Transitional cell carcinoma (TCC), also called urothelial carcinoma, is the most common type of genitourinary cancer. Common risk factors include cigarette smoking and chemical exposure. Worldwide, the annual incidence rates are approximately 9.0 and 2.2 per 100,000 persons for men and women, respectively; the annual mortality rates are reported to be 3.2 and 0.9 per 100,000 persons for men and women, respectively [1
]. However, the incidence and mortality rates of bladder cancer vary across countries. For instance, in Taiwan, higher incidence of bladder cancer was found in black-foot disease endemic areas [2
]. The recommendations on the clinical management of bladder cancer is based on its muscle invasiveness, classified as non-muscle-invasive or muscle-invasive bladder cancer. Approximately 75% of patients with newly diagnosed bladder cancer present with non-muscle-invasive disease and undergo transurethral resection of the bladder followed by adjuvant therapy, such as intravesical chemotherapy or bacillus Calmette–Guérin (BCG) immunotherapy, to reduce the risk of disease recurrence or progression [1
]. In patients with muscle-invasive disease or disease progression, treatment options include radical cystectomy and neoadjuvant chemotherapy, but as patients potentially progressed to metastatic status, the survival rate drops [3
]. The efficacy of chemotherapy may be limited by drug toxicity, resistance, and adverse effects, and so novel therapeutic strategies that enhance treatment efficacy or increase sensitivity to drugs are of high clinical importance.
Epigallocatechin gallate (EGCG) is a major bioactive polyphenolic compound found in green tea leaves, and the effect of EGCG and their synthetic analogs have been reported as potential anti-cancer agents in various cancer types through modulating and regulating multiple signaling pathways [4
]. Regarding the potential effects of EGCG on the urinary tract system, flavonoid antioxidants including EGCG were reported to ameliorate cyclophosphamide-induced cystitis [5
]. In addition, Bazi et al. claimed the protective effect of EGCG against bladder degeneration in rats [6
], and the antioxidant effect against H2
-induced oxidative stress through superoxide was observed in normal and malignant bladder cells [7
]. In bladder cancer, EGCG inhibits proliferation and migration, and induces apoptosis of bladder cancer cells [8
], and it also exerts cytotoxic effect and prevents intravesical tumor growth with similar efficacy to mitomycin C [10
]. Sterilization through radiation could keep EGCG in a stable form and of benefit in treating superficial bladder cancer [11
]. A recent phase II trial of polyphenol consisted primarily of EGCG administered in patients prior to bladder cancer surgery resulted in definite tissue accumulation and biologic activity of polyphenol [12
]. The evidence suggested EGCG as a potential therapeutic agent for prevention of tumor implantation in bladder cancer.
To investigate the role and novel therapeutic targets of EGCG on bladder urothelial carcinoma, we conducted this study to explore the effect of EGCG on altered gene expression and microRNA expression profiles of bladder cancer cells using next generation sequencing (NGS), and further investigate functionally enriched biological themes using different bioinformatics database analyses.
The current study investigated the potential effects of EGCG on bladder urothelial carcinoma using NGS and bioinformatics analysis. A total of 108 differentially expressed genes in EGCG-treated bladder TCC cells were identified, and were mainly involved in NAD biogenesis, inflammatory response, and oxidation-reduction metabolism. In addition, miRNA target prediction analysis identified several miRNA–mRNA interactions that potentially participated in the response of bladder TCC to EGCG treatment, including miR-185-3p-ARRB1
, and miR-22-3p-PPM1K
. Of these genes with potential miRNA interactions, DLG2
was involved in Hippo pathway and inflammatory response, and TNS1
, and MGAT5B
were involved in inflammatory response. A schematic summary of the current study is illustrated in Figure 6
NAD is an essential coenzyme participating in various energy metabolic pathways, cell signaling regulatory pathways and DNA repair, and a potential therapeutic target for oncotherapy [23
]. Higher levels of NAD in cancer cells has been reported [24
]. The high glycolytic rate of cancer cells, called “Warburg effect” requires NAD for glycolytic enzymes, and the NAD salvage pathway is important in cancer biology and an intriguing target to prevent cancer growth [23
]. Nicotinamide nucleotide adenylyltransferase 2 (NMNAT2) is a cytosolic enzyme that assists in maintaining mitochondrial NAD level, although the association between NAD salvage pathway and mitochondrial NAD level remains unclear [23
]. In osteosarcoma cells, knockdown of NMNAT2 reduced p53-mediated cell death upon DNA damage [25
]. Interestingly, previous study has reported overexpression of NMNAT2 enhanced sensitivity of colorectal cancer cells to specific pro-drug and improved therapeutic efficacy [26
]. In addition, reduced NAD level also activates signaling pathways promoting epithelial to mesenchymal transition (EMT), facilitating tumor progression [27
]. In bladder cancer, the role of nicotinamide metabolism has recently been reported. The concentration of NAD in whole blood was decreased in patients with bladder cancer compared to control subjects, and plasma levels of nicotinamide metabolites were also altered [28
]. NQO1, one of the major quinone reductases highly inducible under cellular stress, modulate NAD/NADH redox balance, and specific polymorphism of NQO1 is associated with cancer risk in urinary system [29
]. However, the role of NMNAT2 in bladder cancer cells and whether expression of NMNAT2 affects therapeutic efficacy remains unclear.
Another important issue is reactive oxygen species (ROS) which is toxic to all cells, including cancer cells. Therefore, a good anti-oxidant defense system in cells is essential to prevent cytotoxic damage [23
]. NAD is also involved in the regulation of oxidative stress, and report showed ascorbate induced cell death of neuroblastoma cells through NAD depletion and ROS-induced DNA damage, which suggested that NAD and ROS production are partially responsible for the cancer cell cytotoxicity [30
]. The current evidence suggested the importance of NAD biosynthesis in cancer pathogenesis and NAD as potential biomarkers. Our analytic results also indicated the involvement of NAD biosynthesis and related oxidation-reduction process in EGCG-treated bladder TCC cells, which suggested the potential effect of EGCG in urothelial carcinoma through alteration in NAD and oxidation-reduction related metabolism.
Chronic inflammation plays an important role in cancer. The link between inflammation and development of bladder cancer has also been reported, and the oncogenic changes may potentiate inflammatory microenvironment that leads to angiogenesis and invasion of bladder cancer [31
]. EGCG is a major constituent of green tea polyphenols that is documented as having antioxidant, anti-inflammatory, and anti-cancer effects [33
]. The anti-inflammatory property is mainly through targeting toll-like receptor 4 signaling pathway, which is also associated with cancer progression [34
]. In our current study, results suggested the involvement of inflammatory response among dysregulated genes with potential miRNA interactions, particularly TNS1
, and ARRB1
, and the activation z-score was −0.128, indicating inhibited inflammatory response upon EGCG treatment.
Arrestin beta 1 (ARRB1) is one of the beta arrestins regulating various cellular physiologic processes, and have been demonstrated to have pro-tumorigenic effects [35
]. Kallifatidis et al. recently reported increased ARRB1 expression in bladder cancer cells, and ARRB1 gene knockout reversed the aggressive phenotype of bladder cancer cell lines [37
]. MiR-185-3p is a potential oncosuppressor miRNA evident in nasopharyngeal carcinoma (NPC), and the inhibition of miR-185-3p promoted invasion and metastasis of NPC cells [38
]. In the current study, altered miR-185-3p-ARRB1 interaction was identified in EGCG-treated bladder TCC cells, with 3.92-fold up-regulated miR-185-3p and 4.01-fold suppressed ARRB1. The evidence suggested the potential effect of EGCG in altered miR-185-3p expression that may affect bladder TCC cells.
Down-regulation of tensin 1 (TNS1) potentially regulated by miR-31-5p and miR-642a-5p was also identified in the current study. The expression of TNS1 was found higher in colorectal cancer cell lines and tissues, and suppression of TNS1 decreased proliferation and invasiveness of cancer cells, while the level of TNS1 was positively associated with poor survival in patients with colorectal cancer, indicating TNS1 could potentially be a therapeutic target [39
]. MiR-31-5p serves as an oncosuppressor miRNA and increases the sensitivity of bladder cancer cells to mitomycin C [40
]. EGCG also exerted cytotoxic effect to bladder cancer cells with similar efficacy to mitomycin C [10
]. This suggests that EGCG can be a potent adjunctive compound to mitomycin C in bladder cancer treatment through regulating miR-31-5p and its potential target TNS1. However, the role of miR-642a-5p in bladder cancer remains unclear, and further investigation is needed to clarify the role of miR-642a-5p-TNS1 interaction in bladder cancer in response to EGCG treatment.
In our current study, DLG2 (discs large homolog 2) potentially regulated by miR-1226-3p and miR-484, along with SCRIB and SMAD1, were associated with Hippo signaling (p
= 6.74 × 10−3
). The effect of EGCG in cancer treatment was validated from in vitro and in vivo studies [4
]. The Hippo signaling pathway is found dysregulated in bladder cancer, causing cancer progression and chemotherapy resistance [42
]. EGCG was proposed to affect cell proliferation and apoptosis through Hippo signaling pathway in oral cancer [43
], but has not been studied in bladder cancer. While the role of DLG2 as a tumor suppressor gene in osteosarcoma [44
] and miRNA target in ovarian cancer inhibiting cell migration [45
] were reported, its involvement in bladder cancer remains unclear.
There are some limitations in the current study. Firstly, the current results were based on results from bladder cancer cell line, BFTC-905. Since genomic variability is an important issue in urothelial carcinoma, future study will be conducted on other bladder cancer cell lines and primary cells from patients with urothelial carcinoma for further investigation. In addition, experimental validation of potential targets and miRNAs in EGCG-treated bladder cancer cells is needed. Moreover, it is important to investigate the changes in gene expression in clinical specimens of patients with different bladder cancer stages to determine the therapeutic effect of EGCG.