of Action	Sources
Induction of Nrf2	Kensler et al. 2013 [49]; Dinkova-Kostova et al. 2017 [54]; Subedi et al. 2019 [85]; Wang et al. 2018 [76]; Liu et al. 2019 [69]; Jo et al. 2014 [160]
Increased ROS	Liang et al. 2018 [75]; Sing et al. 2005 [107]; Jo et al. 2014 [160]
Cell cycle arrest	Wang et al. 2015 [48]; Cheng et al. 2016 [103]; Zuryn et al. 2016 [104]
Induction of apoptosis	Wang et al. 2015 [48]; Sharma et al. 2011 [173]; Hahm and Singh 2010 [86]; Sing et al. 2005 [107]; Jo et al. 2015 [160]
Enhanced anti-cancer drug effects	Calcabrini et al. 2020 [168]; Kerr et al. 2018 [167]
Micro-RNA activation	Wang et al. 2016 [50]
Enhanced immune cell function	Thejass and Kuttan 2006 [87]
Suppression of inflammation	Ali et al. 2020 [89]; Pal and Konkimalla 2016 [88]; Heiss et al. 2001 [82]; Shan et al. 2012 [79]; Sharma et al. 2011 [173]; Subedi et al. 2019 [85]; Hahm and Singh 2010 [86]
Downregulation of STAT3	Hahm and Singh 2010 [86]; Subedi et al. 2019 [85]
COX-2 suppression	Woo and Kwon 2007 [84]; Sharma et al. 2011 [173]
Cytochrome P450 inhibition	Maheo et al. 1997 [66]; Barcelo et al. 1996 [67]
Reduction of PD-1 and PD-1L expression	Shen et al. 2021 [93]
HDAC inhibition	Myzak et al. 2007 [94]; Jiang et al. 2016 [101]
Inhibition of DNA adduct formation	Singletary and MacDonald 2000 [110]; Bacon et al. 2003 [185]; Ding et al. 2010 [158]
Reduced AP-1 expression	Dickinson et al. 2009 [186]
Altered DNA methylation profiles	Li et al. 2020 [187]
Reduced polycomb group [170] gene expression	Balasubramanian et al. 2011 [170]

Origin of bladder cancer

The primary function of the bladder is to store urine until it is ready to be excreted [115]. The bladder wall is composed of four layers. From the outside to the inside, there is the adventitia, the muscular layer, the submucosal layer, and finally, the mucosal layer, or urothelium [116,117]. The urothelium is a layer of transitional epithelium consisting of basal cells, intermediate cells, and a superficial layer of umbrella cells that line the urinary bladder cavity and act as an impermeability layer and are held together by tight junctions [118]. Umbrella cells change morphology depending on whether the bladder is full of urine or empty. These cells become flattened and elongated when the urinary bladder is full and, conversely, are taller and more cuboidal once the bladder is emptied [119]. When the bladder is relaxed, it consists of five to seven layers of cells, but when it fills with urine and is distended, the urothelium will become two to three layers [120]. Most bladder cancers begin in the transitional epithelium and can advance to deeper layers with cancer progression [121].

Carcinogen-induced field cancerization

Field cancerization

Field cancerization in which cancers develop in epithelia exposed to environmental carcinogens is well-documented, and histologically detectable precancerous changes are present throughout large sections of the carcinogen-exposed surface [122,123]. Furthermore, evidence of clinically detectable field cancerization in histologically normal epithelia is associated with precancerous changes in the form of mutation-harboring cell groups. These clonal mutations are thought to be the first detectable manifestations of the multi-hit carcinogenic process [124]. In carcinogen-induced cancers, damage resulting in cancer formation predates cancer by decades [124]. For example, driver mutations can be detected in hematological malignancies approximately ten years before the clinical diagnosis [125]. Bladder cancer often develops multifocally, and superficial bladder cancer has high recurrence rates. One possible explanation for these phenomena is field cancerization, wherein carcinogen exposure causes DNA mutations at independent locations in the urothelium [126,127]. This is supported by studies showing genetically-dissimilar primary and recurrent tumors [128].

Chemical carcinogen-induced bladder cancer

Smoking is the greatest risk factor for bladder cancer and contributes to approximately 50% of diagnosed cases [129-131]. In patients with a history of cigarette smoking, bladder cancers generally have higher HER2 amplification mutations that help drive the cancers, whereas those who do not smoke more commonly have PIK3CA mutations [132]. Mutations in the genes TP53, CDKN2B, and TERT are common in both smokers and non-smokers [133-137].

In addition to smoking, occupational exposure to certain carcinogens, such as aromatic amines, fossil fuels, aniline dyes, or certain metals like aluminum, account for approximately 20% of bladder cancer cases [12,138,139]. Certain genetic factors can also contribute to developing bladder cancer. For instance, N-acetyltransferase and glutathione S-transferase gene polymorphisms can affect the abilities of these enzymes to detoxify certain carcinogens that may contribute to bladder cancer [140].

Even with early treatment, there is a significant likelihood of bladder cancer recurrence, and those with the invasive disease face poorer outcomes, even with aggressive treatment. Due to continued poor outcomes for those with bladder cancer, there is a need for new interventions to protect against and treat this disease. SFN is a possible intervention that is well tolerated and has no significant toxicities [141,142].

Smoking carcinogen-induced epithelial cancers

Each year, smoking contributes to 180,000 cancer-related deaths in the US [143]. Cigarette smoke contains over 70 carcinogens that increase cancer risks [144]. While many of the chemicals in cigarette smoke are carcinogenic, many others are converted into carcinogenic compounds by cytochrome P450 enzymes [145]. These carcinogens and their metabolites can cause DNA adducts, contributing to tumorigenesis if not detoxified into harmless compounds, and have been shown to induce soluble factor profiles consistent with NF-κβ expression, including TNF-α, IL-1β, metalloproteases, and leukocyte-stimulating factors, among others, in mice [143,146,147].

Bladder cancer prevention with SFN

SFN in bladder cancer

Early evidence indicates a potential role for isothiocyanates, particularly SFN, in the chemoprevention of bladder cancer. Men who regularly consumed cruciferous vegetables were found to have a lower relative risk (0.49) for bladder cancer than men who did not consume cruciferous vegetables (P = 0.0082). This equated to a 51% risk reduction for those who consumed more than five servings of cruciferous vegetables weekly compared to those who consumed one serving or less [10,148]. Another retrospective case-control study that analyzed isothiocyanate intake and its effects on bladder cancer risk discovered that increased isothiocyanate intake was associated with a 29% decreased risk of bladder cancer in past and current smokers and older individuals [149]. Increased cruciferous vegetable intake may also improve survival outcomes in patients with bladder cancer [150]. Raw cruciferous vegetables appear to provide the greatest risk reduction, most likely due to the higher yield of isothiocyanates [151,152].

SFN and other isothiocyanates are secreted predominantly in the urine, further supporting the potential role of SFN in preventing bladder carcinogenesis [152,153]. Shortly after dosing rats with broccoli sprout extract, Munday et al. observed that urinary isothiocyanate levels were 2-3 times greater in urine than in plasma [154]. Similarly, broccoli sprout consumption in humans led to isothiocyanate levels that were 50 times greater in urine than in plasma eight hours after ingestion [155]. These studies indicate that isothiocyanates are concentrated in the urine and thus contact the bladder epithelium, the primary site for bladder cancer initiation.

In N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer models, Nrf2 regulates carcinogen detoxification genes and interacts with the p53 protein to further aid in chemoprevention. Increased tumor incidence and invasion were observed in Nrf2 null mice treated with BBN [156,157]. SFN inhibits DNA damage in murine bladder epithelium exposed to the cigarette smoke carcinogen 4-aminobiphenyl by activating Nrf2 and downstream phase II enzymes [158]. SFN also inhibits the ubiquitination and breakdown of Nrf2, thereby allowing for increased Nrf2 nuclear translocation and anti-cancer gene induction [159].

Bladder cancer cell culture studies have shown that SFN decreases cell viability and initiates apoptosis. In T24 urinary bladder cancer cells, SFN reduces cell proliferation and leads to chromatin clumping and fragmentation, increased cytochrome c release, caspase-9 and caspase-3 activation, and subsequent T24 cell apoptosis [160]. SFN treatment of T24 cells increased ROS generation, inducing mitochondrial dysfunction and cellular apoptosis. The ROS allowed for Nrf2 release from Keap1, as measured by the induction of heme oxygenase-1. Moreover, SFN treatment increased the endoplasmic reticulum stress response pathway, further contributing to T24 cell apoptosis [160].

In UM-UC-3 bladder cancer cells, sulforaphane also induces DNA fragmentation, caspase-9, and caspase-3 cleavage, mitochondrial damage, and subsequent cellular apoptosis, in addition to G2-M cell cycle arrest in vitro [161,162]. In the UM-UC-3 murine xenograft model, mice treated with SFN showed a tumor volume reduction of 67%, compared to control tumor volume 15 days after initial treatment. At 24 days of treatment, the average tumor volume was still 36% less in SFN-treated mice than in control mice. Interestingly, the tumors in mice treated with SFN decreased angiogenesis compared control tumors and had increased immune cell infiltration. There was also more than a 5-fold increase in the number of apoptotic cells in tumors of SFN-treated mice compared to tumors of control mice [163].

In rats fed broccoli sprout extract with high levels of SFN before and during exposure to BBN, there was decreased bladder cancer occurrence and reduced tumor burden compared to controls given BBN alone. Importantly, no negative impacts were observed in the bladders of rats given SFN alone, indicating not only the potential efficacy of SFN in bladder cancer chemoprevention, but also the safety of SFN treatment [154,164].

Finally, the chemoprotective mechanisms of SFN may play an enhanced role in treating bladder cancer when used in combination with traditional chemotherapeutic drugs and may even prolong the time to therapy resistance. The chemotherapeutic agent, Everolimus, is an mTOR inhibitor and was shown to have some anti-tumor activity in patients with urothelial carcinoma [165]. Justin et al. showed that when combined with Everolimus, SFN improved therapeutic effects by reducing RT112 cancer cell chemotaxis and invasion. There was increased cancer cell therapy resistance, invasion, and migration of tumors treated with Everolimus alone [166]. SFN has also shown promise in combination with cisplatin, doxorubicin, and CAR-T cell therapy and may even help mitigate some of the toxic side effects of certain chemotherapeutics [93,167,168].

Discussion

SFN is an isothiocyanate found in cruciferous vegetables that have shown chemoprotective effects in various cancer models, including those of the bladder [158,160,163,169,170]. SFN induces phase II detoxification enzymes through the activation and nuclear accumulation of Nrf2 while also decreasing phase I enzyme activation, particularly cytochrome P450 isoenzymes, which can convert many compounds into carcinogenic metabolites [60,62,66,67,171]. In the urothelium, SFN also acts to decrease inflammation through the detoxification of ROS, inhibition of NF-κβ and COX-2 expression, and modulation of inflammatory cytokines, including IL-1β, IL-2, IL-6, and IFN-γ [74,75,82-87,172-174]. Finally, SFN reduces DNA damage through HDAC regulation, the induction of cell cycle arrest, and the apoptosis of transformed cells [94,98-101,103,104,107]. SFN reduces tumor cell growth and invasion through the inactivation of sulfatase-2, a key component in the Wnt and fibroblast growth factor signaling pathways in many human cancers [175]. Finally, SFN activates chemoprotective mechanisms on its own and acts synergistically with some traditional chemotherapeutics to reduce tumor burden and limit the toxic side effects of traditional chemotherapy [166-168].

Besides its favorable biological activities, SFN can play a vital role in preventing and treating bladder cancer due to the active excretion and concentration in urine after isothiocyanate consumption [153-155]. Accordingly, there is compelling evidence that SFN inhibits urothelial cell DNA damage induced by cigarette carcinogens and reduces overall bladder cancer risk in those who regularly consume cruciferous vegetables [10,148,158]. SFN-induced Nrf2 activation has also been found to detoxify certain carcinogens from cigarette smoke and upregulate the expression of p53. SFN also induces cell cycle arrest and apoptosis in transformed bladder epithelium [156,157,159-161]. Taken together, these studies provide strong evidence for the chemoprotective nature of SFN in various human epithelial cancers, including those of the bladder.

Future studies

Increased cruciferous vegetable intake has previously been associated with a decreased overall cancer risk [176]. Inverse associations between cruciferous vegetable intake, especially broccoli, and bladder cancer risk, as well as bladder cancer mortality have been reported in epidemiological studies [177,178]. The potent chemopreventive and chemotherapeutic effects of SFN have been well documented in cell culture and animal models of bladder cancer [158,179,180]. However, to translate these findings to the bedside, it is essential to validate these findings in prospective cohort studies and to further investigate the effects of SFN-rich diets in humans, especially those with elevated bladder cancer risks. Further exploration of dietary SFN in the prevention and/or treatment of bladder cancer may help facilitate a paradigm shift in clinicians’ use of food as medicine and encourage enhanced research and prescription of nutrition-based oncology interventions [181]. Currently, a prospective study designed specifically to evaluate the impact of cruciferous vegetable intake on bladder cancer prognosis in the context of polymorphic ITC-metabolizing genes is underway [182]. Clinical trials to examine the effect of SFN or SFN-rich dietary supplements alone or in combination with other therapies in preventing bladder cancer recurrence are also warranted.

Future studies of SFN in bladder cancer can emphasize supplemental cancer therapy and prevention. For cancer treatment, SFN can be used either as an alternative therapeutic agent or as a supplementary agent in combination with other chemo- or immunotherapies. SFN in food extract format, e.g., broccoli sprout extract, is commercially available, facilitating the clinical investigation of SFN in the patient population [183]. While cancer treatment with SFN is possible, the wider implication of SFN as a therapeutic agent may be its potential to prevent primary or secondary cancers among high-risk populations.

Cruciferous vegetables are a rich dietary source of SFN and are widely available in the US. As the initiatives behind food as medicine expand, cruciferous vegetables could serve as a low-cost and low-toxicity regimen for long-term use among populations at high risk of developing cancer due to a history of heavy exposure to smoking and carcinogens. Given the high recurrence risk that early-stage bladder cancer survivors face, studies on how to engage and encourage patients to increase their consumption of cruciferous vegetables to improve cancer outcomes are ongoing [184]. Overall, these lines of research may offer evidence-based dietary recommendations to fight against carcinogen-induced cancers and can also have wider-reaching and paradigm-shifting effects on how modern medicine uses nutrition to tackle prevention and treatment challenges.

Disclosure of conflict of interest

None.

Abbreviations

BBN

N-butyl-N-(4-hydroxybutyl) nitrosamine

MIBC

muscle-invasive bladder cancer

NMIBC

non-muscle invasive bladder cancer

SFN

Sulforaphane

TURBT

transurethral resection of bladder tumor