Introduction
Bladder cancer, which is the third malignancy of the
urinary system of frequency, is responsible for approximately
5% of cancer-related deaths.[] Factors such as
smoking, chemicals, urinary system infection, bladder
stones, radiation exposure, and western style diet are
blamed in the etiology.[]
Etiological factors such as smoking and chemical
agents are known to cause cancer by causing an increase
in reactive oxygen radicals. Free oxygen radicals cause deterioration of many cell components,
especially protein, lipid, and nucleic acid. Increased
free oxygen radicals are associated with cancer risk
through lipid peroxidation and protein oxidation. In
addition, free oxygen radicals and oxidative stress affect
nucleic acids and cause oncogenic mutations and
tumoral growth.[,] However, there is a balance between
oxidant and antioxidant structures. Disruption
of this balance in favor of oxidant contributes to tumor
development. Antioxidants have an anti-tumoral
effect because they prevent oxidation. Along with many minerals and vitamins in the body, many enzymes
have antioxidant properties.[4]
There are endogenous oxidant and antioxidant
structures in the human body. Protein carbonyl (PC),
an oxidant, is the end product of protein oxidation.
It has been shown that this structure is increased in
colon and breast cancer.[] Paraoxanase-1 (PON-1)
and arylesterase (ARE) are endogenous antioxidant
enzymes. These usually act together and act by binding
to the lipoprotein. The levels of these two enzymes
have been shown to decrease in colon cancer.[] Glutathione
reductase (GR) and glutathione S transferase
(GST) are the most well-known antioxidants. These
act on glutathione and inactivate oxidant structures.
It has been shown that the decrease in the levels of
these two enzymes is effective in the development of
colorectal cancer.[]
In this study, we aimed to measure PON-1, ARE,
GST, GR enzyme activities, and PC, which is a protein
oxidation product, which plays a role in antioxidant
mechanisms in the body and in bladder cancer.
Methods
This prospective study was performed between April
2021 and October 2021, after receiving governmental
ethics commission approval (Ethics Number:
31.03.2021-22). Transurethral resection tumor
(TUR-T) was performed in 63 patients with suspected
bladder tumors and not taking any active treatment.
Thirteen patients were excluded from this study because
their pathology results were benign or because
of a pathology other than urothelial carcinoma, and 4
patients were excluded because of additional systemic
diseases. Forty-six patients whose histopathological results
were defined as transitional cell carcinoma after
TUR-T and hospital staff, 50 healthy patients were included
in the study. The control group was formed by
matching the study group with the patients in terms of
gender and age. Among the patients in both groups,
those with active infection, additional systemic disease
(diabetes mellitus, vasculitis, chronic kidney, liver disease,
etc.), chronic alcohol and drug use were excluded
from the study. In addition, patients with additional
cancer in the study group and any cancer in the control
group were excluded from the study.
Blood Collection
All patients in the control and study groups stopped
all food and drink intake at 9 pm. At 9 am, after they had been fasting for 12 h, intravenous blood was
drawn from all patients before the operation. The
drawn blood was kept at +4°C with the help of ice
cubes. Then, after centrifugation at 5000 rpms for 10
min, the serum portion of the study was completed.
The obtained serum samples were kept at -80°C to
await for biochemical analysis.
PC
The Reznick and Packer method (1994) was used to determine
the plasma PC level. This method is based on
the spectrophotometric measurement (Shimadzu UV-
1601 Japan) of the color formed by the proteins in the
plasma with 2.4-dinitrophenylhydrazine.[]
ARE and PON-1
PON-1 activity of 4-nitrophenol, which is formed as a
result of enzymatic hydrolysis when paraoxon (O, Odiethyl-
O-p-nitrophenyl phosphate; Sigma Co, London,
UK.), is used as a substrate. ARE activity, on the
other hand, was determined by measuring the colored
product given by phenol, which is formed as a result
of enzymatic hydrolysis of Phenyl Acetate (Sigma Co,
UK), which is then used as a substrate in Techcomp
8500 II UV/VIS spectrophotometer (Techcomp Ltd.,
China).[]
• UNIT=1nmol 4-nitrophenol/L serum/min for
PON-1 activity
• UNIT=1micromol phenol/ml serum/min for ARE
activity.
GST and GR
The GST activity was determined using the Habig et
al.[] method. The GR level was measured as described
by Beutler.[]
Statistical Analysis
Descriptive statistics for continuous variables; the
mean was expressed as Standard Deviation, while Categorical
Variables were expressed as numbers and percentages.
In comparisons that were made in terms of
continuous variables, Student t-test was used for those
with normal distribution and Mann-Whitney U-test
for those without normal distribution. Pearson correlation
coefficients were calculated separately for the
groups in order to determine the relationship between
continuous variables. The statistical significance level
was taken as 5% in the calculations and the SPSS (Version:
21, IBM Corporation, Armonk, NY) statistical
package program was used for the calculations.
Results
For this study, there were 46 bladder cancer patients
included in the study group, and 50 healthy individuals
in the control group. While the mean age of the
patients in the study group was 62.80±10.74, it was
62.42±10.55 in the control group (p=0.86). The study
group had 8 (17.3%) female patients and 38 (82.6%)
male patients, and the control group had 9 (17.4%) females
and 41 (82.6%) males. While the body mass index
of the patients in the study group was 28.5 ± 3.46, it
was 28.96±3.64 in the control group (p=0.52). The tumor
grade of 30 patients (65.2%) was identified as low
grade, while 16 (34.7%) patients" tumors were identified
as high grade. The tumor invasion rate of 35 (76%)
patients was T0, while 11 (23.9%) patients had a tumor
invasion rate of T1. While all biochemical parameters
were significantly different in the patient group compared
to the healthy control group, when we evaluate
the study group within itself, there were significant differences
in tumor grades and invasion rates in all parameters
except GST and PC enzymes (Tables 1-3).
Table 1: Comparison of biochemical parameters between
bladder cancer and control groups
Table 2: Comparison of biochemical parameters among
patients with bladder tumors according to
tumor grades
Table 3: Comparison of biochemical parameters among
patients with bladder tumors according to
tumor invasion level
Discussion
In this study, it was shown that PC, which is one of the
protein oxidation products, was significantly higher
and antioxidant enzymes ARE, PON-1, GR, and GST
were found to be low in patients with bladder cancer.
In addition, this data show the importance of oxidative
stress factors in bladder cancer patients.
Free oxygen radicals that are formed as a result
of oxidative stress cause the deterioration in lipid,
protein, nucleic acids and carbohydrate structures in
cells. In addition, reactive oxygen species first cause
the peroxidation of unsaturated fatty acids found in
the cell membrane structure. As a result of peroxidation
in the cell membrane structure, deterioration in
the structure and functions of the cell begins to occur.[] Malondialdehyde (MDA), which is released
as a result of lipid peroxidation, is used as a marker of
oxidative stress.[] However, apart from fatty acids,
proteins are also affected by free oxygen radicals. As
a result of the effect of free radicals, separation in the
peptide structures and deterioration in the structure
of the protein occur.[] Compared with MDA, PC,
which is a protein oxidation product, has the advantage
that it is released earlier and that the PC level will
remain stable for a long time, while lipid peroxidation
products disappear within minutes.[] In addition,
PC can be stored for 3 months for analysis if kept at-80°C and is relatively easier to detect when compared
to MDA.[,] Over the past decade, many studies
have been published reporting increased PC levels in
various diseases, and it has been shown to have a good correlation with the severity of these diseases.[-]
In our study, the presence of PC has also shown its
association with bladder cancer.
Oxidative stress, which is effective in cancer development,
occurs as a result of imbalances between
oxidant and antioxidant mechanisms. Whongsiri,
Patcharawalai et al.[] stated that oxidative stress
can trigger urothelial carcinomas and antioxidants
can be used in defense. Wieczorek et al.[] reported
that antioxidant deficiency (catalase, superoxide dismutase,
and glutathione peroxidase) is effective in the
development of bladder tumors and low antioxidant
levels in cases that relapse after TUR-T. Among the
other antioxidant enzymes, we used in our study, both
PON-1 and ARE enzymes are in esterase structures.
PON-1 enzymes that are circulating with high-density
lipoprotein in the circulation protects low-density
lipoproteins against oxidation, as PON-1 interacts
with ARE enzymes in this function. It has also been
observed that decreases in PON-1 and ARE activities
are associated with many diseases, including cancer.
[-] In the study conducted by Michalak et al.,[]
no correlation was found between the presence and
severity of ovarian cancer and the activities of these
enzymes. However, in our study, a decrease in the
activity of these enzymes was detected with both the
presence of bladder cancer and the increase in the degree
and invasion of the disease. Although there was a
difference in enzyme activities between the groups in
the study of Michalak et al., it was thought that there
was no statistically significant difference due to the
small number of patients.
Glutathione is an important point in the oxidative
mechanism. It provides inactivation of intracellular
xenobiotics and increases cell resistance against free
oxygen radicals.[,] In case of intracellular deficiency,
fatty acids and free fatty acids in the cell membrane
are easily oxidized by free oxygen radicals. Two
of the most important enzymes in the glutathione
mechanism are the GR and GST enzymes, as these
enzymes play an important role in cell defense.[-] In the study of Gecit et al.,[] GST enzyme levels
were found to be low in bladder tissues, but the relationship
of these parameters with regards to tumor
grade and invasion rate was not investigated.In our
study, the GR and GST enzyme levels of patients were
significantly lower than those of the control group.
In our study, it was observed that oxidant and
antioxidant levels are important in terms of tumor
progression and aggressiveness, as well as their effect
on the development of bladder tumors. In addition, oxidative stress parameters may be prognostic factors
for bladder cancer and may also contribute to its diagnosis,
and may also lead to the development of therapies
in cancer treatment. Therefore, we think that this
study makes important contributions to the literature.
The limiting factor of our study is that oxidant and
antioxidant structures were not measured in patient
urine and tissue samples.
Conclusion
We think that oxidative stress is effective in the development
as well as in the exacerbation of its degree and
invasion, but that antioxidant mechanisms are protective.
For this reason, we believe that antioxidants can
be used in future treatment modalities. However, this
study should be supported by additional, more voluminous
studies.
Acknowledgment: The authors thank Halit Demir for the
biochemical analysis of the blood.
Peer-review: Externally peer-reviewed.
Conflict of Interest: All authors declared no conflict of interest.
Ethics Committee Approval: The study was approved by
The Van Yüzüncü Yıl University Interventional Clinical Research
Ethics Committee (No: 22, Date: 31/03/2021).
Financial Support: This study has received no financial
support.
Authorship contributions: Concept - M.D.; Design -
M.D., C.D.; Supervision - M.D., C.D.; Funding - M.D., C.D.;
Materials - M.D., C.D.; Data collection and/or processing -
M.D., C.D.; Data analysis and/or interpretation - M.D., C.D.;
Literature search - M.D., C.D.; Writing - M.D., C.D.; Critical
review - M.D., C.D.
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