Natural Phytochemicals in Bladder Cancer Prevention and Therapy

Phytochemicals are natural small-molecule compounds derived from plants that have attracted attention for their anticancer activities. Some phytochemicals have been developed as first-line anticancer drugs, such as paclitaxel and vincristine. In addition, several phytochemicals show good tumor suppression functions in various cancer types. Bladder cancer is a malignant tumor of the urinary system. To date, few specific phytochemicals have been used for bladder cancer therapy, although many have been studied in bladder cancer cells and mouse models. Therefore, it is important to collate and summarize the available information on the role of phytochemicals in the prevention and treatment of bladder cancer. In this review, we summarize the effects of several phytochemicals including flavonoids, steroids, nitrogen compounds, and aromatic substances with anticancer properties and classify the mechanism of action of phytochemicals in bladder cancer. This review will contribute to facilitating the development of new anticancer drugs and strategies for the treatment of bladder cancer using phytochemicals.


INTRODUCTION
Cancer has multiple causes, such as genetic mutation and cellular dysregulation. The disease is characterized by uncontrolled cell growth, abnormal differentiation, proliferation, invasion, and metastasis (1). Several plant compounds inhibit cancer cell phenotypes. Based on this anticancer activity, several phytochemicals have been developed as FDA-approved drugs for cancer therapy (2,3). For example, paclitaxel has been carried out the clinical trial and been a first-line treatment for lung, ovarian, and breast cancer (3). Paclitaxel promotes tubulin polymerization and assembly, and prevents depolymerization, thereby stabilizing tubulin, inhibiting cancer cell mitosis, and preventing cell apoptosis to effectively prevent cancer cell proliferation and exhibit anticancer activity (3). Paclitaxel has also entered the clinical trial stage III for bladder cancer with other chemotherapeutic drugs together -paclitaxel/gemcitabine/cisplatin (PGC): 142 patients were involved into this clinical trial (74 observation and 68 PGC treatment). This study indicated that PGC improves overall survival in high risk invasive bladder cancer (4). However paclitaxel alone has not been carried out clinical trial study on bladder cancer. Another phytochemical-derived antitumor drug, vincristine, is used to treat acute lymphoblastic leukemia and is effective in treating other acute leukemia, lymphatic sarcoma, reticular cell sarcoma, and breast cancer. It exerts these effects via suppression of microtubule formation in mitotic spindles, resulting in cell cycle arrest at the metaphase stage (5,6).
Phytochemicals can be used for cancer treatment and prevention. Furthermore, these compounds can be used to treat patients whose cancer has recurred and can contribute to preventing carcinogenesis in those who do not have cancer during their lifetime. Resveratrol (3,5,4'-trihydroxy-transstilbene), a non-flavonoid polyphenol found in several food plants including grapes, peanuts, soy beans, berries, and pomegranates, possesses cancer prevention benefits via its antiproliferative and anti-oxidative activities (7). Because phytochemicals are abundant in food sources and have low side effects, an increasing number have been developed as health supplements, including resveratrol and curcumin (8).
Bladder cancer is a common cancer of the urinary system. Because muscle invasive bladder cancer (MIBC) has a high mortality rate, and non-muscle invasive bladder cancer (NMIBC) has a high recurrence rate, both cancer types cause considerable physical and physiological pain to patients (9). Although phytochemicals have been studied for the treatment and prevention of bladder cancer, a systematic summary of their uses is lacking. Further, few drugs based on phytochemicals and their derivatives have been developed. Therefore, a comprehensive summary of previous studies on the activity of phytochemicals against bladder cancer is important for focusing on research priorities, achieving novel breakthroughs, and developing new anticancer drugs. In this review, two decades of research on phytochemical-based treatment of bladder cancer are systematically summarized.

FLAVONOIDS
Flavonoids are natural compounds that have a 2-phenylfluorone structure with a ketone carbonyl group and a basic oxygen atom. These compounds form salts in the presence of strong acids and the resulting hydroxyl derivatives are usually yellow. Thus, these compounds are also called flavins or flavones (10). In plants, flavonoids usually combine with sugars to form glycosides with a small portion existing in a free state (aglycone). A considerable number of plants contain flavonoids, which play important roles in growth, development, flowering, fruit development, and disease prevention. Recently, the anticancer activities of flavonoids have been reported (11).
Apigenin, a flavonoid found in celery, is widely recognized for its anticancer activity. Our previous study demonstrated that apigenin suppresses cancer cell invasion by downregulating urokinase-type plasminogen activator receptor (uPAR) expression in bladder cancer T24 cells. In particular, apigenin showed substantial anticancer activity through the suppression of high levels of interleukin (IL)-1b-induced uPAR expression (17). These effects are mediated via the inhibition of mitogenactivated protein kinase (MAPK)-induced nuclear factor (NF)-kB and activator protein (AP)-1 activity, which are essential transcription factors for uPAR (17). Finally, apigenin suppresses T24 bladder cancer cell proliferation by arresting the cell cycle in the G2/M phase (18,19).
Luteolin, widely found in nature, was originally isolated from the leaves, stems, and branches of plants of the Resedaceae family. Recently, luteolin was reported to inhibit bladder cancer cell viability and induce G2/M cell cycle arrest via upregulation of p21/WAF1 and reduction of phosphorylated S6 (p-S6), a key downstream molecule in the mechanistic target of rapamycin (mTOR) signaling pathway. Furthermore, luteolin also has an antioxidant effect that reduces intracellular reactive oxygen species (ROS) by elevating thioredoxin 1. Orally administered luteolin showed antitumor activity by markedly suppressing tumor growth in a xenograft mouse model by upregulating p21/WAF1 and repressing mTOR signaling (20).
Tangeretin is among the most abundant flavones in citrus peels, and its anticancer properties have been described, such as its inhibition of bladder carcinoma BFTC-905 cell viability through apoptosis. Besides, tangeretin induces calcium homeostasis in the mitochondria, activates caspase 3 and caspase-9, and causes cytochrome C release, which in turn, enhances apoptosis (22).
The 3′-hydroxy-flavone kaempferol, widely found in various fruits, vegetables, and beverages, and its anticancer activities have been reported in several studies as follows. Dang et al. (26) showed that kaempferol inhibits bladder cancer proliferation and invasion by downregulating the c-Met/p38 signaling pathway in bladder cancer 5637, T24, 253J, and TCCSUP cells. Wu et al. (27) reported the anti-oxidative and anticancer activities of kaempferol in bladder cancer. Furthermore, kaempferol attenuates ROS-induced hemolysis and decreases cell proliferation by suppressing p-AKT, Bcl lymphoma extra-large (Bcl-xL), cyclin-dependent kinase 4 (CDK4), cyclin D1, and MCL-1. In addition, it enhances p21/WAF1, p38, p53, p-ATM, p-BRCA1 DNA repair-associated, Bax, and BH3 interacting domain death agonist (Bid) expression, leading to S phase arrest and the induction of apoptosis of EJ bladder cancer cells (28).
Fisetin, a flavonoid derived from Rhus succedanea L., induces apoptosis of human bladder cancer by increasing the ratio between pro-apoptotic and anti-apoptotic proteins by upregulating p53 and downregulating NF-kB activity in T24 and EJ bladder cancer cells. Additionally, fisetin elevates p53 and p21/WAF1 protein levels and decreases CDK2, CDK4, cyclin A, and cyclin D1 levels, thereby leading to G0/G1 cell cycle arrest. Moreover, fisetin increases Bcl antagonist/killer (Bak) and Bax expression and decreases Bcl-2 and Bcl-xL expression, which in turn triggers apoptosis (28).
Morinis a pale-yellow pigment extracted from the bark of plants of the mulberry family such as the yellow mulberry, mulberry orange tree, and many Chinese herbs. Morin has been shown to exhibit multiple anticancer activities such as inhibition of bladder cancer cell proliferation and invasion (29). Morin decreases CDK2, CDK4, cyclin D1, and cyclin E via modulation of the p21/WAF1 pathway, suppressing c-Jun Nterminal kinase (JNK) and AKT phosphorylation, and preventing MMP-9 expression by repressing NF-kB, SP-1, and AP-1 in EJ bladder cancer cells (29).
Quercetin is a flavonol that is widely distributed in the plant kingdom and exhibits various biological activities. Several studies have shown that quercetin inhibits cell survival and induces apoptosis of bladder cancer cell lines: This compound also upregulates tumor suppressors by inhibiting CDK inhibitor 2A (CDKN2A) and Ras association domain family member 1A (RASSF1A) gene methylation (30,31). Quercetin inhibits cancer cell growth by arresting the cell cycle at the G0/G1 phase and decreases the protein expression of mutant p53 and survivin in EJ, J82, and T24 bladder cancer cells (30).

Isoflavone
Isoflavones are phenolic compounds formed by cyclization following the extension of the cinnamyl coenzyme A side chain during phenylalanine metabolism in plants (32). The isoflavone genistein mainly exists in legumes such as the locust horn and mountain bean root (33). Genistein inhibits bladder cancer cells proliferation by arresting the cell cycle at the G2/M phase via suppression of cyclin A and cyclin B1, and upregulation of CDKN1A (p21/WAF1). Moreover, genistein also inactivates the PI3K/AKT signaling pathway by increasing ROS accumulation in T24 bladder cancer cells (34). Interestingly, g e n i s t e i n a l s o s e n s i t i z e s b l a d d e r c a n c e r c e l l s t o hydroxycamptothecin treatment in vitro and in vivo through ATM/NF-kB/inhibitor of NF-kB kinase (IKK)-mediated apoptosis (35). Another representative isoflavone is puerarin, which is isolated from Radix puerariae. Ye et al. (36) reported that puerarin inhibits proliferation and triggers apoptosis of T24 bladder cancer cells. The proposed underlying mechanism is the inhibition of the sirtuin 1 (SIRT1)/p53 signaling pathway (36). Puerarin also inhibits bladder cancer cell viability through cell cycle arrest at the G0/G1 phase by a mechanism that involves downregulation of mTOR and p70S6K phosphorylation, without affecting their protein levels (37). Moreover, puerarin upregulates the microRNA miR-16, which subsequently downregulates cyclooxygenase (COX)-2 expression via NF-kB signaling pathway inactivation, thereby decreasing the viability of T24 bladder cancer cells (38).

Flavanone
Flavanones are dihydroflavones found in the peels of citrus fruits such as lemon and pomelo fruits. Naringenin is an example an active flavanone found in citrus fruit extracts, and is known for its pharmacological utility (48,49). Although the anticancer activities of naringenin have been widely reported, recent experimental evidence indicates that naringenin inhibits TSGH-8301 bladder cancer cell migration via deactivation of AKT signaling and MMP-2 downregulation (50).
Orientin is a polyhydroxy-flavanone found in Lophatherum gracile and Trollius chinensis Bunge (56). This compound inhibits cancer cell proliferation, reduces cell viability, causes cell cycle arrest, and represses the expression of inflammatory mediators in T24 bladder cancer cells. In addition, orientin decreases the expression of NF-kB components and blocks the Hedgehog signaling pathway (57).

Biflavone
Ginkgetin, amentoflavone, sotetsuflavone, and hinokiflavone are representative biflavones. Ginkgetin, one of the best known biflavones, has been used to treat breast (58), lung (59), and prostate (60) cancers; however, its effects on bladder cancer have not been reported. Sotetsuflavone and hinokiflavone suppress lung cancer, but not bladder cancer. Amentoflavone markedly inhibits TSGH-8301 bladder cancer cell activity via the induction of mitochondria-dependent intrinsic apoptosis and Fas cell surface death receptor (FAS)/FAS ligand (FASL)-dependent extrinsic apoptosis. Further, amentoflavone reduces the expression of anti-apoptotic proteins including MCL-1 and cellular FLICE-like inhibitory protein, thereby enhancing apoptosis (61).

STEROIDS Phytosterols
Phytosterols are plant sterols and mainly include those found in grain, rapeseed oil, beans, and rapeseed sterols, and corresponding alkanols. Phytosterols are active plant constituents that have numerous human health benefits (62). Studies have found that phytosterols reduce blood cholesterol, prevent and treat prostatic hypertrophy, inhibit tissue hyperplasia, and regulate immunity (62,63). Recently, the anticancer activities of phytosterols have been described (64,65).
Dioscin, a plant-based steroid, mainly comes from the roots of the ginger plant (66). This compound induces cancer cell apoptosis by demethylation of death-associated protein kinase 1 (DAPK-1) and Ras-association domain family 1a (RASSF-1a) genes via antioxidant activity in T24 and 5637 bladder cancer cells. In many carcinomas, DNA is often methylated at CpG rich regions (66), and many tumor suppressor genes are often hypermethylated including RASSF-1a and DAPK-1, whichhave been identified in studies conducted in some western countries.
Solasonine is a steroidal glycoside alkaloid isolated from black nightshade, and its anticancer activity has not been widely studied because of its low water solubility. Miranda et al. (67) developed a nanotechnology-based strategy using poly (D, Llactide) nanoparticles to improve the performance of solasonine for bladder cancer therapy. Interestingly, this study showed high nanoparticle uptake by RT4 bladder and MDA-MB-231 breast cancer cells, but not by normal HaCaT keratinocytes cells, suggesting targeted cancer cell uptake (67).
In addition to bladder and breast cancers, solasonine inhibits hepatocellular carcinoma cells via the non-apoptotic cell death pathway, ferroptosis. Solasonine suppresses glutathione synthetase (GSS) and glutathione peroxidase 4 (GPX4) (68). GSS, the key enzyme in the synthesis of the oxidation inhibitor GSH, suppresses ferroptosis by regulating the intracellular peroxidation environment. GPX4 prevents ferroptosis by converting hydroperoxide lipids into nontoxic lipid alcohols (68).

Monoterpenes
Monoterpenes are terpenoids usually derived from the polymerization of two isoprene molecules, and their oxygencontaining and saturated derivatives (69). Monoterpenes are divided into the following four categories according to the basic carbon skeleton of the molecule: acyclic monoterpenes, single-ring monoterpenes, double-ring monoterpenes, and tricyclic monoterpenes. Monoterpenes are important materials for the pharmaceutical, food, and cosmetic industries (69). Limonene, a monocyclic monoterpene, is found in citrus fruits, especially the peels. Recently, limonene was found to strongly suppress the viability of human T24 bladder cancer cells [halfmaximal inhibitory concentration (IC 50 )= 9 mM] (70). In addition, it caused chromatin concentration and nuclear fragmentation, thereby inducing bladder cancer cell apoptosis by the downregulation of Bcl-2 expression and upregulation of Bax and caspase-3 expression. Moreover, limonene also induces cell cycle arrest in the G2/M phase and inhibits bladder cancer cell migration and invasion (70). The terpenoid linalool exhibits strong anticancer activity against various carcinoma cells, particularly cervical (IC 50 = 0.37 mg/mL), stomach (IC 50 = 14.1 mg/mL), skin (IC 50 = 14.9 mg/mL), lung (IC 50 = 21.5 mg/mL), and bone (IC 50 = 21.7 mg/mL) carcinoma (71).

Sesquiterpenes
Sesquiterpenes are natural terpenes that contain three isoprene units and have various skeletal structures, such as chains and rings. Sesquiterpenes are mostly liquid and are mainly found in volatile plant oils. Curcumol, a natural sesquiterpene with anticancer activity, was originally isolated from Curcuma rhizomes (72). Zhou et al. (73) reported that curcumol inhibits EJ and T24 bladder cancer cell proliferation and induces apoptosis in a dose-dependent manner by decreasing the accumulation of enhancer of zeste homolog 2 (EZH2).
Atractylenolide I, an active sesquiterpene component extracted from Rhizoma atractylodis, inhibits T24 bladder cancer cell proliferation by arresting the cell cycle in the G2/M phase via downregulation of cell cycle-related proteins such as CDK1, cell division cycle 25C (CDC25c), and cyclin B1. Moreover, atractylenolide I promotes apoptosis via activation of the mitochondria-dependent apoptotic pathway and inhibition of the PI3K/AKT pathway in both T24 cells and xenografted tumors (74).
Parthenolide, a sesquiterpene lactone found in the herb, Brachynoma, has antitumor activity. Further, parthenolide substantially decreases bladder cancer cell viability via G1 phase cell cycle arrest by modulation of CDK2 phosphorylation. It also triggers apoptosis by enhancing Bcl-2 degradation and stabilizing PARP in 5637 bladder cancer cells (75).

Diterpenes
Yuanhuacine, an active diterpene from Daphne genkwa, has a wide range of antitumor activities. Zhang et al. (77) discovered that yuanhuacine inhibits T24T bladder cancer cell proliferation by arresting the cell cycle in the G2/M phase in an SP1dependent manner. Yuanhuacine upregulates p21/WAF1 expression through a p38 pathway, but does not affect p53 or activate p53 protein levels.
Triptolide can also be used in combination with hydroxycamptothecin to enhance the anticancer effect of chemotherapeutic drugs on bladder cancer cells. This effect is achieved by increasing apoptosis through the upregulation of apoptosis-related proteins (Bcl-xL and Caspase 8) and inhibition of cyclin D1, CDK4, and CDK6 expression, which arrested the cell cycle of EJ and UMUC3 bladder cancer cells in the G1/S phase (81). In addition, some diterpenes with antitumor activity, such as jolkinolide B, cafestol, and oridonin, exhibited excellent efficacy against prostate, esophageal, and lung cancer, but have not been studied in bladder cancer (82)(83)(84).

Triterpenes
Triterpenes are composed of several isoprenoids joined end to end by the removal of the hydroxyl group, and some of these compounds have antitumor activity. Ursolic acid, a triterpene presents in Prunella vulgaris L and Ilex rotunda Thunb, represses T24 bladder cancer cell growth by regulating anti-apoptotic NF-kB-p65 and AKT signaling, and suppressing IkBa, NF-kB-p65, and AKT phosphorylation. In addition, ursolic acid upregulates pro-apoptotic proteins such as caspase 3, indicating that it could serve as a candidate for the treatment and prevention of bladder cancer (85). Pachymic acid is an antitumor lanostane-type triterpenoid extracted from Poria cocos. Importantly, pachymic acid suppresses the growth of EJ bladder cancer cells by increasing the sub-G1 DNA ratio, with the accumulation of apoptotic bodies and promotion of DNA fragmentation. Molecularly, pachymic acid activates caspase3, caspase8, and caspase9, which in turn, dose-dependently accelerate apoptosis. Furthermore, pachymic acid elevates ROS and decreases the DYm (86). Nimbolide, an active triterpene derived from Azadirachta indica, reportedly has several antitumor activities against bladder cancer. Nimbolide strongly inhibits bladder cancer cell proliferation with an IC 50 of 3.0 mM. In bladder cancer cells treated with nimbolide, the G2/M phase cell cycle was arrested via the checkpoint kinase 2 (Chk2)/Cdc25C and Chk2/p21WAF1-related cyclin B1/Wee1 pathway. Moreover, nimbolide elevates JNK phosphorylation while reducing p38 and AKT phosphorylation in EJ and 5637 bladder cancer cells (87).

Iridoids
Iridoids are plant-derived monoterpenes, which usually form iridoid glycosides with sugars. Catalpol is an active iridoid glucoside that is abundantly present in the traditional Chinese medicinal plant Rehmannia glutinosa. Its anticancer activity was recently described by Jin et al. (88) who found that it strongly inhibited the proliferation, migration, and invasion of T24 bladder cancer cells. Moreover, many proteins involved in apoptosis are upregulated by catalpol such as active caspase 3 and PARP. Catalpol also promotes apoptosis by inducing proapoptotic protein expression and suppressing Bcl-2 protein expression by modulating the PI3K/AKT pathway (88). Genipin, a natural iridoid derived from Gardenia jasminoides fruit, inhibits the growth of T24 and 5637 bladder cancer cell lines in vitro and in vivo (89). Genipin decreases clonogenic growth and cell viability in a dose-dependent manner, arrests the cell cycle at the G0/G1 phase, and increases the percentage of apoptotic cells, leading to loss of the DYm, promotion of cytochrome C release into the cytosol, and suppression of PI3K and AKT phosphorylation (90).

ALKALOIDS
Alkaloids are nitrogen-containing organic compounds found in nature, mainly in various plants and some animals. Most alkaloids have a complex ring structure with nitrogen located in the ring structure, which confers important biological activity. Alkaloids are also important bioactive ingredients of many traditional Chinese herbs. Many alkaloids screened from herbs or medicinal plants have shown anticancer and anti-proliferative effects in vivo and in vitro. Some have already been successfully developed as antitumor drugs, such as vinblastine, vinorelbine, vincristine, and vindesine (91). Goonewardene carried out a phase I/II clinical study using vincristine combined with methotrexate and cisplatin. This clinical trial result showed that vincristine combined chemotherapy significantly prolonged the survival period of invasive bladder cancer patients (92). The neoadjuvant cisplatin, methotrexate, and vinblastine (CMV) chemotherapy in muscle-invasive bladder cancer patients was studied in a randomized phase III trial. CMV treatment showed a statistically significant 16% reduction in the risk of death, indicating CMV chemotherapy improves outcome as first-line adjunctive treatment for invasive bladder cancer (93).
The alkaloid oxymatrine has been extracted from the dried roots, plants, and fruits of the leguminous matrine plant using organic solvents such as ethanol. The anti-bladder cancer activity of oxymatrine was recently discovered: Oxymatrine dose-dependently suppresses the proliferation of bladder cancer cells, arrests the cell cycle, and triggers apoptosis through Bax and caspase 3 upregulation and downregulation of p53, Bcl-2, and survivin expression in T24 bladder cancer cells (94).
Dauricine is a diphenyl isoquinoline alkaloid that exists in the rhizome of the Asiatic Moonseed. Dauricine inhibits the proliferation of urinary system cancer cells, including EJ bladder cancer cells and PC-3M prostate cancer cells. The minimum effective concentration was 3.81-5.15 g/mL, and the anticancer effect was concentration-dependent (95).
Capsaicin is an alkaloid found in chili peppers. Capsaicin markedly inhibited cell migration through the suppression of SIRT1 deacetylase via proteasome-mediated protein degradation, thereby elevating b-catenin acetylation and decreasing MMP-9 and MMP-2 activation (96) in T24 bladder cancer cells.
Other studies have shown that capsaicin inhibits bladder cancer cell migration and suppresses cell growth by enhancing apoptosis and inducing cell cycle arrest of TSGH-8301 and T24 bladder cancer cells. Capsaicin represses ERK, FAK, and paxillin phosphorylation, thereby inhibiting cancer cell migration. Moreover, capsaicin reduces the expression of tumorassociated NADH oxidase and SIRT1, thereby inhibiting proliferation, triggering apoptosis, and delaying cell cycle progression (97).
Piperlongumine, a naturally occurring alkaloid extracted from the longer pepper (Piper longum), was recently discovered to have selective anticancer activity. Piperlongumine inhibits bladder cancer cell proliferation, blocking the cell cycle at the G2/M phase and suppressing cell migration. Additionally, piperlongumine drastically increases ROS levels, which are closely related to the inhibition of cell cycle progression of cancer cells. In in vivo experiments, piperlongumine inhibited cancer cells via downregulation of Slug, N-cadherin, ZEB1, and b-catenin (98).

ORGANIC ACIDS AND ESTERS
Natural organic acids isolated from plants or agricultural byproducts have exhibited certain physiological activities. These acids are widely distributed in the leaves, roots, and fruits of Chinese herbs and fruits, such as black plums, schisandra chinensis and raspberries. Ellagic acid is a polyphenolic organic acid found in green tea, nuts, grapes, pomegranates, and berries, and shows antitumor activity in four different bladder cancer cell lines (UMUC3, 5637, T24, and HT1376). It exerts anti-proliferative activities and enhances the anticancer activity of mitomycin C in bladder cancer therapy. Ellagic acid also suppresses cancer cell invasion and represses chemotaxis, specifically via reduced vascular endothelial growth factor (VEGF)-A-induced VEGF receptor (VEGFR)-2 expression. More interestingly, ellagic acid reduces the expression of programmed cell death 1 ligand 1, an important protein in tumor cell immune escape. In addition, an in vivo study showed the antitumor activity of ellagic acid in human bladder cancer xenografts (99). In another study using a TSGH-8301 bladder cancer cell model, the antitumor activity of ellagic acid was reported to inhibit cell growth, leading to morphological changes, arrest of the cell cycle at the G0/G1 checkpoint, and triggering of apoptosis. Molecularly, ellagic acid enhances Ca 2+ levels, elevates ROS, decreases the DYm, and activates caspases including caspase 9 and caspase 3 (100).
Gallic acid is a polyphenol organic acid found in various plants such as rhubarb, dogwood, and eucalyptus. Gallic acid concentration-dependently inhibited T24 bladder cell viability, with IC 50 values of 21.73, 18.62, and 11.59 µg/mL following treatment for 24, 48, and 72 h, respectively. In addition, gallic acid induces T24 cell apoptosis associated with ROS accumulation. Gallic acid-induced apoptosis involves DYm depolarization and the caspase 3, Bax, p53, and cytoplasmic cytochrome C levels increase, whereas PI3K, AKT, IkBa, IKKa, and NF-kB p65 phosphorylation decrease. These results indicate that gallic acid is a potential anticancer drug candidate that inhibits cell proliferation, promotes apoptosis, and suppresses the PI3K/AKT/NF-kB signaling axis (101). In addition, the antitumor mechanism of action of gallic acid has been shown to be mediated via the MAPK and PI3K/AKT pathways. Finally, gallic acid inhibits fatty acid synthase and increases estrogen receptor (ER) alpha in TSGH-8301 bladder cancer cells (102).

AROMATIC PHYTOCHEMICALS Anthrone and Its Derivatives
Gambogic acid, extracted from gamboges, has an anthronederived structure, induces ROS, and promotes a dramatic autophagic response via the JNK pathway. Gambogic acid induces ROS-mediated caspases activation, leading to the degradation of autophagic proteins, and causes mitochondrial hyperpolarization and caspase activation, which triggers the intrinsic apoptotic pathway. In addition, gambogic acid suppresses NF-kB activation through ROS-mediated suppression of IkBa phosphorylation in T24 and UMUC3 bladder cancer cell lines (103).
Gartanin, a naturally occurring xanthone, has anticancer activity in various bladder cancer cell lines including HT1376, T24, 5637, TCCSUP, RT4, UMUC3and J82 cells (104). The underlying molecular mechanism of gartanin involves a marked suppression of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and p70S6 expression in T24 and RT4 cells. Further, triggering of mTOR pathway-mediated autophagy in T24 and RT4 cells has also been observed. In addition, gartanin downregulates protein expression of the apoptosis inhibitor Bcl-2, whereas it activates the p53 pathway, inducing apoptosis (104). Cinnamate 3-Phenyl-2-acrylate (cinnamate), is an aromatic organic acid isolated from cinnamon bark or benzoin. Curcumin, a representative cinnamate derivative, widely exists in the rhizomes of some plants of the Zingiberaceae and Araceae families (105). The anticancer activity of curcumin has been widely investigated against various cancers, including that of the bladder, where it regulates numerous intracellular signaling molecules (106). The human trophoblast cell surface antigen 2 (Trop2) is a well-known cancer driver that is dysregulated in many cancers. Zhang et al. (107) found that curcumin strongly inhibits T24 and RT4 bladder cancer cell proliferation by decreasing Trop2 expression and its key downstream molecule cyclin E1. Recently, the anticancer effect of curcumin against bladder cancer stem cells has been reported: Curcumin reduces cell sphere formation by suppressing the expression of breast cancer stem cell markers such as CD133, CD44, Nanog, and octamer-binding transcription factor 4 in UMUC3 and EJ bladder cancer cells. In addition, curcumin inhibits proliferation and triggers apoptosis of bladder cancer cells. Interestingly, curcumin suppresses Sonic Hedgehog pathway activation, which is positively correlated with tumor growth (108). In addition, curcumin enhances the antitumor activity of cisplatin against bladder cancer. The apoptotic cell ratio of T24 and 253J-Bv cells cotreated with curcumin and cisplatin was higher than that of cells incubated with either agent alone. The ROS scavenger N-acetyl-L-cysteine and ERK inhibitor U0126 abolished the combined effect of curcumin and cisplatin. Thus, curcumin might induce apoptosis via a ROS-mediated MEK/ ERK-dependent pathway (109). Ferulic acid, another cinnamatetype phytochemical, has also been studied for its activity against bladder cancer. For instance, Peng et al. (110) reported the effects of ferulic acid on T24 bladder cancer cells in two-dimensional (2D) and 3D cell culture systems where they found that it dramatically increased apoptosis, and induced much higher cytotoxicity in 3D than in 2D culture systems. Ferulic acid also upregulates anti-oxidative enzymes such as superoxide dismutase, catalase, and pro-apoptotic proteins such as caspase3, cleaved caspase 9, and Bax (111).

Stilbenes
Piceatannol is a phytochemical, similar to resveratrol, found in grapes, blueberries, passionfruit, and other fruits. Piceatannol inhibits EJ bladder cancer cell proliferation by arresting the cell cycle in the G0/G1 phase. Moreover, piceatannol induces EJ cell apoptosis through a mechanism associated with the increases in protein expression of phosphatase and tensin homolog in concert with a decrease in the phosphorylation of AKT (111).

QUINONE COMPOUNDS Anthraquinones
Emodin is an anthraquinone found in dried rhizomes and roots of Polygonum. This compound has anticancer effects against various cell lines, including bladder cancer cells (112). Ma et al. (113) reported that emodin dose-dependently suppresses proliferation and invasion of T24 and 5637 bladder cancer cells. Further, emodin downregulates the mRNA and protein expression of Notch1, Jagged1, VEGF, VEGFR2, and MMP2 (113). Additionally, Notch1 overexpression rescues emodininduced cell growth suppression, indicating that emodin inhibits the proliferation of bladder cancer cells via the Notch1 pathway (113). Arbutin, which occurs abundantly in the bearberry plant, is a glycosylated hydroquinone that inhibits the proliferation of TCCSUP bladder cancer cells in a concentration-dependent manner. However, arbutin did not show marked cytotoxicity against TCCSUP cells even at concentrations of 500 µg/mL. Arbutin inactivates the ERK pathway and downregulates cell proliferation. Moreover, arbutin markedly elevates p21/ WAF1expression, thereby negatively regulating the cell cycle (114). Juglone is a quinone isolated from the root bark, pericarp, and branch bark of fresh Juglone catalpa and its anticancer activity includes inhibition of proliferation and induction of apoptosis of RT4 and TCCSUP bladder cancer cell lines (115).

Phenanthraquinone
Tanshinone IIA is a lipid-soluble phenanthraquinone compound extracted from Salvia miltiorrhiza Bunge. Tanshinone IIA has been reported to inhibit cell proliferation with an IC 50 < 5 mg/mL against various bladder cancer cells, including T24, 5637, and BFTC-905 cells. Moreover, tanshinone IIA suppresses cancer cell invasion by decreasing the expression and activity of MMP-9 and MMP-2, and suppresses the expression of C-C motif chemokine ligand 2. In addition, tanshinone IIA inhibits EMT by elevating the epithelial marker E-cadherin and reducing mesenchymal markers such as vimentin and N-cadherin. Tanshinone IIA also decreases transcription regulators such as Slug and Snail in bladder cancer cells (116). Naphthoquinone b-Lapachone is a natural naphthoquinone compound extracted from the bark of the lapacho tree that inhibits the viability of T24 bladder cancer cells by inducing apoptosis at the micromolar concentration range. Treatment of T24 cells with b-lapachone downregulated Bcl-2 expression and upregulated that of Bax. b-Lapachone-induced apoptosis is associated with the activation of caspase 9 and caspase 3. Moreover, b-lapachone also inhibits FAS and FASL in a dose-dependent manner in bladder cancer (117).
Based on the above review, phytochemicals exhibited anticancer activities through various mechanisms, including the MAPK, PI3K/AKT, and mTOR, cell cycle checkpoint, and apoptosis regulation signaling pathways (Figure 1). Proteins involved in these signaling pathways are affected by phytochemicals and are interrelated. For example, AKT influences cell proliferation by phosphorylating p21/WAF1 and regulates NF-kB signal transduction by phosphorylating IKKs (118). Caspases are a family of cysteine proteases that are central regulatory proteins of apoptosis (119). FAS is activated by FASL, leading to the activation of downstream caspases (e.g., caspase8 and caspase 9), which in turn, activate downstream molecules (e.g., caspase-3) to perform apoptosis (119). Various phytochemicals, such as nobiletin, tangeretin, genipin, and gallic acid, increase the release of cytochrome C in the mitochondria, triggering mitochondria-dependent apoptosis. The anti-apoptotic proteins Bcl-2 and Bcl-xL are localized in the mitochondrial outer membrane and inhibit the release of cytochrome C (120). Pro-apoptotic proteins, such as Bad, Bax, Bid, and Bim, are localized in the cytoplasm and translocate to the mitochondria after receiving a death signal, and promote the release of cytochrome C. When Bad is transferred to the mitochondria, it forms a pre-apoptotic complex with Bcl-xL, thereby accelerating the release of cytochrome C (120). Following its release, cytochrome C binds to apoptotic peptidase activating factor 1 and forms an apoptotic activation complex with caspase9, thereby accelerating apoptosis (121). The phytochemicals which are able to induce apoptosis have been shown as Figure 2.
Various phytochemicals, such as tanshinone, silibinin, and piperlongumine, effectively inhibit cell invasion via attenuating MMPs and uPAR ( Figure 3) and alleviation of NF-kB. Moreover, EMT is an important mechanism for cell invasion, and the tumor microenvironment and tumor cells secrete cytokines that lead to EMT. These cytokines activate intracellular signal transduction pathways that upregulate specific zinc-finger transcription factors such as Slug, ZEB1, and Snail, thereby enhancing EMT (Figure 3).
MAPKs are a family of protein kinases that are specific to serine, threonine, and tyrosine (122), and play important roles in complicated cellular processes such as embryonic development, cell differentiation, tumorigenesis, and cancer metastasis. Three MAPK families, ERK, JNK and p38 kinase, have been classified in mammalian cells (122), and activation of the ERK and JNK pathways is positively related to tumor cell proliferation (123). Since targeting MAPK has been considered as good anticancer strategy (124), this review summarizes the anticancer activities of numerous phytochemicals, such as arbutin, apigenin, capsaicin, curcumin, kaempferol, morin, naringin, nimbolide, and yuanhuacine, which modulate the MAPK signaling pathway.
The cell cycle is another key process related to cell proliferation and cell cycle related molecules are widely considered as anticancer targets (124). CDK4, CDK6, cyclin D,   and cyclin E are essential for the transition of cells from the G1 phase to S phase, whereas p16Ink4A and p21/WAF1 inhibit cell cycle progression (125). During the G2/M transition, cells critically require CDC2 and cyclin B, and p53 is the checkpoint protein mediating this process (125). We summarized the anticancer activities of various phytochemicals, including fisetin, quercetin, genistein, puerarin, naringin, limonene, atractylenolide I, triptolide, nimbolide, capsaicin, piperlongumine, ellagic acid, piceatannol, and arbutin, which attenuate the cell cycle via different mechanisms, mainly by suppressing CDK and cyclin expression and increasing cell cycle inhibitor expression ( Figure 4). Because of various anti-tumor activities of phytochemicals, intake of plant-derived diets to prevent bladder cancer has also been extensively studied. Epidemiological studies showed high fluid intake reduce 49% bladder cancer risk compared with the low intake group, indicating phytochemical might be used for preventing bladder carcinogenesis (126). Moreover, the bladder cancer preventive function of phytochemical monomer was also studied: EGCG intake could reduce the tumor cell proliferation markers, which was consistent with potential chemo-preventive efficacy in bladder cancer patients (43).

CONCLUSION
In summary, phytochemicals can combat bladder cancer through various mechanisms, such as inhibition of proliferation, migration and invasion; induction of apoptosis; and promotion of autophagy. Table 1 provides an overview of the functions and corresponding molecular mechanisms of different phytochemicals. The use of phytochemicals could be a potentially effective approach for cancer prevention and treatment based on several unique properties. 1) Drug and food homology: many phytochemicals with anticancer activities are derived from vegetables and fruits, and dietary habits are closely linked to cancer. 2) Phytochemicals come from a wide range of sources and can either be extracted from plants or chemically synthesized. 3) Most phytochemicals are metabolized and excreted in the urine and, consequently, are more efficient at reaching the lesion location, the bladder. Overall, this review