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Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group
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Kaohsiung Journal of Medical Sciences (2017) xx, 1e6
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
Original Article
Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group Erdal Benli a,*, Ahmet Bayrak b, Abdullah Cirakoglu a, Tulin Bayrak b, Tevfik Noyan b a b
Department of Urology, Ordu University Faculty of Medicine, Ordu, Turkey Department of Biochemistry, Ordu University Faculty of Medicine, Ordu, Turkey
Received 21 February 2017; accepted 10 May 2017
KEYWORDS Oxidative stress; Prostate cancer; PON1; PAF-AH; Psa
Abstract The aim of the study was to measure platelet-activating factor acetyl hydrolase (PAF-AH) and paraoxonase (PON1) enzyme activity levels in patients with high Psa values to compare with healthy peers and also to determine the efficacy of these parameters in predicting pathologic results of patients with high Psa values. This study included 66 patients with Psa value > 4 ng/dl (Group 1) and 44 patients with Psa <4 ng/dl (Group 2) for a total of 110 patients. Parameters measured in serum of PON1, PAF-AH, and MDA were compared between the groups. Additionally the same parameters were compared between patients with prostate biopsy performed due to high Psa and diagnosed with cancer and the control group with normal Psa values. The PAF-AH activity in Group 1 was 125.17 8.64 and in Group 2 was 120.08 9.23 U/ml (p Z 0.003). The PON1 activity was 63.12 6.74 and 65.91 7.77 U/ml in the groups, respectively (p Z 0.04). Additionally, there were significant differences identified between the control group and PCa diagnosis group in terms of PAF-AH and PON1 activities (p Z 0.004 and p Z 0.02, respectively). The enzyme activity of PAF-AH and PON1 measured in serum of patients with high Psa value and patients with diagnosis of prostate cancer (PCa) were identified to have changed by a significant amount compared to healthy peers with normal Psa value. It was concluded that these parameters may be beneficial markers for use in assessment of patients with high Psa value. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Conflicts of interest: All authors declare no conflicts of interest. * Corresponding author. Department of Urology, Ordu University Faculty of Medicine, 52100, Ordu, Turkey. E-mail address: [email protected] (E. Benli). http://dx.doi.org/10.1016/j.kjms.2017.06.016 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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Introduction Prostate cancer (PCa) is the most commonly diagnosed male cancer in the United States, similar to most European countries [1]. The mechanism of prostate carcinogenesis is not completely understood, but evidence suggests that oxidative stress plays a role [2]. Most cells in healthy individuals have adequate antioxidant defenses to protect them from reactive oxygen species (ROS). However, this capacity diminishes with age and can be overwhelmed by an abundance of ROS from exogenous or endogenous sources, or by enzymatic deficiencies due to polymorphisms and mutations [3]. It is well known that oxidative stress, due to a defect in the antioxidant systems or the presence of excessive reactive oxygen species, plays a role in the etiopathogenesis of PCa [4]. Plasma contains various antioxidant molecules including albumin which is the major circulating antioxidant in both plasma and serum [5]. In addition, the most important extracellular enzymes that are involved in this process are paraoxonase (PON1) and platelet-activating factor acetyl hydrolase (PAF-AH) [6]. PON1 mainly conjugates the intermediates to excretable hydrophilic derivatives and binds to high-density lipoprotein (HDL). Thus, it plays a role in the detoxification process of organophosphorus compounds and carcinogenic lipidsoluble radicals from lipid peroxidation [7]. PAF-AH plays an essential role in lipid metabolism as an antioxidant molecule, which hydrolyzes both PAF (a potent inflammatory mediator) and oxidized phospholipids [7]. Since enzyme levels were found to be decreased or increased after inflammatory stimuli in animal models, it is unclear whether PAF-AH is an acute-phase response protein [8]. Though Psa is commonly used in PCa screening, Psa is known to be affected by many benign situations. As a result the search continues for the ideal tumor marker specific to prostate cancer and not affected by benign situations. To our knowledge, there is no study in the literature measuring the activity of PON1, PAF-AH and oxidative balance in patients with PCa. The aim of this study was to identify oxidative stress in PCa patients, and investigate whether new generation biochemical parameters or those that are previously known are associated with the risk of incidental PCa.
E. Benli et al. and supplying written consent. Serum for laboratory tests was obtained after 12 h starvation in the morning and stored at 80 C until laboratory studies. The patients’ ages, BMI, complaint at application, additional diseases, smoking habit and known laboratory tests like Psa and CRP were recorded. Parameters measured in serum were compared between healthy individuals with normal Psa value (control group), the group with high Psa value and patients with prostate cancer diagnosis.
Biochemical study PON1 activity measurement PON1 activity was measured spectrophotometrically at 270 nm based on phenol formed as a result of enzymatic hydrolysis of phenylacetate used as substrate. The solution reaction environment was created with 50 mM TriseHCl (pH: 8) containing 1 mM calcium chloride (CaCl2). The final concentration of the reaction environment was 1 mM phenylacetate and an appropriate amount of enzyme was added to begin the reaction. At the end of the reaction, the phenol formed was spectrophotometrically measured at 270 nm [9]. One unit of PON1 activity is defined as 1 mmol of substrate hydrolyzed per min, under the defined assay conditions.
PAF-AH activity measurement The PAF-AH activity used 2-thio PAF as substrate. Upon hydrolysis of the acetyl thioester bond at the sn-2 position by PAF-AH, free thiols are detected using DTNB (5,50 -dithiobis(2-nitrobenzoic acid)) and the 5-thio-2 nitrobenzoic acid is spectrophotometrically measured at 412 nm. The activity measurement environment is created with final volume of 1 ml, with 10 mM triseHCl (pH: 7.4), DTNB, synthetic substrate platelet-activating factor (PAF) and the serum to be measured. The 5-thio-2-nitrobenzoic acid formed as a result of the reaction is measured at 412 nm spectrophotometrically. One unit of PAF-AH activity is defined as 1 nmol of substrate hydrolyzed per min, under the defined assay conditions [10].
Level malondialdehyde (MDA) determination
Methods Study design This study received permission from Ordu University local ethics committee (2016/34). The study included a total of 110 individuals over the age of 40; 66 patients with Psa value 4 ng/dl (Group 1) and 44 patients with Psa value < 4 ng/dl as control group (Group 2). Patients with high Psa value had Psa values rechecked. Exclusion criteria included situations like systemic infection, urinary tract infections, recent urologic procedures, presence of ureteral catheter, systemic antibiotic use and prostatitis. Patients with Psa value 4 ng/dl underwent sextant prostate biopsy accompanied by transrectal ultrasound and antibiotic prophylaxis after receiving necessary information
The lipid peroxidation product of one molecule malondialdehyde (MDA) enters a reaction with 2 molecules of thiobarbituric acid (TBA) at pH 2e3 between 95 and 100 C. The pink-colored chromogene formed by the reaction is extracted with n-butyl alcohol and the obtained organic phase is read at 523 nm [11].
Statistical analyses Parameters with normal distribution are presented as mean SD while parameters with non-normal distribution are presented as median IQR. Comparison between groups used the Student t test for parametric data analysis and the ManneWhitney U test for nonparametric data. Correlation analysis of parametric data was completed with
Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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PAF-AH and PON1 values in Prostate Cancer
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the Pearson test, while the Spearman correlation test was used for non-parametic data. All statistical analyses were completed with “SPSS for Windows version 20.0” program. A p value < 0.05 was accepted as significant.
Results The age distribution of patients participating in the study showed the mean age in Group 1 with Psa >4 ng/dl was 64.74 7.82 (50e80 years), while in Group 2, the control group, the mean age was 61.50 9.31 (38e85 years) (p Z 0.06) (Table 1). In terms of mean Psa value when the groups are compared, Group 1 was 6.87 7.27 (3.9e70) ng/dl (median IQR) and Group 2 was 0.91 0.60 (0.2e3.8) ng/ dl (p < 0.001). Similarly the CRP value for Group 1 was 0.21 0.35 mg/dl and for Group 2 was 0.1 0.2 mg/dl (p Z 0.023). The PAF-AH activity showing antiinflammatory capacity was 125.17 8.64 (mean SD) for Group 1 and 120.08 9.23 U/ml for Group 2. The PAF-AH activity was identified to have increased in the group with high Psa values and this increase was statistically significant (p Z 0.003). The serum PON1 activity showing antioxidant capacity was 63.12 6.74 and 65.91 7.77 U/ml, respectively and the PON1 activity in the group with high Psa was identified to have reduced. This reduction was statistically significant (p Z 0.046). Additionally, the MDA values showing oxidative levels were 2.88 0.48 and 2.64 0.48 nmol/ml in the groups, respectively (p Z 0.013) (Table 2). The correlations between Psa values and other parameters were investigated. There were positive correlations
Table 1 Characteristics of patients with high Psa values and the control group. Groups
Group 1 (Psa >4) (n)
Group 2 (Psa <4) (n)
p-Value
Age (mean SD) Hypertension Diabetes Pulmonary disease Heart disease
64.74 7.82 19 9 7 7
61.50 9.31 15 9 6 10
0.056 0.509 0.432 0.594 0.182
Table 2
between Psa and CRP (r Z 0.126; p Z 0.77), MDA (r Z 0.124; p Z 0.61); and PAF-AH (r Z 0.107; p Z 0.103), and negative correlations with albumin (r Z 0.46; p Z 0.584) and PON1 (r Z 0.122; p Z 0.062); however these correlations were not statistically significant. There was a positive correlation between PAF-AH and MDA (r Z 0.205; p Z 0.031). There was a significant positive correlation between CRP and MDA (r Z 0.126; p Z 0.020) and PAF-AH (r Z 0.195; p Z 0.007). In the group with biopsy performed, patients were divided into two groups as malignant or benign according to pathology results. In the two groups according to pathology results, parameters measured in serum and demographic characteristics were compared (Table 3). There were no differences between the groups in terms of PAF-AH activity, PON1 activity, MDA, CRP and Psa levels measured in serum (Table 4). When the correlation between parameters is investigated in the groups according to biopsy pathology results, there was a negative correlation of PAF-AH and Psa (r Z 0.287; p Z 0.023) and a positive correlation of PAF-AH and MDA (r Z 0.317; p Z 0.012). The parameters measured in serum were compared between the patients with prostate cancer diagnosis and the control group with normal Psa (Table 5). There was a negative significant correlation between PAF-AH activity and Psa level (r Z 0.402; p Z 0.018) and a positive correlation between PAF-AH activity and MDA level that was close to significance (r Z 0.326, p Z 0.06).
Discussion In this study; in the group with high Psa, CRP, MDA levels and PAF-AH activity increased, while PON1 activity reduced. Additionally comparing the patients with PCa diagnosis with the control group, there were significant differences identified for the same parameters. We consider the cause of the variations occurring in the measured parameters to be the inflammatory process developing in the prostate. At the same time, this is in accordance with previous studies reporting that Psa elevation and prostate cancer develop within an inflammatory framework. Prostate cancer is a very commonly reported disease in males globally. It takes second place among the most commonly diagnosed cancers in the US [1]. The definite cause
Distribution of parameters measured in serum in groups with normal and high Psa values.
Groups
Group 1 (Psa >4) (mean SD)
Group 2 (Psa <4) (mean SD)
p-Value
Psa (ng/dl) IPSS score Prostate volume (ml) Testosterone (ng/dl) PON1 (U/I) PAF-AH (mmol/min/ml) MDA (nmol/ml) Albumin (g/dl) CRP (mg/dl)
6.87 7.27 13.25 8.211 45.2 18.6 5.5 2.0 63.12 6.74a 125.17 8.64 2.88 0.48 4.40 0.28 0.21 0.35a
0.91 0.60 14.4 6.2 55.1 23,1 5.5 2.0 65.91 7.77a 120.08 9.23 2.64 0.48 4.46 0.29 0.10 0.20a
<0.001* 0.425 0.016* 0.22 0.046* 0.003* 0.013* 0.376 0.023*
*: p < 0.05. a IQR given instead of SD.
Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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E. Benli et al.
Table 3 biopsy.
Characteristics of patients who underwent
Groups
Benign group Prostate cancer group
Age (median std.dev) Hypertension Diabetes Pulmonary disease Heart disease
61.25 7.78 67.42 6.80 0.002* 11 7 1 5
8 2 6 2
p-Value
0.149 0.028* 0.218 0.113
of PCa is unknown. Among the most commonly reported risk factors are genetic causes, family history, diet and environmental factors. However, recently there is increasing evidence suggesting that chronic inflammatory events cause the development of both benign and malignant prostate diseases [12]. Many factors in prostate tissue may cause the occurrence of a variety of mediators and cytokines which may begin a chronic inflammatory process [13]. In this environment, oxygen reduces for a period and the result is that oxidative components such as free oxygen radicals begin to accumulate. These conditions may make physiologic survival of normal cells more difficult [14]. In this situation to increase the survival prospects of prostate epithelium against harmful factors, the synthesis of a variety of factors increases led by growth factors. Thus the hyperproliferative environment developing in prostate tissue forms an appropriate environment for cancer formation [15]. The fact that oncogens related to cancer directly or indirectly target pro-inflammatory pathways supports this view. For example, transcription of a variety of mediators
Table 4
like RAS and inflammatory cytokine IL-8 is activated [16]. Again there is a correlation reported between TLR4 (Tolllike receptor 4) carried on the 9th chromosome and PCa. Mutations occurring in the TLR4 gene cause variations in the immune response signals which are proposed as a cause of PCa development [17]. The TLR4 receptor releases proinflammatory cytokines like IL-1, IL-6, and TNF-a which are reported to be effective on the invasion and metastasis processes of a variety of cancer cells [18]. As mentioned above, the close correlation between inflammation and PCa has been observed in surgically removed PCa specimens. A study on the topic by Nickell et al. identified that according to pathology results of 8224 patients who underwent prostate biopsy for elevated Psa, 80% had evidence of chronic inflammation and nearly 15% had evidence of acute inflammation [19]. Psa commonly used in the diagnosis of prostate cancer is not a unique marker for cancer. Apart from cancer, it is affected by many benign situations like ureteral catheterization, urinary tract infections and prostatitis. PCa diagnosis continues to require a new biomarker specific to prostate cancer and not affected by other situations. Recently inflammatory markers like PON1 and PAF-AH levels have been reported as correlated to a variety of diseases with an inflammatory basis. With evidence of an inflammatory basis, prostate diseases may be correlated with these new markers. PON1 is synthesized in the liver and is an ester hydrolase found in plasma in close relationship with HDL. PON1 is thought to moderate the inflammatory process by suppressing oxidative damage. This effect is related to hydrolysis of oxidative LDL, and suppression of chemotaxis and monocytes. Additionally it increases antioxidant capacity, playing an important role in the oxidanteantioxidant balance. Studies on this topic have suggested a close correlation between diseases based on an
Distribution of parameters measured in serum for biopsy patients.
Parameters
Benign group
Prostate cancer group
p-Value
Psa (ng/dl) PON1 (U/I) PAF-AH (mmol/min/ml) MDA (nmol/ml) Albumin (g/dl) CRP (mg/dl)
6.2 3.94 64.39 6.02 124.10 8.66 2.82 0.53 4.41 0.27 0.20 0.25a
7.6 12.9 62.08 7.21 126.05 8.64 2.92 0.45 4.39 0.30 0.23 0.37a
0.008* 0.250 0.415 0.465 0.763 0.603
a
a
a
Median IQR given instead of mean SD.
Table 5
Comparison of patients diagnosed with PCa and the control group with normal Psa values.
Parameters
Prostate cancer (Pca)
Control group
p-Value
Psa (ng/dl) PON1 (U/I) PAF-AH (mmol/min/ml) MDA (nmol/ml) Albumin (g/dl) CRP (mg/dl)
7.6 12.9 62.08 7.21 126.05 8.64 2.93 0.45 4.39 0.31 0.23 0.37a
0.90 0.53 65.96 7.69 120.14 9.14 2.64 0.48 4.46 0.29 0.10 0.22a
<0.001* 0.023* 0.004* 0.007* 0.763 0.026*
a
a
a
Median IQR given instead of mean SD.
Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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PAF-AH and PON1 values in Prostate Cancer inflammatory process such as stroke, diabetes, Parkinsons, atherosclerosis, familial hypercholesterolemia and Alzheimers and PON1 [20]. There are studies showing a correlation between PON1 activity and the carcinogenetic process. A study showed a close correlation between low PON1 activity and increased oxidative DNA damage [21]. This correlation was a focus to show the protective effect of PON1 especially by detoxifying free oxygen radicals against genotoxic damage. A reduction in serum PON1 activity causes accumulation of oxidative components and may begin the carcinogenic process. Studies on the topic have reported a close relationship between PON1 polymorphism and breast, lung, pancreas, ovarian and prostate cancers [22]. These results are supported by other studies [23]. In conclusion, PON1 has antioxidant and detoxification properties that are understood to protect against the harmful effects of inflammatory damage in tissues. In our study PON1 activity was significantly reduced in the group with high Psa value and the group diagnosed with cancer compared to the control group. PON1 level may be beneficial in the early diagnosis of PCa in terms of showing the antioxidant capacity; in other words for the early identification of an inflammatory process. The other marker investigated in our study, PAF-AH enzyme, affects the phospholipid structure mediator of PAF. PAF activates proinflammatory cells like neutrophils and macrophages and inflammatory pathways. It is reported to mediate many biological functions like epithelial apoptosis, increased vascular permeability and smooth muscle contractions [24]. Additionally due to its correlation with proliferation, anti-apoptosis, migration and DNA repair, it is proposed as possibly related to cancer development [25]. A study suppressing PAF reported reduced adhesion to endothelial cells and metastasis of melanoma and colon cancer cells [26]. The PAF-AH enzyme blocks formation of the oxidative and hyperproliferative environment activated by the carcinogenic process by suppressing PAF and PAF similar oxidizing phospholipids [27]. As a result it is considered to have a protective effect against the carcinogenic process. This opinion is supported by the fact that PAF activates enzymes causing destruction of extracellular matrix and basal membrane which are important in the spread of tumor cells to distant tissue. A study on this topic reported reduced motility of tumor cells with increased PAF-AH expression [28]. Another study identified cessation of in-vivo vascularization and cell proliferation of tumor cells and reported increased survival [29]. In short, the amount of evidence supporting the correlation between PAF-AH and cancer in the literature is increasing. However, some studies have suggested contrarily that PAF-AH is an agent responsible for the development of cancer. The reason for this is the proposal that destructive products like the oxidizing fatty acids and lysophosphatidylcholine occurring due to destruction of PAF and PAF similar phospholipids begin the carcinogenic process. Studies on this topic have shown that these compounds stimulate tumor development in tumors like breast cancer, ovarian cancer and multiple myeloma [25]. Another study reported PAF-AH deficiency reduced intestinal polyposis and colon tumor incidence [30]. In a study investigating the relationship between PAF-AH and PCa, genes for the PAFAH enzyme were reported to irregularly regulate prostate
5 cancer. The oncogenic gene of ERG is re-regulated and commonly increases in all stages of prostate cancer. Gene expression analysis reported ERG regulated the transcription of PAF-AH. When the ERG gene is suppressed, PAF-AH expression reduced and they reported an antiproliferative, proapoptotic and antimigratorial effect occurred. PAF-AH is suggested as beneficial for use as a prognostic tumor marker in some cancers like prostate cancer, melanoma, non-Hodgkins lymphoma and hepatocellular cancer [25,31]. In conclusion, there is debate in the literature about whether PAF-AH is anticarcinogenic or procarcinogenic. In our study in the patient group with high Psa and in the group with diagnosis of prostate cancer, PAF-AH activity was identified to increase compared to the control group. We believe the increased inflammatory process may cause this variation. As proposed by some studies above, we believe it increases in response to the inflammatory process rather than as a cause of cancer. In our study, as supported by other parameters, we consider the increase in PAF-AH activity in patients with prostate cancer and high Psa value to be related to the early period increase to suppress the inflammatory process. In conclusion, PAF-AH may be beneficial to determine the inflammatory process early in early diagnosis of prostate cancer. MDA is an important marker of oxidative stress and increases in serum and tissue as a sign of lipid peroxidation. As a result MDA may be used as an oxidative damage index in the inflammatory process. In our study the MDA levels in the group with high Psa and in the PCa group were identified to significantly increase compared with a control group (p Z 0.013 and p Z 0.007, respectively). The presence of an inflammatory environment in patients with prostate cancer and high Psa values was additionally noted in the serum c-reactive protein (CRP) levels. CRP commonly increases as an acute phase reactant during inflammatory events [32]. In our study, CRP activity was significantly increased in the group with high Psa value and the group with prostate cancer diagnosis compared to the control group. Additionally, our results identified a significant positive correlation between CRP and MDA, PAF-AH and Psa. These results are in accordance with the literature [33]. As a result, we believe the entry into use of markers to identify inflammation before PCa diagnosis will expand the horizons of PCa diagnosis. This study has some limitations. The low number of patients and single center results, in addition to not studying inflammatory cytokines and PAF activity are among important deficiencies. Additionally lack of knowledge about long term results of patients with biopsy performed and benign pathology reported is another limitation. However, to the best of our knowledge, this study is the first to compare serum PAF-AH, MDA and PON1 levels between patients with high Psa values and those with prostate cancer diagnosis with healthy peers and we believe it is important for its noteworthy results. Psa, commonly used for diagnosis of prostate cancer, is not an ideal tumor marker. As a result, the use of inflammatory markers like PON1, PAF-AH and MDA for early diagnosis of prostate cancer, which is believed to be related to a chronic inflammatory process, may be beneficial. Early diagnosis of the inflammatory variations may be beneficial
Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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6 in terms of anti-inflammatory treatment to stop the inflammatory process and taking some precautions like increasing antioxidant intake and life changes.
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Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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Kaohsiung Journal of Medical Sciences (2017) xx, 1e6
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
Original Article
Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group Erdal Benli a,*, Ahmet Bayrak b, Abdullah Cirakoglu a, Tulin Bayrak b, Tevfik Noyan b a b
Department of Urology, Ordu University Faculty of Medicine, Ordu, Turkey Department of Biochemistry, Ordu University Faculty of Medicine, Ordu, Turkey
Received 21 February 2017; accepted 10 May 2017
KEYWORDS Oxidative stress; Prostate cancer; PON1; PAF-AH; Psa
Abstract The aim of the study was to measure platelet-activating factor acetyl hydrolase (PAF-AH) and paraoxonase (PON1) enzyme activity levels in patients with high Psa values to compare with healthy peers and also to determine the efficacy of these parameters in predicting pathologic results of patients with high Psa values. This study included 66 patients with Psa value > 4 ng/dl (Group 1) and 44 patients with Psa <4 ng/dl (Group 2) for a total of 110 patients. Parameters measured in serum of PON1, PAF-AH, and MDA were compared between the groups. Additionally the same parameters were compared between patients with prostate biopsy performed due to high Psa and diagnosed with cancer and the control group with normal Psa values. The PAF-AH activity in Group 1 was 125.17 8.64 and in Group 2 was 120.08 9.23 U/ml (p Z 0.003). The PON1 activity was 63.12 6.74 and 65.91 7.77 U/ml in the groups, respectively (p Z 0.04). Additionally, there were significant differences identified between the control group and PCa diagnosis group in terms of PAF-AH and PON1 activities (p Z 0.004 and p Z 0.02, respectively). The enzyme activity of PAF-AH and PON1 measured in serum of patients with high Psa value and patients with diagnosis of prostate cancer (PCa) were identified to have changed by a significant amount compared to healthy peers with normal Psa value. It was concluded that these parameters may be beneficial markers for use in assessment of patients with high Psa value. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Conflicts of interest: All authors declare no conflicts of interest. * Corresponding author. Department of Urology, Ordu University Faculty of Medicine, 52100, Ordu, Turkey. E-mail address: [email protected] (E. Benli). http://dx.doi.org/10.1016/j.kjms.2017.06.016 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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Introduction Prostate cancer (PCa) is the most commonly diagnosed male cancer in the United States, similar to most European countries [1]. The mechanism of prostate carcinogenesis is not completely understood, but evidence suggests that oxidative stress plays a role [2]. Most cells in healthy individuals have adequate antioxidant defenses to protect them from reactive oxygen species (ROS). However, this capacity diminishes with age and can be overwhelmed by an abundance of ROS from exogenous or endogenous sources, or by enzymatic deficiencies due to polymorphisms and mutations [3]. It is well known that oxidative stress, due to a defect in the antioxidant systems or the presence of excessive reactive oxygen species, plays a role in the etiopathogenesis of PCa [4]. Plasma contains various antioxidant molecules including albumin which is the major circulating antioxidant in both plasma and serum [5]. In addition, the most important extracellular enzymes that are involved in this process are paraoxonase (PON1) and platelet-activating factor acetyl hydrolase (PAF-AH) [6]. PON1 mainly conjugates the intermediates to excretable hydrophilic derivatives and binds to high-density lipoprotein (HDL). Thus, it plays a role in the detoxification process of organophosphorus compounds and carcinogenic lipidsoluble radicals from lipid peroxidation [7]. PAF-AH plays an essential role in lipid metabolism as an antioxidant molecule, which hydrolyzes both PAF (a potent inflammatory mediator) and oxidized phospholipids [7]. Since enzyme levels were found to be decreased or increased after inflammatory stimuli in animal models, it is unclear whether PAF-AH is an acute-phase response protein [8]. Though Psa is commonly used in PCa screening, Psa is known to be affected by many benign situations. As a result the search continues for the ideal tumor marker specific to prostate cancer and not affected by benign situations. To our knowledge, there is no study in the literature measuring the activity of PON1, PAF-AH and oxidative balance in patients with PCa. The aim of this study was to identify oxidative stress in PCa patients, and investigate whether new generation biochemical parameters or those that are previously known are associated with the risk of incidental PCa.
E. Benli et al. and supplying written consent. Serum for laboratory tests was obtained after 12 h starvation in the morning and stored at 80 C until laboratory studies. The patients’ ages, BMI, complaint at application, additional diseases, smoking habit and known laboratory tests like Psa and CRP were recorded. Parameters measured in serum were compared between healthy individuals with normal Psa value (control group), the group with high Psa value and patients with prostate cancer diagnosis.
Biochemical study PON1 activity measurement PON1 activity was measured spectrophotometrically at 270 nm based on phenol formed as a result of enzymatic hydrolysis of phenylacetate used as substrate. The solution reaction environment was created with 50 mM TriseHCl (pH: 8) containing 1 mM calcium chloride (CaCl2). The final concentration of the reaction environment was 1 mM phenylacetate and an appropriate amount of enzyme was added to begin the reaction. At the end of the reaction, the phenol formed was spectrophotometrically measured at 270 nm [9]. One unit of PON1 activity is defined as 1 mmol of substrate hydrolyzed per min, under the defined assay conditions.
PAF-AH activity measurement The PAF-AH activity used 2-thio PAF as substrate. Upon hydrolysis of the acetyl thioester bond at the sn-2 position by PAF-AH, free thiols are detected using DTNB (5,50 -dithiobis(2-nitrobenzoic acid)) and the 5-thio-2 nitrobenzoic acid is spectrophotometrically measured at 412 nm. The activity measurement environment is created with final volume of 1 ml, with 10 mM triseHCl (pH: 7.4), DTNB, synthetic substrate platelet-activating factor (PAF) and the serum to be measured. The 5-thio-2-nitrobenzoic acid formed as a result of the reaction is measured at 412 nm spectrophotometrically. One unit of PAF-AH activity is defined as 1 nmol of substrate hydrolyzed per min, under the defined assay conditions [10].
Level malondialdehyde (MDA) determination
Methods Study design This study received permission from Ordu University local ethics committee (2016/34). The study included a total of 110 individuals over the age of 40; 66 patients with Psa value 4 ng/dl (Group 1) and 44 patients with Psa value < 4 ng/dl as control group (Group 2). Patients with high Psa value had Psa values rechecked. Exclusion criteria included situations like systemic infection, urinary tract infections, recent urologic procedures, presence of ureteral catheter, systemic antibiotic use and prostatitis. Patients with Psa value 4 ng/dl underwent sextant prostate biopsy accompanied by transrectal ultrasound and antibiotic prophylaxis after receiving necessary information
The lipid peroxidation product of one molecule malondialdehyde (MDA) enters a reaction with 2 molecules of thiobarbituric acid (TBA) at pH 2e3 between 95 and 100 C. The pink-colored chromogene formed by the reaction is extracted with n-butyl alcohol and the obtained organic phase is read at 523 nm [11].
Statistical analyses Parameters with normal distribution are presented as mean SD while parameters with non-normal distribution are presented as median IQR. Comparison between groups used the Student t test for parametric data analysis and the ManneWhitney U test for nonparametric data. Correlation analysis of parametric data was completed with
Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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PAF-AH and PON1 values in Prostate Cancer
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the Pearson test, while the Spearman correlation test was used for non-parametic data. All statistical analyses were completed with “SPSS for Windows version 20.0” program. A p value < 0.05 was accepted as significant.
Results The age distribution of patients participating in the study showed the mean age in Group 1 with Psa >4 ng/dl was 64.74 7.82 (50e80 years), while in Group 2, the control group, the mean age was 61.50 9.31 (38e85 years) (p Z 0.06) (Table 1). In terms of mean Psa value when the groups are compared, Group 1 was 6.87 7.27 (3.9e70) ng/dl (median IQR) and Group 2 was 0.91 0.60 (0.2e3.8) ng/ dl (p < 0.001). Similarly the CRP value for Group 1 was 0.21 0.35 mg/dl and for Group 2 was 0.1 0.2 mg/dl (p Z 0.023). The PAF-AH activity showing antiinflammatory capacity was 125.17 8.64 (mean SD) for Group 1 and 120.08 9.23 U/ml for Group 2. The PAF-AH activity was identified to have increased in the group with high Psa values and this increase was statistically significant (p Z 0.003). The serum PON1 activity showing antioxidant capacity was 63.12 6.74 and 65.91 7.77 U/ml, respectively and the PON1 activity in the group with high Psa was identified to have reduced. This reduction was statistically significant (p Z 0.046). Additionally, the MDA values showing oxidative levels were 2.88 0.48 and 2.64 0.48 nmol/ml in the groups, respectively (p Z 0.013) (Table 2). The correlations between Psa values and other parameters were investigated. There were positive correlations
Table 1 Characteristics of patients with high Psa values and the control group. Groups
Group 1 (Psa >4) (n)
Group 2 (Psa <4) (n)
p-Value
Age (mean SD) Hypertension Diabetes Pulmonary disease Heart disease
64.74 7.82 19 9 7 7
61.50 9.31 15 9 6 10
0.056 0.509 0.432 0.594 0.182
Table 2
between Psa and CRP (r Z 0.126; p Z 0.77), MDA (r Z 0.124; p Z 0.61); and PAF-AH (r Z 0.107; p Z 0.103), and negative correlations with albumin (r Z 0.46; p Z 0.584) and PON1 (r Z 0.122; p Z 0.062); however these correlations were not statistically significant. There was a positive correlation between PAF-AH and MDA (r Z 0.205; p Z 0.031). There was a significant positive correlation between CRP and MDA (r Z 0.126; p Z 0.020) and PAF-AH (r Z 0.195; p Z 0.007). In the group with biopsy performed, patients were divided into two groups as malignant or benign according to pathology results. In the two groups according to pathology results, parameters measured in serum and demographic characteristics were compared (Table 3). There were no differences between the groups in terms of PAF-AH activity, PON1 activity, MDA, CRP and Psa levels measured in serum (Table 4). When the correlation between parameters is investigated in the groups according to biopsy pathology results, there was a negative correlation of PAF-AH and Psa (r Z 0.287; p Z 0.023) and a positive correlation of PAF-AH and MDA (r Z 0.317; p Z 0.012). The parameters measured in serum were compared between the patients with prostate cancer diagnosis and the control group with normal Psa (Table 5). There was a negative significant correlation between PAF-AH activity and Psa level (r Z 0.402; p Z 0.018) and a positive correlation between PAF-AH activity and MDA level that was close to significance (r Z 0.326, p Z 0.06).
Discussion In this study; in the group with high Psa, CRP, MDA levels and PAF-AH activity increased, while PON1 activity reduced. Additionally comparing the patients with PCa diagnosis with the control group, there were significant differences identified for the same parameters. We consider the cause of the variations occurring in the measured parameters to be the inflammatory process developing in the prostate. At the same time, this is in accordance with previous studies reporting that Psa elevation and prostate cancer develop within an inflammatory framework. Prostate cancer is a very commonly reported disease in males globally. It takes second place among the most commonly diagnosed cancers in the US [1]. The definite cause
Distribution of parameters measured in serum in groups with normal and high Psa values.
Groups
Group 1 (Psa >4) (mean SD)
Group 2 (Psa <4) (mean SD)
p-Value
Psa (ng/dl) IPSS score Prostate volume (ml) Testosterone (ng/dl) PON1 (U/I) PAF-AH (mmol/min/ml) MDA (nmol/ml) Albumin (g/dl) CRP (mg/dl)
6.87 7.27 13.25 8.211 45.2 18.6 5.5 2.0 63.12 6.74a 125.17 8.64 2.88 0.48 4.40 0.28 0.21 0.35a
0.91 0.60 14.4 6.2 55.1 23,1 5.5 2.0 65.91 7.77a 120.08 9.23 2.64 0.48 4.46 0.29 0.10 0.20a
<0.001* 0.425 0.016* 0.22 0.046* 0.003* 0.013* 0.376 0.023*
*: p < 0.05. a IQR given instead of SD.
Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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Table 3 biopsy.
Characteristics of patients who underwent
Groups
Benign group Prostate cancer group
Age (median std.dev) Hypertension Diabetes Pulmonary disease Heart disease
61.25 7.78 67.42 6.80 0.002* 11 7 1 5
8 2 6 2
p-Value
0.149 0.028* 0.218 0.113
of PCa is unknown. Among the most commonly reported risk factors are genetic causes, family history, diet and environmental factors. However, recently there is increasing evidence suggesting that chronic inflammatory events cause the development of both benign and malignant prostate diseases [12]. Many factors in prostate tissue may cause the occurrence of a variety of mediators and cytokines which may begin a chronic inflammatory process [13]. In this environment, oxygen reduces for a period and the result is that oxidative components such as free oxygen radicals begin to accumulate. These conditions may make physiologic survival of normal cells more difficult [14]. In this situation to increase the survival prospects of prostate epithelium against harmful factors, the synthesis of a variety of factors increases led by growth factors. Thus the hyperproliferative environment developing in prostate tissue forms an appropriate environment for cancer formation [15]. The fact that oncogens related to cancer directly or indirectly target pro-inflammatory pathways supports this view. For example, transcription of a variety of mediators
Table 4
like RAS and inflammatory cytokine IL-8 is activated [16]. Again there is a correlation reported between TLR4 (Tolllike receptor 4) carried on the 9th chromosome and PCa. Mutations occurring in the TLR4 gene cause variations in the immune response signals which are proposed as a cause of PCa development [17]. The TLR4 receptor releases proinflammatory cytokines like IL-1, IL-6, and TNF-a which are reported to be effective on the invasion and metastasis processes of a variety of cancer cells [18]. As mentioned above, the close correlation between inflammation and PCa has been observed in surgically removed PCa specimens. A study on the topic by Nickell et al. identified that according to pathology results of 8224 patients who underwent prostate biopsy for elevated Psa, 80% had evidence of chronic inflammation and nearly 15% had evidence of acute inflammation [19]. Psa commonly used in the diagnosis of prostate cancer is not a unique marker for cancer. Apart from cancer, it is affected by many benign situations like ureteral catheterization, urinary tract infections and prostatitis. PCa diagnosis continues to require a new biomarker specific to prostate cancer and not affected by other situations. Recently inflammatory markers like PON1 and PAF-AH levels have been reported as correlated to a variety of diseases with an inflammatory basis. With evidence of an inflammatory basis, prostate diseases may be correlated with these new markers. PON1 is synthesized in the liver and is an ester hydrolase found in plasma in close relationship with HDL. PON1 is thought to moderate the inflammatory process by suppressing oxidative damage. This effect is related to hydrolysis of oxidative LDL, and suppression of chemotaxis and monocytes. Additionally it increases antioxidant capacity, playing an important role in the oxidanteantioxidant balance. Studies on this topic have suggested a close correlation between diseases based on an
Distribution of parameters measured in serum for biopsy patients.
Parameters
Benign group
Prostate cancer group
p-Value
Psa (ng/dl) PON1 (U/I) PAF-AH (mmol/min/ml) MDA (nmol/ml) Albumin (g/dl) CRP (mg/dl)
6.2 3.94 64.39 6.02 124.10 8.66 2.82 0.53 4.41 0.27 0.20 0.25a
7.6 12.9 62.08 7.21 126.05 8.64 2.92 0.45 4.39 0.30 0.23 0.37a
0.008* 0.250 0.415 0.465 0.763 0.603
a
a
a
Median IQR given instead of mean SD.
Table 5
Comparison of patients diagnosed with PCa and the control group with normal Psa values.
Parameters
Prostate cancer (Pca)
Control group
p-Value
Psa (ng/dl) PON1 (U/I) PAF-AH (mmol/min/ml) MDA (nmol/ml) Albumin (g/dl) CRP (mg/dl)
7.6 12.9 62.08 7.21 126.05 8.64 2.93 0.45 4.39 0.31 0.23 0.37a
0.90 0.53 65.96 7.69 120.14 9.14 2.64 0.48 4.46 0.29 0.10 0.22a
<0.001* 0.023* 0.004* 0.007* 0.763 0.026*
a
a
a
Median IQR given instead of mean SD.
Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016
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PAF-AH and PON1 values in Prostate Cancer inflammatory process such as stroke, diabetes, Parkinsons, atherosclerosis, familial hypercholesterolemia and Alzheimers and PON1 [20]. There are studies showing a correlation between PON1 activity and the carcinogenetic process. A study showed a close correlation between low PON1 activity and increased oxidative DNA damage [21]. This correlation was a focus to show the protective effect of PON1 especially by detoxifying free oxygen radicals against genotoxic damage. A reduction in serum PON1 activity causes accumulation of oxidative components and may begin the carcinogenic process. Studies on the topic have reported a close relationship between PON1 polymorphism and breast, lung, pancreas, ovarian and prostate cancers [22]. These results are supported by other studies [23]. In conclusion, PON1 has antioxidant and detoxification properties that are understood to protect against the harmful effects of inflammatory damage in tissues. In our study PON1 activity was significantly reduced in the group with high Psa value and the group diagnosed with cancer compared to the control group. PON1 level may be beneficial in the early diagnosis of PCa in terms of showing the antioxidant capacity; in other words for the early identification of an inflammatory process. The other marker investigated in our study, PAF-AH enzyme, affects the phospholipid structure mediator of PAF. PAF activates proinflammatory cells like neutrophils and macrophages and inflammatory pathways. It is reported to mediate many biological functions like epithelial apoptosis, increased vascular permeability and smooth muscle contractions [24]. Additionally due to its correlation with proliferation, anti-apoptosis, migration and DNA repair, it is proposed as possibly related to cancer development [25]. A study suppressing PAF reported reduced adhesion to endothelial cells and metastasis of melanoma and colon cancer cells [26]. The PAF-AH enzyme blocks formation of the oxidative and hyperproliferative environment activated by the carcinogenic process by suppressing PAF and PAF similar oxidizing phospholipids [27]. As a result it is considered to have a protective effect against the carcinogenic process. This opinion is supported by the fact that PAF activates enzymes causing destruction of extracellular matrix and basal membrane which are important in the spread of tumor cells to distant tissue. A study on this topic reported reduced motility of tumor cells with increased PAF-AH expression [28]. Another study identified cessation of in-vivo vascularization and cell proliferation of tumor cells and reported increased survival [29]. In short, the amount of evidence supporting the correlation between PAF-AH and cancer in the literature is increasing. However, some studies have suggested contrarily that PAF-AH is an agent responsible for the development of cancer. The reason for this is the proposal that destructive products like the oxidizing fatty acids and lysophosphatidylcholine occurring due to destruction of PAF and PAF similar phospholipids begin the carcinogenic process. Studies on this topic have shown that these compounds stimulate tumor development in tumors like breast cancer, ovarian cancer and multiple myeloma [25]. Another study reported PAF-AH deficiency reduced intestinal polyposis and colon tumor incidence [30]. In a study investigating the relationship between PAF-AH and PCa, genes for the PAFAH enzyme were reported to irregularly regulate prostate
5 cancer. The oncogenic gene of ERG is re-regulated and commonly increases in all stages of prostate cancer. Gene expression analysis reported ERG regulated the transcription of PAF-AH. When the ERG gene is suppressed, PAF-AH expression reduced and they reported an antiproliferative, proapoptotic and antimigratorial effect occurred. PAF-AH is suggested as beneficial for use as a prognostic tumor marker in some cancers like prostate cancer, melanoma, non-Hodgkins lymphoma and hepatocellular cancer [25,31]. In conclusion, there is debate in the literature about whether PAF-AH is anticarcinogenic or procarcinogenic. In our study in the patient group with high Psa and in the group with diagnosis of prostate cancer, PAF-AH activity was identified to increase compared to the control group. We believe the increased inflammatory process may cause this variation. As proposed by some studies above, we believe it increases in response to the inflammatory process rather than as a cause of cancer. In our study, as supported by other parameters, we consider the increase in PAF-AH activity in patients with prostate cancer and high Psa value to be related to the early period increase to suppress the inflammatory process. In conclusion, PAF-AH may be beneficial to determine the inflammatory process early in early diagnosis of prostate cancer. MDA is an important marker of oxidative stress and increases in serum and tissue as a sign of lipid peroxidation. As a result MDA may be used as an oxidative damage index in the inflammatory process. In our study the MDA levels in the group with high Psa and in the PCa group were identified to significantly increase compared with a control group (p Z 0.013 and p Z 0.007, respectively). The presence of an inflammatory environment in patients with prostate cancer and high Psa values was additionally noted in the serum c-reactive protein (CRP) levels. CRP commonly increases as an acute phase reactant during inflammatory events [32]. In our study, CRP activity was significantly increased in the group with high Psa value and the group with prostate cancer diagnosis compared to the control group. Additionally, our results identified a significant positive correlation between CRP and MDA, PAF-AH and Psa. These results are in accordance with the literature [33]. As a result, we believe the entry into use of markers to identify inflammation before PCa diagnosis will expand the horizons of PCa diagnosis. This study has some limitations. The low number of patients and single center results, in addition to not studying inflammatory cytokines and PAF activity are among important deficiencies. Additionally lack of knowledge about long term results of patients with biopsy performed and benign pathology reported is another limitation. However, to the best of our knowledge, this study is the first to compare serum PAF-AH, MDA and PON1 levels between patients with high Psa values and those with prostate cancer diagnosis with healthy peers and we believe it is important for its noteworthy results. Psa, commonly used for diagnosis of prostate cancer, is not an ideal tumor marker. As a result, the use of inflammatory markers like PON1, PAF-AH and MDA for early diagnosis of prostate cancer, which is believed to be related to a chronic inflammatory process, may be beneficial. Early diagnosis of the inflammatory variations may be beneficial
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6 in terms of anti-inflammatory treatment to stop the inflammatory process and taking some precautions like increasing antioxidant intake and life changes.
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Please cite this article in press as: Benli E, et al., Comparison of serum acetyl hydrolase (PAF-AH) and paraoxonase 1 (PON1) values between prostate cancer patients and a control group, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/ j.kjms.2017.06.016