UROLOGICAL ONCOLOGY The Evaluation of the Relationship between Bladder Cancer and Oxidative Stress Using NRF-2/KEAP-1 Pathway, Zinc and Copper Levels Cağri Doğan1*, Cenk Murat Yazıcı1, Murat Akgül1, Polat Türker1 Purpose: It has been shown that Copper and Zinc contribute to the structure of the antioxidant enzymes. In addi- tion, NRF-2 and KEAP-1 complex have a powerful effect on the intracellular organization of the antioxidants. We evaluated the relation of Copper, Zinc, NRF-2, and KEAP-1 complex regarding the oxidative stress with tumor stage - grade in patients with bladder cancer. Materials and Methods: A total of 52 patients (32 bladder cancer and 20 control group) were included in the study. The demographic properties of groups were identical. Serum NRF-2, KEAP-1, Cu, and Zn levels were com- pared by ELISA method between the groups, and tissue NRF-2, KEAP-1, Cu and Zn levels were evaluated also by ELISA method in cancer patients. Results: Serum levels of NRF-2 and KEAP-1 of the bladder cancer patients were found to be higher than the control group (p = 0.004 and p = 0.001, respectively). On the other hand serum levels of Copper and Zinc were found to be lower than the control group (p = 0.008 and p = 0.001, respectively). However, the subgroup analysis according to the stages and grades of the tumour showed no difference. The Copper level obtained from the tissue analysis was detected to be considerably decreased with tumour stage and grade. Conclusion: Bladder cancer patients had higher serum NRF-2 and KEAP-1 levels and lower serum Copper and Zinc levels. In addition, the Copper levels decreased with the tumour stage and grade. Studies with larger number of patients are needed to demonstrate the efficacy of these markers. Keywords: bladder cancer; NRF-2; KEAP-1; urinary markers Department of Urology, Tekirdag Namik Kemal University School of Medicine, Tekirdag, Turkey. *Correspondence: Assistant Professor at Department of Urology, Tekirdağ Namık Kemal University Medical School. e-mail: Phone number:00905558908805. drcagridogan@gmail.com. Adress: Tekirdag Namık Kemal University, Urology Department, Süleymanpaşa, Tekirdağ, Turkey, 59030. Received September 2020 & Accepted April 2021 INTRODUCTION Bladder cancer (BC) is the 9th most frequently de-tected and 13th most-lethal common malignancy worldwide. In addition, non-muscle invasive bladder cancer (NMIBC) accounts approximately for 75% of the patients at admittance(1). The important risk factors known for the aetiology include smoking, exposure to aromatic amines and polycyclic hydrocarbons, genetic predisposition, and chronic irritation(2). The gold stand- ard method for the diagnosis and treatment of BC is cystoscopy. Although several diagnostic markers have also been identified to minimize this invasive proce- dure, none of them could get ahead of cystoscopy(3-4). Exposure to toxic metabolites such as aromatic amines and oxidative stress appear to be important factors in carcinogenesis. The changes caused by these factors at the cellular level lead to the initiation of the oncolog- ical process and chronicity. Reactive oxygen radicals (ROR) are the most important radical type formed by living systems originated from the reduction of the molecular oxygen(5). ROR play a significant role in the different complex course of carcinogenesis. A higher value of ROR concentration is associated with the pres- ence of tumours. There are a lot of epidemiologic evi- dences for ROR in cancer pathogenesis. ROR have also close relationship with BC. It has been demonstrated that ROR is one of the important reasons for the pres- ence and recurrence of BC(6,7). When the cell is exposed to oxidative stress, it activates its own antioxidant systems in the first stage. These systems might be mediated via enzymatic or non-enzy- matic pathways, and exert activity to limit the catabolic effects of the ROR(8). Trace elements in the structure of the enzymes responsible for the antioxidant system as co-factors are as functionally important as the en- zymes themselves against oxidative stress. The imbal- ance of these elements may lead to lack of limitation of the oxidative stress(9). Copper and Zinc are the essential molecules which contribute to the structure of the an- tioxidant enzymes as co-factors and play an important role in the metabolism of oxidative stress(10). The ab- normal changes in the levels of the trace elements may cause mutation and cancer by affecting the structural characteristics of the antioxidant enzymes(11). The pres- ence of an association between trace elements and BC is also observed in the previous studies. The imbalance of these important trace elements might play an impor- tant role in BC induction(12). Total antioxidant capacities have close relationship in the presence of carcinogenesis. Novel studies reported that antioxidants could prevent the initiation and pro- motion of carcinogenesis(7). The physiological role of Urology Journal/Vol 18 No. 4/ July-August 2021/ pp. 422-428. [DOI: 10.22037/uj.v18i.6439] antioxidants is to prevent damage to cellular compo- nents that may arise as a result of chemical reactions involving ROR. Nuclear transcription factor erythroid 2p45 related factor 2 (NRF-2) and Ketch-like ECH re- lated protein 1 (KEAP-1) complex have an important ef- fect on the intracellular organization of the antioxidant system(8). When the cell is exposed to oxidative stress, NRF-2 is separated from KEAP-1 and translocated to the nucleus, and provides the transcriptional activation of the detoxification enzymes and antioxidant molecule genes(13). Schematic diagram of the ‘NRF2 - KEAP1- Antioxidant Response Element’ signaling pathway is shown in Figure 1(14). In addition to protecting the nor- mal cells, this complex may also protect the cancer cells exposed to stress and prolong the survival of the cancer cells(15). While its relation with several types of cancer has been evaluated in the literature, there is no study evaluating the relationship between the BC and NRF-2/ KEAP-1 complex. In this trial, we aimed to assess the level of NRF-2, KEAP-1 molecules and Copper and Zinc levels asso- ciated with oxidative stress. Furthermore, we evaluated the place of NRF-2, KEAP-1, Zinc, and Copper mol- ecules in the diagnosis of BC, which may be possible BC markers. PATIENTS AND METHODS All procedures in this cross-sectional study were per- formed in accordance with the ethical standards of the institutional / national research committee with the standards of Helsinki declaration. This study was ap- proved by the local / national ethics committee (no: 2015/12/01/12) and all patients gave written informed consent prior to surgery and the study. The sample size was calculated based on the formula according to the previously published works(6,7,12). The sample size estimation was 35 cases. For this reason we included 35 patients to the study, however, 3 patients were excluded from the study because of unacceptable values. While patients with < 2 cm of mass in their bladder were included in the trial, patients with a history of non-bladder cancer, chronic inflammatory disease, and rheumatic disease were excluded. Transurethral resec- tion of bladder tumour (TUR-B) was performed under general anaesthesia. Patients diagnosed with a BC type other than transitional cell carcinoma were excluded from the trial. Approximately 0.5cm3 tumour tissue pieces were collected from the bladder mass, and stored for analysis at -86°C in freezer condition. Moreover, 8 mL of blood samples were collected to the serum sep- arating gel tubes and centrifuged at 3000 rpm for 10 minutes. The resultant sera, then, were taken into Ep- pendorf tube and stored in a -86°C freezer to analyse the levels of NRF-2, KEAP-1, Copper, and Zinc. No tissue sample was collected from the patients in this group due to ethical reasons. The control group was composed of totally healthy in- dividuals. Both the patient and the control group were similar patterns for age, sex, smoking status and region of living area. We performed cystoscopy to the control group as a gold standard to exclude BC. The individuals to whom cystoscopy was performed were included as a control group for different evaluations such as benign prostate hypertrophy (BPH) etc. without ethical re- striction. The cystoscopy of these individuals was also normal for BPH and other diseases. The exclusion cri- teria in the study group were also applied to the control group. Bladder tumour tissue samples were homogenized in saline buffer solution. Following the homogenization, NRF-2 levels (Cusabio brand kit with the lot number of CSB-EL 015752HU) and KEAP-1 levels (Cusabio brand kit with the lot number of CSB-EL 012147HU) in the tissue were measured by ELISA method. Tissue samples for measuring Copper and Zinc levels were boiled with 2 mL nitric acid at 100°C for 1 hour and Table 1. The Demographics of Patients and Control Group. Control Bladder group tumour group Number 20 32 Median age (± SD) 60.2 ±11.2 67.7 ± 10.95 Gender (female / male) 5/15 4/28 Comorbidity (DM/HT) 2/2 6/5 Control group average values (Min.-Max.) Bladder tumour group average values (Min.-Max.) P value KEAP-1 (serum) pg/mL 577 (193-1843) 1133 (150-10897) 0.004 NRF-2 (serum) pg/mL 3227 (2340-5150) 5708 (1463-20645) 0.001 Copper serum pg/mL 126 (80-247) 101 (38-219) 0.008 Zinc serum pg/mL 180 (135-326) 146 (61-279) 0.001 Table 2. The Correlation Between Parameters of Serum Levels in Bladder Tumour Group and Control Group. Figure 1. Schematic diagram of the Nrf2-Keap1-ARE signaling pathway. Nrf2 is constantly ubiquitinated through Keap1 and degraded in the proteasome. After exposure to oxidative stress, Keap1 is inactivated and Nrf2 becomes phosphorylated. Phospho- rylated Nrf2 (p-Nrf2) accumulates in the nucleus and binds to An- tioxidant Response Element sites. It subsequently activates other genes including antioxidants, detoxifying enzymes, and transport molecules. Bladder cancer and oxidative stress-Doğan et al. Urological Oncology 423 Vol 18 No 4 July-August 2021 424 digested, and then 2 mL of perchloric acid (60%) was added. The digested material was diluted with deionized water and the levels of Copper and Zinc were measured using flame atomic absorption spectrophotometry (TM Shimadzu AA-6800). For the analysis of the data, SPSS version 20.0 soft- ware was used. The normality of the data was checked using Shapiro-Wilk test. As the variables do not distrib- ute normally, non-parametric statistical tests were pre- ferred. Mann-Whitney U test was used for the compar- ison of two independent groups. For the comparisons of more than two independent groups, Kruskal-Wallis test was used. Mann-Whitney U test was used for the comparison of subgroups. The Receiver Operating Curve analysis (ROC) was performed to detect the pre- dictive accuracy of the studied markers (NRF-2 and KEAP-1). The results were expressed as median and minimum-maximum values using marginal or cross tables. P < 0.05 value was accepted to be statistically significant. RESULTS A total of 32 patients with the diagnosis of BC were included in the trial. Twenty-eight (87.5%) of the pa- tients were male, 4 (12.5%) of them were female, and the mean age of the patients was 67.7 ± 10.9 years. Six (18%) of the patients had diabetes mellitus (DM) and 5 (15%) had hypertension (HT) (Table 1). After the re- section, 2 (6%) of the patients were detected to have carcinoma in situ (CIS), 7 (21%) of them were found to have Ta/low grade, 3 (9%) of them had Ta/high grade, 9 (28%) of them had T1/low grade, 5 (15%) of them had T1/high grade, and 6 (18%) of the patients were found to have T2/high grade tumour. A total of 20 healthy individuals were included in the control group for the trial. Fifteen (75%) of the patients in this group were male and 5 (25%) of them were fe- male. The mean age was 60.2 ± 11.2 years. Two (10%) of the patients in the control group had diabetes mellitus and 2 (%10) patients were detected to have hyperten- sion (Table 1). A serum median levels of NRF-2 and KEAP-1 in the study group were 5708 pg/mL (min:1463-max:20645) and 1133 pg/mL (min:150-max:10897), respective- ly. On the other hand, serum median level of NRF-2 was 3227 pg/mL (min:2340-max:5150) and KEAP- 1 was 577 pg/mL (min:193-max:1843) at the control group. The study group values of NRF-2 and KEAP-1 were found to be significantly higher than the control group (p = 0.001 and p = 0.004). The ROC analysis for the serum NRF-2 and KEAP-1 in BC patients was shown in Figure 2. The cut-off value for serum NRF- 2 was 4291 pg/mL (sensitivity: 75%- specificity: 55%) (AUC=0.800, p < 0.0001, %95 CI (0.679-0.931)). In addition the cut-off value for serum KEAP-1 was 913 pg/mL (sensitivity: 65%- specificity: 60%) (AUC = 0.741, p < 0.004, %95 CI (0.606-0.875)). The normal value of serum total Copper level in healthy population was 63.7-140.12 pg/mL. A serum medi- an level of total Copper value in BC patients was 101 (min:38-max:219) pg/mL (min:1463-max:20645) and in control group was 126 pg/mL (min:80-max:247). A serum median level of Zinc in BC patients was 146 pg/ mL (min:61-max:279) and in control group was 180 pg/mL (min:135-max:326). Serum levels of Copper and Zinc were found to be statistically significantly lower than the control group (p=0.008 and p=0.001, respectively) (Table 2). Subgroup analysis performed based on the stages of the BC showed no difference in the staging and grading of the tumours caused by se- rum levels of Zinc and Copper (p = 0.26 and p = 0.89, respectively). In addition, tissue levels of NRF-2 and KEAP-1 were detected to cause no significant differ- ence in the staging and grading of the tumours. The only significant difference between BC stages/grades was found in the tissue Copper level (p : 0.038). When we analysed the subgroups for tissue Copper level, this significance only occurred between Ta low grade ver- sus T2 (p : 0.001), Ta high grade versus T2 (p : 0.02), and CIS versus T2 (p : 0.04) stages (Table 3). Values and statistical analyses of the trial data for serum and tissue Copper, Zinc, NRF-2, and KEAP-1 according to Table 3. The Comparison of Copper Levels in Bladder Cancer Tissue for Significant Subgroups. Bladder tumour staging/grading N (%) Cu tissue values (pg/mL) P value Ta low grade versus 7 (21%) 9.96 (3.7-119.8) 0.001 T2 high grade 6 (28%) 2.8 (1.1-3.7) CIS versus 2 (6%) 11.1 (10.8-11.3) 0.04 T2 high grade 6 (28%) 2.8 (1.1-3.7) Ta high grade versus 3 (9%) 4.6 (4.3-4.9) 0.02 T2 high grade 6 (28%) 2.8 (1.1-3.7) Bladder tumour staging N (%) Average values (Min-Max) P value KEAP-1 (serum) pg/mL Ta low grade 7 (21%) 1025 (150-10897) 0.67 CIS 2 (6%) 3452 (1154-5750) Ta high grade 3 (9%) 975 (788-1960) T1 low grade 9 (28%) 1088 (383-3050) T1 high grade 5 (15%) 2175 (375-4160) T2 high grade 6 (28%) 1038 (420-4160) KEAP-1 (tissue) pg/mL/mgPrt Ta low grade 7 (21%) 130 (8-600) 0.62 CIS 2 (6%) 166 (68-265) Ta high grade 3 (9%) 37 (25-106) T1 low grade 9 (28%) 50 (19-185) T1 high grade 5 (15%) 111 (22-219) T2 high grade 6 (28%) 70 (22-219) Table 4. The Correlation Between Bladder Tumour Stage and KEAP-1 Levels. Bladder cancer and oxidative stress-Doğan et al. the stages and grades are shown in Tables 4 to 7. DISCUSSION The dysregulation of the ROR mechanism may play a role in the pathology of tumorigenesis. It activates the abnormal introduction of the signalling pathway that triggers the tumor formation. The uncontrolled growth of the cells leads to the development of the cancer mass with the help of the reactive oxygen-nitrogen species, signal transduction, transcription factors, and kinases/ phosphatases cascades(16). Copper and Zinc are some of the fundamental molecules involved in oxidative stress. They contribute to the structure of antioxidant enzymes as co-factors(10). Previous studies have shown that oxidative stress may be an important factor in the development of BC(7,16-19). It has known that the intake of vitamins and antioxidants decreases BC recurrence (20). Mazdak et al. also stated that the distribution of total antioxidant activity did not show normal pattern in pa- tients with BC. They revealed that the patients with BC had a lower level of total antioxidant activity(7). NFR-2/KEAP-1 complex has also an important role in antioxidant activity. NFR-2 is a transcription factor be- longing to Cap’n’Collar family (CNC) in this complex and has a close interaction with KEAP-1. KEAP-1 is another molecule of this complex. It acts as a sensor to identify the oxidant and electrophilic compound(8). When the cell is exposed to oxidative stress, it separates from KEAP-1. The free NRF-2 quantity increases in the cytoplasm. When a certain concentration is achieved, NRF-2 molecules translocate to the nucleus and bind to Antioxidant Response Element (ARE) region. ARE is present in the promotor region of many antioxidant enzymes. This region provides the transcriptional acti- vation of the detoxifying enzymes and antioxidant mol- ecules in the cells exposed to oxidative stress(15). While its relation with several types of cancer has been evaluated in the literature, there is no study evaluating the relationship between the BC and NRF-2/KEAP-1 complex. The somatic mutation of the NRF-2 prevents it from being identified by KEAP-1, and leads to NRF-2 up-regulation. This mechanism has been demonstrated to play a role in pulmonary, head/neck, and oesopha- gus cancer(21-23). Similarly, KEAP-1 mutations had been detected in pulmonary and gallbladder carcinoma tis- sues(24-25). KEAP-1 mutations cause over-expression of NRF-2 in the cancer cells and activation of the cytopro- tective proteins in response. Similar findings are also thought to be in association for BC. In our trial, when the BC and the control groups are compared, serum NRF-2, KEAP-1, Copper and Zinc levels were observed to have statistically significant differences. Being an important part of the oxidative stress, trace elements in the serum were detected to be significantly reduced in patients with BC. We be- lieve that this reduction may be associated with the fact that trace elements such as Zinc and Copper are used as co-factors of the enzymes acting to prevent oxida- tive stress or used for system activation. Furthermore, NRF-2 and KEAP-1 increased in BC patients that show the presence of oxidative stress indirectly. The increase of NRF-2 and KEAP-1 was statistically considerable in patients with BC compared to the control group. It suggests that oxidative stress is an important factor in the development of BC. Similar trials are showing the relationship between BC and Copper and Zinc levels in the serum(10,12,26). When the results of these studies are examined, different data are obtained. In the previous studies, the serum levels of Zinc have been found to be generally low in cases with BC and in the meta-analysis by Song et al. this reduction was found to be significant (10). Consistent with the literature findings, in the pres- Table 5. The Correlation Between Bladder Tumour Stage and NRF-2 Levels. Bladder tumour staging Average values (Min-Max) P value NRF-2 (serum) pg/mL Ta low grade 7500 (1462-206445) 0.73 CIS 17261 (5771-28750) Ta high grade 4875 (3938-9800) T1 low grade 5438 (1916-15250) T1 high grade 10875 (1875-20800) T2 high grade 5188 (2100-20800) NRF-2 (tissue) pg/mL/mgPrt Ta low grade 6598 (1056-74967) 0.65 CIS 20785 (8457-33113) Ta high grade 4685 (3094-13282) T1 low grade 6287 (2358-23114) T1 high grade 13902 (2748-27366) T2 high grade 8801 (2723-27366) Bladder tumour staging Average values (Min-Max) P value Copper (serum) pg/mL Ta low grade 101 (38-146) 0.89 CIS 106 (97-114) Ta high grade 88 (86-1467) T1 low grade 100 (63-219) T1 high grade 115 (98-146) T2 high grade 105 (84-155) Copper (tissue) pg/mL/mgPrt Ta low grade 10 (4-120) 0.03 CIS 11.07 (10.84-11.31) Ta high grade 4.59 (4.29-4.87) T1 low grade 4.32 (1.68-11.50) T1 high grade 3.99 (2.97-13.59) T2 high grade 2.75 (1.06-3.74) Table 6. The Correlation Between Bladder Tumour Stage and Copper Levels. Bladder cancer and oxidative stress-Doğan et al. Urological Oncology 425 Vol 18 No 4 July-August 2021 426 ent trial, the serum levels of Zinc of the cases with BC were found to be significantly lower. The relationship between BC and the serum level of Copper has been reported in varying degrees in the literature. The gen- eral opinion is that the serum levels of Copper in cases with BC are increased compared to the control group. In the meta-analysis by Song et al., serum level of Copper was detected to be increased in patients with BC.(10) In our study, the serum level of Copper was detected to be considerably lower than the control group which is not consistent with the literature data(12). On the other hand, there were some studies that state no relationship between BC and control groups(26,27). The subgroup het- erogeneity of BC for stage and grade might be one rea- son for this inconstancy. We choose most of our cases from high-risk NMIBC and muscle invasive BC. The status of stage and grade might affect the Copper levels. In addition, the Copper level may also be involved in different mechanisms in this patient group. We believe that this inconsistency is associated with the fact that there are not enough studies to conduct a meta-analy- sis on this subject. In our study, we found that only the tissue level of Copper was significant for BC and this significance only occurred between Ta low versus T2, Ta high versus T2, and CIS versus T2 stages. We could not find any correlation between T1 and other stages. It might be due to the invasion of lamina propria (T1) is the transition layer for muscle invasion. For this reason, Table 7. The Correlation Between Bladder Tumour Stage and Zinc Levels. Bladder tumour staging Median values (Min-Max) P value Zinc (serum) pg/mL Ta low grade 140 (102-179) 0.26 CIS 202 (193-211) Ta high grade 130 (112-147) T1 low grade 159 (61-204) T1 high grade 143 (129-164) T2 high grade 150 (116-279) Zinc (tissue) pg/mL/mgPrt Ta low grade 36 (13-148) 0.06 CIS 67 (48-87) Ta high grade 16 (6-36) T1 low grade 21 (4-127) T1 high grade 20 (9-76) T2 high grade 11 (2-25) the results might show only significant results with dis- tinct stages. The serum levels of NFR-2 and KEAP-1 in cases with BC were found to be considerably higher than those of the control group. While there are no data regard- ing the BC, the serum levels of NRF-2 and KEAP-1 were found to be increased in patients with prostate, breast, and pulmonary cancers(28). The blood levels of these molecules that are the active components of the antioxidant system were also found to be increased in our study. We also evaluated the position of serum and tissue Zinc, Copper, NRF-2 and KEAP-1 molecules as BC markers. No relationship was detected between these molecules and the pathological stages except the Copper tissue levels of the BC. While there is no study regarding the BC, there are some studies showing the relationship between NRF-2 and KEAP-1 molecules, and other urological cancers(29). NRF-2 levels differ in benign and malignant prostate tissue with being signif- icantly more expressed in tissues with prostate cancer compared to benign tissue. Furthermore, a correlation was detected between the stage of the prostate cancer and NRF-2 levels(29). Limited number of participants and the non-homoge- neous distribution of the BC stages were the negative aspects of this trial. The highly broad reference ranges of NRF-2 and KEAP-1 were also the other negative as- pects of the study. In addition, the patient and control Figure 2. The ROC curve for serum NRF-2 and KEAP-1 in bladder cancer patients. Bladder cancer and oxidative stress-Doğan et al. group could not have exactly similar patterns for the type of ROR stress, the region of inhabitance of par- ticipants and the duration of exposure to agents. We know that Copper and Zinc had previously been studied in BC, however, as NRF-2 and KEAP-1 had not been studied before, we hope that this trial paves the way for future trials. We think that conducting much larger, comprehensive, new trials on this timely subject will give way to make the follow-up protocol of the patients with BC less invasive and more cost effective. CONCLUSIONS The serum levels of Zinc and Copper were found to be significantly decreased and the serum levels of NRF- 2 and KEAP-1 were found to be increased in patients with BC. For the prediction of the stage of the BC, it was detected that only the tissue level of Copper was significant and this significance only occurred between Ta low grade versus T2, Ta high grade versus T2, and CIS versus T2 stages. Other variables were observed to be non-significant for the prediction of the tumour stage. In this regard, we believe that the tissue level of Copper may aid other markers and cystoscopy in the diagnosis and follow-up of the patients. ACKNOWLEDGEMENT This investigation was supported by Tekirdağ Namık Kemal University Scientific Research Projects Com- mittee with project number NKU-BAP.00.20.TU.15.01. CONFLICT OF INTEREST We have no conflict of interest to declare. REFERENCES 1. Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F. Bladder Cancer Incidence and Mortality: A Global Overview and Recent Trends. Eur Urol. 2017; 71: 96-108. 2. 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